Use of ire1α-XBP1 signaling pathway biomarkers for modulating immune responses

Abstract

To provide use of biomarkers of IRE1α-XBP1 signaling pathway for modulating immune responses.SOLUTION: The invention is based, in part, on the identification of an IRE1α-XBP1-cMyc axis in NK cell immunity. The invention provides compositions and methods for treating conditions that would benefit from modulating (e.g., upregulating or downregulating) an immune response using an agent that modulates the IRE1α-XBP1 pathway, or a composition comprising modified NK cells.SELECTED DRAWING: Figure 4B

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 769,265, filed November 19, 2018, and U.S. Provisional Patent Application No. 62 / 800,706, filed February 04, 2019, the entire contents of each of which are incorporated herein by reference in their entirety.

[0002] Government funding This invention was made with government support under Grant Nos. T32GM007739 and F30 AI136239-01A1 awarded by the National Institutes of Health. The government has certain rights in this invention. [Background technology]

[0003] NK cells are important mediators of host immunity against malignancies and viral infections (Caligiuri et al. (2008) Blood 112:461-469). Although extrinsic regulators of NK cell development and function have been identified, including diverse ligands for major NK cell receptors and microenvironment-derived proinflammatory cytokines (Cooper et al. (2009) Proc. Natl. Acad. Sci. USA 106:1915-1919, Madera et al. (2016) J. Exp. Med. 213:225-33, Zawislak et al. (2013) Proc. Natl. Acad. Sci. USA 110:6967-6972, Sun et al. (2012) J. Exp. Med. 209:947-954), relatively little is known about how NK cells mechanistically translate these signals into critical effector functions (Beaulieu et al. (2014) Nat. Immunol. 15:546-553, Rapp et al. (2014) J. Exp. Med. 209:947-954). al. (2017) Sci. Immunol. 2(18)). Furthermore, novel features of NK cells have emerged in recent years, including their ability to undergo clonal expansion and generate long-lasting memory, although the molecular mechanisms underlying these "adoptive" properties require further characterization. Renewed interest in NK cell-based cancer immunotherapy (Morvan and Lanier (2016) Nat. Rev. Cancer 16:7-19, Vivier et al. (2012) Nat. Rev. Immunol. 12:239-252) and developing new vaccine strategies to control deadly infectious diseases highlights the urgent need to identify new and unique regulators of NK cell-mediated immunity.

[0004] Activation of the ER stress sensor IRE1α and its substrate transcription factor XBP1 (Yoshida et al. (2001) Cell 107:881-891, Lee et al. (2003) Mol. Cell. Biol. 23:7448-7459) is a hallmark of “professional” secretory cells that must constitutively cope with the high demands for protein synthesis, folding, and secretion (Hess et al. (2011) Gastroenterol. 141:1463-1472, Lee et al. (2011) Proc. Natl. Acad. Sci. USA 108:8885-8890, Lee et al. (2008) Science 320:1492-1496, Kaser et al. (2008) Cell 134:743-756). This highly evolutionarily conserved signaling pathway is involved in the regulation of tumor cells (Chen et al. (2014) Nature 508:103-107), myeloid-derived suppressor cells (Condamine et al. (2014) J. Clin. Invest. 124:2626-2639), macrophages (Yan et al. (2014) J. Clin. Invest. 124:2626-2639), and inflammatory cytokines (Yan et al. (2014) J. Clin. Invest. 124:2626-2639). et al. (2016) Cell Rep. 16:2914-2927), T cells (Song et al. (2018) Nature 562:423-428), and dendritic cells (Cubillos-Ruiz et al. (2015) Cell 161:1527-1538). However, it is unclear whether NK cell function is driven by IRE1α-XBP1 signaling and, if so, what specific activity it controls.

[0005] Therefore, there is a great need in the art to uncover the unique pathways that control NK cell responses in order to develop new NK cell-based immunotherapies. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Caligiuri et al. (2008) Blood 112:461-469 [Non-licensed document 2] Cooper et al. (2009) Proc.Natl.Acad.Sci.USA 106:1915-1919 [Non-licensed document 3] Madera et al.(2016)J.Exp.Med.213:225-33

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[0007] The present invention is based, at least in part, on the discovery that the ER stress sensor inositol-requiring enzyme 1 (IRE1α) and its substrate transcription factor X-box binding protein 1 (XBP1) significantly drive NK cell-mediated responses to viral infection and tumors in vivo and promote homeostatic proliferation. IRE1α and XBP1 were found to be important for robust hypertrophy of activated mouse and human NK cells and located downstream of the mTOR signaling pathway. In addition, transcriptome and chromatin immunoprecipitation analyses revealed c-Myc as a novel and direct downstream target of XBP1 for downstream control of NK cell proliferation. Genetic ablation or pharmaceutical blockade of IRE1α downregulated c-Myc, while overexpression of XBP1 caused c-Myc hyperactivation. NK cells haploinsufficient for c-Myc exhibited functional defects comparable to those lacking IRE1α or XBP1. Genetic overexpression of c-Myc significantly rescued the proliferation defect in IRE1α-deficient NK cells. Consistent with its binding to c-Myc, IRE1α / XBP1 also promotes oxidative phosphorylation in NK cells. This study identifies an IRE1α-XBP1-cMyc axis in NK cell immunity and provides new insights into host protection against infection and cancer.

[0008] In one aspect, a composition is provided comprising natural killer (NK) cells modified to upregulate the IRE1α-XBP1 pathway.

[0009] Numerous embodiments are further provided that can be applied to any aspect of the invention described herein. For example, in one embodiment, the IRE1α-XBP1 pathway is upregulated by increasing the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in NK cells. In another embodiment, the copy number, amount, and / or activity of at least one biomarker listed in Table 1 is increased by contacting NK cells with a nucleic acid molecule or fragment thereof encoding at least one biomarker listed in Table 1, a polypeptide or fragment thereof of at least one biomarker listed in Table 1, a small molecule binding to at least one biomarker listed in Table 1, or a pro-inflammatory cytokine. In yet another embodiment, the pro-inflammatory cytokine is IL-2, IL-15, IL-12, and / or IL-18. In yet another embodiment, NK cells have increased splicing of XBP1 to XBP1s transcripts. In another embodiment, expression of XBP1 target genes is upregulated in NK cells. In yet another embodiment, the XBP1 target gene is c-Myc or a canonical XBP1 target gene selected from the group consisting of Hspa5, Dnajb9, Sec24d, Sec63, Hyou1, Sec61a, and P4hb. In yet another embodiment, expression of the c-Myc target gene is upregulated in NK cells. In another embodiment, oxidative phosphorylation (OXPHOS) is upregulated in NK cells. In yet another embodiment, the NK cells are activated NK cells and / or memory NK cells, optionally, the memory NK cells are cytokine-induced memory-like NK cells (CIML). In yet another embodiment, the NK cells are Ly49H-expressing NK cells or CD56 bright In another embodiment, the NK cells are derived from peripheral blood mononuclear cells (PBMCs) or umbilical cord blood (UCB). In yet another embodiment, the composition is derived from an NK cell line.

[0010] In another aspect, a method of treating a subject having a condition that may benefit from upregulation of an immune response is provided, comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0011] As mentioned above, certain embodiments are applicable to any of the methods described herein. For example, in one embodiment, the NK cells are derived from a subject treated with the composition. In another embodiment, the NK cells are derived from a different subject not treated with the composition. In yet another embodiment, the condition is an infection. In yet another embodiment, the infection is a viral infection, a bacterial infection, a protozoan infection, a parasitic infection, a fungal infection, or a helminth infection. In another embodiment, the viral infection is caused by a virus selected from the group consisting of CMV, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), influenza A virus, Epstein-Barr virus (EBV), human herpes simplex virus (HSV) types 1 and 2, respiratory syncytial virus (RSV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), Zika virus, Rift Valley fever virus (RVFV), dengue virus (DENV), chikungunya virus (CHIKV), enterovirus (EV), and human adenovirus (HAdV). In yet another embodiment, the composition promotes antiviral immunity in a subject. In yet another embodiment, the composition reduces viral titers in a subject. In another embodiment, the composition increases overall survival. In another embodiment, the bacterial infection is caused by Listeria monocytogenes, Mycobacterium tuberculosis, or Salmonella typhimurium. In yet another embodiment, the parasitic infection is caused by Plasmodium or Cryptosporidium, optionally wherein the Plasmodium is a malaria parasite. In yet another embodiment, the fungal infection is caused by Aspergillus, optionally wherein the Aspergillus is Aspergillus fumigatus. In another embodiment, the condition is lymphopenia. In yet another embodiment, the subject has undergone hematopoietic cell transplantation (HCT). In yet another embodiment, the condition is cancer. In another embodiment, the cancer is an NK cell-sensitive cancer.In yet another embodiment, the cancer is selected from the group consisting of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, and cervical cancer. In yet another embodiment, the composition promotes anti-tumor immunity in a subject. In another embodiment, the composition increases the amount of NK cells infiltrating a tumor. In yet another embodiment, the composition increases the amount of conventional type 1 dendritic cells and / or CD8+ T cells infiltrating a tumor. In yet another embodiment, the composition reduces the number of proliferating cells in cancer and / or reduces the volume or size of a tumor containing cancer cells. In another embodiment, the methods described herein further include administering immunotherapy and / or cancer therapy to the subject, optionally administering the immunotherapy and / or cancer therapy before, after, or simultaneously with the composition. In yet another embodiment, the immunotherapy is cell-based. In yet another embodiment, the immunotherapy comprises a cancer composition and / or a virus. In another embodiment, the immunotherapy inhibits an immune checkpoint. In yet another embodiment, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilin, and A2aR. In yet another embodiment, the cancer therapy is selected from the group consisting of radiation, radiosensitizers, and chemotherapy.

[0012] In yet another aspect, a method is provided for treating a subject having a condition that may benefit from upregulation of an immune response, the method comprising administering to the subject a therapeutically effective amount of an agent that upregulates the IRE1α-XBP1 pathway such that the condition that may benefit from upregulation of an immune response is treated.

[0013] As mentioned above, certain embodiments are applicable to any of the methods described herein. For example, in one embodiment, the agent upregulates the IRE1α-XBP1 pathway in NK cells. In another embodiment, the NK cells are activated NK cells and / or memory NK cells, and optionally, the memory NK cells are cytokine-induced memory-like NK cells (CIML). In yet another embodiment, the NK cells are Ly49H-expressing NK cells or CD56 brightIn yet another embodiment, the agent is an NK cell. In yet another embodiment, the agent upregulates the IRE1α-XBP1 pathway by increasing the copy number, amount, and / or activity of at least one biomarker listed in Table 1. In another embodiment, the agent is a nucleic acid molecule or fragment thereof encoding at least one biomarker listed in Table 1, a polypeptide or fragment thereof of at least one biomarker listed in Table 1, a small molecule that binds to at least one biomarker listed in Table 1, or a pro-inflammatory cytokine. In yet another embodiment, the pro-inflammatory cytokine is IL-2, IL-15, IL-12, and / or IL-18. In yet another embodiment, the agent promotes splicing of XBP1 to XBP1s transcripts. In another embodiment, the agent upregulates an XBP1 target gene. In yet another embodiment, the XBP1 target gene is c-Myc or a canonical XBP1 target gene selected from the group consisting of Hspa5, Dnajb9, Sec24d, Sec63, Hyou1, Sec61a, and P4hb. In yet another embodiment, the agent upregulates expression of a c-Myc target gene. In another embodiment, the agent upregulates oxidative phosphorylation (OXPHOS) in NK cells. In yet another embodiment, the agent promotes NK cell proliferation. In yet another embodiment, the condition is an infection. In another embodiment, the infection is a viral infection, a bacterial infection, a protozoan infection, or a helminth infection. In yet another embodiment, the viral infection is caused by a virus selected from the group consisting of CMV, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), influenza A virus, Epstein-Barr virus (EBV), human herpes simplex virus (HSV) types 1 and 2, respiratory syncytial virus (RSV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), Zika virus, Rift Valley fever virus (RVFV), dengue virus (DENV), chikungunya virus (CHIKV), enterovirus (EV), and human adenovirus (HAdV). In yet another embodiment, the agent promotes antiviral immunity in a subject.In another embodiment, the agent promotes clonal expansion of NK cells during viral infection. In yet another embodiment, the agent reduces viral titer in the subject. In yet another embodiment, the agent increases overall survival. In another embodiment, the bacterial infection is caused by Listeria monocytogenes, Mycobacterium tuberculosis, or Salmonella typhimurium. In yet another embodiment, the parasitic infection is caused by Plasmodium or Cryptosporidium, optionally where the Plasmodium is Plasmodium. In yet another embodiment, the fungal infection is caused by Aspergillus, optionally where the Aspergillus is Aspergillus fumigatus. In another embodiment, the condition is lymphopenia. In yet another embodiment, the subject has undergone HCT or NK cell adoptive transfer immunotherapy, and optionally the NK cells are genetically modified. In yet another embodiment, the agent promotes homeostatic proliferation of NK cells in vivo. In another embodiment, the condition is cancer. In yet another embodiment, the cancer is an NK cell-sensitive cancer. In yet another embodiment, the cancer is selected from the group consisting of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, and cervical cancer. In another embodiment, the agent promotes NK cell-mediated anti-tumor immunity. In yet another embodiment, the agent increases the number of NK cells infiltrating tumors. In yet another embodiment, the agent increases the number of tumor-infiltrating conventional type 1 dendritic cells and / or tumor-infiltrating CD8+ T cells. In another embodiment, the agent reduces the number of proliferating cells in the cancer and / or reduces the volume or size of tumors containing cancer cells.In yet another embodiment, the method described herein further comprises administering to the subject immunotherapy and / or cancer therapy, optionally administered before, after, or simultaneously with the composition. In yet another embodiment, the immunotherapy is cell-based. In another embodiment, the immunotherapy comprises a cancer vaccine and / or a virus. In yet another embodiment, the immunotherapy inhibits an immune checkpoint. In yet another embodiment, the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilin, and A2aR. In another embodiment, the cancer therapy is selected from the group consisting of radiation, radiosensitizers, and chemotherapy.

[0014] In yet another aspect, a method is provided for treating a subject having a condition that may benefit from downregulation of an immune response, the method comprising administering to the subject a therapeutically effective amount of an agent that downregulates the IRE1α-XBP1 pathway such that the condition that may benefit from downregulation of an immune response is treated.

[0015] As mentioned above, certain embodiments are applicable to any of the methods described herein. For example, in one embodiment, the agent downregulates the IRE1α-XBP1 pathway in NK cells. In another embodiment, the NK cells are activated NK cells and / or memory NK cells, and optionally, the memory NK cells are cytokine-induced memory-like NK cells (CIML). In yet another embodiment, the NK cells are Ly49H-expressing NK cells or CD56 brightNK cells. In yet another embodiment, the agent downregulates the IRE1α-XBP1 pathway by decreasing the copy number, amount, and / or activity of at least one biomarker listed in Table 1. In another embodiment, the agent is a small molecule inhibitor, a CRISPR guide RNA (gRNA), an RNA interference agent, an antisense oligonucleotide, a peptide or peptidomimetic inhibitor, an aptamer, an antibody, or an intrabody. In yet another embodiment, the RNA interference agent is a small interfering RNA (siRNA), a CRISPR RNA (crRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), or a piwi-interacting RNA (piRNA). In yet another embodiment, the agent comprises an antibody and / or intrabody, or an antigen-binding fragment thereof, that specifically binds to at least one biomarker listed in Table 1. In another embodiment, the antibody and / or intrabody, or an antigen-binding fragment thereof, is murine, chimeric, humanized, composite, or human. In yet another embodiment, the antibody and / or intrabody, or antigen-binding fragment thereof, is detectably labeled, comprises an effector domain, comprises an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. In yet another embodiment, the agent is a small molecule inhibitor of the IRE1α RNase domain. In another embodiment, the small molecule inhibitor is 4μ8c (also known as 8-formyl-7-hydroxy-4-methylcoumarin or 7-hydroxy-4-methyl-2-oxo-2H-chromene-8-carbaldehyde or CAS14003-96-4). In yet another embodiment, the agent reduces splicing of XBP1 to XBP1s transcripts. In yet another embodiment, the agent downregulates XBP1 target genes. In another embodiment, the XBP1 target gene is c-Myc or a canonical XBP1 target gene selected from the group consisting of Hspa5, Dnajb9, Sec24d, Sec63, Hyou1, Sec61a, and P4hb. In yet another embodiment, the agent downregulates expression of a c-Myc target gene.In yet another embodiment, the agent downregulates oxidative phosphorylation (OXPHOS) in NK cells. In another embodiment, the agent promotes NK cell proliferation. In yet another embodiment, the agent inhibits proliferation of primary NK cells cultured in vitro with IL-2 and IL-15. In yet another embodiment, the condition is an inflammatory disease, and optionally, the inflammatory disease is an autoimmune disease.

[0016] In another aspect, a method is provided for evaluating the effectiveness of an agent that modulates the IRE1α-XBP1 pathway to treat a condition that may benefit from modulating an immune response in a subject, the method comprising: a) detecting in a subject sample at a first time point the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in NK cells; b) repeating step a) for at least one subsequent time point after administration of the agent; and c) comparing the copy number, amount, and / or activity detected in steps a) and b) relative to the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in the sample at the first time point. and comparing, wherein a presence or significant increase in the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in the subsequent sample compared to the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in the sample at the first time point indicates that the agent treats a condition that would benefit from upregulating an immune response in the subject, and an absence or significant decrease in the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in the subsequent sample compared to the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in the sample at the first time point indicates that the agent treats a condition that would benefit from downregulating an immune response in the subject.

[0017] As mentioned above, certain embodiments are applicable to any of the methods described herein. For example, in one embodiment, the first and / or at least one subsequent sample is selected from the group consisting of an ex vivo sample and an in vivo sample. In another embodiment, the first and / or at least one subsequent sample is a single sample or a portion of a pooled sample obtained from a subject. In yet another embodiment, the agent upregulates the IRE1α-XBP1 pathway to treat a condition that may benefit from upregulating the immune response in a subject. In yet another embodiment, the condition that may benefit from upregulating the immune response is cancer or infection. In another embodiment, the cancer is selected from the group consisting of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, and cervical cancer. In yet another embodiment, between the first time point and the subsequent time point, the subject has undergone treatment, completed treatment, and / or is in cancer remission. In yet another embodiment, the cancer treatment is selected from the group consisting of immunotherapy, targeted therapy, chemotherapy, radiation therapy, hormone therapy, anti-cancer vaccine, anti-cancer virus, and checkpoint inhibitor. In another embodiment, the sample comprises cells, serum, peritumoral tissue, and / or intratumoral tissue obtained from the subject. In yet another embodiment, the agent downregulates the IRE1α-XBP1 pathway to treat a condition that would benefit from downregulating the immune response in the subject. In yet another embodiment, the condition that would benefit from downregulating the immune response is an inflammatory disease. In another embodiment, the agent is administered in a pharmaceutically acceptable formulation. In yet another embodiment, the subject is an animal model of the condition. In yet another embodiment, the animal model is a mouse model. In another embodiment, the subject is a mammal. In yet another embodiment, the mammal is a mouse or a human. In yet another embodiment, the mammal is a human.

[0018] In yet another aspect, a method for promoting NK cell proliferation is provided, comprising contacting NK cells with a therapeutically effective amount of an agent that upregulates the IRE1α-XBP1 pathway in NK cells.

[0019] As mentioned above, certain embodiments are applicable to any of the methods described herein. For example, in one embodiment, the NK cells are activated NK cells and / or memory NK cells, and optionally, the memory NK cells are cytokine-induced memory-like NK cells (CIML). In another embodiment, the NK cells are Ly49H-expressing NK cells or CD56 brightThe agent is an NK cell. In yet another embodiment, the agent upregulates the IRE1α-XBP1 pathway by increasing the copy number, amount, and / or activity of at least one biomarker listed in Table 1. In yet another embodiment, the agent is a nucleic acid molecule or fragment thereof encoding at least one biomarker listed in Table 1, a polypeptide or fragment thereof of at least one biomarker listed in Table 1, a small molecule that binds to at least one biomarker listed in Table 1, or a pro-inflammatory cytokine. In another embodiment, the pro-inflammatory cytokine is IL-2, IL-15, IL-12, and / or IL-18. In yet another embodiment, the agent promotes splicing of XBP1 to XBP1s transcripts. In yet another embodiment, the agent upregulates an XBP1 target gene. In another embodiment, the XBP1 target gene is c-Myc or a canonical XBP1 target gene selected from the group consisting of Hspa5, Dnajb9, Sec24d, Sec63, Hyou1, Sec61a, and P4hb. In yet another embodiment, the agent upregulates expression of a c-Myc target gene. In yet another embodiment, the agent upregulates oxidative phosphorylation (OXPHOS) in NK cells. In another embodiment, the agent increases the level of proliferation marker Ki-67 in NK cells. In yet another embodiment, the agent promotes NK cell proliferation in response to infection. In yet another embodiment, the infection is a viral infection, a bacterial infection, a protozoan infection, a parasitic infection, a fungal infection, or a helminth infection. In another embodiment, the viral infection is caused by a virus selected from the group consisting of CMV, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), influenza A virus, Epstein-Barr virus (EBV), human herpes simplex virus (HSV) types 1 and 2, respiratory syncytial virus (RSV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), Zika virus, Rift Valley fever virus (RVFV), dengue virus (DENV), chikungunya virus (CHIKV), enterovirus (EV), and human adenovirus (HAdV).In yet another embodiment, the bacterial infection is caused by Listeria monocytogenes, Mycobacterium tuberculosis, or Salmonella typhimurium. In yet another embodiment, the parasitic infection is caused by Plasmodium or Cryptosporidium, optionally where the Plasmodium is a malaria parasite. In another embodiment, the fungal infection is caused by Aspergillus, optionally where the Aspergillus is Aspergillus fumigatus. In yet another embodiment, the agent promotes NK cell proliferation in response to lymphopenia. In yet another embodiment, the agent promotes NK cell proliferation in response to cancer. In another embodiment, the cancer is an NK cell-sensitive cancer. In yet another embodiment, the cancer is selected from the group consisting of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, and cervical cancer.

[0020] In yet another aspect, a method for reducing NK cell proliferation, comprising contacting NK cells with a therapeutically effective amount of an agent that downregulates the IRE1α-XBP1 pathway in NK cells.

[0021] As mentioned above, certain embodiments are applicable to any of the methods described herein. For example, in one embodiment, the NK cells are activated NK cells and / or memory NK cells, and optionally, the memory NK cells are cytokine-induced memory-like NK cells (CIML). In another embodiment, the NK cells are Ly49H-expressing NK cells or CD56 brightNK cells. In yet another embodiment, the agent downregulates the IRE1α-XBP1 pathway by decreasing the copy number, amount, and / or activity of at least one biomarker listed in Table 1. In yet another embodiment, the agent is a small molecule inhibitor, a CRISPR guide RNA (gRNA), an RNA interference agent, an antisense oligonucleotide, a peptide or peptidomimetic inhibitor, an aptamer, an antibody, or an intrabody. In another embodiment, the RNA interference agent is a small interfering RNA (siRNA), a CRISPR RNA (crRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), or a piwi-interacting RNA (piRNA). In yet another embodiment, the agent comprises an antibody and / or intrabody, or an antigen-binding fragment thereof, that specifically binds to at least one biomarker listed in Table 1. In yet another embodiment, the antibody and / or intrabody, or an antigen-binding fragment thereof, is murine, chimeric, humanized, composite, or human. In another embodiment, the antibody and / or intrabody, or antigen-binding fragment thereof, is detectably labeled, comprises an effector domain, comprises an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. The agent is a small molecule inhibitor of the RNase domain. In yet another embodiment, the small molecule inhibitor is 4μ8c. In another embodiment, the agent reduces splicing of XBP1 to XBP1s transcripts. In yet another embodiment, the agent downregulates an XBP1 target gene. In yet another embodiment, the XBP1 target gene is c-Myc or a canonical XBP1 target gene selected from the group consisting of Hspa5, Dnajb9, Sec24d, Sec63, Hyou1, Sec61a, and P4hb. In another embodiment, the agent downregulates expression of a c-Myc target gene. In yet another embodiment, the agent downregulates oxidative phosphorylation (OXPHOS) in NK cells. In yet another embodiment, the agent reduced the level of proliferation of the proliferation marker Ki-67 in NK cells. In another embodiment, the agent reduces NK cell proliferation in NK cell-driven cancer. In yet another embodiment, the NK cell-driven cancer is aggressive NK cell leukemia (ANKL). In certain embodiments, for example, the following are provided: (Item 1) A composition comprising natural killer (NK) cells modified to upregulate the IRE1α-XBP1 pathway. (Item 2) 2. The composition of item 1, wherein the IRE1α-XBP1 pathway is upregulated by increasing the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in the NK cells. (Item 3) 3. The composition of item 1 or 2, wherein the copy number, amount, and / or activity of at least one biomarker listed in Table 1 is increased by contacting the NK cells with a nucleic acid molecule or fragment thereof encoding at least one biomarker listed in Table 1, a polypeptide or fragment thereof of at least one biomarker listed in Table 1, a small molecule that binds to at least one biomarker listed in Table 1, or a pro-inflammatory cytokine. (Item 4) 4. The composition of item 3, wherein the pro-inflammatory cytokine is IL-2, IL-15, IL-12 and / or IL-18. (Item 5) 5. The composition of any one of items 1 to 4, wherein the NK cells have increased splicing of XBP1 to XBP1s transcripts. (Item 6) 6. The composition of any one of items 1 to 5, wherein expression of an XBP1 target gene is upregulated in the NK cells. (Item 7) 7. The composition of claim 6, wherein the XBP1 target gene is c-Myc or a canonical XBP1 target gene selected from the group consisting of Hspa5, Dnajb9, Sec24d, Sec63, Hyou1, Sec61a, and P4hb. (Item 8) 8. The composition of claim 7, wherein expression of a c-Myc target gene is upregulated in the NK cells. (Item 9) The composition of item 8, wherein oxidative phosphorylation (OXPHOS) is upregulated in the NK cells. (Item 10) 10. The composition of any one of items 1 to 9, wherein the NK cells are activated NK cells and / or memory NK cells, and optionally the memory NK cells are cytokine-induced memory-like NK cells (CIML). (Item 11) The NK cells are Ly49H-expressing NK cells or CD56 bright 11. The composition according to any one of items 1 to 10, wherein the composition is a NK cell. (Item 12) 12. The composition of any one of items 1 to 11, wherein the NK cells are derived from peripheral blood mononuclear cells (PBMCs) or umbilical cord blood (UCB). (Item 13) 13. The composition according to any one of items 1 to 12, wherein the composition is derived from an NK cell line. (Item 14) 14. A method of treating a subject having a condition that can benefit from upregulation of an immune response, the method comprising administering to the subject a therapeutically effective amount of the composition of any one of items 1 to 13. (Item 15) 15. The method of claim 14, wherein the NK cells are derived from the subject treated with the composition. (Item 16) 15. The method of item 14, wherein the NK cells are from a different subject not treated with the composition. (Item 17) 17. The method of any one of items 14 to 16, wherein the condition is an infection. (Item 18) 18. The method of claim 17, wherein the infection is a viral infection, a bacterial infection, a protozoan infection, a parasitic infection, a fungal infection, or a helminth infection. (Item 19) 19. The method of item 18, wherein the viral infection is caused by a virus selected from the group consisting of CMV, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), influenza A virus, Epstein-Barr virus (EBV), human herpes simplex virus (HSV) types 1 and 2, respiratory syncytial virus (RSV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), Zika virus, Rift Valley fever virus (RVFV), dengue virus (DENV), chikungunya virus (CHIKV), enterovirus (EV), and human adenovirus (HAdV). (Item 20) 20. The method of any one of items 14 to 19, wherein the composition promotes antiviral immunity in the subject. (Item 21) 21. The method of any one of items 14 to 20, wherein the composition reduces viral titer in the subject. (Item 22) 22. The method of any one of items 14 to 21, wherein the composition increases overall survival rate. (Item 23) 19. The method of claim 18, wherein the bacterial infection is caused by Listeria monocytogenes, Mycobacterium tuberculosis, or Salmonella typhimurium. (Item 24) 19. The method of claim 18, wherein the parasitic infection is caused by Plasmodium or Cryptosporidium, and optionally the Plasmodium is a malaria parasite. (Item 25) 19. The method of claim 18, wherein the fungal infection is caused by Aspergillus, and optionally the Aspergillus is Aspergillus fumigatus. (Item 26) 17. The method according to any one of items 14 to 16, wherein the condition is lymphopenia. (Item 27) 17. The method of any one of items 14 to 16, wherein the subject has undergone hematopoietic cell transplantation (HCT). (Item 28) 17. The method according to any one of items 14 to 16, wherein the condition is cancer. (Item 29) 29. The method of claim 28, wherein the cancer is an NK cell-sensitive cancer. (Item 30) 30. The method of item 28 or 29, wherein the cancer is selected from the group consisting of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, and cervical cancer. (Item 31) 31. The method according to any one of items 28 to 30, wherein the composition promotes anti-tumor immunity in the subject. (Item 32) 32. The method according to any one of items 28 to 31, wherein the composition increases the amount of NK cells infiltrating a tumor. (Item 33) 33. The method of any one of items 28 to 32, wherein the composition increases the amount of conventional type 1 dendritic cells and / or CD8+ T cells that infiltrate tumors. (Item 34) 34. The method of any one of items 28 to 33, wherein the composition reduces the number of proliferating cells in the cancer and / or reduces the volume or size of a tumor containing the cancer cells. (Item 35) 35. The method of any one of items 28 to 34, further comprising administering to the subject an immunotherapy and / or cancer therapy, optionally wherein the immunotherapy and / or cancer therapy is administered before, after, or simultaneously with the composition. (Item 36) 36. The method of claim 35, wherein the immunotherapy is cell-based. (Item 37) 36. The method of claim 35, wherein the immunotherapy comprises a cancer composition and / or a virus. (Item 38) 36. The method of claim 35, wherein the immunotherapy inhibits an immune checkpoint. (Item 39) 39. The method of item 38, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilin, and A2aR. (Item 40) 36. The method of claim 35, wherein the cancer therapy is selected from the group consisting of radiation, radiosensitizers, and chemotherapy. (Item 41) A method for treating a subject having a condition that may benefit from upregulation of an immune response, comprising administering to the subject a therapeutically effective amount of an agent that upregulates the IRE1α-XBP1 pathway so that the condition that may benefit from upregulation of an immune response is treated. (Item 42) The method of item 41, wherein the agent upregulates the IRE1α-XBP1 pathway in NK cells. (Item 43) 43. The method of claim 42, wherein the NK cells are activated NK cells and / or memory NK cells, and optionally the memory NK cells are cytokine-induced memory-like NK cells (CIML). (Item 44) The NK cells are Ly49H-expressing NK cells or CD56 bright 43. The method of item 41 or 42, wherein the cells are NK cells. (Item 45) 45. The method of any one of items 41 to 44, wherein the agent upregulates the IRE1α-XBP1 pathway by increasing the copy number, amount, and / or activity of at least one biomarker listed in Table 1. (Item 46) 46. ​​The method of any one of items 41 to 45, wherein the agent is a nucleic acid molecule or fragment thereof encoding at least one biomarker listed in Table 1, a polypeptide or fragment thereof of at least one biomarker listed in Table 1, a small molecule that binds to at least one biomarker listed in Table 1, or a pro-inflammatory cytokine. (Item 47) 47. The method of claim 46, wherein the pro-inflammatory cytokine is IL-2, IL-15, IL-12 and / or IL-18. (Item 48) 48. The method of any one of items 41 to 47, wherein the agent promotes splicing of XBP1 to XBP1s transcripts. (Item 49) 49. The method of any one of items 41 to 48, wherein the agent upregulates an XBP1 target gene. (Item 50) 50. The method of claim 49, wherein the XBP1 target gene is c-Myc or a canonical XBP1 target gene selected from the group consisting of Hspa5, Dnajb9, Sec24d, Sec63, Hyou1, Sec61a, and P4hb. (Item 51) 51. The method of claim 50, wherein the agent upregulates expression of a c-Myc target gene. (Item 52) 52. The method of claim 51, wherein the agent upregulates oxidative phosphorylation in the NK cells. (Item 53) 53. The method according to any one of items 41 to 52, wherein the agent promotes proliferation of NK cells. (Item 54) 54. The method of any one of items 41 to 53, wherein the condition is an infection. (Item 55) 55. The method of claim 54, wherein the infection is a viral infection, a bacterial infection, a protozoan infection, or a helminth infection. (Item 56) 56. The method of item 55, wherein the viral infection is caused by a virus selected from the group consisting of CMV, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), influenza A virus, Epstein-Barr virus (EBV), human herpes simplex virus (HSV) types 1 and 2, respiratory syncytial virus (RSV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), Zika virus, Rift Valley fever virus (RVFV), dengue virus (DENV), chikungunya virus (CHIKV), enterovirus (EV), and human adenovirus (HAdV). (Item 57) 57. The method of any one of items 41 to 56, wherein the agent promotes antiviral immunity in the subject. (Item 58) 58. The method according to any one of items 41 to 57, wherein the agent promotes clonal hypertrophy of NK cells upon viral infection. (Item 59) 59. The method of any one of items 41 to 58, wherein the agent reduces viral titer in the subject. (Item 60) 60. The method of any one of items 41 to 59, wherein the agent increases overall survival. (Item 61) 56. The method of item 55, wherein the bacterial infection is caused by Listeria monocytogenes, Mycobacterium tuberculosis, or Salmonella typhimurium. (Item 62) 56. The method of claim 55, wherein the parasitic infection is caused by Plasmodium or Cryptosporidium, and optionally the Plasmodium is a malaria parasite. (Item 63) 56. The method of claim 55, wherein the fungal infection is caused by Aspergillus, and optionally the Aspergillus is Aspergillus fumigatus. (Item 64) 54. The method of any one of items 41 to 53, wherein the condition is lymphopenia. (Item 65) 54. The method of any one of items 41 to 53, wherein the subject has undergone HCT or NK cell adoptive transfer immunotherapy, and optionally the NK cells are genetically modified. (Item 66) 66. The method of item 64 or 65, wherein the agent promotes homeostatic proliferation of NK cells in vivo. (Item 67) 54. The method according to any one of items 41 to 53, wherein the condition is cancer. (Item 68) Item 68. The method of item 67, wherein the cancer is an NK cell-sensitive cancer. (Item 69) 69. The method of item 67 or 68, wherein the cancer is selected from the group consisting of solid tumor, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, and cervical cancer. (Item 70) 70. The method of any one of items 67 to 69, wherein the agent promotes NK cell-mediated anti-tumor immunity. (Item 71) 71. The method according to any one of items 67 to 70, wherein the agent increases the number of NK cells infiltrating a tumor. (Item 72) 72. The method of any one of items 67 to 71, wherein the agent increases the number of tumor-infiltrating conventional type 1 dendritic cells and / or tumor-infiltrating CD8+ T cells. (Item 73) 73. The method of any one of items 67 to 72, wherein the agent reduces the number of proliferating cells in the cancer and / or reduces the volume or size of a tumor containing the cancer cells. (Item 74) 74. The method of any one of items 67 to 73, further comprising administering to the subject an immunotherapy and / or cancer therapy, optionally wherein the immunotherapy and / or cancer therapy is administered before, after, or simultaneously with the composition. (Item 75) 75. The method of claim 74, wherein the immunotherapy is cell-based. (Item 76) 75. The method of item 74, wherein the immunotherapy comprises a cancer vaccine and / or a virus. (Item 77) 75. The method of claim 74, wherein the immunotherapy inhibits an immune checkpoint. (Item 78) 78. The method of claim 77, wherein the immune checkpoint is selected from the group consisting of CTLA-4, PD-1, VISTA, B7-H2, B7-H3, PD-L1, B7-H4, B7-H6, ICOS, HVEM, PD-L2, CD160, gp49B, PIR-B, KIR family receptors, TIM-1, TIM-3, TIM-4, LAG-3, GITR, 4-IBB, OX-40, BTLA, SIRP alpha (CD47), CD48, 2B4 (CD244), B7.1, B7.2, ILT-2, ILT-4, TIGIT, HHLA2, butyrophilin, and A2aR. (Item 79) 75. The method of item 74, wherein the cancer therapy is selected from the group consisting of radiation, radiosensitizers, and chemotherapy. (Item 80) A method for treating a subject having a condition that may benefit from downregulation of an immune response, comprising administering to the subject a therapeutically effective amount of an agent that downregulates the IRE1α-XBP1 pathway so as to treat the condition that may benefit from downregulation of an immune response. (Item 81) 81. The method of claim 80, wherein the agent downregulates the IRE1α-XBP1 pathway in NK cells. (Item 82) 82. The method of claim 81, wherein the NK cells are activated NK cells and / or memory NK cells, and optionally the memory NK cells are cytokine-induced memory-like NK cells (CIML). (Item 83) The NK cells are Ly49H-expressing NK cells or CD56 bright 82. The method of item 80 or 81, wherein the cells are NK cells. (Item 84) 84. The method of any one of items 80 to 83, wherein the agent downregulates the IRE1α-XBP1 pathway by decreasing the copy number, amount, and / or activity of at least one biomarker listed in Table 1. (Item 85) 85. The method of item 84, wherein the agent is a small molecule inhibitor, a CRISPR guide RNA (gRNA), an RNA interference agent, an antisense oligonucleotide, a peptide or peptidomimetic inhibitor, an aptamer, an antibody, or an intrabody. (Item 86) 86. The method of claim 85, wherein the RNA interference agent is a small interfering RNA (siRNA), CRISPR RNA (crRNA), small hairpin RNA (shRNA), microRNA (miRNA), or piwi-interacting RNA (piRNA). (Item 87) 86. The method of item 85, wherein the agent comprises an antibody and / or intrabody, or an antigen-binding fragment thereof, that specifically binds to at least one biomarker listed in Table 1. (Item 88) 88. The method of claim 87, wherein the antibody and / or intrabody, or antigen-binding fragment thereof, is murine, chimeric, humanized, conjugated, or human. (Item 89) 89. The method of claim 87 or 88, wherein the antibody and / or intrabody, or antigen-binding fragment thereof, is detectably labeled, comprises an effector domain, comprises an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. (Item 90) 86. The method of claim 85, wherein the agent is a small molecule inhibitor of the IRE1α RNase domain. (Item 91) 87. The method of item 86, wherein the small molecule inhibitor is 4μ8c. (Item 92) 92. The method of any one of items 80 to 91, wherein the agent reduces splicing of XBP1 to XBP1s transcripts. (Item 93) 93. The method of any one of items 80 to 92, wherein the agent downregulates an XBP1 target gene. (Item 94) 94. The method of claim 93, wherein the XBP1 target gene is c-Myc or a canonical XBP1 target gene selected from the group consisting of Hspa5, Dnajb9, Sec24d, Sec63, Hyou1, Sec61a, and P4hb. (Item 95) 94. The method of claim 93, wherein the agent downregulates expression of a c-Myc target gene. (Item 96) 96. The method of item 95, wherein the agent downregulates oxidative phosphorylation in the NK cells. (Item 97) 97. The method of any one of items 80 to 96, wherein the agent inhibits proliferation of NK cells. (Item 98) 98. The method of paragraph 97, wherein the agent inhibits proliferation of primary NK cells cultured in vitro with IL-2 and IL-15. (Item 99) 92. The method of any one of items 74 to 91, wherein the condition is an inflammatory disease. (Item 100) Item 94. The method of item 93, wherein the inflammatory disease is an autoimmune disease. (Item 101) 1. A method for assessing the efficacy of an agent that modulates the IRE1α-XBP1 pathway to treat a condition that may benefit from modulating an immune response in a subject, comprising: a) detecting in a subject sample at a first time point the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in NK cells; b) repeating step a) for at least one subsequent time point after administration of the agent; and c) comparing the copy number, amount, and / or activity detected in steps a) and b), wherein a presence of, or a significant increase in, the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in the subsequent sample compared to the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in the sample at the first time point indicates that the agent treats a condition that would benefit from upregulating an immune response in the subject, and an absence of, or a significant decrease in, the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in the subsequent sample compared to the copy number, amount, and / or activity of at least one biomarker listed in Table 1 in the sample at the first time point indicates that the agent treats a condition that would benefit from downregulating an immune response in the subject. (Item 102) 102. The method of claim 101, wherein the first and / or at least one subsequent sample is selected from the group consisting of an ex vivo sample and an in vivo sample. (Item 103) 103. The method of any one of paragraphs 101 or 102, wherein the first and / or at least one subsequent sample is a single sample or part of pooled samples obtained from the subject. (Item 104) The method of any one of items 101 to 103, wherein the agent upregulates the IRE1α-XBP1 pathway to treat a condition that may benefit from upregulating an immune response in a subject. (Item 105) 105. The method of claim 104, wherein the condition that can benefit from upregulating the immune response is cancer or an infection. (Item 106) 106. The method of claim 105, wherein the cancer is selected from the group consisting of solid tumors, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, and cervical cancer. (Item 107) 107. The method of any one of items 101-106, wherein between the first time point and the subsequent time point, the subject has undergone treatment, completed treatment, and / or is in remission of the cancer. (Item 108) 108. The method of claim 107, wherein the cancer treatment is selected from the group consisting of immunotherapy, targeted therapy, chemotherapy, radiation therapy, hormone therapy, anti-cancer vaccine, anti-cancer virus, and checkpoint inhibitor. (Item 109) 109. The method of any one of items 104 to 108, wherein the sample comprises cells, serum, peritumoral tissue, and / or intratumoral tissue obtained from the subject. (Item 110) The method of any one of items 101 to 103, wherein the agent downregulates the IRE1α-XBP1 pathway to treat a condition that may benefit from downregulating the immune response in a subject. (Item 111) 105. The method of claim 104, wherein the condition that can benefit from downregulating the immune response is an inflammatory disease. (Item 112) 112. The method of any one of items 41 to 111, wherein the agent is administered in a pharmaceutically acceptable formulation. (Item 113) 113. The method of any one of items 14 to 112, wherein the subject is an animal model of the condition. (Item 114) Item 114. The method of item 113, wherein the animal model is a mouse model. (Item 115) 115. The method according to any one of items 14 to 114, wherein the subject is a mammal. (Item 116) 116. The method of claim 115, wherein the mammal is a mouse or a human. (Item 117) 117. The method of claim 116, wherein the mammal is a human. (Item 118) A method for promoting the proliferation of NK cells, comprising contacting the NK cells with a therapeutically effective amount of an agent that upregulates the IRE1α-XBP1 pathway in the NK cells. (Item 119) 119. The method of claim 118, wherein the NK cells are activated NK cells and / or memory NK cells, and optionally the memory NK cells are cytokine-induced memory-like NK cells (CIML). (Item 120) The NK cells are Ly49H-expressing NK cells or CD56 bright 110. The method of claim 118 or 119, wherein the cells are NK cells. (Item 121) 121. The method of any one of items 118 to 120, wherein the agent upregulates the IRE1α-XBP1 pathway by increasing the copy number, amount, and / or activity of at least one biomarker listed in Table 1. (Item 122) 122. The method of any one of items 118-121, wherein the agent is a nucleic acid molecule or fragment thereof encoding at least one biomarker listed in Table 1, a polypeptide or fragment thereof of at least one biomarker listed in Table 1, a small molecule that binds to at least one biomarker listed in Table 1, or a pro-inflammatory cytokine. (Item 123) 123. The method of claim 122, wherein the pro-inflammatory cytokine is IL-2, IL-15, IL-12 and / or IL-18. (Item 124) 124. The method of any one of items 118 to 123, wherein the agent promotes splicing of XBP1 to XBP1s transcripts. (Item 125) 125. The method of any one of items 118 to 124, wherein the agent upregulates an XBP1 target gene. (Item 126) 126. The method of claim 125, wherein the XBP1 target gene is c-Myc or a canonical XBP1 target gene selected from the group consisting of Hspa5, Dnajb9, Sec24d, Sec63, Hyou1, Sec61a, and P4hb. (Item 127) Item 127. The method of item 126, wherein the agent upregulates expression of a c-Myc target gene. (Item 128) 128. The method of claim 127, wherein the agent upregulates oxidative phosphorylation in the NK cells. (Item 129) 129. The method of any one of items 118 to 128, wherein the agent increased the level of proliferation of the proliferation marker Ki-67 in the NK cells. (Item 130) 120. The method of any one of items 118 to 129, wherein the agent promotes NK cell proliferation in response to infection. (Item 131) 131. The method of claim 130, wherein the infection is a viral infection, a bacterial infection, a protozoan infection, a parasitic infection, a fungal infection, or a helminth infection. (Item 132) 132. The method of claim 131, wherein the viral infection is caused by a virus selected from the group consisting of CMV, human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), influenza A virus, Epstein-Barr virus (EBV), human herpes simplex virus (HSV) types 1 and 2, respiratory syncytial virus (RSV), human papillomavirus (HPV), lymphocytic choriomeningitis virus (LCMV), Zika virus, Rift Valley fever virus (RVFV), dengue virus (DENV), chikungunya virus (CHIKV), enterovirus (EV), and human adenovirus (HAdV). (Item 133) 132. The method of claim 131, wherein the bacterial infection is caused by Listeria monocytogenes, Mycobacterium tuberculosis, or Salmonella typhimurium. (Item 134) 132. The method of claim 131, wherein the parasitic infection is caused by Plasmodium or Cryptosporidium, and optionally the Plasmodium is a malaria parasite. (Item 135) 132. The method of claim 131, wherein the fungal infection is caused by Aspergillus, and optionally the Aspergillus is Aspergillus fumigatus. (Item 136) 120. The method of any one of items 118 to 129, wherein the agent promotes NK cell proliferation in response to lymphopenia. (Item 137) 120. The method of any one of items 118 to 129, wherein the agent promotes NK cell proliferation in response to cancer. (Item 138) Item 138. The method of item 137, wherein the cancer is an NK cell-sensitive cancer. (Item 139) 139. The method of item 137 or 138, wherein the cancer is selected from the group consisting of solid tumor, blood cancer, bladder cancer, brain cancer, breast cancer, colon cancer, gastric cancer, glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma, myeloma, cervical cancer, ovarian cancer, melanoma, pancreatic cancer, kidney cancer, salivary gland cancer, stomach cancer, thymic epithelial cancer, thyroid cancer, and cervical cancer. (Item 140) A method for reducing the proliferation of NK cells, comprising contacting the NK cells with a therapeutically effective amount of an agent that downregulates the IRE1α-XBP1 pathway in the NK cells. (Item 141) 141. The method of claim 140, wherein the NK cells are activated NK cells and / or memory NK cells, and optionally the memory NK cells are cytokine-induced memory-like NK cells (CIML). (Item 142) The NK cells are Ly49H-expressing NK cells or CD56 bright 142. The method of item 140 or 141, wherein the cells are NK cells. (Item 143) 143. The method of any one of items 140 to 142, wherein the agent downregulates the IRE1α-XBP1 pathway by decreasing the copy number, amount, and / or activity of at least one biomarker listed in Table 1. (Item 144) 144. The method of claim 143, wherein the agent is a small molecule inhibitor, a CRISPR guide RNA (gRNA), an RNA interference agent, an antisense oligonucleotide, a peptide or peptidomimetic inhibitor, an aptamer, an antibody, or an intrabody. (Item 145) 145. The method of claim 144, wherein the RNA interference agent is a small interfering RNA (siRNA), CRISPR RNA (crRNA), small hairpin RNA (shRNA), microRNA (miRNA), or piwi-interacting RNA (piRNA). (Item 146) 145. The method of claim 144, wherein the agent comprises an antibody and / or intrabody, or an antigen-binding fragment thereof, that specifically binds to at least one biomarker listed in Table 1. (Item 147) 147. The method of claim 146, wherein the antibody and / or intrabody, or antigen-binding fragment thereof, is murine, chimeric, humanized, conjugated, or human. (Item 148) 148. The method of claim 146 or 147, wherein the antibody and / or intrabody, or antigen-binding fragment thereof, is detectably labeled, comprises an effector domain, comprises an Fc domain, and / or is selected from the group consisting of Fv, Fav, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabody fragments. (Item 149) Item 145. The method of item 144, wherein the agent is a small molecule inhibitor of the IRE1α RNase domain. (Item 150) 150. The method of claim 149, wherein the small molecule inhibitor is 4μ8c. (Item 151) 151. The method of any one of items 140 to 150, wherein the agent reduces splicing of XBP1 to XBP1s transcripts. (Item 152) 152. The method of any one of items 140 to 151, wherein the agent downregulates an XBP1 target gene. (Item 153) 153. The method of claim 152, wherein the XBP1 target gene is c-Myc or a canonical XBP1 target gene selected from the group consisting of Hspa5, Dnajb9, Sec24d, Sec63, Hyou1, Sec61a, and P4hb. (Item 154) 153. The method of claim 152, wherein the agent downregulates expression of a c-Myc target gene. (Item 155) 155. The method of claim 154, wherein the agent downregulates oxidative phosphorylation in the NK cells. (Item 156) 156. The method of any one of items 140 to 155, wherein the agent reduced the level of the proliferation marker Ki-67 in the NK cells. (Item 157) 156. The method of any one of items 140 to 155, wherein the agent reduces NK cell proliferation in NK cell-driven cancers. (Item 158) Item 158. The method of item 157, wherein the cancer caused by NK cells is aggressive NK cell leukemia (ANKL). [Brief explanation of the drawings]

[0022] [Figure 1A]Figures 1A-1G show the induction of the IRE1α / XBP1 UPR in mouse and human activated NK cells in vitro and in vivo. Figure 1A shows a gene ontology (GO) analysis of the top 500 differentially expressed genes in Ly49H+ wild-type splenic NK cells collected from either naive or MCMV-infected mice at 1.5 days post-infection (PI). The top 10 enriched GO clusters with the keyword "stimulation" are shown. Figure 1B shows a heatmap of RNA-seq analysis showing the expression of canonical IRE1α / XBP1 target genes over a period of time after MCMV infection. Figure 1C shows flow cytometry analysis of Venus reporter expression in splenic NK cells from ERAI transgenic reporter mice at 2 days post-infection (PI). Figure 1D shows a heatmap of RNA-seq analysis demonstrating the expression of canonical IRE1α / XBP1 target genes in splenic NK cells harvested from wild-type mice and cultured for 16 hours in either the presence or absence of IL-12 and IL-18 (GSE106138). Figure 1E shows flow cytometry analysis of Venus reporter expression in sorted, purified splenic NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of IL-12 and IL-18. Representative histogram plots (left) and quantification (right) are shown. Figure 1F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 hours of culture in the presence or absence of human recombinant IL-12 and IL-18, or IL-15. β-ACTIN was used as a reference, and data are presented as fold changes normalized to unstimulated levels. Figure 1G shows flow cytometry analysis of Venus reporter expression in sorted purified spleen and BM NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml), or IL-15 (100 ng / ml). Representative histogram plots (top) and quantification (bottom). p values ​​are as indicated, and ns indicates "not significant."For Figures 1C and 1E, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figure 1F, a one-sample t-test was performed. From biological replicates, error bars in Figures 1C and 1E indicate standard deviation, and in Figure 1F, values ​​are shown from minimum to maximum. Each column in Figures 1B and 1D represents a different mouse. Experiments in Figures 1C and 1E contained 2-3 mice per group and were independently repeated three times. N equaled 6 PBMC donors in Figure 1F, and experiments were independently repeated two times. [Figure 1B]Figures 1A-1G show the induction of the IRE1α / XBP1 UPR in mouse and human activated NK cells in vitro and in vivo. Figure 1A shows a gene ontology (GO) analysis of the top 500 differentially expressed genes in Ly49H+ wild-type splenic NK cells collected from either naive or MCMV-infected mice at 1.5 days post-infection (PI). The top 10 enriched GO clusters with the keyword "stimulation" are shown. Figure 1B shows a heatmap of RNA-seq analysis showing the expression of canonical IRE1α / XBP1 target genes over a period of time after MCMV infection. Figure 1C shows flow cytometry analysis of Venus reporter expression in splenic NK cells from ERAI transgenic reporter mice at 2 days post-infection (PI). Figure 1D shows a heatmap of RNA-seq analysis demonstrating the expression of canonical IRE1α / XBP1 target genes in splenic NK cells harvested from wild-type mice and cultured for 16 hours in either the presence or absence of IL-12 and IL-18 (GSE106138). Figure 1E shows flow cytometry analysis of Venus reporter expression in sorted, purified splenic NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of IL-12 and IL-18. Representative histogram plots (left) and quantification (right) are shown. Figure 1F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 hours of culture in the presence or absence of human recombinant IL-12 and IL-18, or IL-15. β-ACTIN was used as a reference, and data are presented as fold changes normalized to unstimulated levels. Figure 1G shows flow cytometry analysis of Venus reporter expression in sorted purified spleen and BM NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml), or IL-15 (100 ng / ml). Representative histogram plots (top) and quantification (bottom). p values ​​are as indicated, and ns indicates "not significant."For Figures 1C and 1E, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figure 1F, a one-sample t-test was performed. From biological replicates, error bars in Figures 1C and 1E indicate standard deviation, and in Figure 1F, values ​​are shown from minimum to maximum. Each column in Figures 1B and 1D represents a different mouse. Experiments in Figures 1C and 1E contained 2-3 mice per group and were independently repeated three times. N equaled 6 PBMC donors in Figure 1F, and experiments were independently repeated two times. [Figure 1C]Figures 1A-1G show the induction of the IRE1α / XBP1 UPR in mouse and human activated NK cells in vitro and in vivo. Figure 1A shows a gene ontology (GO) analysis of the top 500 differentially expressed genes in Ly49H+ wild-type splenic NK cells collected from either naive or MCMV-infected mice at 1.5 days post-infection (PI). The top 10 enriched GO clusters with the keyword "stimulation" are shown. Figure 1B shows a heatmap of RNA-seq analysis showing the expression of canonical IRE1α / XBP1 target genes over a period of time after MCMV infection. Figure 1C shows flow cytometry analysis of Venus reporter expression in splenic NK cells from ERAI transgenic reporter mice at 2 days post-infection (PI). Figure 1D shows a heatmap of RNA-seq analysis demonstrating the expression of canonical IRE1α / XBP1 target genes in splenic NK cells harvested from wild-type mice and cultured for 16 hours in either the presence or absence of IL-12 and IL-18 (GSE106138). Figure 1E shows flow cytometry analysis of Venus reporter expression in sorted, purified splenic NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of IL-12 and IL-18. Representative histogram plots (left) and quantification (right) are shown. Figure 1F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 hours of culture in the presence or absence of human recombinant IL-12 and IL-18, or IL-15. β-ACTIN was used as a reference, and data are presented as fold changes normalized to unstimulated levels. Figure 1G shows flow cytometry analysis of Venus reporter expression in sorted purified spleen and BM NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml), or IL-15 (100 ng / ml). Representative histogram plots (top) and quantification (bottom). p values ​​are as indicated, and ns indicates "not significant."For Figures 1C and 1E, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figure 1F, a one-sample t-test was performed. From biological replicates, error bars in Figures 1C and 1E indicate standard deviation, and in Figure 1F, values ​​are shown from minimum to maximum. Each column in Figures 1B and 1D represents a different mouse. Experiments in Figures 1C and 1E contained 2-3 mice per group and were independently repeated three times. N equaled 6 PBMC donors in Figure 1F, and experiments were independently repeated two times. [Figure 1D]Figures 1A-1G show the induction of the IRE1α / XBP1 UPR in mouse and human activated NK cells in vitro and in vivo. Figure 1A shows a gene ontology (GO) analysis of the top 500 differentially expressed genes in Ly49H+ wild-type splenic NK cells collected from either naive or MCMV-infected mice at 1.5 days post-infection (PI). The top 10 enriched GO clusters with the keyword "stimulation" are shown. Figure 1B shows a heatmap of RNA-seq analysis showing the expression of canonical IRE1α / XBP1 target genes over a period of time after MCMV infection. Figure 1C shows flow cytometry analysis of Venus reporter expression in splenic NK cells from ERAI transgenic reporter mice at 2 days post-infection (PI). Figure 1D shows a heatmap of RNA-seq analysis demonstrating the expression of canonical IRE1α / XBP1 target genes in splenic NK cells harvested from wild-type mice and cultured for 16 hours in either the presence or absence of IL-12 and IL-18 (GSE106138). Figure 1E shows flow cytometry analysis of Venus reporter expression in sorted, purified splenic NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of IL-12 and IL-18. Representative histogram plots (left) and quantification (right) are shown. Figure 1F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 hours of culture in the presence or absence of human recombinant IL-12 and IL-18, or IL-15. β-ACTIN was used as a reference, and data are presented as fold changes normalized to unstimulated levels. Figure 1G shows flow cytometry analysis of Venus reporter expression in sorted purified spleen and BM NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml), or IL-15 (100 ng / ml). Representative histogram plots (top) and quantification (bottom). p values ​​are as indicated, and ns indicates "not significant."For Figures 1C and 1E, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figure 1F, a one-sample t-test was performed. From biological replicates, error bars in Figures 1C and 1E indicate standard deviation, and in Figure 1F, values ​​are shown from minimum to maximum. Each column in Figures 1B and 1D represents a different mouse. Experiments in Figures 1C and 1E contained 2-3 mice per group and were independently repeated three times. N equaled 6 PBMC donors in Figure 1F, and experiments were independently repeated two times. [Figure 1E]Figures 1A-1G show the induction of the IRE1α / XBP1 UPR in mouse and human activated NK cells in vitro and in vivo. Figure 1A shows a gene ontology (GO) analysis of the top 500 differentially expressed genes in Ly49H+ wild-type splenic NK cells collected from either naive or MCMV-infected mice at 1.5 days post-infection (PI). The top 10 enriched GO clusters with the keyword "stimulation" are shown. Figure 1B shows a heatmap of RNA-seq analysis showing the expression of canonical IRE1α / XBP1 target genes over a period of time after MCMV infection. Figure 1C shows flow cytometry analysis of Venus reporter expression in splenic NK cells from ERAI transgenic reporter mice at 2 days post-infection (PI). Figure 1D shows a heatmap of RNA-seq analysis demonstrating the expression of canonical IRE1α / XBP1 target genes in splenic NK cells harvested from wild-type mice and cultured for 16 hours in either the presence or absence of IL-12 and IL-18 (GSE106138). Figure 1E shows flow cytometry analysis of Venus reporter expression in sorted, purified splenic NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of IL-12 and IL-18. Representative histogram plots (left) and quantification (right) are shown. Figure 1F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 hours of culture in the presence or absence of human recombinant IL-12 and IL-18, or IL-15. β-ACTIN was used as a reference, and data are presented as fold changes normalized to unstimulated levels. Figure 1G shows flow cytometry analysis of Venus reporter expression in sorted purified spleen and BM NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml), or IL-15 (100 ng / ml). Representative histogram plots (top) and quantification (bottom). p values ​​are as indicated, and ns indicates "not significant."For Figures 1C and 1E, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figure 1F, a one-sample t-test was performed. From biological replicates, error bars in Figures 1C and 1E indicate standard deviation, and in Figure 1F, values ​​are shown from minimum to maximum. Each column in Figures 1B and 1D represents a different mouse. Experiments in Figures 1C and 1E contained 2-3 mice per group and were independently repeated three times. N equaled 6 PBMC donors in Figure 1F, and experiments were independently repeated two times. [Figure 1F]Figures 1A-1G show the induction of the IRE1α / XBP1 UPR in mouse and human activated NK cells in vitro and in vivo. Figure 1A shows a gene ontology (GO) analysis of the top 500 differentially expressed genes in Ly49H+ wild-type splenic NK cells collected from either naive or MCMV-infected mice at 1.5 days post-infection (PI). The top 10 enriched GO clusters with the keyword "stimulation" are shown. Figure 1B shows a heatmap of RNA-seq analysis showing the expression of canonical IRE1α / XBP1 target genes over a period of time after MCMV infection. Figure 1C shows flow cytometry analysis of Venus reporter expression in splenic NK cells from ERAI transgenic reporter mice at 2 days post-infection (PI). Figure 1D shows a heatmap of RNA-seq analysis demonstrating the expression of canonical IRE1α / XBP1 target genes in splenic NK cells harvested from wild-type mice and cultured for 16 hours in either the presence or absence of IL-12 and IL-18 (GSE106138). Figure 1E shows flow cytometry analysis of Venus reporter expression in sorted, purified splenic NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of IL-12 and IL-18. Representative histogram plots (left) and quantification (right) are shown. Figure 1F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 hours of culture in the presence or absence of human recombinant IL-12 and IL-18, or IL-15. β-ACTIN was used as a reference, and data are presented as fold changes normalized to unstimulated levels. Figure 1G shows flow cytometry analysis of Venus reporter expression in sorted purified spleen and BM NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml), or IL-15 (100 ng / ml). Representative histogram plots (top) and quantification (bottom). p values ​​are as indicated, and ns indicates "not significant."For Figures 1C and 1E, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figure 1F, a one-sample t-test was performed. From biological replicates, error bars in Figures 1C and 1E indicate standard deviation, and in Figure 1F, values ​​are shown from minimum to maximum. Each column in Figures 1B and 1D represents a different mouse. Experiments in Figures 1C and 1E contained 2-3 mice per group and were independently repeated three times. N equaled 6 PBMC donors in Figure 1F, and experiments were independently repeated two times. [Figure 1G]Figures 1A-1G show the induction of the IRE1α / XBP1 UPR in mouse and human activated NK cells in vitro and in vivo. Figure 1A shows a gene ontology (GO) analysis of the top 500 differentially expressed genes in Ly49H+ wild-type splenic NK cells collected from either naive or MCMV-infected mice at 1.5 days post-infection (PI). The top 10 enriched GO clusters with the keyword "stimulation" are shown. Figure 1B shows a heatmap of RNA-seq analysis showing the expression of canonical IRE1α / XBP1 target genes over a period of time after MCMV infection. Figure 1C shows flow cytometry analysis of Venus reporter expression in splenic NK cells from ERAI transgenic reporter mice at 2 days post-infection (PI). Figure 1D shows a heatmap of RNA-seq analysis demonstrating the expression of canonical IRE1α / XBP1 target genes in splenic NK cells harvested from wild-type mice and cultured for 16 hours in either the presence or absence of IL-12 and IL-18 (GSE106138). Figure 1E shows flow cytometry analysis of Venus reporter expression in sorted, purified splenic NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of IL-12 and IL-18. Representative histogram plots (left) and quantification (right) are shown. Figure 1F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 hours of culture in the presence or absence of human recombinant IL-12 and IL-18, or IL-15. β-ACTIN was used as a reference, and data are presented as fold changes normalized to unstimulated levels. Figure 1G shows flow cytometry analysis of Venus reporter expression in sorted purified spleen and BM NK cells from ERAI transgenic reporter mice after 16 hours of culture in the presence or absence of murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml), or IL-15 (100 ng / ml). Representative histogram plots (top) and quantification (bottom). p values ​​are as indicated, and ns indicates "not significant."For Figures 1C and 1E, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figure 1F, a one-sample t-test was performed. From biological replicates, error bars in Figures 1C and 1E indicate standard deviation, and in Figure 1F, values ​​are shown from minimum to maximum. Each column in Figures 1B and 1D represents a different mouse. Experiments in Figures 1C and 1E contained 2-3 mice per group and were independently repeated three times. N equaled 6 PBMC donors in Figure 1F, and experiments were independently repeated two times. [Figure 2A]Figures 2A-2D show qPCR validation of the upregulation of the IRE1α / XBP1 pathway and ER stress markers in activated NK cells, as well as the requirement for STAT4 and mTOR signaling pathways in driving IRE1α / XBP1. Figure 2A shows quantitative real-time PCR analysis of the indicated UPR genes in sorted, purified splenic NK cells from wild-type mice 1 day post-MCMV infection. β-ACTIN was used as a reference, and data were normalized to uninfected control expression levels. Figure 2B shows quantitative real-time PCR analysis of the indicated UPR genes in sorted, purified splenic NK cells from wild-type mice after 16 hours of culture in the presence or absence of IL-12 and IL-18. Figure 2C shows a heatmap of an RNA-seq analysis (GSE106138) demonstrating the expression of canonical IRE1α / XBP1 target genes in STAT4-deficient Ly49H+ NK cells sorted from the spleens of wild-type and mixed-BM chimeric mice 2 days post-MCMV infection. Figure 2D shows flow cytometry analysis of IRE1α activation in cytokine-activated NK cells after pharmaceutical inhibition of mTOR. NK cells from ERAI reporter mice were stimulated for 6 hours with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) in the presence of the mTOR inhibitors rapamycin (Rapa, 5 nM and 10 nM for low and high doses, respectively), Ku-0063794 (Ku, 1.5 μM and 3 μM for low and high doses, respectively), PP242 (PP, 0.5 μM and 1 μM for low and high doses, respectively), or the IRE1 inhibitor 4μ8C (2.5 μM and 5 μM for low and high doses, respectively), or DMSO control. Representative flow cytometry plots (top) and quantification of relative inhibitory efficiency (bottom) are shown. ns indicates "not significant," * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.0001, and **** indicates p<0.0001. For Figure 2A, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 2B, a two-tailed unpaired Student's t-test was performed. All error bars indicate standard deviation from biological replicates. Data are representative of three independent experiments.N equals 3 mice / group in Figure 2A. N equals 3 mice in Figure 2B, with 3 technical replicates for ex vivo culture. Each column is a different mouse in Figure 2C. N equals 3 ERAI mice in Figure 2D. The experiment was repeated three times (Figures 2A and 2B) and twice (Figure 2D) independently. [Figure 2B]Figures 2A-2D show qPCR validation of the upregulation of the IRE1α / XBP1 pathway and ER stress markers in activated NK cells, as well as the requirement for STAT4 and mTOR signaling pathways in driving IRE1α / XBP1. Figure 2A shows quantitative real-time PCR analysis of the indicated UPR genes in sorted, purified splenic NK cells from wild-type mice 1 day post-MCMV infection. β-ACTIN was used as a reference, and data were normalized to uninfected control expression levels. Figure 2B shows quantitative real-time PCR analysis of the indicated UPR genes in sorted, purified splenic NK cells from wild-type mice after 16 hours of culture in the presence or absence of IL-12 and IL-18. Figure 2C shows a heatmap of an RNA-seq analysis (GSE106138) demonstrating the expression of canonical IRE1α / XBP1 target genes in STAT4-deficient Ly49H+ NK cells sorted from the spleens of wild-type and mixed-BM chimeric mice 2 days post-MCMV infection. Figure 2D shows flow cytometry analysis of IRE1α activation in cytokine-activated NK cells after pharmaceutical inhibition of mTOR. NK cells from ERAI reporter mice were stimulated for 6 hours with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) in the presence of the mTOR inhibitors rapamycin (Rapa, 5 nM and 10 nM for low and high doses, respectively), Ku-0063794 (Ku, 1.5 μM and 3 μM for low and high doses, respectively), PP242 (PP, 0.5 μM and 1 μM for low and high doses, respectively), or the IRE1 inhibitor 4μ8C (2.5 μM and 5 μM for low and high doses, respectively), or DMSO control. Representative flow cytometry plots (top) and quantification of relative inhibitory efficiency (bottom) are shown. ns indicates "not significant," * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.0001, and **** indicates p<0.0001. For Figure 2A, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 2B, a two-tailed unpaired Student's t-test was performed. All error bars indicate standard deviation from biological replicates. Data are representative of three independent experiments.N equals 3 mice / group in Figure 2A. N equals 3 mice in Figure 2B, with 3 technical replicates for ex vivo culture. Each column is a different mouse in Figure 2C. N equals 3 ERAI mice in Figure 2D. The experiment was repeated three times (Figures 2A and 2B) and twice (Figure 2D) independently. [Figure 2C]Figures 2A-2D show qPCR validation of the upregulation of the IRE1α / XBP1 pathway and ER stress markers in activated NK cells, as well as the requirement for STAT4 and mTOR signaling pathways in driving IRE1α / XBP1. Figure 2A shows quantitative real-time PCR analysis of the indicated UPR genes in sorted, purified splenic NK cells from wild-type mice 1 day post-MCMV infection. β-ACTIN was used as a reference, and data were normalized to uninfected control expression levels. Figure 2B shows quantitative real-time PCR analysis of the indicated UPR genes in sorted, purified splenic NK cells from wild-type mice after 16 hours of culture in the presence or absence of IL-12 and IL-18. Figure 2C shows a heatmap of an RNA-seq analysis (GSE106138) demonstrating the expression of canonical IRE1α / XBP1 target genes in STAT4-deficient Ly49H+ NK cells sorted from the spleens of wild-type and mixed-BM chimeric mice 2 days post-MCMV infection. Figure 2D shows flow cytometry analysis of IRE1α activation in cytokine-activated NK cells after pharmaceutical inhibition of mTOR. NK cells from ERAI reporter mice were stimulated for 6 hours with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) in the presence of the mTOR inhibitors rapamycin (Rapa, 5 nM and 10 nM for low and high doses, respectively), Ku-0063794 (Ku, 1.5 μM and 3 μM for low and high doses, respectively), PP242 (PP, 0.5 μM and 1 μM for low and high doses, respectively), or the IRE1 inhibitor 4μ8C (2.5 μM and 5 μM for low and high doses, respectively), or DMSO control. Representative flow cytometry plots (top) and quantification of relative inhibitory efficiency (bottom) are shown. ns indicates "not significant," * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.0001, and **** indicates p<0.0001. For Figure 2A, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 2B, a two-tailed unpaired Student's t-test was performed. All error bars indicate standard deviation from biological replicates. Data are representative of three independent experiments.N equals 3 mice / group in Figure 2A. N equals 3 mice in Figure 2B, with 3 technical replicates for ex vivo culture. Each column is a different mouse in Figure 2C. N equals 3 ERAI mice in Figure 2D. The experiment was repeated three times (Figures 2A and 2B) and twice (Figure 2D) independently. [Figure 2D]Figures 2A-2D show qPCR validation of the upregulation of the IRE1α / XBP1 pathway and ER stress markers in activated NK cells, as well as the requirement for STAT4 and mTOR signaling pathways in driving IRE1α / XBP1. Figure 2A shows quantitative real-time PCR analysis of the indicated UPR genes in sorted, purified splenic NK cells from wild-type mice 1 day post-MCMV infection. β-ACTIN was used as a reference, and data were normalized to uninfected control expression levels. Figure 2B shows quantitative real-time PCR analysis of the indicated UPR genes in sorted, purified splenic NK cells from wild-type mice after 16 hours of culture in the presence or absence of IL-12 and IL-18. Figure 2C shows a heatmap of an RNA-seq analysis (GSE106138) demonstrating the expression of canonical IRE1α / XBP1 target genes in STAT4-deficient Ly49H+ NK cells sorted from the spleens of wild-type and mixed-BM chimeric mice 2 days post-MCMV infection. Figure 2D shows flow cytometry analysis of IRE1α activation in cytokine-activated NK cells after pharmaceutical inhibition of mTOR. NK cells from ERAI reporter mice were stimulated for 6 hours with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) in the presence of the mTOR inhibitors rapamycin (Rapa, 5 nM and 10 nM for low and high doses, respectively), Ku-0063794 (Ku, 1.5 μM and 3 μM for low and high doses, respectively), PP242 (PP, 0.5 μM and 1 μM for low and high doses, respectively), or the IRE1 inhibitor 4μ8C (2.5 μM and 5 μM for low and high doses, respectively), or DMSO control. Representative flow cytometry plots (top) and quantification of relative inhibitory efficiency (bottom) are shown. ns indicates "not significant," * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.0001, and **** indicates p<0.0001. For Figure 2A, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 2B, a two-tailed unpaired Student's t-test was performed. All error bars indicate standard deviation from biological replicates. Data are representative of three independent experiments.N equals 3 mice / group in Figure 2A. N equals 3 mice in Figure 2B, with 3 technical replicates for ex vivo culture. Each column is a different mouse in Figure 2C. N equals 3 ERAI mice in Figure 2D. The experiment was repeated three times (Figures 2A and 2B) and twice (Figure 2D) independently. [Figure 3A]Figures 3A-3F show that IRE1α / XBP1 is involved in NK cell development and maturation. Figure 3A shows validation of IRE1NK knockout efficiency. XBP1 splicing assay (upper panel) and quantitative real-time PCR analysis (lower panel) of the indicated UPR genes in sorted purified splenic NK cells from IRE1NK and IRE1f / f littermate mice after 4 hours of ex vivo incubation with or without tunicamycin, a pharmacological inducer of ER stress. Quantitative real-time PCR data are shown as relative expression to β-actin. Figure 3B shows flow cytometry analysis of NK cell development in the bone marrow of IRE1NK and XBP1NK naive mice. The percentages of NK precursor (NKP, DX5-NK1.1-), immature (iNK, DX5-NK1.1+), and mature (mNK, DX5-NK1.1+) NK cells are shown. Figure 3C shows flow cytometry analysis of NK cell mutations in the spleens of IRE1NK and XBP1NK mice. The absolute number and percentage of NK cells (Lin-NK1.1+) and the percentage of Ly49H or KLRG1 expression on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice are shown (upper panel). The percentage of immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) NK cells is shown (lower panel). Figure 3D shows representative histogram plots showing the surface expression of cytokine receptors and activating receptors on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice. Figure 3E shows flow cytometry analysis of NK cell repopulation in mixed bone marrow chimeric mice. WT(CD45.1):IRE1NK(CD45.2) and WT(CD45.1):IRE1vav1(CD45.2) bone marrow chimeras were generated as shown in the schematic diagram (left). NK cell repopulation in irradiated recipient mice was assessed in peripheral blood 8 weeks after bone marrow transplantation (right).Figure 3F shows flow cytometry analysis of the percentage and absolute number of NK cells in the BM, spleen, and lungs of IRE1NK and XBP1NK naive mice compared with their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. ns indicates "not significant," * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. A two-tailed unpaired Student's t-test was performed for Figure 3A. All error bars indicate standard deviation. N equals 3 mice / group in Figures 3A and 3D. N equals 5 mice / group in Figures 3B-3C and 3F. N equals 10 mice in Figure 3E. The experiment was repeated three times (Figures 3A-3C and 3E-3F) and twice (Figure 3A and Figure 3D) independently. [Figure 3B]Figures 3A-3F show that IRE1α / XBP1 is involved in NK cell development and maturation. Figure 3A shows validation of IRE1NK knockout efficiency. XBP1 splicing assay (upper panel) and quantitative real-time PCR analysis (lower panel) of the indicated UPR genes in sorted purified splenic NK cells from IRE1NK and IRE1f / f littermate mice after 4 hours of ex vivo incubation with or without tunicamycin, a pharmacological inducer of ER stress. Quantitative real-time PCR data are shown as relative expression to β-actin. Figure 3B shows flow cytometry analysis of NK cell development in the bone marrow of IRE1NK and XBP1NK naive mice. The percentages of NK precursor (NKP, DX5-NK1.1-), immature (iNK, DX5-NK1.1+), and mature (mNK, DX5-NK1.1+) NK cells are shown. Figure 3C shows flow cytometry analysis of NK cell mutations in the spleens of IRE1NK and XBP1NK mice. The absolute number and percentage of NK cells (Lin-NK1.1+) and the percentage of Ly49H or KLRG1 expression on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice are shown (upper panel). The percentage of immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) NK cells is shown (lower panel). Figure 3D shows representative histogram plots showing the surface expression of cytokine receptors and activating receptors on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice. Figure 3E shows flow cytometry analysis of NK cell repopulation in mixed bone marrow chimeric mice. WT(CD45.1):IRE1NK(CD45.2) and WT(CD45.1):IRE1vav1(CD45.2) bone marrow chimeras were generated as shown in the schematic diagram (left). NK cell repopulation in irradiated recipient mice was assessed in peripheral blood 8 weeks after bone marrow transplantation (right).Figure 3F shows flow cytometry analysis of the percentage and absolute number of NK cells in the BM, spleen, and lungs of IRE1NK and XBP1NK naive mice compared with their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. ns indicates "not significant," * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. A two-tailed unpaired Student's t-test was performed for Figure 3A. All error bars indicate standard deviation. N equals 3 mice / group in Figures 3A and 3D. N equals 5 mice / group in Figures 3B-3C and 3F. N equals 10 mice in Figure 3E. The experiment was repeated three times (Figures 3A-3C and 3E-3F) and twice (Figure 3A and Figure 3D) independently. [Figure 3C]Figures 3A-3F show that IRE1α / XBP1 is involved in NK cell development and maturation. Figure 3A shows validation of IRE1NK knockout efficiency. XBP1 splicing assay (upper panel) and quantitative real-time PCR analysis (lower panel) of the indicated UPR genes in sorted purified splenic NK cells from IRE1NK and IRE1f / f littermate mice after 4 hours of ex vivo incubation with or without tunicamycin, a pharmacological inducer of ER stress. Quantitative real-time PCR data are shown as relative expression to β-actin. Figure 3B shows flow cytometry analysis of NK cell development in the bone marrow of IRE1NK and XBP1NK naive mice. The percentages of NK precursor (NKP, DX5-NK1.1-), immature (iNK, DX5-NK1.1+), and mature (mNK, DX5-NK1.1+) NK cells are shown. Figure 3C shows flow cytometry analysis of NK cell mutations in the spleens of IRE1NK and XBP1NK mice. The absolute number and percentage of NK cells (Lin-NK1.1+) and the percentage of Ly49H or KLRG1 expression on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice are shown (upper panel). The percentage of immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) NK cells is shown (lower panel). Figure 3D shows representative histogram plots showing the surface expression of cytokine receptors and activating receptors on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice. Figure 3E shows flow cytometry analysis of NK cell repopulation in mixed bone marrow chimeric mice. WT(CD45.1):IRE1NK(CD45.2) and WT(CD45.1):IRE1vav1(CD45.2) bone marrow chimeras were generated as shown in the schematic diagram (left). NK cell repopulation in irradiated recipient mice was assessed in peripheral blood 8 weeks after bone marrow transplantation (right).Figure 3F shows flow cytometry analysis of the percentage and absolute number of NK cells in the BM, spleen, and lungs of IRE1NK and XBP1NK naive mice compared with their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. ns indicates "not significant," * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. A two-tailed unpaired Student's t-test was performed for Figure 3A. All error bars indicate standard deviation. N equals 3 mice / group in Figures 3A and 3D. N equals 5 mice / group in Figures 3B-3C and 3F. N equals 10 mice in Figure 3E. The experiment was repeated three times (Figures 3A-3C and 3E-3F) and twice (Figure 3A and Figure 3D) independently. [Figure 3D]Figures 3A-3F show that IRE1α / XBP1 is involved in NK cell development and maturation. Figure 3A shows validation of IRE1NK knockout efficiency. XBP1 splicing assay (upper panel) and quantitative real-time PCR analysis (lower panel) of the indicated UPR genes in sorted purified splenic NK cells from IRE1NK and IRE1f / f littermate mice after 4 hours of ex vivo incubation with or without tunicamycin, a pharmacological inducer of ER stress. Quantitative real-time PCR data are shown as relative expression to β-actin. Figure 3B shows flow cytometry analysis of NK cell development in the bone marrow of IRE1NK and XBP1NK naive mice. The percentages of NK precursor (NKP, DX5-NK1.1-), immature (iNK, DX5-NK1.1+), and mature (mNK, DX5-NK1.1+) NK cells are shown. Figure 3C shows flow cytometry analysis of NK cell mutations in the spleens of IRE1NK and XBP1NK mice. The absolute number and percentage of NK cells (Lin-NK1.1+) and the percentage of Ly49H or KLRG1 expression on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice are shown (upper panel). The percentage of immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) NK cells is shown (lower panel). Figure 3D shows representative histogram plots showing the surface expression of cytokine receptors and activating receptors on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice. Figure 3E shows flow cytometry analysis of NK cell repopulation in mixed bone marrow chimeric mice. WT(CD45.1):IRE1NK(CD45.2) and WT(CD45.1):IRE1vav1(CD45.2) bone marrow chimeras were generated as shown in the schematic diagram (left). NK cell repopulation in irradiated recipient mice was assessed in peripheral blood 8 weeks after bone marrow transplantation (right).Figure 3F shows flow cytometry analysis of the percentage and absolute number of NK cells in the BM, spleen, and lungs of IRE1NK and XBP1NK naive mice compared with their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. ns indicates "not significant," * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. A two-tailed unpaired Student's t-test was performed for Figure 3A. All error bars indicate standard deviation. N equals 3 mice / group in Figures 3A and 3D. N equals 5 mice / group in Figures 3B-3C and 3F. N equals 10 mice in Figure 3E. The experiment was repeated three times (Figures 3A-3C and 3E-3F) and twice (Figure 3A and Figure 3D) independently. [Figure 3E]Figures 3A-3F show that IRE1α / XBP1 is involved in NK cell development and maturation. Figure 3A shows validation of IRE1NK knockout efficiency. XBP1 splicing assay (upper panel) and quantitative real-time PCR analysis (lower panel) of the indicated UPR genes in sorted purified splenic NK cells from IRE1NK and IRE1f / f littermate mice after 4 hours of ex vivo incubation with or without tunicamycin, a pharmacological inducer of ER stress. Quantitative real-time PCR data are shown as relative expression to β-actin. Figure 3B shows flow cytometry analysis of NK cell development in the bone marrow of IRE1NK and XBP1NK naive mice. The percentages of NK precursor (NKP, DX5-NK1.1-), immature (iNK, DX5-NK1.1+), and mature (mNK, DX5-NK1.1+) NK cells are shown. Figure 3C shows flow cytometry analysis of NK cell mutations in the spleens of IRE1NK and XBP1NK mice. The absolute number and percentage of NK cells (Lin-NK1.1+) and the percentage of Ly49H or KLRG1 expression on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice are shown (upper panel). The percentage of immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) NK cells is shown (lower panel). Figure 3D shows representative histogram plots showing the surface expression of cytokine receptors and activating receptors on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice. Figure 3E shows flow cytometry analysis of NK cell repopulation in mixed bone marrow chimeric mice. WT(CD45.1):IRE1NK(CD45.2) and WT(CD45.1):IRE1vav1(CD45.2) bone marrow chimeras were generated as shown in the schematic diagram (left). NK cell repopulation in irradiated recipient mice was assessed in peripheral blood 8 weeks after bone marrow transplantation (right).Figure 3F shows flow cytometry analysis of the percentage and absolute number of NK cells in the BM, spleen, and lungs of IRE1NK and XBP1NK naive mice compared with their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. ns indicates "not significant," * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. A two-tailed unpaired Student's t-test was performed for Figure 3A. All error bars indicate standard deviation. N equals 3 mice / group in Figures 3A and 3D. N equals 5 mice / group in Figures 3B-3C and 3F. N equals 10 mice in Figure 3E. The experiment was repeated three times (Figures 3A-3C and 3E-3F) and twice (Figure 3A and Figure 3D) independently. [Figure 3F]Figures 3A-3F show that IRE1α / XBP1 is involved in NK cell development and maturation. Figure 3A shows validation of IRE1NK knockout efficiency. XBP1 splicing assay (upper panel) and quantitative real-time PCR analysis (lower panel) of the indicated UPR genes in sorted purified splenic NK cells from IRE1NK and IRE1f / f littermate mice after 4 hours of ex vivo incubation with or without tunicamycin, a pharmacological inducer of ER stress. Quantitative real-time PCR data are shown as relative expression to β-actin. Figure 3B shows flow cytometry analysis of NK cell development in the bone marrow of IRE1NK and XBP1NK naive mice. The percentages of NK precursor (NKP, DX5-NK1.1-), immature (iNK, DX5-NK1.1+), and mature (mNK, DX5-NK1.1+) NK cells are shown. Figure 3C shows flow cytometry analysis of NK cell mutations in the spleens of IRE1NK and XBP1NK mice. The absolute number and percentage of NK cells (Lin-NK1.1+) and the percentage of Ly49H or KLRG1 expression on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice are shown (upper panel). The percentage of immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) NK cells is shown (lower panel). Figure 3D shows representative histogram plots showing the surface expression of cytokine receptors and activating receptors on splenic NK cells from naive IRE1NK and IRE1f / f littermate control mice. Figure 3E shows flow cytometry analysis of NK cell repopulation in mixed bone marrow chimeric mice. WT(CD45.1):IRE1NK(CD45.2) and WT(CD45.1):IRE1vav1(CD45.2) bone marrow chimeras were generated as shown in the schematic diagram (left). NK cell repopulation in irradiated recipient mice was assessed in peripheral blood 8 weeks after bone marrow transplantation (right).Figure 3F shows flow cytometry analysis of the percentage and absolute number of NK cells in the BM, spleen, and lungs of IRE1NK and XBP1NK naive mice compared with their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. ns indicates "not significant," * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. A two-tailed unpaired Student's t-test was performed for Figure 3A. All error bars indicate standard deviation. N equals 3 mice / group in Figures 3A and 3D. N equals 5 mice / group in Figures 3B-3C and 3F. N equals 10 mice in Figure 3E. The experiment was repeated three times (Figures 3A-3C and 3E-3F) and twice (Figure 3A and Figure 3D) independently. [Figure 4A]Figures 4A-4H show that IRE1α is required for optimal protective antiviral NK cell responses. IRE1NK and littermate control mice were infected with a lethal dose of MCMV. Figure 4A shows the viral titer in the blood at 4 days PI. Figure 4B shows the survival curve. Figure 4C shows a schematic of the co-transplantation experiment in Figures 4D-4H. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 4D shows quantification of the percentage of engrafted wild-type and IRE1NK Ly49H+ NK cells in the peripheral blood at predetermined time points after MCMV infection. Figure 4E is similar to Figure 4D, except that the relative percentages within the engrafted Ly49H+ NK cells are shown. Figure 4F shows the relative percentages of transplanted wild-type and IRE1NKLy49H+ NK cells in various organs at day 8 (LN) or day 10 (all other tissues) after infection with MCMV. BM indicates bone marrow, and LN indicates lymph node. Figure 4G is the same as Figure 4D, except that the knockout donor was XBP1NK. Figure 4H is the same as Figure 4E, except that the knockout donor was XBP1NK. For Figure 4A, a two-tailed unpaired Student's t-test was performed, and for Figure 4B, a Gehan-Breslow-Wilcoxon test was performed. For Figures 4D–4H, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. p values ​​are as indicated, and ns indicates "not significant." Error bars indicate the standard error of the mean for Figure 4A or the standard deviation for Figures 4E, 4F, and 4H. The experiment was independently repeated three times (Figures 4A, 4F, 4G, and 4H) and four times (Figures 4D-4E). Data are cumulative from three experiments (Figure 4B). In all experiments, N equaled 4 mice / group or more than 4 mice / group. [Figure 4B]Figures 4A-4H show that IRE1α is required for optimal protective antiviral NK cell responses. IRE1NK and littermate control mice were infected with a lethal dose of MCMV. Figure 4A shows the viral titer in the blood at 4 days PI. Figure 4B shows the survival curve. Figure 4C shows a schematic of the co-transplantation experiment in Figures 4D-4H. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 4D shows quantification of the percentage of engrafted wild-type and IRE1NK Ly49H+ NK cells in the peripheral blood at predetermined time points after MCMV infection. Figure 4E is similar to Figure 4D, except that the relative percentages within the engrafted Ly49H+ NK cells are shown. Figure 4F shows the relative percentages of transplanted wild-type and IRE1NKLy49H+ NK cells in various organs at day 8 (LN) or day 10 (all other tissues) after infection with MCMV. BM indicates bone marrow, and LN indicates lymph node. Figure 4G is the same as Figure 4D, except that the knockout donor was XBP1NK. Figure 4H is the same as Figure 4E, except that the knockout donor was XBP1NK. For Figure 4A, a two-tailed unpaired Student's t-test was performed, and for Figure 4B, a Gehan-Breslow-Wilcoxon test was performed. For Figures 4D–4H, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. p values ​​are as indicated, and ns indicates "not significant." Error bars indicate the standard error of the mean for Figure 4A or the standard deviation for Figures 4E, 4F, and 4H. The experiment was independently repeated three times (Figures 4A, 4F, 4G, and 4H) and four times (Figures 4D-4E). Data are cumulative from three experiments (Figure 4B). In all experiments, N equaled 4 mice / group or more than 4 mice / group. [Figure 4C]Figures 4A-4H show that IRE1α is required for optimal protective antiviral NK cell responses. IRE1NK and littermate control mice were infected with a lethal dose of MCMV. Figure 4A shows the viral titer in the blood at 4 days PI. Figure 4B shows the survival curve. Figure 4C shows a schematic of the co-transplantation experiment in Figures 4D-4H. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 4D shows quantification of the percentage of engrafted wild-type and IRE1NK Ly49H+ NK cells in the peripheral blood at predetermined time points after MCMV infection. Figure 4E is similar to Figure 4D, except that the relative percentages within the engrafted Ly49H+ NK cells are shown. Figure 4F shows the relative percentages of transplanted wild-type and IRE1NKLy49H+ NK cells in various organs at day 8 (LN) or day 10 (all other tissues) after infection with MCMV. BM indicates bone marrow, and LN indicates lymph node. Figure 4G is the same as Figure 4D, except that the knockout donor was XBP1NK. Figure 4H is the same as Figure 4E, except that the knockout donor was XBP1NK. For Figure 4A, a two-tailed unpaired Student's t-test was performed, and for Figure 4B, a Gehan-Breslow-Wilcoxon test was performed. For Figures 4D–4H, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. p values ​​are as indicated, and ns indicates "not significant." Error bars indicate the standard error of the mean for Figure 4A or the standard deviation for Figures 4E, 4F, and 4H. The experiment was independently repeated three times (Figures 4A, 4F, 4G, and 4H) and four times (Figures 4D-4E). Data are cumulative from three experiments (Figure 4B). In all experiments, N equaled 4 mice / group or more than 4 mice / group. [Figure 4D]Figures 4A-4H show that IRE1α is required for optimal protective antiviral NK cell responses. IRE1NK and littermate control mice were infected with a lethal dose of MCMV. Figure 4A shows the viral titer in the blood at 4 days PI. Figure 4B shows the survival curve. Figure 4C shows a schematic of the co-transplantation experiment in Figures 4D-4H. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 4D shows quantification of the percentage of engrafted wild-type and IRE1NK Ly49H+ NK cells in the peripheral blood at predetermined time points after MCMV infection. Figure 4E is similar to Figure 4D, except that the relative percentages within the engrafted Ly49H+ NK cells are shown. Figure 4F shows the relative percentages of transplanted wild-type and IRE1NKLy49H+ NK cells in various organs at day 8 (LN) or day 10 (all other tissues) after infection with MCMV. BM indicates bone marrow, and LN indicates lymph node. Figure 4G is the same as Figure 4D, except that the knockout donor was XBP1NK. Figure 4H is the same as Figure 4E, except that the knockout donor was XBP1NK. For Figure 4A, a two-tailed unpaired Student's t-test was performed, and for Figure 4B, a Gehan-Breslow-Wilcoxon test was performed. For Figures 4D–4H, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. p values ​​are as indicated, and ns indicates "not significant." Error bars indicate the standard error of the mean for Figure 4A or the standard deviation for Figures 4E, 4F, and 4H. The experiment was independently repeated three times (Figures 4A, 4F, 4G, and 4H) and four times (Figures 4D-4E). Data are cumulative from three experiments (Figure 4B). In all experiments, N equaled 4 mice / group or more than 4 mice / group. [Figure 4E]Figures 4A-4H show that IRE1α is required for optimal protective antiviral NK cell responses. IRE1NK and littermate control mice were infected with a lethal dose of MCMV. Figure 4A shows the viral titer in the blood at 4 days PI. Figure 4B shows the survival curve. Figure 4C shows a schematic of the co-transplantation experiment in Figures 4D-4H. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 4D shows quantification of the percentage of engrafted wild-type and IRE1NK Ly49H+ NK cells in the peripheral blood at predetermined time points after MCMV infection. Figure 4E is similar to Figure 4D, except that the relative percentages within the engrafted Ly49H+ NK cells are shown. Figure 4F shows the relative percentages of transplanted wild-type and IRE1NKLy49H+ NK cells in various organs at day 8 (LN) or day 10 (all other tissues) after infection with MCMV. BM indicates bone marrow, and LN indicates lymph node. Figure 4G is the same as Figure 4D, except that the knockout donor was XBP1NK. Figure 4H is the same as Figure 4E, except that the knockout donor was XBP1NK. For Figure 4A, a two-tailed unpaired Student's t-test was performed, and for Figure 4B, a Gehan-Breslow-Wilcoxon test was performed. For Figures 4D–4H, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. p values ​​are as indicated, and ns indicates "not significant." Error bars indicate the standard error of the mean for Figure 4A or the standard deviation for Figures 4E, 4F, and 4H. The experiment was independently repeated three times (Figures 4A, 4F, 4G, and 4H) and four times (Figures 4D-4E). Data are cumulative from three experiments (Figure 4B). In all experiments, N equaled 4 mice / group or more than 4 mice / group. [Figure 4F]Figures 4A-4H show that IRE1α is required for optimal protective antiviral NK cell responses. IRE1NK and littermate control mice were infected with a lethal dose of MCMV. Figure 4A shows the viral titer in the blood at 4 days PI. Figure 4B shows the survival curve. Figure 4C shows a schematic of the co-transplantation experiment in Figures 4D-4H. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 4D shows quantification of the percentage of engrafted wild-type and IRE1NK Ly49H+ NK cells in the peripheral blood at predetermined time points after MCMV infection. Figure 4E is similar to Figure 4D, except that the relative percentages within the engrafted Ly49H+ NK cells are shown. Figure 4F shows the relative percentages of transplanted wild-type and IRE1NKLy49H+ NK cells in various organs at day 8 (LN) or day 10 (all other tissues) after infection with MCMV. BM indicates bone marrow, and LN indicates lymph node. Figure 4G is the same as Figure 4D, except that the knockout donor was XBP1NK. Figure 4H is the same as Figure 4E, except that the knockout donor was XBP1NK. For Figure 4A, a two-tailed unpaired Student's t-test was performed, and for Figure 4B, a Gehan-Breslow-Wilcoxon test was performed. For Figures 4D–4H, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. p values ​​are as indicated, and ns indicates "not significant." Error bars indicate the standard error of the mean for Figure 4A or the standard deviation for Figures 4E, 4F, and 4H. The experiment was independently repeated three times (Figures 4A, 4F, 4G, and 4H) and four times (Figures 4D-4E). Data are cumulative from three experiments (Figure 4B). In all experiments, N equaled 4 mice / group or more than 4 mice / group. [Figure 4G]Figures 4A-4H show that IRE1α is required for optimal protective antiviral NK cell responses. IRE1NK and littermate control mice were infected with a lethal dose of MCMV. Figure 4A shows the viral titer in the blood at 4 days PI. Figure 4B shows the survival curve. Figure 4C shows a schematic of the co-transplantation experiment in Figures 4D-4H. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 4D shows quantification of the percentage of engrafted wild-type and IRE1NK Ly49H+ NK cells in the peripheral blood at predetermined time points after MCMV infection. Figure 4E is similar to Figure 4D, except that the relative percentages within the engrafted Ly49H+ NK cells are shown. Figure 4F shows the relative percentages of transplanted wild-type and IRE1NKLy49H+ NK cells in various organs at day 8 (LN) or day 10 (all other tissues) after infection with MCMV. BM indicates bone marrow, and LN indicates lymph node. Figure 4G is the same as Figure 4D, except that the knockout donor was XBP1NK. Figure 4H is the same as Figure 4E, except that the knockout donor was XBP1NK. For Figure 4A, a two-tailed unpaired Student's t-test was performed, and for Figure 4B, a Gehan-Breslow-Wilcoxon test was performed. For Figures 4D–4H, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. p values ​​are as indicated, and ns indicates "not significant." Error bars indicate the standard error of the mean for Figure 4A or the standard deviation for Figures 4E, 4F, and 4H. The experiment was independently repeated three times (Figures 4A, 4F, 4G, and 4H) and four times (Figures 4D-4E). Data are cumulative from three experiments (Figure 4B). In all experiments, N equaled 4 mice / group or more than 4 mice / group. [Figure 4H]Figures 4A-4H show that IRE1α is required for optimal protective antiviral NK cell responses. IRE1NK and littermate control mice were infected with a lethal dose of MCMV. Figure 4A shows the viral titer in the blood at 4 days PI. Figure 4B shows the survival curve. Figure 4C shows a schematic of the co-transplantation experiment in Figures 4D-4H. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 4D shows quantification of the percentage of engrafted wild-type and IRE1NK Ly49H+ NK cells in the peripheral blood at predetermined time points after MCMV infection. Figure 4E is similar to Figure 4D, except that the relative percentages within the engrafted Ly49H+ NK cells are shown. Figure 4F shows the relative percentages of transplanted wild-type and IRE1NKLy49H+ NK cells in various organs at day 8 (LN) or day 10 (all other tissues) after infection with MCMV. BM indicates bone marrow, and LN indicates lymph node. Figure 4G is the same as Figure 4D, except that the knockout donor was XBP1NK. Figure 4H is the same as Figure 4E, except that the knockout donor was XBP1NK. For Figure 4A, a two-tailed unpaired Student's t-test was performed, and for Figure 4B, a Gehan-Breslow-Wilcoxon test was performed. For Figures 4D–4H, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. p values ​​are as indicated, and ns indicates "not significant." Error bars indicate the standard error of the mean for Figure 4A or the standard deviation for Figures 4E, 4F, and 4H. The experiment was independently repeated three times (Figures 4A, 4F, 4G, and 4H) and four times (Figures 4D-4E). Data are cumulative from three experiments (Figure 4B). In all experiments, N equaled 4 mice / group or more than 4 mice / group. [Figure 5A]Figures 5A-5D show the distributable functions of IRE1α / XBP1 in NK cell priming, cytokine production, or cytotoxicity. Figure 5A shows the gating strategy applied in the analysis of co-transplantation and mixed bone marrow chimera experiments. Figure 5B shows flow cytometry analysis of intracellular IFN-γ in splenic WT or IRE1NKLy49H+ NK cells from mixed bone marrow chimeric mice as shown in Figure 3E at day 2 post-infection with MCMV. Figure 5C shows flow cytometry analysis of CD69 and intracellular granzyme B in splenic WT or IRE1NKLy49H+ NK cells from mixed bone marrow chimeric mice at day 2 post-infection with MCMV. Figure 5D shows flow cytometry analysis of co-transplanted WT or IRE1NKLy49H+ NK cells from the spleen of a Ly49H-deficient recipient at 2 days post-infection. Quantification of pan-Akt, p-Akt, and p-S6 was normalized to WT expression levels as 100%. A two-tailed unpaired Student's t-test was performed for Figures 5B and 5C. All error bars indicate standard deviation. Data were independently repeated three times. N equals 6 mice in Figure 5B. N equals 4 mice in Figure 5C. N equals 3 mice in Figure 5D. [Figure 5B]Figures 5A-5D show the distributable functions of IRE1α / XBP1 in NK cell priming, cytokine production, or cytotoxicity. Figure 5A shows the gating strategy applied in the analysis of co-transplantation and mixed bone marrow chimera experiments. Figure 5B shows flow cytometry analysis of intracellular IFN-γ in splenic WT or IRE1NKLy49H+ NK cells from mixed bone marrow chimeric mice as shown in Figure 3E at day 2 post-infection with MCMV. Figure 5C shows flow cytometry analysis of CD69 and intracellular granzyme B in splenic WT or IRE1NKLy49H+ NK cells from mixed bone marrow chimeric mice at day 2 post-infection with MCMV. Figure 5D shows flow cytometry analysis of co-transplanted WT or IRE1NKLy49H+ NK cells from the spleen of a Ly49H-deficient recipient at 2 days post-infection. Quantification of pan-Akt, p-Akt, and p-S6 was normalized to WT expression levels as 100%. A two-tailed unpaired Student's t-test was performed for Figures 5B and 5C. All error bars indicate standard deviation. Data were independently repeated three times. N equals 6 mice in Figure 5B. N equals 4 mice in Figure 5C. N equals 3 mice in Figure 5D. [Figure 5C]Figures 5A-5D show the distributable functions of IRE1α / XBP1 in NK cell priming, cytokine production, or cytotoxicity. Figure 5A shows the gating strategy applied in the analysis of co-transplantation and mixed bone marrow chimera experiments. Figure 5B shows flow cytometry analysis of intracellular IFN-γ in splenic WT or IRE1NKLy49H+ NK cells from mixed bone marrow chimeric mice as shown in Figure 3E at day 2 post-infection with MCMV. Figure 5C shows flow cytometry analysis of CD69 and intracellular granzyme B in splenic WT or IRE1NKLy49H+ NK cells from mixed bone marrow chimeric mice at day 2 post-infection with MCMV. Figure 5D shows flow cytometry analysis of co-transplanted WT or IRE1NKLy49H+ NK cells from the spleen of a Ly49H-deficient recipient at 2 days post-infection. Quantification of pan-Akt, p-Akt, and p-S6 was normalized to WT expression levels as 100%. A two-tailed unpaired Student's t-test was performed for Figures 5B and 5C. All error bars indicate standard deviation. Data were independently repeated three times. N equals 6 mice in Figure 5B. N equals 4 mice in Figure 5C. N equals 3 mice in Figure 5D. [Figure 5D]Figures 5A-5D show the distributable functions of IRE1α / XBP1 in NK cell priming, cytokine production, or cytotoxicity. Figure 5A shows the gating strategy applied in the analysis of co-transplantation and mixed bone marrow chimera experiments. Figure 5B shows flow cytometry analysis of intracellular IFN-γ in splenic WT or IRE1NKLy49H+ NK cells from mixed bone marrow chimeric mice as shown in Figure 3E at day 2 post-infection with MCMV. Figure 5C shows flow cytometry analysis of CD69 and intracellular granzyme B in splenic WT or IRE1NKLy49H+ NK cells from mixed bone marrow chimeric mice at day 2 post-infection with MCMV. Figure 5D shows flow cytometry analysis of co-transplanted WT or IRE1NKLy49H+ NK cells from the spleen of a Ly49H-deficient recipient at 2 days post-infection. Quantification of pan-Akt, p-Akt, and p-S6 was normalized to WT expression levels as 100%. A two-tailed unpaired Student's t-test was performed for Figures 5B and 5C. All error bars indicate standard deviation. Data were independently repeated three times. N equals 6 mice in Figure 5B. N equals 4 mice in Figure 5C. N equals 3 mice in Figure 5D. [Figure 6A]Figures 6A-6F show that IRE1α / XBP1 regulates infection-induced NK cell proliferation, but not survival. Figure 6A shows a schematic diagram of the assay used to assess infection-induced NK cell proliferation and apoptosis in Figures 6B-6F. Equal numbers of Ly49H+ NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transferred into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 6B shows the relative percentages of transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice at predetermined time points after MCMV infection. Figure 6C shows a representative plot (left) and quantification (right) of flow cytometry analysis showing CTV dilution of transplanted wild-type and IRE1NK Ly49H+ (responder) and Ly49H- (bystander) NK cells in the spleens 4 days after infection with MCMV. Flow cytometry analysis of EdU (Figure 6D) and Annexin V (Figure 6E) in cotransplanted Ly49H+ NK cells 3.5 days after infection with MCMV. Mice were intraperitoneally injected with EdU 12 hours before measurement. Figure 6F shows a representative flow cytometry plot (left) of FLICA+ cells and quantification of their percentage (right) in cotransplanted Ly49H+ NK cells similar to those in Figure 6D. p values ​​are as indicated, and ns indicates "not significant." For Figure 6B, a two-way analysis of variance (ANOVA) with Sidak's post-hoc test was performed. For Figure 6C, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 6D–6F, a two-tailed unpaired Student's t-test was performed. All error bars indicate standard deviation. Data were independently repeated three times (Figures 6B, 6C, and 6F) and twice (Figures 6D and 6E). N equals 4 mice / group for all experiments except for Figure 6C, where n equals 3 mice / group. [Figure 6B]Figures 6A-6F show that IRE1α / XBP1 regulates infection-induced NK cell proliferation, but not survival. Figure 6A shows a schematic diagram of the assay used to assess infection-induced NK cell proliferation and apoptosis in Figures 6B-6F. Equal numbers of Ly49H+ NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transferred into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 6B shows the relative percentages of transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice at predetermined time points after MCMV infection. Figure 6C shows a representative plot (left) and quantification (right) of flow cytometry analysis showing CTV dilution of transplanted wild-type and IRE1NK Ly49H+ (responder) and Ly49H- (bystander) NK cells in the spleens 4 days after infection with MCMV. Flow cytometry analysis of EdU (Figure 6D) and Annexin V (Figure 6E) in cotransplanted Ly49H+ NK cells 3.5 days after infection with MCMV. Mice were intraperitoneally injected with EdU 12 hours before measurement. Figure 6F shows a representative flow cytometry plot (left) of FLICA+ cells and quantification of their percentage (right) in cotransplanted Ly49H+ NK cells similar to those in Figure 6D. p values ​​are as indicated, and ns indicates "not significant." For Figure 6B, a two-way analysis of variance (ANOVA) with Sidak's post-hoc test was performed. For Figure 6C, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 6D–6F, a two-tailed unpaired Student's t-test was performed. All error bars indicate standard deviation. Data were independently repeated three times (Figures 6B, 6C, and 6F) and twice (Figures 6D and 6E). N equals 4 mice / group for all experiments except for Figure 6C, where n equals 3 mice / group. [Figure 6C]Figures 6A-6F show that IRE1α / XBP1 regulates infection-induced NK cell proliferation, but not survival. Figure 6A shows a schematic diagram of the assay used to assess infection-induced NK cell proliferation and apoptosis in Figures 6B-6F. Equal numbers of Ly49H+ NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transferred into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 6B shows the relative percentages of transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice at predetermined time points after MCMV infection. Figure 6C shows a representative plot (left) and quantification (right) of flow cytometry analysis showing CTV dilution of transplanted wild-type and IRE1NK Ly49H+ (responder) and Ly49H- (bystander) NK cells in the spleens 4 days after infection with MCMV. Flow cytometry analysis of EdU (Figure 6D) and Annexin V (Figure 6E) in cotransplanted Ly49H+ NK cells 3.5 days after infection with MCMV. Mice were intraperitoneally injected with EdU 12 hours before measurement. Figure 6F shows a representative flow cytometry plot (left) of FLICA+ cells and quantification of their percentage (right) in cotransplanted Ly49H+ NK cells similar to those in Figure 6D. p values ​​are as indicated, and ns indicates "not significant." For Figure 6B, a two-way analysis of variance (ANOVA) with Sidak's post-hoc test was performed. For Figure 6C, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 6D–6F, a two-tailed unpaired Student's t-test was performed. All error bars indicate standard deviation. Data were independently repeated three times (Figures 6B, 6C, and 6F) and twice (Figures 6D and 6E). N equals 4 mice / group for all experiments except for Figure 6C, where n equals 3 mice / group. [Figure 6D]Figures 6A-6F show that IRE1α / XBP1 regulates infection-induced NK cell proliferation, but not survival. Figure 6A shows a schematic diagram of the assay used to assess infection-induced NK cell proliferation and apoptosis in Figures 6B-6F. Equal numbers of Ly49H+ NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transferred into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 6B shows the relative percentages of transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice at predetermined time points after MCMV infection. Figure 6C shows a representative plot (left) and quantification (right) of flow cytometry analysis showing CTV dilution of transplanted wild-type and IRE1NK Ly49H+ (responder) and Ly49H- (bystander) NK cells in the spleens 4 days after infection with MCMV. Flow cytometry analysis of EdU (Figure 6D) and Annexin V (Figure 6E) in cotransplanted Ly49H+ NK cells 3.5 days after infection with MCMV. Mice were intraperitoneally injected with EdU 12 hours before measurement. Figure 6F shows a representative flow cytometry plot (left) of FLICA+ cells and quantification of their percentage (right) in cotransplanted Ly49H+ NK cells similar to those in Figure 6D. p values ​​are as indicated, and ns indicates "not significant." For Figure 6B, a two-way analysis of variance (ANOVA) with Sidak's post-hoc test was performed. For Figure 6C, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 6D–6F, a two-tailed unpaired Student's t-test was performed. All error bars indicate standard deviation. Data were independently repeated three times (Figures 6B, 6C, and 6F) and twice (Figures 6D and 6E). N equals 4 mice / group for all experiments except for Figure 6C, where n equals 3 mice / group. [Figure 6E]Figures 6A-6F show that IRE1α / XBP1 regulates infection-induced NK cell proliferation, but not survival. Figure 6A shows a schematic diagram of the assay used to assess infection-induced NK cell proliferation and apoptosis in Figures 6B-6F. Equal numbers of Ly49H+ NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transferred into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 6B shows the relative percentages of transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice at predetermined time points after MCMV infection. Figure 6C shows a representative plot (left) and quantification (right) of flow cytometry analysis showing CTV dilution of transplanted wild-type and IRE1NK Ly49H+ (responder) and Ly49H- (bystander) NK cells in the spleens 4 days after infection with MCMV. Flow cytometry analysis of EdU (Figure 6D) and Annexin V (Figure 6E) in cotransplanted Ly49H+ NK cells 3.5 days after infection with MCMV. Mice were intraperitoneally injected with EdU 12 hours before measurement. Figure 6F shows a representative flow cytometry plot (left) of FLICA+ cells and quantification of their percentage (right) in cotransplanted Ly49H+ NK cells similar to those in Figure 6D. p values ​​are as indicated, and ns indicates "not significant." For Figure 6B, a two-way analysis of variance (ANOVA) with Sidak's post-hoc test was performed. For Figure 6C, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 6D–6F, a two-tailed unpaired Student's t-test was performed. All error bars indicate standard deviation. Data were independently repeated three times (Figures 6B, 6C, and 6F) and twice (Figures 6D and 6E). N equals 4 mice / group for all experiments except for Figure 6C, where n equals 3 mice / group. [Figure 6F]Figures 6A-6F show that IRE1α / XBP1 regulates infection-induced NK cell proliferation, but not survival. Figure 6A shows a schematic diagram of the assay used to assess infection-induced NK cell proliferation and apoptosis in Figures 6B-6F. Equal numbers of Ly49H+ NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transferred into recipient Ly49H-deficient mice 1 day before infection with MCMV. Figure 6B shows the relative percentages of transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice at predetermined time points after MCMV infection. Figure 6C shows a representative plot (left) and quantification (right) of flow cytometry analysis showing CTV dilution of transplanted wild-type and IRE1NK Ly49H+ (responder) and Ly49H- (bystander) NK cells in the spleens 4 days after infection with MCMV. Flow cytometry analysis of EdU (Figure 6D) and Annexin V (Figure 6E) in cotransplanted Ly49H+ NK cells 3.5 days after infection with MCMV. Mice were intraperitoneally injected with EdU 12 hours before measurement. Figure 6F shows a representative flow cytometry plot (left) of FLICA+ cells and quantification of their percentage (right) in cotransplanted Ly49H+ NK cells similar to those in Figure 6D. p values ​​are as indicated, and ns indicates "not significant." For Figure 6B, a two-way analysis of variance (ANOVA) with Sidak's post-hoc test was performed. For Figure 6C, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 6D–6F, a two-tailed unpaired Student's t-test was performed. All error bars indicate standard deviation. Data were independently repeated three times (Figures 6B, 6C, and 6F) and twice (Figures 6D and 6E). N equals 4 mice / group for all experiments except for Figure 6C, where n equals 3 mice / group. [Figure 7A]Figures 7A-7J show that IRE1α / XBP1 supports NK homeostatic proliferation. Figure 7A shows a schematic diagram of the lymphopenia-induced homeostatic proliferation experiment in Figures 7B-7D. Equal numbers of NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transplanted into lymphocyte-deficient recipient Rag2- / -Il2rg- / - mice. Flow cytometry assessment of NK cell hypertrophy and proliferation was performed in the spleens of recipient mice 3-5 days after transplantation. Figure 7B shows the relative percentages of transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at the indicated time points after transplantation. Figure 7C shows representative flow cytometry plots (left) and quantification (center) of CTV dilution and Ki-67 levels (right) of transplanted wild-type and IRE1NKNK cells in the spleen of recipient Rag2- / -Il2rg- / - mice on day 4 posttransplant. Figure 7D shows the relative percentages (left) and absolute numbers (right) of wild-type and IRE1NKNK cells after ex vivo co-incubation with IL-2 and IL-15 for the indicated days. Figure 7E shows representative flow cytometry plots (top) and quantification (bottom) of FSC and SSC of the cells in Figure 7D at the indicated time points during culture. The left panel of Figure 7F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 h of ex vivo incubation with IL-2 and IL-15 in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. β-actin was used as a reference, and data are presented as the ratio of XBP1 to total XBP1. The center panel of Figure 7F shows flow cytometry analysis of CTV dilution of human primary NK cells at designated time points during culture with IL-2 and IL-15 in the presence or absence of IRE1i. Plots show representative data from three different PBMC donors. The right panel of Figure 7F shows quantification of the percentage of proliferating cells at day 6 in culture. Figure 7G shows representative flow cytometry plots and quantification of CTV dilution, and Figure 7H shows representative flow plots of Ki-67 levels at designated time points.IRE1NK and IRE1f / fNK cells from littermate animals were pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15 for the indicated days. Figure 7I shows representative flow cytometry plots and quantification of CTV dilutions on day 6 during ex vivo culture with IL-2 and IL-15, similar to Figure 7F, except that CD56bright and CD56dimNK cells were plotted separately. Figure 7J shows representative flow cytometry plots and quantification of XBP1s protein levels in CD56bright and CD56dimNK cells before and after 16 hours of cytokine stimulation. p values ​​are as indicated. For Figures 7B and 7D, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 7C, a two-tailed unpaired Student's t-test was performed. For Figure 7G, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 7F, 7I, and 7J, a two-tailed paired Student's t-test was performed. All error bars indicate standard deviation. Data are representative of three (Figures 7B and 7C) and two (Figures 7D-7E and 7G-7H) independent repeats. Data in Figures 7F, 7I, and 7J are pooled from two independent repeats. N equals 2 mice / group in Figure 7B. N equals 4 mice / group in Figures 7C, 7G, and 7H. N equals 3 mice / group (plus technical repeats for ex vivo culture) in Figures 7D and 7E. N equals 6, 5, and 5 PBMC donors in Figures 7F, 7I, and 7J. [Figure 7B]Figures 7A-7J show that IRE1α / XBP1 supports NK homeostatic proliferation. Figure 7A shows a schematic diagram of the lymphopenia-induced homeostatic proliferation experiment in Figures 7B-7D. Equal numbers of NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transplanted into lymphocyte-deficient recipient Rag2- / -Il2rg- / - mice. Flow cytometry assessment of NK cell hypertrophy and proliferation was performed in the spleens of recipient mice 3-5 days after transplantation. Figure 7B shows the relative percentages of transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at the indicated time points after transplantation. Figure 7C shows representative flow cytometry plots (left) and quantification (center) of CTV dilution and Ki-67 levels (right) of transplanted wild-type and IRE1NKNK cells in the spleen of recipient Rag2- / -Il2rg- / - mice on day 4 posttransplant. Figure 7D shows the relative percentages (left) and absolute numbers (right) of wild-type and IRE1NKNK cells after ex vivo co-incubation with IL-2 and IL-15 for the indicated days. Figure 7E shows representative flow cytometry plots (top) and quantification (bottom) of FSC and SSC of the cells in Figure 7D at the indicated time points during culture. The left panel of Figure 7F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 h of ex vivo incubation with IL-2 and IL-15 in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. β-actin was used as a reference, and data are presented as the ratio of XBP1 to total XBP1. The center panel of Figure 7F shows flow cytometry analysis of CTV dilution of human primary NK cells at designated time points during culture with IL-2 and IL-15 in the presence or absence of IRE1i. Plots show representative data from three different PBMC donors. The right panel of Figure 7F shows quantification of the percentage of proliferating cells at day 6 in culture. Figure 7G shows representative flow cytometry plots and quantification of CTV dilution, and Figure 7H shows representative flow plots of Ki-67 levels at designated time points.IRE1NK and IRE1f / fNK cells from littermate animals were pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15 for the indicated days. Figure 7I shows representative flow cytometry plots and quantification of CTV dilutions on day 6 during ex vivo culture with IL-2 and IL-15, similar to Figure 7F, except that CD56bright and CD56dimNK cells were plotted separately. Figure 7J shows representative flow cytometry plots and quantification of XBP1s protein levels in CD56bright and CD56dimNK cells before and after 16 hours of cytokine stimulation. p values ​​are as indicated. For Figures 7B and 7D, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 7C, a two-tailed unpaired Student's t-test was performed. For Figure 7G, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 7F, 7I, and 7J, a two-tailed paired Student's t-test was performed. All error bars indicate standard deviation. Data are representative of three (Figures 7B and 7C) and two (Figures 7D-7E and 7G-7H) independent repeats. Data in Figures 7F, 7I, and 7J are pooled from two independent repeats. N equals 2 mice / group in Figure 7B. N equals 4 mice / group in Figures 7C, 7G, and 7H. N equals 3 mice / group (plus technical repeats for ex vivo culture) in Figures 7D and 7E. N equals 6, 5, and 5 PBMC donors in Figures 7F, 7I, and 7J. [Figure 7C]Figures 7A-7J show that IRE1α / XBP1 supports NK homeostatic proliferation. Figure 7A shows a schematic diagram of the lymphopenia-induced homeostatic proliferation experiment in Figures 7B-7D. Equal numbers of NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transplanted into lymphocyte-deficient recipient Rag2- / -Il2rg- / - mice. Flow cytometry assessment of NK cell hypertrophy and proliferation was performed in the spleens of recipient mice 3-5 days after transplantation. Figure 7B shows the relative percentages of transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at the indicated time points after transplantation. Figure 7C shows representative flow cytometry plots (left) and quantification (center) of CTV dilution and Ki-67 levels (right) of transplanted wild-type and IRE1NKNK cells in the spleen of recipient Rag2- / -Il2rg- / - mice on day 4 posttransplant. Figure 7D shows the relative percentages (left) and absolute numbers (right) of wild-type and IRE1NKNK cells after ex vivo co-incubation with IL-2 and IL-15 for the indicated days. Figure 7E shows representative flow cytometry plots (top) and quantification (bottom) of FSC and SSC of the cells in Figure 7D at the indicated time points during culture. The left panel of Figure 7F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 h of ex vivo incubation with IL-2 and IL-15 in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. β-actin was used as a reference, and data are presented as the ratio of XBP1 to total XBP1. The center panel of Figure 7F shows flow cytometry analysis of CTV dilution of human primary NK cells at designated time points during culture with IL-2 and IL-15 in the presence or absence of IRE1i. Plots show representative data from three different PBMC donors. The right panel of Figure 7F shows quantification of the percentage of proliferating cells at day 6 in culture. Figure 7G shows representative flow cytometry plots and quantification of CTV dilution, and Figure 7H shows representative flow plots of Ki-67 levels at designated time points.IRE1NK and IRE1f / fNK cells from littermate animals were pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15 for the indicated days. Figure 7I shows representative flow cytometry plots and quantification of CTV dilutions on day 6 during ex vivo culture with IL-2 and IL-15, similar to Figure 7F, except that CD56bright and CD56dimNK cells were plotted separately. Figure 7J shows representative flow cytometry plots and quantification of XBP1s protein levels in CD56bright and CD56dimNK cells before and after 16 hours of cytokine stimulation. p values ​​are as indicated. For Figures 7B and 7D, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 7C, a two-tailed unpaired Student's t-test was performed. For Figure 7G, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 7F, 7I, and 7J, a two-tailed paired Student's t-test was performed. All error bars indicate standard deviation. Data are representative of three (Figures 7B and 7C) and two (Figures 7D-7E and 7G-7H) independent repeats. Data in Figures 7F, 7I, and 7J are pooled from two independent repeats. N equals 2 mice / group in Figure 7B. N equals 4 mice / group in Figures 7C, 7G, and 7H. N equals 3 mice / group (plus technical repeats for ex vivo culture) in Figures 7D and 7E. N equals 6, 5, and 5 PBMC donors in Figures 7F, 7I, and 7J. [Figure 7D]Figures 7A-7J show that IRE1α / XBP1 supports NK homeostatic proliferation. Figure 7A shows a schematic diagram of the lymphopenia-induced homeostatic proliferation experiment in Figures 7B-7D. Equal numbers of NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transplanted into lymphocyte-deficient recipient Rag2- / -Il2rg- / - mice. Flow cytometry assessment of NK cell hypertrophy and proliferation was performed in the spleens of recipient mice 3-5 days after transplantation. Figure 7B shows the relative percentages of transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at the indicated time points after transplantation. Figure 7C shows representative flow cytometry plots (left) and quantification (center) of CTV dilution and Ki-67 levels (right) of transplanted wild-type and IRE1NKNK cells in the spleen of recipient Rag2- / -Il2rg- / - mice on day 4 posttransplant. Figure 7D shows the relative percentages (left) and absolute numbers (right) of wild-type and IRE1NKNK cells after ex vivo co-incubation with IL-2 and IL-15 for the indicated days. Figure 7E shows representative flow cytometry plots (top) and quantification (bottom) of FSC and SSC of the cells in Figure 7D at the indicated time points during culture. The left panel of Figure 7F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 h of ex vivo incubation with IL-2 and IL-15 in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. β-actin was used as a reference, and data are presented as the ratio of XBP1 to total XBP1. The center panel of Figure 7F shows flow cytometry analysis of CTV dilution of human primary NK cells at designated time points during culture with IL-2 and IL-15 in the presence or absence of IRE1i. Plots show representative data from three different PBMC donors. The right panel of Figure 7F shows quantification of the percentage of proliferating cells at day 6 in culture. Figure 7G shows representative flow cytometry plots and quantification of CTV dilution, and Figure 7H shows representative flow plots of Ki-67 levels at designated time points.IRE1NK and IRE1f / fNK cells from littermate animals were pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15 for the indicated days. Figure 7I shows representative flow cytometry plots and quantification of CTV dilutions on day 6 during ex vivo culture with IL-2 and IL-15, similar to Figure 7F, except that CD56bright and CD56dimNK cells were plotted separately. Figure 7J shows representative flow cytometry plots and quantification of XBP1s protein levels in CD56bright and CD56dimNK cells before and after 16 hours of cytokine stimulation. p values ​​are as indicated. For Figures 7B and 7D, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 7C, a two-tailed unpaired Student's t-test was performed. For Figure 7G, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 7F, 7I, and 7J, a two-tailed paired Student's t-test was performed. All error bars indicate standard deviation. Data are representative of three (Figures 7B and 7C) and two (Figures 7D-7E and 7G-7H) independent repeats. Data in Figures 7F, 7I, and 7J are pooled from two independent repeats. N equals 2 mice / group in Figure 7B. N equals 4 mice / group in Figures 7C, 7G, and 7H. N equals 3 mice / group (plus technical repeats for ex vivo culture) in Figures 7D and 7E. N equals 6, 5, and 5 PBMC donors in Figures 7F, 7I, and 7J. [Figure 7E]Figures 7A-7J show that IRE1α / XBP1 supports NK homeostatic proliferation. Figure 7A shows a schematic diagram of the lymphopenia-induced homeostatic proliferation experiment in Figures 7B-7D. Equal numbers of NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transplanted into lymphocyte-deficient recipient Rag2- / -Il2rg- / - mice. Flow cytometry assessment of NK cell hypertrophy and proliferation was performed in the spleens of recipient mice 3-5 days after transplantation. Figure 7B shows the relative percentages of transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at the indicated time points after transplantation. Figure 7C shows representative flow cytometry plots (left) and quantification (center) of CTV dilution and Ki-67 levels (right) of transplanted wild-type and IRE1NKNK cells in the spleen of recipient Rag2- / -Il2rg- / - mice on day 4 posttransplant. Figure 7D shows the relative percentages (left) and absolute numbers (right) of wild-type and IRE1NKNK cells after ex vivo co-incubation with IL-2 and IL-15 for the indicated days. Figure 7E shows representative flow cytometry plots (top) and quantification (bottom) of FSC and SSC of the cells in Figure 7D at the indicated time points during culture. The left panel of Figure 7F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 h of ex vivo incubation with IL-2 and IL-15 in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. β-actin was used as a reference, and data are presented as the ratio of XBP1 to total XBP1. The center panel of Figure 7F shows flow cytometry analysis of CTV dilution of human primary NK cells at designated time points during culture with IL-2 and IL-15 in the presence or absence of IRE1i. Plots show representative data from three different PBMC donors. The right panel of Figure 7F shows quantification of the percentage of proliferating cells at day 6 in culture. Figure 7G shows representative flow cytometry plots and quantification of CTV dilution, and Figure 7H shows representative flow plots of Ki-67 levels at designated time points.IRE1NK and IRE1f / fNK cells from littermate animals were pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15 for the indicated days. Figure 7I shows representative flow cytometry plots and quantification of CTV dilutions on day 6 during ex vivo culture with IL-2 and IL-15, similar to Figure 7F, except that CD56bright and CD56dimNK cells were plotted separately. Figure 7J shows representative flow cytometry plots and quantification of XBP1s protein levels in CD56bright and CD56dimNK cells before and after 16 hours of cytokine stimulation. p values ​​are as indicated. For Figures 7B and 7D, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 7C, a two-tailed unpaired Student's t-test was performed. For Figure 7G, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 7F, 7I, and 7J, a two-tailed paired Student's t-test was performed. All error bars indicate standard deviation. Data are representative of three (Figures 7B and 7C) and two (Figures 7D-7E and 7G-7H) independent repeats. Data in Figures 7F, 7I, and 7J are pooled from two independent repeats. N equals 2 mice / group in Figure 7B. N equals 4 mice / group in Figures 7C, 7G, and 7H. N equals 3 mice / group (plus technical repeats for ex vivo culture) in Figures 7D and 7E. N equals 6, 5, and 5 PBMC donors in Figures 7F, 7I, and 7J. [Figure 7F]Figures 7A-7J show that IRE1α / XBP1 supports NK homeostatic proliferation. Figure 7A shows a schematic diagram of the lymphopenia-induced homeostatic proliferation experiment in Figures 7B-7D. Equal numbers of NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transplanted into lymphocyte-deficient recipient Rag2- / -Il2rg- / - mice. Flow cytometry assessment of NK cell hypertrophy and proliferation was performed in the spleens of recipient mice 3-5 days after transplantation. Figure 7B shows the relative percentages of transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at the indicated time points after transplantation. Figure 7C shows representative flow cytometry plots (left) and quantification (center) of CTV dilution and Ki-67 levels (right) of transplanted wild-type and IRE1NKNK cells in the spleen of recipient Rag2- / -Il2rg- / - mice on day 4 posttransplant. Figure 7D shows the relative percentages (left) and absolute numbers (right) of wild-type and IRE1NKNK cells after ex vivo co-incubation with IL-2 and IL-15 for the indicated days. Figure 7E shows representative flow cytometry plots (top) and quantification (bottom) of FSC and SSC of the cells in Figure 7D at the indicated time points during culture. The left panel of Figure 7F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 h of ex vivo incubation with IL-2 and IL-15 in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. β-actin was used as a reference, and data are presented as the ratio of XBP1 to total XBP1. The center panel of Figure 7F shows flow cytometry analysis of CTV dilution of human primary NK cells at designated time points during culture with IL-2 and IL-15 in the presence or absence of IRE1i. Plots show representative data from three different PBMC donors. The right panel of Figure 7F shows quantification of the percentage of proliferating cells at day 6 in culture. Figure 7G shows representative flow cytometry plots and quantification of CTV dilution, and Figure 7H shows representative flow plots of Ki-67 levels at designated time points.IRE1NK and IRE1f / fNK cells from littermate animals were pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15 for the indicated days. Figure 7I shows representative flow cytometry plots and quantification of CTV dilutions on day 6 during ex vivo culture with IL-2 and IL-15, similar to Figure 7F, except that CD56bright and CD56dimNK cells were plotted separately. Figure 7J shows representative flow cytometry plots and quantification of XBP1s protein levels in CD56bright and CD56dimNK cells before and after 16 hours of cytokine stimulation. p values ​​are as indicated. For Figures 7B and 7D, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 7C, a two-tailed unpaired Student's t-test was performed. For Figure 7G, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 7F, 7I, and 7J, a two-tailed paired Student's t-test was performed. All error bars indicate standard deviation. Data are representative of three (Figures 7B and 7C) and two (Figures 7D-7E and 7G-7H) independent repeats. Data in Figures 7F, 7I, and 7J are pooled from two independent repeats. N equals 2 mice / group in Figure 7B. N equals 4 mice / group in Figures 7C, 7G, and 7H. N equals 3 mice / group (plus technical repeats for ex vivo culture) in Figures 7D and 7E. N equals 6, 5, and 5 PBMC donors in Figures 7F, 7I, and 7J. [Figure 7G]Figures 7A-7J show that IRE1α / XBP1 supports NK homeostatic proliferation. Figure 7A shows a schematic diagram of the lymphopenia-induced homeostatic proliferation experiment in Figures 7B-7D. Equal numbers of NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transplanted into lymphocyte-deficient recipient Rag2- / -Il2rg- / - mice. Flow cytometry assessment of NK cell hypertrophy and proliferation was performed in the spleens of recipient mice 3-5 days after transplantation. Figure 7B shows the relative percentages of transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at the indicated time points after transplantation. Figure 7C shows representative flow cytometry plots (left) and quantification (center) of CTV dilution and Ki-67 levels (right) of transplanted wild-type and IRE1NKNK cells in the spleen of recipient Rag2- / -Il2rg- / - mice on day 4 posttransplant. Figure 7D shows the relative percentages (left) and absolute numbers (right) of wild-type and IRE1NKNK cells after ex vivo co-incubation with IL-2 and IL-15 for the indicated days. Figure 7E shows representative flow cytometry plots (top) and quantification (bottom) of FSC and SSC of the cells in Figure 7D at the indicated time points during culture. The left panel of Figure 7F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 h of ex vivo incubation with IL-2 and IL-15 in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. β-actin was used as a reference, and data are presented as the ratio of XBP1 to total XBP1. The center panel of Figure 7F shows flow cytometry analysis of CTV dilution of human primary NK cells at designated time points during culture with IL-2 and IL-15 in the presence or absence of IRE1i. Plots show representative data from three different PBMC donors. The right panel of Figure 7F shows quantification of the percentage of proliferating cells at day 6 in culture. Figure 7G shows representative flow cytometry plots and quantification of CTV dilution, and Figure 7H shows representative flow plots of Ki-67 levels at designated time points.IRE1NK and IRE1f / fNK cells from littermate animals were pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15 for the indicated days. Figure 7I shows representative flow cytometry plots and quantification of CTV dilutions on day 6 during ex vivo culture with IL-2 and IL-15, similar to Figure 7F, except that CD56bright and CD56dimNK cells were plotted separately. Figure 7J shows representative flow cytometry plots and quantification of XBP1s protein levels in CD56bright and CD56dimNK cells before and after 16 hours of cytokine stimulation. p values ​​are as indicated. For Figures 7B and 7D, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 7C, a two-tailed unpaired Student's t-test was performed. For Figure 7G, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 7F, 7I, and 7J, a two-tailed paired Student's t-test was performed. All error bars indicate standard deviation. Data are representative of three (Figures 7B and 7C) and two (Figures 7D-7E and 7G-7H) independent repeats. Data in Figures 7F, 7I, and 7J are pooled from two independent repeats. N equals 2 mice / group in Figure 7B. N equals 4 mice / group in Figures 7C, 7G, and 7H. N equals 3 mice / group (plus technical repeats for ex vivo culture) in Figures 7D and 7E. N equals 6, 5, and 5 PBMC donors in Figures 7F, 7I, and 7J. [Figure 7H]Figures 7A-7J show that IRE1α / XBP1 supports NK homeostatic proliferation. Figure 7A shows a schematic diagram of the lymphopenia-induced homeostatic proliferation experiment in Figures 7B-7D. Equal numbers of NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transplanted into lymphocyte-deficient recipient Rag2- / -Il2rg- / - mice. Flow cytometry assessment of NK cell hypertrophy and proliferation was performed in the spleens of recipient mice 3-5 days after transplantation. Figure 7B shows the relative percentages of transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at the indicated time points after transplantation. Figure 7C shows representative flow cytometry plots (left) and quantification (center) of CTV dilution and Ki-67 levels (right) of transplanted wild-type and IRE1NKNK cells in the spleen of recipient Rag2- / -Il2rg- / - mice on day 4 posttransplant. Figure 7D shows the relative percentages (left) and absolute numbers (right) of wild-type and IRE1NKNK cells after ex vivo co-incubation with IL-2 and IL-15 for the indicated days. Figure 7E shows representative flow cytometry plots (top) and quantification (bottom) of FSC and SSC of the cells in Figure 7D at the indicated time points during culture. The left panel of Figure 7F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 h of ex vivo incubation with IL-2 and IL-15 in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. β-actin was used as a reference, and data are presented as the ratio of XBP1 to total XBP1. The center panel of Figure 7F shows flow cytometry analysis of CTV dilution of human primary NK cells at designated time points during culture with IL-2 and IL-15 in the presence or absence of IRE1i. Plots show representative data from three different PBMC donors. The right panel of Figure 7F shows quantification of the percentage of proliferating cells at day 6 in culture. Figure 7G shows representative flow cytometry plots and quantification of CTV dilution, and Figure 7H shows representative flow plots of Ki-67 levels at designated time points.IRE1NK and IRE1f / fNK cells from littermate animals were pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15 for the indicated days. Figure 7I shows representative flow cytometry plots and quantification of CTV dilutions on day 6 during ex vivo culture with IL-2 and IL-15, similar to Figure 7F, except that CD56bright and CD56dimNK cells were plotted separately. Figure 7J shows representative flow cytometry plots and quantification of XBP1s protein levels in CD56bright and CD56dimNK cells before and after 16 hours of cytokine stimulation. p values ​​are as indicated. For Figures 7B and 7D, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 7C, a two-tailed unpaired Student's t-test was performed. For Figure 7G, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 7F, 7I, and 7J, a two-tailed paired Student's t-test was performed. All error bars indicate standard deviation. Data are representative of three (Figures 7B and 7C) and two (Figures 7D-7E and 7G-7H) independent repeats. Data in Figures 7F, 7I, and 7J are pooled from two independent repeats. N equals 2 mice / group in Figure 7B. N equals 4 mice / group in Figures 7C, 7G, and 7H. N equals 3 mice / group (plus technical repeats for ex vivo culture) in Figures 7D and 7E. N equals 6, 5, and 5 PBMC donors in Figures 7F, 7I, and 7J. [Figure 7I]Figures 7A-7J show that IRE1α / XBP1 supports NK homeostatic proliferation. Figure 7A shows a schematic diagram of the lymphopenia-induced homeostatic proliferation experiment in Figures 7B-7D. Equal numbers of NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transplanted into lymphocyte-deficient recipient Rag2- / -Il2rg- / - mice. Flow cytometry assessment of NK cell hypertrophy and proliferation was performed in the spleens of recipient mice 3-5 days after transplantation. Figure 7B shows the relative percentages of transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at the indicated time points after transplantation. Figure 7C shows representative flow cytometry plots (left) and quantification (center) of CTV dilution and Ki-67 levels (right) of transplanted wild-type and IRE1NKNK cells in the spleen of recipient Rag2- / -Il2rg- / - mice on day 4 posttransplant. Figure 7D shows the relative percentages (left) and absolute numbers (right) of wild-type and IRE1NKNK cells after ex vivo co-incubation with IL-2 and IL-15 for the indicated days. Figure 7E shows representative flow cytometry plots (top) and quantification (bottom) of FSC and SSC of the cells in Figure 7D at the indicated time points during culture. The left panel of Figure 7F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 h of ex vivo incubation with IL-2 and IL-15 in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. β-actin was used as a reference, and data are presented as the ratio of XBP1 to total XBP1. The center panel of Figure 7F shows flow cytometry analysis of CTV dilution of human primary NK cells at designated time points during culture with IL-2 and IL-15 in the presence or absence of IRE1i. Plots show representative data from three different PBMC donors. The right panel of Figure 7F shows quantification of the percentage of proliferating cells at day 6 in culture. Figure 7G shows representative flow cytometry plots and quantification of CTV dilution, and Figure 7H shows representative flow plots of Ki-67 levels at designated time points.IRE1NK and IRE1f / fNK cells from littermate animals were pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15 for the indicated days. Figure 7I shows representative flow cytometry plots and quantification of CTV dilutions on day 6 during ex vivo culture with IL-2 and IL-15, similar to Figure 7F, except that CD56bright and CD56dimNK cells were plotted separately. Figure 7J shows representative flow cytometry plots and quantification of XBP1s protein levels in CD56bright and CD56dimNK cells before and after 16 hours of cytokine stimulation. p values ​​are as indicated. For Figures 7B and 7D, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 7C, a two-tailed unpaired Student's t-test was performed. For Figure 7G, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 7F, 7I, and 7J, a two-tailed paired Student's t-test was performed. All error bars indicate standard deviation. Data are representative of three (Figures 7B and 7C) and two (Figures 7D-7E and 7G-7H) independent repeats. Data in Figures 7F, 7I, and 7J are pooled from two independent repeats. N equals 2 mice / group in Figure 7B. N equals 4 mice / group in Figures 7C, 7G, and 7H. N equals 3 mice / group (plus technical repeats for ex vivo culture) in Figures 7D and 7E. N equals 6, 5, and 5 PBMC donors in Figures 7F, 7I, and 7J. [Figure 7J]Figures 7A-7J show that IRE1α / XBP1 supports NK homeostatic proliferation. Figure 7A shows a schematic diagram of the lymphopenia-induced homeostatic proliferation experiment in Figures 7B-7D. Equal numbers of NK cells from wild-type and IRE1NK donors were labeled with the cell proliferation tracing dye CTV and then co-transplanted into lymphocyte-deficient recipient Rag2- / -Il2rg- / - mice. Flow cytometry assessment of NK cell hypertrophy and proliferation was performed in the spleens of recipient mice 3-5 days after transplantation. Figure 7B shows the relative percentages of transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at the indicated time points after transplantation. Figure 7C shows representative flow cytometry plots (left) and quantification (center) of CTV dilution and Ki-67 levels (right) of transplanted wild-type and IRE1NKNK cells in the spleen of recipient Rag2- / -Il2rg- / - mice on day 4 posttransplant. Figure 7D shows the relative percentages (left) and absolute numbers (right) of wild-type and IRE1NKNK cells after ex vivo co-incubation with IL-2 and IL-15 for the indicated days. Figure 7E shows representative flow cytometry plots (top) and quantification (bottom) of FSC and SSC of the cells in Figure 7D at the indicated time points during culture. The left panel of Figure 7F shows quantitative real-time PCR analysis of XBP1 splicing activity in sorted, purified human primary NK cells derived from PBMCs after 16 h of ex vivo incubation with IL-2 and IL-15 in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. β-actin was used as a reference, and data are presented as the ratio of XBP1 to total XBP1. The center panel of Figure 7F shows flow cytometry analysis of CTV dilution of human primary NK cells at designated time points during culture with IL-2 and IL-15 in the presence or absence of IRE1i. Plots show representative data from three different PBMC donors. The right panel of Figure 7F shows quantification of the percentage of proliferating cells at day 6 in culture. Figure 7G shows representative flow cytometry plots and quantification of CTV dilution, and Figure 7H shows representative flow plots of Ki-67 levels at designated time points.IRE1NK and IRE1f / fNK cells from littermate animals were pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15 for the indicated days. Figure 7I shows representative flow cytometry plots and quantification of CTV dilutions on day 6 during ex vivo culture with IL-2 and IL-15, similar to Figure 7F, except that CD56bright and CD56dimNK cells were plotted separately. Figure 7J shows representative flow cytometry plots and quantification of XBP1s protein levels in CD56bright and CD56dimNK cells before and after 16 hours of cytokine stimulation. p values ​​are as indicated. For Figures 7B and 7D, a two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed. For Figure 7C, a two-tailed unpaired Student's t-test was performed. For Figure 7G, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was performed. For Figures 7F, 7I, and 7J, a two-tailed paired Student's t-test was performed. All error bars indicate standard deviation. Data are representative of three (Figures 7B and 7C) and two (Figures 7D-7E and 7G-7H) independent repeats. Data in Figures 7F, 7I, and 7J are pooled from two independent repeats. N equals 2 mice / group in Figure 7B. N equals 4 mice / group in Figures 7C, 7G, and 7H. N equals 3 mice / group (plus technical repeats for ex vivo culture) in Figures 7D and 7E. N equals 6, 5, and 5 PBMC donors in Figures 7F, 7I, and 7J. [Figure 8A]Figures 8A-8H show that IRE1NK RNA-seq analysis highlights Myc as an XBP1 target gene. Figure 8A shows a Venn diagram of differentially expressed genes in Ly49HIRE1NK NK cells compared to WT NK cells during infection with MCMV. Cells were harvested from IRE1NK (CD45.2):WT (CD45.1) mixed BM chimeras at three time points: days 0, 1.5, and 7 post-infection. Figure 8B shows an IPA upstream analysis (predicting Myc regulation in IRE1NK NK cells) derived from the RNA-seq analysis (day 1.5) shown in Figure 8A. Figure 8C shows an IPA analysis (functional overlap between IRE1- and c-Myc-regulated genes derived from RNA-seq analysis of Ly49HIRE1NK NK cells at day 1.5 post-infection with MCMV). Figure 8D shows quantitative real-time PCR analysis of canonical c-Myc target genes in transplanted Ly49H+WT or IRE1NK NK cells sorted and purified from the spleens of recipient Ly49H-deficient mice 1.5 days after infection with MCMV. β-actin was used as a reference, and data are shown as relative expression normalized to transplanted WT NK cells. Figure 8E shows a schematic of putative XBP1 binding sites in the Myc promoter region (based on SABiosciences' proprietary database, ENCODE). Figure 8F shows quantitative real-time PCR and flow cytometry analysis of basal levels of c-Myc mRNA and protein in naive NK cells from IRE1NK and IRE1f / f littermate control mice. Figure 8G shows flow cytometry analysis of the kinetics of XBP1s, p-S6, p-Akt, and pan-Akt expression in primary human NK cells after stimulation with IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for the indicated times. Figure 8H shows purified splenic NK cells from IRE1NK and IRE1f / f littermate control mice stimulated with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for 1 and 16 hours in the presence or absence of the mTOR inhibitor rapamycin (10 nM). c-Myc levels were quantified by flow cytometry.ns indicates "not significant," * indicates p<0.05, and ** indicates p<0.01. One-sample t-tests performed on the data in Figure 8D (Figure 8D) are cumulative from three experiments. All error bars are standard deviations from biological replicates. N equals 3-5 mice / group for all experiments, and data were independently repeated two (Figures 8G and 8H) or three times (Figures 8D and 8F). [Figure 8B]Figures 8A-8H show that IRE1NK RNA-seq analysis highlights Myc as an XBP1 target gene. Figure 8A shows a Venn diagram of differentially expressed genes in Ly49HIRE1NK NK cells compared to WT NK cells during infection with MCMV. Cells were harvested from IRE1NK (CD45.2):WT (CD45.1) mixed BM chimeras at three time points: days 0, 1.5, and 7 post-infection. Figure 8B shows an IPA upstream analysis (predicting Myc regulation in IRE1NK NK cells) derived from the RNA-seq analysis (day 1.5) shown in Figure 8A. Figure 8C shows an IPA analysis (functional overlap between IRE1- and c-Myc-regulated genes derived from RNA-seq analysis of Ly49HIRE1NK NK cells at day 1.5 post-infection with MCMV). Figure 8D shows quantitative real-time PCR analysis of canonical c-Myc target genes in transplanted Ly49H+WT or IRE1NK NK cells sorted and purified from the spleens of recipient Ly49H-deficient mice 1.5 days after infection with MCMV. β-actin was used as a reference, and data are shown as relative expression normalized to transplanted WT NK cells. Figure 8E shows a schematic of putative XBP1 binding sites in the Myc promoter region (based on SABiosciences' proprietary database, ENCODE). Figure 8F shows quantitative real-time PCR and flow cytometry analysis of basal levels of c-Myc mRNA and protein in naive NK cells from IRE1NK and IRE1f / f littermate control mice. Figure 8G shows flow cytometry analysis of the kinetics of XBP1s, p-S6, p-Akt, and pan-Akt expression in primary human NK cells after stimulation with IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for the indicated times. Figure 8H shows purified splenic NK cells from IRE1NK and IRE1f / f littermate control mice stimulated with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for 1 and 16 hours in the presence or absence of the mTOR inhibitor rapamycin (10 nM). c-Myc levels were quantified by flow cytometry.ns indicates "not significant," * indicates p<0.05, and ** indicates p<0.01. One-sample t-tests performed on the data in Figure 8D (Figure 8D) are cumulative from three experiments. All error bars are standard deviations from biological replicates. N equals 3-5 mice / group for all experiments, and data were independently repeated two (Figures 8G and 8H) or three times (Figures 8D and 8F). [Figure 8C]Figures 8A-8H show that IRE1NK RNA-seq analysis highlights Myc as an XBP1 target gene. Figure 8A shows a Venn diagram of differentially expressed genes in Ly49HIRE1NK NK cells compared to WT NK cells during infection with MCMV. Cells were harvested from IRE1NK (CD45.2):WT (CD45.1) mixed BM chimeras at three time points: days 0, 1.5, and 7 post-infection. Figure 8B shows an IPA upstream analysis (predicting Myc regulation in IRE1NK NK cells) derived from the RNA-seq analysis (day 1.5) shown in Figure 8A. Figure 8C shows an IPA analysis (functional overlap between IRE1- and c-Myc-regulated genes derived from RNA-seq analysis of Ly49HIRE1NK NK cells at day 1.5 post-infection with MCMV). Figure 8D shows quantitative real-time PCR analysis of canonical c-Myc target genes in transplanted Ly49H+WT or IRE1NK NK cells sorted and purified from the spleens of recipient Ly49H-deficient mice 1.5 days after infection with MCMV. β-actin was used as a reference, and data are shown as relative expression normalized to transplanted WT NK cells. Figure 8E shows a schematic of putative XBP1 binding sites in the Myc promoter region (based on SABiosciences' proprietary database, ENCODE). Figure 8F shows quantitative real-time PCR and flow cytometry analysis of basal levels of c-Myc mRNA and protein in naive NK cells from IRE1NK and IRE1f / f littermate control mice. Figure 8G shows flow cytometry analysis of the kinetics of XBP1s, p-S6, p-Akt, and pan-Akt expression in primary human NK cells after stimulation with IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for the indicated times. Figure 8H shows purified splenic NK cells from IRE1NK and IRE1f / f littermate control mice stimulated with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for 1 and 16 hours in the presence or absence of the mTOR inhibitor rapamycin (10 nM). c-Myc levels were quantified by flow cytometry.ns indicates "not significant," * indicates p<0.05, and ** indicates p<0.01. One-sample t-tests performed on the data in Figure 8D (Figure 8D) are cumulative from three experiments. All error bars are standard deviations from biological replicates. N equals 3-5 mice / group for all experiments, and data were independently repeated two (Figures 8G and 8H) or three times (Figures 8D and 8F). [Figure 8D]Figures 8A-8H show that IRE1NK RNA-seq analysis highlights Myc as an XBP1 target gene. Figure 8A shows a Venn diagram of differentially expressed genes in Ly49HIRE1NK NK cells compared to WT NK cells during infection with MCMV. Cells were harvested from IRE1NK (CD45.2):WT (CD45.1) mixed BM chimeras at three time points: days 0, 1.5, and 7 post-infection. Figure 8B shows an IPA upstream analysis (predicting Myc regulation in IRE1NK NK cells) derived from the RNA-seq analysis (day 1.5) shown in Figure 8A. Figure 8C shows an IPA analysis (functional overlap between IRE1- and c-Myc-regulated genes derived from RNA-seq analysis of Ly49HIRE1NK NK cells at day 1.5 post-infection with MCMV). Figure 8D shows quantitative real-time PCR analysis of canonical c-Myc target genes in transplanted Ly49H+WT or IRE1NK NK cells sorted and purified from the spleens of recipient Ly49H-deficient mice 1.5 days after infection with MCMV. β-actin was used as a reference, and data are shown as relative expression normalized to transplanted WT NK cells. Figure 8E shows a schematic of putative XBP1 binding sites in the Myc promoter region (based on SABiosciences' proprietary database, ENCODE). Figure 8F shows quantitative real-time PCR and flow cytometry analysis of basal levels of c-Myc mRNA and protein in naive NK cells from IRE1NK and IRE1f / f littermate control mice. Figure 8G shows flow cytometry analysis of the kinetics of XBP1s, p-S6, p-Akt, and pan-Akt expression in primary human NK cells after stimulation with IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for the indicated times. Figure 8H shows purified splenic NK cells from IRE1NK and IRE1f / f littermate control mice stimulated with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for 1 and 16 hours in the presence or absence of the mTOR inhibitor rapamycin (10 nM). c-Myc levels were quantified by flow cytometry.ns indicates "not significant," * indicates p<0.05, and ** indicates p<0.01. One-sample t-tests performed on the data in Figure 8D (Figure 8D) are cumulative from three experiments. All error bars are standard deviations from biological replicates. N equals 3-5 mice / group for all experiments, and data were independently repeated two (Figures 8G and 8H) or three times (Figures 8D and 8F). [Figure 8E]Figures 8A-8H show that IRE1NK RNA-seq analysis highlights Myc as an XBP1 target gene. Figure 8A shows a Venn diagram of differentially expressed genes in Ly49HIRE1NK NK cells compared to WT NK cells during infection with MCMV. Cells were harvested from IRE1NK (CD45.2):WT (CD45.1) mixed BM chimeras at three time points: days 0, 1.5, and 7 post-infection. Figure 8B shows an IPA upstream analysis (predicting Myc regulation in IRE1NK NK cells) derived from the RNA-seq analysis (day 1.5) shown in Figure 8A. Figure 8C shows an IPA analysis (functional overlap between IRE1- and c-Myc-regulated genes derived from RNA-seq analysis of Ly49HIRE1NK NK cells at day 1.5 post-infection with MCMV). Figure 8D shows quantitative real-time PCR analysis of canonical c-Myc target genes in transplanted Ly49H+WT or IRE1NK NK cells sorted and purified from the spleens of recipient Ly49H-deficient mice 1.5 days after infection with MCMV. β-actin was used as a reference, and data are shown as relative expression normalized to transplanted WT NK cells. Figure 8E shows a schematic of putative XBP1 binding sites in the Myc promoter region (based on SABiosciences' proprietary database, ENCODE). Figure 8F shows quantitative real-time PCR and flow cytometry analysis of basal levels of c-Myc mRNA and protein in naive NK cells from IRE1NK and IRE1f / f littermate control mice. Figure 8G shows flow cytometry analysis of the kinetics of XBP1s, p-S6, p-Akt, and pan-Akt expression in primary human NK cells after stimulation with IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for the indicated times. Figure 8H shows purified splenic NK cells from IRE1NK and IRE1f / f littermate control mice stimulated with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for 1 and 16 hours in the presence or absence of the mTOR inhibitor rapamycin (10 nM). c-Myc levels were quantified by flow cytometry.ns indicates "not significant," * indicates p<0.05, and ** indicates p<0.01. One-sample t-tests performed on the data in Figure 8D (Figure 8D) are cumulative from three experiments. All error bars are standard deviations from biological replicates. N equals 3-5 mice / group for all experiments, and data were independently repeated two (Figures 8G and 8H) or three times (Figures 8D and 8F). [Figure 8F]Figures 8A-8H show that IRE1NK RNA-seq analysis highlights Myc as an XBP1 target gene. Figure 8A shows a Venn diagram of differentially expressed genes in Ly49HIRE1NK NK cells compared to WT NK cells during infection with MCMV. Cells were harvested from IRE1NK (CD45.2):WT (CD45.1) mixed BM chimeras at three time points: days 0, 1.5, and 7 post-infection. Figure 8B shows an IPA upstream analysis (predicting Myc regulation in IRE1NK NK cells) derived from the RNA-seq analysis (day 1.5) shown in Figure 8A. Figure 8C shows an IPA analysis (functional overlap between IRE1- and c-Myc-regulated genes derived from RNA-seq analysis of Ly49HIRE1NK NK cells at day 1.5 post-infection with MCMV). Figure 8D shows quantitative real-time PCR analysis of canonical c-Myc target genes in transplanted Ly49H+WT or IRE1NK NK cells sorted and purified from the spleens of recipient Ly49H-deficient mice 1.5 days after infection with MCMV. β-actin was used as a reference, and data are shown as relative expression normalized to transplanted WT NK cells. Figure 8E shows a schematic of putative XBP1 binding sites in the Myc promoter region (based on SABiosciences' proprietary database, ENCODE). Figure 8F shows quantitative real-time PCR and flow cytometry analysis of basal levels of c-Myc mRNA and protein in naive NK cells from IRE1NK and IRE1f / f littermate control mice. Figure 8G shows flow cytometry analysis of the kinetics of XBP1s, p-S6, p-Akt, and pan-Akt expression in primary human NK cells after stimulation with IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for the indicated times. Figure 8H shows purified splenic NK cells from IRE1NK and IRE1f / f littermate control mice stimulated with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for 1 and 16 hours in the presence or absence of the mTOR inhibitor rapamycin (10 nM). c-Myc levels were quantified by flow cytometry.ns indicates "not significant," * indicates p<0.05, and ** indicates p<0.01. One-sample t-tests performed on the data in Figure 8D (Figure 8D) are cumulative from three experiments. All error bars are standard deviations from biological replicates. N equals 3-5 mice / group for all experiments, and data were independently repeated two (Figures 8G and 8H) or three times (Figures 8D and 8F). [Figure 8G]Figures 8A-8H show that IRE1NK RNA-seq analysis highlights Myc as an XBP1 target gene. Figure 8A shows a Venn diagram of differentially expressed genes in Ly49HIRE1NK NK cells compared to WT NK cells during infection with MCMV. Cells were harvested from IRE1NK (CD45.2):WT (CD45.1) mixed BM chimeras at three time points: days 0, 1.5, and 7 post-infection. Figure 8B shows an IPA upstream analysis (predicting Myc regulation in IRE1NK NK cells) derived from the RNA-seq analysis (day 1.5) shown in Figure 8A. Figure 8C shows an IPA analysis (functional overlap between IRE1- and c-Myc-regulated genes derived from RNA-seq analysis of Ly49HIRE1NK NK cells at day 1.5 post-infection with MCMV). Figure 8D shows quantitative real-time PCR analysis of canonical c-Myc target genes in transplanted Ly49H+WT or IRE1NK NK cells sorted and purified from the spleens of recipient Ly49H-deficient mice 1.5 days after infection with MCMV. β-actin was used as a reference, and data are shown as relative expression normalized to transplanted WT NK cells. Figure 8E shows a schematic of putative XBP1 binding sites in the Myc promoter region (based on SABiosciences' proprietary database, ENCODE). Figure 8F shows quantitative real-time PCR and flow cytometry analysis of basal levels of c-Myc mRNA and protein in naive NK cells from IRE1NK and IRE1f / f littermate control mice. Figure 8G shows flow cytometry analysis of the kinetics of XBP1s, p-S6, p-Akt, and pan-Akt expression in primary human NK cells after stimulation with IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for the indicated times. Figure 8H shows purified splenic NK cells from IRE1NK and IRE1f / f littermate control mice stimulated with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for 1 and 16 hours in the presence or absence of the mTOR inhibitor rapamycin (10 nM). c-Myc levels were quantified by flow cytometry.ns indicates "not significant," * indicates p<0.05, and ** indicates p<0.01. One-sample t-tests performed on the data in Figure 8D (Figure 8D) are cumulative from three experiments. All error bars are standard deviations from biological replicates. N equals 3-5 mice / group for all experiments, and data were independently repeated two (Figures 8G and 8H) or three times (Figures 8D and 8F). [Figure 8H]Figures 8A-8H show that IRE1NK RNA-seq analysis highlights Myc as an XBP1 target gene. Figure 8A shows a Venn diagram of differentially expressed genes in Ly49HIRE1NK NK cells compared to WT NK cells during infection with MCMV. Cells were harvested from IRE1NK (CD45.2):WT (CD45.1) mixed BM chimeras at three time points: days 0, 1.5, and 7 post-infection. Figure 8B shows an IPA upstream analysis (predicting Myc regulation in IRE1NK NK cells) derived from the RNA-seq analysis (day 1.5) shown in Figure 8A. Figure 8C shows an IPA analysis (functional overlap between IRE1- and c-Myc-regulated genes derived from RNA-seq analysis of Ly49HIRE1NK NK cells at day 1.5 post-infection with MCMV). Figure 8D shows quantitative real-time PCR analysis of canonical c-Myc target genes in transplanted Ly49H+WT or IRE1NK NK cells sorted and purified from the spleens of recipient Ly49H-deficient mice 1.5 days after infection with MCMV. β-actin was used as a reference, and data are shown as relative expression normalized to transplanted WT NK cells. Figure 8E shows a schematic of putative XBP1 binding sites in the Myc promoter region (based on SABiosciences' proprietary database, ENCODE). Figure 8F shows quantitative real-time PCR and flow cytometry analysis of basal levels of c-Myc mRNA and protein in naive NK cells from IRE1NK and IRE1f / f littermate control mice. Figure 8G shows flow cytometry analysis of the kinetics of XBP1s, p-S6, p-Akt, and pan-Akt expression in primary human NK cells after stimulation with IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for the indicated times. Figure 8H shows purified splenic NK cells from IRE1NK and IRE1f / f littermate control mice stimulated with murine recombinant IL-12 (20 ng / ml) and IL-18 (10 ng / ml) for 1 and 16 hours in the presence or absence of the mTOR inhibitor rapamycin (10 nM). c-Myc levels were quantified by flow cytometry.ns indicates "not significant," * indicates p<0.05, and ** indicates p<0.01. One-sample t-tests performed on the data in Figure 8D (Figure 8D) are cumulative from three experiments. All error bars are standard deviations from biological replicates. N equals 3-5 mice / group for all experiments, and data were independently repeated two (Figures 8G and 8H) or three times (Figures 8D and 8F). [Figure 9A]Figures 9A-9J show that XBP1 promotes NK proliferation, at least in part, through direct regulation of c-Myc. Figure 9A shows the top 10 enriched GSEA gene clusters in RNA-seq analysis of IRE1NK relative to wild-type Ly49H+ splenic NK cells harvested from IRE1NK bone marrow chimeric mice at day 1.5 postinfection. Figure 9B shows a heatmap of Myc target genes differentially expressed in IRE1NK relative to wild-type NK cells. Genes were clustered by functional annotation. Figure 9C shows flow cytometry analysis of c-Myc induction at the transcriptional (top row) and translational (middle row) levels in splenic NK cells from either naive or wild-type mice at day 1 postinfection with MCMV. The bottom row of Figure 9C shows representative flow cytometry plots demonstrating the simultaneous induction of c-Myc protein and Venus reporter expression in NK cells from the indicated organs of ERAI transgenic mice at day 2 postinfection with MCMV. Figure 9D shows quantitative real-time PCR and flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice 1 day after infection with MCMV. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection. Figure 9E shows flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at predetermined time points after transplantation. Figure 9F shows representative flow cytometry histograms of c-Myc expression in splenic NK cells from either XBP1tg mice or wild-type littermates. Figure 9G shows a chromatin immunoprecipitation assay using the NK cell lines MNK-1 (mouse, left graph), NKL, and KHYG-1 (human, right graph) to assess XBP1 binding to the c-Myc locus. Anti-XBP1s Ab was used, and IgG was used as a mock control. Figure 9H shows a representative flow cytometry plot (left) and quantification of c-Myc reporter expression.Prior to evaluation, splenic NK cells from c-Myc reporter mice (MycGFP) were treated with the indicated cytokines for 16 hours in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. The upper panel of Figure 9I shows the percentage of engrafted wild-type and MycNK Ly49H+ NK cells in peripheral blood at the indicated time points after MCMV infection (excluding the spleen endpoint). Equal numbers of Ly49H+ NK cells from wild-type and MycNK donors were co-transferred into recipient Ly49H-deficient mice 1 day before infection. The lower panel of Figure 9I shows the relative percentages within engrafted Ly49H+ NK cells. Figure 9J shows representative immunoblots and quantification of c-Myc protein in IRE1NK and WT NK cells sorted from the same mixed BM chimeric mice at day 1.5 postinfection. Quantification shows cumulative data from four different mixed BM chimeras. p values ​​are as indicated, and ns indicates "not significant." A one-sample t-test was performed for Figure 9J. A two-tailed unpaired Student's t-test was performed for Figures 9D and 9G. A two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed for Figures 9E and 9I. All error bars indicate standard deviation. Each column in Figure 9B is from a different mouse. Data are representative of three (Figures 9C-9E and 9G) and two independent repeats (Figures 9F, 9H, 9I, and 9J). Data were three (Figures 9D and 9H) and two (Figures 9G-9J) independent repeats. N equals 3 or 4 mice / group in Figures 9D and 9J; n equals 3 technical repeats / group in Figure 9G; n equals 3 technical repeats / treatment in ex vivo culture in Figure 9H; and n equals 5 mice / group in Figure 9I. [Figure 9B]Figures 9A-9J show that XBP1 promotes NK proliferation, at least in part, through direct regulation of c-Myc. Figure 9A shows the top 10 enriched GSEA gene clusters in RNA-seq analysis of IRE1NK relative to wild-type Ly49H+ splenic NK cells harvested from IRE1NK bone marrow chimeric mice at day 1.5 postinfection. Figure 9B shows a heatmap of Myc target genes differentially expressed in IRE1NK relative to wild-type NK cells. Genes were clustered by functional annotation. Figure 9C shows flow cytometry analysis of c-Myc induction at the transcriptional (top row) and translational (middle row) levels in splenic NK cells from either naive or wild-type mice at day 1 postinfection with MCMV. The bottom row of Figure 9C shows representative flow cytometry plots demonstrating the simultaneous induction of c-Myc protein and Venus reporter expression in NK cells from the indicated organs of ERAI transgenic mice at day 2 postinfection with MCMV. Figure 9D shows quantitative real-time PCR and flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice 1 day after infection with MCMV. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection. Figure 9E shows flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at predetermined time points after transplantation. Figure 9F shows representative flow cytometry histograms of c-Myc expression in splenic NK cells from either XBP1tg mice or wild-type littermates. Figure 9G shows a chromatin immunoprecipitation assay using the NK cell lines MNK-1 (mouse, left graph), NKL, and KHYG-1 (human, right graph) to assess XBP1 binding to the c-Myc locus. Anti-XBP1s Ab was used, and IgG was used as a mock control. Figure 9H shows a representative flow cytometry plot (left) and quantification of c-Myc reporter expression.Prior to evaluation, splenic NK cells from c-Myc reporter mice (MycGFP) were treated with the indicated cytokines for 16 hours in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. The upper panel of Figure 9I shows the percentage of engrafted wild-type and MycNK Ly49H+ NK cells in peripheral blood at the indicated time points after MCMV infection (excluding the spleen endpoint). Equal numbers of Ly49H+ NK cells from wild-type and MycNK donors were co-transferred into recipient Ly49H-deficient mice 1 day before infection. The lower panel of Figure 9I shows the relative percentages within engrafted Ly49H+ NK cells. Figure 9J shows representative immunoblots and quantification of c-Myc protein in IRE1NK and WT NK cells sorted from the same mixed BM chimeric mice at day 1.5 postinfection. Quantification shows cumulative data from four different mixed BM chimeras. p values ​​are as indicated, and ns indicates "not significant." A one-sample t-test was performed for Figure 9J. A two-tailed unpaired Student's t-test was performed for Figures 9D and 9G. A two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed for Figures 9E and 9I. All error bars indicate standard deviation. Each column in Figure 9B is from a different mouse. Data are representative of three (Figures 9C-9E and 9G) and two independent repeats (Figures 9F, 9H, 9I, and 9J). Data were three (Figures 9D and 9H) and two (Figures 9G-9J) independent repeats. N equals 3 or 4 mice / group in Figures 9D and 9J; n equals 3 technical repeats / group in Figure 9G; n equals 3 technical repeats / treatment in ex vivo culture in Figure 9H; and n equals 5 mice / group in Figure 9I. [Figure 9C]Figures 9A-9J show that XBP1 promotes NK proliferation, at least in part, through direct regulation of c-Myc. Figure 9A shows the top 10 enriched GSEA gene clusters in RNA-seq analysis of IRE1NK relative to wild-type Ly49H+ splenic NK cells harvested from IRE1NK bone marrow chimeric mice at day 1.5 postinfection. Figure 9B shows a heatmap of Myc target genes differentially expressed in IRE1NK relative to wild-type NK cells. Genes were clustered by functional annotation. Figure 9C shows flow cytometry analysis of c-Myc induction at the transcriptional (top row) and translational (middle row) levels in splenic NK cells from either naive or wild-type mice at day 1 postinfection with MCMV. The bottom row of Figure 9C shows representative flow cytometry plots demonstrating the simultaneous induction of c-Myc protein and Venus reporter expression in NK cells from the indicated organs of ERAI transgenic mice at day 2 postinfection with MCMV. Figure 9D shows quantitative real-time PCR and flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice 1 day after infection with MCMV. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection. Figure 9E shows flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at predetermined time points after transplantation. Figure 9F shows representative flow cytometry histograms of c-Myc expression in splenic NK cells from either XBP1tg mice or wild-type littermates. Figure 9G shows a chromatin immunoprecipitation assay using the NK cell lines MNK-1 (mouse, left graph), NKL, and KHYG-1 (human, right graph) to assess XBP1 binding to the c-Myc locus. Anti-XBP1s Ab was used, and IgG was used as a mock control. Figure 9H shows a representative flow cytometry plot (left) and quantification of c-Myc reporter expression.Prior to evaluation, splenic NK cells from c-Myc reporter mice (MycGFP) were treated with the indicated cytokines for 16 hours in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. The upper panel of Figure 9I shows the percentage of engrafted wild-type and MycNK Ly49H+ NK cells in peripheral blood at the indicated time points after MCMV infection (excluding the spleen endpoint). Equal numbers of Ly49H+ NK cells from wild-type and MycNK donors were co-transferred into recipient Ly49H-deficient mice 1 day before infection. The lower panel of Figure 9I shows the relative percentages within engrafted Ly49H+ NK cells. Figure 9J shows representative immunoblots and quantification of c-Myc protein in IRE1NK and WT NK cells sorted from the same mixed BM chimeric mice at day 1.5 postinfection. Quantification shows cumulative data from four different mixed BM chimeras. p values ​​are as indicated, and ns indicates "not significant." A one-sample t-test was performed for Figure 9J. A two-tailed unpaired Student's t-test was performed for Figures 9D and 9G. A two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed for Figures 9E and 9I. All error bars indicate standard deviation. Each column in Figure 9B is from a different mouse. Data are representative of three (Figures 9C-9E and 9G) and two independent repeats (Figures 9F, 9H, 9I, and 9J). Data were three (Figures 9D and 9H) and two (Figures 9G-9J) independent repeats. N equals 3 or 4 mice / group in Figures 9D and 9J; n equals 3 technical repeats / group in Figure 9G; n equals 3 technical repeats / treatment in ex vivo culture in Figure 9H; and n equals 5 mice / group in Figure 9I. [Figure 9D]Figures 9A-9J show that XBP1 promotes NK proliferation, at least in part, through direct regulation of c-Myc. Figure 9A shows the top 10 enriched GSEA gene clusters in RNA-seq analysis of IRE1NK relative to wild-type Ly49H+ splenic NK cells harvested from IRE1NK bone marrow chimeric mice at day 1.5 postinfection. Figure 9B shows a heatmap of Myc target genes differentially expressed in IRE1NK relative to wild-type NK cells. Genes were clustered by functional annotation. Figure 9C shows flow cytometry analysis of c-Myc induction at the transcriptional (top row) and translational (middle row) levels in splenic NK cells from either naive or wild-type mice at day 1 postinfection with MCMV. The bottom row of Figure 9C shows representative flow cytometry plots demonstrating the simultaneous induction of c-Myc protein and Venus reporter expression in NK cells from the indicated organs of ERAI transgenic mice at day 2 postinfection with MCMV. Figure 9D shows quantitative real-time PCR and flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice 1 day after infection with MCMV. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection. Figure 9E shows flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at predetermined time points after transplantation. Figure 9F shows representative flow cytometry histograms of c-Myc expression in splenic NK cells from either XBP1tg mice or wild-type littermates. Figure 9G shows a chromatin immunoprecipitation assay using the NK cell lines MNK-1 (mouse, left graph), NKL, and KHYG-1 (human, right graph) to assess XBP1 binding to the c-Myc locus. Anti-XBP1s Ab was used, and IgG was used as a mock control. Figure 9H shows a representative flow cytometry plot (left) and quantification of c-Myc reporter expression.Prior to evaluation, splenic NK cells from c-Myc reporter mice (MycGFP) were treated with the indicated cytokines for 16 hours in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. The upper panel of Figure 9I shows the percentage of engrafted wild-type and MycNK Ly49H+ NK cells in peripheral blood at the indicated time points after MCMV infection (excluding the spleen endpoint). Equal numbers of Ly49H+ NK cells from wild-type and MycNK donors were co-transferred into recipient Ly49H-deficient mice 1 day before infection. The lower panel of Figure 9I shows the relative percentages within engrafted Ly49H+ NK cells. Figure 9J shows representative immunoblots and quantification of c-Myc protein in IRE1NK and WT NK cells sorted from the same mixed BM chimeric mice at day 1.5 postinfection. Quantification shows cumulative data from four different mixed BM chimeras. p values ​​are as indicated, and ns indicates "not significant." A one-sample t-test was performed for Figure 9J. A two-tailed unpaired Student's t-test was performed for Figures 9D and 9G. A two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed for Figures 9E and 9I. All error bars indicate standard deviation. Each column in Figure 9B is from a different mouse. Data are representative of three (Figures 9C-9E and 9G) and two independent repeats (Figures 9F, 9H, 9I, and 9J). Data were three (Figures 9D and 9H) and two (Figures 9G-9J) independent repeats. N equals 3 or 4 mice / group in Figures 9D and 9J; n equals 3 technical repeats / group in Figure 9G; n equals 3 technical repeats / treatment in ex vivo culture in Figure 9H; and n equals 5 mice / group in Figure 9I. [Figure 9E]Figures 9A-9J show that XBP1 promotes NK proliferation, at least in part, through direct regulation of c-Myc. Figure 9A shows the top 10 enriched GSEA gene clusters in RNA-seq analysis of IRE1NK relative to wild-type Ly49H+ splenic NK cells harvested from IRE1NK bone marrow chimeric mice at day 1.5 postinfection. Figure 9B shows a heatmap of Myc target genes differentially expressed in IRE1NK relative to wild-type NK cells. Genes were clustered by functional annotation. Figure 9C shows flow cytometry analysis of c-Myc induction at the transcriptional (top row) and translational (middle row) levels in splenic NK cells from either naive or wild-type mice at day 1 postinfection with MCMV. The bottom row of Figure 9C shows representative flow cytometry plots demonstrating the simultaneous induction of c-Myc protein and Venus reporter expression in NK cells from the indicated organs of ERAI transgenic mice at day 2 postinfection with MCMV. Figure 9D shows quantitative real-time PCR and flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice 1 day after infection with MCMV. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection. Figure 9E shows flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at predetermined time points after transplantation. Figure 9F shows representative flow cytometry histograms of c-Myc expression in splenic NK cells from either XBP1tg mice or wild-type littermates. Figure 9G shows a chromatin immunoprecipitation assay using the NK cell lines MNK-1 (mouse, left graph), NKL, and KHYG-1 (human, right graph) to assess XBP1 binding to the c-Myc locus. Anti-XBP1s Ab was used, and IgG was used as a mock control. Figure 9H shows a representative flow cytometry plot (left) and quantification of c-Myc reporter expression.Prior to evaluation, splenic NK cells from c-Myc reporter mice (MycGFP) were treated with the indicated cytokines for 16 hours in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. The upper panel of Figure 9I shows the percentage of engrafted wild-type and MycNK Ly49H+ NK cells in peripheral blood at the indicated time points after MCMV infection (excluding the spleen endpoint). Equal numbers of Ly49H+ NK cells from wild-type and MycNK donors were co-transferred into recipient Ly49H-deficient mice 1 day before infection. The lower panel of Figure 9I shows the relative percentages within engrafted Ly49H+ NK cells. Figure 9J shows representative immunoblots and quantification of c-Myc protein in IRE1NK and WT NK cells sorted from the same mixed BM chimeric mice at day 1.5 postinfection. Quantification shows cumulative data from four different mixed BM chimeras. p values ​​are as indicated, and ns indicates "not significant." A one-sample t-test was performed for Figure 9J. A two-tailed unpaired Student's t-test was performed for Figures 9D and 9G. A two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed for Figures 9E and 9I. All error bars indicate standard deviation. Each column in Figure 9B is from a different mouse. Data are representative of three (Figures 9C-9E and 9G) and two independent repeats (Figures 9F, 9H, 9I, and 9J). Data were three (Figures 9D and 9H) and two (Figures 9G-9J) independent repeats. N equals 3 or 4 mice / group in Figures 9D and 9J; n equals 3 technical repeats / group in Figure 9G; n equals 3 technical repeats / treatment in ex vivo culture in Figure 9H; and n equals 5 mice / group in Figure 9I. [Figure 9F]Figures 9A-9J show that XBP1 promotes NK proliferation, at least in part, through direct regulation of c-Myc. Figure 9A shows the top 10 enriched GSEA gene clusters in RNA-seq analysis of IRE1NK relative to wild-type Ly49H+ splenic NK cells harvested from IRE1NK bone marrow chimeric mice at day 1.5 postinfection. Figure 9B shows a heatmap of Myc target genes differentially expressed in IRE1NK relative to wild-type NK cells. Genes were clustered by functional annotation. Figure 9C shows flow cytometry analysis of c-Myc induction at the transcriptional (top row) and translational (middle row) levels in splenic NK cells from either naive or wild-type mice at day 1 postinfection with MCMV. The bottom row of Figure 9C shows representative flow cytometry plots demonstrating the simultaneous induction of c-Myc protein and Venus reporter expression in NK cells from the indicated organs of ERAI transgenic mice at day 2 postinfection with MCMV. Figure 9D shows quantitative real-time PCR and flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice 1 day after infection with MCMV. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection. Figure 9E shows flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at predetermined time points after transplantation. Figure 9F shows representative flow cytometry histograms of c-Myc expression in splenic NK cells from either XBP1tg mice or wild-type littermates. Figure 9G shows a chromatin immunoprecipitation assay using the NK cell lines MNK-1 (mouse, left graph), NKL, and KHYG-1 (human, right graph) to assess XBP1 binding to the c-Myc locus. Anti-XBP1s Ab was used, and IgG was used as a mock control. Figure 9H shows a representative flow cytometry plot (left) and quantification of c-Myc reporter expression.Prior to evaluation, splenic NK cells from c-Myc reporter mice (MycGFP) were treated with the indicated cytokines for 16 hours in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. The upper panel of Figure 9I shows the percentage of engrafted wild-type and MycNK Ly49H+ NK cells in peripheral blood at the indicated time points after MCMV infection (excluding the spleen endpoint). Equal numbers of Ly49H+ NK cells from wild-type and MycNK donors were co-transferred into recipient Ly49H-deficient mice 1 day before infection. The lower panel of Figure 9I shows the relative percentages within engrafted Ly49H+ NK cells. Figure 9J shows representative immunoblots and quantification of c-Myc protein in IRE1NK and WT NK cells sorted from the same mixed BM chimeric mice at day 1.5 postinfection. Quantification shows cumulative data from four different mixed BM chimeras. p values ​​are as indicated, and ns indicates "not significant." A one-sample t-test was performed for Figure 9J. A two-tailed unpaired Student's t-test was performed for Figures 9D and 9G. A two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed for Figures 9E and 9I. All error bars indicate standard deviation. Each column in Figure 9B is from a different mouse. Data are representative of three (Figures 9C-9E and 9G) and two independent repeats (Figures 9F, 9H, 9I, and 9J). Data were three (Figures 9D and 9H) and two (Figures 9G-9J) independent repeats. N equals 3 or 4 mice / group in Figures 9D and 9J; n equals 3 technical repeats / group in Figure 9G; n equals 3 technical repeats / treatment in ex vivo culture in Figure 9H; and n equals 5 mice / group in Figure 9I. [Figure 9G]Figures 9A-9J show that XBP1 promotes NK proliferation, at least in part, through direct regulation of c-Myc. Figure 9A shows the top 10 enriched GSEA gene clusters in RNA-seq analysis of IRE1NK relative to wild-type Ly49H+ splenic NK cells harvested from IRE1NK bone marrow chimeric mice at day 1.5 postinfection. Figure 9B shows a heatmap of Myc target genes differentially expressed in IRE1NK relative to wild-type NK cells. Genes were clustered by functional annotation. Figure 9C shows flow cytometry analysis of c-Myc induction at the transcriptional (top row) and translational (middle row) levels in splenic NK cells from either naive or wild-type mice at day 1 postinfection with MCMV. The bottom row of Figure 9C shows representative flow cytometry plots demonstrating the simultaneous induction of c-Myc protein and Venus reporter expression in NK cells from the indicated organs of ERAI transgenic mice at day 2 postinfection with MCMV. Figure 9D shows quantitative real-time PCR and flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice 1 day after infection with MCMV. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection. Figure 9E shows flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at predetermined time points after transplantation. Figure 9F shows representative flow cytometry histograms of c-Myc expression in splenic NK cells from either XBP1tg mice or wild-type littermates. Figure 9G shows a chromatin immunoprecipitation assay using the NK cell lines MNK-1 (mouse, left graph), NKL, and KHYG-1 (human, right graph) to assess XBP1 binding to the c-Myc locus. Anti-XBP1s Ab was used, and IgG was used as a mock control. Figure 9H shows a representative flow cytometry plot (left) and quantification of c-Myc reporter expression.Prior to evaluation, splenic NK cells from c-Myc reporter mice (MycGFP) were treated with the indicated cytokines for 16 hours in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. The upper panel of Figure 9I shows the percentage of engrafted wild-type and MycNK Ly49H+ NK cells in peripheral blood at the indicated time points after MCMV infection (excluding the spleen endpoint). Equal numbers of Ly49H+ NK cells from wild-type and MycNK donors were co-transferred into recipient Ly49H-deficient mice 1 day before infection. The lower panel of Figure 9I shows the relative percentages within engrafted Ly49H+ NK cells. Figure 9J shows representative immunoblots and quantification of c-Myc protein in IRE1NK and WT NK cells sorted from the same mixed BM chimeric mice at day 1.5 postinfection. Quantification shows cumulative data from four different mixed BM chimeras. p values ​​are as indicated, and ns indicates "not significant." A one-sample t-test was performed for Figure 9J. A two-tailed unpaired Student's t-test was performed for Figures 9D and 9G. A two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed for Figures 9E and 9I. All error bars indicate standard deviation. Each column in Figure 9B is from a different mouse. Data are representative of three (Figures 9C-9E and 9G) and two independent repeats (Figures 9F, 9H, 9I, and 9J). Data were three (Figures 9D and 9H) and two (Figures 9G-9J) independent repeats. N equals 3 or 4 mice / group in Figures 9D and 9J; n equals 3 technical repeats / group in Figure 9G; n equals 3 technical repeats / treatment in ex vivo culture in Figure 9H; and n equals 5 mice / group in Figure 9I. [Figure 9H]Figures 9A-9J show that XBP1 promotes NK proliferation, at least in part, through direct regulation of c-Myc. Figure 9A shows the top 10 enriched GSEA gene clusters in RNA-seq analysis of IRE1NK relative to wild-type Ly49H+ splenic NK cells harvested from IRE1NK bone marrow chimeric mice at day 1.5 postinfection. Figure 9B shows a heatmap of Myc target genes differentially expressed in IRE1NK relative to wild-type NK cells. Genes were clustered by functional annotation. Figure 9C shows flow cytometry analysis of c-Myc induction at the transcriptional (top row) and translational (middle row) levels in splenic NK cells from either naive or wild-type mice at day 1 postinfection with MCMV. The bottom row of Figure 9C shows representative flow cytometry plots demonstrating the simultaneous induction of c-Myc protein and Venus reporter expression in NK cells from the indicated organs of ERAI transgenic mice at day 2 postinfection with MCMV. Figure 9D shows quantitative real-time PCR and flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice 1 day after infection with MCMV. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection. Figure 9E shows flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at predetermined time points after transplantation. Figure 9F shows representative flow cytometry histograms of c-Myc expression in splenic NK cells from either XBP1tg mice or wild-type littermates. Figure 9G shows a chromatin immunoprecipitation assay using the NK cell lines MNK-1 (mouse, left graph), NKL, and KHYG-1 (human, right graph) to assess XBP1 binding to the c-Myc locus. Anti-XBP1s Ab was used, and IgG was used as a mock control. Figure 9H shows a representative flow cytometry plot (left) and quantification of c-Myc reporter expression.Prior to evaluation, splenic NK cells from c-Myc reporter mice (MycGFP) were treated with the indicated cytokines for 16 hours in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. The upper panel of Figure 9I shows the percentage of engrafted wild-type and MycNK Ly49H+ NK cells in peripheral blood at the indicated time points after MCMV infection (excluding the spleen endpoint). Equal numbers of Ly49H+ NK cells from wild-type and MycNK donors were co-transferred into recipient Ly49H-deficient mice 1 day before infection. The lower panel of Figure 9I shows the relative percentages within engrafted Ly49H+ NK cells. Figure 9J shows representative immunoblots and quantification of c-Myc protein in IRE1NK and WT NK cells sorted from the same mixed BM chimeric mice at day 1.5 postinfection. Quantification shows cumulative data from four different mixed BM chimeras. p values ​​are as indicated, and ns indicates "not significant." A one-sample t-test was performed for Figure 9J. A two-tailed unpaired Student's t-test was performed for Figures 9D and 9G. A two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed for Figures 9E and 9I. All error bars indicate standard deviation. Each column in Figure 9B is from a different mouse. Data are representative of three (Figures 9C-9E and 9G) and two independent repeats (Figures 9F, 9H, 9I, and 9J). Data were three (Figures 9D and 9H) and two (Figures 9G-9J) independent repeats. N equals 3 or 4 mice / group in Figures 9D and 9J; n equals 3 technical repeats / group in Figure 9G; n equals 3 technical repeats / treatment in ex vivo culture in Figure 9H; and n equals 5 mice / group in Figure 9I. [Figure 9I]Figures 9A-9J show that XBP1 promotes NK proliferation, at least in part, through direct regulation of c-Myc. Figure 9A shows the top 10 enriched GSEA gene clusters in RNA-seq analysis of IRE1NK relative to wild-type Ly49H+ splenic NK cells harvested from IRE1NK bone marrow chimeric mice at day 1.5 postinfection. Figure 9B shows a heatmap of Myc target genes differentially expressed in IRE1NK relative to wild-type NK cells. Genes were clustered by functional annotation. Figure 9C shows flow cytometry analysis of c-Myc induction at the transcriptional (top row) and translational (middle row) levels in splenic NK cells from either naive or wild-type mice at day 1 postinfection with MCMV. The bottom row of Figure 9C shows representative flow cytometry plots demonstrating the simultaneous induction of c-Myc protein and Venus reporter expression in NK cells from the indicated organs of ERAI transgenic mice at day 2 postinfection with MCMV. Figure 9D shows quantitative real-time PCR and flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice 1 day after infection with MCMV. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection. Figure 9E shows flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at predetermined time points after transplantation. Figure 9F shows representative flow cytometry histograms of c-Myc expression in splenic NK cells from either XBP1tg mice or wild-type littermates. Figure 9G shows a chromatin immunoprecipitation assay using the NK cell lines MNK-1 (mouse, left graph), NKL, and KHYG-1 (human, right graph) to assess XBP1 binding to the c-Myc locus. Anti-XBP1s Ab was used, and IgG was used as a mock control. Figure 9H shows a representative flow cytometry plot (left) and quantification of c-Myc reporter expression.Prior to evaluation, splenic NK cells from c-Myc reporter mice (MycGFP) were treated with the indicated cytokines for 16 hours in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. The upper panel of Figure 9I shows the percentage of engrafted wild-type and MycNK Ly49H+ NK cells in peripheral blood at the indicated time points after MCMV infection (excluding the spleen endpoint). Equal numbers of Ly49H+ NK cells from wild-type and MycNK donors were co-transferred into recipient Ly49H-deficient mice 1 day before infection. The lower panel of Figure 9I shows the relative percentages within engrafted Ly49H+ NK cells. Figure 9J shows representative immunoblots and quantification of c-Myc protein in IRE1NK and WT NK cells sorted from the same mixed BM chimeric mice at day 1.5 postinfection. Quantification shows cumulative data from four different mixed BM chimeras. p values ​​are as indicated, and ns indicates "not significant." A one-sample t-test was performed for Figure 9J. A two-tailed unpaired Student's t-test was performed for Figures 9D and 9G. A two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed for Figures 9E and 9I. All error bars indicate standard deviation. Each column in Figure 9B is from a different mouse. Data are representative of three (Figures 9C-9E and 9G) and two independent repeats (Figures 9F, 9H, 9I, and 9J). Data were three (Figures 9D and 9H) and two (Figures 9G-9J) independent repeats. N equals 3 or 4 mice / group in Figures 9D and 9J; n equals 3 technical repeats / group in Figure 9G; n equals 3 technical repeats / treatment in ex vivo culture in Figure 9H; and n equals 5 mice / group in Figure 9I. [Figure 9J]Figures 9A-9J show that XBP1 promotes NK proliferation, at least in part, through direct regulation of c-Myc. Figure 9A shows the top 10 enriched GSEA gene clusters in RNA-seq analysis of IRE1NK relative to wild-type Ly49H+ splenic NK cells harvested from IRE1NK bone marrow chimeric mice at day 1.5 postinfection. Figure 9B shows a heatmap of Myc target genes differentially expressed in IRE1NK relative to wild-type NK cells. Genes were clustered by functional annotation. Figure 9C shows flow cytometry analysis of c-Myc induction at the transcriptional (top row) and translational (middle row) levels in splenic NK cells from either naive or wild-type mice at day 1 postinfection with MCMV. The bottom row of Figure 9C shows representative flow cytometry plots demonstrating the simultaneous induction of c-Myc protein and Venus reporter expression in NK cells from the indicated organs of ERAI transgenic mice at day 2 postinfection with MCMV. Figure 9D shows quantitative real-time PCR and flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK Ly49H+ NK cells in the spleens of recipient Ly49H-deficient mice 1 day after infection with MCMV. Equal numbers of Ly49H+ NK cells from wild-type and knockout donors were co-transplanted into recipient Ly49H-deficient mice 1 day before infection. Figure 9E shows flow cytometry analysis of c-Myc expression in transplanted wild-type and IRE1NK NK cells in the spleens of recipient Rag2- / -Il2rg- / - mice at predetermined time points after transplantation. Figure 9F shows representative flow cytometry histograms of c-Myc expression in splenic NK cells from either XBP1tg mice or wild-type littermates. Figure 9G shows a chromatin immunoprecipitation assay using the NK cell lines MNK-1 (mouse, left graph), NKL, and KHYG-1 (human, right graph) to assess XBP1 binding to the c-Myc locus. Anti-XBP1s Ab was used, and IgG was used as a mock control. Figure 9H shows a representative flow cytometry plot (left) and quantification of c-Myc reporter expression.Prior to evaluation, splenic NK cells from c-Myc reporter mice (MycGFP) were treated with the indicated cytokines for 16 hours in the presence or absence of the IRE1 inhibitor (IRE1i) 4μ8c. The upper panel of Figure 9I shows the percentage of engrafted wild-type and MycNK Ly49H+ NK cells in peripheral blood at the indicated time points after MCMV infection (excluding the spleen endpoint). Equal numbers of Ly49H+ NK cells from wild-type and MycNK donors were co-transferred into recipient Ly49H-deficient mice 1 day before infection. The lower panel of Figure 9I shows the relative percentages within engrafted Ly49H+ NK cells. Figure 9J shows representative immunoblots and quantification of c-Myc protein in IRE1NK and WT NK cells sorted from the same mixed BM chimeric mice at day 1.5 postinfection. Quantification shows cumulative data from four different mixed BM chimeras. p values ​​are as indicated, and ns indicates "not significant." A one-sample t-test was performed for Figure 9J. A two-tailed unpaired Student's t-test was performed for Figures 9D and 9G. A two-way analysis of variance (ANOVA) with Sidak post-hoc test was performed for Figures 9E and 9I. All error bars indicate standard deviation. Each column in Figure 9B is from a different mouse. Data are representative of three (Figures 9C-9E and 9G) and two independent repeats (Figures 9F, 9H, 9I, and 9J). Data were three (Figures 9D and 9H) and two (Figures 9G-9J) independent repeats. N equals 3 or 4 mice / group in Figures 9D and 9J; n equals 3 technical repeats / group in Figure 9G; n equals 3 technical repeats / treatment in ex vivo culture in Figure 9H; and n equals 5 mice / group in Figure 9I. [Figure 10A]Figures 10A-10I demonstrate the specific requirement of IRE1α / XBP1 for NK cell-mediated antitumor immunity. Figure 10A shows the gross morphology of lungs and individual lung lobes from IREf / f and IRE1NK mice at day 10 after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown on the right. Figure 10B shows H&E microscopy analysis of lungs from IREf / f and IRE1NK mice described in Figure 10A. Figure 10C shows survival curves of B16F10-inoculated IRE1f / f and IRE1NK mice described in Figure 10A. Figure 10D is similar to Figure 10A and shows quantification of total extrapulmonary metastatic nodules from IRE1f / f and XBP1NK mice at day 20 after intravenous injection of B16F10 melanoma. Figures 10E-10G show flow cytometry analysis of lungs from mice inoculated with the B16F10 tumor described in Figure 10A. Graphs shown are (Figure 10E) the percentage and absolute number of NK cells in the total lymphocyte population, (Figure 10F) Ki-67, and (Figure 10G) c-Myc expression in lung-infiltrating NK cells. p values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 10A and 10D-10G. Gehan-Breslow-Wilcoxon test was performed for Figure 10C. All error bars indicate the standard error of the mean (s.e.m.). Each dot represents a different mouse. Data are pooled from three (Figure 10A) and two independent experiments (Figures 10C-10E) or are representative of two independent repeats (Figures 10B, 10F, and 10G). Figure 10H shows the gross morphology of lungs and individual lung lobes from Myc+ / -Ncr1Cre+ (MycNK) and Myc+ / -Ncr1Cre- (Cre-Ctrl) littermate mice 12 days after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown in the histogram. Figure 10I shows the results of flow cytometry analysis of lungs from mice inoculated with B16F10 tumors as described in Figure 10H. Graphs shown are representative flow cytometry plots (upper panel) and quantification (histogram) of the proportion of NK cells in the total lymphocyte population in the lung. p values ​​are as indicated. A two-tailed, unpaired Student's t-test was performed.All error bars indicate the standard error of the mean (sem). Each dot represents a different mouse. Data are representative of two independent repeats. [Figure 10B]Figures 10A-10I demonstrate the specific requirement of IRE1α / XBP1 for NK cell-mediated antitumor immunity. Figure 10A shows the gross morphology of lungs and individual lung lobes from IREf / f and IRE1NK mice at day 10 after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown on the right. Figure 10B shows H&E microscopy analysis of lungs from IREf / f and IRE1NK mice described in Figure 10A. Figure 10C shows survival curves of B16F10-inoculated IRE1f / f and IRE1NK mice described in Figure 10A. Figure 10D is similar to Figure 10A and shows quantification of total extrapulmonary metastatic nodules from IRE1f / f and XBP1NK mice at day 20 after intravenous injection of B16F10 melanoma. Figures 10E-10G show flow cytometry analysis of lungs from mice inoculated with the B16F10 tumor described in Figure 10A. Graphs shown are (Figure 10E) the percentage and absolute number of NK cells in the total lymphocyte population, (Figure 10F) Ki-67, and (Figure 10G) c-Myc expression in lung-infiltrating NK cells. p values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 10A and 10D-10G. Gehan-Breslow-Wilcoxon test was performed for Figure 10C. All error bars indicate the standard error of the mean (s.e.m.). Each dot represents a different mouse. Data are pooled from three (Figure 10A) and two independent experiments (Figures 10C-10E) or are representative of two independent repeats (Figures 10B, 10F, and 10G). Figure 10H shows the gross morphology of lungs and individual lung lobes from Myc+ / -Ncr1Cre+ (MycNK) and Myc+ / -Ncr1Cre- (Cre-Ctrl) littermate mice 12 days after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown in the histogram. Figure 10I shows the results of flow cytometry analysis of lungs from mice inoculated with B16F10 tumors as described in Figure 10H. Graphs shown are representative flow cytometry plots (upper panel) and quantification (histogram) of the proportion of NK cells in the total lymphocyte population in the lung. p values ​​are as indicated. A two-tailed, unpaired Student's t-test was performed.All error bars indicate the standard error of the mean (sem). Each dot represents a different mouse. Data are representative of two independent repeats. [Figure 10C]Figures 10A-10I demonstrate the specific requirement of IRE1α / XBP1 for NK cell-mediated antitumor immunity. Figure 10A shows the gross morphology of lungs and individual lung lobes from IREf / f and IRE1NK mice at day 10 after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown on the right. Figure 10B shows H&E microscopy analysis of lungs from IREf / f and IRE1NK mice described in Figure 10A. Figure 10C shows survival curves of B16F10-inoculated IRE1f / f and IRE1NK mice described in Figure 10A. Figure 10D is similar to Figure 10A and shows quantification of total extrapulmonary metastatic nodules from IRE1f / f and XBP1NK mice at day 20 after intravenous injection of B16F10 melanoma. Figures 10E-10G show flow cytometry analysis of lungs from mice inoculated with the B16F10 tumor described in Figure 10A. Graphs shown are (Figure 10E) the percentage and absolute number of NK cells in the total lymphocyte population, (Figure 10F) Ki-67, and (Figure 10G) c-Myc expression in lung-infiltrating NK cells. p values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 10A and 10D-10G. Gehan-Breslow-Wilcoxon test was performed for Figure 10C. All error bars indicate the standard error of the mean (s.e.m.). Each dot represents a different mouse. Data are pooled from three (Figure 10A) and two independent experiments (Figures 10C-10E) or are representative of two independent repeats (Figures 10B, 10F, and 10G). Figure 10H shows the gross morphology of lungs and individual lung lobes from Myc+ / -Ncr1Cre+ (MycNK) and Myc+ / -Ncr1Cre- (Cre-Ctrl) littermate mice 12 days after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown in the histogram. Figure 10I shows the results of flow cytometry analysis of lungs from mice inoculated with B16F10 tumors as described in Figure 10H. Graphs shown are representative flow cytometry plots (upper panel) and quantification (histogram) of the proportion of NK cells in the total lymphocyte population in the lung. p values ​​are as indicated. A two-tailed, unpaired Student's t-test was performed.All error bars indicate the standard error of the mean (sem). Each dot represents a different mouse. Data are representative of two independent repeats. [Figure 10D]Figures 10A-10I demonstrate the specific requirement of IRE1α / XBP1 for NK cell-mediated antitumor immunity. Figure 10A shows the gross morphology of lungs and individual lung lobes from IREf / f and IRE1NK mice at day 10 after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown on the right. Figure 10B shows H&E microscopy analysis of lungs from IREf / f and IRE1NK mice described in Figure 10A. Figure 10C shows survival curves of B16F10-inoculated IRE1f / f and IRE1NK mice described in Figure 10A. Figure 10D is similar to Figure 10A and shows quantification of total extrapulmonary metastatic nodules from IRE1f / f and XBP1NK mice at day 20 after intravenous injection of B16F10 melanoma. Figures 10E-10G show flow cytometry analysis of lungs from mice inoculated with the B16F10 tumor described in Figure 10A. Graphs shown are (Figure 10E) the percentage and absolute number of NK cells in the total lymphocyte population, (Figure 10F) Ki-67, and (Figure 10G) c-Myc expression in lung-infiltrating NK cells. p values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 10A and 10D-10G. Gehan-Breslow-Wilcoxon test was performed for Figure 10C. All error bars indicate the standard error of the mean (s.e.m.). Each dot represents a different mouse. Data are pooled from three (Figure 10A) and two independent experiments (Figures 10C-10E) or are representative of two independent repeats (Figures 10B, 10F, and 10G). Figure 10H shows the gross morphology of lungs and individual lung lobes from Myc+ / -Ncr1Cre+ (MycNK) and Myc+ / -Ncr1Cre- (Cre-Ctrl) littermate mice 12 days after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown in the histogram. Figure 10I shows the results of flow cytometry analysis of lungs from mice inoculated with B16F10 tumors as described in Figure 10H. Graphs shown are representative flow cytometry plots (upper panel) and quantification (histogram) of the proportion of NK cells in the total lymphocyte population in the lung. p values ​​are as indicated. A two-tailed, unpaired Student's t-test was performed.All error bars indicate the standard error of the mean (sem). Each dot represents a different mouse. Data are representative of two independent repeats. [Figure 10E]Figures 10A-10I demonstrate the specific requirement of IRE1α / XBP1 for NK cell-mediated antitumor immunity. Figure 10A shows the gross morphology of lungs and individual lung lobes from IREf / f and IRE1NK mice at day 10 after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown on the right. Figure 10B shows H&E microscopy analysis of lungs from IREf / f and IRE1NK mice described in Figure 10A. Figure 10C shows survival curves of B16F10-inoculated IRE1f / f and IRE1NK mice described in Figure 10A. Figure 10D is similar to Figure 10A and shows quantification of total extrapulmonary metastatic nodules from IRE1f / f and XBP1NK mice at day 20 after intravenous injection of B16F10 melanoma. Figures 10E-10G show flow cytometry analysis of lungs from mice inoculated with the B16F10 tumor described in Figure 10A. Graphs shown are (Figure 10E) the percentage and absolute number of NK cells in the total lymphocyte population, (Figure 10F) Ki-67, and (Figure 10G) c-Myc expression in lung-infiltrating NK cells. p values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 10A and 10D-10G. Gehan-Breslow-Wilcoxon test was performed for Figure 10C. All error bars indicate the standard error of the mean (s.e.m.). Each dot represents a different mouse. Data are pooled from three (Figure 10A) and two independent experiments (Figures 10C-10E) or are representative of two independent repeats (Figures 10B, 10F, and 10G). Figure 10H shows the gross morphology of lungs and individual lung lobes from Myc+ / -Ncr1Cre+ (MycNK) and Myc+ / -Ncr1Cre- (Cre-Ctrl) littermate mice 12 days after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown in the histogram. Figure 10I shows the results of flow cytometry analysis of lungs from mice inoculated with B16F10 tumors as described in Figure 10H. Graphs shown are representative flow cytometry plots (upper panel) and quantification (histogram) of the proportion of NK cells in the total lymphocyte population in the lung. p values ​​are as indicated. A two-tailed, unpaired Student's t-test was performed.All error bars indicate the standard error of the mean (sem). Each dot represents a different mouse. Data are representative of two independent repeats. [Figure 10F]Figures 10A-10I demonstrate the specific requirement of IRE1α / XBP1 for NK cell-mediated antitumor immunity. Figure 10A shows the gross morphology of lungs and individual lung lobes from IREf / f and IRE1NK mice at day 10 after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown on the right. Figure 10B shows H&E microscopy analysis of lungs from IREf / f and IRE1NK mice described in Figure 10A. Figure 10C shows survival curves of B16F10-inoculated IRE1f / f and IRE1NK mice described in Figure 10A. Figure 10D is similar to Figure 10A and shows quantification of total extrapulmonary metastatic nodules from IRE1f / f and XBP1NK mice at day 20 after intravenous injection of B16F10 melanoma. Figures 10E-10G show flow cytometry analysis of lungs from mice inoculated with the B16F10 tumor described in Figure 10A. Graphs shown are (Figure 10E) the percentage and absolute number of NK cells in the total lymphocyte population, (Figure 10F) Ki-67, and (Figure 10G) c-Myc expression in lung-infiltrating NK cells. p values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 10A and 10D-10G. Gehan-Breslow-Wilcoxon test was performed for Figure 10C. All error bars indicate the standard error of the mean (s.e.m.). Each dot represents a different mouse. Data are pooled from three (Figure 10A) and two independent experiments (Figures 10C-10E) or are representative of two independent repeats (Figures 10B, 10F, and 10G). Figure 10H shows the gross morphology of lungs and individual lung lobes from Myc+ / -Ncr1Cre+ (MycNK) and Myc+ / -Ncr1Cre- (Cre-Ctrl) littermate mice 12 days after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown in the histogram. Figure 10I shows the results of flow cytometry analysis of lungs from mice inoculated with B16F10 tumors as described in Figure 10H. Graphs shown are representative flow cytometry plots (upper panel) and quantification (histogram) of the proportion of NK cells in the total lymphocyte population in the lung. p values ​​are as indicated. A two-tailed, unpaired Student's t-test was performed.All error bars indicate the standard error of the mean (sem). Each dot represents a different mouse. Data are representative of two independent repeats. [Figure 10G]Figures 10A-10I demonstrate the specific requirement of IRE1α / XBP1 for NK cell-mediated antitumor immunity. Figure 10A shows the gross morphology of lungs and individual lung lobes from IREf / f and IRE1NK mice at day 10 after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown on the right. Figure 10B shows H&E microscopy analysis of lungs from IREf / f and IRE1NK mice described in Figure 10A. Figure 10C shows survival curves of B16F10-inoculated IRE1f / f and IRE1NK mice described in Figure 10A. Figure 10D is similar to Figure 10A and shows quantification of total extrapulmonary metastatic nodules from IRE1f / f and XBP1NK mice at day 20 after intravenous injection of B16F10 melanoma. Figures 10E-10G show flow cytometry analysis of lungs from mice inoculated with the B16F10 tumor described in Figure 10A. Graphs shown are (Figure 10E) the percentage and absolute number of NK cells in the total lymphocyte population, (Figure 10F) Ki-67, and (Figure 10G) c-Myc expression in lung-infiltrating NK cells. p values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 10A and 10D-10G. Gehan-Breslow-Wilcoxon test was performed for Figure 10C. All error bars indicate the standard error of the mean (s.e.m.). Each dot represents a different mouse. Data are pooled from three (Figure 10A) and two independent experiments (Figures 10C-10E) or are representative of two independent repeats (Figures 10B, 10F, and 10G). Figure 10H shows the gross morphology of lungs and individual lung lobes from Myc+ / -Ncr1Cre+ (MycNK) and Myc+ / -Ncr1Cre- (Cre-Ctrl) littermate mice 12 days after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown in the histogram. Figure 10I shows the results of flow cytometry analysis of lungs from mice inoculated with B16F10 tumors as described in Figure 10H. Graphs shown are representative flow cytometry plots (upper panel) and quantification (histogram) of the proportion of NK cells in the total lymphocyte population in the lung. p values ​​are as indicated. A two-tailed, unpaired Student's t-test was performed.All error bars indicate the standard error of the mean (sem). Each dot represents a different mouse. Data are representative of two independent repeats. [Figure 10H]Figures 10A-10I demonstrate the specific requirement of IRE1α / XBP1 for NK cell-mediated antitumor immunity. Figure 10A shows the gross morphology of lungs and individual lung lobes from IREf / f and IRE1NK mice at day 10 after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown on the right. Figure 10B shows H&E microscopy analysis of lungs from IREf / f and IRE1NK mice described in Figure 10A. Figure 10C shows survival curves of B16F10-inoculated IRE1f / f and IRE1NK mice described in Figure 10A. Figure 10D is similar to Figure 10A and shows quantification of total extrapulmonary metastatic nodules from IRE1f / f and XBP1NK mice at day 20 after intravenous injection of B16F10 melanoma. Figures 10E-10G show flow cytometry analysis of lungs from mice inoculated with the B16F10 tumor described in Figure 10A. Graphs shown are (Figure 10E) the percentage and absolute number of NK cells in the total lymphocyte population, (Figure 10F) Ki-67, and (Figure 10G) c-Myc expression in lung-infiltrating NK cells. p values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 10A and 10D-10G. Gehan-Breslow-Wilcoxon test was performed for Figure 10C. All error bars indicate the standard error of the mean (s.e.m.). Each dot represents a different mouse. Data are pooled from three (Figure 10A) and two independent experiments (Figures 10C-10E) or are representative of two independent repeats (Figures 10B, 10F, and 10G). Figure 10H shows the gross morphology of lungs and individual lung lobes from Myc+ / -Ncr1Cre+ (MycNK) and Myc+ / -Ncr1Cre- (Cre-Ctrl) littermate mice 12 days after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown in the histogram. Figure 10I shows the results of flow cytometry analysis of lungs from mice inoculated with B16F10 tumors as described in Figure 10H. Graphs shown are representative flow cytometry plots (upper panel) and quantification (histogram) of the proportion of NK cells in the total lymphocyte population in the lung. p values ​​are as indicated. A two-tailed, unpaired Student's t-test was performed.All error bars indicate the standard error of the mean (sem). Each dot represents a different mouse. Data are representative of two independent repeats. [Figure 10I]Figures 10A-10I demonstrate the specific requirement of IRE1α / XBP1 for NK cell-mediated antitumor immunity. Figure 10A shows the gross morphology of lungs and individual lung lobes from IREf / f and IRE1NK mice at day 10 after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown on the right. Figure 10B shows H&E microscopy analysis of lungs from IREf / f and IRE1NK mice described in Figure 10A. Figure 10C shows survival curves of B16F10-inoculated IRE1f / f and IRE1NK mice described in Figure 10A. Figure 10D is similar to Figure 10A and shows quantification of total extrapulmonary metastatic nodules from IRE1f / f and XBP1NK mice at day 20 after intravenous injection of B16F10 melanoma. Figures 10E-10G show flow cytometry analysis of lungs from mice inoculated with the B16F10 tumor described in Figure 10A. Graphs shown are (Figure 10E) the percentage and absolute number of NK cells in the total lymphocyte population, (Figure 10F) Ki-67, and (Figure 10G) c-Myc expression in lung-infiltrating NK cells. p values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 10A and 10D-10G. Gehan-Breslow-Wilcoxon test was performed for Figure 10C. All error bars indicate the standard error of the mean (s.e.m.). Each dot represents a different mouse. Data are pooled from three (Figure 10A) and two independent experiments (Figures 10C-10E) or are representative of two independent repeats (Figures 10B, 10F, and 10G). Figure 10H shows the gross morphology of lungs and individual lung lobes from Myc+ / -Ncr1Cre+ (MycNK) and Myc+ / -Ncr1Cre- (Cre-Ctrl) littermate mice 12 days after intravenous injection of B16F10 melanoma. Quantification of total extrapulmonary metastatic nodules is shown in the histogram. Figure 10I shows the results of flow cytometry analysis of lungs from mice inoculated with B16F10 tumors as described in Figure 10H. Graphs shown are representative flow cytometry plots (upper panel) and quantification (histogram) of the proportion of NK cells in the total lymphocyte population in the lung. p values ​​are as indicated. A two-tailed, unpaired Student's t-test was performed.All error bars indicate the standard error of the mean (sem). Each dot represents a different mouse. Data are representative of two independent repeats. [Figure 11A] Figures 11A-11D show that IRE1α-induced NK cell hypertrophy is associated with the presentation of immune cell types beneficial to tumor control, and that IRE1α in NK cells promotes IFN-γ production in tumor-infiltrating lymphocytes. Figure 11A shows the gating strategy. Figure 11B shows the percentages of conventional type 1 dendritic cells (cDC1) (left), CD8+ T cells (center), and CD4+ T cells (right) in the lungs and spleens of IRE1NK and IRE1f / f littermate control mice at day 20 after intravenous injection of B16F10 melanoma cells. Figure 11C shows representative flow cytometry plots and quantification of baseline levels and relative percentages of NK cell numbers and Ki-67 and c-Myc expression in the lungs of naive IRE1NK and IRE1f / f littermate control mice. * indicates p<0.05, and ** indicates p<0.01. A two-tailed, unpaired Student's t-test was performed on Figure 11B. All error bars represent the standard error of the mean (sem). For all experiments, n = 4-5 mice per group, and each date was independently repeated three times. Figure 11D shows that IRE1α in NK cells promotes IFN-γ production in tumor-infiltrating lymphocytes. Bar graphs show the percentage of IFN-γ-producing cells among NK cells, CD4+ T cells, and CD8+ T cells in the lungs (upper panel) and spleens (lower panel) of IRE1NK and IRE1f / f littermate control mice on day 20 after intravenous injection of B16F10 melanoma cells. * indicates p<0.05, ** indicates p<0.01. Two-tailed unpaired Student's t-test was performed. All error bars represent the standard error of the mean (sem). n = 4-6 mice per group, and each date was independently repeated three times. [Figure 11B]Figures 11A-11D show that IRE1α-induced NK cell hypertrophy is associated with the presentation of immune cell types beneficial to tumor control, and that IRE1α in NK cells promotes IFN-γ production in tumor-infiltrating lymphocytes. Figure 11A shows the gating strategy. Figure 11B shows the percentages of conventional type 1 dendritic cells (cDC1) (left), CD8+ T cells (center), and CD4+ T cells (right) in the lungs and spleens of IRE1NK and IRE1f / f littermate control mice at day 20 after intravenous injection of B16F10 melanoma cells. Figure 11C shows representative flow cytometry plots and quantification of baseline levels and relative percentages of NK cell numbers and Ki-67 and c-Myc expression in the lungs of naive IRE1NK and IRE1f / f littermate control mice. * indicates p<0.05, and ** indicates p<0.01. A two-tailed, unpaired Student's t-test was performed on Figure 11B. All error bars represent the standard error of the mean (sem). For all experiments, n = 4-5 mice per group, and each date was independently repeated three times. Figure 11D shows that IRE1α in NK cells promotes IFN-γ production in tumor-infiltrating lymphocytes. Bar graphs show the percentage of IFN-γ-producing cells among NK cells, CD4+ T cells, and CD8+ T cells in the lungs (upper panel) and spleens (lower panel) of IRE1NK and IRE1f / f littermate control mice on day 20 after intravenous injection of B16F10 melanoma cells. * indicates p<0.05, ** indicates p<0.01. Two-tailed unpaired Student's t-test was performed. All error bars represent the standard error of the mean (sem). n = 4-6 mice per group, and each date was independently repeated three times. [Figure 11C]Figures 11A-11D show that IRE1α-induced NK cell hypertrophy is associated with the presentation of immune cell types beneficial to tumor control, and that IRE1α in NK cells promotes IFN-γ production in tumor-infiltrating lymphocytes. Figure 11A shows the gating strategy. Figure 11B shows the percentages of conventional type 1 dendritic cells (cDC1) (left), CD8+ T cells (center), and CD4+ T cells (right) in the lungs and spleens of IRE1NK and IRE1f / f littermate control mice at day 20 after intravenous injection of B16F10 melanoma cells. Figure 11C shows representative flow cytometry plots and quantification of baseline levels and relative percentages of NK cell numbers and Ki-67 and c-Myc expression in the lungs of naive IRE1NK and IRE1f / f littermate control mice. * indicates p<0.05, and ** indicates p<0.01. A two-tailed, unpaired Student's t-test was performed on Figure 11B. All error bars represent the standard error of the mean (sem). For all experiments, n = 4-5 mice per group, and each date was independently repeated three times. Figure 11D shows that IRE1α in NK cells promotes IFN-γ production in tumor-infiltrating lymphocytes. Bar graphs show the percentage of IFN-γ-producing cells among NK cells, CD4+ T cells, and CD8+ T cells in the lungs (upper panel) and spleens (lower panel) of IRE1NK and IRE1f / f littermate control mice on day 20 after intravenous injection of B16F10 melanoma cells. * indicates p<0.05, ** indicates p<0.01. Two-tailed unpaired Student's t-test was performed. All error bars represent the standard error of the mean (sem). n = 4-6 mice per group, and each date was independently repeated three times. [Figure 11D]Figures 11A-11D show that IRE1α-induced NK cell hypertrophy is associated with the presentation of immune cell types beneficial to tumor control, and that IRE1α in NK cells promotes IFN-γ production in tumor-infiltrating lymphocytes. Figure 11A shows the gating strategy. Figure 11B shows the percentages of conventional type 1 dendritic cells (cDC1) (left), CD8+ T cells (center), and CD4+ T cells (right) in the lungs and spleens of IRE1NK and IRE1f / f littermate control mice at day 20 after intravenous injection of B16F10 melanoma cells. Figure 11C shows representative flow cytometry plots and quantification of baseline levels and relative percentages of NK cell numbers and Ki-67 and c-Myc expression in the lungs of naive IRE1NK and IRE1f / f littermate control mice. * indicates p<0.05, and ** indicates p<0.01. A two-tailed, unpaired Student's t-test was performed on Figure 11B. All error bars represent the standard error of the mean (sem). For all experiments, n = 4-5 mice per group, and each date was independently repeated three times. Figure 11D shows that IRE1α in NK cells promotes IFN-γ production in tumor-infiltrating lymphocytes. Bar graphs show the percentage of IFN-γ-producing cells among NK cells, CD4+ T cells, and CD8+ T cells in the lungs (upper panel) and spleens (lower panel) of IRE1NK and IRE1f / f littermate control mice on day 20 after intravenous injection of B16F10 melanoma cells. * indicates p<0.05, ** indicates p<0.01. Two-tailed unpaired Student's t-test was performed. All error bars represent the standard error of the mean (sem). n = 4-6 mice per group, and each date was independently repeated three times. [Figure 12]Figure 12 shows that IRE1α depletion has minimal impact on RIDD in infected-activated NK cells. Volcano plot of RNA-seq analysis showing all genes in IRE1NK (CD45.2) versus WT Ly49H+ splenic NK cells harvested from IRE1NK (CD45.2) vs. WT (CD45.1) mixed BM chimeric mice at day 1.5 PI. RIDD target genes (So et al. (2012) Cell Metab. 16:487-499) and XBP1 target genes (So et al. (2012) Cell Metab. 16:487-499) are highlighted in red and blue, respectively. [Figure 13] Figure 13 shows representative electron microscopy of IRE1NK versus WT Ly49H+ splenic NK cells harvested from IRE1NK(CD45.2):WT(CD45.1) mixed BM chimeric mice at day 7 PI. The lower high-resolution plot shows mitochondrial morphology. n=3 mixed BM chimeric mice. [Figure 14A]Figures 14A-C show that IRE1 supports NK cell OXPHOS and mitochondrial function. Figure 14A shows the top 10 enriched GSEA signature gene sets in the RNA-seq analysis at day 1.5 PI, as shown in Figure 8A. Figure 14B shows a heatmap of differentially expressed OXPHOS target genes in IRE1NK cells relative to WT NK cells at day 1.5 PI. Genes were clustered by functional annotation. c-Myc-regulated genes (Morrish and Hockenbery (2014) Cold Spring Harb. Perspect. Med. 4:a014225) are indicated in black text on the right. Figure 14C shows an analysis of NK cell oxygen consumption rate (OCR) to assess the rate of OXPHOS and maximal respiration. Primary human NK cells isolated from PBMCs were incubated with IL-12 and IL-18 for 16 hours in the presence or absence of the IRE1α inhibitor 4μ8C (5μM). Approximately 600,000 cells were seeded per well, and data were normalized to total protein quantification. Oligo (oligomycin, 1 μM), FCCP (carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone, 1 μM), R (rotenone, 0.5 μM), and A (antimycin, 0.5 μM). The representative plot in Figure 14C shows data from one PBMC donor along with three technical controls. Quantification in Figure 14B shows combined data from three PBMC donors and two independent experiments. A two-tailed unpaired Student's t-test was performed, and p values ​​are as indicated. [Figure 14B]Figures 14A-C show that IRE1 supports NK cell OXPHOS and mitochondrial function. Figure 14A shows the top 10 enriched GSEA signature gene sets in the RNA-seq analysis at day 1.5 PI, as shown in Figure 8A. Figure 14B shows a heatmap of differentially expressed OXPHOS target genes in IRE1NK cells relative to WT NK cells at day 1.5 PI. Genes were clustered by functional annotation. c-Myc-regulated genes (Morrish and Hockenbery (2014) Cold Spring Harb. Perspect. Med. 4:a014225) are indicated in black text on the right. Figure 14C shows an analysis of NK cell oxygen consumption rate (OCR) to assess the rate of OXPHOS and maximal respiration. Primary human NK cells isolated from PBMCs were incubated with IL-12 and IL-18 for 16 hours in the presence or absence of the IRE1α inhibitor 4μ8C (5μM). Approximately 600,000 cells were seeded per well, and data were normalized to total protein quantification. Oligo (oligomycin, 1 μM), FCCP (carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone, 1 μM), R (rotenone, 0.5 μM), and A (antimycin, 0.5 μM). The representative plot in Figure 14C shows data from one PBMC donor along with three technical controls. Quantification in Figure 14B shows combined data from three PBMC donors and two independent experiments. A two-tailed unpaired Student's t-test was performed, and p values ​​are as indicated. [Figure 14C]Figures 14A-C show that IRE1 supports NK cell OXPHOS and mitochondrial function. Figure 14A shows the top 10 enriched GSEA signature gene sets in the RNA-seq analysis at day 1.5 PI, as shown in Figure 8A. Figure 14B shows a heatmap of differentially expressed OXPHOS target genes in IRE1NK cells relative to WT NK cells at day 1.5 PI. Genes were clustered by functional annotation. c-Myc-regulated genes (Morrish and Hockenbery (2014) Cold Spring Harb. Perspect. Med. 4:a014225) are indicated in black text on the right. Figure 14C shows an analysis of NK cell oxygen consumption rate (OCR) to assess the rate of OXPHOS and maximal respiration. Primary human NK cells isolated from PBMCs were incubated with IL-12 and IL-18 for 16 hours in the presence or absence of the IRE1α inhibitor 4μ8C (5μM). Approximately 600,000 cells were seeded per well, and data were normalized to total protein quantification. Oligo (oligomycin, 1 μM), FCCP (carbonyl cyanide-p-(trifluoromethoxy)phenylhydrazone, 1 μM), R (rotenone, 0.5 μM), and A (antimycin, 0.5 μM). The representative plot in Figure 14C shows data from one PBMC donor along with three technical controls. Quantification in Figure 14B shows combined data from three PBMC donors and two independent experiments. A two-tailed unpaired Student's t-test was performed, and p values ​​are as indicated. [Figure 15A]Figures 15A-E show that restoration of c-Myc in the absence of IRE1 rescues the NK cell proliferation defect. Figure 15A shows real-time PCR analysis of c-Myc mRNA in naive NK cells from MycOE (Mycfsf / +Ncr1Cre+) and littermate control (Mycfsf / +Ncr1Cre-) mice. Figure 15B shows representative flow cytometry plots of CTV dilutions after 3 days of ex vivo culture of NK cells with IL-2 and IL-15, as in Figure 15A, in the presence or absence of the IRE1α inhibitor 4μ8C (5μM). Figures 15C-15E show WT (Mycfsf / +IRE1f / fNcr1Cre-), IRE1NK (Myc+ / +IRE1f / fNcr1Cre+), MycOE (Mycfsf / +IRE1+ / +Ncr1Cre+), and MycOEIRE1NK (Mycfsf / +IRE1f / fNcr1Cre+) NK cells pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15. Figure 15C shows a representative flow cytometry plot and quantification of CTV dilution on day 3, and Figure 15D shows a representative flow cytometry plot of Ki-67 levels on day 3 and quantification of the percentage of proliferating cells (defined as Ki-67+ CTVlo). Figure 15E shows the absolute number of NK cells on day 6. P values ​​are as indicated; ns = not significant. One-sample t-tests are performed for Figure 15A. One-way analysis of variance (ANOVA) with Tukey post-hoc test was performed for Figures 15C-15E. All error bars are sem. In Figure 15A, N = 5 mice / group, in Figure 15B, n = 2 mice / group, and in Figures 15C-15E, n = 4 mice / group. Technical replicates in culture per mouse are shown in Figures 15B-15E. Figure 15A shows cumulative data from two independent experiments. The experiments in Figures 15B-15E were repeated three times independently. [Figure 15B]Figures 15A-E show that restoration of c-Myc in the absence of IRE1 rescues the NK cell proliferation defect. Figure 15A shows real-time PCR analysis of c-Myc mRNA in naive NK cells from MycOE (Mycfsf / +Ncr1Cre+) and littermate control (Mycfsf / +Ncr1Cre-) mice. Figure 15B shows representative flow cytometry plots of CTV dilutions after 3 days of ex vivo culture of NK cells with IL-2 and IL-15, as in Figure 15A, in the presence or absence of the IRE1α inhibitor 4μ8C (5μM). Figures 15C-15E show WT (Mycfsf / +IRE1f / fNcr1Cre-), IRE1NK (Myc+ / +IRE1f / fNcr1Cre+), MycOE (Mycfsf / +IRE1+ / +Ncr1Cre+), and MycOEIRE1NK (Mycfsf / +IRE1f / fNcr1Cre+) NK cells pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15. Figure 15C shows a representative flow cytometry plot and quantification of CTV dilution on day 3, and Figure 15D shows a representative flow cytometry plot of Ki-67 levels on day 3 and quantification of the percentage of proliferating cells (defined as Ki-67+ CTVlo). Figure 15E shows the absolute number of NK cells on day 6. P values ​​are as indicated; ns = not significant. One-sample t-tests are performed for Figure 15A. One-way analysis of variance (ANOVA) with Tukey post-hoc test was performed for Figures 15C-15E. All error bars are sem. In Figure 15A, N = 5 mice / group, in Figure 15B, n = 2 mice / group, and in Figures 15C-15E, n = 4 mice / group. Technical replicates in culture per mouse are shown in Figures 15B-15E. Figure 15A shows cumulative data from two independent experiments. The experiments in Figures 15B-15E were repeated three times independently. [Figure 15C]Figures 15A-E show that restoration of c-Myc in the absence of IRE1 rescues the NK cell proliferation defect. Figure 15A shows real-time PCR analysis of c-Myc mRNA in naive NK cells from MycOE (Mycfsf / +Ncr1Cre+) and littermate control (Mycfsf / +Ncr1Cre-) mice. Figure 15B shows representative flow cytometry plots of CTV dilutions after 3 days of ex vivo culture of NK cells with IL-2 and IL-15, as in Figure 15A, in the presence or absence of the IRE1α inhibitor 4μ8C (5μM). Figures 15C-15E show WT (Mycfsf / +IRE1f / fNcr1Cre-), IRE1NK (Myc+ / +IRE1f / fNcr1Cre+), MycOE (Mycfsf / +IRE1+ / +Ncr1Cre+), and MycOEIRE1NK (Mycfsf / +IRE1f / fNcr1Cre+) NK cells pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15. Figure 15C shows a representative flow cytometry plot and quantification of CTV dilution on day 3, and Figure 15D shows a representative flow cytometry plot of Ki-67 levels on day 3 and quantification of the percentage of proliferating cells (defined as Ki-67+ CTVlo). Figure 15E shows the absolute number of NK cells on day 6. P values ​​are as indicated; ns = not significant. One-sample t-tests are performed for Figure 15A. One-way analysis of variance (ANOVA) with Tukey post-hoc test was performed for Figures 15C-15E. All error bars are sem. In Figure 15A, N = 5 mice / group, in Figure 15B, n = 2 mice / group, and in Figures 15C-15E, n = 4 mice / group. Technical replicates in culture per mouse are shown in Figures 15B-15E. Figure 15A shows cumulative data from two independent experiments. The experiments in Figures 15B-15E were repeated three times independently. [Figure 15D]Figures 15A-E show that restoration of c-Myc in the absence of IRE1 rescues the NK cell proliferation defect. Figure 15A shows real-time PCR analysis of c-Myc mRNA in naive NK cells from MycOE (Mycfsf / +Ncr1Cre+) and littermate control (Mycfsf / +Ncr1Cre-) mice. Figure 15B shows representative flow cytometry plots of CTV dilutions after 3 days of ex vivo culture of NK cells with IL-2 and IL-15, as in Figure 15A, in the presence or absence of the IRE1α inhibitor 4μ8C (5μM). Figures 15C-15E show WT (Mycfsf / +IRE1f / fNcr1Cre-), IRE1NK (Myc+ / +IRE1f / fNcr1Cre+), MycOE (Mycfsf / +IRE1+ / +Ncr1Cre+), and MycOEIRE1NK (Mycfsf / +IRE1f / fNcr1Cre+) NK cells pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15. Figure 15C shows a representative flow cytometry plot and quantification of CTV dilution on day 3, and Figure 15D shows a representative flow cytometry plot of Ki-67 levels on day 3 and quantification of the percentage of proliferating cells (defined as Ki-67+ CTVlo). Figure 15E shows the absolute number of NK cells on day 6. P values ​​are as indicated; ns = not significant. One-sample t-tests are performed for Figure 15A. One-way analysis of variance (ANOVA) with Tukey post-hoc test was performed for Figures 15C-15E. All error bars are sem. In Figure 15A, N = 5 mice / group, in Figure 15B, n = 2 mice / group, and in Figures 15C-15E, n = 4 mice / group. Technical replicates in culture per mouse are shown in Figures 15B-15E. Figure 15A shows cumulative data from two independent experiments. The experiments in Figures 15B-15E were repeated three times independently. [Figure 15E]Figures 15A-E show that restoration of c-Myc in the absence of IRE1 rescues the NK cell proliferation defect. Figure 15A shows real-time PCR analysis of c-Myc mRNA in naive NK cells from MycOE (Mycfsf / +Ncr1Cre+) and littermate control (Mycfsf / +Ncr1Cre-) mice. Figure 15B shows representative flow cytometry plots of CTV dilutions after 3 days of ex vivo culture of NK cells with IL-2 and IL-15, as in Figure 15A, in the presence or absence of the IRE1α inhibitor 4μ8C (5μM). Figures 15C-15E show WT (Mycfsf / +IRE1f / fNcr1Cre-), IRE1NK (Myc+ / +IRE1f / fNcr1Cre+), MycOE (Mycfsf / +IRE1+ / +Ncr1Cre+), and MycOEIRE1NK (Mycfsf / +IRE1f / fNcr1Cre+) NK cells pre-labeled with CTV and cultured ex vivo with IL-2 and IL-15. Figure 15C shows a representative flow cytometry plot and quantification of CTV dilution on day 3, and Figure 15D shows a representative flow cytometry plot of Ki-67 levels on day 3 and quantification of the percentage of proliferating cells (defined as Ki-67+ CTVlo). Figure 15E shows the absolute number of NK cells on day 6. P values ​​are as indicated; ns = not significant. One-sample t-tests are performed for Figure 15A. One-way analysis of variance (ANOVA) with Tukey post-hoc test was performed for Figures 15C-15E. All error bars are sem. In Figure 15A, N = 5 mice / group, in Figure 15B, n = 2 mice / group, and in Figures 15C-15E, n = 4 mice / group. Technical replicates in culture per mouse are shown in Figures 15B-15E. Figure 15A shows cumulative data from two independent experiments. The experiments in Figures 15B-15E were repeated three times independently. [Figure 16A]Figures 16A-G show characterization of NK cell development and maturation in Myc+ / - heterozygous mice (Het, referred to in the text as "MycNK") and Myc- / - (KO) mice. Figure 16A shows quantitative real-time PCR validation of c-Myc expression in Het and KO NK cells before and after ex vivo IL-12 and IL-18 stimulation. Figure 16B shows the results of flow cytometry analysis of the percentage and absolute number of NK cells in the BM and spleen of MycHet mice compared to their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. Figure 16C shows similar results to Figure 16B, except that data for Myc KO animals are shown. Figure 16D shows the results of flow cytometry analysis of NK cell development in the BM and NK cell mutations in the spleen of MycHet mice. For the BM (upper panel), the percentages of NK cells that are NK precursors (NKP, DX5-NK1.1-), immature NK cells (iNK, DX5-NK1.1+), or mature NK cells (mNK, DX5-NK1.1+) are shown. For the spleen (lower panel), the percentages of NK cells that are immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) are shown. Figure 16E shows the results of flow cytometry analysis similar to that shown in Figure 16D, except that data for Myc KO animals are shown. Figure 16F shows representative histogram plots showing the surface expression of cytokine and activating receptors on splenic NK cells from Myc Het mice compared with their Cre littermate controls. Figure 16G shows the same results as Figure 16F, except that data for Myc KO animals are shown. P values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 16A-16E. All error bars are sem. Data are representative of three (Figures 16A-16E) and two (Figures 16F and 16G) independent experiments. N = 3 mice / group for Figures 16A, 16F, and 16G. N = 4-5 mice / group for all experiments in Figures 16B-16E.Data were independently repeated three times (Figures 16B-16E) or twice (Figures 16F and 16G). [Figure 16B]Figures 16A-G show characterization of NK cell development and maturation in Myc+ / - heterozygous mice (Het, referred to in the text as "MycNK") and Myc- / - (KO) mice. Figure 16A shows quantitative real-time PCR validation of c-Myc expression in Het and KO NK cells before and after ex vivo IL-12 and IL-18 stimulation. Figure 16B shows the results of flow cytometry analysis of the percentage and absolute number of NK cells in the BM and spleen of MycHet mice compared to their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. Figure 16C shows similar results to Figure 16B, except that data for Myc KO animals are shown. Figure 16D shows the results of flow cytometry analysis of NK cell development in the BM and NK cell mutations in the spleen of MycHet mice. For the BM (upper panel), the percentages of NK cells that are NK precursors (NKP, DX5-NK1.1-), immature NK cells (iNK, DX5-NK1.1+), or mature NK cells (mNK, DX5-NK1.1+) are shown. For the spleen (lower panel), the percentages of NK cells that are immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) are shown. Figure 16E shows the results of flow cytometry analysis similar to that shown in Figure 16D, except that data for Myc KO animals are shown. Figure 16F shows representative histogram plots showing the surface expression of cytokine and activating receptors on splenic NK cells from Myc Het mice compared with their Cre littermate controls. Figure 16G shows the same results as Figure 16F, except that data for Myc KO animals are shown. P values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 16A-16E. All error bars are sem. Data are representative of three (Figures 16A-16E) and two (Figures 16F and 16G) independent experiments. N = 3 mice / group for Figures 16A, 16F, and 16G. N = 4-5 mice / group for all experiments in Figures 16B-16E.Data were independently repeated three times (Figures 16B-16E) or twice (Figures 16F and 16G). [Figure 16C]Figures 16A-G show characterization of NK cell development and maturation in Myc+ / - heterozygous mice (Het, referred to in the text as "MycNK") and Myc- / - (KO) mice. Figure 16A shows quantitative real-time PCR validation of c-Myc expression in Het and KO NK cells before and after ex vivo IL-12 and IL-18 stimulation. Figure 16B shows the results of flow cytometry analysis of the percentage and absolute number of NK cells in the BM and spleen of MycHet mice compared to their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. Figure 16C shows similar results to Figure 16B, except that data for Myc KO animals are shown. Figure 16D shows the results of flow cytometry analysis of NK cell development in the BM and NK cell mutations in the spleen of MycHet mice. For the BM (upper panel), the percentages of NK cells that are NK precursors (NKP, DX5-NK1.1-), immature NK cells (iNK, DX5-NK1.1+), or mature NK cells (mNK, DX5-NK1.1+) are shown. For the spleen (lower panel), the percentages of NK cells that are immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) are shown. Figure 16E shows the results of flow cytometry analysis similar to that shown in Figure 16D, except that data for Myc KO animals are shown. Figure 16F shows representative histogram plots showing the surface expression of cytokine and activating receptors on splenic NK cells from Myc Het mice compared with their Cre littermate controls. Figure 16G shows the same results as Figure 16F, except that data for Myc KO animals are shown. P values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 16A-16E. All error bars are sem. Data are representative of three (Figures 16A-16E) and two (Figures 16F and 16G) independent experiments. N = 3 mice / group for Figures 16A, 16F, and 16G. N = 4-5 mice / group for all experiments in Figures 16B-16E.Data were independently repeated three times (Figures 16B-16E) or twice (Figures 16F and 16G). [Figure 16D]Figures 16A-G show characterization of NK cell development and maturation in Myc+ / - heterozygous mice (Het, referred to in the text as "MycNK") and Myc- / - (KO) mice. Figure 16A shows quantitative real-time PCR validation of c-Myc expression in Het and KO NK cells before and after ex vivo IL-12 and IL-18 stimulation. Figure 16B shows the results of flow cytometry analysis of the percentage and absolute number of NK cells in the BM and spleen of MycHet mice compared to their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. Figure 16C shows similar results to Figure 16B, except that data for Myc KO animals are shown. Figure 16D shows the results of flow cytometry analysis of NK cell development in the BM and NK cell mutations in the spleen of MycHet mice. For the BM (upper panel), the percentages of NK cells that are NK precursors (NKP, DX5-NK1.1-), immature NK cells (iNK, DX5-NK1.1+), or mature NK cells (mNK, DX5-NK1.1+) are shown. For the spleen (lower panel), the percentages of NK cells that are immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) are shown. Figure 16E shows the results of flow cytometry analysis similar to that shown in Figure 16D, except that data for Myc KO animals are shown. Figure 16F shows representative histogram plots showing the surface expression of cytokine and activating receptors on splenic NK cells from Myc Het mice compared with their Cre littermate controls. Figure 16G shows the same results as Figure 16F, except that data for Myc KO animals are shown. P values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 16A-16E. All error bars are sem. Data are representative of three (Figures 16A-16E) and two (Figures 16F and 16G) independent experiments. N = 3 mice / group for Figures 16A, 16F, and 16G. N = 4-5 mice / group for all experiments in Figures 16B-16E.Data were independently repeated three times (Figures 16B-16E) or twice (Figures 16F and 16G). [Figure 16E]Figures 16A-G show characterization of NK cell development and maturation in Myc+ / - heterozygous mice (Het, referred to in the text as "MycNK") and Myc- / - (KO) mice. Figure 16A shows quantitative real-time PCR validation of c-Myc expression in Het and KO NK cells before and after ex vivo IL-12 and IL-18 stimulation. Figure 16B shows the results of flow cytometry analysis of the percentage and absolute number of NK cells in the BM and spleen of MycHet mice compared to their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. Figure 16C shows similar results to Figure 16B, except that data for Myc KO animals are shown. Figure 16D shows the results of flow cytometry analysis of NK cell development in the BM and NK cell mutations in the spleen of MycHet mice. For the BM (upper panel), the percentages of NK cells that are NK precursors (NKP, DX5-NK1.1-), immature NK cells (iNK, DX5-NK1.1+), or mature NK cells (mNK, DX5-NK1.1+) are shown. For the spleen (lower panel), the percentages of NK cells that are immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) are shown. Figure 16E shows the results of flow cytometry analysis similar to that shown in Figure 16D, except that data for Myc KO animals are shown. Figure 16F shows representative histogram plots showing the surface expression of cytokine and activating receptors on splenic NK cells from Myc Het mice compared with their Cre littermate controls. Figure 16G shows the same results as Figure 16F, except that data for Myc KO animals are shown. P values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 16A-16E. All error bars are sem. Data are representative of three (Figures 16A-16E) and two (Figures 16F and 16G) independent experiments. N = 3 mice / group for Figures 16A, 16F, and 16G. N = 4-5 mice / group for all experiments in Figures 16B-16E.Data were independently repeated three times (Figures 16B-16E) or twice (Figures 16F and 16G). [Figure 16F]Figures 16A-G show characterization of NK cell development and maturation in Myc+ / - heterozygous mice (Het, referred to in the text as "MycNK") and Myc- / - (KO) mice. Figure 16A shows quantitative real-time PCR validation of c-Myc expression in Het and KO NK cells before and after ex vivo IL-12 and IL-18 stimulation. Figure 16B shows the results of flow cytometry analysis of the percentage and absolute number of NK cells in the BM and spleen of MycHet mice compared to their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. Figure 16C shows similar results to Figure 16B, except that data for Myc KO animals are shown. Figure 16D shows the results of flow cytometry analysis of NK cell development in the BM and NK cell mutations in the spleen of MycHet mice. For the BM (upper panel), the percentages of NK cells that are NK precursors (NKP, DX5-NK1.1-), immature NK cells (iNK, DX5-NK1.1+), or mature NK cells (mNK, DX5-NK1.1+) are shown. For the spleen (lower panel), the percentages of NK cells that are immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) are shown. Figure 16E shows the results of flow cytometry analysis similar to that shown in Figure 16D, except that data for Myc KO animals are shown. Figure 16F shows representative histogram plots showing the surface expression of cytokine and activating receptors on splenic NK cells from Myc Het mice compared with their Cre littermate controls. Figure 16G shows the same results as Figure 16F, except that data for Myc KO animals are shown. P values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 16A-16E. All error bars are sem. Data are representative of three (Figures 16A-16E) and two (Figures 16F and 16G) independent experiments. N = 3 mice / group for Figures 16A, 16F, and 16G. N = 4-5 mice / group for all experiments in Figures 16B-16E.Data were independently repeated three times (Figures 16B-16E) or twice (Figures 16F and 16G). [Figure 16G]Figures 16A-G show characterization of NK cell development and maturation in Myc+ / - heterozygous mice (Het, referred to in the text as "MycNK") and Myc- / - (KO) mice. Figure 16A shows quantitative real-time PCR validation of c-Myc expression in Het and KO NK cells before and after ex vivo IL-12 and IL-18 stimulation. Figure 16B shows the results of flow cytometry analysis of the percentage and absolute number of NK cells in the BM and spleen of MycHet mice compared to their Cre littermate controls. BM NK cells were identified by Lin-CD122, and splenic NK cells were identified by Lin-NK1.1+. Figure 16C shows similar results to Figure 16B, except that data for Myc KO animals are shown. Figure 16D shows the results of flow cytometry analysis of NK cell development in the BM and NK cell mutations in the spleen of MycHet mice. For the BM (upper panel), the percentages of NK cells that are NK precursors (NKP, DX5-NK1.1-), immature NK cells (iNK, DX5-NK1.1+), or mature NK cells (mNK, DX5-NK1.1+) are shown. For the spleen (lower panel), the percentages of NK cells that are immature (CD27+CD11b-), mature (CD27-CD11b+), or intermediate (CD27+CD11b+) are shown. Figure 16E shows the results of flow cytometry analysis similar to that shown in Figure 16D, except that data for Myc KO animals are shown. Figure 16F shows representative histogram plots showing the surface expression of cytokine and activating receptors on splenic NK cells from Myc Het mice compared with their Cre littermate controls. Figure 16G shows the same results as Figure 16F, except that data for Myc KO animals are shown. P values ​​are as indicated. Two-tailed unpaired Student's t-tests were performed for Figures 16A-16E. All error bars are sem. Data are representative of three (Figures 16A-16E) and two (Figures 16F and 16G) independent experiments. N = 3 mice / group for Figures 16A, 16F, and 16G. N = 4-5 mice / group for all experiments in Figures 16B-16E.Data were independently repeated three times (Figures 16B-16E) or twice (Figures 16F and 16G). [Figure 17A] Figures 17A-C show that IRE1α is important for IFN-γ production in cytokine-induced memory-like (CIML) human primary NK cells (e.g., cells described in Romee et al. (2012) Blood 120:4751-4760). Figure 17A provides a schematic illustrating the experimental approach. Briefly, NK cells were purified from human PBMCs and preactivated for 16 hours with rhIL-12 + rhIL-18 + rhIL-15, or control conditions (rhIL-15), in the presence or absence of an IRE1 inhibitor (IRE1i) called MKC8866. The cells were then washed three times to remove cytokines and cultured in complete medium supplemented with rhIL-15 to support survival for 7 days. The cells were restimulated with rhIL-12 + rhIL-18 for 6 hours, and the readout was IFN-γ by flow cytometry. Figure 17B shows the gating strategy used in the flow cytometry analysis. FIG. 17C is a heat map showing the percentage of IFN-γ-producing CIML NK cells generated in the presence or absence of the IRE1 inhibitor MKC8866 during preactivation after restimulation. [Figure 17B]Figures 17A-C show that IRE1α is important for IFN-γ production in cytokine-induced memory-like (CIML) human primary NK cells (e.g., cells described in Romee et al. (2012) Blood 120:4751-4760). Figure 17A provides a schematic illustrating the experimental approach. Briefly, NK cells were purified from human PBMCs and preactivated for 16 hours with rhIL-12 + rhIL-18 + rhIL-15, or control conditions (rhIL-15), in the presence or absence of an IRE1 inhibitor (IRE1i) called MKC8866. The cells were then washed three times to remove cytokines and cultured in complete medium supplemented with rhIL-15 to support survival for 7 days. The cells were restimulated with rhIL-12 + rhIL-18 for 6 hours, and the readout was IFN-γ by flow cytometry. Figure 17B shows the gating strategy used in the flow cytometry analysis. FIG. 17C is a heat map showing the percentage of IFN-γ-producing CIML NK cells generated in the presence or absence of the IRE1 inhibitor MKC8866 during preactivation after restimulation. [Figure 17C] Figures 17A-C show that IRE1α is important for IFN-γ production in cytokine-induced memory-like (CIML) human primary NK cells (e.g., cells described in Romee et al. (2012) Blood 120:4751-4760). Figure 17A provides a schematic illustrating the experimental approach. Briefly, NK cells were purified from human PBMCs and preactivated for 16 hours with rhIL-12 + rhIL-18 + rhIL-15, or control conditions (rhIL-15), in the presence or absence of an IRE1 inhibitor (IRE1i) called MKC8866. The cells were then washed three times to remove cytokines and cultured in complete medium supplemented with rhIL-15 to support survival for 7 days. The cells were restimulated with rhIL-12 + rhIL-18 for 6 hours, and the readout was IFN-γ by flow cytometry. Figure 17B shows the gating strategy used in the flow cytometry analysis. FIG. 17C is a heat map showing the percentage of IFN-γ-producing CIML NK cells generated in the presence or absence of the IRE1 inhibitor MKC8866 during preactivation after restimulation.

[0023] Note that for each figure containing a histogram, the left to right bar for each individual measurement corresponds to the box in the figure from top to bottom in the legend for the figure shown. DETAILED DESCRIPTION OF THE INVENTION

[0024] The present invention is based, at least in part, on the discovery that the ER stress sensor inositol-requiring enzyme 1 (IRE1α) and its substrate transcription factor X-box binding protein 1 (XBP1) significantly mediate NK cell-mediated responses to viral infection, homeostatic growth, and tumors in vivo. Using an IRE1-activated reporter mouse line, we demonstrated that activated NK cells upregulate the IRE1α / XBP1 pathway after exposure to pro-inflammatory cytokines in vitro and after viral infection in vivo. Using a newly engineered genetic mouse model, we demonstrated that the IRE1α / XBP1 pathway is a positive-cell-specific regulator of NK cell proliferation and hypertrophy during viral infection or lymphopenia, and of NK cell-mediated antitumor protection. We also showed that IRE1α / XBP1 is induced by the mTOR and Stat4 signaling pathways in activated NK cells. In addition, we found that XBP1 promotes NK cell hypertrophy, in part by directly binding to and activating the c-Myc promoter, upregulating key c-Myc target genes required for NK cell hypertrophy, and regulating mitochondrial respiration. Furthermore, we determined that IRE1α / XBP1 regulates NK cell memory, particularly the generation of cytokine-induced memory-like NK cells (CIML). This study elucidates the role of the IRE1α / XBP1 pathway and the transcription factor c-Myc in NK cell-mediated immunity.

[0025] Accordingly, the present invention relates, in part, to compositions and methods for treating conditions that may benefit from modulating an immune response using compositions, e.g., modified NK cells, or agents that modulate the IRE1α / XBP1 pathway.

[0026] I. Definition The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. As an example, "an element" is used. "element" means one element or more than one element.

[0027] The term "altered amount" or "altered level" refers to an increase or decrease in the copy number (e.g., germline and / or somatic) of a biomarker nucleic acid in a disease sample, e.g., an increase or decrease in expression level, compared to the expression level or copy number of the biomarker nucleic acid in a control sample. The term "altered amount" of a biomarker also includes an increase or decrease in the protein level of a biomarker protein in a sample (e.g., a disease sample) compared to the corresponding protein level of the biomarker protein in a normal control sample. Furthermore, an altered amount of a biomarker protein can be determined by detecting post-translational modifications, such as the methylation status of the marker, which can affect the expression or activity of the biomarker protein.

[0028] The amount of a biomarker in a subject is "significantly" higher or lower than the normal amount of the biomarker if the amount of the biomarker is, respectively, higher or lower than the normal level by an amount that exceeds the standard error of the assay used to assess the amount, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 350%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% higher or lower than that amount. Alternatively, the amount of a biomarker in a subject may be considered "significantly" higher or lower than the normal amount if the amount is at least about 2-fold, preferably at least about 3-fold, 4-fold, or 5-fold higher or lower, respectively, than the normal amount of the biomarker.

[0029] The term "altered expression level" of a biomarker refers to an expression level or copy number of a biomarker in a test sample, e.g., a sample from a patient suffering from a condition that would benefit from modulating an immune response (e.g., cancer or viral infection), that is higher or lower than the standard error of the assay used to assess expression or copy number, preferably at least 2-fold, more preferably 3-fold, 4-fold, 5-fold, or 10-fold or more, of the expression level or copy number of the biomarker in a control sample (e.g., a sample from a healthy subject without the relevant disease), and preferably the average expression level or copy number of the biomarker in several control samples. An altered expression level is higher or lower than the standard error of the assay used to assess expression or copy number, preferably at least 2-fold, more preferably 3-fold, 4-fold, 5-fold, or 10-fold or more, of the expression level or copy number of the biomarker in a control sample (e.g., a sample from a healthy subject without the relevant disease), and preferably the average expression level or copy number of the biomarker in several control samples.

[0030] The term "altered activity" of a biomarker refers to the activity of the biomarker that, for example, increases or decreases a disease state in a disease sample compared to the activity of the biomarker in a normal control sample. Altered activity of a biomarker can be the result of, for example, altered expression of the biomarker, altered protein levels of the biomarker, altered structure of the biomarker, or altered interaction with other proteins involved in the same or different pathways as the biomarker, or altered interaction with transcriptional activators or inhibitors, for example.

[0031] The term "altered structure" of a marker refers to the presence of a mutation or allelic variant in a biomarker nucleic acid or protein, e.g., a mutation that affects the expression or activity of the biomarker nucleic acid or protein compared to a normal or wild-type gene or protein. For example, mutations include, but are not limited to, substitution, deletion, or addition mutations. Mutations can be present in coding or non-coding regions of the biomarker nucleic acid.

[0032] Unless otherwise specified herein, the terms "antibody" and "antibodies" broadly encompass naturally occurring forms of antibodies (e.g., IgG, IgA, IgM, IgE) and recombinant antibodies, such as single-chain antibodies, chimeric and humanized antibodies, and multispecific antibodies, as well as fragments and derivatives of all of the foregoing, which fragments and derivatives contain at least an antigen-binding site. Antibody derivatives may include proteins or chemical moieties conjugated to the antibody.

[0033] The term "antibody" as used herein also includes an "antigen-binding portion" (or simply "antibody portion") of an antibody. The term "antigen-binding portion," as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a biomarker polypeptide or fragment thereof). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL domain, the VH domain, the CL domain, and the CH1 domain; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH domain and the CH1 domain; (iv) a Fv fragment consisting of the VL domain and the VH domain of a single arm of an antibody; and (v) a dAb fragment (Ward et al., (1989) Nature 341:544-546), and (vi) isolated complementarity-determining regions (CDRs). Furthermore, although the two domains, VL and VH, of an Fv fragment are encoded by separate genes, they can be linked by a synthetic linker using recombinant techniques, allowing them to be produced as a single protein chain in which the VL and VH regions pair to form a monovalent polypeptide (known as single-chain Fvs (scFvs), see, e.g., Bird et al. (1988) Science 242:423-426, and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883, and Osbourn et al. 1998, Nature Biotechnology 16:778). Such single-chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody. Any VH and VL sequence of a specific scFv can be linked to human immunoglobulin constant region cDNA or genomic sequences to generate expression vectors encoding complete IgG polypeptides or other isotypes. VH and VL can also be used to generate Fab, Fv, or other fragments of immunoglobulins using either protein chemistry or recombinant DNA technology. Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing them to pair with the complementary domains of another chain and create two antigen-binding sites (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).

[0034] Furthermore, an antibody or antigen-binding portion thereof may be part of a larger immunoadhesive polypeptide formed by covalent or noncovalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesive polypeptides include the use of streptavidin core regions to generate tetrameric scFv polypeptides (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101) and the use of cysteine ​​residues, biomarker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv polypeptides (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab fragments and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies, respectively. Furthermore, antibodies, antibody portions, and immunoadhesive polypeptides can be obtained using standard recombinant DNA techniques as described herein.

[0035] Antibodies can be polyclonal or monoclonal, xenogeneic, allogeneic, or syngeneic, or modified forms thereof (e.g., humanized, chimeric, etc.). Antibodies can also be fully human. Preferably, antibodies of the present invention specifically bind or substantially specifically bind to a biomarker polypeptide or a fragment thereof. As used herein, the terms "monoclonal antibody" and "monoclonal antibody composition" refer to a population of antibody polypeptides that contain only one species of antigen-binding site capable of immunoreacting with a particular epitope of an antigen, while the terms "polyclonal antibody" and "polyclonal antibody composition" refer to a population of antibody polypeptides that contain multiple species of antigen-binding sites capable of interacting with a particular antigen. A monoclonal antibody composition typically exhibits a single binding affinity for the particular antigen with which it immunoreacts.

[0036] An antibody may be "humanized," which is intended to include antibodies made by non-human cells with variable and constant regions modified to more closely resemble antibodies that can be made by human cells. For example, the amino acid sequence of a non-human antibody is modified to incorporate amino acids found in human germline immunoglobulin sequences. The humanized antibodies of the present invention may include, for example, amino acid residues in the CDRs that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term "humanized antibody" also includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0037] A "blocking" antibody or antibody "antagonist" is one that inhibits or reduces at least one biological activity of the antigen(s) to which it binds. In certain embodiments, the blocking or antagonist antibodies or fragments thereof described herein substantially or completely inhibit a given biological activity of the antigen(s).

[0038] The term "antisense" nucleic acid polypeptide includes a nucleotide sequence that is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA polypeptide, complementary to an mRNA sequence, or complementary to the coding strand of a gene. Thus, an antisense nucleic acid polypeptide can hydrogen bond to a sense nucleic acid polypeptide.

[0039] The term "body fluid" refers to fluids that are excreted or secreted by the body, as well as fluids that are not normally excreted or secreted by the body (e.g., amniotic fluid, aqueous humor, bile, blood and plasma, cerebrospinal fluid, dry and wet earwax, Cowper's fluid or pre-ejaculate, chyle, chyme, feces, female ejaculate, interstitial fluid, intracellular fluid, lymph, menses, breast milk, mucus, pleural fluid, pus, saliva, sebum, semen, serum, sweat, synovial fluid, tears, urine, vaginal mucous fluid, vitreous humor, vomit).

[0040] The term "conditions that may benefit from an increased immune response" refers to conditions in which upregulation of an immune response is desirable. Such conditions are well known in the art and include, but are not limited to, disorders that require increased production or function of NK cells, such as fighting cancer, infection (e.g., parasitic, bacterial, helminth, fungal, or viral infection), and the like. The term "conditions that may benefit from a decreased immune response" refers to conditions in which downregulation of an immune response is desirable. Such conditions are also well known in the art and include, but are not limited to, inflammatory diseases, e.g., autoimmune diseases. The term "conditions that may benefit from modulation of the immune response" refers to conditions in which upregulation or downregulation of an immune response is desirable.

[0041] The terms "cancer" or "tumor" or "hyperproliferative" refer to the presence of cells that have properties characteristic of cancer-causing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, and certain distinctive morphological features. Cancer cells are often in the form of tumors, but such cells may exist alone in ...

Claims

[Claim 1] A composition comprising natural killer (NK) cells modified to upregulate the IRE1α-XBP1 pathway.

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