Hyperactive dendritic cells enable sustained Anti-tumor immunity based on adoptive cell transfer

Abstract

To provide a pharmaceutical composition used for cancer immunotherapy.SOLUTION: A pharmaceutical composition for inducing immune responses in a subject, the pharmaceutical composition comprising a population of therapeutic dentritic cells, wherein the therapeutic dentritic cells are obtained by culturing progenitor cells obtained from a cell-donor exvivo in presence of transforming growth factor-beta (TGF - β), stem-cell factor (SCF), fms-like tyrosine kinase 3 ligand (FLT3 - L), or a combination thereof, under conditions effective to induce differentiation; Provided is a pharmaceutical composition produced by a method comprising obtaining live dendritic cells thereby; priming the live dendritic cells exvivo with a TLR ligand; culturing the primed dendritic cells exvivo with a non-canonical inflammasome-activating lipid; and loading the dendritic cells with an immunogen thereby producing a population of therapeutic dendritic cells.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Priority claim This application claims the benefit of U.S. Provisional Application No. 62 / 937,075, filed November 18, 2019, the entire contents of which are incorporated herein by reference.

[0002] Federally funded research or development This invention was made with government support under Grant No. AI116550 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0003] Technical Field This application relates to cancer immunotherapy, for example, stimulation of T cell-mediated anti-tumor therapy. [Background technology]

[0004] Background technology Central to understanding protective immunity against infection and cancer are dendritic cells (DCs), migratory phagocytes that patrol the body's tissues (D. Alvarez, EH et al., Immunity, Vol. 29, No. 3, pp. 325-42, September 2008). DCs survey the environment for threats to the host, most commonly for evidence of infection or tissue damage. This surveillance is achieved through the action of a superfamily of threat assessment receptors, known as pattern recognition receptors (PRRs), which typically recognize microbial products or host-encoded molecules indicative of tissue injury (S.W. Brubaker et al., Annu. Rev. Immunol., Vol. 33, pp. 257-90, 2015; C.A. Janeway and R. Medzhitov, Annu. Rev. Immunol., Vol. 20, pp. 197-216, January 2002). Microbial ligands for PRRs are classified as pathogen-associated molecular patterns (PAMPs), whereas host-derived PRR ligands are damage-associated molecular patterns (DAMPs) (P. Matzinger, Science, Vol. 296, No. 5566, pp. 301-305, April 2002).

[0005] Upon detecting PAMPs, PRRs unleash signaling pathways that fundamentally alter the physiology of DCs that express them (A. Iwasaki and R. Medzhitov, Nat. Immunol., Vol. 16, No. 4, pp. 343-53, April 2015; O. Joffre et al., Immunol. Rev., Vol. 227, No. 1, pp. 234-247, January 2009). For example, before PRR activation, DCs are typically considered non-inflammatory cells. Upon encountering extracellular PAMPs, PRRs stimulate the rapid and robust upregulation of numerous inflammatory mediators, including cytokines, chemokines, and interferons. Concurrent with these gene expressions is the upregulation of factors important for DC migration to draining lymph nodes (dLNs) and T cell activation, such as MHC and costimulatory molecules. Thus, PRR signaling processes shift DC activity from an unstimulated (naive) state to an "activated" state (K. Inaba et al., J. Exp. Med., Vol. 191, No. 6, pp. 927-36, March 2000; I. Mellman and R.M. Steinman, Cell, Vol. 106, No. 3, pp. 255-8, August 2001). Summary of the Invention [Problem to be solved by the invention]

[0006] overview There is a need to diversify current approaches to cancer immunotherapy. The present application is directed to methods for generating populations of therapeutic dendritic cells, methods for inducing an immune response in a subject, methods for treating cancer, and methods for superactivating dendritic cells (DCs) that induce type I T helper (TH1) and cytotoxic T lymphocyte (CTL) responses in the absence of TH2 immunity. The superactivating stimulus promotes T cell responses that protect against tumors that are sensitive or resistant to PD-1 blockade. This protective response is dependent on inflammasomes in DCs and can be elicited using tumor lysate as an immunogen. [Means for solving the problem]

[0007] In certain embodiments, a method for inducing a protective immune response to an immunogen in a subject includes obtaining dendritic cells, exposing the dendritic cells to an effective amount of a non-canonical inflammasome-activating lipid. and administering to the subject live dendritic cells in an amount effective to enhance a protective immune response, thereby inducing a protective immune response. In some embodiments, a therapeutically effective amount of a non-canonical inflammasome-activating lipid superactivates the dendritic cells.

[0008] In certain embodiments, the dendritic cells are optionally cultured ex vivo with an immunogen. In certain embodiments, the dendritic cells are optionally cultured ex vivo with a cytokine.

[0009] In certain embodiments, the non-canonical inflammasome-activating lipid comprises 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC), oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC), oxPAPC species, components thereof, or combinations thereof.

[0010] In certain embodiments, the method further comprises administering a chemotherapeutic agent.

[0011] In certain embodiments, the treatment approaches disclosed herein may also be combined with any of the following therapies: radiation, chemotherapy, surgery, therapeutic antibodies, immunomodulators, proteasome inhibitors, pan-deacetylase (DAC) inhibitors, histone deacetylase (HDAC) inhibitors, checkpoint inhibitors, adoptive cell therapy, including CAR-T cell therapy and NK cell therapy, and vaccines.

[0012] Preferably, the method described herein inhibits the growth or progression of cancer, such as tumor, or viral infection in a subject.For example, the method described herein inhibits tumor growth by at least 1%, for example, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100%. In other cases, the methods described herein reduce the size of the tumor by at least 1 mm in diameter, e.g., at least 2 mm in diameter, at least 3 mm in diameter, at least 4 mm in diameter, at least 5 mm in diameter, at least 6 mm in diameter, at least 7 mm in diameter, at least 8 mm in diameter, at least 9 mm in diameter, at least 10 mm in diameter, at least 11 mm in diameter, at least 12 mm in diameter, at least 13 mm in diameter, at least 14 mm in diameter, at least 15 mm in diameter, at least 20 mm in diameter, at least 25 mm in diameter, at least 30 mm in diameter, at least 40 mm in diameter, at least 50 mm in diameter, or more. In some cases, the subject has had a majority of the tumor resected.

[0013] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention. Other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0014] Other features and advantages of the invention will become apparent from the following detailed description, drawings, and claims. [Brief explanation of the drawings]

[0015] [Figure 1A] Figure 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1A: Release of cytokines, IL-1β and TNFα, was monitored by ELISA. Means and SDs from four mice are shown, and each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1B] This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figures 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h; or BMDCs were primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1B: Cell mortality was measured by LDH release into the cell supernatant. The mean and SD of four mice are shown; each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1C]This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figures 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1C: BMDCs treated with the indicated stimuli as in Figure 1A were stained for CD11c and CD40 using a live-dead violet kit. The mean fluorescence intensity (MFI) of surface CD40 (among live CD11c+ cells) was measured by flow cytometry. The mean and SD of four mice are shown, and each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1D] This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figures 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1D: BMDCs pretreated with the indicated stimuli were transferred onto CD40-coated plates and cultured for 24 h. IL-12p70 cytokine release was measured by ELISA. Mean and SD of four mice are shown; each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1E]Figure 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1E: BMDCs pretreated with the indicated stimuli as in Figure 1A were incubated with OVA protein for 2 h or with FITC-labeled OVA for 45 min. OVA-FITC uptake (left panel) was assessed by flow cytometry. Data are expressed as the percentage of OVA-associated CD11c BMDCs at 37°C and normalized to OVA-associated CD11c BMDCs at 4°C. OVA peptide presentation on MHC-I (right panel) was monitored using a PE-conjugated antibody against H-2Kb bound to the OVA peptide SIINFEKL (SEQ ID NO: 1). Data are expressed as the frequency of SIINFEKL (SEQ ID NO: 1)-associated DCs among live CD11c+ cells. Mean and SD from three replicates are shown, and data are representative of at least three independent experiments. Mean and SD from four mice are shown, and each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1F]This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figures 1A-1F: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 1F: BMDCs treated with the indicated stimuli as in Figure 1A were loaded (or not) with OVA protein or the OVA peptide SIINFEKL for 1 h and then incubated with splenic OT-II naive CD4+ T cells or OT-I naive CD8+ T cells for 4 days. Figure 1F: Supernatants were collected on day 4, and the release of cytokines IFNγ, IL-2, IL-10, TNFα, and IL-13 was measured by ELISA. The mean and SD of four mice are shown, and each panel is representative of two independent experiments. *P<0.05;**P<0.01;***P<0.005. [Figure 1G] This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figure 1G: BMDCs were left untreated (none) or treated with LPC for 24 h, or BMDCs were primed with LPS for 3 h and then treated with PGPC or alum for 21 h. Treated BMDCs were co-cultured with splenic OT-I or OT-II T cells as in Figure 1F. Four days after co-culture, CD4+ and CD8+ T cells were stimulated with PMA and ionomycin for 5 h in the presence of brefeldin A and monensin. The frequency of TH1 cells (TNFα+IFNγ+) and the frequency of TH2 cells (Gata3+IL-4+IL-10+) among CD4+ T cells were measured by intracellular staining. Data are presented as the ratio of TH1 cells to TH2 cells (left panel). The frequency of IFNγ+ among CD8+ T cells is presented in the right panel. The mean and SD from three replicates are shown, and each panel is representative of at least two independent experiments. The mean and SD from four mice are shown, and each panel is representative of two independent experiments. *P<0.05; **P<0.01; ***P<0.005. [Figure 1H] (Figure 1H) is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. C57BL / 6 mice were subcutaneously injected with endofit-OVA protein alone or together with LPS, emulsified in either incomplete Freud's adjuvant (IFA) or alum, as indicated, into the right flank. Alternatively, mice were injected with endofit-OVA protein and LPS plus OxPAPC or PGPC, both emulsified in IFA. Forty days after immunization, CD4+ and CD8+ T cells were isolated from skin-draining lymph nodes (dLNs). T cells were then cultured with naive BMDCs loaded with or without OVA or SIINFEKL peptide for 5 days. IFNγ, IL-10, and IL-13 secretion were measured by ELISA. Means and SDs from four mice are shown, and each panel is representative of two independent experiments. *P<0.05;**P<0.01;***P<0.005. [Figure 2] C57BL / 6 WT mice were subcutaneously inoculated with 5 × 10 live B16OVA cells in the upper left dorsal region. On days 7, 14, and 21 after tumor challenge, mice were subcutaneously injected with 5 × 10 untreated WT BMDCs (DC naive), activated WT BMDCs treated with LPS for 23 h and then pulsed with B16OVA WTL for 1 h (DCLPS), or WT, NLRP3 − / −, or casp1 / 11 − / − BMDCs primed with LPS for 3 h, treated with PGPC for 20 h, and then pulsed with B16OVA WTL for 1 h (DCLPS + PGPC). Survival was monitored daily (n = 5 mice per group). [Figure 3A]Figure 3A and Figure 3B are a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. BMDCs generated with GMCSF were left untreated (none) or treated with MPLA alone, alum alone, OxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with MPLA for 3 h and then treated with the indicated stimuli for 21 h. Figure 5A: Release of cytokines IL-1β and TNFα was monitored by ELISA. The mean and SD of three replicates are shown, and all panels are representative of at least three independent experiments. *P<0.05. [Figure 3B] Figure 3A and Figure 3B are a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. BMDCs generated with GMCSF were left untreated (none) or treated with MPLA alone, alum alone, OxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with MPLA for 3 h and then treated with the indicated stimuli for 21 h. Figure 3B: Cell mortality was measured by LDH release into the cell supernatant. The mean and SD of three replicates are shown, and all panels are representative of at least three independent experiments. *P<0.05. [Figure 3C] Figure 3C is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figure 3C, Figure 3D: Splenic CD11c+ cells were sorted and left untreated (none), or treated with LPS alone, alum alone, or PGPC alone; or DCs were primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 3C: Release of cytokines IL-1β and TNFα was monitored by ELISA. Means and SD of three replicates are shown; all panels are representative of at least three independent experiments. *P<0.05. [Figure 3D]This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figures 3C and 3D: Splenic CD11c+ cells were sorted and left untreated (none), or treated with LPS alone, alum alone, or PGPC alone; or DCs were primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 3D: Cell mortality was measured by LDH release into the cell supernatant. The mean and SD of three replicates are shown; all panels are representative of at least three independent experiments. *P<0.05. [Figure 3E] This is a series of graphs showing that superactivated DCs are excellent antigen-presenting cells and promote TH1-dominated immune responses without evidence of TH2 immunity. Figures 3E-3F: BMDCs generated with GMCSF and treated with the indicated stimuli as in A were stained with anti-CD11c, anti-CD80, anti-CD69, and anti-H2kb antibodies using a live-dead violet kit. Figure 3E: The mean fluorescence intensity (MFI) of surface CD80 (left panel), CD69 (middle panel), and H2kb (right panel) among live CD11c+ cells was measured by flow cytometry. The mean and SD of three replicates are shown, and all panels are representative of at least three independent experiments. *P<0.05. [Figure 4A]Figure 4A-4C: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, OxPAPC alone, or PGPC alone for 24 h. BMDCs were primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 4A: BMDCs were incubated with fixable FITC-labeled OVA at 37°C or 4°C for 45 min. BMDCs were then stained with a live-dead violet kit. Gating strategy for flow cytometry to identify the frequency of OVA-FITC-associated BMDCs at 37°C compared with OVA-associated BMDCs at 4°C. The mean and SD of four mice are shown, and each panel is representative of two independent experiments. ***P<0.005. [Figure 4B] Figure 4A-4C: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, OxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 4B: BMDCs were incubated with endofit-OVA protein for 2 h. Gating strategy for identifying the frequency of SIINFEKL (SEQ ID NO: 1) peptide bound to H2kb on the surface of viable BMDCs, as measured by flow cytometry using a PE-conjugated antibody against H-2kb bound to the OVA peptide SIINFEKL. Each panel represents three replicates from one of three experiments. The mean and SD of four mice are shown, and each panel is representative of two independent experiments. ***P<0.005. [Figure 4C]Figure 4A-4C: WT BMDCs were left untreated (none) or treated with LPS alone, alum alone, OxPAPC alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. Figure 4C: C57BL / 6 mice were injected subcutaneously into the right flank with endofit-OVA protein alone or with LPS, emulsified in either incomplete Freud's adjuvant (IFA) or alum, as indicated. Alternatively, mice were injected with endofit-OVA protein and LPS plus OxPAPC or PGPC, both emulsified in IFA. Forty days after immunization, CD4+ T cells were isolated from skin-draining lymph nodes (dLNs). T cells were then cultured with naive BMDCs loaded with or without OVA for 5 days. IL-4 secretion was measured by ELISA. The mean and SD of four mice are shown, and each panel is representative of two independent experiments. ***P<0.005. [Figure 5] This is a series of plots showing that superactivated DCs are excellent antigen-presenting cells and promote a TH1-dominated immune response without evidence of TH2 immunity. BMDCs were left untreated (none), treated with LPC for 24 h, or primed with LPS for 3 h and then treated with PGPC or alum for 21 h. Treated BMDCs were then cocultured with splenic OT-II T cells at a 1:5 (BMDC:T cell) ratio. Four days after coculture, CD4+ T cells were stimulated with PMA+ionomycin in the presence of brefeldin A and monensin for 5 h. Gating strategy for identifying the frequency of IL-4+IL-10+ TH2 cells among live CD4+ T cells, as measured by intracellular staining. Each panel represents three replicates from one of three experiments. [Figure 6A]A series of graphs and immunostaining experiments demonstrate that cDC1 and cDC2 cells achieve a hyperactivated state in vitro. (Figures 6A-6B) Splenic DCs (left panel) or FLT3-generated DCs (right panel) were sorted as cDC1 (CD11c+CD24+) or cDC2 (CD11c+Sirpa+). DCs were left untreated (none) or treated with LPS alone, alum alone, OxPAPC, or PGPC alone for 24 h. DCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. (Figure 6A) Cell death was measured by LDH release into the supernatant. [Figure 6B] A series of graphs and immunostaining experiments demonstrate that cDC1 and cDC2 cells achieve a hyperactivated state in vitro. (Figures 6A-6B) Splenic DCs (left panel) or FLT3-generated DCs (right panel) were sorted as cDC1 (CD11c+CD24+) or cDC2 (CD11c+Sirpa+). DCs were left untreated (none) or treated with LPS alone, alum alone, OxPAPC, or PGPC alone for 24 h. DCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. (Figure 6B) IL-1β and TNFα cytokine release was monitored by ELISA. Means and SD of three independent experiments are shown. [Figure 6C] A series of graphs and immunostainings showing that cDC1 and cDC2 cells achieve a hyperactivated state in vitro (Figure 6C). FLT3-DCs treated with the indicated stimuli were stained with phalloidin-FITC and DAPI. Images were obtained using a 40x oil immersion lens on a Zeiss confocal microscope. [Figure 6D] A series of graphs and immunostainings showing that cDC1 and cDC2 cells achieve a hyperactivated state in vitro (Figure 6D). FLT3-DCs treated with the indicated stimuli were stained with Live-dead Violet Kit, CCR7 PE, and CD11c-APC. The mean fluorescence intensity (MFI) of CCR7 (gated on live CD11c+ cells) was measured by flow cytometry. [Figure 7] Figure 1 shows a diagram and plots demonstrating that superactivated cDC1s control tumor rejection induced by superactivation-based immunotherapy. WT mice were inoculated subcutaneously with 3 × 10 live B16OVA cells in the dorsum of the mouse. On days 7, 14, and 21 after tumor challenge, mice were left untreated or subcutaneously injected with 1 × 10 cells of untreated WT cDC1s, WT cDC1s treated with LPS for 23 h (active cDC1s), or WT cDC1s primed with LPS for 3 h and then treated with PGPC for 20 h (superactive cDC1s) in the right flank. All DCs were pulsed with tumor lysate for 1 h before injection. Survival was monitored daily (n = 5 mice per group). [Figure 8A] Figures 8A-8B show a series of graphs, plots, and diagrams demonstrating that superactive cDC1s control tumor rejection and enhance tumor infiltration of antitumor-specific T cells. Batf3- / - mice were inoculated subcutaneously into the dorsum of the back with 3 x 10 live B16OVA cells. On days 7, 14, and 21 after tumor challenge, mice were subcutaneously injected into the right flank with 1 x 10 untreated WT cDC1s, or WT cDC1s treated with LPS for 23 h and then pulsed with B16OVA tumor lysate for 1 h (active cDC1s), or WT cDC1s primed with LPS for 3 h, then treated with PGPC for 20 h, and then pulsed with tumor lysate for 1 h (superactive cDC1s). (Figure 8C) Survival was monitored daily (n = 5 mice per group). [Figure 8B]Figure 8A-8B: Batf3- / - mice were subcutaneously inoculated with 3 x 10 live B16OVA cells into the dorsum. On days 7, 14, and 21 after tumor challenge, mice were subcutaneously injected with 1 x 10 cells of untreated WT cDC1s, WT cDC1s treated with LPS for 23 h and then pulsed with B16OVA tumor lysate for 1 h (active cDC1s), or WT cDC1s primed with LPS for 3 h, then treated with PGPC for 20 h, and then pulsed with tumor lysate for 1 h (superactive cDC1s) (Figure 8B). Skin-draining lymph nodes (dLNs), tumors, and spleen tissues were excised from immunized mice 15 days after tumor inoculation. The percentages of antigen-specific CD8+ and CD4+ T cells were measured using SIINFEKL and AAHAEINEA tetramer staining, respectively (n=5 mice per group). [Figure 8C] Figures 8A-8B show that superactive cDC1s control tumor rejection and enhance tumor infiltration of antitumor-specific T cells. Figures 8A-8B: Batf3- / - mice were inoculated subcutaneously into the dorsum of the mouse with 3 x 10 live B16OVA cells. On days 7, 14, and 21 after tumor challenge, the right flank of the mouse was subcutaneously injected with 1 x 10 untreated WT cDC1s, or WT cDC1s treated with LPS for 23 h and then pulsed with B16OVA tumor lysate for 1 h (active cDC1s), or WT cDC1s primed with LPS for 3 h, then treated with PGPC for 20 h, and then pulsed with tumor lysate for 1 h (superactive cDC1s). (Figure 8C) Representative plots of SIINFEKL+CD8+ T cells in the tumor and dLN of treated mice. [Figure 9A]A series of diagrams and plots show that superactive cDC1s regulate tumor rejection in an inflammasome-dependent manner. (Figure 9A) Casp 1 / 11- / - mice and (Figure 9B) NLRP3- / - mice were inoculated subcutaneously into the dorsum of 3.105 live B16OVA cells. On days 7, 14, and 21 after tumor challenge, mice were subcutaneously injected into the right flank with 1.106 cells of either untreated WT cDC1s (naive cDC1s), WT cDC1s treated with LPS for 23 h and then pulsed with B16OVA tumor lysate for 1 h (active cDC1s), or WT or Casp 1 / 11- / - cDC1s primed with LPS for 3 h and then treated with PGPC for 20 h (superactive cDC1s). All DCs were pulsed with tumor lysate for 1 h before injection. Survival was monitored daily in (Figure 9A) Casp1 / 11- / - mice and (Figure 9B) NLRP3 mice (n=5 mice per group). [Figure 9B] A series of diagrams and plots show that superactive cDC1s regulate tumor rejection in an inflammasome-dependent manner. (Figure 9A) Casp 1 / 11- / - mice and (Figure 9B) NLRP3- / - mice were inoculated subcutaneously into the dorsum of 3.105 live B16OVA cells. On days 7, 14, and 21 after tumor challenge, mice were subcutaneously injected into the right flank with 1.106 cells of either untreated WT cDC1s (naive cDC1s), WT cDC1s treated with LPS for 23 h and then pulsed with B16OVA tumor lysate for 1 h (active cDC1s), or WT or Casp 1 / 11- / - cDC1s primed with LPS for 3 h and then treated with PGPC for 20 h (superactive cDC1s). All DCs were pulsed with tumor lysate for 1 h before injection. Survival was monitored daily in (Figure 9A) Casp1 / 11- / - mice and (Figure 9B) NLRP3 mice (n=5 mice per group). [Figure 10A] Mass spectrometry of synthetic lipids: Mass spectrometry of non-oxidized PAPC (FIG. 10A). [Figure 10B] Mass spectrometry of synthetic lipids: Mass spectrometry of oxPAPC (FIG. 10B). [Figure 10C]Mass spectrometry of synthetic lipids: Mass spectrometry of PEIPC-enriched oxPAPC (FIG. 10C). [Figure 10D] Mass spectrometry of synthetic lipids: Mass spectrometry of biotin-labeled oxPAPC (FIG. 10D). [Figure 11A] Oxidized phospholipids induce hyperactive cDC1 and cDC2 cells that exhibit a hypermigratory phenotype. (A) Wild-type, NLRP3- / -, or Casp1 / 11- / - BMDCs generated using FLT3L were left untreated (none) or treated with LPS alone, alum alone, or PGPC alone for 24 h. BMDCs were also primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. IL-1β and TNFα release was monitored by ELISA. Cell mortality was measured by LDH release into the cell supernatant. Means and SD from three replicates are shown; data are representative of at least three independent experiments. [Figure 11B] Oxidized phospholipids induce hyperactive cDC1 and cDC2 cells that exhibit a hypermigratory phenotype. (B) Wild-type BMDCs generated using FLT3L were sorted as cDC1 or cDC2 cells and then treated with the indicated stimuli as in A. IL-1β and TNFα release was monitored by ELISA. Cell mortality was measured by LDH release into the cell supernatant. Mean and SD from three independent experiments performed in two different laboratories. [Figure 12A]Superactivated DCs induce significant CTL responses and durable antitumor immunity dependent on CCR7 expression and inflammasome activation. (A-B) Wild-type BMDCs generated using FLT3L were left untreated (DC naive) or treated with LPS alone (DC activated) for 18 h. Alternatively, BMDCs were primed with LPS for 3 h and then treated with PGPCs (DC superactivated) or alum (DC pyroptotic) for 15 h. Alternatively, BMDCs derived from NLRP3- / - or CCR7- / - mice were primed with LPS for 3 h and then treated with PGPCs for 15 h. 1.10e6 BMDCs were incubated with OVA protein for 1 h before subcutaneous injection into wild-type mice. Injection of BMDCs without OVA protein served as a control. Seven days after BMDC injection, skin-draining lymph nodes were dissected and stained with OVA peptide tetramer antibody, anti-CD45, anti-CD3, anti-CD8a, and anti-CD4 using a live-dead violet kit. (A) The percentage of SIINFEKL+CD8+ T cells (upper panel) and the percentage of live AAHAEINEA+CD4+ T cells were measured by flow cytometry. [Figure 12B]Superactivated DCs induce significant CTL responses and durable antitumor immunity dependent on CCR7 expression and inflammasome activation. (A-B) Wild-type BMDCs generated using FLT3L were left untreated (DC naive) or treated with LPS alone (DC activated) for 18 h. Alternatively, BMDCs were primed with LPS for 3 h and then treated with PGPCs (DC superactivated) or alum (DC pyroptotic) for 15 h. Alternatively, BMDCs derived from NLRP3- / - or CCR7- / - mice were primed with LPS for 3 h and then treated with PGPCs for 15 h. 1.10e6 BMDCs were incubated with OVA protein for 1 h before subcutaneous injection into wild-type mice. Injection of BMDCs without OVA protein served as a control. Seven days after BMDC injection, skin-draining lymph nodes were dissected and stained with OVA peptide tetramer antibody, anti-CD45, anti-CD3, anti-CD8a, and anti-CD4 using a live-dead violet kit. (B) The absolute numbers of SIINFEKL+CD8+ T cells (upper panel) and live AAHAEINEA+CD4+ T cells were measured by flow cytometry using CounterBright beads. [Figure 13A] Superactive stimulation induces significant CTL responses in an inflammasome-dependent manner. (A) C57BL / 6 mice were subcutaneously injected with OVA alone, together with LPS, with PGPC, or with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), into the right flank. Seven or 40 days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (A) Percentages of CD44-low CD62L-low effector T cells (Teff), CD44-high CD62L-low effector memory T cells (TEM), and CD44-high CD62L-high central memory T cells (TCM) are shown among live CD3+CD8+ cells. [Figure 13B](B) Superactive stimulation induces significant CTL responses in an inflammasome-dependent manner. CD8+ T cells were sorted from the dLN 7 days after immunization and then treated with PMA and ionomycin for 5 h or cocultured with B16OVA cells (target cells) at a 1:3 (effector:target) ratio. The percentage of CD107a+ cells among live CD8+ T cells was monitored using flow cytometry to assess CD8+ T cell degranulation. Mean and SD for 5-10 mice are shown. [Figure 13C] (C) Superactive stimulation induces significant CTL responses in an inflammasome-dependent manner. (D) Mice were subcutaneously injected with OVA alone, together with LPS, or together with LPS plus oxPAPC or PGPC, all emulsified in IFA, or with LPS plus alum. Alternatively, NLRP3- / - mice were injected with OVA emulsified in IFA together with LPS plus PGPC. Seven days after immunization, CD8+ T cells were sorted from the cutaneous dLNs of immunized mice and cocultured with OVA-loaded (or unloaded) BMDCs at a ratio of 1:10 (DC:T cells) for 7 days. The percentage of SIINFEKL+IFNγ+ among live CD8+ T cells was measured using OVA peptide tetramer staining followed by intracellular IFNγ staining. [Figure 13D]Superactive stimulation induces significant CTL responses in an inflammasome-dependent manner. (D-E) CD45.1 mice were irradiated and then bone marrow reconstituted with either WT, NLRP3- / -, Casp1 / 11- / -, or CCR7- / - mice (5:1 ratio), all on a CD45.2 C57BL / 6 background. Six weeks after reconstitution, chimeric mice were injected with tamoxifen every other day for 7 days. Chimeric mice were then immunized subcutaneously in the right flank with OVA and LPS plus PGPC emulsified in IFA. Seven days after immunization, CD8+ T cells were isolated from the skin-draining lymph nodes (dLN) or spleen by magnetic enrichment using anti-CD8 beads. (D) The percentages of Teff, TEM, TCM, and naive T cells in the skin dLN were measured by flow cytometry. [Figure 13E] Superactive stimulation induces significant CTL responses in an inflammasome-dependent manner. (D-E) CD45.1 mice were irradiated and then bone marrow reconstituted with either WT, NLRP3- / -, Casp1 / 11- / -, or CCR7- / - mice (5:1 ratio), all on a CD45.2 C57BL / 6 background. Six weeks after reconstitution, chimeric mice were injected with tamoxifen every other day for 7 days. Chimeric mice were then subcutaneously immunized in the right flank with OVA and LPS plus PGPC emulsified in IFA. Seven days after immunization, CD8+ T cells were isolated from the skin-draining lymph nodes (dLNs) or spleens by magnetic enrichment using anti-CD8 beads. (E) The percentage of SIINFEKL+ among live CD8+ T cells in the dLN (left panel) or spleen (right panel) was measured by flow cytometry using OVA peptide tetramer staining. Total CD8+ T cells were sorted from the dLN and cocultured with naive BMDCs (with or without OVA loading) at a ratio of 1:10 (DC:T cells) for 7 days. [Figure 14A]Immunization with superactive stimuli eradicates tumors with immunogenicity ranging from highly immunogenic (hot) to icy tumors. (A) C57BL / 6 mice were inoculated subcutaneously with 5 × 10 live MC38OVA cells in the upper left dorsum. 14 days later, mice were left untreated (naive) or subcutaneously injected in the right flank with syngeneic MC38OVA whole tumor lysate (WTL) plus LPS and PGPC, with or without intravenous (iv) injection of neutralizing anti-IL-1β antibody or intraperitoneal injection of anti-CD4 or anti-CD8a antibodies. Mice received two boost injections with WTL and LPS plus PGPC on days 37 and 55 after tumor inoculation. Tumors were allowed to reach 20 mm in diameter. Survival rates are shown (n = 10 mice per group). [Figure 14B] Immunization with superactive stimuli eradicates tumors with immunogenicity ranging from highly immunogenic (hot) to icy (icy) tumors. (B) C57BL / 6 mice were inoculated subcutaneously with 3 × 10 live B16OVA cells in the upper left dorsum. Ten days later, mice were left untreated (naive) or intraperitoneally injected with anti-PD1 antibody. Alternatively, mice were injected subcutaneously in the right flank with syngeneic B16OVA WTL + LPS and PGPC, with or without intravenous (i.v.) injections of neutralizing anti-IL-1β antibodies or intraperitoneal injections of anti-CD4 or anti-CD8a antibodies. Mice received two boost injections with B16OVA WTL + LPS and PGPC on days 17 and 24 after tumor inoculation. Survival rates are shown (n = 10 mice per group). [Figure 14C]Immunization with superactive stimuli eradicates tumors with immunogenicity ranging from highly immunogenic (hot) to icy (icy) tumors. (C) C57BL / 6 mice were inoculated subcutaneously with 3 × 10 live B16-F10 cells in the upper left dorsal region. Seven days later, mice were left untreated (naive) or injected intraperitoneally with anti-PD1 antibody. Alternatively, mice were immunized subcutaneously in the right flank with syngeneic B16-F10 wild-type T cells plus LPS and PGPC, with or without intravenous (i.v.) injections of neutralizing anti-IL-1β antibodies or intraperitoneal injections of anti-CD4 or anti-CD8a antibodies. Mice received two booster injections on days 14 and 21 after tumor inoculation. Survival rates are shown (n = 10 mice per group). [Figure 14D] Immunization with superactive stimuli eradicates tumors with immunogenicity ranging from hot to icy tumors. (D) BALB / c WT mice were inoculated subcutaneously with 3 × 10 live CT26 cells in the left dorsal region. Seven days later, mice were left untreated (naive) or intraperitoneally injected with anti-PD1 antibody. Alternatively, mice were injected subcutaneously in the right flank with syngeneic CT26 WT plus LPS and PGPC, with or without intravenous (i.v.) injections of neutralizing anti-IL-1β antibodies or intraperitoneal injections of anti-CD4 or anti-CD8a antibodies. Mice received two booster injections on days 14 and 21 after tumor inoculation. Survival rates are shown (n = 10 mice per group). [Figure 15A] Figure 1: Superactive cDC1s can use multiple antigen sources to stimulate T cell-mediated antitumor immunity. (A) Zbtb46DTR mice were subcutaneously injected with B16OVA cells. Mice were injected with diphtheria toxin (DTx) every other day for four consecutive injections, or with PBS. Seven days after tumor injection, all mice were immunized with B16OVA WTL + LPS and PGPC followed by two boost injections. Mouse survival is shown (n = 10 mice per group). [Figure 15B](B) Superactive cDC1s can utilize multiple antigen sources to stimulate T cell-mediated antitumor immunity. (C) CD45.1 mice were irradiated and then reconstituted with mixed BM derived from Zbtb46DTR mice plus either WT, Nlrp3- / -, Casp1 / 11- / -, or Ccr7- / - mice. Six weeks after reconstitution, chimeric mice were subcutaneously injected with B16OVA cells, and all mice received DTx injections three times a week for a total of 12 consecutive injections. Seven days after tumor inoculation, chimeric mice were immunized with B16OVA WTL and LPS+PGPC, followed by two boost injections. Mouse survival rates are shown (n = 5 mice per group). [Figure 15C] Superactive cDC1s can use multiple antigen sources to stimulate T cell-mediated antitumor immunity. (C-D) WT or Batf3- / - mice were subcutaneously injected with B16OVA cells. Seven days after tumor inoculation, mice were either left untreated or immunized with B16OVA WTL and LPS+PGPC followed by two boost injections. (C) Mouse survival rate (n = 10 mice per group). [Figure 15D] Superactive cDC1s can use multiple antigen sources to stimulate T cell-mediated antitumor immunity. (C-D) WT or Batf3- / - mice were subcutaneously injected with B16OVA cells. Seven days after tumor inoculation, mice were either left untreated or WT and Batf3- / - mice were immunized with B16OVA WTL and LPS+PGPC followed by two boost injections. (D) Twenty-one days after tumor inoculation, the percentage of OVA-specific CD8+ and CD4+ T cells was assessed using tetramer staining (n = 5 mice per group). [Figure 15E]Superactivated cDC1s can use multiple antigen sources to stimulate T cell-mediated antitumor immunity. (E-F) Batf3- / - mice were subcutaneously injected with B16OVA cells into the right flank. Seven days after tumor inoculation, mice were either left untreated (no cDC1 injection) or subcutaneously injected with FLT3-derived naive cDC1s, activated cDC1s treated with LPS, or superactivated cDC1s pretreated with LPS and PGPC into the left flank. All cDC1s were loaded with B16OVA WTL 1 hour before injection. (E) Mouse survival rate (n = 5 mice per group). [Figure 15F] Superactivated cDC1s can use multiple antigen sources to stimulate T cell-mediated antitumor immunity. (E–F) Batf3- / - mice were subcutaneously injected with B16OVA cells into the right flank. Seven days after tumor inoculation, mice were either left untreated (no cDC1 injection) or subcutaneously injected with FLT3-derived naive cDC1s, activated cDC1s treated with LPS, or superactivated cDC1s pretreated with LPS and PGPC into the left flank. All cDC1s were loaded with B16OVA WTL 1 h before injection. (F) Twenty-one days after tumor inoculation, OVA-specific CD8+ and CD4+ T cells were assessed using tetramer staining (n = 5 mice per group). [Figure 16A] Oxidized phospholipids induce inflammasome-dependent IL-1β secretion and promote a hypermigratory DC phenotype by cDC1 and cDC2 cells. (A) Wild-type BMDCs generated using FLT3L were left untreated (none) or treated with CpG1806 or PGPC alone for 24 h, or BMDCs were primed with CpG1806 for 3 h and then treated with the indicated stimuli for 21 h. IL-1β and TNFα release was monitored by ELISA. Cell mortality was measured by LDH release into the cell supernatant. Means and SD from three replicates are shown, and data are representative of at least three independent experiments. [Figure 16B](B) Gating strategy for isolating FLT3L-producing BMDC-derived or spleen-derived cDC1 or cDC2 from wild-type mice. The purity after sorting is shown for spleen cDCs or FLT3L DCs. (C) Oxidized phospholipids induce inflammasome-dependent IL-1β secretion by cDC1 and cDC2 cells and promote a hypermigratory DC phenotype. (D) Gating strategy for isolating FLT3L-producing or spleen-derived cDC1 or cDC2 from wild-type mice. The purity after sorting is shown for spleen cDCs or FLT3L DCs. [Figure 16C] Oxidized phospholipids induce inflammasome-dependent IL-1β secretion and promote a hypermigratory DC phenotype by cDC1 and cDC2 cells. (C) Splenic cDC1 or cDC2 cells were left untreated (none) or treated with LPS alone, alum alone, oxPAPC alone, or PGPC alone for 18 h, or BMDCs were primed with LPS for 3 h and then treated with the indicated stimuli for 15 h. IL-1β and TNFα release was monitored by ELISA. Cell mortality was measured by LDH release into the cell supernatant. Mean and SD from three independent experiments performed in two different laboratories. [Figure 17A]Superactivated DCs induce significant CTL responses and durable antitumor immunity dependent on CCR7 expression and inflammasome activation. Wild-type BMDCs generated using FLT3L were left untreated (DC naive) or treated with LPS alone (DC activated) for 18 h. Alternatively, BMDCs were primed with LPS for 3 h before PGPCs (DC superactivated) or alum (DC pyroptotic) were added to the culture medium for 15 h. Alternatively, BMDCs derived from NLRP3- / - or CCR7- / - mice were primed with LPS for 3 h before PGPCs were added to the culture medium for 15 h. BMDCs were washed and then incubated with FITC-labeled OVA for 45 min or non-fluorescent OVA protein for 2 h. (A) OVA peptide presentation on MHC-I was monitored using a PE-conjugated antibody against H-2Kb bound to the OVA peptide SIINFEKL. Data are expressed as the frequency of SIINFEKL-associated DCs among live CD11c+ cells. The mean and SD from three replicates are shown and data are representative of three independent experiments. [Figure 17B](B) Superactivated DCs induce significant CTL responses and durable antitumor immunity dependent on CCR7 expression and inflammasome activation. Wild-type BMDCs generated using FLT3L were left untreated (DC naive) or treated with LPS alone (DC activated) for 18 h. Alternatively, BMDCs were primed with LPS for 3 h before PGPCs (DC superactivated) or alum (DC pyroptotic) were added to the culture medium for 15 h. Alternatively, BMDCs derived from NLRP3- / - or CCR7- / - mice were primed with LPS for 3 h before PGPCs were added to the culture medium for 15 h. BMDCs were washed and then incubated with FITC-labeled OVA for 45 min or non-fluorescent OVA protein for 2 h. (C) Wild-type, NLRP3- / -, or CCR7- / - BMDCs generated using FLT3L were stimulated as in A. BMDCs were washed and then stained with a live-dead violet kit, CD11c, and CD40. The mean fluorescence intensity (MFI) of surface CD40 (among live CD11c+ cells) was measured by flow cytometry. [Figure 17C]Superactivated DCs induce significant CTL responses and durable antitumor immunity dependent on CCR7 expression and inflammasome activation. Wild-type BMDCs generated using FLT3L were left untreated (DC naive) or treated with LPS alone (DC activated) for 18 h. Alternatively, BMDCs were primed with LPS for 3 h and then cultured with PGPCs (DC superactivated) or alum (DC pyroptotic) for 15 h. Alternatively, BMDCs derived from NLRP3- / - or CCR7- / - mice were primed with LPS for 3 h and then cultured with PGPCs for 15 h. BMDCs were washed and then incubated with FITC-labeled OVA for 45 min or with non-fluorescent OVA protein for 2 h. (C) CCR7- / - BMDCs generated using FLT3L were left untreated (none) or treated with LPS alone, alum alone, or PGPC alone for 24 h. Alternatively, BMDCs were primed with LPS for 3 h and then treated with the indicated stimuli for 21 h. IL-1β and TNFα release was monitored by ELISA. Cell mortality was measured by LDH release into the cell supernatant. Means and SD from three replicates are shown, and data are representative of at least three independent experiments. [Figure 18A] Superactivating stimuli enhance the generation of memory T cells and augment antigen-specific IFNγ effector responses in an inflammasome-dependent manner. C57BL / 6 mice were subcutaneously injected with OVA alone, together with LPS, with PGPC, or with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), into the right flank. Seven days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (A) Gating strategy for identifying the percentages of CD44-low CD62L-low effector T cells (Teff), CD44-high CD62L-low effector memory T cells (Tem), and CD44-high CD62L-high central memory T cells (Tcm). Each panel is representative of five mice. *P<0.05; **P<0.01. [Figure 18B] (B) Superactivating stimuli enhance the generation of memory T cells and augment antigen-specific IFNγ effector responses in an inflammasome-dependent manner. C57BL / 6 mice were subcutaneously injected with OVA alone, together with LPS, together with PGPC, or together with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), into the right flank. Seven days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (C) The absolute numbers of Teff or TEM cells among total CD3+ viable cells in skin dLNs per mouse were assessed by flow cytometry. Each panel is representative of five mice. *P<0.05; **P<0.01. [Figure 18C] (C) Superactivating stimulation enhances the generation of memory T cells and antigen-specific IFNγ effector responses in an inflammasome-dependent manner. OVA alone, together with LPS, with PGPC, or with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), was injected subcutaneously into the right flank of C57BL / 6 mice. Seven days after immunization, T cells were isolated from skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (C) Seven days after immunization, CD8+ T cells were sorted from dLNs and then cultured with naive BMDCs loaded with or without serial dilutions of OVA protein starting at 1000 μg / ml. IFNγ cytokine secretion was measured by ELISA. The mean and SD of five mice are shown. Each panel is representative of five mice. *P<0.05; **P<0.01. [Figure 18D](D) Superactivating stimulation enhances the generation of memory T cells and antigen-specific IFNγ effector responses in an inflammasome-dependent manner. C57BL / 6 mice were subcutaneously injected with OVA alone, together with LPS, with PGPC, or with LPS plus oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA), into the right flank. Seven days after immunization, T cells were isolated from the skin-draining lymph nodes (dLNs) by magnetic enrichment using anti-CD8 beads. (E) CD8+ T cells were sorted from the dLNs 7 days after immunization and then treated with PMA plus ionomycin for 5 h or cocultured with B16OVA cells (target cells) at a 1:3 (effector:target) ratio. Gating strategy for determining the percentage of CD107a+ cells among live CD8+ T cells by flow cytometry. Each panel is representative of five mice. *P<0.05; **P<0.01. [Figure 19A] Superactive stimulation enhances the generation of memory T cells and antigen-specific IFNγ effector responses in an inflammasome-dependent manner. (A-B) CD45.1 mice were irradiated and then bone marrow reconstituted with either WT, NLRP3- / -, Casp1 / 11- / -, or CCR7- / - mice (5:1 ratio), all on a CD45.2 C57BL / 6 background. Six weeks after reconstitution, chimeric mice were injected with tamoxifen every other day for 7 days. Chimeric mice were then subcutaneously immunized in the right flank with OVA and LPS plus PGPC emulsified in IFA. Seven days after immunization, CD8+ T cells were isolated from the skin-draining lymph nodes (dLN) or spleen by magnetic enrichment using anti-CD8 beads. (A) The percentages of Teff, TEM, TCM, and naive T cells in the skin dLN were measured by flow cytometry. Each panel is representative of 5 mice. [Figure 19B]Superactive stimulation enhances the generation of memory T cells and antigen-specific IFNγ effector responses in an inflammasome-dependent manner. (A-B) CD45.1 mice were irradiated and then bone marrow reconstituted with either WT or ZBTB46DTR mice (5:1 ratio) on a CD45.2 C57BL / 6 background plus either NLRP3- / -, Casp1 / 11- / -, or CCR7- / - mice. Six weeks after reconstitution, chimeric mice were injected with tamoxifen every other day for 7 days. Chimeric mice were then subcutaneously immunized in the right flank with OVA and LPS plus PGPC emulsified in IFA. Seven days after immunization, CD8+ T cells were isolated from the skin-draining lymph nodes (dLNs) or spleens by magnetic enrichment using anti-CD8 beads. (B) The percentage of SIINFEKL+ among live CD8+ T cells in the dLN (upper panel) or spleen (lower panel) was measured by flow cytometry using OVA peptide tetramer staining. [Figure 20A] (A-B) Mice were injected subcutaneously (sc) into the right flank with PBS (naive), B16OVA cell lysate alone (none), or with LPS, or B16OVA lysate plus LPS and oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). Fifteen days after immunization, mice were challenged subcutaneously with 3 × 10 live B16OVA cells in the upper left back. Tumor-free mice were rechallenged subcutaneously 150 days later with 5 × 10 live B16OVA cells in the back. (A) Tumor growth was monitored every 2 days (upper panel). For survival experiments (lower panel), tumors were allowed to reach a diameter of 20 mm (n = 8–15 mice per group). [Figure 20B](A-B) Mice were injected subcutaneously (sc) into the right flank with PBS (unimmunized), B16OVA cell lysate alone (none), or with LPS, or B16OVA lysate plus LPS and oxPAPC or PGPC, all emulsified in incomplete Freud's adjuvant (IFA). Fifteen days after immunization, mice were challenged subcutaneously with 3 × 10 live B16OVA cells in the upper left back. Tumor-free mice were rechallenged subcutaneously 150 days later with 5 × 10 live B16OVA cells in the back. (B-C) At the end of tumor growth, tumors were harvested and dissociated to obtain single tumor cell suspensions. (B) The percentage of tumor-infiltrating CD3+CD4+ and CD3+CD8+ T cells among enriched live CD45+ cells was assessed by flow cytometry. [Figure 20C] (B-C) Tumors were harvested at the end of tumor growth and dissociated to obtain a single tumor cell suspension. (C) Tumor-infiltrating CD3+ T cells were sorted and then stimulated for 24 h in the presence of anti-CD3 and anti-CD28 Dynabeads. IFNγ release was measured by ELISA (lower panel) (n = 4 mice per group). [Figure 21A] (A-B) The absolute numbers of CD8+ T cells and CD69+CD103+ T-resident memory CD8+ T cells were assessed at the immunization site or tumor injection site in survivor mice and measured by flow cytometry (n=4 mice). [Figure 21B] (A-B) The absolute numbers of CD8+ T cells and CD69+CD103+ T-resident memory CD8+ T cells were assessed at the immunization site or tumor injection site in survivor mice and measured by flow cytometry (n=4 mice). [Figure 21C](C-D) Circulating memory CD8+ T cells (TCM) were isolated from the spleens of survivor mice or age-matched, tumor-naive mice, and resident memory CD8+ T cells (TRM) were isolated from the inguinal adipose tissue. (C) TCM and TRM from survivor mice were co-cultured with B16OVA, B16-F10, or CT26 tumor cells at a 1:5 (tumor cell:T cell) ratio for 5 h. Cell death mediated by cytolytic CD8+ T cells was measured by LDH release into the supernatant. [Figure 21D] (C-D) Circulating memory CD8+ T cells (TCM) were isolated from the spleens of survivor mice or age-matched, unimmunized tumor-bearing mice, and resident memory CD8+ T cells (TRM) were isolated from the cutaneous inguinal adipose tissue. (D) Mice were left untreated (no Tx) or were inoculated intravenously (iv) with 5 x 105 CD8+ TCM cells isolated from survivor mice or age-matched, unimmunized tumor-bearing mice and / or intradermally with 5 x 105 CD8+ TRM cells. Seven days later, all mice were challenged with 3 x 105 live B16OVA cells. Survival was monitored every 2 days. Tumors were allowed to reach 20 mm in diameter (n = 5 mice per group). DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description The innate immune system has traditionally been viewed as operating in an all-or-none fashion, with DCs either acting to initiate inflammatory responses that promote adaptive immunity or not. Thus, the Toll-like receptors (TLRs) expressed by DCs appear to be of central importance in determining the immunogenic potential of DCs. The mammalian immune system is responsible for detecting microorganisms and activating defensive responses that limit infection. Central to this task are dendritic cells, which promote T-cell activation following microbial sensing. It has been proposed that dendritic cells can assess any infectious threat and direct a proportionate response (Blander, JM (2014) Nat Rev Immunol 14, pp. 601-618; Vance, RE et al. (2009) Cell hostµbe 6, pp. 10-21), the mechanism that may cause its immunomodulatory activity is unknown.

[0017] PRRs act directly or indirectly to detect molecules common to a wide range of microorganisms, traditionally referred to as pathogen-associated molecular patterns (PAMPs), including factors such as bacterial lipopolysaccharide (LPS), bacterial flagellin, or viral double-stranded RNA, among others.

[0018] A key characteristic of PRRs as regulators of immunity is their ability to recognize specific microbial products. Therefore, PRR-mediated signaling events should provide a definitive indication of infection. It has been postulated that a "GO" signal is activated by PRRs expressed on DCs that promote inflammation and T cell-mediated immunity. Interestingly, several research groups have recently proposed that DCs are not simply capable of acting in an all-or-none manner (Blander, JM and Sander, LE (2012). Nat Rev Immunol 12, 215-225; Vance, RE et al. (2009) Cell hostµbe 6, 10-21). Rather, DCs are capable of activating any The most commonly considered means by which pathogenicity can be assessed are: This is based on the ability of virulent pathogens to activate a wider variety of PRRs than nonpathogens. However, not all microorganisms share a common set of PRR activators, and not all PRR activators are equally potent. Therefore, the number of PRRs activated during infection is not an ideal assessment of virulence. Furthermore, an increase in the number of PRRs activated during infection generally results in a significant inflammatory response, which may indirectly promote a significant T cell response. Situations previously proposed to enhance DC activation (e.g., by using virulent pathogens as stimuli) are also expected to enhance MΦ activation (Vance, RE et al., (2009) Cell Host Microbiology 6, 10–21). Therefore, it remains unclear whether mechanisms exist for the immune system (i.e., DCs) to specifically assess the threat of infection.

[0019] One possible means by which infection threat can be assessed is by coincidence detection, a well-known process in which independent inputs result in a response that differs from that elicited by any single input. For PRRs, one such input must be a microbial product as an indicator of infection, regardless of pathogenic threat. To assess virulence threat, a second input must be present. Without wishing to be bound by theory, this putative second input is thought to be a molecule produced at the site of tissue injury, since cellular damage is a feature frequently associated with highly pathogenic microorganisms. Candidate molecules that could provide a second stimulus for DCs are a diverse family of molecules called damage-associated molecular patterns (DAMPs), also known as alarmins (Kono, H. and Rock, KL (2008) Nat Rev Immunol 8, 279-289; Pradue, T. and Cooper, EL (2012) Front Immunol 3, 287). DAMPs are found at sites of infectious and non-infectious tissue injury and have been proposed to regulate inflammatory responses, although their mechanism of action remains unclear. One such class of DAMPs is represented by oxidized phospholipids derived from 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC), collectively known as oxPAPC. These lipids are produced at sites of both infectious and non-infectious tissue injury (Berliner, JA and Watson, AD (2005)). Engl J Med 353, pp. 9-11; Imai, Y. et al. (2008) Cell 133, pp. 235-249; Shirey, KA et al. (2013) Nature 497, pp. 498-502) and is found at high levels in the membranes of dying cells (Chang, MK et al. (2004) J Exp Med 200, pp. 1359-1370). oxPAPC is also an active component of oxidized low-density lipoprotein (oxLDL) aggregates that promote inflammation in atherosclerotic tissue (Leitinger, N. (2003) Curr Opin Lipidol 14, pp. 421-430), and local concentrations can be 10-100 μM (Oskolkova, OV et al. (2010) J Immunol 185, pp. 7706-7712). The association between oxPAPC and dying cells raises the possibility that this lipid could be used as a standard indicator of tissue health, and thus, oxPAPC may represent an increased infectious threat in the presence of microbial products.

[0020] Because of these characteristics, activated DCs have a superior ability to stimulate antigen-specific T cell responses, and numerous strategies have been attempted to promote DC activation in order to stimulate protective immunity. These strategies generally involve the use of synthetic or natural microbial products that stimulate PRRs of the Toll-like receptor (TLR) family. A notable example is the monophosphoryl lipid A (MPLA) molecule, an FDA-approved TLR4 ligand that is being used to adjuvant an increasing number of vaccines (J. Paavonen, Lancet, Vol. 374, No. 9686, pp. 301-314, July 2009; M. Kundi, Expert Rev. Vaccines, Vol. 6, No. 2, pp. 133-140, April 2007; A.M. Diierlaurent et al., J. Immunol., Vol. 183, No. 10, pp. 6186-6197). Page, November 2009). Notably, TLRs alone do not upregulate all the molecular signals necessary to promote T cell-mediated immunity. Members of the interleukin-1 (IL-1) family of cytokines are critical regulators of many aspects of T cell differentiation, persistent memory T cell generation, and effector function (S.Z. Ben-Sasson et al., Proc. Natl. Acad. Sci. USA, Vol. 106, No. 17, pp. 7119-24, April 2009; S.Z. Ben-Sasson et al., J. Exp. Med., Vol. 210, No. 3, pp. 491-502, March 2013; A. Jain et al., Nat. Commun., Vol. 9, No. 1, pp. 1-13, 2018). Expression of IL-1β, a well-characterized family member, is highly induced by TLR signaling, but this cytokine is not released from cells via the conventional biosynthetic pathway because it lacks an N-terminal secretory signal. Rather, IL-1β accumulates in an inactive state in the cytosol of DCs activated by TLR ligands (C. Garlanda et al., Immunity, Vol. 39, No. 6, pp. 1003–1018, December 2013). The lack of IL-1β release from activated DCs raises the possibility that TLR signaling alone is not sufficient to maximally stimulate T cell responses and protective immunity.

[0021] The DC activation state is not the only cell fate that DCs can achieve upon PRR signaling. Indeed, different PRRs stimulate different DC fates. One such fate is the inflammatory form of cell death known as pyroptosis. Pyroptosis is a regulated process resulting from the action of inflammasomes, supramolecular assembly centers (SMOCs) that assemble in the cytosol of DCs and other cells (A. Lu et al., Cell, Vol. 156, No. 6, pp. 1193-1206, March 2014; J.C. Kagan et al., Nat. Rev. Immunol., Vol. 14, No. 12, pp. 821-826, December 2014). Inflammasome assembly is typically stimulated in response to the detection of PAMPs or DAMPs in the host cell cytosol; therefore, cytosolic PRRs are responsible for linking cytosolic threat assessment to inflammasome-dependent pyroptosis (KJ Kieser and JC Kagan, Nat. Rev. Immunol., Vol. 17, No. 6, pp. 376–390, May 2017; M. Lamkanfi and V.M. Dixit, Cell, Vol. 157, No. 5, pp. 1013–22, May 2014). The pyroptotic process results in the release of IL-1β and other IL-1 family members from the cell, providing a signal to T cells that TLRs cannot provide. Despite this increased activity in promoting IL-1β release, pyroptotic cells die and therefore lose their ability to participate in the multi-day process required to stimulate and differentiate naive T cells in the dLN (TR Mempel et al., Nature, Vol. 427, No. 6970, pp. 154-159, January 2004).Indeed, stimuli that promote pyroptosis, such as the commonly used vaccine adjuvant alum (SC Isenbarth et al., Nature, Vol. 453, No. 7198, pp. 1122-1126, June 2008; M. Kool et al., J. Immunol., Vol. 181, No. 6, pp. 3755-3759, September 2008), are well understood for their ability to stimulate type 2 immune responses (P. Marrack et al., Nat. Rev. Immunol., Vol. 9, No. 4, pp. 287-293, April 2009), which are inappropriate for the elimination of many microbial infections or cancers.

[0022] Adoptive cell therapy (ACT), including allogeneic and autologous hematopoietic stem cell transplantation (HSCT) and recombinant cell (i.e., CAR T) therapy, is the treatment of choice for many malignant diseases (for a review of HSCT and adoptive cell therapy approaches, see Rager & Porter, Ther Adv Hematol (2011) Vol. 2 (No. 6) pp. 409-428; Roddie & Peggs, Expert Opin. Biol. Ther. (2011) Vol. 11 (No. 4): pp. 473-487; Wang et al., Int. J. Cancer. (2015) Vol. 136, pp. 1751-1768 (See Ji; and Chang, YJ and XJ Huang, Blood Rev, 2013, 27(1):55-62.) Such adoptive cell therapies include, but are not limited to, allogeneic and autologous hematopoietic stem cell transplantation, donor leukocyte (or lymphocyte) infusion (DLI), adoptive transfer of tumor-infiltrating lymphocytes, or adoptive transfer of T cells or NK cells (including engineered cells, i.e., CAR T, CAR NK, gene-edited T cells or NK cells; see Hu et al., Acta Pharmacologica Sinica (2018) 39:167-176; Irving et al., Front Immunol. (2017) 8:267). Beyond the need for donor-derived cells to reconstitute hematopoiesis after radiation and chemotherapy, immune reconstitution from the transplanted cells is important for eliminating residual tumor cells. The effectiveness of ACT as a treatment option for malignancies is influenced by several factors, including the origin, composition, and phenotype (lymphocyte subsets, activation state) of donor cells, the underlying disease, the pre-transplant conditioning regimen and post-transplant immune support (i.e., IL-2 therapy), and the graft-versus-tumor (GVT) effect mediated by donor cells within the graft. Furthermore, these factors must be balanced against transplant-associated mortality, which typically results from the conditioning regimen and / or excessive immune activity of donor cells within the host (i.e., graft-versus-host disease, cytokine release syndrome, etc.).

[0023] This application is based, in part, on the discovery that stimuli that activate dendritic cells (DCs) or promote DC pyroptosis induce mixed T cell responses consisting of type I and type II T helper (Th) cells. In contrast, stimuli that superactivate DCs selectively stimulate TH1 and cytotoxic T lymphocyte (CTL) immune responses, without evidence of TH2-induced immunity. The TH1-biased immunity elicited by superactivated DCs confers the unique ability of TH1 cells to mediate long-term protective antitumor immunity, even when complex antigen sources (e.g., tumor cell lysates) are used. As shown herein, superactivated DCs can be generated ex vivo and used, for example, for adoptive cell therapy.

[0024] Thus, provided herein is a method for generating a population of therapeutic dendritic cells, comprising obtaining live dendritic cells from a cell donor, priming the dendritic cells ex vivo with a TLR ligand, culturing the primed dendritic cells ex vivo with a non-canonical inflammasome-activating lipid, and loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells.

[0025] Also provided herein is a method of inducing an immune response in a subject, comprising obtaining live dendritic cells from a cell donor, priming the dendritic cells ex vivo with a TLR ligand, culturing the primed dendritic cells ex vivo with a non-canonical inflammasome-activating lipid, loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells, and administering the population of therapeutic dendritic cells to a subject, thereby inducing an immune response in the subject.

[0026] Also provided herein are methods of treating cancer, comprising obtaining live dendritic cells from a cell donor, priming the dendritic cells ex vivo with a TLR ligand, culturing the primed dendritic cells ex vivo with a non-canonical inflammasome-activating lipid, and loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells, and administering the population of therapeutic dendritic cells to a subject, thereby treating cancer in the subject.

[0027] dendritic cells The methods disclosed herein include obtaining viable dendritic cells from a cell donor. The dendritic cells obtained from the cell donor can be immature or mature. The dendritic cells can be differentiated in vivo or in vitro.

[0028] In some embodiments, obtaining viable dendritic cells from a cell donor comprises harvesting progenitor cells from the cell donor and exposing the progenitor cells to conditions effective to induce differentiation. and culturing the cells in vivo to obtain dendritic cells from the cell donor. Methods for differentiating precursor cells into dendritic cells in vitro are known in the art; see, for example, Ardavin et al., "Origin and Differentiation of Dendritic Cells," TRENDS in Immunol, 22(12):691-700 (2001).

[0029] In some embodiments, the progenitor cells are lymphoid progenitor cells, in some embodiments, the progenitor cells are myeloid progenitor cells, in some embodiments, the progenitor cells are blood monocytes.

[0030] In some embodiments, the progenitor cells are derived from bone marrow. In some embodiments, the progenitor cells are derived from blood. In some embodiments, the progenitor cells are derived from peripheral blood mononuclear cells. In some embodiments, the progenitor cells are derived from umbilical cord blood.

[0031] In some embodiments, culturing the progenitor cells ex vivo under conditions effective to induce differentiation comprises culturing the progenitor cells in the presence of one or more cytokines.

[0032] In some embodiments, culturing the progenitor cells ex vivo under conditions effective to induce differentiation comprises culturing the progenitor cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), tumor necrosis factor alpha (TNF-α), transforming growth factor beta (TGF-β), interleukin 7 (IL-7), stem cell factor (SCF), fms-like tyrosine kinase 3 ligand (FLT3-L), interleukin 1 (IL-1), or a combination thereof.

[0033] In some embodiments, the progenitor cells are cultured ex vivo for about 1 to about 48 hours. In some embodiments, the progenitor cells are cultured ex vivo for about 6 to about 48 hours, about 12 to about 48 hours, about 18 to about 48 hours, about 24 to about 48 hours, about 30 to about 48 hours, about 36 to about 48 hours, about 42 to about 48 hours, about 1 to about 42 hours, about 6 to about 42 hours, about 12 to about 42 hours, about 18 to about 42 hours, about 24 to about 42 hours, about 30 to about 42 hours, about 36 to about 42 hours, about 1 to about 36 hours, about 6 to about 36 hours, about 12 to about 36 hours. The culture is carried out for about 18 to about 36 hours, about 24 to about 36 hours, about 30 to about 36 hours, about 1 to about 30 hours, about 6 to about 30 hours, about 12 to about 30 hours, about 18 to about 30 hours, about 24 to about 30 hours, about 1 to about 24 hours, about 6 to about 24 hours, about 12 to about 24 hours, about 18 to about 24 hours, about 1 to about 18 hours, about 6 to about 18 hours, about 12 to about 18 hours, about 1 to about 12 hours, about 6 to about 12 hours, or about 1 to about 6 hours.

[0034] In some embodiments, the progenitor cells are blood monocytes. In some embodiments, the blood monocytes are cultured in the presence of GM-CSF and / or IL-4.

[0035] In some embodiments, obtaining dendritic cells from the cell donor comprises harvesting in vivo differentiated dendritic cells from the cell donor. In some embodiments, the in vivo differentiated dendritic cells are immature dendritic cells. In some embodiments, the in vivo differentiated dendritic cells are immature dendritic cells. Dendritic cells that have differentiated in vivo are called mature dendritic cells.

[0036] In some embodiments, the in vivo differentiated dendritic cells are harvested from the spleen of the cell donor. In some embodiments, the in vivo differentiated dendritic cells are harvested from the lymph nodes of the cell donor. In some embodiments, the in vivo differentiated dendritic cells are harvested from the thymus of the cell donor. In some embodiments, the in vivo differentiated dendritic cells are harvested from the blood of the cell donor. In some embodiments, the in vivo differentiated dendritic cells are harvested from the skin of the cell donor.

[0037] In some embodiments, obtaining dendritic cells from a subject comprises freezing progenitor cells and / or in vivo differentiated dendritic cells.

[0038] The methods disclosed herein include priming dendritic cells ex vivo with a TLR ligand. Suitable TLR ligands are described herein. Priming dendritic cells can include incubating precursor cells, differentiating dendritic cells ex vivo, and / or differentiating dendritic cells in vivo in the presence of a TLR ligand.

[0039] In some embodiments, dendritic cells are primed ex vivo with a TLR ligand for about 1 to about 24 hours. In some embodiments, dendritic cells are primed ex vivo with a TLR ligand for about 3 to about 24 hours, about 6 to about 24 hours, about 9 to about 24 hours, about 12 to about 24 hours, about 15 to about 24 hours, about 18 to about 24 hours, about 21 to about 24 hours, about 1 to about 21 hours, about 3 to about 21 hours, about 6 to about 21 hours, about 9 to about 21 hours, about 12 to about 21 hours, about 15 to about 21 hours, about 18 to about 21 hours, about 1 to about 18 hours, about 3 to about 18 hours, about Primed with a TLR ligand ex vivo for 6 to about 18 hours, about 9 to about 18 hours, about 12 to about 18 hours, about 15 to about 18 hours, about 1 to about 15 hours, about 3 to about 15 hours, about 6 to about 15 hours, about 9 to about 15 hours, about 12 to about 15 hours, about 1 to about 12 hours, about 3 to about 12 hours, about 6 to about 12 hours, about 9 to about 12 hours, about 1 to about 9 hours, about 3 to about 9 hours, about 6 to about 9 hours, about 1 to about 6 hours, about 3 to about 6, or about 1 to about 3 hours.

[0040] In embodiments in which progenitor cells are differentiated ex vivo, priming of dendritic cells may occur prior to culturing the progenitor cells ex vivo under conditions effective to induce differentiation. In some embodiments, priming of dendritic cells may occur after culturing the progenitor cells ex vivo under conditions effective to induce differentiation. In some embodiments, priming of dendritic cells may occur simultaneously with culturing the progenitor cells ex vivo under conditions effective to induce differentiation.

[0041] The methods disclosed herein include culturing primed dendritic cells ex vivo with a non-canonical inflammasome-activating lipid. Suitable non-canonical inflammasome-activating lipids are described herein. Culturing primed dendritic cells can include incubating precursor cells, ex vivo differentiated dendritic cells, and / or in vivo differentiated dendritic cells in the presence of the non-canonical inflammasome-activating lipid.

[0042] In some embodiments, the step of culturing primed dendritic cells ex vivo with a non-canonical inflammasome-activating lipid is carried out for about 1 to about 48 hours. In some embodiments, the progenitor cells are cultured for about 6 to about 48 hours, about 12 to about 48 hours, about 18 to about 48 hours, about 24 to about 48 hours, about 30 to about 48 hours, about 36 to about 48 hours, about 42 to about 48 hours, about 1 to about 42 hours, about 6 to about 42 hours, about 12 to about 42 hours, about 18 to about 42 hours, about 24 to about 42 hours, about 30 to about 42 hours, about 36 to about 42 hours, about 1 to about 42 hours. About 36 hours, about 6 to about 36 hours, about 18 to about 36 hours, about 24 to about 36 hours, about 30 to about 36 hours, about 1 to about 30 hours, about 6 to about 30 hours, about 12 to about 30 hours, about 18 to about 30 hours, about 24 to about 30 hours, about 1 to about 24 hours, about 6 to about 24 hours, about 12 to about 24 hours, about 18 to about 24 hours, about 1 to about 18 hours, about 6 to about 18 hours, about 12 to about 18 hours The cells are cultured for about 1 to about 12 hours, about 6 to about 12 hours, or about 1 to about 6 hours.

[0043] In some embodiments, culturing primed dendritic cells ex vivo with a non-canonical inflammasome-activating lipid is performed simultaneously with priming dendritic cells ex vivo with a TLR ligand, hi some embodiments, culturing primed dendritic cells ex vivo with a non-canonical inflammasome-activating lipid is performed after priming dendritic cells ex vivo with a TLR ligand.

[0044] The methods disclosed herein include loading dendritic cells with an immunogen. Suitable immunogens are disclosed herein. Loading dendritic cells with the immunogen can include culturing the dendritic cells with the immunogen.

[0045] In some embodiments, the step of loading dendritic cells with an immunogen can be carried out over a period of about 1 to about 24 hours. In some embodiments, the step of loading dendritic cells with an immunogen can be carried out over a period of about 3 to about 24 hours, about 6 to about 24 hours, about 9 to about 24 hours, about 12 to about 24 hours, about 15 to about 24 hours, about 18 to about 24 hours, about 21 to about 24 hours, about 1 to about 21 hours, about 3 to about 21 hours, about 6 to about 21 hours, about 9 to about 21 hours, about 12 to about 21 hours, about 15 to about 21 hours, about 18 to about 21 hours, about 1 to about 18 hours, or about 3 to about 18 hours. The reaction may be carried out over a period of about 6 to about 18 hours, about 9 to about 18 hours, about 12 to about 18 hours, about 15 to about 18 hours, about 1 to about 15 hours, about 3 to about 15 hours, about 6 to about 15 hours, about 9 to about 15 hours, about 12 to about 15 hours, about 1 to about 12 hours, about 3 to about 12 hours, about 6 to about 12 hours, about 9 to about 12 hours, about 1 to about 9 hours, about 3 to about 9 hours, about 6 to about 9 hours, about 1 to about 6 hours, about 3 to about 6 hours, or about 1 to about 3 hours.

[0046] In some embodiments, loading the dendritic cells with an immunogen occurs simultaneously with culturing the dendritic cells ex vivo with a non-canonical inflammasome-activating lipid, hi some embodiments, loading the dendritic cells with an immunogen occurs after culturing the dendritic cells ex vivo with a non-canonical inflammasome-activating lipid.

[0047] In some embodiments of the methods for generating a population of therapeutic dendritic cells and / or for inducing an adaptive immune response, the dendritic cells and / or progenitor cells are frozen. In some embodiments, the dendritic cells and / or progenitor cells are frozen before loading with an immunogen. In some embodiments, the dendritic cells are frozen after loading with an immunogen.

[0048] Methods for obtaining dendritic cells and loading them with, for example, cancer immunogens are known in the art, e.g., U.S. Patent Application Publication No. 20060134067(A1), U.S. Patent No. 9694059(B2), U.S. Patent No. 9962433(B2), U.S. Patent Application Publication No. 20080254537(A1), U.S. Patent No. 9701942(B2), U.S. Patent Application Publication No. 20060057129(A1), U.S. Patent Application Publication No. 20080254537(A1), U.S. Patent No. 9701942(B2 ... No. 0160263206(A1), U.S. Patent Application Publication No. 20150352200(A1), U.S. Patent Application Publication No. 20070292448(A1), U.S. Patent No. 6,251,665(B1), WO 2003010292(A3), U.S. Patent Application Publication No. 2017036325(A1), U.S. Patent Application Publication No. 20040197903(A1), and U.S. Patent No. 10,731,130(B2).

[0049] TLR ligands As used herein, the term "pattern recognition receptor ligand" refers to a receptor that is a member of the Toll-like receptor (TLR) family, the RIG-I-like receptor (RLR) family, the nucleotide-binding leucine-rich repeat-containing (NLR) family, cGAS, STING, or AIM2. The present invention relates to molecular compounds that activate one or more members of the pattern recognition receptor (ALR) family. Specific examples of pattern recognition receptor ligands include natural or synthetic bacterial lipopolysaccharides (LPS), natural or synthetic bacterial lipoproteins, natural or synthetic DNA or RNA sequences, natural or synthetic cyclic dinucleotides, and natural or synthetic carbohydrates. Cyclic dinucleotides include cyclic GMP-AMP (cGAMP), cyclic diAMP, and cyclic diGMP.

[0050] In some embodiments, the TLR ligand is selected from a TLR1 ligand, a TLR2 ligand, a TLR3 ligand, a TLR4 ligand, a TLR5 ligand, a TLR6 ligand, a TLR7 ligand, a TLR8 ligand, a TLR9 ligand, a TLR10 ligand, a TLR11 ligand, a TLR12 ligand, a TLR13 ligand, and combinations thereof.

[0051] In some embodiments, the TLR ligand is a TLR4 ligand. In some embodiments, the TLR4 ligand is LPS. In some embodiments, the TLR4 ligand is MPLA.

[0052] oxidized phospholipids The term "non-canonical inflammasome-activating lipids," as used herein, refers to lipids that can induce inflammatory responses in cellular caspase-11-dependent inflammasomes. Exemplary "non-canonical inflammasome-activating lipids" include PAPC, oxPAPC, and oxPAPC species (e.g., HOdiA-PC, KOdiA-PC, HOOA-PC, KOOA-PC, POVPC, PGPG), as well as RhodoLPS (LPS-RS, or LPS derived from Rhodobacter sphaeroides).

[0053] As used herein, the term "oxPAPC" or "oxidized PAPC" refers to lipids produced by oxidation of 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (PAPC), resulting in a mixture of oxidized phospholipids containing either fragmented or full-length oxygenated sn-2 residues. Well-characterized oxidative fragmentation species contain 5-carbon sn-2 residues bearing omega-aldehyde or omega-carboxyl groups. Oxidation of arachidonic acid residues also produces phospholipids containing esterified isoprostanes. oxPAPC includes HOdiA-PC, KOdiA-PC, HOOA-PC, and KOOA-PC species, among other oxidation products present in oxPAPC.

[0054] In some embodiments, the non-canonical inflammasome-activating lipid comprises a species of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC).

[0055] oxPAPC species are known and described in the art. See, e.g., Ni et al., "Evaluation of Air Oxidized PAPC: A Multi Laboratory Study by LC-MS / MS," Free Radical Biology and Medicine 144:156-66 (2019); see Table 1.

[0056] In some embodiments, the non-canonical inflammasome-activating lipid is 2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl]2-(trimethylazanyl)-2-hydroxypropyl]-2-hydroxypropyl ... trimethylazaniumyl)ethyl phosphate (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxo-octenoyl)-sn-glycero-3-phosphorylcholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (KOOA-PC), [(2R)-3-hexadecanoyloxy-2-(5-oxopentanoyloxy)propyl]2-(trimethylazaniumyl)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl]2-(trimethylazaniumyl)ethyl phosphate (POVPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-en-1-ylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazaniumyl)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-en-1-ylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazaniumyl)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butanoyloxy]propyl]2-(trimethylazaniumyl)ethyl phosphate (PEIPC), or a combination thereof.

[0057] In some embodiments, the non-canonical inflammasome-activating lipid is [(2R)-2- (4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl]-2-( Contains trimethylazaniumyl)ethyl phosphate (PGPC).

[0058] In some embodiments, the oxPAPC species is an oxPAPC species listed in Table 1 or a combination thereof.

[0059] [Table 1-1]

[0060] Table 1-2

[0061] Table 1-3

[0062] Table 1-4

[0063] Table 1-5

[0064] Table 1-6

[0065] Table 1-7

[0066] Table 1-8

[0067] Table 1-9

[0068] Table 1-10

[0069] Immunogen "Immunogen" and "antigen" are used interchangeably and refer to any compound that is the subject of a cellular or humoral immune response. Non-viable immunogens include, for example, killed immunogens, subunit vaccines, recombinant proteins or peptides, etc. The adjuvants disclosed herein can be used with any suitable immunogen. Exemplary immunogens of interest include those composed of or derived from viruses, mycoplasmas, parasites, protozoans, prions, etc. Thus, immunogens of interest may be derived, without limitation, from human papillomavirus, herpesviruses, such as herpes simplex virus or varicella zoster virus, retroviruses, such as human immunodeficiency virus 1 or 2, hepatitis virus, influenza virus, rhinovirus, respiratory syncytial virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae, bacteria of the genera Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, amoeba, malaria parasites, and / or Trypanosoma cruzi.

[0070] The immunogen of interest is expressed in disease target cells (e.g., neoplastic cells, infected cells) and is expressed at lower levels or not at all in other tissues. Examples of target cells include cells derived from neoplastic diseases, including, but not limited to, sarcoma, lymphoma, leukemia, carcinoma, melanoma, breast cancer, prostate cancer, ovarian cancer, cervical cancer, colon cancer, lung cancer, glioblastoma, and astrocytoma. Alternatively, the target cells may be infected with, for example, a virus, mycoplasma, bacteria, parasite, protozoan, prion, etc. Thus, immunogens of interest may be derived, without limitation, from human papillomavirus (see below), herpesviruses, e.g., herpes simplex virus or varicella zoster virus, retroviruses, e.g., human immunodeficiency virus 1 or 2, hepatitis virus, influenza virus, rhinovirus, respiratory syncytial virus, cytomegalovirus, adenovirus, Mycoplasma pneumoniae, bacteria of the genera Salmonella, Staphylococcus, Streptococcus, Enterococcus, Clostridium, Escherichia, Klebsiella, Vibrio, Mycobacterium, amoeba, malaria parasites, and Trypanosoma cruzi.

[0071] In some embodiments, infection with an infectious agent is associated with the development of cancer. See, e.g., Kuper et al., "Infections as a Major Preventable Cause of Human Cancer," Journal of International Medicine 249(S741):61-74 (2001).

[0072] In addition to tumor antigens and antigens of infectious agents, mutant forms of tumor suppressor gene products, including but not limited to p53, BRCA1, BRCA2, retinoblastoma, and TSG101, or oncogene products such as, for example, without limitation, RAS, WT, MYC, ERK, and TRK, may also provide target antigens for use with the present disclosure. Target antigens may also be autoantigens, such as those associated with cancer or neoplastic disease. In one embodiment, the immunogen is a peptide derived from a heat shock protein (hsp)-peptide complex of a diseased cell, or the hsp-peptide complex itself.

[0073] "Cancer," as used herein, as known in the art, means a disease, condition, trait, genotype, or phenotype characterized by uncontrolled cell growth or replication, and includes, for example, colorectal cancer, as well as leukemias such as acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), and chronic lymphocytic leukemia, AIDS-related cancers such as Kaposi's sarcoma; breast cancer; bone cancers such as osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, giant cell tumor, adamantinoma, and chordoma; brain cancers such as meningioma, glioblastoma, low-grade astrocytoma, oligodendrocytoma, pituitary tumor, schwannoma, and metastatic brain cancer; various lymphomas such as mantle cell lymphoma, leukemia, and lymphomas of the thyroid gland. "carcinoma" means cancers of the head and neck, including lymphoma, non-Hodgkin's lymphoma, adenoma, squamous cell carcinoma, laryngeal cancer, gallbladder and bile duct cancer, retinal cancers such as retinoblastoma, esophageal cancer, gastric cancer, multiple myeloma, ovarian cancer, uterine cancer, thyroid cancer, testicular cancer, endometrial cancer, melanoma, lung cancer, bladder cancer, prostate cancer, lung cancer (including non-small cell lung cancer), pancreatic cancer, sarcoma, Wilms' tumor, cervical cancer, head and neck cancer, skin cancer, nasopharyngeal carcinoma, liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladder adenocarcinoma, parotid gland cancer, endometrial sarcoma, multidrug resistant cancer; and proliferative diseases and conditions, such as tumor angiogenesis-associated neovascularization, macular degeneration (e.g., wet / dry AMD), corneal neovascularization, diabetic retinopathy, neovascular glaucoma, myopic degeneration, and other proliferative diseases and conditions.

[0074] Immunogens, e.g., cancer immunogens, and their use in, e.g., loading dendritic cells, are known and described in the art. See, e.g., Michael J.P. Lawman and Patricia D. Lawman (eds.), "Cancer Vaccines, Methods and Protocols," Methods in Molecular Biol. 1136 (2014); Chiang et al., "Whole Tumor Antigen Vaccines: Where Are We?,” Vaccines (Basel) 3(2): 344-72 (2015); Thumann et al., “Antigen Loading of Dendritic Cells with Whole Tumor Cell Preparations,” J. Immunol. Methods 277: 1-16 (2003); Kamigaki et al., “Immunotherapy of Autologous Tumor Lysate-Loaded Dendritic Cell Vaccines by a See "Closed-Flow Electroporation System for Solid Tumors," Anticancer Res. 33:2971-6 (2013); U.S. Pat. Nos. 3,823,126; 3,960,827; and 4,160,018.

[0075] In some embodiments, the immunogen is a cancer antigen. In some embodiments, the cancer antigen is selected from tumor lysate, apoptotic bodies, peptides, tumor RNA, tumor-derived exosomes, tumor DC fusion, or a combination thereof.

[0076] In some embodiments, the immunogen is a whole tumor lysate.

[0077] In some embodiments, the whole tumor lysate is subjected to irradiation, boiling, and / or freeze-thaw lysis. Prepared from:

[0078] In some embodiments, the immunogen is autologous. In some embodiments, the immunogen is allogeneic.

[0079] In some embodiments of the methods of inducing an immune response in a subject, the immunogen is a tumor lysate from a cell donor.

[0080] Cell donors and subjects The terms "patient" or "individual" or "subject" are used interchangeably herein and refer to a mammalian subject to be treated, with human patients being preferred. In some cases, the methods disclosed herein are used in laboratory animals, veterinary applications, and in the development of animal models for disease, including, but not limited to, rodents, including mice, rats, hamsters, and primates.

[0081] In some embodiments, the cell donor and / or the subject is a mammalian subject. The term "mammal," as used herein, is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals.

[0082] In some embodiments, the cell donor and / or the subject is a human subject.

[0083] In some embodiments of the methods of inducing an immune response in a subject, the cell donor is the subject. In some embodiments, the cell donor is not the subject. In some embodiments, the precursor cells and / or in vivo differentiated dendritic cells are autologous. In some embodiments, the precursor cells and / or in vivo differentiated dendritic cells are allogeneic.

[0084] Administration In the methods disclosed herein, a population of therapeutic dendritic cells is administered to a subject. In some embodiments, a therapeutically effective amount of live dendritic cells is administered to a subject.

[0085] The dose of the therapeutic dendritic cell populations disclosed herein can vary depending on the nature of the immunogen and the context of the dendritic cells, but should be sufficient to enhance the effectiveness of live dendritic cells in eliciting an immunogenic response. For therapeutic or prophylactic treatments, the amount of live dendritic cells administered is typically 1 x 10 cells per dose. 3 pieces, 1×10 4 pieces, 1×10 5 pieces, 1×10 6 pieces, 1×10 7 pieces, 1×10 8 pieces, 1×10 9 pieces, 1×10 10 pcs or 1 x 10 11 The dendritic cells of the present disclosure are generally non-toxic and can generally be administered as live cells in relatively large amounts without causing life-threatening side effects.

[0086] Among the methods are general-purpose methods, which in some embodiments involve isolating, preparing, treating, culturing cells from a subject, and reintroducing them into the same patient, before or after cryopreservation, as described herein.

[0087] Administration of the populations of therapeutic dendritic cells disclosed herein is by any suitable means that results in a concentration of cells that is effective in ameliorating, reducing, or stabilizing cancer. The populations of therapeutic dendritic cells may be provided in a dosage form suitable for parenteral (e.g., subcutaneous, intravenous, intramuscular, intravesicular, intratumoral, or intraperitoneal) administration routes.

[0088] Dosages for humans were initially determined based on the data used in mice or non-human primates, as described herein. The dose is determined by extrapolation based on the amount of the disclosed therapeutic dendritic cell population, as those skilled in the art will recognize that it is routine in the art to modify doses for humans compared to animal models. For example, the dose may be about 1 x 10 cells per administration. 3 pieces, 1×10 4 pieces, 1×10 5 pieces, 1×10 5pieces, 1×10 6 pieces, 1×10 7 pieces, 1×10 8 pieces, 1×10 9 From about 1 x 10 11 The number of ions may vary between one or more.

[0089] An "appropriate dose level" refers to a dose level that provides a reasonable therapeutic balance between beneficial effects and adverse effects (e.g., sufficient immunostimulatory activity provided by the administered dendritic cells disclosed herein, with a sufficiently low level of macrophage stimulation). For example, this dose level can refer to the peak or mean serum level in a subject of anti-immunogen antibodies produced after administration of an immunogenic composition (including the dendritic cells described herein) at a particular dose level.

[0090] As defined herein, a "therapeutically effective" amount (i.e., an effective dose) of a compound or agent means an amount sufficient to produce a therapeutically (e.g., clinically) desired result. The composition may be administered one or more times per day, including once every other day, up to one or more times per week. One of skill in the art will appreciate that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the subject's general health and / or age, and other current illnesses, may influence the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of live dendritic cells disclosed herein can include a single treatment or a series of treatments.

[0091] Therapeutic dendritic cell populations disclosed herein are administered parenterally by injection, infusion, or implantation (subcutaneous, intravenous, intramuscular, intratumoral, intravesicular, intraperitoneal) of a formulation, or via a suitable delivery device or implant containing a conventional, non-toxic, pharmaceutically acceptable carrier. The formulation and preparation of such carriers are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

[0092] As used herein, a "pharmaceutically acceptable" ingredient / carrier, etc. is one that is suitable for use in humans and / or animals without undue adverse side effects (e.g., toxicity, irritation, or allergic response) and for a reasonable benefit / risk ratio.

[0093] Provided herein are methods for treating cancer or symptoms thereof, comprising administering a population of therapeutic dendritic cells. Accordingly, one embodiment is a method of treating a subject suffering from or susceptible to cancer. The method may comprise administering to the subject a therapeutic amount of a population of therapeutic dendritic cells disclosed herein, under conditions that treat the disease or disorder, and in a dose sufficient to treat the disease or disorder, or symptoms thereof.

[0094] As used herein, "effective amount" means an amount that provides a therapeutic or prophylactic benefit.

[0095] "Treating" a disease, as that term is used herein, means reducing the frequency or severity of at least one sign or symptom of a disease or disorder, e.g., cancer, experienced by a subject.

[0096] "Treatment" is an intervention made to prevent the occurrence of a disorder or to alter the pathology or symptoms of a disorder. Thus, "treatment" relates to both therapeutic treatment and prophylactic or preventative measures. "Treatment" may also be designated as palliative care. Those in need of treatment include those already with the disorder as well as those in whom the disorder is to be prevented. Thus, "treating" or "treatment" of a condition, disorder, or condition includes (1) preventing or delaying the appearance of clinical symptoms of the condition, disorder, or condition occurring in a human or other mammal that may be afflicted with or prone to the condition, disorder, or condition but that has not yet experienced or displayed clinical or subclinical symptoms of the condition, disorder, or condition; (2) inhibiting the condition, disorder, or condition, i.e., arresting, alleviating, or delaying the onset of the disease or its recurrence (if treatment is maintained) or at least one clinical or subclinical symptom thereof; or (3) palliating the disease, i.e., causing regression of at least one of the condition, disorder, or condition, or its clinical or subclinical symptoms. The benefit to the individual to be treated will be statistically significant or at least perceptible to the patient or physician.

[0097] Thus, in the case of cancer, "treatment" can include: (1) reducing the size and / or number of tumors; (2) reducing the number of circulating tumor cells; (3) reducing the risk of metastasis; and (4) reducing the risk of cancer development and / or recurrence.

[0098] For example, "modulation" of a symptom, molecular level, biological activity, etc., refers to, for example, a detectably increased or decreased symptom or activity, etc. Such an increase or decrease can be observed in a subject treated with superactivated DCs compared to a subject not treated with the superactivated DCs, provided that the untreated subject (e.g., a subject administered the immunogen in the absence of an adjuvant lipid) has the same or a similar disease or infection as the treated subject, or is prone to develop the same or a similar disease or infection as the treated subject. Such an increase or decrease can be at least about 2%, 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 100%, 150%, 200%, 250%, 300%, 400%, 500%, 1000%, or more, or within any range between any two of these values. Modulation can be ascertained subjectively or objectively, e.g., by subject self-assessment, by clinician evaluation, or by performance of a suitable assay or measurement, including, e.g., assessing the degree and / or quality of immune stimulation in a subject achieved by administration of the dendritic cells disclosed herein. Modulation can be transient, long-term, or permanent, and can vary in time during or after administration of the dendritic cells disclosed herein to a subject, or during or after use of the dendritic cells disclosed herein in an assay or other method described herein or in the cited references, e.g., within the time periods described below, or about 12 to 24 or 48 hours after administration or use of an adjuvant lipid disclosed herein, about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 21, 28 days, or 1, 3, 6, 9, or more months after the subject has received such immune stimulatory composition / treatment.

[0099] The present disclosure includes methods of inducing an immune response. In some embodiments, the immune response is an adaptive immune response.

[0100] In some embodiments, the immune response is a therapeutic immune response. The term "therapeutic immune response" as used herein refers to an increase in humoral and / or cellular immunity against a target antigen, as measured by standard techniques. Preferably, the level of immunity induced against the target antigen is at least four times, preferably at least five times, the level before administration of the immunogen. Furthermore, the immune response can also be measured qualitatively. In this case, the inhibition of progression or remission of neoplastic disease or infectious disease in a subject using an appropriate in vitro or in vivo assay is considered to indicate the induction of a therapeutic immune response.

[0101] The methods herein may include administering to a subject (including subjects identified as in need of such treatment) an effective amount of a population of therapeutic dendritic cells disclosed herein that produces such an effect. Identification of a subject in need of such treatment may be the judgment of the subject or a medical professional, and may be subjective (e.g., opinion) or objective (e.g., measurable by a testing or diagnostic method).

[0102] Therapeutic methods (including prophylactic treatments) disclosed herein generally involve administration of a therapeutically effective amount of a population of therapeutic dendritic cells disclosed herein to a subject (e.g., animal, human), including a mammal, particularly a human, in need thereof. Such treatments would be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for cancer or a condition thereof. Determination of such an "at risk" subject can be made by any objective or subjective determination, such as by diagnostic testing or the opinion of the subject or health care provider (e.g., genetic testing, enzyme or protein markers, markers (described herein), family history, etc.).

[0103] The present disclosure also provides methods for monitoring the course of treatment. The methods may include identifying the level of a diagnostic marker (e.g., any target, protein, or indicator thereof described herein that is modulated by a compound herein), or a diagnostic measurement (e.g., a screen, assay) in a subject suffering from or susceptible to a cancer-related disorder or symptom thereof, wherein the subject is receiving a therapeutic amount of a compound herein sufficient to treat the disorder or symptom thereof. The level of the marker identified by this method can be compared to known levels of the marker in healthy controls or other affected patients to establish the subject's condition. In some cases, a second level of the marker in the subject is determined at a later time point than the first level, and the two levels are compared to monitor the course of disease or the effectiveness of therapy. In certain embodiments, a pre-treatment level of the marker in the subject is determined before the initiation of a treatment disclosed herein. This pre-treatment level of the marker may then be compared to the level of the marker in the subject after the initiation of treatment to determine the effectiveness of the treatment.

[0104] In some embodiments, the populations of therapeutic dendritic cells disclosed herein are administered as part of a pharmaceutical composition.

[0105] In some embodiments, the pharmaceutical composition is administered systemically, e.g., formulated in a pharmaceutically acceptable buffer, e.g., saline. Preferred routes of administration include, for example, intravesical instillation, subcutaneous injection, intravenous injection, intraperitoneal injection, intramuscular injection, intratumoral injection, or intradermal injection, which provide continuous, sustained, or effective levels of the composition in the patient. Human patients or other animals are treated using a therapeutically effective amount of the therapeutic agent identified herein in a physiologically acceptable carrier. Suitable carriers and their formulations are described, for example, in Remington's Pharmaceutical Sciences by E.W. Martin. The amount of therapeutic agent to be administered will vary depending on the method of administration, the age and weight of the patient, and the clinical symptoms of the cancer. Generally, the amount will be in the range of amounts used for other agents used in the treatment of other diseases associated with cancer, although in certain cases, smaller amounts may be required due to the increased specificity of the compound. Live dendritic cells are administered at a dose that enhances the subject's immune response or reduces the proliferation, survival, or invasiveness of neoplastic or infected cells, as determined by methods known to those skilled in the art.

[0106] The compositions comprising the populations of therapeutic dendritic cells disclosed herein can be administered cutaneously, subcutaneously, intravenously, intramuscularly, parenterally, pulmonary, vaginally, rectally, nasally, or topically. The compositions may also be delivered by injection, orally, by spray, or by particle bombardment.

[0107] Pharmaceutical compositions of the therapeutic dendritic cell populations disclosed herein can be included in a kit, container, pack, or dispenser together with instructions for administration.

[0108] Combination therapy As used herein, the term "in combination" refers to the administration of a therapy to a subject, and refers to the use of two or more therapies for therapeutic benefit. The administration term "in combination" can also refer to the prophylactic use of a therapy to a subject when used with at least one additional therapy. The use of the term "in combination" does not limit the order in which the therapies (e.g., a first and a second therapy) are administered to a subject. Therapy can be administered to the subject with cancer, with cancer, or susceptible to cancer before (for example, 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), at the same time, or after (for example, 1 minute, 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after).The therapy is administered to the subject in order and within a period that allows the therapy to act together. In certain embodiments, the therapies are administered to a subject in a sequence and within a time period that results in increased efficacy than if the therapies were administered otherwise. Any additional therapy may be administered in any order and with other additional therapies.

[0109] As used herein, the term "cancer therapy" refers to a therapy useful for treating cancer. Examples of anti-cancer therapeutic agents include, for example, surgical procedures, chemotherapeutic agents, immunotherapy, growth inhibitors, cytotoxic agents, agents used in radiotherapy, anti-angiogenic agents, apoptotic agents, anti-tubulin agents, and other agents for treating cancer, such as anti-HER-2 antibodies (e.g., HERCEPTIN™), anti-CD20 antibodies, epidermal growth factor receptor (EGFR) antagonists (e.g., tyrosine kinase inhibitors), HER1 / EGFR inhibitors (e.g., erlotinib (TARCEVA™)), These include, but are not limited to, platelet-derived growth factor inhibitors (e.g., GLEEVEC™ (imatinib mesylate)), COX-2 inhibitors (e.g., celecoxib), interferons, cytokines, antagonists (e.g., neutralizing antibodies) that bind to one or more of the following receptors: ErbB2, ErbB3, ErbB4, PDGFRβ, BlyS, APRIL, BCMA, or VEGF, TRAIL / Apo2, and other bioactive and organic chemical agents. Combinations thereof are also contemplated for use in the methods described herein.

[0110] Some embodiments of the method of inducing an immune response in a subject include administering to the subject an anti-cancer agent. In some embodiments, the anti-cancer agent is a chemotherapeutic agent. In some embodiments, the anti-cancer agent is an immune checkpoint modulator.

[0111] Anti-cancer agents: In certain embodiments, the method further comprises administering an anti-cancer agent. In some embodiments, the anti-cancer agent is a chemotherapeutic agent, a growth inhibitory agent, a chimeric antigen receptor-expressing T cell, an antibody or antigen-binding fragment thereof, an antibody-drug conjugate, an angiogenesis inhibitor, and combinations thereof.

[0112] In some embodiments, the anticancer agent is a chemotherapeutic agent or growth inhibitor.For example, the chemotherapeutic agent or growth inhibitor can include alkylating agents, anthracyclines, antihormones, aromatase inhibitors, antiandrogens, protein kinase inhibitors, lipid kinase inhibitors, antisense oligonucleotides, ribozymes, antimetabolites, topoisomerase inhibitors, cytotoxic agents, antitumor antibiotics, proteasome inhibitors, microtubule inhibitors, EGFR antagonists, retinoids, tyrosine kinase inhibitors, histone deacetylase inhibitors, and combinations thereof.

[0113] A "chemotherapeutic agent" is a compound useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA™, Genentech / OSI Pharm.), bortezomib (VELCADE™, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX™, AstraZeneca), sunitinib (SUTENT™, Pfizer / Sugen), letrozole (FEMARA™, Novartis), imatinib mesylate (GLEEVEC™, Novartis), and rifacilin. rtis), finasunate (VATALANIB™, Novartis), oxaliplatin (ELOXATIN™, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (sirolimus, RAPAMUNE™, Wyeth), lapatinib (TYKERB™, GSK572016, GlaxoSmithKline), lonafamib (SCH66336), sorafenib (NEXAVAR™, Bayer Labs.), gefitinib (IRESSA™, AstraZeneca), AG1478, alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethyleneimines and methylameramines, including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylmelamine; acetogenins (especially bullatacin and bullatacinone); camptothecins (including topotecan and irinotecan); bryostatin; kallistatin; CC-1065 (including its adozelesin, carzelesin, and beizelesin synthetic analogs) cryptophycins (especially cryptophycin 1 and cryptophycin 8); corticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-deductases including finasteride and dutasteride; vorinostat, romidepsin, panobinostat, valproic acid, mocetinostat, dolastatins; aldesleukin, talc, duocarmycins (including synthetic analogs, KW-2189 and CBI-TM1); eleutherobin; pancratistatin; sarcodictin; spongistatins; antibiotics such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin γ1I and calicheamicin ω1I (Angew Chem. Intl. Ed. Engl.1994, 33:183-186); dynemicins, including dynemicin A; bisphosphonates, such as clodronate; esperamicin; as well as neocarzinostatin chromophores and related chromoprotein enediyne antibiotic chromophores, aclacinomycin, actinomycin, anthramycin, azaserine, bleomycin, cactinomycin, carabimycin, caminomycin, carzinov leucine, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN™ (doxorubicin) (morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolinodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycofecin, Nolic acids, nogalamycin, olivomycin, peplomycin, porfiromycin, puromycin, chelamycin, rhodrubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purines analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; antiadrenal agents, such as a. Minoglutethimide, mitotane, trilostane; folic acid replenishers such as furoic acid; aceglatone; aldophosphamide glycosides; aminolevulinic acid; eniluracil; amsacrine; bestravcil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; epothilone; etoglucide; gallium nitrate; hydroxyurea; lentinan; lonidynin; maytansinoids, such as maytansine and ansamitocin; mitoguazone; mitoxantrone; mopidamol; nitraelin; pentostatin; phenamet; pirarubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK™ polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; schizofuran; spirogermanium; tenuazonic acid; triazicone; 2,2',2''-trichlorotriethylamine; trichothecenes (especially T-2 toxin, veracrine A, roridin A, and anguidine); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, such as TAXOL (paclitaxel; Bristol-Myers Squibb Oncology, Princeton, NJ), ABRAXANE™ (Cremophor-free), an albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), and TAXOTERE™ (docetaxel; Sanofi-Aventis); chlorambucil; GEMZAR™ (gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE™ (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA™); ibandronate; CPT-11; the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0114] In some embodiments, chemotherapeutic agents may include alkylating agents (including monofunctional and bifunctional alkylating agents), such as thiotepa, CYTOXAN™, cyclophosphamide, nitrogen mustards, such as chlorambucil, chlomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembitine, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; temozolomide; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0115] In some embodiments, chemotherapeutic agents may include anthracyclines such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone, valrubicin, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0116] In some embodiments, chemotherapeutic agents may include antihormonal agents (e.g., antiestrogens and selective estrogen receptor modulators (SERMs), including tamoxifen (NOLVADEX™; including tamoxifen citrate), raloxifene, droloxifene, iodoxifene, 4-hydroxytamoxifen, trioxifene, ketoxifene, LY117018, onapristone, and FARESTON™ (toremifene citrate)); and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0117] In some embodiments, chemotherapeutic agents may include aromatase inhibitors, which inhibit the aromatase enzyme that controls estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, MEGASE™ (megestrol acetate), AROMASIN™ (exemestane; Pfizer), formestany, fadrozole, RIVISOR™ (vorozole), FEMARA™ (letrozole; Novartis), and ARIMIDEX™ (anastrozole; AstraZeneca), as well as pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0118] In some embodiments, chemotherapeutic agents may include antiandrogens, such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans-retinoic acid, fenretinide; and troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0119] In some embodiments, the chemotherapeutic agent may include a protein kinase inhibitor, a lipid kinase inhibitor, or an antisense oligonucleotide, particularly one that inhibits the expression of genes in signal transduction pathways involved in cell proliferation disorders, such as PKC-α, Ralf, and H-Ras.

[0120] In some embodiments, chemotherapeutic agents may include ribozymes, such as VEGF expression inhibitors (eg, ANGIOZYME™) and HER2 expression inhibitors.

[0121] In some embodiments, chemotherapeutic agents may include cytotoxic agents or antitumor antibiotics, such as dactinomycin, actinomycin, bleomycin, plicamycin, mitomycin, e.g., mitomycin C, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0122] In some embodiments, chemotherapeutic agents may include proteasome inhibitors such as bortezomib (VELCADE™, Millennium Pharm.), epoxomicins such as carfilzomib (KYPROLIS™, Onyx Pharm.), marizomib (NPI-0052), MLN2238, CEP-18770, oprozomib, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0123] In some embodiments, chemotherapeutic agents may include microtubule inhibitors, such as vinca alkaloids, including vincristine, vinblastine, vindesine, and vinorelbine; taxanes, including paclitaxel and docetaxel; podophyllotoxins; and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0124] In some embodiments, chemotherapeutic agents may include "EGFR antagonists," which refer to compounds that bind to or otherwise directly interact with EGFR and prevent or reduce its signaling activity, alternatively referred to as "EGFRi." Examples of such agents include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb579 (ATCC CRL HB 8506), MAb455 (ATCC CRL HB8507), MAb225 (ATCC CRL 8508), MAb528 (ATCC CRL 8509) (see U.S. Pat. No. 4,943,533, Mendelsohn et al.) and their variants, such as chimeric 225 (C225 or cetuximab; ERBUTIX) and reshaped human 225 (H225) (see WO 96 / 40210, Imclone Systems Inc.). IMC-11F8, an antibody targeted to fully human EGFR (Imclone); antibodies that bind to type II mutant EGFR (U.S. Pat. No. 5,212,290); humanized and chimeric antibodies that bind to EGFR, as described in U.S. Pat. No. 5,891,996; and human antibodies that bind to EGFR, such as ABX-EGF or panitumumab (see WO 98 / 50433, Abgenix / Amgen); EMD55900 (Stragliotto et al., Eur. J. Cancer 32A:636-640 (1996); EMD7200 (matuzumab), a humanized EGFR antibody against EGFR that competes with both EGF and TGF-α for binding to EGFR (EMD / Merck); human EGFR antibodies, HuMax-EGFR (GenMab); fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3, and E7.6.3, and described in U.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb806 or humanized mAb806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). The anti-EGFR antibody may be conjugated to a cytotoxic agent, thus forming an immunoconjugate (see, for example, EP 659439, Merck Patent GmbH).EGFR antagonists can be small molecules, e.g., those described in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, and 6,140,332. Nos. 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, and the following PCT publications: WO 98 / 14451, WO 98 / 50038, WO 99 / 09016, and WO 99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA™ Genentech / OSI Pharmaceuticals); PD183805 (CI1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA™) 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca; ZM105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim; PKI-166 ((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine; CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide); EKB-569 (N-[4-[(3-chloro-4-fluorophenyl)amino]-3- cyano-7-ethoxy-6-quinolinyl]-4-(-dimethylamino)-2-butenamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU5271; Pfizer); dual EGFR / HER2 tyrosine kinase inhibitors, such as lapatinib (TYKERB™, GSK572016 or N-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

[0125] In some embodiments, the chemotherapeutic agent may comprise a tyrosine kinase inhibitor, such as an EGFR-targeted drug as noted in the preceding paragraph; a small molecule HER2 tyrosine kinase inhibitor, e.g., TAK165 available from Takeda; CP-724,714, a selective oral inhibitor of ErbB2 receptor tyrosine kinase (Pfizer and OSI); a dual HER inhibitor, e.g., EKB-569 (available from Wyeth), which preferably binds to EGFR but inhibits both HER2-overexpressing and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); a pan-HER inhibitor, e.g., canertinib (CI-1033; Pharmacia); a Raf-1 inhibitor, e.g., ISIS, which inhibits Raf-1 signaling. antisense agent ISIS-5132 available from GlaxoSmithKline Pharmaceuticals; non-HER-targeted TK inhibitors such as imatinib mesylate (GLEEVEC™, available from GlaxoSmithKline); multi-targeted tyrosine kinase inhibitors such as sunitinib (SUTENT™, available from Pfizer); VEGF receptor tyrosine kinase inhibitors such as vatalanib (PTK787 / ZK222584, Novartis / Schering available from AG); MAPK extracellular regulated kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, e.g., PD153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines, e.g., CGP59326, CGP60261, and CGP62706; pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); tyrphostins containing a nitrothiophene moiety; PD-0183805 (Warner-Lambert); antisense molecules (e.g., those that bind to HER-encoding nucleic acids); quinoxalines (U.S. Pat. No. 5,804,396); tyrphostins (U.S. Pat. No. 5,804,396); ZD6474 (Astra Zeneca);PTK-787 (Novartis / Schering AG); pan-HER inhibitors, such as CI-1033 (Pfizer); Affinitac (ISIS3521; Isis / Lilly); imatinib mesylate (GLEEVEC™); PKI166 (Novartis); GW2016 (GlaxoSmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), including rapamycin (sirolimus, RAPAMUNE™); or the following patent publications: U.S. Pat. No. 5,804,396; WO 1999 / 09016 (American Cyanamid); WO 1998 / 43960 (American Cyanamid; 1997 / 38983 (Warner Lambert); 1999 / 06378 (Warner Lambert); 1999 / 06396 (Warner Lambert); 1996 / 30347 (Pfizer, Inc); 1996 / 33978 (Zeneca); 1996 / 3397 (Zeneca) and 1996 / 33980 (Zeneca);

[0126] In some embodiments, the chemotherapeutic agent may include a retinoid, such as retinoic acid, as well as pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0127] In some embodiments, the chemotherapeutic agent may comprise an antimetabolite. Examples of an antimetabolite include folic acid analogs and antifolates, such as denopterin, methotrexate, pteropterin, and trimethotrexate; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs, such as 5-fluorouracil (5-FU), ancitabine, azacitidine, 6-azauridine, carmofur, and citabine. These may include tarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; nucleoside analogs; and nucleotide analogs.

[0128] In some embodiments, the chemotherapeutic agent can include a topoisomerase inhibitor.Examples of topoisomerase inhibitors can include topoisomerase 1 inhibitors, such as LURTOTECAN™ and ABARELIX™ rmRH; topoisomerase II inhibitors, such as doxorubicin, epirubicin, etoposide, and bleomycin; and topoisomerase inhibitor RFS 2000.

[0129] In some embodiments, the chemotherapeutic agent may include a histone deacetylase (HDAC) inhibitor, such as vorinostat, romidepsin, belinostat, mocetinostat, valproic acid, panobinostat, and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing.

[0130] Chemotherapeutic agents further include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone 17-butyrate, 17- hydrocortisone valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone 17-butyrate, clobetasol 17-propionate, fluocortolone caproate, fluocortolone pivalate, and fluprednidene acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer form (feG) (IMULAN) BioTherapeutics, LLC; antirheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salts, hydroxychloroquine, leflunomide minocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), cetolithumab pegol (Cimzia), golimumab (Simponi), interleukin 1 (IL-1) blockers such as anakinra (K ineret), T cell costimulation blockers, e.g., abatacept (Orencia), interleukin 6 (IL-6) blockers, e.g., tocilizumab (ACTEMERA™); interleukin 13 (IL-13) blockers, e.g., lebrikizumab; interferon alpha (IFN) blockers, e.g., rontalizumab; beta 7 integrin blockers, e.g., rhuMAb beta 7; IgE pathway blockers, e.g., anti-M1 prime; secreted homotrimeric LTa3 and membrane-bound heterotrimeric LTa1 / β2 blockers, e.g., anti-lymphotoxin alpha (LTa); radioisotopes (e.g., 211 At, 131 I, 125 I, 90Y, 186 Re, 188 Re, 212 Bi, 32 P, 212 Pb, and Lu radioisotopes); various investigational agents, such as thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols, such as quercetin, resveratrol, piceatannol, epigallocatechin gallate, theaflavin, flavanols, procyanidins, betulinic acid and its derivatives; autophagy inhibitors, such as chloroquine; delta-9-tetrahydrocannabinol steroids (dronabinol, MARINOL™); beta-lapachone; lapachol; colchicine; betulinic acid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin; podophyllotoxin; tegafur (UFTORAL™); bexarotene (TARGRETIN™); bisphosphonates, such as clodronate (e.g., BONEFOS™ or OSTAC™), etidronate (DIDROCAL™), NE-58095, zoledronic acid / zoledronate (ZOMETA™), alendronate (FOSAMAX™), pamidronate (AREDIA™), tiludronate (SKELID™), or risedronate (ACTONEL™); and epidermal growth factor receptor (EGF-R); vaccines, such as the THERATOPE™ vaccine; perifosine, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; tipifarnib (R11577); orafenib, ABT510; Bcl-2 inhibitors, such as ob It may also include rimersen sodium (GENASENSE™); pixantrone; farnesyltransferase inhibitors, such as lonafarnib (SCH6636, SARASAR™); and pharmaceutically acceptable salts, acids, and derivatives of any of the foregoing; and combinations of two or more of the foregoing, such as CHOP (short for the combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone); and FOLFOX (short for the treatment regimen using oxaliplatin (ELOXATIN™) in combination with 5-FU and leucovorin).

[0131] Chemotherapeutic agents can also include non-steroidal anti-inflammatory drugs with analgesic, antipyretic, and anti-inflammatory effects.NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase.Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, and naproxen, acetic acid derivatives such as indomethacin, sulindac, etodolac, diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, rofecoxib, and valdecoxib. NSAIDs may be indicated for the symptomatic relief of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthropathy, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headache and migraine, postoperative pain, mild to moderate pain due to inflammation and tissue injury, fever, intestinal obstruction, and renal colic.

[0132] Immune checkpoint modulation: In certain embodiments, immune checkpoint modulators are co-administered with superactivated dendritic cells. Immune checkpoints relate to inhibitory pathways of the immune system responsible for maintaining self-tolerance and regulating the duration and breadth of physiological immune responses.

[0133] Certain cancer cells thrive by exploiting immune checkpoint pathways, which are a major mechanism of immune tolerance, particularly for T cells specific to tumor antigens. For example, certain cancer cells overexpress one or more immune checkpoint proteins that are responsible for inhibiting cytotoxic T cell responses. Therefore, immune checkpoint regulators can be administered to overcome the inhibitory signals and allow and / or enhance immune attacks against cancer cells. Immune checkpoint regulators can promote immune cell responses against cancer cells by reducing, inhibiting, or suppressing signaling by negative immune cell response regulators (e.g., CTLA4), or can stimulate or enhance signaling by positive regulators of the immune response (e.g., CD28).

[0134] Immunotherapeutic agents targeting immune checkpoint regulators can be administered to promote targeted immune attack on cancer cells. The immunotherapeutic agent can be or include an antibody agent that targets (e.g., is specific for) an immune checkpoint regulator. Examples of immunotherapeutic agents include antibody agents that target one or more of CTLA-4, PD-1, PD-L1, GITR, OX40, LAG-3, KIR, TIM-3, CD28, CD40; and CD137. Specific examples of antibody agents can include monoclonal antibodies. Specific monoclonal antibodies targeting immune checkpoint regulators For example, ipilimumab targets CTLA-4, tremelimumab targets CTLA-4, pembrolizumab targets PD-1, etc.

[0135] The programmed death 1 (PD-1) protein is an inhibitory member of the extended CD28 / CTLA-4 family of T cell regulators (Okazaki et al. (2002) Curr Opin Immunol 14:391-779-82; Bennett et al. (2003) J. Immunol. 170:711-718). Other members of the CD28 family include CD28, CTLA-4, ICOS, and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified: programmed death-ligand 1 (PD-L1) and programmed death-ligand 2 (PD-L2). PD-L1 and PD-L2 have been shown to bind to PD-1 and downregulate T cell activation and cytokine secretion (Freeman et al. (2000) J Exp Med 192:1027-34; Latchman et al. (2001) Nat Immunol 2:261-8; Carter et al. (2002) Eur J Immunol 32:634-43; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53).

[0136] PD-L1 (also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1)) is a 40-kDa type 1 transmembrane protein. PD-L1 binds to its receptor PD-1, found on activated T cells, B cells, and myeloid cells, to regulate activation or inhibition. Both PD-L1 and PD-L2 are B7 homologs that bind to PD-1 but not to CD28 or CTLA-4 (Blank et al. (2005) Cancer Immunol Immunother. 54:307-14). PD-L1 binding to its receptor PD-1 on T cells delivers a signal that inhibits TCR-mediated activation of IL-2 production and T cell proliferation. The mechanism involves inhibition of ZAP70 phosphorylation and its association with CD3 zeta (Sheppard et al. (2004) FEBS Lett. 574:37-41). PD-1 signaling attenuates TCR signaling-induced PKC-θ activation loop phosphorylation, which is required for activation of the transcription factors NF-κB and AP-1 and production of IL-2. PD-L1 also binds to the costimulatory molecule CD80 (B7-1), but not CD86 (B7-2) (Butte et al. (2008) Mol Immunol. 45:3567-72).

[0137] Expression of PD-L1 on the cell surface has been shown to be upregulated via IFN-γ stimulation. PD-L1 expression is found in many human cancers, including lung, ovarian, and colon cancers and various myelomas, and is frequently associated with poor prognosis (Iwai et al. (2002) PNAS 99:12293-7; Ohigashi et al. (2005) Clin Cancer Res 11:2947-53; Okazaki et al. (2007) Intern. Immun. 19:813-24; Thompson et al. (2006) Cancer Res. 66:3381-5). PD-L1 has been proposed to be important in tumor immunity by increasing apoptosis of antigen-specific T cell clones (Dong et al. (2002) Nat Med 8:793-800). PD-L1 may be involved in intestinal mucosal inflammation, and it has been proposed that inhibition of PD-L1 suppresses colitis-associated wasting (Kanai et al. (2003) J Immunol 171:4156-63).

[0138] Exemplary anti-PD1 antibodies include pembrolizumab (MK-3475, Merck), nivolumab (BMS-936558, Bristol-Myers Squibb), and pidilizumab (CT-011, Curetech LTD.). Anti-PD1 antibodies are available, for example, from ABCAM™ (AB137132), BIOLEGEND™ ​​(EH12.2 H7, RMP1-14) and Affymetrix Ebioscience (J105, J116, MIH4).

[0139] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA, genetics, immunology, cell biology, cell culture, and transgenic biology, which are within the skill of the art. See, e.g., Maniatis et al., 1982, Molecular Cloning (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Sambrook et al., 1989, Molecular Cloning, 2nd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Spring Harbor, NY; Sambrook and Russell, 2001, Molecular Cloning, 3rd ed. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Ausubel et al., 1992, Current Protocols in Molecular Biology (John Wiley & Sons, with regular updates); Glover, 1985, DNA Cloning (IRL Press, Oxford); Anand, 1992; Guthrie and Fink, 1991; Harlow and Lane, 1988, Antibodies (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY); Jakoby and Pastan, 1979; Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins, eds., 1984); Transcription And Translation (B.D. Hames & S.J. Higgins, eds., 1984); Culture Of Animal Cells (R.I. Freshney, Alan R. Liss, Inc., 1987);Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the monograph, Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (JH Miller and MPCalos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, vols. 154 and 155 (Wu et al., eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Handbook Of Experimental Immunology, vols. I-IV (D.M. Weir and C.C. Blackwell, eds., 1986); Riott, Essential Immunology, 6th ed., Blackwell Scientific Publications, Oxford, 1988; Hogan et al., Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986); Westerfield, M., The zebrafish book.A guide for See The Laboratory Use of Zebrafish (Danio rerio), (4th ed., University of Oregon Press, Eugene, 2000).

[0140] Genes: All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species to which the compositions and methods disclosed herein are applicable. When disclosing a gene or gene product from a particular species, the disclosure is intended to be illustrative only and not limiting unless the context in which it appears clearly dictates otherwise. Thus, for example, with respect to the genes or gene products disclosed herein, it is intended to include homologous and / or orthologous genes and gene products from other species.

[0141] Ranges: Throughout this disclosure, various aspects of the disclosure may be presented in a range format. The description in range format is understood to be merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range is considered to have all possible subranges explicitly disclosed, as well as individual numerical values ​​within that range. For example, a description of a range, such as 1 to 6, is considered to have explicitly stated subranges, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, such as 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This is true regardless of the breadth of the range.

[0142] Any composition or method provided herein can be combined with any one or more of the other compositions and methods provided herein.

[0143] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, and suitable methods and materials are described below.It is understood and expected that those skilled in the art can make modifications to the principles of the invention disclosed herein, and such modifications are intended to be included within the scope of this invention. [Example]

[0144] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0145] Example 1: Superactive dendritic cells stimulate long-lasting antitumor immunity against complex antigen mixtures An ideal strategy for stimulating protective immunity would combine the benefits of activated and pyroptotic DCs, allowing activated cells to maintain viability while retaining the ability to release IL-1β. We recently identified a novel DC activation state that exhibits these characteristics. DCs achieve a persistent state of "hyperactivation" upon exposure to PAMPs (e.g., TLR ligands) and a group of oxidized phospholipids (DAMPs) released from dying cells (I. Zanoni et al., Science, Vol. 352, No. 6290, pp. 1232-1236, 2016; I. Zanoni et al., Immunity, Vol. 47, No. 4, pp. 697-709 e3, 2017). This group of oxidized lipids is known as oxPAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine). Superactivated DCs exhibit the activity of activated DCs with respect to cytokine release (e.g., TNFα) but have also acquired the ability to release IL-1β over several days. Consistent with their designation as "superactivated" DCs, superactivated DCs are superior to their activated counterparts in their ability to stimulate T cell responses against model antigens.

[0146] The ability of this DAMP (oxPAPC) to bind and stimulate the cytosolic PRR caspase-11 defined the mechanism underlying the hyperactive state of DCs (I. Zanoni et al., 2016). Stimulation of caspase-11 triggers activation of NLRP3 and assembly of inflammasomes, which does not result in pyroptosis but rather in the release of IL-1β from live cells. IL-1β release from hyperactive cells is mediated by the pore-forming protein gasdermin D, which serves as a conduit for secreting these cytokines (C.E. Vavold et al., Immunity, 2018 Jan. 16;48(1):35-44, pp. e6; X. Liu et al., Nature, Vol. 535). , No. 7610, pp. 153-158, 2016; R.A. Aglietti et al., Proc. Natl. Acad. Sci. USA, Vol. 113, No. 28, pp. 7858-63, July 2016; N. Kayagaki et al., Nature, Vol. 526, No. 7575, pp. 666-671, September 2015). To remove gasdermin D pores, the cell membrane is thought to be repaired in a manner that ensures cell viability (S. Ruehl et al., Science, Vol. 362, No. 6417, pp. 956-960, November 2018). However, in other instances (e.g., alum stimulation), membrane repair pathways are overwhelmed, and pyroptosis ensues. Despite this insight into how IL-1β can be released from live cells, the physiological benefits of a hyperactive cellular state for the command of adaptive immunity remain poorly defined.

[0147] material and method Mouse strains and tumor cell lines: C57BL / 6J (Jax000664), caspase-1 / -11 dKO mice (Jax016621), NLRP3KO (Jax021302), Casp11KO (Jax024698), OT-I (Jax003831) and OT-II (Jax004194), and BALB / c (Jax000651) mice were purchased from Jackson Labs. For the syngeneic tumor model in C57BL / 6J, two melanoma cell lines were used: the parental cell line, B16.F10, and the OVA-expressing cell line, B16.F10OVA. For the syngeneic colorectal model, the OVA-expressing MC-38 cell line, derived from C57BL6 mouse colon adenocarcinoma cells, was used. These cell lines were a gift from the Arlene Sharpe Laboratory. For the syngeneic colon cancer model in BALB / c mice, the CT26 cell line was used (a gift from the Jeff Karp laboratory).

[0148] Reagents: E. coli LPS (serotype O55:B5-TLRGRADE™) was purchased from Enzo and used at 1 μg / ml for cell culture or 10 μg / mouse for in vivo use. Monophosphoryl lipid A (MPLA) from S. minnesota R595 was purchased from Invivogen and used at 1 μg / ml for cell culture or 20 μg / mouse for in vivo use. OxPAPC was purchased from Invivogen, resuspended in prewarmed serum-free medium, and used at 100 μg / ml for cell stimulation or 65 μg / mouse for in vivo use. POVPC and PGPC were purchased from Cayman Chemical. Reconstitution of commercially available POVPC and PGPC was performed as previously described (CL Evavold et al., Immunity, 2018, January 16;48(1):35-44). Briefly, the ethanol solvent was evaporated using a gentle stream of nitrogen gas. Prewarmed serum-free medium was then immediately added to the dried lipids to a final concentration of 1 mg / ml. The reconstituted lipids were incubated at 37°C for 5–10 minutes and sonicated for 20 seconds before adding to the cells. POVPC or PGPC was used at 100 μg / ml for cell stimulation or 65 μg / mouse for in vivo use. EndoFit chicken egg ovalbumin protein, with endotoxin levels <1 EU / mg, and OVA257–264 peptide were purchased from Invivogen at a concentration of 200 μg / mouse for in vivo use or 500 μg or 100 μg / ml for in vitro use. Incomplete Freud's adjuvant (F5506) was purchased from Sigma and used at a working concentration of 1:4 (IFA:antigen emulsion) for in vivo immunization. Alum hydrogel was obtained from Accurate. Antigen was purchased from Chemist Chemical and used at a working concentration of 2 mg / mouse for in vivo immunizations. In some experiments, Addavax, a squalene-oil-in-water adjuvant, was used in place of IFA at a working concentration of 1:2 (AddVax:antigen).

[0149] Cell culture: BMDCs were cultured in IMDM (Gibco), 10% B16-GM-CSF-derived supernatant, 2 μM 2-mercaptoethanol, 100 U / ml penicillin, 100 μg / ml streptomycin (Sigma-Aldrich), and 10% FBS. After 6 days of culture, BMDCs were washed with PBS and resuspended in IMDM containing 10% FBS at 1 × 10 6 The cells were replated in a final volume of 100 μl at a concentration of 100 cells / ml. CD11c was detected by flow cytometry using a BD Fortessa. + DC purity was assessed and was typically >80%. Splenic DCs from mice injected with B16-FLT3 for 15 days were cultured using CD11c + MHC + Purified as live cells and then cultured at 1 x 10 in complete IMDM. 6 DCs were plated at a concentration of 100 cells / ml in a final volume of 100 μl. To induce hyperactivated or pyroptotic BMDCs, DCs were primed with LPS (1 μg / ml) for 3 hours and then stimulated with OxPAPC or PGPC (100 μg / ml) or alum (100 μg / ml) in complete IMDM for 21 hours. In some cases, activated BMDCs were restimulated for an additional 24 hours on plate-bound agonist anti-CD40 using Ultra-LEAF anti-mouse CD40 (clone 1C10; BioLegend). T cells were cultured in RPMI-1640 (Gibco) supplemented with 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin (Sigma-Aldrich), and 50 μM β-mercaptoethanol (Sigma-Aldrich). All tumor cell lines were cultured in DMEM supplemented with 10% FBS. For OVA-expressing cell lines, puromycin (2 μg / ml) was added to the medium.

[0150] LDH assay and ELISA: After BMDC stimulation, fresh supernatants were clarified by centrifugation and then subjected to LDH release assays using the Pierce LDH Cytotoxicity Colorimetric Assay Kit (Life Technologies) according to the manufacturer's protocol. Absorbance measurements were performed at wavelengths of 490 nm and 680 nm using a Tecan plate reader. To measure secreted cytokines, supernatants were collected, clarified by centrifugation, and stored at -20°C. ELISAs for IL-1β, TNFα, IL-10, IL-12p70, IFNγ, IL-2, IL-13, IL-4, and IL-17 were performed using eBioscience Ready-SET-Go! (now ThermoFisher) ELISA kits according to the manufacturer's protocol.

[0151] Flow cytometry: After FcR blockade, day 7 BMDCs were resuspended in MACS buffer (PBS containing 1% FCS and 2 mM EDTA) and stained with the following fluorescently labeled antibodies (BioLegend): anti-CD11c (clone N418), anti-IA / IE (clone M5 / 114.15.2), anti-CD40 (clone 3 / 23), anti-CD80 (16-10A1), anti-CD69 clone (H1.2F3), and anti-H-2Kb (clone AF6-88.5). Single-cell suspensions from tumors, draining inguinal lymph nodes, or skin-inguinal adipose tissue were resuspended in MACS buffer (PBS containing 1% FCS and 2 mM EDTA) and stained with the following fluorescently labeled antibodies (BioLegend): anti-CD8α (clone 53-6.7), anti-CD4 (clone RM4-5), anti-CD44 (clone IM7), anti-CD62L (MEL-14), anti-CD3 (17A2), anti-CD103 (2E7), anti-CD69 clone (H1.2F3), and anti-CD45 (A20 or 30F11). To determine cell viability, cells were stained in PBS at 4°C for 20 minutes using the LIVE / DEAD™ Fixable Violet Dead Cell Stain Kit (Molecular Probes). T cells from draining inguinal lymph nodes were stained with OVA peptide tetramer for 1 hour at room temperature. PE-conjugated H2K(b)SIINFEKL (OVA257-264; SEQ ID NO: 1) and APC-conjugated IA(b)AAHAEINEA (OVA329-337; SEQ ID NO: 2) were used. IA(b) and H2K(b) associated with the CLIP peptide were used as isotype controls. Tetramers were purchased from the NIH Tetramer Core Facility. In some experiments, FITC anti-CD8.1 (clone Lyt-2.1 CD8-E1) purchased from Accurate Chemical was used with the tetramers. To determine absolute cell numbers, COUNTBRIGHT counting beads (Molecular probes) were used according to the manufacturer's protocol. Appropriate isotype controls were used as staining controls. Data were acquired using a BD FACS ARIA or BD Fortessa. Data were analyzed using FlowJo software.

[0152] Antigen uptake assay: FITC-labeled chicken OVA (FITC-OVA) (Invitrogen-Molecular Probes) was used to examine the antigen uptake and endocytosis capacity of BMDCs in different activation states (activated, superactivated, or pyroptotic). Briefly, pretreated BMDCs were incubated with either FITC-OVA or AF488-dextran (0.5 mg / ml) for 45 min at 37°C or 4°C (as a control for antigen surface binding). BMDCs were then washed and stained with the Live / Dead Fixable Violet Dead Cell Stain Kit (Molecular Probes) to distinguish live from dead cells. Cells were then fixed with BD fixative and resuspended in MACS buffer (PBS containing 1% FCS and 2 mM EDTA). FITC fluorescence of live cells was measured every 15 min using a Fortessa flow cytometer (Becton-Dickenson). Fluorescence values ​​of BMDCs incubated at 37°C were reported as the percentage of OVA-FITC- or Dextran-AF488-associated cells, and data were normalized to the percentage of OVA-FITC-associated cells incubated at 4°C.

[0153] OVA antigen presentation assay: To measure the efficiency of OVA antigen presentation on MHC-I, 0.5 × 10 cells were treated with activation (LPS), superactivation (LPS + PGPC or LPS + OxPAPC), or pyroptotic stimulation (LPS + alum). 6 BMDCs were incubated with Endofit-OVA protein (0.5 mg / ml) at 37°C for 2 hours. The cells were then washed with MACS buffer and incubated on ice for 20-30 minutes with APC anti-mouse H-2K. b H-2K bound to antibody (clone AF6-88.5, BioLegend) and OVA peptide SIINFEKL b The cells were stained with a PE-conjugated antibody (SEQ ID NO: 1; clone 25-D1.16, BioLegend) that binds to total surface H-2K. Appropriate isotype controls were used as staining controls. bThe percentage of cells expressing OVA peptide on MHC-I was calculated. Data were acquired using a Fortessa flow cytometer (Becton-Dickenson) and analyzed with FlowJo software (Tree Star).

[0154] In vitro T cell stimulation of OT-I and OT-II: splenic CD8 + T cells and CD4 + T cells were sorted from OT-I and OT-II mice by magnetic cell sorting using anti-CD8 or anti-CD4 beads (Miltenyi Biotech), respectively. Sorted T cells were then seeded onto 96-well plates at a concentration of 100,000 cells per well in the presence of 20,000 or 10,000 DCs (5:1 or 10:1 ratios) pretreated with either LPS (activating stimulus), LPS + PGPC (superactivating stimulus), or LPS + alum (pyroptotic stimulus) and pulsed (or not) with OVA protein or SIINFEKL (SEQ ID NO: 1) peptide at 100 μg / ml for 2 hours. After 5 days of culture, supernatants were collected and clarified by centrifugation for short-term storage at -20°C and cytokine measurement by ELISA.

[0155] Intracellular staining: For intracellular cytokine staining, cells were stimulated with 50 ng / ml phorbol 12-myristate 13-acetate (PMA) and 500 ng / ml ionomycin (Sigma-Aldrich) for 4-5 h in the presence of GolgiStop (BD) and Brefeldin A. Cells were then washed twice with PBS and stained with LIVE / DEAD™ Fixable Violet or Green Dead Cell Stain Kit (Molecular probes) in PBS for 20 min at 4°C. Cells were then resuspended in MACS buffer. Cells were washed twice and stained for appropriate surface markers at 4°C for 20 minutes. After two washes, cells were fixed and permeabilized using the BD Cytofix / Cytoperm kit according to the manufacturer's protocol for 20 minutes at 4°C, then washed with 1X permeabilization wash buffer (BD). Intracellular cytokine staining was performed in 1X permeabilization buffer for 20–30 minutes at 4°C using the following conjugated antibodies, all purchased from BioLegend: anti-Ki67 (clone 16A8), anti-IFN-γ (clone XMG1.2), anti-TNFα (clone MP6-XT22), anti-Gata3 (16E10A23), anti-IL4 (11B11), and anti-IL10 (clone JES5-16E3). Data were acquired using a BD FACS ARIA or BD Fortessa. Data were analyzed using FlowJo software.

[0156] CD107a degranulation assay: CD8 + To assess the effector antitumor activity of T cells, we assessed the surface exposure of the lysosome-associated protein CD107a by flow cytometry. Briefly, CD8 T cells were isolated from the skin-draining lymph nodes of immunized mice by magnetic cell enrichment using anti-CD8 beads and columns (Miltenyi Biotech). + T cells were isolated and then analyzed using a FACS ARIA (BD) for CD3 + CD8 + Sorted as live cells. Freshly sorted CD8 + T cells were cultured at 1 x 10 in complete RPMI. 6 The T cells were resuspended at a concentration of 100,000 cells / ml. PerCP / Cy5.5 anti-mouse CD107a (LAMP-1) antibody (clone 1D4B, BioLegend) was added to the medium at a concentration of 1 μg / ml in the presence of GolgiStop (BD). The T cells were then immediately seeded at 100,000 cells onto 10,000 MC38OVA or B16OVA tumor cells / well in a 96-well plate. Alternatively, CD8 +T cells were plated alone and stimulated with 50 ng / ml phorbol 12-myristate 13-acetate (PMA) and 500 ng / ml ionomycin (Sigma-Aldrich). After 5 hours of culture, cells were washed with MACS buffer and stained with the LIVE / DEAD™ Fixable Violet Dead Cell Stain Kit (Molecular probes) and APC anti-CD8 (clone 53-6.7, BioLegend). Cells were then fixed with BD fixative for 20 minutes at 4°C and resuspended in MACS buffer. CD107a and CD107b were detected by flow cytometry using a Fortessa flow cytometer (BD). + The percentage of cells was determined.

[0157] In vitro cytotoxicity assay: CD8 derived from spleen or skin-inguinal adipose tissue of survivor mice + T cells were isolated using anti-CD8 MACS beads and columns (Miltenyi Biotec). Enriched T cells were then analyzed using a FACS ARIA for CD45 + CD3 + CD8 + Viable cells were sorted. The purity after sorting was >97%. Tumor cell lines, e.g., B16OVA, B16F-10, or CT26 cells, were plated in 96-well plates (2 × 10 cells) at least 5 hours before co-culture with T cells. 4 Cells were seeded in complete DMEM on 10 wells (10 cells / well). 5 CD8 + Twelve hours after seeding the T cells onto the tumor cells, cytotoxicity was assessed by LDH release assay using the Pierce LDH Cytotoxicity Colorimetric Assay Kit (Life Technologies) according to the manufacturer's protocol.

[0158] Preparation of whole tumor cell lysates: To prepare whole tumor cell lysates (WTL) for immunization, tumor cell lines were cultured in complete DMEM for 4–5 days. When cells reached confluence, the supernatant was collected, and the cells were washed and dissociated using trypsin-EDTA (Gibco). Then, tumor cell lines were cultured in the collected culture supernatant at 5 × 106 Cells were resuspended at 1000 cells / ml and lysed by three freeze-thaw cycles.

[0159] Tumor infiltration: To evaluate the frequency of tumor-infiltrating lymphocytes (TILs) in immunized mice, tumors were collected when they reached a size of 1.8–2 cm. Tumors were dissociated using a gentleMACS dissociator (Milteny Biotec) and a gentleMACS dissociator according to the manufacturer's protocol. After digestion, tumors were washed with PBS and passed through a 70 μm filter and a 30 μm filter. CD45 microbeads (Milteny Biotec) were used to dissociate CD45. + Cells were positively selected, and T cell infiltration was assessed by flow cytometry. Tumor-infiltrating T cells were cultured with Dynabeads Mouse T Activator CD3 / CD28 (Gibco) to activate and expand T cells.

[0160] Adoptive cell transfer: For T cell transfer, CD8 T cells derived from the spleen or skin and inguinal adipose tissue of survivor mice were used. + T cells were isolated using anti-CD8 MACS beads and columns (Milteny Biotec). The enriched cells were then analyzed using a FACS ARIA for CD45 + CD3 + CD8 + Viable cells were sorted. The purity after sorting was >97%. The sorted T cells were then plated onto 24-well plates (~2 × 10 6 5 × 10 cells / well) and stimulated for 24 h in the presence of IL-2 (50 ng / ml). 5 Activated circulating splenic CD8 + T cell or cutaneous inguinal fat resident CD8 + T cells were transferred into naive recipient mice by iv or intradermal (id) injection, respectively, with some mice receiving both T cell subsets.

[0161] For DC transplantation, BMDCs were harvested on day 6 and 5 × 10 6 DCs were seeded in 6-well plates. DC activation was induced by incubation with a superactivating stimulus (LPS + PGPC) or an activating stimulus (LPS). Tumor lysate was added to the DC culture plate at a ratio of 1 DC to 2 tumor cells (i.e., 1:2) for 1 h. Unloaded naive DCs were used as a negative control.

[0162] Statistical analysis: Statistical significance for experiments with three or more groups was tested by two-way analysis of variance with Tukey's multiple comparison test correction. Adjusted p values ​​calculated using Prism (Graphpad) are indicated by an asterisk: <0.05 ( * );<0.0005( *** );≦0.0001( **** )

[0163] result Superactivating stimuli upregulate several activities important for DCs to stimulate T cell immunity Virtually all studies of DC superactivation have focused on the ability of DCs to release IL-1β while maintaining viability. The extent to which DC function is affected by superactivating stimuli has not been defined. To investigate this scope, bone marrow-derived DCs (BMDCs) were primed with LPS and subsequently treated with oxPAPC or a specific, pure lipid component of oxPAPC called PGPC (I. Zanoni et al., Science, Vol. 352, No. 6290, pp. 1232–1236, 2016). The resulting superactivated cells were compared with conventionally activated BMDCs (treated with LPS) or pyroptotic BMDCs (primed with LPS and then treated with alum). In contrast to the activating stimuli that did not induce IL-1β release as expected, pyroptotic or superactivating stimuli promoted IL-1β release into the extracellular medium (Figure 1A). All stimuli examined promoted secretion of the cytokine TNFα (Figure 1A). This finding is consistent with previous studies (I. Zanoni et al., 2016) that established that LPS-activated BMDCs release TNFα but not IL-1β. IL-1β secretion, assessed by the release of the cytosolic enzyme lactate dehydrogenase (LDH) (Figure 1B), coincided with cell death in pyroptotic DCs. In contrast, IL-1β secretion occurred in the absence of LDH release in superactivated cells (Figure 1B). Similar behavior of BMDCs was observed when LPS was replaced with MPLA, an FDA-approved TLR4 ligand used in vaccines against human papillomavirus (HPV) and hepatitis B virus (HBV) (Figures 3A and 3B). The secretion of hyperactivated BMDCs was significantly suppressed by IL-1β secretion, as assessed by the release of the cytosolic enzyme lactate dehydrogenase (LDH) (Figure 1B). To confirm whether this behavior extends to DCs differentiated in vivo, we isolated CD11c DCs from the spleens of mice injected with B16-FLT3. + Similar to the behavior of GMCSF-derived BMDCs, treatment with LPS and PGPC increased splenic CD11c activity in the absence of cell death as assessed by LDH release. +This resulted in the release of TNFα and IL-1β from DCs (Figures 3C and 3D). These results demonstrate that the superactivating stimulator PGPC can be used to induce IL-1β release from viable DCs differentiated in vitro or in vivo.

[0164] We examined several signals important for T cell differentiation, such as the expression of the costimulatory molecules CD80, CD69, and CD40, as well as the secretion of the p70 subunit of IL-12. Surface expression of CD80 was similar in DCs responding to all activating stimuli (Figure 5E). In contrast, CD40 expression was significantly affected by the activating stimuli. Compared with the activating stimulus LPS, superactivating stimuli significantly induced CD40 expression (Figure 1C). Pyroptotic stimuli were very weak inducers of CD40 and CD69, even within the range of 20–30% of viable cells after LPS-alum treatment (Figures 1C and 3E). When cultured on agonist anti-CD40-coated plates, differential expression of CD40 correlated with superactivated DCs, which had the greatest ability to secrete IL-12p70 (Figure 1D).

[0165] Although superactivated BMDCs were not superior to their activated counterparts in antigen capture, as assessed by comparable internalization of fluorescent ovalbumin (OVA-FITC) (Figures 4A and 4B), the former cell populations displayed significantly higher amounts of the OVA-derived SIINFEKL peptide on cell surface MHC-I molecules (Figures 1E and 4C). The total amount of surface MHC-I did not differ between activated and superactivated cells (Figure 3E). Collectively, compared with other stimuli of DCs, superactivated BMDCs exhibit enhanced several activities important for T cell differentiation.

[0166] Superactive DCs stimulate a TH1-focused immune response without evidence of TH2 immunity To assess the effect of DC activation state on T cell command, BMDCs were treated as described above and then loaded with OVA. These cells were then exposed to naive OT-II or OT-I T cells. OT-II cells express T cell receptors (TCRs) specific for the MHC-II-restricted OVA peptide (OVA323-339), whereas OT-I cells express TCRs specific for the MHC-I-restricted OVA peptide (OVA257-264) (KA Hogquist et al., Cell, Vol. 76, No. 1, pp. 17-27, January 1994; MJ Burnden et al., Immunol. Cell Biol., Vol. 76, No. 1, pp. 34-40, February 1998). To identify T cell polarization toward a TH1 response (IFNγ production) or a TH2 response (IL-10, IL-4, or IL-13 production), the activity of responding T cells was assessed by ELISA. Regardless of DC activation status, OVA-treated BMDCs stimulated IFNγ production from OT-II T cells. The magnitude of IFNγ production varied slightly among the activation stimuli tested (Fig. 1F). Similarly, TNFα production by OT-II cell responses was comparable across all DC activation statuses (Fig. 1F). These results indicate that in vitro TH1 responses are generally induced regardless of the activation status of antigen-presenting cells (APCs). In contrast, TH2 responses differed significantly across DC activation statuses. Stimuli inducing BMDC activation (LPS) or pyroptosis (LPS + alum) promoted the release of large amounts of IL-10 and IL-13, whereas superactivating stimuli resulted in minimal production of TH2-associated cytokines (Figure 1F). Intracellular staining of single cells for TH1 cytokines (IFNγ and TNFα) and TH2 cytokines (IL-4 and IL-10) and the TH2 lineage-defining transcription factor GATA3 allowed calculation of the ratio of TH1 to TH2 cells generated by different DC activation stimuli. This analysis demonstrated that superactivated BMDCs significantly biased individual T cells toward the IFNγ-producing TH1 lineage. We found that pyroptotic stimulation induced a TH1-TH2 cell ratio of >100:1 under hyperactivation conditions (Fig. 1G and Fig. 5). In contrast, all other activation stimuli induced mixed T cell responses, with pyroptotic stimulation resulting in a nearly 1:1 ratio of TH1 to TH2 cells (Fig. 1G).

[0167] CD8 + Similar studies performed with OT-I T cells revealed a slight enhancement of IFNγ production by superactivating BMDCs compared with activating or pyroptotic stimuli (Figure 1F). IL-2 production in response to OT-I cells was comparable across all DC activation states (Figure 1F). Together, these results suggest that in We show that a hyperactive BMDC state in vitro results in a significantly TH1-biased T cell response and a slightly enhanced CD8 T cell response, whereas activating or pyroptotic stimulation results in a mixed TH1 and TH2 response.

[0168] Hyperactivation of DC inflammasome components is sufficient to confer protective antitumor immunity. Because DCs are the primary cells responsible for promoting de novo T cell-mediated immunity, we sought to determine whether conditions that specifically activate DCs are sufficient to confer antitumor immunity. We addressed this possibility by adoptively transferring BMDCs stimulated ex vivo with different activation stimuli and with wild-type T cells into mice. BMDCs were chosen because 1) their hyperactivation state has been well characterized and 2) they are considered a model for monocyte-derived DCs, the most common APCs used in DC-based immunotherapy in humans (RL Sabado et al., Cell Res., Vol. 27, No. 1, pp. 74–95, January 2017).

[0169] BMDCs were treated with various activation stimuli along with WTL and then injected sc into B16OVA tumor-bearing mice every 7 days for 3 weeks. LPS-activated BMDCs pulsed with B16OVA WTL conferred modest protection from B16OVA-induced lethality compared with mice injected with naive BMDCs, and 25–30% of mice receiving DC transplants rejected tumors and remained tumor-free long after the final / third DC transplant (Figure 2). Notably, hyperactivated BMDCs induced complete rejection of B16OVA tumors in 100% of tumor-bearing mice (Figure 2). The antitumor activity of hyperactivated DCs depended on the inflammasome in these cells because of the NLRP3 gene. - / - and Casp1 - / - 11 - / - Transplantation of BMDCs induced only minor rejection, comparable to that of activated DCs (Figure 2). Thus, these data demonstrate that superactivated DCs are sufficient to induce long-lasting, protective antitumor immunity and that inflammasomes within DCs are essential for this process.

[0170] Consideration This study expanded the range of immunological activities upregulated by treatment of DCs with superactivating stimuli. Superactivating stimuli not only elicit IL-1β release from live cells, but also surpass other activating stimuli in their ability to induce CD40 expression and IL-12p70 secretion. Furthermore, cells exposed to superactivating stimuli exhibit elevated surface expression of MHC-peptide complexes. Together, these findings underscore the superactivating nature of DCs exposed to oxPAPC or its pure component, PGPC, and provide evidence of their enhanced ability to promote adaptive immunity. While superactivated DCs are indeed superior stimulators of T cell responses to activated or pyroptotic cells, the most notable aspect of their activity may be their ability to stimulate both TH1-focused and CTL-focused responses. Indeed, superactivating DC stimuli resulted in a 100:1 ratio of TH1:TH2 cells, and no other DC activation strategy has induced such a polarized T cell response.

[0171] It is noteworthy that alum, a clear inflammasome stimulator, does not exhibit the same activity as oxPAPC or PGPC. Indeed, alum often induces TH2 immunity. It is known that alum treatment or alum plus LPS treatment induced robust TH2 immunity in the present study. A possible explanation for the lack of TH1-focused immunity in alum-treated cells is based on the present findings that alum is a weak inducer of several signals essential for TH1 differentiation, such as CD40 expression and IL-12p70 secretion. Notably, CD40 expression was significantly lower even when DCs that did not undergo pyroptosis in response to alum plus LPS were examined. Perhaps due to the lack of high-level expression of this factor, pyroptotic stimulation is a weak inducer of TH1 responses and, therefore, antitumor immunity. Without wishing to be bound by theory, it has been proposed that the TH1-focused immunity induced by superactivated DCs is due to the action of inflammasomes as well as several other characteristics of superactivated DCs. These additional characteristics include increased antigen-presenting capacity, CD40 expression, IL-12p70 expression, and increased survival. Presumably, each of these increased activities is important for DC function as APCs and contributes to the marked TH1-focused immune responses observed under conditions of DC hyperactivation.

[0172] These results may explain why certain chemotherapeutic agents (e.g., oxaliplatin) induce cell death and inflammasome-dependent antitumor T cell immunity (F. Ghiringhelli et al., Nat. Med., Vol. 15, No. 10, pp. 1170-1178, 2009). Oxaliplatin is a robust stimulator of reactive oxygen species (ROS) production, which can oxidize cell membranes and generate a complex mixture of different oxidized phospholipid species, including PGPC. Therefore, the protective immunity induced by oxaliplatin may be due to the action of hyperactivated DCs, which prime antitumor T cell responses.

[0173] We have discovered that superactivating stimuli can be utilized as immunotherapy using complex mixtures of antigens. WTLs are an intriguing source of antigens for several reasons, particularly important from a practical standpoint. A key benefit of a WTL-based approach is that it alleviates the need for neoantigen identification. Despite the potential utility offered by WTL-based immunotherapy, previous studies in this field have yielded mixed results. Our discovery that superactivating stimuli can uniquely support WTLs to elicit potent antitumor immunity may explain the lack of success in previous studies, as the DC activation strategy discovered here has not been previously considered. Regarding the latter point, it is noteworthy that the DC superactivation strategy can protect mice from the lethality associated with tumors sensitive to and resistant to PD-1 inhibition. While the full range of tumors susceptible to treatment with superactivating stimuli has not yet been defined, this study urges further investigation into the importance of DC-centered strategies for cancer immunotherapy.

[0174] Example 2: Hyperactive cDC1 regulates tumor rejection in an inflammasome-dependent manner Conventional dendritic cells (cDCs) are adept at presenting exogenous and endogenous antigens to T cells and regulating T cell proliferation, survival, and effector function. cDCs are divided into two major subsets, cDC1 and cDC2. Resident cDC1 in the spleen and lymph nodes (LNs) express CD8α, CD24, and XCR1, whereas cDC2 express CD4 and Sirpα. cDC1 cross-present tumor-associated antigens and mediate TH1 immunity and anti-tumor CD8 + Classical DCs stimulate T cells to efficiently reject tumors, whereas cDC2s are responsible for type 2 immune responses against parasites where they activate Th2 immunity.

[0175] To examine whether resident cDCs can achieve a superactivated state, we sorted cDC1 or cDC2 from the spleens of WT mice and either left untreated, treated with LPS for 24 h, or primed with LPS for 3 h, followed by treatment with the superactivating stimuli oxPAPC or PGPC, or with the pyroptosis stimuli alum for 21 h. In contrast to splenic cDC2, splenic cDC1 were rapidly killed after isolation, as measured by LDH release (Figure 6). As a result, cDC1 cells were not primed with LPS and failed to produce TNFα cytokines in response to activating stimuli (LPS), superactivating stimuli (LPS + OxPAPC / PGPC), or pyroptotic stimuli (LPS + Alum) (Figure 6B, left panel). Only minimal IL-1β release was observed in response to the superactivating stimulus (LPS + PGPC) (Figure 6B, left panel). In contrast, splenic cDC2 cells were efficiently primed with LPS and achieved a superactivated state, identified by their ability to produce IL-1β without undergoing cell death (Figures 6A-B, left panels). Because resident cDC1 cells are highly sensitive to ex vivo isolation and in vitro stimulation, we alternatively generated cDCs from bone marrow (BM) progenitors using the cytokine FLT3 ligand (FLT3 L). FLT3L-generated cDC1s and cDC2s were sorted on day 9 of culture and then treated conventionally with activating, superactivating, or pyroptotic stimuli. In contrast to resident cDC1 cells isolated from the spleen, FLT3L-generated cDC1s and cDC2s were efficiently primed with LPS and achieved a superactivated state in response to stimulation with LPS + PGPC, but not LPS + oxPAPC, as measured by the release of large amounts of IL-1β and TNFα while maintaining their viability (Figures 6A-6B, right panels). These data indicate that PGPC, a pure form of oxidized phospholipid, can superactivate cDC1 and cDC2 subsets.

[0176] Hyperactive FLT3L-generated cDC1s exhibited more stellate dendrites compared to their naive, activated, or pyroptotic counterparts, indicating a higher migratory potential. Indeed, hyperactive cDC1s and cDC2s upregulated the chemokine receptor CCR7, which guides migratory DCs to lymph nodes for T cell stimulation (Figure 6C-6D). Taken together, these results demonstrate that cDC1 and cDC2 subsets achieve a hyperactivated state in vitro and exhibit unique properties compared to their classically activated counterparts.

[0177] The unique function of cDC1 cells is crucial in the context of cancer, where cDC1s take up tumor antigens and cross-present them to T cells within the tumor microenvironment (TME) or after migration to draining lymph nodes. The fact that cDC1s are hyperactivated in vitro raises the need to further explore the value of the cDC hyperactivated state for cancer immunotherapy. To this end, to investigate the role of the cDC1 hyperactivated state in controlling tumor rejection, mice were inoculated subcutaneously (sc) with B16OVA cells in the left dorsum of the mouse. On days 7, 14, and 21 after tumor challenge, mice were left untreated or injected with 1 × 10 untreated WT cDC1s (cDC1 naive), WT cDC1s treated with LPS for 23 h (cDC1 active), or WT cDC1s primed with LPS for 3 h and then treated with PGPC for 20 h (cDC1 hyperactive). 6 cDC1s were injected subcutaneously into the right flank. All cDCs were pulsed with B16OVA tumor lysate 1 h prior to injection. Surprisingly, adoptive transfer of superactivated cDC1s conferred potent and long-lasting protection against tumor growth in mice, whereas transfer of naive or activated cDC1s induced only modest tumor rejection (Figure 7). These data provide the first evidence of a prominent role for the superactivated state of cDC1s in sustained tumor rejection.

[0178] To further confirm the important role of hyperactive cDC1 in tumor control, we investigated the role of CD8 + Batf3 lacking cDC1 and defective in cross-presentation - / -I used a mouse, but the result was Batf3 - / - Mice expressing anti-tumor antigen-specific CD8 + Therefore, Batf3 cells inoculated with B16OVA cells are defective in T cell responses. - / - Mice were unable to control tumor growth compared to WT mice (Figure 8A). However, tumor-bearing Baft3 - / - When mice were replenished with WT cDC1s by subcutaneous injection on days 7, 14, and 21 after tumor challenge, we observed no significant differences between naive or activated cDC1s and WT cDC1s. In contrast, we found that only superactive cDC1s could completely eradicate tumors. This protection was not observed when WT superactive cDC1s were injected, but when Batf3 - / - Batf3 recovered in mice injected with WT activated or naive cDC1 cells - / - Tumor-infiltrating OVA-specific CD8 is not restored in mice + and CD4 + This correlated with a high frequency of T cells (Figures 8B-8C). Taken together, these data provide strong evidence that superactive cDC1s regulate tumor rejection by enhancing tumor infiltration of anti-tumor-specific T cells.

[0179] The mechanism underlying the hyperactive state of DCs is the oxidized phospholipids in question (oxPAPC / PGPC It has been well documented that caspase-1 / 11 can bind to and stimulate the cytoplasmic pathogen recognition receptor (PRR) caspase-11. Caspase-1 / 11 stimulation leads to the activation and assembly of the NLRP3 inflammasome, leading to the release of IL-1β from live cells via the gasdermin-D pore. To evaluate the role of IL-1β in the antitumor activity of hyperactive cDC1s, we investigated the role of Casp1 / 11 in IL-1β secretion. - / - Mouse or NLRP3 - / - The mice used were Casp1 / 11 - / - or NLRP3 - / - Mice were inoculated with B16OVA cells in the left dorsal region. On days 7, 14, and 21 after tumor challenge, mice were left untreated (no DC injection) or injected with untreated WT cDC1 (cDC1ナイーブ ) or WT cDC1 treated with LPS for 23 h (cDC1 活性 ), or WT or Casp1 / 11 primed with LPS for 3 h and then treated with PGPC for 20 h - / - cDC1(cDC1 超活性 ) 1.10 6 DCs were injected subcutaneously into the right flank. All DCs were pulsed with B16OVA tumor lysate 1 h prior to injection. Interestingly, we found that the Casp1 / 11 expression of WT hyperactivated cDC1 was significantly higher than that of WT naive or activated cDC1, which induced only modest protection. - / - and NLRP3 - / - Adoptive transfer into recipient mice was found to completely block tumor growth in 100% of tumor-bearing mice. Caspase 1 / 11 is unable to induce IL-1β secretion in response to a superactivating stimulus (LPS + PGPC). - / - or NLRP3 - / - This protection was dependent on the inflammasome machinery, as the derived cDC1s were unable to induce tumor rejection. Collectively, adoptive transfer of superactivated DCs is sufficient to induce durable antitumor responses and recapitulate the protection observed with superactivation-based vaccines.

[0180] Taken together, the data herein have the potential to shift the paradigm in DC-based immunotherapy with respect to the activation state used in adoptive cell transfer-based immunotherapy, and thus may reinvigorate efforts to "pre-condition" DCs in vitro to generate effective cancer immunotherapies.

[0181] Example 3: Oxidized phospholipids induce hyperactive cDC1 and cDC2 cells Virtually all studies evaluating the state of cell superactivation have focused on the ability of bone marrow-derived DCs (BMDCs) generated with the cytokine granulocyte-macrophage colony-stimulating factor (GM-CSF) to release IL-1β while maintaining viability [24, 31, 32, 33, 34]. Recent studies have demonstrated that monocyte-derived macrophages, rather than DCs, are responsible for inflammasome activation and IL-1β secretion

[35] . To examine whether conventional DCs can achieve a superactivation state, we used BMDCs generated using the DC hematopoietin Fms-like tyrosine kinase 3 ligand (Flt3L). To assess superactivation, FLT3-DCs were primed with LPS and subsequently treated with the oxidized phospholipid oxPAPC, or the pure lipid component of oxPAPC, known as PGPC

[36] . Alternatively, FLT3-DCs were stimulated with conventional activating stimuli, such as LPS alone, or primed with LPS followed by treatment with a pyroptotic stimulus, such as alum. In contrast to activation stimuli that did not induce IL-1β release from DCs, pyroptotic DCs promoted the release of IL-1β into the extracellular medium (Figure 14A). IL-1β secretion coincided with cell death in pyroptotic DCs, as assessed by the release of the cytosolic enzyme lactate dehydrogenase (LDH) (Figure 14B). Interestingly, the superactivating stimulus LP Stimulation with S+PGPC, or to a lesser extent LPS+oxPAPC, induced IL-1β secretion from DCs in the absence of LDH release (Figure 14A). All DCs primed or stimulated with LPS promoted the secretion of the cytokine TNFα (Figure 14A). IL-1β secretion in pyroptotic or hyperactivated DCs depended on the inflammasome components NLRP3 and caspase-1 / 11 in both cases (Figure 14A). These findings are consistent with previous studies that defined the mechanism underlying the hyperactivated state of DCs, in which oxPAPC binds to and stimulates the cytosolic PRR caspase-11, resulting in NLRP3 activation and nonpyroptic inflammasome assembly, leading to IL-1β release from live cells

[24] . Similar behavior of DCs was observed when they were primed with other TLR agonists, such as the TLR9 agonist CpG (Figure 19A). Thus, these data indicate that FLT3 DCs can achieve a hyperactivated state. DCs are divided into two major subsets, cDC1 and cDC2. cDC1 are typical DCs that can cross-present tumor-associated antigens and prime CD8+ T cells

[37] ,

[38] . On the other hand, cDC2 regulate type 2 immune responses to parasites and activate Th2 immunity. To confirm whether the behavior of hyperactivated DCs extends to cDC1 or cDC2, we isolated cDC1 or cDC2 from wild-type naive FLT3-DCs or from the spleen (Figure 19B). Similar to the behavior of FLT3-derived DCs, treatment with LPS and PGPC, and to a lesser extent LPS and oxPAPC, resulted in the release of TNFα and IL-1β from FLT3-derived cDC1s and cDC2 in the absence of cell death as assessed by LDH release (Figure 14A). These data indicate that PGPC is the bioactive component of oxPAPC that induces the superactivation of cDC1 and cDC2. Furthermore, a similar behavior was observed in splenic cDC2, which produced IL-1β in response to the pyroptotic stimuli LPS and alum, concomitant with pyroptotic cell death, but in response to the superactivating stimuli LPS and PGPC, produced IL-1β in the absence of cell death (Fig. 16C).In contrast, splenic cDC1s produced minimal amounts of IL-1β in response to pyroptotic or superactivating stimuli. This is because splenic cDC1s were highly susceptible to cell death after sorting and could not be primed with LPS (Figure 16C). Overall, these results demonstrate that superactivating stimuli can be used to induce IL-1β release from viable DCs differentiated in vitro or in vivo. For practical reasons, we continued to use FLT3-derived DCs as the DC source in this paper.

[0182] Example 4: Superactivated DCs enhance CTL responses in an inflammasome-dependent manner IL-1β is a critical regulator of T cell differentiation, persistent memory T cell generation, and effector function [12–14]. We were interested to determine whether superactivated DCs, which produce IL-1β for several days in the dLN, could enhance CD8+ T cell stimulation. To test this, we sought to measure OVA-specific CD8+ T cells in the dLN after subcutaneous (sc) adoptive transfer of DCs containing OVA protein. First, we examined the ability of different DC conditions to internalize OVA protein and cross-present the OVA peptide SIINFEKL on H2kb molecules. We found that all DCs in different conditions internalized OVA to a similar extent, as indicated by comparable internalization of fluorescent ovalbumin (OVA-FITC). However, we found that both activated and superactivated DCs primed with LPS or CpG exhibited enhanced SIINFEKL cross-presentation when loaded with OVA protein compared to naive counterparts. This is consistent with previous studies showing that DC maturation increases their antigen-presenting capacity. Surprisingly, pyroptotic stimulation significantly reduced the cross-presentation capacity of DCs, suggesting that pyroptotic DCs are not suitable for optimal T cell stimulation. Thus, when 1.10e6 DCs of OVA-loaded naive, activated, pyroptotic, or superactivated DCs were injected into WT mice, the superactivated DCs exhibited the highest frequency and absolute number of DCs in the dLN of recipient mice compared with naive, activated, and pyroptotic DCs. The induction of SIINFEKL+CD8+ T cells was observed (Figures 12A and 17B). The increased CD8+ T cell response mediated by superactivated DCs was dependent on inflammasome activation, because injection of LPS+PGPC-treated NLRP3- / - DCs induced weak OVA-specific T cell responses.

[0183] Example 5: Superactivating stimuli enhance the generation of memory T cells and augment antigen-specific IFNγ effector responses in an inflammasome-dependent manner We hypothesized that superactivating stimuli might be a potent adjuvant that could enhance the effects of superactivated DC injections. To investigate this possibility, mice were immunized sc with OVA alone, OVA plus an activating stimuli (LPS), or OVA plus a superactivating stimuli (LPS plus oxPAPC or PGPC). Seven and 40 days after immunization, memory and effector T cell generation in the dLN was assessed by flow cytometry using CD44 and CD62L markers, which distinguish between CD44-low CD62L-low effector T cells (Teff), CD44-high CD62L-low effector memory T cells (TEM), and CD44-high CD62L-high central memory T cells (TCM)

[47] . Seven days after immunization, superactivating stimuli were superior to activating stimuli in inducing CD8+ Teff cells (Figure 13A, upper panel and Figures 18A-B). Furthermore, at this early time point, the superactivating stimulus induced the highest amount of CD8+ TEM cells (Figure 13A, middle panel and Figures 18A-18B). At 40 days after immunization, mice exposed to the superactivating stimulus had abundant TCM cells, whereas mice immunized with OVA alone or LPS were less abundant (Figure 13A, bottom panel). Conversely, Teff and TEM cells were more abundant at 40 days after immunization in mice immunized with OVA alone or LPS compared with mice immunized with OVA+. Thus, these data indicate that the superactivating stimuli oxPAPC and PGPC enhance the magnitude of effector and memory T cell generation. Furthermore, the increased frequency of Teff cells at 7 days after immunization correlated with enhanced IFNγ responses of CD8+ T cells upon ex vivo restimulation in the presence of OVA-loaded naive BMDCs isolated from the dLNs of mice immunized with the OVA+ superactivating stimulus (Figure 18C). Furthermore, when total CD8+ T cells were isolated from mice immunized with the superactivating stimulus and cocultured with the OVA-expressing B16 tumor cell line (B16OVA), the CD8+ T cells exhibited enhanced degranulation activity compared with CD8+ T cells isolated from mice immunized with OVA alone or OVA plus LPS (Figures 13B and 18D), indicating that the superactivating stimulus enhances CTL function.

[0184] To evaluate the antigen specificity of T cells resulting from sc immunization with different activation stimuli, mice were injected with OVA alone, together with an activation stimulus (LPS), together with a pyroptotic stimulus (LPS + alum), or together with a superactivating stimulus (LPS + oxPAPC or PGPC). Alternatively, mice were immunized sc with LPS + PGPC without the OVA antigen. Seven days after immunization, CD8+ T cells were isolated from the cutaneous dLN of immunized mice and restimulated ex vivo for 7 days with naive BMDCs loaded with or without OVA to enrich for OVA-specific T cell subsets. T cell effector function of OVA-specific T cells was assessed by intracellular staining for IFNγ. TCR specificity was assessed by staining with an MHC-restricted OVA peptide tetramer. An H2kb-restricted SIINFEKL (OVA257-264) peptide tetramer was used. The frequencies of tetramer+IFNγ+ double-positive cells were measured for CD4+ and CD8+ T cell subsets. Strikingly, OVA with superactivating stimulation was superior in inducing antigen-specific T cells, because oxPAPC-based or PGPC-based immunization resulted in the induction of the highest frequency of tetramer+IFNγ+ responses upon CD8+ T cell restimulation with OVA antigen (Fig. 13C). In contrast, pyroptotic stimulation (LPS+Alum) was the weakest inducer of antigen-specific IFNγ responses (Fig. 13C). These results suggest that Alum exerts a potent inhibitory effect on the body fluids. This is consistent with previous studies showing that it is an effective adjuvant for promoting sexual immunity and Th2 responses, but not Th1 or CTL responses [39, 42, 43].

[0185] Previous studies have shown that co-immunization with recombinant IL-1β, a cytokine whose physiological activity is naturally regulated by the inflammasome, can enhance antigen-specific T cell responses [47, 13]. However, despite both superactivating and pyroptotic stimulation inducing IL-1β secretion, how is it that superactivating stimulation induces high antigen-specific T cell activity, while pyroptotic stimulation does not? To determine whether inflammasome-mediated events regulate T cell responses triggered by superactivating stimulation, we performed a controlled comparison of T cell activity in WT and NLRP3- / - mice. Notably, we found that NLRP3 is required for the superactivation-induced increase in antigen-specific responses by CD8+ T cells (Figure 13C). Thus, these data indicate that pyroptotic versus nonpyroptic inflammasome activation following immunization with superactivating or pyroptotic stimulation, respectively, induces distinctly different adaptive immune T cell regulation.

[0186] Our previous results using a DC injection strategy showed that DCs stimulated with pyroptotic stimuli lost their ability to migrate to the adjacent dLN and stimulate T cell activation, whereas DCs exposed to superactive stimuli supermigrated to the dLN and enhanced CTL responses (Figures 12A-B). However, it is unknown whether endogenous DCs achieve in vivo superactivation following immunization with superactive stimuli. To assess this, chimeric mice were generated using Zbtb46DTR and WT mice, Zbtb46DTR and NLRP3- / - mice, or Zbtb46DTR and Casp1 / 11- / - mice. To this end, 4-week-old CD45.1-irradiated mice were reconstituted on a CD45.2 background using mixed bone marrow from 80% Zbtb46DTR mice and 20% WT, 20% NLRP3- / -, or 20% Casp1 / 11- / - mice, as previously described

[51] . Six weeks after reconstitution, the efficacy of BM reconstitution was assessed in all mice by flow cytometry using CD45.1 versus CD45.2 markers. To eliminate Zbtb46+ conventional DCs, chimeric mice were treated with diphtheria toxin (DT) every other day, resulting in either WT DCs capable of hyperactivation or inflammasome-deficient (NLRP3- / - or Casp1 / 11- / -) DCs that could not hyperactivate, respectively. To test the effect of endogenous DC hyperactivation on CD8+ T cell responses, all chimeric mice received three consecutive DT injections followed by sc immunization with OVA, LPS, and PGPC. Seven days after immunization, we evaluated the CD8+ T cell responses from dLNs. Interestingly, we found that the amount of Teff CD8+ T cells was significantly reduced in chimeric mice containing DCs that could not become hyperactive, such as NLRP3- / - and Casp1 / 11- / - chimeric mice, compared with chimeric mice containing WT DCs that could become hyperactive (Figure 13D, Figure 19A).Furthermore, we found that the frequency of SIINFEKL+CD8+ T cells in the dLN or spleen was reduced in NLRP3- / - and Casp1 / 11- / - chimeric mice containing DCs that could not become hyperactivated, whereas abundant SIINFEKL+CD8+ cells were observed in chimeric mice containing WT DCs (Fig. 13E, Fig. 19B). Thus, these data clearly demonstrate that 1) endogenous DCs can reach a state of hyperactivation in vivo and enhance CTL responses following immunization with hyperactivating stimuli, and 2) inflammasome activation in endogenous DCs is important for hyperactivation-mediated protective CTL responses.

[0187] Example 6: Infiltration of superactivated DCs into lymphoid tissues is essential for superactivation-mediated CTL responses The present inventors previously demonstrated that DCs stimulated with superactive stimuli supermigrate to the dLN and induce CTL responses. We demonstrated that hyperactivation-mediated CTL responses enhanced the infiltration of endogenous hyperactive DCs into the dLN (Figures 12A-12B). To assess whether hyperactivation-mediated CTL responses require the entry of endogenous hyperactive DCs into the dLN, chimeric mice were generated as described above using Zbtb46DTR and WT or Zbtb46DTR and CCR7- / - BM (Figure 13D). Overall, endogenous transport of hyperactive DCs into the dLN is essential for hyperactivation-mediated CTL function.

[0188] Example 7: Superactive cDC1 can use complex antigen sources to stimulate preventative T cell-mediated antitumor immunity Current efforts to stimulate antitumor immunity include strategies to stimulate resident T cell populations (e.g., PD-1 blockade) or personalized cancer vaccine strategies to stimulate de novo T cell responses against tumor-specific antigens (TSAs)

[46] . The latter approach has been hampered by the unavailability of tumor cell lysates, a source of TSAs, also known as neoantigens. Therefore, efforts are underway to advance the identification of neoantigens that can be used in pure form to elicit T cell-mediated antitumor immunity. While these efforts have yielded success, the path to neoantigen identification requires the discovery of mutated TSAs as well as aberrantly expressed TSAs

[54] , which are challenging and do not represent the natural history of events. As discussed above, WTLs are an intriguing alternative source of antigens because this lysate provides the numerous antigens required for personalized antitumor immune responses. However, fundamental questions remain unanswered, such as what the most effective types of adjuvants (including those associated with different types of antigens) are that can be used in cancer vaccines.

[0189] To address the possibility that the superactivating stimulus could be an adjuvant against WTL, mice were immunized in the right flank with WTL alone, WTL mixed with the activating stimulus LPS, or WTL mixed with the superactivating stimuli LPS + oxPAPC or LPS + PGPC. The source of WTL was B16OVA cells. Fifteen days after immunization, mice were subcutaneously challenged in the upper left back with B16OVA parental cells. Naive mice or mice immunized with WTL alone did not show any protection, and all mice contained large tumors and died by 24 days after tumor inoculation (Figure 20A). Similarly, WTL + LPS immunization showed minimal protection. Although two of eight mice immunized with WTL + LPS were tumor-free, they relapsed quickly after rechallenge with B16OVA (Figure 20A), indicating that a stimulus that simply activates DCs does not confer protective immunity. In contrast, WTL immunization in the presence of LPS and oxPAPC induced a significant delay in tumor growth and provided significant protection against subsequent lethal rechallenge with B16OVA parental tumor cells, with 50% of immunized mice being completely protected (Figure 20A). To determine whether the protective response induced by oxPAPC correlated with T cell responses, tumors were harvested from mice that received each activating stimulus. Tumors from mice immunized with LPS + oxPAPC contained substantially more CD4+ and CD8+ T cells than those immunized with LPS (Figure S7B). Furthermore, when comparing the same number of T cells derived from their tumors, oxPAPC-based immunization resulted in intratumoral T cells that secreted the greatest amounts of IFNγ upon anti-CD3 and anti-CD28 stimulation (Figure 20C). Thus, the predominant restriction of tumor growth induced by the superactivating stimulus (LPS + oxPAPC) was consistent with the infiltration of inflammatory T cells into the tumor.

[0190] Notably, the protective phenotype induced by PGPC, a pure oxPAPC component, was superior to that of oxPAPC. WTL immunization in the presence of LPS + PGPC rendered 100% of mice tumor-free for 150 days after tumor challenge. These mice completely rejected a lethal rechallenge with B16OVA cells and remained tumor-free for 300 days after the initial tumor challenge (Figure 20A). Because these mice never relapsed, we were intrigued by how tumor cell growth remained controlled at the tumor injection site in mice immunized with WTL + LPS + PGPC.

[0191] Among memory T cell subsets, resident memory T cells (TRM) are defined by the expression of the CD103 integrin in addition to the C-type lectin CD69, which contributes to their resident properties in peripheral tissues

[55] . CD8+ TRM cells have recently attracted considerable attention because they accumulate at tumor sites in various human cancer tissues and correlate with more favorable clinical outcomes [54, 55, 56]. In an experimental cutaneous melanoma model, cutaneous CD8+ TRM cells promoted sustained protection against melanoma progression

[58] .

[0192] We examined the presence of TRM cells at tumor injection sites and in immunized skin biopsies of survivor mice previously immunized with the superactivating stimulus LPS+PGPC. Interestingly, 200 days after tumor inoculation, CD8+CD69+CD103+ TRM cells were highly enriched at the tumor injection site but were scarce at the immunization sites of all survivor mice (Figures 21A-B). These data are consistent with clinical and experimental reports linking high levels of TRM to long-term tumor control, potentially resulting in long-term maintenance of TRM cells at the tumor injection site [56, 57]. Thus, immunization with both WTL and superactivating stimuli appears to generate TRM cells that can stave off tumor cells.

[0193] To examine the functional specificity of these T cells, we monitored their cytotoxic lymphocyte (CTL) activity ex vivo. Circulating memory CD8+ T cells and TRM cells were isolated from the spleens or skin adipose tissue of survivor mice that had previously received a superactivating stimulus. These cells were cultured with B16OVA cells, non-OVA-expressing B16 cells, or the unrelated cancer cell line CT26. CTL activity, assessed by LDH release, was observed only when CD8+ T cells were mixed with B16OVA cells or B16 cells (Figure 21C). Killing of CT26 cells was not observed (Figure 21C), thus demonstrating the functional and antigen-specific nature of the superactivation-induced T cell response.

[0194] Based on the antigen-specific T cell responses induced by the superactivating stimulation, we confirmed whether T cells were sufficient to protect against tumor progression. CD8+ T cells were transferred from survivor mice to naive mice, which were subsequently challenged with the parental tumor cell line used as the primary immunogen. Transfer of CD8+ TRM cells or circulating CD8+ T cells from survivor mice to naive recipient mice conferred significant protection from subsequent tumor challenge, with the TRM subset playing a dominant role in protection (Figure 21D). Transfer of both T cell subsets from survivor mice to naive mice 1 week before tumor inoculation 100% protected recipient mice from subsequent tumor challenge (Figure 21D). Together, these data indicate that PGPC-based superactivating stimulation confers optimal protection in the B16 melanoma model by significantly inducing circulating and resident antitumor CD8+ T cell responses.

[0195] Example 8: Superactive stimulation provides protection against anti-PD1 resistant established tumors To determine whether superactivation stimuli could be used as a cancer immunotherapy, we examined antitumor responses in mice bearing tumors that were still growing before any additional treatment. For this study, rather than using cultured tumor cells as the antigen source, we used syngeneic tumors from unimmunized mice and generated ex vivo WTLs by dissociating harvested 10 mm tumors and then depleting CD45+ cells. Mice were inoculated subcutaneously (sc) with tumor cells in the upper left dorsum. When tumors reached a size of 3–4 mm, tumor-bearing mice were either left untreated (unimmunized) or received a therapeutic injection consisting of ex vivo WTLs and LPS+PGPC in the right flank. Two subsequent sc boosts of the therapeutic injection were administered (Figure 14A). Interestingly, superactivation-based therapeutic injections induced tumor eradication in a wide range of tumors, including B16OVA and B16F10 melanoma models and MC38OVA and CT26 colon cancer tumor models (Figures 14B–D). In all of these models, a high percentage of mice receiving the immunotherapy regimen remained tumor-free long after tumor inoculation (Figures 14B-14D). The efficacy of immunotherapy was confirmed by IL-1 in all tumor models tested. The protection conferred by the superactivation stimulus plus ex vivo WTL was dependent on IL-1β, as neutralization of IL-1β abolished the protection conferred by the superactivation stimulus plus ex vivo WTL (Figures 14B-D). Furthermore, CD8+ T cells were important for protection against immunogenic tumor models, such as B16OVA or MC38OVA tumors, whereas both CD4+ and CD8+ T cells were required for protection against less immunogenic tumors, such as CT26 and B16F-10 tumors (Figures 14B-D)

[63] . To determine the efficacy of superactivation-based immunotherapy compared with PD-1 blockade-based therapy, a control evaluation was performed. Superactivation-based immunotherapy was as efficient as anti-PD-1 therapy in the immunogenic B16OVA model but was even more efficient in tumor models insensitive to anti-PD-1 treatment, such as CT26 and B16F-10 (Figures 14B-D).

[0196] Example 9: Endogenous hyperactive DCs stimulate long-lasting T cell-mediated antitumor immunity Adoptive transfer of superactivated DCs into tumor-bearing mice induces significant antitumor immunity (Figures 12A-B). To test whether endogenous DCs can initiate hyperactivation-mediated antitumor responses, we used Zbtb46DTR mice, in which conventional DCs had been eliminated by DT injection. Zbtb46DTR or WT mice were injected sc with B16OVA cells. DT was then injected every other day to completely eliminate resident DCs in Zbtb46DTR mice before immunization. When tumors reached a size of 4 mm, Zbtb46DTR or WT mice were immunized with B16OVA WT + superactivating stimuli LPS + PGPC. In contrast to WT mice, which rejected tumors in 90% of mice, Zbtb46DTR mice (lacking DCs) failed to reject tumors. These data confirm that DCs are the initiators of hyperactivation-mediated defense (Figure 15A).

[0197] Example 10: Superactive cDC1 can use complex antigen sources to stimulate T cell-mediated antitumor immunity We demonstrated in vitro that both cDC1 and cDC2 cells can reach a hyperactivated state in response to LPS+PGPC, producing IL-1β while maintaining viability. These data necessitate further definition of the specific DC subsets that initiate hyperactivation-mediated antitumor responses in vivo. Given the importance of the cDC1 subset in tumor rejection, we hypothesized that cDC1 cells play a central role in inducing hyperactivation-mediated antitumor defense. To test this idea, we used Batf3- / - mice (which lack cDC1 but contain cDC2 cells)

[65] . To this end, we immunized Batf3- / - or WT tumor-bearing mice (containing 3–4 mm B16 OVA) with LPS+PGPC and WT. These mice received two sc boost injections 7 days apart. Unimmunized Batf3 − / − mice exhibited more severe tumor growth than unimmunized WT mice, and all mice died of tumors shortly after 18 days post-tumor inoculation. This data supports previous studies showing that rejection of highly immunogenic tumors is significantly impaired in Batf3 − / − mice lacking cDC1 cells

[65] . Interestingly, although immunization of Batf3 − / − mice improved their survival by several days compared with unimmunized Batf3 − / − mice, all Batf3 − / − mice died of tumor growth by 25 days post-tumor inoculation. In contrast, WT mice rejected tumors in 100% of tumor-bearing mice (Figure 15C). Thus, cDC1s play a crucial role in hyperactivation-mediated antitumor immunity. Furthermore, immunized WT mice induced high frequencies of antigen-specific CD8+ and CD4+ T cells in the TME and skin dLNs, whereas immunized Batf3- / - mice induced slightly reduced antigen-specific CD4+ T cells in the TME, but notably, no antigen-specific CD8+ T cells (Fig. 15D).

[0198] To further confirm the role of superactive cDC1s in inducing durable antitumor defense, we sought to assess the ability of superactive cDC1s to restore antitumor defense in Batf3- / - mice. We adoptively transferred naive, activated, or superactivated cDC1 cells into Batf3- / - mice. To this end, FLT3-derived cDC1 cells were sorted from C57BL / 6J mice as B220-MHC-II+CD11c+CD24+ cells as previously described. cDC1 cells were treated in vitro and loaded with B16OVA WTL as described above, and 1.10e6 cells were then injected sc into tumor-bearing Batf3- / - mice. In contrast to naive or activated cDC1s, which conferred only a slight improvement in mouse survival compared with uninjected mice, superactivated cDC1s induced tumor rejection in 100% of tumor-bearing mice, which remained tumor-free for more than 60 days after tumor inoculation (Figure 15E). It is noteworthy that injection of superactivated cDC1s restored CD8+ T cell responses in Batf3- / - tumors, as measured by SIINFEKL tetramer staining in tumors and skin dLNs (Figure 15F). In contrast, injection of naive or activated cDC1s failed to restore antigen-specific CD8+ T cells. cDC1-mediated tumor rejection depended on inflammasome activation, because injection of LPS+PGPC-treated NLRP3- / - cDC1s did not confer any antitumor protection and abolished the ability of superactivated cDC1s to restore CD8+ T cell responses (Figure 15F).

[0199] In addition to their ability to produce IL-1 from live cells, superactivated DCs highly migrated to adjacent dLNs and enhanced CD8+ T cell responses (FIGS. 12A-B).

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Claims

1. 1. A method for generating a population of therapeutic dendritic cells, comprising: obtaining viable dendritic cells from a cell donor; priming the dendritic cells ex vivo with a TLR ligand; The primed dendritic cells were ex- pressed with non-canonical inflammasome-activating lipids. culturing in vivo; and loading said dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells. A method comprising:

2. 1. A method of inducing an immune response in a subject, comprising: obtaining viable dendritic cells from a cell donor; priming the dendritic cells ex vivo with a TLR ligand; culturing the primed dendritic cells ex vivo with a non-canonical inflammasome-activating lipid; loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells; and administering said live dendritic cells to said subject, thereby inducing an immune response in the subject. A method comprising:

3. 1. A method of treating cancer in a subject, comprising: obtaining viable dendritic cells from a cell donor; priming the dendritic cells ex vivo with a TLR ligand; culturing the primed dendritic cells ex vivo with a non-canonical inflammasome-activating lipid; loading the dendritic cells with an immunogen, thereby generating a population of therapeutic dendritic cells; and administering said live dendritic cells to said subject, thereby treating cancer in the subject.

4. The method of any one of claims 1 to 3, wherein the cell donor and / or the subject is a mammalian subject.

5. The method of claim 4, wherein the cell donor and / or the subject is a human subject.

6. obtaining dendritic cells from a cell donor, harvesting progenitor cells from the cell donor; and culturing said precursor cells ex vivo under conditions effective to induce differentiation, thereby obtaining dendritic cells from said cell donor. The method according to any one of claims 1 to 5, comprising:

7. The method according to any one of claims 1 to 5, wherein the step of obtaining dendritic cells from a subject comprises a step of harvesting in vivo differentiated dendritic cells from the cell donor.

8. The method according to any one of claims 1 to 7, wherein the immunogen is derived from an infectious agent associated with the development of cancer.

9. The method according to any one of claims 1 to 7, wherein the immunogen is a cancer antigen.

10. The method of any one of claims 1 to 7, wherein the immunogen is a whole tumor lysate.

11. 11. The method of claim 9 or 10, wherein the immunogen is autologous.

12. The method according to any one of claims 1 to 11, wherein the priming and the culturing are carried out simultaneously.

13. The method according to any one of claims 1 to 11, wherein the priming is carried out before the culturing.

14. 14. The method of any one of claims 1 to 13, wherein the TLR ligand is selected from a TLR1 ligand, a TLR2 ligand, a TLR3 ligand, a TLR4 ligand, a TLR5 ligand, a TLR6 ligand, a TLR7 ligand, a TLR8 ligand, a TLR9 ligand, a TLR10 ligand, a TLR11 ligand, a TLR12 ligand, a TLR13 ligand, and combinations thereof.

15. The method of any one of claims 1 to 13, wherein the TLR ligand is a TLR4 ligand.

16. 16. The method of claim 15, wherein the TLR4 ligand is selected from monophosphoryl lipid A (MPLA), lipopolysaccharide (LPS), or a combination thereof.

17. 17. The method of any one of claims 1 to 16, wherein the non-canonical inflammasome-activating lipid comprises a form of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (oxPAPC).

18. The non-canonical inflammasome-activating lipid is 2-[[(2R)-2-[(E)-7-carboxy-5-hydroxyhept-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (HOdiA-PC), [(2R)-2-[(E)-7-carboxy-5-oxohept-6-enoyl]oxy-3-hexadecanoyloxypropyl]2-(trimethylazaniumyl)ethyl phosphate (KOdiA-PC), 1-palmitoyl-2-(5-hydroxy-8-oxo-octenoyl)-sn-glycero-3-phosphorylcholine (HOOA-PC), 2-[[(2R)-2-[(E)-5,8-dioxooct-6-enoyl]oxy-3-hexadecanoyloxypropoxy]-hydroxyphosphoryl]oxyethyl-trimethylazanium (KOOA-PC), [(2R)-3-hexadecanoyloxy-2-(5-oxopentanoyloxy)propoxy] propyl]2-(trimethylazaniumyl)ethyl phosphate (POVPC), [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl]2-(trimethylazaniumyl)ethyl phosphate (PGPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[2-[(Z)-oct-2-enyl]-5-oxocyclopent-3-en-1-ylidene]methyl]oxiran-2-yl]butanoyloxypropyl 17. The method of any one of claims 1 to 16, comprising the step of: [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butanoyloxy]propyl] 2-(trimethylazaniumyl)ethyl phosphate (PECPC), [(2R)-3-hexadecanoyloxy-2-[4-[3-[(E)-[3-hydroxy-2-[(Z)-oct-2-enyl]-5-oxocyclopentylidene]methyl]oxiran-2-yl]butanoyloxy]propyl] 2-(trimethylazaniumyl)ethyl phosphate (PEIPC), or a combination thereof.

19. The non-canonical inflammasome-activating lipid is [(2R)-2-(4-carboxybutanoyloxy)-3-hexadecanoyloxypropyl]2-(trimethylazaniumyl) 17. The method of any one of claims 1 to 16, comprising PGPC.

20. The method of any one of claims 2 to 19, further comprising administering an anti-cancer agent to the subject.

21. 21. The method of claim 20, wherein the anti-cancer agent is a chemotherapeutic agent.

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