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Immunogenic tumors grow progressively even when heavily infiltrated by CD8+ T cells. We investigated how to rescue CD8+ T-cell function in long-established immunogenic melanomas that contained a high percentage of endogenous PD-1+ tumor-specific CD8+ T cells that were dysfunctional. Treatment with αPD-L1– and αCTLA-4–blocking antibodies did not prevent tumors from progressing rapidly. We then tested exogenous tumor-specific antigen delivery into tumors using Salmonella Typhimurium A1-R (A1-R) to increase antigen levels and generate a proinflammatory tumor microenvironment. Antigen-producing A1-R rescued the endogenous tumor-specific CD8+ T-cell response: Proliferation was induced in the lymphoid organs and effector function was recovered in the tumor. Treatment with antigen-producing A1-R led to improved mouse survival and resulted in 32% rejection of long-established immunogenic melanomas. Following treatment with antigen-producing A1-R, the majority of tumor-specific CD8+ T cells still expressed a high level of PD-1 in the tumor. Combining antigen-producing A1-R with αPD-L1-blocking antibody enhanced the expansion of tumor-specific CD8+ T cells and resulted in 80% tumor rejection. Collectively, these data show a powerful new therapeutic approach to rescue dysfunctional endogenous tumor-specific CD8+ T cells and eradicate advanced immunogenic tumors. Cancer Immunol Res; 1(2); 123–33. ©2013 AACR.

Suppl. Figure 1. A. Tumors that escape an equilibrium caused by CD8 T cells targeting tumor stroma are not Ag-loss variants. B. The Ag-specific lytic function of 2C cells in vivo has not declined in mice bearing escaping tumors. C. Equilibrium can be achieved in mice lacking perforin Suppl. Figure 2. PD-L1 but not Tim3 blocking in vitro partially reverses the ability of tumor-infiltrating CD8+ T cells to produce cytokines. Suppl. Figure 3. A second transfer of CD8+ T cells following tumor irradiation or anti-Gr1 antibody therapy cannot prevent tumor escape. Suppl. Figure 4. Cancer cells are negative for MHC-II in vitro but can up-regulate MHC-II in tumors in vivo.

Adoptively transferred CD8+ T cells can stabilize the size of solid tumors over long periods of time by exclusively recognizing antigen cross-presented on tumor stroma. However, these tumors eventually escape T-cell–mediated growth control. The aim of this study was to eradicate such persistent cancers. In our model, the SIYRYYGL antigen is expressed by cancer cells that lack the MHC-I molecule Kb needed for direct presentation, but the antigen is picked up and cross-presented by tumor stroma. A single injection of antigen-specific 2C CD8+ T cells caused long-term inhibition of tumor growth, but without further intervention, tumors started to progress after approximately 3 months. Escape was associated with reduced numbers of circulating 2C cells. Tumor-infiltrating 2C cells produced significantly less TNFα and expressed more of the “exhaustion” markers PD-1 and Tim-3 than T cells from lymphoid organs. High-dose local ionizing radiation, depletion of myeloid-derived suppressor cells, infusions of additional 2C cells, and antibodies blocking PD-L1 did not prevent tumor escape. In contrast, adoptive transfer of allogeneic CD4+ T cells restored the numbers of circulating Ag-specific CD8+ T cells and their intratumoral function, resulting in tumor eradication. These CD4+ T cells had no antitumor effects in the absence of CD8+ T cells and recognized the alloantigen cross-presented on tumor stroma. CD4+ T cells might also be effective in cancer patients when PD-1/PD-L1 blockade does not rescue intratumoral CD8+ T-cell function and tumors persist. Cancer Immunol Res; 5(2); 127–36. ©2017 AACR.

PDF file - 291K, Figure S1. A1-R SIINF preferentially accumulates in established B16-OVA tumors. Figure S2. A1-R SIINF induces systemic SIINF-specific CD8+ T cell proliferation. Figure S3. Weekly A1-R SIINF treatment leads to rejection of established B16-OVA tumors. Figure S4. The majority of intratumoral SIINF-specific CD8+ T cells still express a high level of PD-1 following A1-R OVA treatment.

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Cytokine production by 2C CD8+ T cells from mice treated or not with allogeneic CD4+ T cells

Supplementary Methods - Contains detailed information on whole exome and RNA sequencing, variant calling, TCR gene transfer, and longitudinal imaging Supplementary Fig. S1 - Pipeline for exome/RNA analysis and neoepitope prediction Supplementary Fig. S2 - Pipeline for exome and clustering analysis of autochthonous 8101 tumor fragments Supplementary Fig. S3 - Generation of 1D9 T cells Supplementary Fig. S4 - Generation of mp68-expressing tumor cell lines Supplementary Fig. S5 - Expansion of 1D9 T cells is antigen-specific and not driven by lymphopenia-induced proliferation Supplementary Fig. S6 - 1D9 T cells do not infiltrate in tumors if mp68 is not expressed Supplementary Table S1 - Results of whole exome sequencing of Bulk tumor cells and Bulk reisolates after 1D9 T cell therapy Supplementary Table S2 - Results of whole exome sequencing of fragments derived from the autochthonous 8101 tumor

Purpose: Solid tumors that have grown two weeks or longer in mice and have diameters larger than 1 cm are histologically indistinguishable from autochthonous human cancers. When experimental tumors reach this clinically relevant size, they are usually refractory to most immunotherapies but may be destroyed by adoptive T-cell transfer. However, TCR-transgenic T cells and/or tumor cells overexpressing antigens are frequently used in these experiments. Here we studied the requirements for destroying clinical size, unmanipulated 8101 tumors by adoptive cell therapy.Experimental Design: 8101 arose in an old mouse after chronic exposure to UV light. A cancer line was established, which was never serially transplanted. The immunodominant CD8+ T cell-recognized antigen of this tumor is caused by a somatic tumor–specific mutation in the RNA helicase p68. 8101 tumors were treated with spleen cells from young naive, or young and old immunized mice to ascertain the characteristics of immune cells that lead to rejection.Results: Here we show that the mutant p68 peptide has an exceptionally high affinity to the presenting MHC class I molecule Kb and that spleen cells from immunized young syngeneic mice adoptively transferred to Rag−/− or cancer-suppressed euthymic mice eradicate 8101 tumors larger than 1 cm in average diameter and established for several weeks. Spleen cells from naive young mice or from old and boosted (reimmunized) mice were ineffective.Conclusions: Relapse-free destruction of large and long-established tumors expressing a genuine very high-affinity tumor-specific antigen can be achieved by using adoptive transfer of lymphocytes from immunized young individuals. Clin Cancer Res; 18(9); 2526–33. ©2012 AACR.

(A) Quantification of bands from Western gel shown in Main Figure 3A using Image J. The pixel units obtained for each protein band was normalized to the pixel units calculated from their respective b-Actin loading control. (B-G) Clonogenic survival of various human and mice tumor cell lines with stable shSTING knockdown in response to increasing dose of IR. (H-I) Cell viability assays of primary and immortalized MEFs isolated from WT and STING-/- mice in response to increasing IR dose. Data are representative of at least three experiments. P-values were determined using unpaired Student's t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

PDF file, 33KB, Percentages and absolute numbers of regulatory CD4+ T cells (Treg) in the peripheral blood of young (5 month-old) and old (15 monthold) mice.

(A) Quantification of bands from Western gel shown in Main Figure 5D using Image J. The pixel units obtained for each protein band was normalized to the pixel units calculated from their respective b-Actin loading control. (B-E) Kinetic analysis of WT MEFs (B) as well as shScrambled D54 (C), HCT116 (D), and SCC61 (E) proliferation in vitro were measured over time in response to WEE1 inhibitor MK1775 {plus minus} IR (6 Gy) using the IncuCyte live cell imaging system. In vitro growth curve data are representative of at least three experiments, each with n = 3 per group. P-values were determined using unpaired Student's t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

(A-D) Quantification of bands from Western gels shown in Main Figures 3H (A), 3I (B), 3J (C), and 3K (D) using Image J. The pixel units obtained for each protein band was normalized to the pixel units calculated from their respective b-Actin loading control. (E) Western analysis of lysates harvested from HEK293 cells that were transiently transfected with either an empty vector of recombinant STING at 48 hours following exposure to 3Gy IR show that the protein levels of p21 and cyclin D1 were restored following overexpression of STING in HEK293 cells. (F) Western analysis of lysates harvested from siCDKN1A HCT116 cells at 48 hours following exposure to increasing dose of IR demonstrate that depletion of p21 did not affect STING expression, and only affected proteins that it regulates downstream of the pathway. (G) Western analysis of lysates harvested from MEFs that were transiently transfected with siRNA targeting STING and/or p21 at 48 hours reveal that depletion of STING led to down-regulation of p21, but not vice versa. Quantified bands are shown right next to each Western blot panels. (H-J) Kinetic analysis of STING-depleted human tumor cell lines D54 (H), HCT116 (I), and SCC61 (J) that were transiently transfected with siRNA targeting p21. Proliferation of nuclei-stained cells were measured in vitro over time using the IncuCyte live cell imaging system. The number of nuclei-stained cells were calculated using the Incucyte software and data are representative of three experiments, each with n = 3 per group. P-values were determined using unpaired Student's t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

PDF file, 196KB, Loss of expression of the mutant p68 (mp68) rejection antigen by all 8101 variants that grew progressively in na�ve young mice but retention by 8101 tumor that developed in the old recipient.

WT and STING-/- MEFs examined by cytokinesis-block micronucleus (CBMN) assay by treatment with IR, then cytochalasin at 24 hours and then fixed and stained for DNA and mitochondrial/cytoplasmic compartments using Draq5 and MitoTracker Red at 48 hours. Scale bar, 10 µm.

(A-C) BrdU/PI cell cycle profile of MEFs (A), p53-/- HCT116 (B), and CDKN1A-depleted HCT116 cells (C) at 24 hours post-IR. Flow cytometry was used to measure the percent cell population in G1, S, and G2/M phase of single cells stained with FITC-conjugated anti-BrdU (y-axis) and PI (x-axis). (D-F) Western analyses of lysates from WT and STING-/- immortalized MEFs (D), p53-negative HCT116 (E), and siCDKN1A HCT116 (F) probed for mitotic proteins BUB1 and MAD2L1 at 48 hours post-exposure to increasing IR doses. Quantified bands are shown below each of their respective Western blot panels. (G) ChIP analyses reveal that in the absence of either p21 or STING, E2F4 does not bind to the promoter region of BUB1 and MAD2L1 in HCT116 tumor cells. Data are representative of at least two experiments. P-values were determined using unpaired Student's t-test. Error bars are SEM. *P < 0.05, **P < 0.01, ***P < 0.005.

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Given the integral role of stimulator of interferon genes (STING, TMEM173) in the innate immune response, its loss or impairment in cancer is thought to primarily affect antitumor immunity. Here we demonstrate a role for STING in the maintenance of cellular homeostasis through regulation of the cell cycle. Depletion of STING in human and murine cancer cells and tumors resulted in increased proliferation compared with wild-type controls. Microarray analysis revealed genes involved in cell-cycle regulation are differentially expressed in STINGko compared with WT MEFs. STING-mediated regulation of the cell cycle converged on NFκB- and p53-driven activation of p21. The absence of STING led to premature activation of cyclin-dependent kinase 1 (CDK1), early onset to S-phase and mitosis, and increased chromosome instability, which was enhanced by ionizing radiation. These results suggest a pivotal role for STING in maintaining cellular homeostasis and response to genotoxic stress.Significance:These findings provide clear mechanistic understanding of the role of STING in cell-cycle regulation, which may be exploited in cancer therapy because most normal cells express STING, while many tumor cells do not.See related commentary by Gius and Zhu, p. 1295

Purpose: Cancers usually contain multiple unique tumor-specific antigens produced by single amino acid substitutions (AAS) and encoded by somatic nonsynonymous single nucleotide substitutions. We determined whether adoptively transferred T cells can reject large, well-established solid tumors when engineered to express a single type of T-cell receptor (TCR) that is specific for a single AAS.Experimental Design: By exome and RNA sequencing of an UV-induced tumor, we identified an AAS in p68 (mp68), a co-activator of p53. This AAS seemed to be an ideal tumor-specific neoepitope because it is encoded by a trunk mutation in the primary autochthonous cancer and binds with highest affinity to the MHC. A high-avidity mp68-specific TCR was used to genetically engineer T cells as well as to generate TCR-transgenic mice for adoptive therapy.Results: When the neoepitope was expressed at high levels and by all cancer cells, their direct recognition sufficed to destroy intratumor vessels and eradicate large, long-established solid tumors. When the neoepitope was targeted as autochthonous antigen, T cells caused cancer regression followed by escape of antigen-negative variants. Escape could be thwarted by expressing the antigen at increased levels in all cancer cells or by combining T-cell therapy with local irradiation. Therapeutic efficacies of TCR-transduced and TCR-transgenic T cells were similar.Conclusions: Gene therapy with a single TCR targeting a single AAS can eradicate large established cancer, but a uniform expression and/or sufficient levels of the targeted neoepitope or additional therapy are required to overcome tumor escape. Clin Cancer Res; 22(11); 2734–43. ©2015 AACR.See related commentary by Liu, p. 2602

PDF file, 174KB, A cancer progressor variant is selected by a young mouse upon injection of cryopreserved 8101 original tumor fragments.

Cell cycle profile of pulse EdU-labeled (A) and chase EdU-labeled (B) WT and STING-/- MEFs at different time points post-IR treatment. Flow cytometry was used to measure the percent cell population in G1, S, and G2/M phase of single cells stained with AlexaFluor 647-conjugated anti-EdU (y-axis) and PI (x-axis).

Western blot analysis of multiple stable D54 (A), HCT116 (B), and MC-38 (C) cell lines expressing short hairpin RNAs targeting STING or scrambled control (SHCOO2). Quantified bands are shown below each of their respective Western blot panels.

The pixel units obtained for each protein band from Western gels shown in Main Figures 4B (A), 4C (B), 4D (C), and 4E (D) was normalized to the pixel units calculated from their respective b-Actin loading control. For phosphorylated proteins, we then quantified the fraction of phosphorylated proteins to the absolute total amount of protein (total protein + phosphorylated protein). For dose-dependent and stimulation assays, the variables tested were normalized to their respective wild-type unstimulated controls.

Supplementary Figure 2 from Low Surface Expression of B7-1 (CD80) Is an Immunoescape Mechanism of Colon Carcinoma

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Supplementary Figure 3 from Low Surface Expression of B7-1 (CD80) Is an Immunoescape Mechanism of Colon Carcinoma

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Supplementary Figure 1 from Low Surface Expression of B7-1 (CD80) Is an Immunoescape Mechanism of Colon Carcinoma

Artificially enforced expression of CD80 (B7-1) and CD86 (B7-2) on tumor cells renders them more immunogenic by triggering the CD28 receptor on T cells. The enigma is that such B7s interact with much higher affinity with CTLA-4 (CD152), an inhibitory receptor expressed by activated T cells. We show that unmutated CD80 is spontaneously expressed at low levels by mouse colon carcinoma cell lines and other transplantable tumor cell lines of various tissue origins. Silencing of CD80 by interfering RNA led to loss of tumorigenicity of CT26 colon carcinoma in immunocompetent mice, but not in immunodeficient Rag−/− mice. CT26 tumor cells bind CTLA-4Ig, but much more faintly with a similar CD28Ig chimeric protein, thus providing an explanation for the dominant inhibitory effects on tumor immunity displayed by CD80 at that expression level. Interestingly, CD80-negative tumor cell lines such as MC38 colon carcinoma and B16 melanoma express CD80 at dim levels during in vivo growth in syngeneic mice. Therefore, low CD80 surface expression seems to give an advantage to cancer cells against the immune system. Our findings are similar with the inhibitory role described for the dim CD80 expression on immature dendritic cells, providing an explanation for the low levels of CD80 expression described in various human malignancies. (Cancer Res 2006; 66(4): 2442-50)

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In the era of cancer immunotherapy, there is significant interest in combining conventional cancer therapies, such as radiotherapy, with drugs that stimulate the immune system. The observation that ionizing radiation applied to murine tumors delays the growth of distant tumors (“abscopal effect”) and that this effect is potentiated by immunostimulatory drugs, led to clinical trials in which often only one lesion is irradiated in combination with immunotherapy drugs. The results of these initial clinical trials combining radio therapy and immunotherapy show that a meaningful abscopal effect is still infrequent. Recent preclinical data suggest that preexistent intratumoral T cells can survive radiation and contribute to its therapeutic effect. In this review, we discuss possible mechanisms underlying the preclinical/clinical discrepancies regarding the abscopal effect, and we propose the irradiation of multiple or all tumor sites in combination with systemic immunotherapy as a possible avenue to increase the efficacy of radio-immunotherapy.

Ineffective antigen cross-presentation in the tumor microenvironment compromises the generation of antitumor immune responses. Radiotherapy-radiodynamic therapy (RT-RDT) with nanoscale metal-organic frameworks (nMOFs) induces robust adaptive immune responses despite modest activation of canonical antigen presenting dendritic cells. Here, using transplantable and autochthonous murine tumor models, we demonstrate that RT-RDT induces antitumor immune responses via early neutrophil infiltration and reprogramming. Intravenous or intratumoral injection of nMOFs recruited peripheral CD11b+Ly6G+CD11c- neutrophils into tumors. The activation of nMOFs by low-dose X-rays significantly increased the population of CD11b+Ly6G+CD11c+ hybrid neutrophils with upregulated expression of the co-stimulatory molecules CD80 and CD86 as well as major histocompatibility complex class II molecules. Thus, nMOF-enabled RT-RDT reshapes a favorable tumor microenvironment for antitumor immune responses by reprogramming tumor-infiltrating neutrophils to function as non-canonical antigen presenting cells for effective cross-presentation of tumor antigens.

Successful combinations of radiotherapy and immunotherapy depend on the presence of live T cells within the tumor; however, radiotherapy is believed to damage T cells. Here, based on longitudinal in vivo imaging and functional analysis, we report that a large proportion of T cells survive clinically relevant doses of radiation and show increased motility, and higher production of interferon gamma, compared with T cells from unirradiated tumors. Irradiated intratumoral T cells can mediate tumor control without newly-infiltrating T cells. Transcriptomic analysis suggests T cell reprogramming in the tumor microenvironment and similarities with tissue-resident memory T cells, which are more radio-resistant than circulating/lymphoid tissue T cells. TGFβ is a key upstream regulator of T cell reprogramming and contributes to intratumoral Tcell radio-resistance. These findings have implications for the design of radio-immunotherapy trials in that local irradiation is not inherently immunosuppressive, and irradiation of multiple tumors might optimize systemic effects of radiotherapy., Lymphocytes are considered one of the most radiosensitive cell types in the body. Here the authors show that unlike circulating lymphocytes, tumor-infiltrating T cells survive therapeutic doses of irradiation, remaining functional and contributing to radiotherapy induced anti-tumor immunity.

Tumor-induced CD45(−)Ter119(+)CD71(+) erythroid progenitor cells (EPCs), termed “Ter-cells,” promote tumor progression by secreting artemin, a neurotrophic peptide that activates REarranged during Transfection (RET) signaling. We demonstrate that both local tumor ionizing radiation (IR) and anti-Programmed death ligand 1 (PD-L1) treatment decreased tumor-induced Ter-cell abundance in mouse spleen and artemin secretion outside the irradiation field in an interferon- (IFN) and CD8(+) T cell-dependent manner. Recombinant erythropoietin (EPO) promoted resistance to radiotherapy or anti-PD-L1 therapies by restoring Ter-cell numbers and serum artemin concentration. Blockade of artemin or potential artemin signaling partners, or depletion of Ter-cells augmented the anti-tumor effects of both IR and anti-PD-L1 therapies in mice. Analysis of samples from patients who received radio-immunotherapy demonstrated that IR-mediated reduction of Ter-cells, artemin, and GFRα3, an artemin signaling partner, were each associated with tumor regression. Patients with melanoma who received immunotherapy exhibited favorable outcomes associated with decreased expression of GFRα3. These findings demonstrate an out-of-field, or “abscopal,” effect mediated by adaptive immunity, which is induced during local tumor irradiation. This effect, in turn, governs the therapeutic effects of radiation and immunotherapy. Therefore, our results identify multiple targets to potentially improve outcomes following radiotherapy and immunotherapy.

The relative contribution of the effector molecules produced by T cells to tumour rejection is unclear, but interferon-γ (IFNγ) is critical in most of the analysed models1. Although IFNγ can impede tumour growth by acting directly on cancer cells2,3, it must also act on the tumour stroma for effective rejection of large, established tumours4,5. However, which stroma cells respond to IFNγ and by which mechanism IFNγ contributes to tumour rejection through stromal targeting have remained unknown. Here we use a model of IFNγ induction and an IFNγ–GFP fusion protein in large, vascularized tumours growing in mice that express the IFNγ receptor exclusively in defined cell types. Responsiveness to IFNγ by myeloid cells and other haematopoietic cells, including T cells or fibroblasts, was not sufficient for IFNγ-induced tumour regression, whereas responsiveness of endothelial cells to IFNγ was necessary and sufficient. Intravital microscopy revealed IFNγ-induced regression of the tumour vasculature, resulting in arrest of blood flow and subsequent collapse of tumours, similar to non-haemorrhagic necrosis in ischaemia and unlike haemorrhagic necrosis induced by tumour necrosis factor. The early events of IFNγ-induced tumour ischaemia resemble non-apoptotic blood vessel regression during development, wound healing or IFNγ-mediated, pregnancy-induced remodelling of uterine arteries6–8. A better mechanistic understanding of how solid tumours are rejected may aid the design of more effective protocols for adoptive T-cell therapy.

Adoptively transferred CD8+ T cells can stabilize the size of solid tumors over long periods of time by exclusively recognizing antigen cross-presented on tumor stroma. However, these tumors eventually escape T-cell–mediated growth control. The aim of this study was to eradicate such persistent cancers. In our model, the SIYRYYGL antigen is expressed by cancer cells that lack the MHC-I molecule Kb needed for direct presentation, but the antigen is picked up and cross-presented by tumor stroma. A single injection of antigen-specific 2C CD8+ T cells caused long-term inhibition of tumor growth, but without further intervention, tumors started to progress after approximately 3 months. Escape was associated with reduced numbers of circulating 2C cells. Tumor-infiltrating 2C cells produced significantly less TNFα and expressed more of the “exhaustion” markers PD-1 and Tim-3 than T cells from lymphoid organs. High-dose local ionizing radiation, depletion of myeloid-derived suppressor cells, infusions of additional 2C cells, and antibodies blocking PD-L1 did not prevent tumor escape. In contrast, adoptive transfer of allogeneic CD4+ T cells restored the numbers of circulating Ag-specific CD8+ T cells and their intratumoral function, resulting in tumor eradication. These CD4+ T cells had no antitumor effects in the absence of CD8+ T cells and recognized the alloantigen cross-presented on tumor stroma. CD4+ T cells might also be effective in cancer patients when PD-1/PD-L1 blockade does not rescue intratumoral CD8+ T-cell function and tumors persist. Cancer Immunol Res; 5(2); 127–36. ©2017 AACR.

Fibroblasts are common cell types in cancer stroma and lay down collagen required for survival and growth of cancer cells. Although some cancer therapy strategies target tumor fibroblasts, their origin remains controversial. Multiple publications suggest circulating mesenchymal precursors as a source of tumor-associated fibroblasts. However, we show by three independent approaches that tumor fibroblasts derive primarily from local, sessile precursors. First, transplantable tumors developing in a mouse expressing green fluorescent reporter protein (EGFP) under control of the type I collagen (Col-I) promoter (COL-EGFP) had green stroma, whereas we could not find COL-EGFP+ cells in tumors developing in the parabiotic partner lacking the fluorescent reporter. Lack of incorporation of COL-EGFP+ cells from the circulation into tumors was confirmed in parabiotic pairs of COL-EGFP mice and transgenic mice developing autochthonous intestinal adenomas. Second, transplantable tumors developing in chimeric mice reconstituted with bone marrow cells from COL-EGFP mice very rarely showed stromal fibroblasts expressing EGFP. Finally, cancer cells injected under full-thickness COL-EGFP skin grafts transplanted in nonreporter mice developed into tumors containing green stromal cells. Using multicolor in vivo confocal microscopy, we found that Col-I–expressing fibroblasts constituted approximately one-third of the stromal mass and formed a continuous sheet wrapping the tumor vessels. In summary, tumors form their fibroblastic stroma predominantly from precursors present in the local tumor microenvironment, whereas the contribution of bone marrow-derived circulating precursors is rare.

Purpose: Cancers usually contain multiple unique tumor-specific antigens produced by single amino acid substitutions (AAS) and encoded by somatic nonsynonymous single nucleotide substitutions. We determined whether adoptively transferred T cells can reject large, well-established solid tumors when engineered to express a single type of T-cell receptor (TCR) that is specific for a single AAS. Experimental Design: By exome and RNA sequencing of an UV-induced tumor, we identified an AAS in p68 (mp68), a co-activator of p53. This AAS seemed to be an ideal tumor-specific neoepitope because it is encoded by a trunk mutation in the primary autochthonous cancer and binds with highest affinity to the MHC. A high-avidity mp68-specific TCR was used to genetically engineer T cells as well as to generate TCR-transgenic mice for adoptive therapy. Results: When the neoepitope was expressed at high levels and by all cancer cells, their direct recognition sufficed to destroy intratumor vessels and eradicate large, long-established solid tumors. When the neoepitope was targeted as autochthonous antigen, T cells caused cancer regression followed by escape of antigen-negative variants. Escape could be thwarted by expressing the antigen at increased levels in all cancer cells or by combining T-cell therapy with local irradiation. Therapeutic efficacies of TCR-transduced and TCR-transgenic T cells were similar. Conclusions: Gene therapy with a single TCR targeting a single AAS can eradicate large established cancer, but a uniform expression and/or sufficient levels of the targeted neoepitope or additional therapy are required to overcome tumor escape. Clin Cancer Res; 22(11); 2734–43. ©2015 AACR. See related commentary by Liu, p. 2602

Given the integral role of stimulator of interferon genes (STING, TMEM173) in the innate immune response, its loss or impairment in cancer is thought to primarily affect antitumor immunity. Here we demonstrate a role for STING in the maintenance of cellular homeostasis through regulation of the cell cycle. Depletion of STING in human and murine cancer cells and tumors resulted in increased proliferation compared with wild-type controls. Microarray analysis revealed genes involved in cell-cycle regulation are differentially expressed in STINGko compared with WT MEFs. STING-mediated regulation of the cell cycle converged on NFκB- and p53-driven activation of p21. The absence of STING led to premature activation of cyclin-dependent kinase 1 (CDK1), early onset to S-phase and mitosis, and increased chromosome instability, which was enhanced by ionizing radiation. These results suggest a pivotal role for STING in maintaining cellular homeostasis and response to genotoxic stress. Significance: These findings provide clear mechanistic understanding of the role of STING in cell-cycle regulation, which may be exploited in cancer therapy because most normal cells express STING, while many tumor cells do not. See related commentary by Gius and Zhu, p. 1295

Purpose Stereotactic body radiotherapy (SBRT) may stimulate innate and adaptive immunity to augment immunotherapy response. Multisite SBRT is an emerging paradigm for treating metastatic disease. Anti-PD-1–treatment outcomes may be improved with lower disease burden. In this context, we conducted a phase I study to evaluate the safety of pembrolizumab with multisite SBRT in patients with metastatic solid tumors. Patients and Methods Patients progressing on standard treatment received SBRT to two to four metastases. Not all metastases were targeted, and metastases > 65 mL were partially irradiated. SBRT dosing varied by site and ranged from 30 to 50 Gy in three to five fractions with predefined dose de-escalation if excess dose-limiting toxicities were observed. Pembrolizumab was initiated within 7 days after completion of SBRT. Pre- and post-SBRT biopsy specimens were analyzed in a subset of patients to quantify interferon-γ–induced gene expression. Results A total of 79 patients were enrolled; three patients did not receive any treatment and three patients only received SBRT. Patients included in the analysis were treated with SBRT and at least one cycle of pembrolizumab. Most (94.5%) of patients received SBRT to two metastases. Median follow-up for toxicity was 5.5 months (interquartile range, 3.3 to 8.1 months). Six patients experienced dose-limiting toxicities with no radiation dose reductions. In the 68 patients with imaging follow-up, the overall objective response rate was 13.2%. Median overall survival was 9.6 months (95% CI, 6.5 months to undetermined) and median progression-free survival was 3.1 months (95% CI, 2.9 to 3.4 months). Expression of interferon-γ–associated genes from post–SBRT tumor biopsy specimens significantly correlated with nonirradiated tumor response. Conclusion Multisite SBRT followed by pembrolizumab was well tolerated with acceptable toxicity. Additional studies exploring the clinical benefit and predictive biomarkers of combined multisite SBRT and PD-1–directed immunotherapy are warranted.