Methods and agents for enhancing the immune response

Abstract

Methods are provided for enhancing an immune response in a subject by (a) identifying one or more immune response associated cell types in the subject or one or more immune response associated cell types to be administered to the subject; and (b) suppressing autophagy in the one or more immune response associated cell types in the subject or in the one or more immune response associated cell types to be administered to the subject, wherein the suppressing is performed in vivo, ex vivo, in vitro, or by any combination thereof on one or more of either or both cell types. Such methods enhance immune function in vivo, ex vivo or in vitro, thus enhancing the immune response in the subject.

Description

METHODS AND AGENTS FOR ENHANCING THE IMMUNE RESPONSECROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. provisional patent application serial no. 63 / 569,551, filed March 25, 2024, and is incorporated here by reference in its entirety.GOVERNMENT SUPPORT

[0002] This invention was made with government support under W81XWH-22- 1-0920, awarded by the Medical Research and Development Command, and CA221296 awarded by the National Institutes of Health. The government has certain rights in the invention.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing conforming the rules of WIPO Standard ST.26 which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML copy, created on March 23, 2025, is named P.628185-PC_SQL_23MAR25.xml, and is 3,472 bytes in size.BACKGROUND

[0004] Despite significant advances in cancer treatment, cancer prognosis remains poor for many tumor types. Over 600,000 Americans die of cancer each year. Targeted therapies can be highly effective, but are only valuable for a limited subset of tumors with specific mutations.

[0005] In addition to targeted therapies, strategies designed to activate the immune response by modulating immune checkpoints have benefited cancer patients by eliminating barriers to T cell activation. For example, programmed death- 1 (PD-1), a receptor expressed on activated T and B cells, inhibits T cell receptor signaling when activated by its ligand PD-L1. CTLA4 is also expressed on activated T cells and can repress T cell signaling when bound to CD80 or CD86. FDA-approved monoclonal antibodies nivolumab (OPDIVO) and pembrolizumab (KEYTRUDA) that target PD-1 and ipilimumab (YERVOY) that targets CTLA4 can improvepatient survival, but they are only effective in some patients. Patients who respond to immunotherapy arc more likely to have “hot” tumors characterized by high pretreatment tumor PD-L1 expression, high pretreatment infiltrating CD8+T-cells, and high cytokine levels. For both targeted therapy and immune checkpoint inhibitors, resistance can develop, and the treatment itself can cause serious side effects.

[0006] Allogeneic hematopoietic stem cell transplantation can be a curative tool for patients with hematological malignancies. These transplants can eradicate the patient’s tumor- inducing hematopoietic stem cells (HSCs). In addition, the mismatched HSCs can activate an antileukemia immune response. Unfortunately, this strategy also results in graft- versus-host disease, damage inflicted by introduced T cells to other tissues. As a result, stem cell transplantation has limited effectiveness for solid tumors.

[0007] Approval of autologous chimeric antigen receptor (CAR)-T cell therapy for B cell malignancy has spurred rapid advancement in cell-based cancer treatment. T cells can be collected from a patient, modified to express chimeric antigen receptors (CAR), and returned to the same patient, thus eliminating graft-versus-host disease. While CAR T-cells are effective against some types of hard-to-treat hematological tumors, they, too, have had more limited success for solid tumors for reasons that include a failure to accumulate at the active site.

[0008] The ability to activate an anti-tumor immune response that is localized to the tumor, rather than systemic, thereby breaking the concordance between graft-versus-host disease and graft-versus-tumor activity, could revolutionize cancer treatment.SUMMARY

[0009] In one aspect, a method is provided for enhancing an immune response in a subject comprising the steps of: a. identifying one or more immune response associated cell types in the subject or one or more immune response associated cell types to be administered to the subject; andb. suppressing autophagy in the one or more immune response associated cell types in the subject or in the one or more immune response associated cell types to be administered to the subject, wherein the suppressing is performed in vivo, ex vivo, in vitro, or by any combination thereof on one or more of either or both of the cell types in the subject or to be administered to the subject; wherein an immune function is enhanced in vivo, ex vivo or in vitro by the one or more immune response associated cell types in the subject or in the one or more immune response associated cell types after administration to the subject, thus enhancing the immune response in the subject.

[0010] In one aspect, a method is provided for enhancing an immune response in a subject comprising the steps of: a. identifying one or more immune response associated cell types in the subject; and b. suppressing autophagy in the one or more immune response associated cell types in the subject, wherein the suppressing is performed in vivo, ex vivo, in vitro, or by any combination thereof; wherein an immune function is enhanced in vivo, ex vivo or in vitro by the one or more immune response associated cell types in the subject, thus enhancing the immune response in the subject.

[0011] In one aspect, a method is provided for enhancing an immune response in a subject comprising the steps of: a. identifying one or more immune response associated cell types to be administered to the subject; and b. suppressing autophagy in the one or more immune response associated cell types to be administered to the subject, wherein the suppressing is performed in vivo, ex vivo, in vitro, or by any combination thereof;wherein an immune function is enhanced in vivo, ex vivo or in vitro by the one or more immune response associated cell types after administration to the subject, thus enhancing the immune response in the subject.

[0012] In some embodiments, the immune response associated cell types in the subject are one or more of dendritic cells, macrophages, monocytes, cancer-associated fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells. In some embodiments, the immune response associated cell types to be administered to the subject are one or more of dendritic cells, macrophages, monocytes, cancer-associated fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells. In some embodiments, the immune response associated cell types to be administered to the subject are obtained from the subject, obtained from a donor, or obtained from banked immune response associated cells or blood products, or induced in vitro or ex vivo from any of the foregoing. In some embodiments, an immune response associated cell type comprises a tertiary lymphoid structure, a lymph node, a secondary lymphoid organ, a B cell follicle, a tumor, or a wound. In some embodiments, the cell types are fibroblast reticular cells, follicular dendritic cells or lymphoid organizer cells.

[0013] In some embodiments, the suppressing autophagy is performed on the one or more immune response associated cell types in, or administered to, the subject, independently by any one or more of: a. exposing the one or more immune response associated cell types in vivo, ex vivo or in vitro to one or more agents that suppresses autophagy; and / or b. inactivating one or more genes responsible for or associated with autophagy in the one or more immune response associated cell types in vivo, ex vivo or in vitro; and / orc. activating one or more genes responsible for suppressing autophagy in the one or more immune response associated cell types in vivo, ex vivo or in vitro; and / or d. any combination thereof.

[0014] In some embodiments of any of the foregoing, the autophagy is non-canonical autophagy. In some embodiments, the agent that suppresses autophagy inhibits LC3 mediated processing. In some embodiments, the agent that inhibits LC3 mediated processing inhibits LC3 lipidation. In some embodiments, the agent that inhibits LC3 lipidation is selected from 5-(2,3- dichlorophenyl)-2-(((2-morpholinoethyl)amino)methylene) cyclohexane- 1 ,3-dione (DC-LC3in- D5) or an analogue thereof. In some embodiments, the agent that inhibits LC3 lipidation is a V- ATPase inhibitor. In some embodiments, the V-ATPase inhibitor is BafAl, concanamycin A (ConA), or any combination thereof, and may further comprise diphenyleneiodonium (DPI), GSK2795039, or any combination thereof.

[0015] In some embodiments, the suppressing autophagy comprises suppressing Atg7 expression or function. In some embodiments, the Atg7 gene is inactivated in the cell type. The inactivation may be in vitro, ex vivo or in vivo. In some embodiments, the suppressing Atg7 expression or function comprises exposing the cell type to a siRNA targeting Atg7. In some embodiments, inactivating autophagy comprises exposing the cell type to a Cas9 with a guide RNA against ATG7. In some embodiments, the inactivating Atg7 is Atg7 knockout.

[0016] In some embodiments, the gene associated with autophagy is ATG7.

[0017] In some embodiments, a combination of any two or more of (a), (b) and / or (c) above synergistically provides a suppression of autophagy.

[0018] In some embodiments, the enhancing the immune response comprises one or more of: enhanced antigen presentation by dendritic cells; enhanced cytokine production; enhanced chemokine production; elevated inflammatory gene expression; elevated macrophage activation; elevated T cell recruitment; elevated T cell activation, or any combination thereof. In some embodiments, the enhanced antigen presentation comprises enhanced presentation of a cancer antigen or a fragment thereof.

[0019] In some embodiments, autophagy is suppressed in a cell type in vitro or ex vivo, the cell type is exposed to an antigen in vitro or ex vivo, and the cell type is infused into the subject.

[0020] In some embodiments, the autophagy is suppressed in dendritic cells in vitro or ex vivo, the dendritic cells are exposed to an antigen in vitro or ex vivo, and the dendritic cells are infused into the subject.

[0021] In some embodiments, the autophagy is suppressed in macrophages or monocytes in vitro or ex vivo, the macrophages or monocytes are exposed to an antigen in vitro or ex vivo, and the macrophages or monocytes are infused into the subject.

[0022] In some embodiments, a tumor is infused with an agent that suppresses autophagy.

[0023] In some embodiments, autophagy is suppressed in fibroblasts which are infused into a tumor.

[0024] In one aspect, a method is provided for enhancing an immune response in a subject comprising the steps of: a. identifying one or more immune response associated cell types in the subject or one or more immune response associated cell types to be administered to the subject; and b. suppressing or inactivating Atg7 in the one or more immune response associated cell types in the subject or in the one or more immune response associated cell types to be administered to the subject, wherein the suppressing or inactivating is performed in vivo, ex vivo, in vitro, or by any combination thereof on one or more of either or both of the cell types in the subject or to be administered to the subject; wherein an immune function is enhanced in vivo, ex vivo or in vitro by the one or more immune response associated cell types in the subject or in the one or more immune response associated cell types after administration to the subject, thus enhancing the immune response in the subject.

[0025] In one aspect, a method is provided for enhancing an immune response in a subject comprising the steps of: a. identifying one or more immune response associated cell types in the subject; and b. suppressing or inactivating Atg7 in the one or more immune response associated cell types in the subject, wherein the suppressing or inactivating is performed in vivo, ex vivo, in vitro, or by any combination thereof; wherein an immune function is enhanced in vivo, ex vivo or in vitro by the one or more immune response associated cell types in the subject, thus enhancing the immune response in the subject.

[0026] In one aspect, a method is provided for enhancing an immune response in a subject comprising the steps of: a. identifying one or more immune response associated cell types to be administered to the subject; and b. suppressing or inactivating Atg7 in the one or more immune response associated cell types to be administered to the subject, wherein the suppressing or inactivating is performed in vivo, ex vivo, in vitro, or by any combination thereof; wherein an immune function is enhanced in vivo, ex vivo or in vitro by the one or more immune response associated cell types after administration to the subject, thus enhancing the immune response in the subject.

[0027] In some embodiments, the immune response associated cell types in the subject are one or more of dendritic cells, macrophages, monocytes, cancer-associated fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells. In some embodiments, the immune response associated cell types to be administered to the subject are one or more of dendritic cells, macrophages, monocytes, cancer-associated fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrowderived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells. In some embodiments, the immune response associated cell types to be administered to the subject are obtained from the subject, obtained from a donor, or obtained from banked immune response associated cells or blood products, or induced in vitro or ex vivo from any of the foregoing. In some embodiments, an immune response associated cell type comprises a tertiary lymphoid structure, a lymph node, a secondary lymphoid organ, a B cell follicle, a tumor, or a wound. In some embodiments, the cell types are fibroblast reticular cells, follicular dendritic cells or lymphoid organizer cells.

[0028] In some embodiments, the suppressing or inactivating Atg7 is performed on the one or more immune response associated cell types in, or administered to, the subject, independently by any one or more of: a. exposing the one or more immune response associated cell types in vivo, ex vivo or in vitro to one or more agents that suppresses or inhibit Atg7; and / or b. inactivating Atg7 in the one or more immune response associated cell types in vivo, ex vivo or in vitro; and / or c. the combination thereof.

[0029] In some embodiments, the Atg7 gene is inactivated in the cell type. The inactivation may be in vitro, ex vivo or in vivo. In some embodiments, the suppressing Atg7 expression or function comprises exposing the cell type to a siRNA targeting Atg7. In some embodiments, inactivating ATG7 comprises exposing the cell type to a Cas9 with a guide RNA against ATG7. In some embodiments, the inactivating Atg7 is Atg7 knockout.

[0030] In some embodiments, a combination of (a) and (b) synergistically provides an enhancement of the immune response.

[0031] In some embodiments, the enhancing the immune response comprises one or more of: enhanced antigen presentation by dendritic cells; enhanced cytokine production; enhanced chemokine production; elevated inflammatory gene expression; elevated macrophage activation; elevated T cell recruitment; elevated T cell activation, or any combination thereof. In someembodiments, the enhanced antigen presentation comprises enhanced presentation of a cancer antigen or a fragment thereof.

[0032] In some embodiments, Atg7 is suppressed or inactivated in a cell type in vitro or ex vivo, the cell type is exposed to an antigen in vitro or ex vivo, and the cell type is infused into the subject.

[0033] In some embodiments, Atg7 is suppressed or inactivated in dendritic cells in vitro or ex vivo, the dendritic cells are exposed to an antigen in vitro or ex vivo, and the dendritic cells are infused into the subject.

[0034] In some embodiments, Atg7 is suppressed or inactivated in macrophages or monocytes in vitro or ex vivo, the macrophages or monocytes are exposed to an antigen in vitro or ex vivo, and the macrophages or monocytes are infused into the subject.

[0035] In some embodiments, a tumor is infused with an agent that suppresses or inactivates Atg7.

[0036] In some embodiments, Atg7 is suppressed or inactivated in fibroblasts which are infused into a tumor.

[0037] In some embodiments of any of the foregoing methods, the subject has an immunogen- associated disease or condition for which enhanced immunity directed thereto is therapeutically beneficial, or wherein the subject is at risk for an immunogen-associated disease or condition for which enhanced immunity directed thereto is preventatively beneficial. In some embodiments, the disease is cancer. In some embodiments, the cancer is melanoma.

[0038] In some embodiments or any of the foregoing methods, the subject is also administered immune checkpoint blockade. In some embodiments, the immune checkpoint blocks or inhibits CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN- 15049, CHK 1 kinase, CHK2 kinase, A2aR, or a B-7 family ligand.

[0039] In one aspect, an immune response associated cell type is provided in which autophagy is suppressed. In some embodiments, the immune response associated cell type comprises dendritic cells, macrophages, monocytes or fibroblasts. In some embodiments, the cell type is from a subject, from a donor, or from banked cells or blood products. In some embodiments, the autophagy is non-canonical autophagy.

[0040] In some embodiments, the immune response associated cell type cell is for administering to a subject having an immunogen-associated disease or condition for which enhanced immunity directed thereto is therapeutically beneficial, or administering to a subject at risk for an immunogen-associated disease or condition for which enhanced immunity directed thereto is preventatively beneficial.BRIEF DESCRIPTIONS OF THE FIGURES

[0041] Figures 1A-1D. Reduced melanoma growth in Atg7-deficient mice. Figure 1A.Immunoblots of skin. n=3, mean ± SD, ANOVA, female. Figure IB. Autophagosome counts in electron micrographs. Mean ± SEM (for all figs unless noted), n=ll-38 images, t-test, male. Figure 1C. Tumor volumes and weights. n=16-18, ANOVA, male and female. Figure ID.YUMM1.1 tumor volumes in Ubc.Atg7F / F and Atg7F / F mice. n=6-7, t-test, male. For this and all figures: *, p<0.05; **, p<0.01 ; ***, p< 0.001 ; and ****, p< 0.0001.

[0042] Figures 2A-2C. ATG7 loss in bone-marrow-derived cells reduces tumor growth.Figure 2A. ATG7 Immunoblot after transplantation. Figure 2B. Skin from mice with Ubc.LSL- ZsGreen bone marrow. Scale bar=50pm. Figure 2C. Tumor volumes and weights in mice with indicated genotypes in bone marrow cells (BMCs) and host. n=10-12, ANOVA, female.

[0043] Figure 3. Elevated macrophages in autophagy-deficient mice. Macrophages (CD45+Zombie'B220’CD3'CDllb+CDllc‘F4 / 80+) in Atg7F / Fmice with Ubc.Atg7F / For Atg7F / Fbone marrow. n=5, t-test, female.

[0044] Figures 4A-4D. Increased T cells in melanomas in autophagy deficient mice. Figure 4A. CD8+T cells (CD8+TCRP+NK1.1‘) in mice without tumors, n = 5, t-test, female. Figure 4B. CD8+T cells in melanomas in Ubc. A tg7F / Fmice. n=4, t-test, female. Figure 4C. CD8+T cells inMC38 tumors. n=5-6, Mann-Whitney, female. Figure 4D. Granzyme B in CD8+T cells in MC38 tumors. n=5-6, Mann- hitney, female.

[0045] Figures 5A-5I. T cells mediate reduced tumor growth in ATG7-deficient mice.Figure 5A. Volumes and weights of tumors in mice with a-TCRB or isotype control. n=31-52, t- test, female. Figure 5B. Volumes and weights of MC38 tumors in mice with a-TCRB or isotype control. n=14-52, ANOVA, female. Figure 5C. A DC-T cell axis mediates the smaller tumor size in autophagy-deficient mice and sensitizes to ICI. Effector memory (CD62L’CD44hlgh), central memory (CD62L+CD44lllgh), and naive (CD62L+CD44low) T cells in spleens of mice with B16- F10 melanomas. n=4-5, t-test, female. Volumes of YUMM1.1 tumors in S100a4.Atg7F / F(n=7, t- test, male) or in Figure 5D or Ubc.Atg7F / F(n=3, t-test, male). Figure 5E. Mice and controls with a-TCRb or isotype control. Figure 5F. Tumor volumes of B16-F10 tumors in Zbtb46.Atg7F / F (zDC / +) and control mice. n=7-8 mice. One outlier tumor and 2 ulcerated tumors were eliminated from analysis. Male mice. Figure 5G. Weights of B16-F10 tumors in Zbtb46.Atg7F / F (zDC / +) and control mice. n=7-8 mice. One outlier tumor and 2 ulcerated tumors were eliminated from analysis. Male mice. Figure 5H. Tumor volumes of B16-F10 tumors in Zbtb46.Atg7F / F (zDC / +) and control mice. n=7-8 mice. One necrotic tumor was eliminated from analysis. Male and female mice. Figure 51. Weights of B16-F10 tumors in Zbtb46.Atg7F / F (zDC / +) and control mice. n=7-8 mice. One necrotic tumor was eliminated from analysis. Male and female mice.

[0046] Figures 6A-6B. Loss of autophagy combines with immune checkpoint inhibition to further reduce melanoma growth. Figure 6A. Weights of melanomas in A / gZ-dclicicnt or control mice treated with a-PDl / CTLA-4 or isotype control. n=23-25, ANOVA, female. Figure 6B. Weights of melanomas in mice treated with HCQ or PBS and oc-PDl / CTLA-4 or isotype control. n= 16-20, ANOVA, female.

[0047] Figure 7 depicts a model for autophagy in the host.

[0048] Figure 8 depicts reduced division, increased apoptosis, and decreased vasculature in melanomas in Ubc.Atg7F / Fmice. IHC staining (left) and quantification (right) for Ki-67, cleaved caspase-3, and CD31 in B16-F10 melanomas. n=3 slides, up to 12 images per slide, ANOVA, male and female. Scale bar = 50 pm.

[0049] Figures 9A-9K show that Atg7 inactivation in S100A4+cells reduces tumor growth. Figure 9A. S100A4 IHC staining. Figure 9B. Immunoblotting MEFs for ATG7. Figure 9C. Volumes and weights of melanomas. n=13-14, ANOVA, male and female. Figure 9D. IHC for cleaved caspase 3. n=8-10, ANOVA. Scale bar=50 pm. Figure 9E. Volumes of MC38-derived tumors. n=26-28, t-test, female. Figure 9F. Left: S100A4 IB in bone marrow cells (BMCs), dendritic cells (DC), and bone marrow-derived macrophages (BMMs). Middle and right: ATG7 and LC3 in BMMs (middle) and DCs (right) from Atg7F / F, S100a4.Atg7F / For Ubc.Atg7F / Fmice. Figure 9G. Volumes of YUMM 1.1 -derived melanomas. n=6-7, t-test, male. Figure 9H. DC (CDllc+of CD45+CD3'B220‘ cells) in B16-F10 tumors and spleens of S100a4.Atg7F / Fand Atg7F / Fmice. n=7-8, female. Figure 91. CD8+ T cells in B16-F10 melanomas in S100a4.Atg7F / F(n=8) mice, female. Figure 9J. Granzyme B in CD8+ T cells in B16-F10 melanomas. n=4-6, female. Figure 9K Fraction of CD4+ T cells in B16-F10 melanomas that are Foxp3+. n=5, Mann- Whitney, female.

[0050] Figures 10A-10B depicts increased inflammatory gene expression in melanomas in At 7-deficient mice. Figure 10A. RNA-seq experimental design (left). Heat map of differentially expressed genes (right). FDR < 0.05. n=3. One outlier mouse was excluded. Figure 10B. Gene Ontology enrichment of genes upregulated in melanomas in S100a4.Atg7F / Fmice.

[0051] Figures 11A-11E depict elevated monocytes and macrophages in autophagydeficient mice. Figure HA. Fraction of CD45+cells that are monocytes (CD45+Zombie’B220‘ CD3 CDllb+CDllc’F4 / 80 ) or macrophages (CD45+Zombie B220 CD3 CDllb+CDllc F4 / 80+). n=4, t-test, female. (All flow cytometry data is fraction of CD45+unless noted). Figure 11B. Cells with monocyte marker CDllb or macrophage marker F4 / 80 in mice with melanomas. n=5, t-test, female. Figure 11C. Monocytes (CD45+Zombie’B220‘CD3'CDllb+CDllc’F4 / 80') in Ubc. A tg7F / Fmice with Ubc.Atg7F / Fbone marrow and Atg7F / Fmice with Atg7F / Fbone marrow with melanomas. n=4-8, t-test, female. Figure 11D. Macrophages (CD45+Zombie’B220'CD3‘ CDllb+CDllc‘F4 / 80+) in Atg7F / Fmice with Ubc.Atg7F / For Atg 7F / Fbone marrow. n=5, t-test, female. Figure HE. Fraction of Ml macrophages (CD45+Zombie'B220‘CD3‘CDllb+CDllc' F4 / 80+CDllc+CD206 ) in spleens of mice with melanomas. n=7-8, t-test, female.

[0052] Figures 12A-12D depict elevated cytokines in 4 / «7-deficient mice with melanomas. Figures 12A-12C. Multiplex ELISA cytokine levels in plasma of mice without (Figure 12A) or with melanomas (Figures 12B, 12C). Fold change in S100a4.Atg7F / Fto Atg7F / F(Figure 12B) or Ubc.Atg7F / i' to Atg7F / t(Figure 12C). n=2-4, t-test, male and female. Figure 12D. Cytokine arrays of YUMM1.1 melanomas. n=4, t-test, female.

[0053] Figures 13A-13H depicts increased T cells in melanomas in autophagy deficient mice. Figure 13A. CD8+T cells (CD8+TCRP+NK1.1‘) in mice without tumors, n = 5, t-test, female. Figure 13B. T cells in mice with melanomas. n=5, t-test, female. Figures 13C-13D. CD8+ T cells in melanomas in S100a4.Atg7F / F(Figure 13C, n=8) or Ubc.Atg7F / F(Figure 13D, n=4) mice, t-test, female. Figure 13E. Granzyme B in CD8+T cells in melanomas. n=4-6, t-test, female. Figure 13F. Fraction of CD4+T cells in melanomas that are Foxp3+. n=5, Mann- Whitney, female. Figure 13G. CD8+T cells in MC38 tumors. n=5-6, Mann- Whitney, female. Figure 13H. Granzyme B in CD8+T cells in MC38 tumors. n=5-6, Mann- Whitney, female.

[0054] Figures 14A-14C shows T cells transition from naive to effector in Atg7-deficient mice. Figures 14A-14B. Effector memory (CD62L’CD44hlgh), central memory (CD62L+CD44hlgh), and naive (CD62L+CD44low) T cells in spleens (Figure 14A) and blood (Figure 14B) of mice with melanomas. n=4-5, t-test, female. Figure 14C. Fraction of CD8+T cells that are effector memory, central memory, or naive in blood of transplanted mice with melanomas. Bone marrow cell (BMC) and host genotypes are shown. n=5, t-test, female.

[0055] Figures 15A-15D show T cells mediate reduced tumor growth in ATG7-deficient mice. Figurel5A. T cells in melanomas in mice with anti-TCRp or isotype control antibodies. n=5, ANOVA, male and female. Figure 15B. Volumes and weights of tumors in mice with a- TCRP or isotype control. n=31 -52, t-test, female. Figure 15C. Volumes of tumors in mice with a-TCRp or isotype control. n=6-8, ANOVA, female. Figure 15D. Volumes and weights of MC38 tumors in mice with a-TCRP or isotype control. n=14-52, ANOVA, female.

[0056] Figures 16A-16D depict loss of autophagy combined with immune checkpoint inhibition to further reduce melanoma growth. Figures 16A-16B. Weights of melanomas in Atg7-deficient or control mice treated with a-PDl / CTLA-4 or isotype control. (Figurel6A) Ubc.Atg7F / F. n=23-25, ANOVA, female. (Figure 16B) S100a4.Atg7F / F. n=12-14, ANOVA,female. Figure 16C. Tmmunoblots (left) and quantification (right) of SQSTMl / p62 in livers from mice treated with HCQ (60 mg / kg, i.p. daily) or PBS vehicle control, and a-PDl / CTLA-4 (100 pg each i.p. every 2-3 days) or isotype control. n=4, t-test. Figure 16D. HCQ. n=16-20, ANOVA, female.

[0057] Figure 17 shows that autophagy-deficient DC have higher levels of noncanonical NF-kB signaling. IB of DC from Ubc.Atg7F / Fand Atg7F / Fmice with or without bafilomycin Al treatment (100 nM for 3 hr) for canonical and noncanonical NF-kB components with -actin as a loading control.

[0058] Figures 18A-18F show melanoma cells induce autophagy in surrounding cells via TGFb signaling. Figure 18A. Tumor-remote skin (left) or a primary melanoma biopsy sample (right) stained with H&E and analyzed with immunohistochemistry for LC3. Scale bar = 50 pm. Figure 18B. LC3 staining quantified in 23 melanomas for 3-7 regions per tumor. Student’s t-test. Figure 18C. IB of LC3-II in fibroblasts co-cultured with four melanoma cell lines or melanocytes (PCS, M: medium). Figure 18D. IB of LC3-II with A2058 melanoma co-culture in the presence of Baf-IA. Figure 18E. TGF- responsive luciferase activity in fibroblasts cultured with TGFp or A2058 melanoma cell conditioned medium (CM) and a TGFp or control IgG. Figure 18F. IB for LC3 in human fibroblasts incubated with or without A2058 conditioned medium, SMAD inhibitor LY-364947, and Baf-A 1 .

[0059] Figure 19 shows CRISPR / Cas9 editing. IB of CRISPR editing of Atg7 in fibroblasts with Cas9 endonuclease, an Atg7 guide RNA, with and without enhancer carrier DNA, or fibroblasts that were electroporated with Cas9 endonuclease, but the Atg7 guide RNA was omitted.

[0060] Figure 20 shows that autophagy-deficient dendritic cells are more effective at activating T cells than autophagy-proficient dendritic cells. Bone marrow was isolated from Atg7F / F and Ubc.Atg7F / F mice and induced to differentiate into dendritic cells. T cells were introduced into the culture and allowed to proliferate. Ten days later, wild-type dendritic cells pre-loaded with B16-F10 lysate were introduced into the cultures. The number of effector T cells (CD45+NK1.1’TCR+CD8+CD4‘CD44+CD62L‘) in each culture was determined with flow cytometry. n=2, t-test.DETAILED DESCRIPTION

[0061] The (macro) autophagy pathway is a pathway that captures and degrades cytoplasmic material in the lysosome. Autophagy can serve as a mechanism to protect against the accumulation of cellular’ and genetic damage. Inactivation of autophagy in the entire mouse or bone marrow-derived cells resulted in reduced growth of melanoma allografts. Loss of autophagy in the entire mouse also resulted in reduced growth of introduced colorectal carcinoma cells. Elevated inflammatory gene expression, monocytes, macrophages, cytokines, chemokines, T cell recruitment, and T cell activation was observed when tumors were grown in autophagy-deficient mice. Depleting T cells resulted in increased growth of melanomas and colon adenocarcinoma allografts, demonstrating CD8+T cell-mediated killing in tumors hosted in autophagy-deficient mice. When autophagy inhibition in the host was combined with immune checkpoint blockade, melanomas were smaller than achieved by either treatment individually.

[0062] The autophagy pathway responds to signals including nutrient depletion to create autophagosomes that capture cytoplasmic material and fuse with lysosomes for metabolite recycling and energy generation. Autophagy degrades damage- associated molecules to reduce the release of pro-inflammatory cytokines. As will be described herein, inhibiting the autophagy pathway in the nontumor cells in the tumor microenvironment results in activation of an antitumor immune response that reduces tumor growth. Autophagy inactivation in the host resulted in elevated inflammation-associated gene expression and pro-inflammatory cytokines, a transition of T cells from naive to effector, and higher levels of CD8+T cells at the tumor. Depleting T cells resulted in increased growth of tumors hosted in autophagy-deficient mice, demonstrating the importance of CD8+T cell- mediated killing for the slower growth of these tumors. In some embodiments, this disclosure forms the basis for translating these observations to benefit cancer patients with an emphasis on patients from diverse backgrounds.

[0063] Based on multiple lines of experimentation, in some embodiments, loss of autophagy in dendritic cells contributes to reduced tumor growth in autophagy-deficient hosts. By culturing macrophages and dendritic cells from autophagy- deficient mice, elevated levels were found of key transcription factors in the NF-KB pathway, a pathway closely associated with the activation of dendritic cells that allows them to generate effector CD8+ T cells. Such observation in someembodiments represents a potentially important connection between metabolism and the inflammatory response, which may affect immune cell activation in the context of cancer.

[0064] The interplay between autophagy and the NF-KB pathway in macrophages and dendritic cells in culture, in mouse models of cancer and in primary tumors from a diverse cancer patients provides the basis for the methods disclosed herein. In one embodiment, decreased autophagy in immune cells enhances the immune response. As will be described herein, such decreased autophagy, by any one or combination of several methods, in one or more immune cell types, and at various stages in modulating the immune response, such as in vitro, ex vivo or in vivo, enhancement of the immune response may be achieved. Use of such methods in cancer therapy among other conditions and diseases is fully embraced herein.Inhibiting autophagy

[0065] In some embodiments, autophagy is inhibited by any of the methods disclosed herein in any one or more of the cell types disclosed herein. In some embodiments, canonical autophagy is inhibited. In some embodiments, non-canonical autophagy is inhibited. In some embodiments, both canonical and non-canonical autophagy are inhibited. In some embodiments, canonical autophagy, non-canonical autophagy, or both, are inhibited in a cell type. In some embodiments, canonical autophagy, non-canonical autophagy, or both, are inhibited in multiple cell types. Such inhibition, in some embodiments, is achieved by exposing one or more cell types to an agent that inhibits autophagy, herein referred to as chemical or pharmacological inhibition, by a chemical or pharmacological agent. In some embodiments, inhibition of autophagy is achieved by genetic manipulation of one or more cell types, by e.g., knockout, gene inactivation, RNA interference, PROTAC, dTAG or any other method. In some embodiments both chemical inhibition and genetic manipulation are performed on the same or different cell types. Non-limiting examples are described below.Chemical inhibition

[0066] In some embodiments, non-canonical autophagy is chemically inhibited. In some embodiments, a chemical agent inhibits LC3 mediated processing. In some embodiments, the chemical agent that inhibits LC3 mediated processing inhibits LC3 lipidation. Non-limitingexamples of an agent that inhibits LC3 lipidation includes 5-(2,3-dichlorophenyl)-2-(((2- moi'pholinocthyl)amino)mcthylcnc) cyclohexane- 1,3-dionc (DC-LC3in-D5) or an analogue thereof. In some embodiments, the agent that inhibits LC3 lipidation is a V-ATPase inhibitor, such as but not limited to BafAl and concanamycin A (ConA). In some embodiments, diphenyleneiodonium (DPI) or GSK2795039 are used.

[0067] In some embodiments, Atg7 is chemically inhibited. In one embodiment, a pyrazolopyrimidine sulfamate is used, such as described in Huang et al., Discovery and optimization of pyrazolopyrimidine sulfamates as ATG7 inhibitors, Bioorg Med Chem 2020 Oct 1; 28(19): 115681. Other compounds are described in WO2018089786. These and any other references cited herein are incorporated herein by reference in their entireties.

[0068] In some embodiments, canonical autophagy is chemically inhibited. Compound such as chloroquine, hydroxychloroquine, SAR405 (see Ronan B, Flamand O, Vescovi L, Dureuil C, Durand L, Fassy F, et al. A highly potent and selective Vps34 inhibitor alters vesicle trafficking and autophagy. Nat Chem Biol. 2014; 10: 1013-9), bafilomycin Al, EACC (see Vats S, Manjithaya R. A reversible autophagy inhibitor blocks autophagosome-lysosome fusion by preventing Stxl7 loading onto autophagosomes. Mol Biol Cell. 2019; 30: 2283-95).

[0069] As noted herein, any of the foregoing agents may be used singly or in any combination with another chemical agent, or in combination with a genetic inhibition method, to practice the teaching disclosed herein.Genetic inhibition

[0070] In some embodiments, canonical or non-canonical autophagy is inhibited or suppressed by genetic means, by inactivating a gene involved in autophagy, by any of any method known in the art, such as but not limited to knockout, inactivation, RNA interference (e.g., siRNA, shRNA), etc. In some embodiments, CRISPR / Cas9 and a guide is used to inactivate one or more genes involved in autophagy. Such gene may be, by way of non-limiting example, ATG7.

[0071] The inactivation of a gene involved in autophagy, such as but not limited to ATG7, may be performed in vitro, ex vivo or in vivo. In some embodiments, cells such as from a cell line are treated in vitro with an agent to inhibit or suppress autophagy. In some embodiments, cells froma subject or patient are treated ex vivo with an agent to inhibit or suppress autophagy, after which the cells arc returned to the subject or patient. In some embodiments, a subject or patient is treated in vivo with an agent that inhibits or suppresses autophagy. In some embodiments, the in vivo treatment is targeted to cells of interest. In some embodiments, the in vivo treatment is provided at the site of cells of interest. In some embodiments the in vivo treatment is systemic.

[0072] In one embodiment, suppressing autophagy comprises suppressing Atg7 expression or function. In one embodiment, the ATG7 gene is inactivated in the cell type. In one embodiment, suppressing Atg7 expression or function comprises exposing the cell type to a siRNA targeting ATG7. In one embodiment, inactivating Atg7 is by Atg7 knockout. In some embodiments, CRISPR / Cas9 is used to inactivate Atg7.

[0073] Thus, inactivation of Atg7 may be performed in vitro, ex vivo or in vivo. In some embodiments, cells such as from a cell line are treated in vitro with an agent to inactivate ATG7. In some embodiments, cells from a subject or patient are treated ex vivo with an agent to inactivate ATG7, after which the cells are returned to the subject or patient. In some embodiments, a subject or patient is treated in vivo with an agent that inactivates ATG7. In some embodiments, the in vivo treatment to inactivate ATG7 is targeted to cells of interest. In some embodiments, the in vivo treatment to inactivate ATG7 is provided at the site of cells of interest. In some embodiments the in vivo treatment is systemic. Such inactivation of ATG7 may be achieved using a siRNA targeting Atg7, or a Cas9 with a guide RNA against ATG7.

[0074] Thus, inactivation of Atg7 using siRNA may be performed in vitro, ex vivo or in vivo. In some embodiments, cells such as from a cell line are treated in vitro with siRNA to inactivate ATG7. In some embodiments, cells from a subject or patient are treated ex vivo with siRNA to inactivate ATG7, after which the cells are returned to the subject or patient. In some embodiments, a subject or patient is treated in vivo with siRNA that inactivates ATG7. In some embodiments, the in vivo treatment using siRNA to inactivate ATG7 is targeted to cells of interest. In some embodiments, the in vivo treatment to inactivate ATG7 is provided at the site of cells of interest. In some embodiments the in vivo treatment is systemic.

[0075] Thus, inactivation of Atg7 using a Cas9 with a guide RNA against ATG7 may be performed in vitro, ex vivo or in vivo. In some embodiments, cells such as from a cell line aretreated in vitro with a Cas9 with a guide RNA against ATG7. In some embodiments, cells from a subject or patient arc treated ex vivo using a Cas9 with a guide RNA against ATG7, after which the cells are returned to the subject or patient. In some embodiments, a subject or patient is treated in vivo using a Cas9 with a guide RNA against ATG7. In some embodiments, the in vivo treatment using a Cas9 with a guide RNA against ATG7 is targeted to cells of interest. In some embodiments, the in vivo treatment using a Cas9 with a guide RNA against ATG7 is provided at the site of cells of interest. In some embodiments the in vivo treatment using a Cas9 with a guide RNA against ATG7 is systemic.PROTAC or dTAG

[0076] Inactivation of Atg7 may be achieved, by way of non-limiting examples, using PROTAC or dTAG.

[0077] PROTACs are heterobifunctional molecules that degrade target proteins by hijacking the ubiquitin-proteasome system. Proteolysis targeting chimeras (PROTACs) contain three components: the protein-of-interest (POI) binding moiety, a linker, and E3 ubiquitin ligase binding moiety. PROTAC molecules can bind with E3 ligase and the target protein to form POI- PROTAC-E3 ligase ternary complex. Hijacking the ubiquitin-protease system (UPS) subsequently causes the target protein to be polyubiquitinated, which is then followed by the protcasomal degradation of protein. In eukaryotic cells, the UPS is the primary mechanism for maintaining protein homeostasis removing defective and damaged proteins.

[0078] The dTAG system is a dual component platform requiring the expression of FKBP12F36V in-frame with a gene-of-interest and treatment with a heterobifunctional dTAG molecule (dTAG-13) that engages FKBP12F36V and cereblon (CRBN), an E3 ubiquitin ligase. This interaction leads to exclusive degradation of the FKBP12F36V-tagged protein.Combinations of autophagy inhibitors

[0079] As noted herein, any two or more methods may be used to inhibit autophagy in one or more cell type. In some embodiments, two chemical agents work synergistically to suppress autophagy in a cell type. In some embodiments, two chemical agents work synergistically tosuppress canonical autophagy in a cell type. In some embodiments, two chemical agents work synergistically to suppress non-canonical autophagy in a cell type.

[0080] In some embodiments, a chemical method and a genetic method are used to inhibit autophagy in one or more cell type. In some embodiments, two methods work synergistically to suppress autophagy in a cell type. In some embodiments, two methods work synergistically to suppress canonical autophagy in a cell type. In some embodiments, two methods work synergistically to suppress non-canonical autophagy in a cell type.Cell types

[0081] The disclosure is not so limiting regarding the one or more types of cells in which autophagy is suppressed or Atg7 is inactivated or suppressed. Non-limiting examples include dendritic cells, macrophages, monocytes, cancer-associated fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells.Dendritic cells

[0082] In one embodiment, cells in which autophagy is inhibited are dendritic cells. In some embodiments, the dendritic cells are classical dendritic cells. In some embodiments, the dendritic cells are bone marrow derived dendritic cells. In some embodiments, the dendritic cells are plasmacytoid dendritic cells. In some embodiments, the dendritic cells are resident dendritic cells. In some embodiments, the dendritic cells are follicular dendritic cells.Fibroblasts

[0083] In some embodiments, cells in which autophagy is inhibited are fibroblasts. In some embodiments, the fibroblasts are cancer-associated fibroblasts. In some embodiments, the fibroblasts are fibroblast reticular cells.Macrophages and Monocytes

[0084] In some embodiments, cells in which autophagy is inhibited are macrophages or monocytes.T cells

[0085] In some embodiments, cells in which autophagy is inhibited are T cells.B cells

[0086] In some embodiments, the cells in which autophagy is inhibited are B cells.Other Cell Types

[0087] In some embodiments, the cells in which autophagy is inhibited is a lymphoid organizer cell. In some embodiments, the cells in which autophagy is inhibited is endothelial cells. In some embodiments, the cells in which autophagy is inhibited is myeloid derived suppressor cells.Cell Sources

[0088] In any of the forgoing description of cells, such cells may be located within the patient, derived from the patient, grown from cells isolated from the patient, differentiated from cells obtained from the patient, obtained from a donor, obtained from a cell line, obtained from a cell bank, or when multiple cell types are used, any combination thereof.Sites for Inhibition of Autophagy

[0089] As noted herein, the methods disclosed herein may be performed on cells in vitro or ex vivo to be administered, or to or on cells in vivo, or a combination thereof. In one embodiment, cells obtained from a patient, donor, cell line, or any other source are modified to have autophagy inhibited, then administered to a subject. In some embodiments, before or after inhibition of autophagy, the cells, such as dendritic cells or macrophages, are exposed to an antigen. In some embodiments, antigen presentation by the dendritic cells or macrophages in increased in vitro, ex vivo or in vivo. These and other non-limiting variations in the ways of carrying out the methods disclosed herein are described herein.Atg7

[0090] As used herein, Atg7 and ATG7 are used interchangeably to refer to the ATG7 gene or its gene product, Atg7. In any of the embodiments described herein, enhancement of the immune response for the purposes disclosed herein may be achieved by suppressing or inactivating Atg7. Such suppressing or inactivating may be carried out in vitro, ex vivo or in vitro, and may be performed in any one or more of dendritic cells, macrophages, monocytes, cancer-associated fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells.

[0091] Thus, a method is provided for enhancing an immune response in a subject comprising the steps of: a. identifying one or more immune response associated cell types in the subject or one or more immune response associated cell types to be administered to the subject; and b. suppressing or inactivating Atg7 in the one or more immune response associated cell types in the subject or in the one or more immune response associated cell types to be administered to the subject, wherein the suppressing or inactivating is performed in vivo, ex vivo, in vitro, or by any combination thereof on one or more of either or both cell types in the subject or to be administered to the subject; wherein an immune function is enhanced in vivo, ex vivo or in vitro by the one or more immune response associated cell types in the subject or in the one or more immune response associated cell types after administration to the subject, thus enhancing the immune response in the subject.

[0092] In some embodiments, the immune response associated cell types in the subject are one or more of dendritic cells, macrophages, monocytes, cancer-associated fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells. In. In some embodiments, the immune response associated cell types to be administered to the subject are one or more of dendritic cells,macrophages, monocytes or fibroblasts. In some embodiments, the immune response associated cell types to be administered to the subject arc obtained from the subject, obtained from a donor, or obtained from banked immune response associated cells or blood products, or induced in vitro or ex vivo from any of the foregoing. In some embodiments, an immune response associated cell type comprises a tertiary lymphoid structure, a lymph node, a secondary lymphoid organ, a B cell follicle, a tumor, or a wound. In some embodiments, the cell types are fibroblast reticular cells, follicular dendritic cells or lymphoid organizer cells.

[0093] In some embodiments, the suppressing or inactivating Atg7 is performed on the one or more immune response associated cell types in, or administered to, the subject, independently by any one or more of: a. exposing the one or more immune response associated cell types in vivo, ex vivo or in vitro to one or more agents that suppresses or inhibit Atg7; and / or b. inactivating Atg7 in the one or more immune response associated cell types in vivo, ex vivo or in vitro; and / or c. the combination thereof.

[0094] In some embodiments, the Atg7 gene is inactivated in the cell type. The inactivation may be in vitro, ex vivo or in vivo. In some embodiments, the suppressing Atg7 expression or function comprises exposing the cell type to a siRNA targeting Atg7. In some embodiments, inactivating ATG7 comprises exposing the cell type to a Cas9 with a guide RNA against ATG7. In some embodiments, the inactivating Atg7 is Atg7 knockout.

[0095] In some embodiments, a combination of (a) and (b) synergistically provides an enhancement of the immune response.

[0096] In some embodiments, the enhancing the immune response comprises one or more of: enhanced antigen presentation by dendritic cells; enhanced cytokine production; enhanced chemokine production; elevated inflammatory gene expression; elevated macrophage activation; elevated T cell recruitment; elevated T cell activation, or any combination thereof. In someembodiments, the enhanced antigen presentation comprises enhanced presentation of a cancer antigen or a fragment thereof.

[0097] In some embodiments, Atg7 is suppressed or inactivated in dendritic cells in vitro or ex vivo, the dendritic cells are exposed to an antigen in vitro or ex vivo, and the dendritic cells are infused into the subject.

[0098] In some embodiments, Atg7 is suppressed or inactivated in macrophages or monocytes in vitro or ex vivo, the macrophages or monocytes are exposed to an antigen in vitro or ex vivo, and the macrophages or monocytes are infused into the subject.

[0099] In some embodiments, a tumor is infused with an agent that suppresses or inactivates Atg7.

[0100] In some embodiments, Atg7 is suppressed or inactivated in fibroblasts which are infused into a tumor.Antigens

[0101] In some embodiments, a cell type such as a dendritic cell is exposed to an antigen that is processed and presented. In some embodiments, the antigen is a cancer antigen. In some embodiments, the antigen is an infectious microorganism associated antigen.Cancer antigens

[0102] Cancer antigens as embraced herein comprises peptide, protein, glycoprotein, and other components expressed by cancer cells, cancer stroma, and other components of a tumor that targeting following the methods disclosed herein is beneficial.

[0103] Non-limiting examples of cancer antigens useful for the purposes herein comprise any approved or experimental cancer antigen undergoing clinical trials, or other cancer antigens identified as potentially useful in inducing an immune response to the cancer. Such cancer antigens include any treatment, formulation, composition, or other administered agent comprising a cancer antigen or a method for enhancing the immune response against a cancer. Non-limiting examples include sipleucel-T (PROVENGE), CAR-T therapies, antibodies againstcancer antigens, neoantigen vaccines, tumor associated antigen vaccines, antigens such as alphafetoprotein (AFP), carcinocmbryonic antigen (CEA), CA-125, MUC-1, epithelial tumor antigen (ETA), tyrosinase, NY-ESO-1, melanoma-associate antigen (MAGE), Wilms tumor antigens, and abnormal products of ras, mutant KRAS, and mutant p53. Also see for example Xie et al., 2023, Signal Transduction and Targeted Therapy, 8, article 9; Shemesh et al., 2020, Mol Ther.29(2):555-570; Elaen and Rammensee, 2013, Current Opinion in Immunology, 25:277-283; each of which is incorporated herein by reference in its entirety.Infectious microorganism antigens

[0104] Non-limiting examples include antigens from various infectious microorganisms such as causative agents of: a fungal infection (e.g., aspergillus, coccidioidomycosis, tinea pedis (foot), tinea corporis (body), tinea cruris (groin), tinea capitis (scalp), and tinea unguium (nail)), a bacterial infection (e.g., methicillin-resistant Staphylococcus aureus [MRSA], localized skin infections, abscesses, necrotizing facsciitis, pulmonary bacterial infections [e.g., pneumonia], bacterial meningitis, bacterial sinus infections, bacterial cellulitis, such as due to Staphylococcus aureus (MRSA), bacterial vaginosis, gonorrhea, chlamydia, syphilis, Clostridium difficile (C. diff) , tuberculosis, cholera, botulism, tetanus, anthrax, pneumococcal pneumonia, bacterial meningitis, Lyme disease), a viral infection (e.g., varicella-zoster / herpes zoster [shingles], Herpes simplex I [e.g., cold sores / fever blisters], Herpes simplex II [genital herpes], or human papilloma virus [e.g., cervical cancer, throat cancer, esophageal cancer, mouse cancer], Epstein-Ban’ virus [e.g., nasopharyngeal cancer], encephalitis viruses [e.g., brain inflammation], or hepatitis viruses [e.g., liver disease; hepatitis A, hepatitis B, hepatitis C, hepatitis D, hepatitis E, hepatitis F, hepatitis G] or COVID-19), aparasitic infection (e.g., an area infected by scabies, Chagas, Hypoderma tarandi, amoebae, roundworm, Toxoplasma gondii).Immune Checkpoint Inhibition

[0105] In some embodiments, in addition to the inhibition of autophagy in one or more cell types in accordance with the teaching herein, the patient may be administered an immune checkpoint inhibitor. Non-limiting examples of immune checkpoints that may be inhibited include CTLA-4, PD-1, PD LI, PD-L2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN- 15049, CHK 1 kinase, CHK2 kinase, A2aR, or a B-7 familyligand. Inhibitors of each of these checkpoints are known in the art; non-limiting examples include antibodies, small molecules, and fusion polypeptides including antibodies or antigenbinding components fused to other molecules. Non-limiting examples include those listed below.

[0106] In various embodiments, the anti-PD-1 antibody inhibits or blocks binding of the PD-1 receptor to one or both of its ligands, PD-L1 and PD-L2. In exemplary aspects, the monoclonal antibody that specifically binds to PD-1 is nivolumab (BMS936558; Bristol Meyers Squibb), pembrolizumab (MK-3475; Merck), pidilizumab (CT-011; CureTech), lambrolizumab, BMS-936559, atezolizumab, or AMP-224 (GSK / Amplimmune), AMP224 (Medlmmune);AUNP12 (Dr. Reddy's Laboratories Ltd.); BGB108 (BeiGene); MCLA134 (Merus BV); MEDIO68O (Medlmmune); PDR001 (Novartis); REGN2810 (Regeneron / Sanofi); SHR1210 (Jiangsu Hengrui Medicine / Incyte); STIA110X (Sorrento); STIA1110 (Sorrento); TSR042 (AnaptysBio / Tesaro).

[0107] In exemplary aspects, the monoclonal antibody that specifically binds to PD-L1 is BMS-936559 (BMS / Ono), MPDL3280A (Roche / Genentech), or MEDI-4736 (Medlmmune), MSB0010718C (Merck / Serono), ALN-PDL (Alnylam); BGBA317 (BeiGene); KD033 (Kadmon Corp.); KY1003 (Kymab Ltd.); STIA100X (Sorrento); STIA1010 (Sorrento); STIA1011 (Sorrento); STIA1012 (Sorrento);and STIA1014 (Sorrento).

[0108] Antibodies that bind CTLA-4 include ipilimumab and tremilimumab.Chimeric Antigen Receptors (CAR) and CAR-T cells

[0109] Chimeric antigen receptors (CARs), also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors, are receptor proteins that have been engineered to give T cells the ability to target a specific antigen. The receptors are chimeric in that they combine both antigen-binding and T cell activating functions into a single receptor. In some embodiments, CAR-T cell therapy uses T cells engineered with CARs to treat cancer. To prepare CAR-T cells, T cells may be harvested from patients, genetically altered, then infused into patients to attack their tumors. CAR T cells can be derived either autologously from T cells in a patient's own blood or allogeneically from those of a donor.

[0110] Once isolated, these T cells are genetically engineered to express a specific CAR,using a vector derived from an engineered lentivirus such as HIV. The CAR programs the T cells to target an antigen present on the tumor cell surface.

[0111] In some embodiments, the efficacy of such CAR-T cells in a patient is enhanced for example against tumors by suppressing autophagy in the T cells. Chemical or genetic methods as described herein may be used.Cancers

[0112] Non-limiting examples of cancers include esophageal cancer, pancreatic cancer, metastatic pancreatic cancer, metastatic adenocarcinoma of the pancreas, bladder cancer, stomach cancer, fibrotic cancer, glioma, malignant glioma, diffuse intrinsic pontine glioma, recurrent childhood brain neoplasm renal cell carcinoma, clear-cell metastatic renal cell carcinoma, kidney cancer, prostate cancer, metastatic castration resistant prostate cancer, stage IV prostate cancer, metastatic melanoma, melanoma, malignant melanoma, recurrent melanoma of the skin, melanoma brain metastases, stage IIIA skin melanoma; stage IIIB skin melanoma, stage IIIC skin melanoma; stage IV skin melanoma, malignant melanoma of head and neck, lung cancer, non-small cell lung cancer (NSCLC), squamous cell non-small cell lung cancer, breast cancer, recurrent metastatic breast cancer, hepatocellular carcinoma, Hodgkin’s lymphoma, follicular lymphoma, non-Hodgkin’s lymphoma, advanced B-cell NHL, HL including diffuse large B-ccll lymphoma (DLBCL), multiple myeloma, chronic myeloid leukemia, adult acute myeloid leukemia in remission; adult acute myeloid leukemia with Inv(16)(pl3.1q22); CBFB- MYH11; adult acute myeloid leukemia with t(l 6; 16)(p 13.1 ;q22); CBFB-MYH11; adult acute myeloid leukemia with t(8;21)(q22;q22); RUNX1-RUNX1T1; adult acute myeloid leukemia with t(9;l l)(p22;q23); MLLT3-MLL; adult acute promyelocytic leukemia with t(15;17)(q22;ql2); PML-RARA; alkylating agent-related acute myeloid leukemia, chronic lymphocytic leukemia, Richter’s syndrome; Waldenstrom’s macroglobulinemia, adult glioblastoma; adult gliosarcoma, recurrent glioblastoma, recurrent childhood rhabdomyosarcoma, recurrent Ewing sarcoma / peripheral primitive neuroectodermal tumor, recurrent neuroblastoma; recurrent osteosarcoma, colorectal cancer, MSI positive colorectal cancer; MSI negative colorectal cancer, nasopharyngeal nonkeratinizing carcinoma; recurrent nasopharyngeal undifferentiated carcinoma, cervical adenocarcinoma; cervical adenosquamousT1carcinoma; cervical squamous cell carcinoma; recurrent cervical carcinoma; stage IVA cervical cancer; stage IVB cervical cancer, anal canal squamous cell carcinoma; metastatic anal canal carcinoma; recurrent anal canal carcinoma, recurrent head and neck cancer; carcinoma, squamous cell of head and neck, head and neck squamous cell carcinoma (HNSCC), ovarian carcinoma, colon cancer, gastric cancer, advanced GI cancer, gastric adenocarcinoma; gastroesophageal junction adenocarcinoma, bone neoplasms, soft tissue sarcoma; bone sarcoma, thymic carcinoma, urothelial carcinoma, recurrent Merkel cell carcinoma; stage III Merkel cell carcinoma; stage IV Merkel cell carcinoma, myelodysplastic syndrome and recurrent mycosis fungoides and Sezary syndrome. In another related aspect, the tumor or cancer comprises a metastasis of a tumor or cancer. In some embodiments, a solid tumor treated using a method described herein, originated as a blood tumor or diffuse tumor.Treatment of Disease

[0113] In some embodiments, methods of treating described herein for promoting clearance of or alleviating localized symptoms of the autoimmune disease, allergic reaction, hypersensitivity reaction, infection or infectious disease; for facilitating healing and / or preventing or inhibiting infection or rejection of a localized site of an injury or other damage, a transplant or other surgical site; for reducing or eliminating a blood clot causing or at risk for causing a myocardial infarction, an ischemic stroke, or a pulmonary embolism; or for alleviating localized symptoms thereof; or for a combination thereof.

[0114] In some embodiments, methods of treating described herein reduce the size of the tumor, eliminate said tumor, slow the growth or regrowth of the tumor, or prolong survival of said subject, or any combination thereof. In some embodiments, treating reduces or eliminates inflammation or another symptom of the autoimmune-targeted or symptomatic focus of an autoimmune disease, prolongs survival of the subject, or any combination thereof; reduces or eliminates inflammation or another symptom of allergic reaction or hypersensitivity reaction at the reactive focus of an allergic reaction or hypersensitivity reaction, prolongs survival of the subject, or any combination thereof; reduces or eliminates infection or symptoms at the focus of infection or symptoms of a localized infection or infectious disease, prolongs survival of the subject, or any combination thereof; reduces, eliminates, inhibits or prevents structural, organ,tissue, or cell damage, inflammation, infection, or another symptom at a site of injury or a site of chronic damage, improves structural, organ, tissue, or cell function at a site of injury or a site of chronic damage, improves mobility of the subject, prolongs survival of the subject, or any combination thereof; reduces, eliminates, inhibits, or prevents structural, organ, tissue, or cell damage, inflammation, infection, or another symptom at a surgical site, improves structural, organ, tissue, or cell function at a surgical site, improves mobility of the subject, prolongs survival of the subject, or any combination thereof; reduces, eliminates, inhibits or prevents transplanted organ, tissue, or cell damage or rejection, inflammation, infection or another symptom at a transplant site, improves mobility of the subject, prolongs survival of a transplanted organ, tissue, or cell, prolongs survival of the subject, or any combination thereof; or reduces or eliminates a blood clot causing or at risk for causing a myocardial infarction, an ischemic stroke, or a pulmonary embolism in the subject, improves function or survival of a heart, brain, or lung organ, tissue, or cell in the subject, reduces damage to a heart, brain, or lung organ, tissue, or cell in the subject, prolongs survival of a heart, brain, or lung organ, tissue, or cell in the subject, prolongs survival of the subject, or any combination thereof.Exemplary Methods

[0115] The following descriptions of methods for carrying out the disclosure are provided to illustrate the various methods whereby inhibiting autophagy in or more cell type is effective in enhancing the immune response, in any number of different bodily sites, purposes, etc. These examples are not to be construed as limiting the embodiments that may be achieved by the teachings disclosed herein.Enhancing antigen presentation by dendritic cells in vitro or ex vivo

[0116] In one embodiment, autophagy is suppressed in dendritic cells isolated from a patient, prepared from PMBCs obtained from a patient, isolated from or prepared from a donor, or dendritic cells from a cell line. The dendritic cells are exposed to an antigen. The suppression or autophagy and exposure to the antigen may be in any order or concurrently. Any of the methods of combination of methods for suppressing autophagy may be used. After such treatment, the dendritic cells are infused into the patient. Such dendritic cells present antigen more efficiently and enhance the immune response thereto.

[0117] In some embodiments, a checkpoint inhibitor is administered to the patient concurrently or before or after administration of the dendritic cells. Further enhancement of the immune response to the antigen is achieved.Enhancing CAR-T cells

[0118] In one embodiment, T cells collected from a patient and modified to express chimeric antigen receptors (CAR) and returned to the same patient, can be enhanced by also suppressing autophagy in the T cells. Chemical or genetic methods may be used as described elsewhere herein. Such CAR-T cells have enhanced activity at targeting tumors.Enhancing Cells Derived from Hematopoietic Stem Cells

[0119] In one embodiment, autophagy is suppressed in hematopoietic stem cells (HSC) by any one or more methods such as but not limited to those described herein. Such HSC are infused into the patient. In some embodiments, the autophagy-suppresses HSCs are differentiated into macrophages ex vivo before being administered to the patient.

[0120] In some embodiments, the HSCs are engineered to suppress autophagy in the tumor microenvironment. In some embodiments, the suppression of autophagy is driven by the expression of S100a4 in the tumor microenvironment. Thus, in some embodiments, suppression of autophagy occurs in stem cells and their differentiated progeny only near a tumor.Enhancing immune response by macrophages in vitro or ex vivo

[0121] In one embodiment, autophagy is suppressed in macrophages isolated from a patient, prepared from PMBCs obtained from a patient, isolated from or prepared from a donor, or macrophages or monocytes obtained from a cell line. The macrophages are exposed infused into the patient. Such macrophages enhance the immune response thereto.

[0122] In some embodiments, a checkpoint inhibitor is administered to the patient concurrently or before or after administration of the macrophages. Further enhancement of the immune response is achieved.Enhancing tertiary lymphoid structure (TLS) immune response

[0123] Autophagy is suppressed in tertiary lymphoid structures (TLS) in vivo by exposing TLS in vivo to one or more agents that suppress autophagy, or inactivating at least one gene that suppresses autophagy in the TLS in vivo, or activating at least one gene that suppresses autophagy in the TLS in vivo. In some embodiments, a combination of any of the forgoing are used. The immune response by the TLS is enhanced.

[0124] In some embodiments, a checkpoint inhibitor is administered to the patient concurrently or before or after autophagy suppression in TLS is carried out. Further enhancement of the immune response is achieved.Methods of Treatment

[0125] As disclosed herein, suppression of autophagy in any one of more cell types may be performed in vitro, in which the cells may subsequently be administered to the subject. As disclosed herein, suppression of autophagy in any one of more cell types may be performed ex vivo on cells from the patient, after which the cells may subsequently be administered to the subject. As disclosed herein, suppression of autophagy in any one of more cell types may be performed in vivo. In such methods, the cells modified in vitro or ex vivo may be infused, implanted, or otherwise administered to the patient to enhance the immune response.Modification of cells in vivo comprises administering to the subject, or to a site in the subject, a means for suppressing autophagy in desired cell types locally or systemically.

[0126] Such administration of cells or of means for suppressing autophagy, may be administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermally, subcutaneously, intraperitoneally, intraventricularly, intravaginally, intracranially, paracancerally or intratumorally. In some embodiments, the cells for administration, or the agent for suppressing autophagy, may be provided in a suitable pharmaceutical composition or formulation to achieve the desired suppression in vitro, ex vivo or in vivo.

[0127] Thus, in some embodiments, to enhance the immune response in a subject, autophagy is suppressed in vitro in dendritic cells, autophagy is suppressed in vitro in macrophages, autophagy is suppressed in vitro in monocytes, autophagy is suppressed in vitro incancer-associated fibroblasts, autophagy is suppressed in vitro in fibroblast reticular cells, autophagy is suppressed in vitro in B cells, autophagy is suppressed in vitro in endothelial cells, autophagy is suppressed in vitro in myeloid derived suppressor cells, autophagy is suppressed in vitro in follicular dendritic cells, autophagy is suppressed in vitro in bone marrow derived dendritic cells, autophagy is suppressed in vitro in resident dendritic cells, autophagy is suppressed in vitro in classical dendritic cells, autophagy is suppressed in vitro in plasmacytoid dendritic cells, or autophagy is suppressed in vitro in lymphoid organizer cells. Such cells in which autophagy is suppressed are administered to the subject.

[0128] Thus, in some embodiments, to enhance the immune response in a subject, autophagy is suppressed ex vivo in dendritic cells, autophagy is suppressed ex vivo in macrophages, autophagy is suppressed ex vivo in monocytes, autophagy is suppressed ex vivo in cancer-associated fibroblasts, autophagy is suppressed ex vivo in fibroblast reticular cells, autophagy is suppressed ex vivo in B cells, autophagy is suppressed ex vivo in endothelial cells, autophagy is suppressed ex vivo in myeloid derived suppressor cells, autophagy is suppressed ex vivo in follicular dendritic cells, autophagy is suppressed ex vivo in bone marrow derived dendritic cells, autophagy is suppressed ex vivo in resident dendritic cells, autophagy is suppressed ex vivo in classical dendritic cells, autophagy is suppressed ex vivo in plasmacytoid dendritic cells, or autophagy is suppressed ex vivo in lymphoid organizer cells. Such cells in which autophagy is suppressed are administered to the subject.

[0129] Thus, in some embodiments, to enhance the immune response in a subject, autophagy is suppressed in vivo in dendritic cells, autophagy is suppressed in vivo in macrophages, autophagy is suppressed in vivo in monocytes, autophagy is suppressed in vivo in cancer-associated fibroblasts, autophagy is suppressed in vivo in fibroblast reticular cells, autophagy is suppressed in vivo in B cells, autophagy is suppressed in vivo in endothelial cells, autophagy is suppressed in vivo in myeloid derived suppressor cells, autophagy is suppressed in vivo in follicular dendritic cells, autophagy is suppressed in vivo in bone marrow derived dendritic cells, autophagy is suppressed in vivo in resident dendritic cells, autophagy is suppressed in vivo in classical dendritic cells, autophagy is suppressed in vivo in plasmacytoid dendritic cells, or autophagy is suppressed in vivo in lymphoid organizer cells.

[0130] In any of the foregoing methods, autophagy may be suppressed by exposing the cell types in vivo, ex vivo or in vitro to one or more agents that suppresses autophagy and / or inactivating one or more genes responsible for or associated with autophagy in the one or more immune response associated cell types in vivo, ex vivo or in vitro; and / or activating one or more genes responsible for suppressing autophagy in the one or more immune response associated cell types in vivo, ex vivo or in vitro. Non-limiting examples include exposing the cells to an agent that suppresses autophagy, inactivating Atg7 in the cells such as by siRNA or Cas9 with a guide RNA against ATG7, knocking out Atg7, or any combination thereof.

[0131] Thus, in some embodiments, to enhance the immune response in a subject, Atg7 is inactivated or suppressed in vitro in dendritic cells, Atg7 is inactivated or suppressed in vitro in macrophages, Atg7 is inactivated or suppressed in vitro in monocytes, Atg7 is inactivated or suppressed in vitro in cancer-associated fibroblasts, Atg7 is inactivated or suppressed in vitro in fibroblast reticular cells, Atg7 is inactivated or suppressed in vitro in B cells, Atg7 is inactivated or suppressed in vitro in endothelial cells, Atg7 is inactivated or suppressed in vitro in myeloid derived suppressor cells, Atg7 is inactivated or suppressed in vitro in follicular’ dendritic cells, Atg7 is inactivated or suppressed in vitro in bone marrow derived dendritic cells, Atg7 is inactivated or suppressed in vitro in resident dendritic cells, Atg7 is inactivated or suppressed in vitro in classical dendritic cells, Atg7 is inactivated or suppressed in vitro in plasmacytoid dendritic cells, or Atg7 is inactivated or suppressed in vitro in lymphoid organizer cells. Such cells in which autophagy is suppressed are administered to the subject.

[0132] Thus, in some embodiments, to enhance the immune response in a subject, Atg7 is inactivated or suppressed ex vivo in dendritic cells, Atg7 is inactivated or suppressed ex vivo in macrophages, Atg7 is inactivated or suppressed ex vivo in monocytes, Atg7 is inactivated or suppressed ex vivo in cancer-associated fibroblasts, Atg7 is inactivated or suppressed ex vivo in fibroblast reticular cells, Atg7 is inactivated or suppressed ex vivo in B cells, Atg7 is inactivated or suppressed ex vivo in endothelial cells, Atg7 is inactivated or suppressed ex vivo in myeloid derived suppressor cells, Atg7 is inactivated or suppressed ex vivo in follicular dendritic cells, Atg7 is inactivated or suppressed ex vivo in bone marrow derived dendritic cells, Atg7 is inactivated or suppressed ex vivo in resident dendritic cells, Atg7 is inactivated or suppressed ex vivo in classical dendritic cells, Atg7 is inactivated or suppressed ex vivo in plasmacytoiddendritic cells, or Atg7 is inactivated or suppressed ex vivo in lymphoid organizer cells. Such cells in which autophagy is suppressed arc administered to the subject.

[0133] Thus, in some embodiments, to enhance the immune response in a subject, Atg7 is inactivated or suppressed in vivo in dendritic cells, Atg7 is inactivated or suppressed in vivo in macrophages, Atg7 is inactivated or suppressed in vivo in monocytes, Atg7 is inactivated or suppressed in vivo in cancer-associated fibroblasts, Atg7 is inactivated or suppressed in vivo in fibroblast reticular cells, Atg7 is inactivated or suppressed in vivo in B cells, Atg7 is inactivated or suppressed in vivo in endothelial cells, Atg7 is inactivated or suppressed in vivo in myeloid derived suppressor cells, Atg7 is inactivated or suppressed in vivo in follicular dendritic cells, Atg7 is inactivated or suppressed in vivo in bone marrow derived dendritic cells, Atg7 is inactivated or suppressed in vivo in resident dendritic cells, Atg7 is inactivated or suppressed in vivo in classical dendritic cells, Atg7 is inactivated or suppressed in vivo in plasmacytoid dendritic cells, or Atg7 is inactivated or suppressed in vivo in lymphoid organizer cells.

[0134] In any of the foregoing methods, by way of non-limiting examples, Atg7 is inactivated or suppressed using an siRNA targeting Atg7 or a Cas9 with a guide RNA against ATG7.EXAMPLESMaterials and Methods

[0135] Multiplex immunofluorescence (MIF). MIF analysis is performed with multiple, validated 7-plex antibody panels on adjacent slides for melanoma cells and melanocytes (S100), DC (CDllb, CDllc, MHC II), DC activation (CD80, CD86), macrophages (CDllb, F4 / 80), T cells (CD4 and CD8), autophagosomes (LC3+ and p62+ puncta),127 autolysosomes (LC3+lampl+ puncta), newly formed autophagosomes (WIPI2+LC3+p62+ puncta), noncanonical NF-kB (NFKB2, NIK, phospho-IKKa), canonical NF-kB (phopho-p65), and proliferation (Ki-67). Data includes mean, median, and maximum autophagy measures in DC near the tumor, remote from tumors, and adjacent and far from melanocytic nevi. Two-sample t- tests or one-way ANOVA are used.

[0136] Spatial transcriptomics. Spatial transcriptomics are used to investigate gene expression patterns in DC and other immune cells near melanomas. The Nanostring CosMx Digital Spatial Profiling Platform is used to shine UV light on the sample to release photo- cleavable gene barcodes for quantification with next generation sequencing. The Nanostring AtoMx Spatial Informatics Platform is also used for cell typing. GeoDiff is used to identify genes differentially expressed in DC near versus far from melanomas, in DC with high versus low autophagy, and in DC with high versus low noncanonical NF-kB.

[0137] In vivo, B16-F10 and YUMM1.1 melanoma cell models are used as examples of melanomas not highly responsive to ICI to determine the impact of DC-specific autophagy inactivation on tumor growth, mouse survival, DC and T cell activation, and spatial changes in the TME. Tumors are introduced into mice with DC-specific inactivation of Atg7 and Nfkb2 and controls

[0138] Bone marrow is isolated and differentiate the cells into DC (semi-adherent cells after treatment with 20 ng / mL GM-CSF and 20 ng / mL IL-4) or cDCl (semiadherent cells after treatment with 100 pg / mL m-FLT3L and 50 pg / ml m-GM-CSF). Cells are FACS-sorted for CDllb+CDllc+MHC IIhlghto isolate DC. To activate the noncanonical NF-kB pathway, 4 pg / ml a-LTbR polyclonal antibody or 10 pg / mL a-CD40 mAb are used. To stimulate the canonical NF-kB pathway, 10 pg / ml LPS140 or 20 ng / ml TNF-a are used.

[0139] Identifying autophagy-responsive elements in the noncanonical NF-kB pathway. DC or cDCl differentiated from bone marrow of Ubc. Atg7171' and Atg7l / I' mice are treated with one of the 4 stimulatory compounds. Samples with each treatment are collected over a time course of 0, 1, 3, 6, 12, 16, 24, 48 and 72 hours. Protein lysates from Ubc.Atg7F / F and Atg7F / F DC are analyzed with IB for noncanonical NF-kB pathway components plOO, p52, IKKa, NIK, cIAPl / 2, and TRAF2 / 3 to identify candidate autophagy-responsive proteins. Because changes in protein abundance in Ubc.Atg7F / F DC could reflect direct or indirect effects of ATG7 knockout, wild-type DC or cDCl, untreated or treated with a stimulatory trigger are analyzed over a time course, treated with DMSO or bafilomycin Al (100 nM) for 3 hours prior to harvest.

[0140] Monitoring noncanonical NF-kB activity. Total RNA is isolated from DC from Ubc.Atg7F / F and Atg7F / F mice treated with a-LTbR, CD40L, LPS, or TNF-a at three timepointsselected based on the time course above, or untreated. cDNA libraries are generated and sequenced on an Illumina Nova Scq X with TCGB using 100-bp paired-end sequencing to generate at least 100 million reads per sample. Reads are mapped to the human transcriptome using Salmon DESeq2 to determine the impact of autophagy inactivation on NF-kB target genes. In addition, serum-free CM are collected from DC or cDCl from Ubc.Atg7F / F and Atg7F / F mice treated with stimulating factors over a time course at 0 (untreated), 1, 3, 6, 12, 16, 24, 48 and 72 hours. Multiplex cytokine analysis (MSD) is used to monitor the levels of noncanonical NF-kB targets including IF-12p40, CXCF12, CXCF13, CCF19 and CCF21.

[0141] DC or cDCl are derived from bone marrow cells of Ubc.Atg7F / F or Atg7F / F mice as described above. OT-I TCR transgenic CD8+ T cells or OT-II TCR transgenic CD4+ T cells are isolated from spleens of OT I or OT II mice by negative selection with Miltenyi kits. DC are pulsed for 5 h at 37°C with ovalbumin (OVA) — 10 pM OVA257-264 SIINFEKF (SEQ ID NO:1) peptide for MHC I presentation or OVA323-339 ISQAVHAAHAEINEAGR (SEQ ID NO:2) peptide for MHC II presentation. DC or cDCl are cocultures with purified carboxyfluorescein succinimidyl ester (CFSE)-labeled OT-I CD8+ or OT II CD4+ T cells with or without a-CD40 or EPS stimulus. With flow cytometry, cell surface levels are monitored of DC activation markers CD80, CD83, and CD86, and T cell proliferation by the dilution of CFSE. We will collect cell supernatant on day 3 to evaluate levels of cytokines secreted by T cells, DC, or both, including IFN-g, TNF-a, IE-2, CXCL10, and IL-12p40, with MSD. In addition, T cells are isolated from pmel-1 mice (Jackson #005023) that recognize pmel-l / gpl00 presented by B16- F10.145. pmel T cells are cocultured with Ubc.Atg7F / F and Atg7F / F DC pulsed with gplOO peptide KVPRNQDWL (SEQ ID NOG) and B16-F10 melanoma cells engineered to express luciferase to determine T cell-mediated killing of B16-F10 with luminescence. Effects of autophagy inactivation are determined on DC and eDC 1 activation and antigen presentation, including the capacity to activate T cells to kill co-cultured B16-F10.

[0142] Test the importance of noncanonical NF-kB signaling for DC activation. Autophagy-deficient DC exhibit increased activation, cytokine expression, and antigen presentation as a result of elevated noncanonical NF-kB activity. CRISPR / Cas9 is used to inactivate Nfkb2, Nik, or RelB to inhibit noncanonical NF-kB signaling or RelA / p65 for canonical NF-kB signaling in Ubc.Atg7F / F and Atg7F / F DC. Inactivating the noncanonical NF-kB pathway in Ubc.Atg7F / F DC reverses phenotypes identified above is tested. Figure 19 shows CRISPR / Cas9-mcdiatcd knockouts of Atg7. DC will be electroporated with recombinant Cas9 (10 pmol) and one of 2 short guides per gene (90 pmol) against Nik, Nfkb2, RelB or RelA, or a control short guide, using the Neon transfection system with 1 pulse of 1700 volts for 20 ms. Nik, Nfkb2, RelB or RelA knockout will be confirmed with sequencing and IB. DC activation is monitored with immunophenotyping of activation markers, cytokine and chemokine secretion with MSD, and antigen presentation as described above in Ubc.Atg7F / F and Atg7F / F DC with Nik, Nfkb2, RelB, or RelA knockout. In addition, with Zbtb46.Atg7F / FNfkb2F / F mice and genotype controls their DC and cDCl will be evaluated.

[0143] Test the role of autophagy and the noncanonical NF-kB pathway in DC for melanoma progression, tumor and non-tumor histology, and mouse survival. Cohorts of >10 mice will be raised with autophagy and noncanonical NF-kB inactivation in DC and controls: Zbtb46.Atg7F / FNfkb2F / F, Zbtb46.Atg7F / F, Zbtb46.Nfkb2F / F, and Atg7F / F mice.Zbtb46.Atg7F / F mice will breed with Nfkb2F / F mice (Jackson #028720). 0.5 x 106 B16-F10 or YUMM 1.1 mouse melanoma cells will be introduced intradermally to develop orthotopic melanomas. Tumor volumes will be monitored every 2-3 days and final tumor weights after 15 days of tumor growth. FFPE tumor sections are stained with H&E and analyzed histologically by UCLA board-certified dermatopathologists. Non-tumor tissues will also be collected including liver, spleen, lungs, skin, and lymph nodes and monitor for signs of toxicity including immune cell infiltration, necrosis or apoptosis by H&E and activated caspase-3 staining. In separate experiments, survival will be assessed by monitoring mice with tumors until they reach a euthanasia endpoint (deep ulcer, large tumor size, failing health).

[0144] Tumor immunophenotyping. On days 10-12 of tumor growth, tumors, tumor draining lymph nodes (TDLN), and spleens will be collected, and flow cytometry performed for DC (CDllc+CDllb+HLA-DR),147, 148 DC activation (CD80, CD86, CD83), and DC subtypes (CD207, XCR1, CD103, and CD172a);149 monocytes (CDllb), macrophages (CDllb+F4 / 80+), Ml macrophages (CD80, CD86), M2 macrophages (CD163, CD206), and macrophage activation (CD80, CD86, CD68); 147, 150, 151 CD4+ and CD8+ T cells (CD45+NK1.1-TCR +); naive, central memory and central effector T cells (CD44 and CD62L); intracellular cytokines (IFN-g, TNF-a); killing effectors (perforin, granzyme B); and Foxp3+ regulatory T cells.149 FACS-sorted DC will be evaluated for noncanonical NF-kB gene expression with real-time PCR for targets including IL-12p40, CXCL12, CXCL13, CCL19 and CCL21.

[0145] Tumor spatial transcriptomics. Spatial transcriptomics is performed on at least 5 samples each of representative B16-F10 and YUMM1.1 tumors and TDLN in Zbtb46- Cre.Atg7F / F and Atg7F / F mice.

[0146] Bone marrow-derived DC will be generated from autophagy-proficient and - deficient mice. DC or cDCl will be pulsed with B16F10 antigen gplOO, then infused into B16F10 (gplOO+) or YUMM1.1 (gplOO-, included to test the importance of the pulse), melanoma-bearing mice intravenously (IV) to assess DC or cDCl-mediated anti-tumor T cell responses against a known antigen. Alternatively, unpulsed DC or cDCl will be directly injected intratumorally (IT) to assess DC or cDCl -induced anti-tumor T cell activation against available tumor antigens in the TME. In preliminary studies, in vivo trafficking will be monitored of introduced DC with flow cytometry. With IT DC or cDCl introduction. Optimal dosing and timing will be determined that induce a robust anti-tumor immune response that reduces tumor growth, while limiting toxicity to non-tumor tissues.

[0147] In vivo trafficking and kinetics of injected DC and cDCl. DC or cDCl will be generated from CD45.2 Ubc.Atg7F / F or Atg7F / F mice. Autophagy-proficient and -deficient DC will be FACS-sortcd for purity. 0.5 x 106 B16-F10 or YUMM1.1 melanoma cells will be introduced intradermally into cohorts of >10 syngeneic C57BL / 6 CD45.1 mice. Autophagydeficient or -proficient DC or cDCl, or PBS control, will be IT injected into B16-F10 or YUMM1.1 intradermal tumors. Alternatively, DC or cDCl will be pulsed with gplOO peptide for 5 h prior to IV injection into tumor-bearing mice. Tumors and TDLN will be harvested 2, 24, and 48 h post injection, and DC or cDCl levels will be assessed by flow cytometry for DC and T cells using CD45.1 and CD45.2 to distinguish introduced vs endogenous DC. The extent of autophagy-proficient and -deficient DC trafficking, persistence and activation under the different conditions, will be assessed.

[0148] Anti-tumor efficacy of Atg7-proficient and -deficient DC. B16F10 or YUMM1.1 will be introduced intradermally and introduce autophagy-proficient or -deficient DC or cDCl, or no DC, IT or IV, as described above, with >10 mice per condition. DC or cDCl will beadministered on days 4, 6, and 8 post tumor inoculation. Tumor volumes will be monitored every 2-3 days and tumor weights will be determined on day 15. In separate experiments, mouse survival will be compared by maintaining the mice until they reach a euthanasia endpoint. H&E staining will be used to assess tumor histology and evaluate any DC-induced toxicity to other tissues including liver, spleen, lungs, lymph nodes, and skin.

[0149] DC therapy-induced immune changes. To understand local and systemic immune changes in response to DC or cDCl cellular therapy, comprehensive immune monitoring studies of mice will be performed, with introduced DC and controls. FFPE tumor sections will be subjected to MIF studies with validated 7-plex antibody panels, including activated caspase-3 (apoptosis), CD8 and CD4 (T cells), granzyme B (T cell activation), Ki67 (proliferation), CDllc (DC), CD86 (DC activation), F4 / 80 (macrophages), CD31 (vasculature), and DAPI (DNA) (divided into two panels). Antibodies will be added for NIK, phospho-IKKa, and (nuclear) NFKB2 for the noncanonical NF-kB pathway. MIF studies will compare the abundance, proliferative state, activation, and spatial distribution of T cells and DC, and readouts for noncanonical NF-kB pathway signaling, when autophagy-proficient or autophagy-deficient DC are introduced. In addition, on days 10-12 of tumor growth, tumors and TDLN will be collected and flow cytometry performed for DC (CDllb+CDllc+MHC II), CD4+ and CD8+ T cells (CD45+NK1.1-TCRP+); naive, central memory and central effector T cells (CD44 and CD62L); intracellular cytokines (IFN-g, TNF-a); killing effectors (perforin, granzyme B); and Foxp3+ regulatory T cells.

[0150] Autophagy-deficient DC or cDCl will induce pro-inflammatory cytokines and chemokines, T cell recruitment and activation, T cell-mediated tumor cell killing, and reduced tumor growth. Statistics. The p values shown in all figures are as follows: *, p<0.05; **, p<0.01; ***, p< 0.001; and ****, p< 0.0001.EXAMPLE 1Atg7 ablation in the host reduces growth of melanoma allografts.

[0151] Ubc.Atg7F / F mice were generated with ubiquitin-driven CreER that undergo systemic Atg7 deletion upon tamoxifen treatment. Ubc.Atg7F / F mice contained lower levels of ATG7, ATG5-ATG12, and LC3B-II, which runs further on SDS-PAGE due to the lipid,consistent with ATG7’s molecular functions (Fig 1 A). The presence of fewer autophagosomes in dermal fibroblasts was confirmed in Ubc.Atg7F / F mice with transmission electron microscopy (Fig IB). Autophagy-proficient, low immunogenicity B16-F10 melanoma cells were injected intradermally into the flanks of syngeneic, tamoxifen-treated Ubc.Atg7F / F and controls.(Tamoxifen treatment assumed hereafter). Volumes and weights of melanomas in Ubc.Atg7F / F mice were significantly reduced (Fig 1C). Melanomas in Ubc.Atg7F / F mice exhibited reduced proliferation (Ki-67), increased apoptosis (cleaved caspase-3) and reduced vasculature (CD31). Thus, ATG7 in the host is important for melanoma cell viability, proliferation, vasculature, and progression.

[0152] Fig. 1A. Immunoblots of skin. n=3, mean ± SD, ANOVA, female. (IB) Autophagosome counts in electron micrographs. Mean ± SEM (for all figs unless noted), n=l l- 38 images, t-test, male. (1C) Tumor volumes and weights. n=16-18, ANOVA, male and female.

[0153] Figure ID shows that melanomas were also smaller when 0.5 x 106YUMM1.1 melanoma cells (which contain common human mutations BrafV600E / +;Pten- / -;CDKN2A- / -) were intradermally introduced into Ubc.Atg7F / F compared with Atg7F / F mice.EXAMPLE 2Autophagy deficiency in bone marrow-derived cells reduces melanoma growth.

[0154] Using bone marrow transplantation, we generated mice that were ATG7-deficient in bone marrow-derived cells, non-bone-marrow-derived cells, or both, and confirmed successful reconstitution with immunoblotting (Fig 2A) and imaging (Fig 2B). Autophagy inactivation only in bone marrow-derived cells resulted in a significant reduction in melanoma growth (Fig. 2C, white vs black circles).EXAMPLE 3Elevated monocytes and macrophages in mice with autophagy inactivation.

[0155] RNA-seq of B16-F10 melanomas in autophagy-proficient and autophagydeficient mice revealed 505 differentially-expressed genes. Genes upregulated in tumors in autophagy-deficient mice were enriched for inflammatory processes and response to interferon-' / ,a cytokine secreted by T cells. Further, macrophages were more abundant in tumors of Atg7F / F mice with Ubc.Atg7F / F than control bone marrow (Fig 3).EXAMPLE 4 Increased effector T cells in tumors in ATG7-deficient mice.

[0156] hi blood, spleen and skin of Ubc.Atg7F / F mice without tumors, we observed no changes in levels of CD4+ (not shown) or CD8+ T cells (Fig 4A). CD8+ T cells were more abundant in melanomas in Ubc.Atg7F / F (Fig 4B) mice. In MC38-derived tumors, Ubc.Atg7F / F mice also had increased CD8+ T cells (Fig 4C) and elevated granzyme B (Fig 4D). Thus, loss of autophagy in the host increased tumor-associated CD8+ T cells.EXAMPLE 5T cells reduce melanoma growth in Atg7-deficient hosts.

[0157] We sought to test the functional role of T cells in melanoma growth in autophagy-deficient mice. Treatment with an a-TCR antibody that depletes T cells had little effect on B 16-F10 melanomas in Atg7F / F mice, but significantly increased their growth in Ubc.Atg7F / F (Fig 5A) mice. For MC38 tumors, treating Atg7F / F mice with a-TCRp antibody had no significant effect, whereas treating Ubc.Atg7F / F mice partially reversed the tumors’ smaller size (Fig 5B). Thus, the smaller size of B16-F10 and MC38 tumors in autophagydeficient mice is at least partially T-cell-mediated.

[0158] In spleens of S100a4.Atg7F / F mice with B16-F10 melanomas, we observed more central and effector memory CD4+ and CD8+ T cells (Figure 5C). A DC-T cell axis mediates the smaller tumor size in autophagy-deficient mice and sensitizes to ICI. Effector memory (CD62L- CD44high), central memory (CD62L+CD44high), and naive (CD62L+CD441ow) T cells in spleens of mice with B16-F10 melanomas. n=4-5, t-test, female. Volumes of YUMM1.1 tumors in S100a4.Atg7F / F (n=7, t-test, male) or in Figure 5D or Ubc.Atg7F / F (n=3, t-test, male). Figure 5E. Mice and controls with a-TCRb or isotype control. Figure 5F. Tumor volumes of B 16-F10 tumors in Zbtb46.Atg7F / F (zDC / +) and control mice. n=7-8 mice. One outlier tumor and 2 ulcerated tumors were eliminated from analysis. Male mice. Figure 5G. Weights of B16-F10 tumors in Zbtb46.Atg7F / F (zDC / +) and control mice. n=7-8 mice. One outlier tumor and 2ulcerated tumors were eliminated from analysis. Male mice. Figure 5H. Tumor volumes of Bl 6- F10 tumors in Zbtb46.Atg7F / F (zDC / +) and control mice. n=7-8 mice. One outlier tumor and 2 ulcerated tumors were eliminated from analysis. Male and female mice. Figure 51. Weights of B16-F10 tumors in Zbtb46.Atg7F / F (zDC / +) and control mice. n=7-8 mice. One outlier tumor and 2 ulcerated tumors were eliminated from analysis. Male and female mice.

[0159] Autophagy inactivation in Zbtb46+ cells reduces melanoma growth. To assess the role of autophagy in DC, mice with Zbtb46-Cre (Jackson #028538)118 were crossed with Atg7F / F mice to generate Zbtb46.Atg7F / F mice which lose autophagy in classical DC. When 0.5 x 106 B16-F10 melanoma cells were intradermally introduced into Atg7F / F or Zbtb46.Atg7F / F mice, melanomas were significantly smaller in Zbtb46.Atg7F / F mice (Fig. 5F). This finding indicates autophagy in dendritic cells supports melanoma progression.EXAMPLE 6Autophagy inactivation combines with immune checkpoint inhibitors to reduce melanoma growth

[0160] When B16F10 melanomas were grown in Ubc.Atg7F / F mice treated with a- PD1 / CTLA4 or control antibodies, tumors in A( 7-knockout mice that were also treated with immune checkpoint inhibitors were smallest by weight and volume, indicating autophagy inactivation may synergize with immune checkpoint blockade (Fig 6A). Systemic treatment with HCQ33 that increased levels of autophagy client p62 had little effect on tumor size by itself, but a combination of HCQ and a-PDl / CTLA4 resulted in smaller melanomas (Fig 6B). Therefore, Atg7 inactivation was more effective than HCQ at reducing melanoma growth.EXAMPLE 7 Autophagy Model

[0161] A model for autophagy in the host is shown in Fig. 7. Robust melanoma growth occurs because Atg7-proficient stroma comprising autophagy active T cells and monocytes suppress production of effector cytokines, thereby creating a tumor-promoting microenvironment. In contrast, in Atg7 deficient stroma, T cells and monocytes lackingautophagy results in increased effector cytokine production, increased T cells and monocytes, and reduced melanoma growth.EXAMPLE 8Reduced division, increased apoptosis, and decreased vasculature in melanomas in Ubc.Atg7F / F mice

[0162] B16-F10 melanomas grown in Ubc.Atg7F / F, Ubc.Atg7+ / + and Atg7F / F mice were collected and immunostained for Ki-67, cleaved caspase-3 or CD31. IHC staining (Fig. 8, left) and quantification (Fig. 8, right) for Ki-67, cleaved caspase-3, and CD31 in B16-F10 melanomas. n=3 slides, up to 12 images per slide, ANOVA, male and female. Scale bar = 50 pm.Example 9Atg7 inactivation in S100A4+ cells reduces tumor growth

[0163] Fig. 9A shows S100A4 IHC staining, and Fig. 9B, immunoblotting MEFs for ATG7. Fig. 9C shows volumes and weights of melanomas. n=13-14, ANOVA, male and female. Fig. 9D shows IHC for cleaved caspase 3. n=8-10, ANOVA. Scale bar=50 pm. (Fig. 9E) Volumes of MC38-derived tumors. n=26-28, t-test, female.

[0164] Loss of autophagy could affect tumor growth through systemic or local signals.S100a4 transcripts are expressed in fibroblasts cultured macrophages and dendritic cells (Fig.9F) allowing us to test the role of local autophagy inactivation. S100a4.Atg7F / F mice are healthy and fertile. Figure 9F shows, Left: S100A4 IB in bone marrow cells (BMCs), dendritic cells (DC), and bone marrow-derived macrophages (BMMs). Middle and right: ATG7 and LC3 in BMMs (middle) and DCs (right) from Atg7F / F, S100a4.Atg7F / F or Ubc.Atg7F / F mice.

[0165] When 0.5 x 106 YUMM1.1 melanoma cells were injected intradermally, autophagy inactivation in S100A4+ cells resulted in smaller tumors (Fig. 9G) (volumes of YUMM 1.1 -derived melanomas. n=6-7, t-test, male).

[0166] Furthermore, B16-F10 tumors contained lower levels of CD11C+ DC and spleens contained higher levels of CDllc+ DC in S100a4.Atg7F / F compared with Atg7F / F mice (Fig.9H), consistent with DC mobilization in the S100a4.Atg7F / F model. MC38-derived tumors inUbc.Atg7F / F mice also had increased CD8+ T cells (Fig. 91). CD8+ T cells contained higher levels of granzymc B in S100a4.Atg7F / F tumors consistent with increased killing (Fig. 9J). In contrast, anti-inflammatory, regulatory Foxp3+ CD4+ T cells were less abundant in B16-F10 tumors in S100a4.Atg7F / F mice (Fig. 9K).Example 10Increased inflammatory gene expression in melanomas in 4tg7-deficient mice

[0167] B16-F10 melanomas grown in SI 00a4.Atg7F / For Atg7F / Fmice were collected.Total RNA was isolated and analyzed with RNA-seq. RNA-seq experimental design (left). Heat map of differentially expressed genes (Fig. 10A, right). FDR < 0.05. n=3. One outlier mouse was excluded. (Fig. 10B) Gene Ontology enrichment of genes upregulated in melanomas in S100ci4.Atg7F / F mice.Example 11Elevated monocytes and macrophages in autophagy-deficient mice.

[0168] (Fig- HA) Fraction of CD45+ cells that are monocytes (CD45+Zombie-B220- CD3-CDllb+CDllc-F4 / 80-) or macrophages (CD45+Zombie-B220-CD3-CDllb+CDllc- F4 / 80+). n=4, t-test, female. (All flow cytometry data is fraction of CD45+ unless noted). (Fig. 11B) Cells with monocyte marker CD 11b or macrophage marker F4 / 80 in mice with melanomas. n=5, t-test, female. (Fig. 11C) Monocytes (CD45+Zombie-B220-CD3-CDllb+CDllc-F4 / 80-) in Ubc.Atg7F / F mice with Ubc.Atg7F / F bone marrow and Atg7F / F mice with Atg7F / F bone marrow with melanomas. n=4-8, t-test, female. (Fig. HD) Macrophages (CD45+Zombie-B220- CD3-CDllb+CDllc-F4 / 80+) in Atg7F / F mice with Ubc.Atg7F / F or Atg7F / F bone marrow. n=5, t-test, female. (Fig. HE) Fraction of Ml macrophages (CD45+Zombie-B220-CD3- CDllb+CDllc-F4 / 80+CDllc-i-CD206-) in spleens of mice with melanomas. n=7-8, t-test, female.Example 12 Elevated cytokines in ,4 / «7-deficienl mice with melanomas.

[0169] (Figs. 12A-C) Multiplex ELISA cytokine levels in plasma of mice without (A) or with melanomas (Figs. 12B, C). Fold change in S100a4.Atg7F / F to Atg7F / F (B) or Ubc.Atg7F / Fto Atg7F / F (Fig. 12C). n=2-4, t-test, male and female (Fig. 12D). Cytokine arrays of YUMM1 .1 melanomas. n=4, t-test, female. * ** *** ****Example 13 Increased T cells in melanomas in autophagy deficient mice.

[0170] (Fig. 13A) CD8+ T cells (CD8+TCRP+NK1.1-) in mice without tumors, n = 5, t- test, female. (Fig. 13B) T cells in mice with melanomas. n=5, t-test, female. (Figs. 13C, D) CD8+ T cells in melanomas in S100a4.Atg7F / F (C, n=8) or Ubc.Atg7F / F (D, n=4) mice, t-test, female. (Fig. 13E) Granzyme B in CD8+ T cells in melanomas. n=4-6, t-test, female. (Fig. 13F) Fraction of CD4+ T cells in melanomas that are Foxp3+. n=5, Mann- Whitney, female. (Fig. 13G) CD8+ T cells in MC38 tumors. n=5-6, Mann- Whitney, female. (Fig. 13H) Granzyme B in CD8+ T cells in MC38 tumors. n=5-6, Mann- Whitney, female.Example 14 T cells transition from naive to effector in Atg7-deficient mice.

[0171] (Figs. 14A, B) Effector memory (CD62L-CD44high), central memory (CD62L+CD44high), and naive (CD62L+CD441ow) T cells in spleens (Fig. 14A) and blood (Fig. 14B) of mice with melanomas. n=4-5, t-test, female. (Fig. 14C) Fraction of CD8+ T cells that arc effector memory, central memory, or naive in blood of transplanted mice with melanomas. Bone marrow cell (BMC) and host genotypes are shown. n=5, t-test, female.Example 15T cells mediate reduced tumor growth in ATG7-deficient mice.

[0172] (Fig. 15A) T cells in melanomas in mice with anti-TCRp or isotype control antibodies. n=5, ANOVA, male and female. (Fig. 15B) Volumes and weights of tumors in mice with a-TCRp or isotype control. n=31-52, t-test, female. (Fig. 15C) Volumes of tumors in mice with a-TCRp or isotype control. n=6-8, ANOVA, female. (Fig. 15D) Volumes and weights of MC38 tumors in mice with a-TCRp or isotype control. n=14-52, ANOVA, female.Example 16Loss of autophagy combines with immune checkpoint inhibition to further reduce melanoma growth.

[0173] (Figs. 16A, B) Weights of melanomas in AtgZ-deficient or control mice treated with a-PDl / CTLA-4 or isotype control. (Fig. 16A) Ubc.Atg7F / F. n=23-25, ANOVA, female. (Fig. 16B) S100a4.Atg7F / F. n= 12- 14, ANOVA, female. (Fig. 16C) Immunoblots (left) and quantification (right) of SQSTMl / p62 in livers from mice treated with HCQ (60 mg / kg, i.p. daily) or PBS vehicle control, and a-PDl / CTLA-4 (100 pg each i.p. every 2-3 days) or isotype control. n=4, t-test. (Fig. 16D) HCQ. n=16-20, ANOVA, female.Example 17 The noncanonical NF-kB pathway is activated in autophagy-deficient DC.

[0174] Little is known about the noncanonical NF-kB pathway in DC and the TME. Because noncanonical NF-kB in DC regulates cross-priming to CD8+ T cells, it may play a role in reduced tumor progression in autophagy-deficient mice. We generated monocyte-derived DC from bone marrow cells from Ubc.Atg7F / F and Atg7F / F mice by culturing for 7 days in mouse GM-CSF (20 ng / ml) and IL-4 (10 ng / ml). Autophagy inactivation was confirmed by low ATG7 and LC3-II and LC3-II increased further when bafilomycin Al inhibited autophagolysosome degradation (Fig. 17). DC from Ubc.Atg7F / F contained higher levels of NFKB2 p52 isoform, but not elevated levels of phospho-p65S536, an indicator of high activity of the canonical NF-kB pathway (Fig. 17). This finding supports autophagy ablation activating the noncanonical NF-kB pathway in DC.Example 18Autophagy is induced in melanoma-associated fibroblasts in patient samples.

[0175] De-identified melanoma patient samples were collected from the UCLA Translational Pathology Laboratory and the Cedars-Sinai Tissue Biobank. A dermatopathologist identified regions rich in myofibroblasts based on the fibroblasts’ characteristic shape and nearby collagen deposition adjacent to the melanoma and distant from the melanoma. Samples from patients with Clark level 3-5 melanomas were immunostained with an antibody against LC3 (I and II) as an autophagy marker. LC3 levels in the peritumoral region (within 0.5 cm of the tumormargin) and tumor-remote areas (at least 1 cm away from tumors) of the same tissue slide were compared. Among the 23 samples analyzed, 19 cases showed significantly higher LC3 in melanoma-associated fibroblasts compared with fibroblasts in tumor-remote areas (combined p < 0.0001) (Fig. 18A, 18B).

[0176] Melanoma cells activate autophagy in co-cultured fibroblasts via TGF-P signaling. Using a transwell model, four human melanoma cell lines, but not primary human melanocytes (PCS), significantly induced autophagy marker LC3-II. (Fig. 18C) When cocultured fibroblasts were treated with bafilomycin, LC3-II levels increased further, confirming that melanoma cells increase autophagic flux in co-cultured fibroblasts (Fig. 18D). TGF-P can induce autophagy by activating transcription of autophagy genes. Treating fibroblasts expressing TGF-b responsive luciferase with melanoma conditioned medium (CM) increased luciferase activity while antiTGF-P antibody reduced luciferase activity (Fig 18E). Incubating fibroblasts in melanoma CM and a small molecule SMAD pathway inhibitor, LY-364947 (ref 126) reversed the autophagy induction by A2058 CM (Fig. 18F). These results together support a model in which TGF-P secreted by melanoma cells induces autophagy in surrounding cells.Example 19 Noncanonical NF-KB signaling for DC activation.

[0177] Autophagy-deficient DC exhibit increased activation, cytokine expression, and antigen presentation as a result of elevated noncanonical NF-KB activity. CRISPR / Cas9 is used to inactivate Nfkb2, Nik, or RelB to inhibit noncanonical NF-KB signaling or RelA / p65 for canonical NF-KB signaling in Ubc.Atg7F / F and Atg7F / F DC. Inactivating the noncanonical NF- KB pathway in Ubc.Atg7F / F DC I is expected to reverse phenotypes identified above.Experience with CRISPR / Cas9-mediated knockouts is shown in Figure 19. DC will be electroporated with recombinant Cas9 (10 pmol) and one of 2 short guides per gene (90 pmol) against Nik, Nfkb2, RelB or RelA, or a control short guide, using the Neon transfection system with 1 pulse of 1700 volts for 20 ms. Nik, Nfkb2, RelB or RelA knockout will be confirmed with sequencing and IB. We will monitor DC activation with immunophenotyping of activation markers, cytokine and chemokine secretion with MSD, and antigen presentation as described above in Ubc.Atg7F / F and Atg7F / F DC with Nik, Nfkb2, RelB, or RelA knockout. In addition,when Zbtb46.Atg7F / FNfkb2F / F mice and genotype controls are available, we will test DC and cDCl from them as well. We will determine whether inactivation of the canonical or noncanonical NF-KB pathway partially or fully reverses the phenotypes observed in Ubc.Atg7F / F DC.Example 20Dendritic cell autophagy as a regulator of noncanonical NF-KB in the melanoma microenvironment

[0178] As described herein, data show that loss of autophagy in the entire mouse, the tumor microenvironment, bone marrow-derived cells, or dendritic cells results in reduced growth of orthotopic melanomas. The smaller melanomas in mice with an autophagy-deficient tumor microenvironment likely reflects the activity of T cells, as T cell depletion results in larger tumors. Application of the role of autophagy in dendritic cells in clinical melanomas, to investigate the effects of autophagy deficiency on dendritic cell activity, shows that introducing autophagy-deficient dendritic cells into tumors can reduce their growth.

[0179] Data on the function of autophagy-deficient dendritic cells in T cell co-cultures is shown in Figure 20. Bone marrow-derived dendritic cells were generated from Atg7F / Fand Ubc.Atg7F / Fmice by incubating bone marrow cells with 20 ng / mL GM-CSF and 20 ng / mL IL-4 for 6 days. Ten thousand dendritic cells were incubated with 100,000 total T cells isolated from spleens of naive wild-type mice and the co-cultures were grown in dendritic cell culture medium along with 100,00 units / ml IL-2. Ten days post co-culture, wild-type dendritic cells pulsed with B16-F10 protein lysate were introduced into the cultures for 6 hours to assess T cell responses. Flow cytometry analysis revealed the number of effector CD8+ T cells based on cell surface markers CD44 and CD62L (Figure 20). When dendritic cells isolated from Ubc.Atg7F / Fmice were utilized in the original culture, a significant increase was observed in the number of effector CD8+ T cells compared with dendritic cells from Atg7F / Fmice, demonstrating that autophagydeficient dendritic cells are more effective at generating effector CD8+ T cells and promoting CD8+ T cell proliferation. These data support our central hypothesis that autophagy-deficient dendritic cells promote T cell activation that results in a heightened immune response and reduced melanoma growth.Example 21Enhancing antigen presentation by dendritic cells in vitro or ex vivo

[0180] Autophagy is suppressed in dendritic cells isolated from a patient, prepared from PMBCs obtained from a patient, isolated from or prepared from a donor, or dendritic cells from a cell line. The dendritic cells are exposed to an antigen. The suppression or autophagy and exposure to the antigen may be in any order or concurrently. Any of the methods of combination of methods for suppressing autophagy may be used. After such treatment, the dendritic cells are infused into the patient. Such dendritic cells present antigen more efficiently and enhance the immune response thereto.

[0181] A checkpoint inhibitor is administered to the patient concurrently or before or after administration of the dendritic cells. Further enhancement of the immune response to the antigen is achieved.Example 22 Enhancing CAR-T cells

[0182] T cells collected from a patient and modified to express chimeric antigen receptors (CAR) and returned to the same patient, can be enhanced by also suppressing autophagy in the T cells. Chemical or genetic methods may be used as described elsewhere herein. Such CAR-T cells have enhanced activity at targeting tumors.Example 23 Enhancing Cells Derived from Hematopoietic Stem Cells

[0183] Autophagy is suppressed in hematopoietic stem cells (HSC) by any one or more methods such as but not limited to those described herein. Such HSC are infused into the patient. In some embodiments, the autophagy-suppresses HSCs are differentiated into macrophages ex vivo before being administered to the patient.

[0184] The HSCs are engineered to suppress autophagy in the tumor microenvironment. In some embodiments, the suppression of autophagy is driven by the expression of S100a4 in thetumor microenvironment. Thus, suppression of autophagy occurs in stem cells and their differentiated progeny only near a tumor.Example 24Enhancing immune response by macrophages in vitro or ex vivo

[0185] Autophagy is suppressed in macrophages isolated from a patient, prepared from PMBCs obtained from a patient, isolated from or prepared from a donor, or macrophages or monocytes obtained from a cell line. The macrophages are exposed infused into the patient. Such macrophages enhance the immune response thereto.

[0186] A checkpoint inhibitor is administered to the patient concurrently or before or after administration of the macrophages. Further enhancement of the immune response gen is achieved.Example 25Enhancing tertiary lymphoid structure (TLS) immune response

[0187] Autophagy is suppressed in tertiary lymphoid structures (TLS) in vivo by exposing TLS in vivo to one or more agents that suppress autophagy, or inactivating at least one gene that suppresses autophagy in the TLS in vivo, or activating at least one gene that suppresses autophagy in the TLS in vivo. In some embodiments, a combination of any of the forgoing are used. The immune response by the TLS is enhanced.

[0188] A checkpoint inhibitor is administered to the patient concurrently or before or after autophagy suppression in TLS is carried out. Further enhancement of the immune response is achieved.Example 26Testing the importance of noncanonical NF-kB signaling for DC activation

[0189] Autophagy-deficient DC exhibit increased activation, cytokine expression, and antigen presentation as a result of elevated noncanonical NF-kB activity. CRISPR / Cas9 is used to inactivate Nfkb2, Nik, or RelB to inhibit noncanonical NF-kB signaling or RelA / p65 for canonical NF-kB signaling in Ubc.Atg7F / F and Atg7F / F DC. Inactivating the noncanonical NF-kB pathway in Ubc.Atg7F / F DC reverses phenotypes identified above is tested. Figure 19 shows CRISPR / Cas9-mcdiatcd knockouts of Atg7. DC will be electroporated with recombinant Cas9 (10 pmol) and one of 2 short guides per gene (90 pmol) against Nik, Nfkb2, RelB or RelA, or a control short guide, using the Neon transfection system with 1 pulse of 1700 volts for 20 ms. Nik, Nfkb2, RelB or RelA knockout will be confirmed with sequencing and IB. DC activation is monitored with immunophenotyping of activation markers, cytokine and chemokine secretion with MSD, and antigen presentation as described above in Ubc.Atg7F / F and Atg7F / F DC with Nik, Nfkb2, RelB, or RelA knockout. In addition, with Zbtb46.Atg7F / FNfkb2F / F mice and genotype controls their DC and cDCl will be evaluated. Inactivation of the canonical or noncanonical NF-kB pathway will be shown to partially or fully reverse the phenotypes observed in Ubc.Atg7F / F DC.Example 27Testing the role of autophagy and the noncanonical NF-kB pathway in DC for melanoma progression, tumor and non-tumor histology, and mouse survivalL00190J Cohorts of >10 mice will be raised with autophagy and noncanonical NF-kB inactivation in DC and controls: Zbtb46.Atg7F / FNfkb2F / F, Zbtb46.Atg7F / F, Zbtb46.Nfkb2F / F, and Atg7F / F mice. Zbtb46.Atg7F / F mice will breed with Nfkb2F / F mice (Jackson #028720). 0.5 x 106 Bl 6-F10 or YUMM1 .1 mouse melanoma cells will be introduced intradermally to develop orthotopic melanomas. Tumor volumes will be monitored every 2-3 days and final tumor weights after 15 days of tumor growth. FFPE tumor sections are stained with H&E and analyzed histologically by UCLA board-certified dermatopathologists. Non-tumor tissues will also be collected including liver, spleen, lungs, skin, and lymph nodes and monitor for signs of toxicity including immune cell infiltration, necrosis or apoptosis by H&E and activated caspase-3 staining. In separate experiments, survival will be assessed by monitoring mice with tumors until they reach a euthanasia endpoint (deep ulcer, large tumor size, failing health).

[0191] Autophagy inactivation in DC in anticipated to result in increased levels of noncanonical NF-kB components, elevated NF-kB target gene expression, T cell activation, and killing of tumor cells. Involvement of canonical vs. noncanonical NF-kB signaling will be resolved in mediating the effect of autophagy on DC biology and function. Smaller tumors inmice are expected with autophagy inactivation in DC, which will be accompanied by activated DCs that sccrctc higher levels of cytokines and more activated CD8+ T cells. Autophagy loss in DC will improve mouse survival.Example 28 Anti-tumor efficacy of autophagy-deficient DC as a potential cellular therapy using murine models

[0192] Loss of autophagy in DC results in a heightened CD8+ T cell-mediated antimelanoma response. Introducing autophagy-deficient DC into growing melanomas will reduce tumor growth. Bone marrow-derived DC will be generated from autophagy -proficient and - deficient mice. DC or cDCl will be pulsed with B16F10 antigen gplOO, then infused into B16F10 (gpl00+) or YUMM1.1 (gplOO-, included to test the importance of the pulse), melanoma-bearing mice intravenously (IV) to assess DC or cDCl-mediated anti-tumor T cell responses against a known antigen. In this scenario, DC or cDCl are expected to prime and activate gplOO- specific T cells in the lymph node without the need to penetrate the tumor. Alternatively, unpulsed DC or cDCl will be directly injected intratumorally (IT) to assess DC or cDCl-induced anti-tumor T cell activation against available tumor antigens in the TME. In preliminary studies, in vivo trafficking will be monitored of introduced DC with flow cytometry. With IT DC or cDCl introduction, reduced growth is anticipated of both B16F10 and YUMM1 .1 melanomas. Optimal dosing and timing will be determined that induce a robust anti-tumor immune response that reduces tumor growth, while limiting toxicity to non-tumor tissues. If introducing pulsed DC IV is the most effective strategy, to translate such findings to the clinic, human DC will be pulsed with established melanoma antigens that can be validated in a specific patient’s tumor with IHC.Example 29 In vivo trafficking and kinetics of injected DC and cDCl

[0193] DC or cDCl will be generated from CD45.2 Ubc.Atg7F / F or Atg7F / F mice. Autophagy-proficient and -deficient DC will be FACS-sorted for purity. 0.5 x 106 B16-F10 or YUMM1.1 melanoma cells will be introduced intradermally into cohorts of >10 syngeneic C57BL / 6 CD45.1 mice. Autophagy-deficient or -proficient DC or cDCl, or PBS control, will beIT injected into B 16-F 10 or YUMM 1 .1 intradermal tumors. Alternatively, DC or eDC 1 will be pulsed with gplOO peptide for 5 h prior to IV injection into tumor-bearing mice. Tumors and TDLN will be harvested 2, 24, and 48 h post injection, and DC or cDCl levels will be assessed by flow cytometry for DC and T cells as in Aim 2 using CD45.1 and CD45.2 to distinguish introduced vs endogenous DC. The extent of autophagy-proficient and -deficient DC trafficking, persistence and activation under the different conditions, will be assessed.Example 30 Anti-tumor efficacy of Atg7-proficient and -deficient DC

[0194] B 16F 10 or YUMM 1.1 will be introduced intradermally and introduce autophagyproficient or -deficient DC or cDCl, or no DC, IT or IV, as described above, with >10 mice per condition. DC or cDCl will be administered on days 4, 6, and 8 post tumor inoculation. Tumor volumes will be monitored every 2-3 days and tumor weights will be determined on day 15. In separate experiments, mouse survival will be compared by maintaining the mice until they reach a euthanasia endpoint. H&E staining will be used to assess tumor histology and evaluate any DC-induced toxicity to other tissues including liver, spleen, lungs, lymph nodes, and skin.Example 31 DC therapy-induced immune changes

[0195] To understand local and systemic immune changes in response to DC or cDCl cellular therapy, comprehensive immune monitoring studies of mice will be performed, with introduced DC and controls. FFPE tumor sections will be subjected to MIF studies with validated 7-plex antibody panels, including activated caspase-3 (apoptosis), CD8 and CD4 (T cells), granzyme B (T cell activation), Ki67 (proliferation), CD 11c (DC), CD86 (DC activation), F4 / 80 (macrophages), CD31 (vasculature), and DAPI (DNA) (divided into two panels).Antibodies will be added for NIK, phospho-IKKa, and (nuclear) NFKB2 for the noncanonical NF-kB pathway. MIF studies will compare the abundance, proliferative state, activation, and spatial distribution of T cells and DC, and readouts for noncanonical NF-kB pathway signaling, when autophagy-proficient or autophagy-deficient DC are introduced. In addition, on days 10-12 of tumor growth, tumors and TDLN will be collected and flow cytometry performed for DC (CDllb+CDllc+MHC II), CD4+ and CD8+ T cells (CD45+NK1.1-TCRP+); naive, centralmemory and central effector T cells (CD44 and CD62L); intracellular cytokines (IFN-g, TNF-a); killing effectors (perforin, granzyme B); and Foxp3+ regulatory T cells.

[0196] Autophagy-deficient DC or cDCl will induce pro-inflammatory cytokines and chemokines, T cell recruitment and activation, T cell-mediated tumor cell killing, and reduced tumor growth. Mice that receive autophagy-deficient DC or cDCl will survive longer after melanoma cells are introduced, and that toxicity to nontumor tissue from the introduced DC or cDCl will be minimal.

[0197] While certain features disclosed herein have been illustrated and described herein, many modifications, substitutions, changes, and equivalents will now occur to those of ordinary skill in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the guidance herein.

Claims

What is claimed is:

1. A method for enhancing an immune response in a subject comprising the steps of: a. identifying one or more immune response associated cell types in the subject or one or more immune response associated cell types to be administered to the subject; and b. suppressing autophagy in the one or more immune response associated cell types in the subject or in the one or more immune response associated cell types to be administered to the subject, wherein the suppressing is performed in vivo, ex vivo, in vitro, or by any combination thereof on one or more of either or both cell types in the subject or to be administered to the subject; c. wherein an immune function is enhanced in vivo, ex vivo or in vitro by the one or more immune response associated cell types in the subject or in the one or more immune response associated cell types after administration to the subject, thus enhancing the immune response in the subject.

2. The method of claim 1 , wherein the immune response associated cell types in the subject are one or more of dendritic cells, macrophages, monocytes, cancer-associated fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells.

3. The method of claim 1, wherein the immune response associated cell types to be administered to the subject are one or more of dendritic cells, macrophages, monocytes, cancer-associated fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells.

4. The method of claim 3, wherein the immune response associated cell types to be administered to the subject arc obtained from the subject, obtained from a donor, or obtained from banked immune response associated cells or blood products, or induced in vitro or ex vivo from any of the foregoing.

5. The method of any one of claims 1-4, wherein an immune response associated cell type comprises a tertiary lymphoid structure, a lymph node, a secondary lymphoid organ, a B cell follicle, a tumor, or a wound.

6. The method of claim 3, wherein the cell types are fibroblast reticular cells, follicular dendritic cells or lymphoid organizer cells.

7. The method of claim 1, wherein the suppressing autophagy is performed on the one or more immune response associated cell types in, or administered to, the subject, independently by any one or more of: a. exposing the one or more immune response associated cell types in vivo, ex vivo or in vitro to one or more agents that suppresses autophagy; and / or b. inactivating one or more genes responsible for or associated with autophagy in the one or more immune response associated cell types in vivo, ex vivo or in vitro; and / or c. activating one or more genes responsible for suppressing autophagy in the one or more immune response associated cell types in vivo, ex vivo or in vitro; and / or d. any combination thereof.

8. The method of any one of claims 1-7, wherein the autophagy is non-canonical autophagy.

9. The method of claim 8, wherein the agent that suppresses autophagy inhibits LC3 mediated processing.

10. The method of claim 9, wherein the agent that inhibits LC3 mediated processing inhibits LC3 lipidation.11 . The method of claim 10, wherein the agent that inhibits LC3 lipidation is selected from 5- (2,3-dichlorophcnyl)-2-(((2-morpholinocthyl)amino)mcthylcnc) cyclohexane- 1 ,3-dionc (DC-LC3in-D5) or an analogue thereof.

12. The method of claim 10, wherein the agent that inhibits LC3 lipidation is selected from a V-ATPase inhibitor.

13. The method of claim 12, wherein the V-ATPase inhibitor is BafAl, concanamycin A (ConA), or any combination thereof.

14. The method of claim 13, further comprising diphenyleneiodonium (DPI), GSK2795039, or any combination thereof.

15. The method of claim 1, wherein the suppressing autophagy comprises suppressing Atg7 expression or function.

16. The method of claim 15, wherein the Atg7 gene is inactivated in the cell type in vitro, ex vivo or in vitro.

17. The method of claim 16, wherein the suppressing Atg7 expression or function comprises exposing the cell type to a siRNA targeting Atg7 or a Cas9 with a guide RNA against ATG7.

18. The method of claim 16, wherein the inactivating Atg7 is Atg7 knockout.

19. The method of claim 7, wherein the gene associated with autophagy is ATG7.

20. The method of claim 7, wherein a combination of any two or more of (a), (b) and / or (c) synergistically provides a suppression of autophagy.

21. The method of claim 1, wherein the enhancing the immune response comprises one or more of: enhanced antigen presentation by dendritic cells; enhanced cytokine production; enhanced chemokine production; elevated inflammatory gene expression; elevated macrophage activation; elevated T cell recruitment; elevated T cell activation, or any combination thereof.

22. The method of claim 21 , wherein the enhanced antigen presentation comprises enhanced presentation of a cancer antigen or a fragment thereof.

23. The method of claim 1, wherein the autophagy is suppressed in dendritic cells in vitro or ex vivo, the dendritic cells are exposed to an antigen in vitro or ex vivo, and the dendritic cells are infused into the subject.

24. The method of claim 1, wherein the autophagy is suppressed in macrophages or monocytes in vitro or ex vivo, the macrophages or monocytes are exposed to an antigen in vitro or ex vivo, and the macrophages or monocytes are infused into the subject.

25. The method of claim 1, wherein a tumor is infused with an agent that suppresses autophagy.

26. The method of claim 1, wherein autophagy is suppressed in fibroblasts which are infused into a tumor.

27. The method of claim 1, wherein the subject has an immunogen-associated disease or condition for which enhanced immunity directed thereto is therapeutically beneficial, or wherein the subject is at risk for an immunogen-associated disease or condition for which enhanced immunity directed thereto is preventatively beneficial.

28. The method of claim 27, wherein the disease is cancer.

29. The method of claim 28, wherein the cancer is melanoma.

30. The method of claim 1, wherein the subject is also administered immune checkpoint blockade.

31. The method of claim 30, wherein the immune checkpoint blocks or inhibits CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK 1 kinase, CHK2 kinase, A2aR, or a B-7 family ligand.

32. An immune response associated cell type in which autophagy is suppressed.

33. The immune response associated cell type of claim 32, comprising dendritic cells, macrophages, monocytes, canccr-associatcd fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells.

34. The cell type of claim 33, wherein the cell type is from a subject, from a donor, or from banked cells or blood products.

35. The immune response associated cell of claim 32, wherein the autophagy is non- canonical autophagy.

36. The immune response associated cell type cell of claim 32, for administering to a subject having an immunogen-associated disease or condition for which enhanced immunity directed thereto is therapeutically beneficial, or administering to a subject at risk for an immunogen-associated disease or condition for which enhanced immunity directed thereto is preventatively beneficial.

37. A method for enhancing an immune response in a subject comprising the steps of: a. identifying one or more immune response associated cell types in the subject or one or more immune response associated cell types to be administered to the subject; and b. suppressing or inactivating Atg7 in the one or more immune response associated cell types in the subject or in the one or more immune response associated cell types to be administered to the subject, wherein the suppressing or inactivating is performed in vivo, ex vivo, in vitro, or by any combination thereof on one or more of either or both cell types in the subject or to be administered to the subject; wherein an immune function is enhanced in vivo, ex vivo or in vitro by the one or more immune response associated cell types in the subject or in the one or more immune response associated cell types after administration to the subject, thus enhancing the immune response in the subject.

38. The method of claim 37, wherein the immune response associated cell types in the subject arc one or more of dendritic cells, macrophages, monocytes, cancer-associated fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells.

39. The method of claim 37, wherein the immune response associated cell types to be administered to the subject are one or more of dendritic cells, macrophages, monocytes, cancer-associated fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells.

40. The method of claim 37, wherein the immune response associated cell types to be administered to the subject are obtained from the subject, obtained from a donor, or obtained from banked immune response associated cells or blood products, or induced in vitro or ex vivo from any of the foregoing.41 . The method of claim 37, wherein an immune response associated cell type comprises a tertiary lymphoid structure, a lymph node, a secondary lymphoid organ, a B cell follicle, a tumor, or a wound.

42. The method of claim 37, wherein the cell types are fibroblast reticular cells, follicular dendritic cells or lymphoid organizer cells.

43. The method of claim 37, wherein the suppressing or inactivating Atg7 is performed on the one or more immune response associated cell types in, or administered to, the subject, independently by any one or more of: a. exposing the one or more immune response associated cell types in vivo, ex vivo or in vitro to one or more agents that suppresses or inhibit Atg7; and / orb. inactivating Atg7 in the one or more immune response associated cell types in vivo, ex vivo or in vitro; and / or c. the combination thereof.

44. The method of claim 37, wherein the Atg7 gene is inactivated in the cell type.

45. The method of claim 37, wherein the inactivation may be in vitro, ex vivo or in vivo.

46. The method of claim 37, wherein the suppressing Atg7 expression or function comprises exposing the cell type to a siRNA targeting Atg7.

47. The method of claim 37, wherein the inactivating ATG7 comprises exposing the cell type to a Cas9 with a guide RNA against ATG7.

48. The method of claim 37, wherein the inactivating Atg7 is Atg7 knockout.

49. The method of claim 43, wherein the combination of (a) and (b) synergistically provides an enhancement of the immune response.

50. The method of any one of claims 37-49, wherein the enhancing the immune response comprises one or more of: enhanced antigen presentation by dendritic cells; enhanced cytokine production; enhanced chemokine production; elevated inflammatory gene expression; elevated macrophage activation; elevated T cell recruitment; elevated T cell activation, or any combination thereof. In some embodiments, the enhanced antigen presentation comprises enhanced presentation of a cancer antigen or a fragment thereof.

51. The method of claim 50, wherein Atg7 is suppressed or inactivated in dendritic cells in vitro or ex vivo, the dendritic cells are exposed to an antigen in vitro or ex vivo, and the dendritic cells are infused into the subject.

52. The method of claim 50, wherein Atg7 is suppressed or inactivated in macrophages or monocytes in vitro or ex vivo, the macrophages or monocytes are exposed to an antigen in vitro or ex vivo, and the macrophages or monocytes are infused into the subject.

53. The method of claim 50, wherein a tumor is infused with an agent that suppresses or inactivates Atg7.

54. The method of claim 50, wherein Atg7 is suppressed or inactivated in fibroblasts which are infused into a tumor.

55. The method of claim 50, wherein the enhancing the immune response comprises one or more of: enhanced antigen presentation by dendritic cells; enhanced cytokine production; enhanced chemokine production; elevated inflammatory gene expression; elevated macrophage activation; elevated T cell recruitment; elevated T cell activation, or any combination thereof.

56. The method of claim 55, wherein the enhanced antigen presentation comprises enhanced presentation of a cancer antigen or a fragment thereof.

57. The method of claim 37, wherein the subject has an immunogen-associated disease or condition for which enhanced immunity directed thereto is therapeutically beneficial, or wherein the subject is at risk for an immunogen-associated disease or condition for which enhanced immunity directed thereto is preventatively beneficial.

58. The method of claim 57, wherein the disease is cancer.

59. The method of claim 58, wherein the cancer is melanoma.

60. The method of claim 37, wherein the subject is also administered immune checkpoint blockade.61 . The method of claim 60, wherein the immune checkpoint blocks or inhibits CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD 160, CGEN- 15049, CHK 1 kinase, CHK2 kinase, A2aR, or a B-7 family ligand.

62. An immune response associated cell type in which Atg7 is inactivated or suppressed.

63. The immune response associated cell type of claim 62, comprising dendritic cells, macrophages, monocytes, canccr-associatcd fibroblasts, fibroblast reticular cells, B cells, endothelial cells, myeloid derived suppressor cells, follicular dendritic cells, bone marrow derived dendritic cells, resident dendritic cells, classical dendritic cells, plasmacytoid dendritic cells or lymphoid organizer cells.

64. The cell type of claim 63, wherein the cell type is from a subject, from a donor, or from banked cells or blood products.

65. The immune response associated cell type cell of claim 63, for administering to a subject having an immunogen-associated disease or condition for which enhanced immunity directed thereto is therapeutically beneficial, or administering to a subject at risk for an immunogen-associated disease or condition for which enhanced immunity directed thereto is preventatively beneficial.

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