Antitumor Activity Of CD8 Research Articles

Abstract 1519: Effect of obesity on CD4+ T cells in the tumor immune microenvironment of colorectal cancer

Abstract Background: The number of new cases and deaths from colorectal cancer (CRC) is increasing rapidly, and obesity is considered one of the risk factors. Obesity-associated cancers are related to the overproduction of hormones, induction of reactive oxygen species (ROS) production by free fatty acids, and changes in the gut microbiome. Obesity also promotes immune cell dysfunction in the tumor immune microenvironment (TIME). The increase of fatty acid levels due to obesity causes metabolic reprogramming and decreases the cytotoxic activity of natural killer (NK) cells and CD8+ T cells, which accelerate tumor progression. However, the impact of obesity on CD4+ T cells in the TIME remains unclear. Therefore, this study aimed to clarify how obesity affected the number and the anti-tumor activity of CD4+ T cells in CRC using mouse models. Methods: Seven-week-old male C57BL/6J mice were randomly divided into two groups and fed the control diet (CD) or high-fat diet (HFD) for nine weeks. Blood samples were collected from the buccal vein of the mice every four weeks after feeding for analysis. MC38 CRC cells of 4 × 106 were subcutaneously inoculated in the middle of the backs of mice to analyze tumor progression. Three weeks after tumor cell inoculation, the mice were sacrificed, and the immune cells of the tumor, blood, and various organs were analyzed. Results: After nine weeks of feeding, the HFD-fed mice had a significantly increased body weight compared to CD-fed mice. Flow cytometry results revealed that the number and frequency of CD4+ T cells in the blood were significantly reduced in HFD-fed mice compared to those in CD-fed mice, and the expression of programmed death-1 (PD-1) in CD4+ T cells in HFD-fed mice was significantly higher than that in CD-fed mice at eight weeks. Three weeks after tumor inoculation, HFD feeding accelerated tumor growth and decreased the survival rate. The number of CD4+ T cells in the tumors was significantly reduced in HFD-fed mice compared to that in CD-fed mice (55.9 vs 18.2 cells/mg; 95% CI 14.9-60.3 cells/mg; P = 0.006). The number of CD8+ T cells in the tumors was also reduced, which was more pronounced for CD4+ T cells than for CD8+ T cells. In addition, the production of cytokines such as IFN-γ and TNF-α was significantly reduced in CD4+ T cells and CD8+ T cells of HFD-fed mice. In a depletion analysis of CD4+ T cells, the feeding-dependent acceleration of tumor growth was not observed in each group. This result indicated that CD4+ T cells were involved in accelerating tumor growth due to HFD-induced obesity. Conclusion: We showed that the reduced number and dysfunction of CD4+ T cells due to obesity led to a decreased anti-tumor response of both CD4+ and CD8+ T cells, ultimately accelerating the progression of colorectal cancer. Our findings may elucidate the pathogenesis for poor outcomes of colorectal cancer associated with obesity. Citation Format: Masayuki Ando, Kota Yamada, Masafumi Saito, Kimihiro Yamashita, Takao Tsuneki, Yuri Adachi, Tomoki Abe, Tomosuke Mukoyama, Ryuichiro Sawada, Yasufumi Koterazawa, Hitoshi Harada, Naoki Urakawa, Hironobu Goto, Hiroshi Hasegawa, Shingo Kanaji, Takeru Matsuda, Yoshihiro Kakeji. Effect of obesity on CD4+ T cells in the tumor immune microenvironment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1519.

Spatial and Single-Cell Transcriptomics Reveal a Cancer-Associated Fibroblast Subset in HNSCC That Restricts Infiltration and Antitumor Activity of CD8+ T Cells.

Spatial analysis identifies IFN-induced MHC-IhiGal9+ CAFs that form a trap for CD8+ T cells, providing insights into the complex networks in the tumor microenvironment that regulate T-cell infiltration and function.

CTLA-4 blockade induces a microglia-Th1 cell partnership that stimulates microglia phagocytosis and anti-tumor function in glioblastoma

The limited efficacy of immunotherapies against glioblastoma underscores the urgency of better understanding immunity in the central nervous system. We found that treatment with αCTLA-4, but not αPD-1, prolonged survival in a mouse model of mesenchymal-like glioblastoma. This effect was lost upon the depletion of CD4+ Tcells but not CD8+ Tcells. αCTLA-4 treatment increased frequencies of intratumoral IFNγ-producing CD4+ Tcells, and IFNγ blockade negated the therapeutic impact of αCTLA-4. The anti-tumor activity of CD4+ Tcells did not require tumor-intrinsic MHC-II expression but rather required conventional dendritic cells as well as MHC-II expression on microglia. CD4+ Tcells interacted directly with microglia, promoting IFNγ-dependent microglia activation and phagocytosis via the AXL/MER tyrosine kinase receptors, which were necessary fortumor suppression. Thus, αCTLA-4 blockade in mesenchymal-like glioblastoma promotes a CD4+ Tcell-microgliacircuit wherein IFNγ triggers microglia activation and phagocytosis and microglia in turn act as antigen-presenting cells fueling the CD4+ Tcell response.

Abstract 6163: Targeting glucose metabolism through inhibition of pyruvate dehydrogenase kinase to improve response to immune-checkpoint inhibition in melanoma

Abstract Immune-checkpoint inhibitors have improved survival of patients with advanced melanoma, however, 40-50% of patients do not benefit sufficiently. Melanoma tumor lesions have high glucose uptake due to metabolic reprogramming, which supports tumor cell growth and is partially mediated through elevated expression of mitochondrial pyruvate dehydrogenase kinases (PDK1-4). Resultant decreased glucose levels and low pH in the tumor microenvironment are detrimental to antitumor immune cell function. Here, we aimed to reverse metabolic reprogramming in melanoma through PDK inhibition, thereby inhibiting tumor cell growth while maintaining or potentially enhancing antitumor immunity. We used a panel of four melanoma cell lines with genetic backgrounds similar to those most frequently found in patients, namely the BRAF-mutant A375 and S-MEL-28 cell lines, the NRAS-mutant SK-MEL-2 cell line and the BRAF/NRAS wild-type MeWo cell line. These were then treated with PDK inhibitor dichloroacetate (DCA) to determine effects on viability and metabolic phenotype. Furthermore, we investigated whether DCA synergized with treatment with the glutaminase inhibitor CB-839. Finally, we determined the effect of DCA on viability, interferon-γ (IFN-γ) secretion and antitumor activity of CD8+ T cells. MeWo cells were most sensitive to DCA, while SK-MEL-2 was the least sensitive, with IC50 values ranging from 13.3 to 27.0 mM. DCA led to an up to 6-fold increase in oxygen consumption rate:extracellular acidification rate (OCR:ECAR) ratio in all cell lines. SK-MEL-28 cells were not sensitive to CB-839, while the IC50 values in the other cell lines ranged from 7.9 nM in MeWo to 139.2 nM in SK-MEL-2 cells. DCA synergized with CB-839 in 2D and 3D culture, ranging from 2-fold sensitization compared to either drug alone in MeWo up to 5-fold sensitization in SK-MEL-2. In activated CD8+ T cells, viability was not affected by DCA treatment of up to 21 mM, whereas proliferation was only mildly inhibited. These cells also showed a 2.5-fold increase in OCR:ECAR ratio after DCA treatment. Interestingly, IFN-γ secretion by CD8+ T cells was increased 2.8-fold after DCA treatment and tumor cell killing by CD8+ T cells in a coculture with A375 cells was not impaired by DCA or CB-839 treatment. We conclude that DCA can indeed reprogram cellular metabolism in melanoma and synergizes with other metabolically targeted drugs in antitumor activity, while keeping the antitumor reactivity of CD8+ T cells intact. Supported by the Dutch Cancer Foundation (10913/2017-1). Citation Format: Jiske F. Tiersma, Bernardus Evers, Barbara M. Bakker, Steven de Jong, Mathilde Jalving. Targeting glucose metabolism through inhibition of pyruvate dehydrogenase kinase to improve response to immune-checkpoint inhibition in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6163.

Crosstalk among T-cell CX3CR1, immune checkpoints and toll-like receptor 4 signaling pathway in BCG-unresponsive nonmuscle invasive bladder cancer: Mechanism of action of OncoTherad Nano-Immunotherapy.

e16551 Background: This study detailed and characterized the mechanisms of action of OncoTherad nano-immunotherapy based on modulation of Toll-like Receptor 4 (TLR4) signaling pathway, CX3C chemokine receptor 1 (CX3CR1, a marker of T-cell differentiation) and immune checkpoints in patients with Bacillus Calmette-Guérin (BCG)-unresponsive high-grade non-muscle invasive bladder cancer. Methods: A single-center open-label (Paulinia Municipal Hospital, Brazil) and single-arm phase 1/2 study (Clinical Trial: RBR-6swqd2) was applied in 44 patients (30 male and 14 female) with BCG-unresponsive NMIBC (≥ 1 prior course of BCG therapy) submitted to OncoTherad immunotherapy for 24 months. Patient follow-ups were performed with systematic mapping biopsies of the bladder every 3 months for the first year and every 6 months thereafter for up to 2 years. Bladder biopsies of each patient (n = 44) were divided into 2 groups: Group 1 (initial biopsy, before OncoTherad treatment); and Group 2 (bladder biopsy after OncoTherad treatment). Subsequently, the samples were submitted to immunohistochemistry analysis: TLR4-mediated IFN-γ production signaling pathway (TRIF, TBK1, IRF-3), CX3CR1+CD8+ T-cells, immune checkpoints (PD-1/PD-L1 CTLA-4) and Regulatory T cells (FOXP3). Results: After 24-months follow-up, pathological complete response rate was 72.7% (95% CI) and recurrence-free survival of 21.4 months. Bladder samples from patients submitted to OncoTherad treatment (Group 2) showed intensified TLR4, TRIF, TBK1, IRF-3 and IFN-γ immunoreactivities when compared (p < 0.01) to initial biopsies (Group 1). Furthermore, as result of interferon signaling pathway (TRIF-dependent pathway) induction by OncoTherad, intensified CX3CR1 immunoreactivities (p < 0.01) were found in the Group 2. In contrast, PD-1/PD-L1 immunoreactivities were decreased (p < 0.01) in the Group 2 when compared to Group 1. No statistical differences were found between the two groups for FOXP3 and CTLA4. Conclusions: OncoTherad nano-immunotherapy led to activation of TLR4-mediated innate immune system, resulting in increased interferon signaling pathway, which was fundamental in the activation of antitumor CD8+ T-cells and decrease of PD-1/PD-L1 expression in the bladder microenvironment. These important findings are relevant concerning the treatment of patients with NMIBC presenting high-risk of progression that are BCG-unresponsive.

Ultrasound combined with nanobubbles promotes systemic anticancer immunity and augments anti-PD1 efficacy

BackgroundThe poor immunogenicity of solid tumors limits the efficacy ofanti-programmed cell death protein 1 (anti-PD1)-based immune checkpoint blockade (ICB); thus, less than 30% of patients with cancer exhibit a response....

SIRT1 induces the accumulation of TAMs at colorectal cancer tumor sites via the CXCR4/CXCL12 axis

Our previous work suggested that high SIRT1 expression by cancer cells predicted a poor colorectal cancer (CRC) prognosis, but its role in the tumor microenvironment was unclear.Here, we examined tumor-infiltrating lymphocytes (TILs) in CRC expressing different levels of SIRT1. We also established a co-culture system with monocytes, CD8+ T cells and patient-derived tumor organoids (PDOs) to study the relationships between immune cells and cancer cells. The percentage of CD8+ T cells was decreased and the percentage of macrophages was increased in SIRT1-high (SIRT1-hi) CRC. Co-culture results showed that tumor-associated macrophages (TAMs) from SIRT1-hi CRC inhibited the proliferation and anti-tumor activity of CD8+ T cells. Importantly, SIRT1-hi CRC were shown to modulate the migration and the activity of TAMs. RNA sequencing revealed that CD14+ monocytes in SIRT1-hi patients expressed higher levels of CXCR4. Mechanistically, SIRT1 expression was shown to promote CXCL12 expression by inhibiting the acetylation of p53. Our findings indicate that SIRT1 in CRC induces TAM migration through the CXCR4/CXCL12 pathway, and inhibits the proliferation and activity of CD8+ T cells, resulting in promotion of CRC progression.

JAML promotes CD8 and γδ T cell antitumor immunity and is a novel target for cancer immunotherapy.

T cells are critical mediators of antitumor immunity and a major target for cancer immunotherapy. Antibody blockade of inhibitory receptors such as PD-1 can partially restore the activity of tumor-infiltrating lymphocytes (TILs). However, the activation signals required to promote TIL responses are less well characterized. Here we show that the antitumor activity of CD8 and γδ TIL is supported by interactions between junctional adhesion molecule-like protein (JAML) on T cells and its ligand coxsackie and adenovirus receptor (CXADR) within tumor tissue. Loss of JAML through knockout in mice resulted in accelerated tumor growth that was associated with an impaired γδ TIL response and increased CD8 TIL dysfunction. In mouse tumor models, therapeutic treatment with an agonistic anti-JAML antibody inhibited tumor growth, improved γδ TIL activation, decreased markers of CD8 TIL dysfunction, and significantly improved response to anti-PD-1 checkpoint blockade. Thus, JAML represents a novel therapeutic target to enhance both CD8 and γδ TIL immunity.

Reprogramming CD8 + T cells

MEK1/2 inhibition enhances both the persistence and antitumor activity of CD8 + T cells in vivo.

Insights into P-Glycoprotein Inhibitors: New Inducers of Immunogenic Cell Death.

Doxorubicin is a strong inducer of immunogenic cell death (ICD), but it is ineffective in P-glycoprotein (Pgp)-expressing cells. Indeed, Pgp effluxes doxorubicin and impairs the immunesensitizing functions of calreticulin (CRT), an “eat-me” signal mediating ICD. It is unknown if classical Pgp inhibitors, designed to reverse chemoresistance, may restore ICD. We addressed this question by using Pgp-expressing cancer cells, treated with Tariquidar, a clinically approved Pgp inhibitor, and R-3 compound, a N,N-bis(alkanol)amine aryl ester derivative with the same potency of Tariquidar as Pgp inhibitor. In Pgp-expressing/doxorubicin-resistant cells, Tariquidar and R-3 increased doxorubicin accumulation and toxicity, reduced Pgp activity, and increased CRT translocation and ATP and HMGB1 release. Unexpectedly, only R-3 promoted phagocytosis by dendritic cells and activation of antitumor CD8+T-lymphocytes. Although Tariquidar did not alter the amount of Pgp present on cell surface, R-3 promoted Pgp internalization and ubiquitination, disrupting its interaction with CRT. Pgp knock-out restores doxorubicin-induced ICD in MDA-MB-231/DX cells that recapitulated the phenotype of R-3-treated cells. Our work demonstrates that plasma membrane-associated Pgp prevents a complete ICD notwithstanding the release of ATP and HMGB1, and the exposure of CRT. Pharmacological compounds reducing Pgp activity and amount may act as promising chemo- and immunesensitizing agents.

Tumors metabolically paralyze T cells

Immunology The cytokine transforming growth factor–β (TGF-β) suppresses both immune and tumor cells. Dimeloe et al. found that TGF-β from tumor effusions suppressed the antitumor activity of CD4+ T cells by inhibiting their production of the inflammatory cytokine interferon-γ (IFN-γ). The effects of TGF-β were mediated by Smad proteins in the mitochondria and led to decreased mitochondrial respiration. Indeed, IFN-γ production by CD4+ T cells was suppressed by inhibiting a mitochondrial electron transport chain complex. These data suggest that TGF-β suppresses antitumor immunity by metabolically paralyzing T cells. Sci. Signal. 12 , eaav3334 (2019).

Increasing intratumor C/EBP-\u03b2 LIP and nitric oxide levels overcome resistance to doxorubicin in triple negative breast cancer

BackgroundTriple negative breast cancer (TNBC) easily develops resistance to the first-line drug doxorubicin, because of the high levels of the drug efflux transporter P-glycoprotein (Pgp) and the activation of pro-survival pathways dependent on endoplasmic reticulum (ER). Interfering with these mechanisms may overcome the resistance to doxorubicin, a still unmet need in TNBC.MethodsWe analyzed a panel of human and murine breast cancer cells for their resistance to doxorubicin, Pgp expression, lysosome and proteasome activity, nitrite production, ER-dependent cell death and immunogenic cell death parameters. We evaluated the efficacy of genetic (C/EBP-β LIP induction) and pharmacological strategies (lysosome and proteasome inhibitors), in restoring the ER-dependent and immunogenic-dependent cell death induced by doxorubicin, in vitro and in syngeneic mice bearing chemoresistant TNBC. The results were analyzed by one-way analysis of variance test.ResultsWe found that TNBC cells characterized by high levels of Pgp and resistance to doxorubicin, had low induction of the ER-dependent pro-apoptotic factor C/EBP-β LIP upon doxorubicin treatment and high activities of lysosome and proteasome that constitutively destroyed LIP. The combination of chloroquine and bortezomib restored doxorubicin sensitivity by activating multiple and interconnected mechanisms. First, chloroquine and bortezomib prevented C/EBP-β LIP degradation and activated LIP-dependent CHOP/TRB3/caspase 3 axis in response to doxorubicin. Second, C/EBP-β LIP down-regulated Pgp and up-regulated calreticulin that triggered the dendritic cell (DC)-mediated phagocytosis of tumor cell, followed by the activation of anti-tumor CD8+T-lymphocytes upon doxorubicin treatment. Third, chloroquine and bortezomib increased the endogenous production of nitric oxide that further induced C/EBP-β LIP and inhibited Pgp activity, enhancing doxorubicin’s cytotoxicity. In orthotopic models of resistant TNBC, intratumor C/EBP-β LIP induction - achieved by a specific expression vector or by chloroquine and bortezomib - effectively reduced tumor growth and Pgp expression, increased intra-tumor apoptosis and anti-tumor immune-infiltrate, rescuing the efficacy of doxorubicin.ConclusionsWe suggest that preventing C/EBP-β LIP degradation by lysosome and proteasome inhibitors triggers multiple virtuous circuitries that restore ER-dependent apoptosis, down-regulate Pgp and re-activate the DC/CD8+T-lymphocytes response against TNBC. Lysosome and proteasome inhibitors associated with doxorubicin may overcome the resistance to the drug in TNBC.

Tandem immunotherapy achieves synergy

Cancer Immunology Immune checkpoint–inhibitor therapies bolster the antitumor activity of CD8+ T lymphocytes. Wang et al. performed single-cell analysis of tumor-infiltrating lymphocytes in mouse cancer models in which inhibitory anti–PD-1 (programmed cell death protein 1) and stimulatory anti–GITR (glucocorticoid-induced tumor necrosis factor receptor–related protein) antibodies together enhanced tumor control. This combination immunotherapy led to a synergistic increase in tumor antigen–specific memory precursor effector T cells dependent on the CD226 costimulatory pathway. Biochemical studies in liposomes identified CD226 as an additional target of dephosphorylation mediated by the PD-1–SHP2 (Src homology region 2) complex. Thus, further clinical trials could usefully test the efficacy of combined anti-GITR and anti–PD-1 immunotherapy in human cancer. Sci. Immunol. 3 , eaat7061 (2018).

Reinforce the antitumor activity of CD8+ T cells via glutamine restriction.

The antitumor activity of activated CD8+ T cells in the tumor microenvironment seems to be limited due to their being metabolically unfit. This metabolic unfitness is closely associated with T‐cell exhaustion and impairment of memory formation, which are barriers to successful antitumor adoptive immunotherapy. We therefore assessed the role of glutamine metabolism in the antitumor activity of CD8+ T cells using a tumor‐inoculated mouse model. The adoptive transfer of tumor‐specific CD8+ T cells cultured under glutamine‐restricted (dGln) conditions or CD8+ T cells treated with specific inhibitors of glutamine metabolism efficiently eliminated tumors and led to better survival of tumor‐inoculated mice than with cells cultured under control (Ctrl) conditions. The decreased expression of PD‐1 and increased Ki67 positivity among tumor‐infiltrating CD8+ T cells cultured under dGln conditions suggested that the inhibition of glutamine metabolism prevents CD8+ T‐cell exhaustion in vivo. Furthermore, the transferred CD8+ T cells cultured under dGln conditions expanded more efficiently against secondary OVA stimulation than did CD8+ T cells under Ctrl conditions. We found that the expression of a pro‐survival factor and memory T cell‐related transcription factors was significantly higher in CD8+ T cells cultured under dGln conditions than in those cultured under Ctrl conditions. Given these findings, our study uncovered an important role of glutamine metabolism in the antitumor activity of CD8+ T cells. The novel adoptive transfer of tumor‐specific CD8+ T cells cultured in glutamine‐restricted conditions may be a promising approach to improve the efficacy of cell‐based adoptive immunotherapy.

Abstract IA02: Engineering T cells to eradicate tumors in the age of synthetic biology

Abstract Unlike cancer vaccines and immune modulators such as checkpoint inhibitors that seek to harness patient immune responses, adoptive therapy with genetically engineered T cells seeks to create responses that don’t exist in the patient’s immune system. Molecular technologies now make it feasible to not only create T cells with specificity for the tumor by introduction of a selected antigen-specific receptor, but also with qualities not naturally found, including improved function and resistance to immunosuppression. We have been exploring in preclinical models and clinical trials methods to reproducibly provide therapeutic T cell responses by transfer of genetically engineered T cells. For human acute myelogenous leukemia (AML), we have pursued targeting WT1, a gene overexpressed in human leukemic stem cells that is associated with promoting leukemic transformation. Preclinical studies performed in a mouse model demonstrated that CD8 T cells expressing a high affinity TCR specific for this oncogene can be safely administered, with no evidence of toxicity to the normal tissues known to express low but detectable levels of WT1. We have advanced this approach to a clinical trial in leukemia patients with poor prognostic factors that place them at high risk of relapse after hematopoietic cell transplant (HCT), using a high-affinity human TCR specific for WT1 to transduce CD8 cells and reproducibly create high-avidity T cells that recognize leukemic cells. Our clinical results demonstrate that such T cells can prevent leukemic relapse and sustain long-term remissions, and can mediate antileukemic activity in patients who have relapsed. This therapy is now being tested in AML patients who have minimal residual disease after induction therapy and are not candidates for HCT, as well as in solid tumors that similarly overexpress WT1. Unfortunately, there are substantive obstacles in targeting established tumors that can preclude even a T cell expressing a high-affinity TCR from being effective. These impediments include the development of T cell dysfunction, particularly within the microenvironment of solid tumors, and we are using genetically engineered mouse models to elucidate the cellular and molecular pathways that need to be modulated to achieve meaningful therapeutic benefit in a variety of hematologic and solid tumor settings, including pancreatic and ovarian cancer. Our preclinical therapy studies reveal promising antitumor activity, but demonstrate that repeated infusions of functional T cells are required to sustain a therapeutic response in the context of the immunosuppressive tumor microenvironment, and we are engineering T cells to overcome these inhibitory signals and enhance efficacy. In place of current strategies that disrupt inhibitory pathways by systemic administration of blocking mAbs, which globally disrupt immune regulation and thus can have significant toxicity to the host, we are creating synthetic immunomodulatory fusion proteins that take advantage of the expression of inhibitory ligands by tumors by still binding the inhibitory ligand but alternatively delivering a costimulatory rather than inhibitory signal. Additionally, as the antitumor activity of CD8 T cells is enhanced by a concurrent CD4 T cell response, we are engineering CD4 T cells as well as CD8 T cells to create an orchestrated antitumor response. The results suggest that cancer therapy with engineered T cells can provide effective antitumor responses and will likely find an increasing role in the treatment of human cancers. Citation Format: Philip D. Greenberg, Kristin G. Anderson, Dan Egan, Sunil R. Hingorani, Shannon K. Oda, Rachel Perret, Tom M. Schmitt, Ingunn M. Stromnes, Leah Schmidt, Aude G. Chapuis. Engineering T cells to eradicate tumors in the age of synthetic biology [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA02.

Abstract IA29: Novel insights into the dose-dependency of radiation immunogenicity

Abstract Recruitment to the tumor of a Batf3-dependent subset of dendritic cells (DCs) specialized in cross-presentation of tumor-derived antigens to CD8+ cytotoxic T cells is driven by type I interferon (IFN-I) and is essential for activation of antitumor CD8+ T cells. We have recently found that in tumors refractory to treatment with immune checkpoint blockers (ICBs), radiotherapy induces cancer cell-intrinsic activation of IFN-I pathway and release of interferon-beta, mimicking a viral infection, and resulting in recruitment of Batf3-dependent DCs (Vanpouille-Box et al., Nat Commun 2017). This process is mediated by the accumulation of dsDNA in the cancer cell cytoplasm, which is sensed by cGAS and leads to downstream activation of STING. Importantly, the dose and fractionation of radiation are critical for optimal induction of IFN-I production by irradiated cancer cells. For most carcinoma cells there is a single radiation dose threshold of 12-15 Gy above which the DNA exonuclease TREX1 is upregulated. The latter degrades cytoplasmic dsDNA precluding IFN-I activation. Fractionation, i.e., repeated (three times) daily delivery of radiation therapy at doses below this threshold, amplifies the IFN-I pathway activation in the carcinoma cells, an effect that requires upregulation of IFNRA. Furthermore, the synergy of RT with ICBs (anti-CTLA-4 or anti-PD-1) and the induction of abscopal effects (i.e., immune-mediated rejection of nonirradiated synchronous tumors) are completely dependent on the ability of radiotherapy to induce cancer cell-intrinsic IFN-I. These findings have critical implications for the use of radiotherapy to increase the response to ICBs in the clinic, a strategy currently being tested in over one hundred ongoing and planned trials. Supported by NIH 1R01CA201246, Breast Cancer Research Foundation BCRF-016-054, and The Chemotherapy Foundation. Citation Format: Sandra Demaria. Novel insights into the dose-dependency of radiation immunogenicity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr IA29.

Identification of an immune gene expression signature associated with favorable clinical features in Treg-enriched patient tumor samples

Immune heterogeneity within the tumor microenvironment undoubtedly adds several layers of complexity to our understanding of drug sensitivity and patient prognosis across various cancer types. Within the tumor microenvironment, immunogenicity is a favorable clinical feature in part driven by the antitumor activity of CD8+ T cells. However, tumors often inhibit this antitumor activity by exploiting the suppressive function of regulatory T cells (Tregs), thus suppressing the adaptive immune response. Despite the seemingly intuitive immunosuppressive biology of Tregs, prognostic studies have produced contradictory results regarding the relationship between Treg enrichment and survival. We therefore analyzed RNA-seq data of Treg-enriched tumor samples to derive a pan-cancer gene signature able to help reconcile the inconsistent results of Treg studies, by better understanding the variable clinical association of Tregs across alternative tumor contexts. We show that increased expression of a 32-gene signature in Treg-enriched tumor samples (n = 135) is able to distinguish a cohort of patients associated with chemosensitivity and overall survival. This cohort is also enriched for CD8+ T cell abundance, as well as the antitumor M1 macrophage subtype. With a subsequent validation in a larger TCGA pool of Treg-enriched patients (n = 626), our results reveal a gene signature able to produce unsupervised clusters of Treg-enriched patients, with one cluster of patients uniquely representative of an immunogenic tumor microenvironment. Ultimately, these results support the proposed gene signature as a putative biomarker to identify certain Treg-enriched patients with immunogenic tumors that are more likely to be associated with features of favorable clinical outcome.

IL-33 inhibits tumor growth through the activation of anti-tumor CD8+ T cell response in hepatocellular carcinoma

Abstract IL-33 secreted by epithelial cells and activated innate immune cells is a member of IL-1 family with dual-immunological effects. It can induce both type 1 and type 2 immune responses and its function as an alarmin has been well demonstrated. However, the role of IL-33 in the development of hepatocellular carcinoma (HCC) is not clear. Here, we examined the function of IL-33 in various murine HCC models by hydrodynamically injecting either IL-33-expressing tumor cells or IL-33-expressing plasmids into the liver of tumor-bearing mice. Tumor growth was greatly inhibited by IL-33 expression. Similarly, in DEN-induced HCC model, we also demonstrated that IL-33 expression inhibited tumor development. This effect was shown to be dependent on extracellular IL-33 and ST2 expression, because it was diminished in ST2 KO mice. Further study showed there were increased percentages and numbers of CD8+ T cells in both spleen and liver in IL-33-expressing tumor-bearing mice. The percentages and numbers of activated and effector CD4+ and CD8+ T cells were also increased in the liver with IL-33 expression. Moreover, IFN-γ production of the CD8+ T cells was upregulated in both spleen and liver by IL-33. The cytotoxicity of CTLs against tumor cells was also enhanced. Depletion of CD8+ and CD4+T cells diminished the antitumor activity of IL-33, suggesting the anti-tumor function of IL-33 was T cell-dependent. In vitro IL-33 treatment could preferentially expand CD8+ T cells and promote their activation and IFN-γ production. Taken together, we demonstrated in murine HCC models that IL-33 could inhibit tumor development through its interaction with ST2 to promote anti-tumor CD8+ T cell response.

Developing a screening platform for genetic and small molecule targets of cancer immunotherapy.

66 Background: The success of checkpoint blockade therapy is often dependent on CD8 T cell activation against tumor antigens. However, clinical benefit is only seen in a subset of patients, suggesting that there are other possibly targetable immunosuppressive pathways that are allowing the tumor to escape immune surveillance. Methods: A CD8 T cell that recognizes the EGFP200-208 peptide epitope allowed for the use of EGFP as a model tumor antigen while monitoring expression levels at the single cell resolution. Using a lymphoma line expressing EGFP or mCherry as our antigen-positive and -negative cancer models, we employed 3 screening strategies: 1) a forward genetics approach in which we selected for tumor cells that had naturally developed resistance to killing by CD8 T cells; 2) a reverse genetics approach that involved the use of pooled CRISPR libraries to identify knockout clones with a selection advantage or disadvantage when pressured by activated T cells; and 3) small molecule libraries, including FDA-approved drugs, to identify compounds that increased antigen-specific CD8 T cell killing. Results: Despite antigen recognition and early activation in response to the resistant tumor line, T cells failed to produce effector cytokines and underwent apoptosis in a PD-L1 and CTLA-4 independent manner. Candidate genes mediating this phenotype were derived from expression differences between the original susceptible tumor line and the immunoedited resistant tumor line. The pooled CRISPR approach was validated in a curated library by identifying genes with known roles in T cell-mediated killing and antigen presentation, such as Fas, B2m, and Tap1, as well as known suppressive molecules such as BTLA and PD-L1. LDL receptor expressed on the cancer cell emerged as a possible novel suppressor of T cells. Decitabine and 4-cinnolinethiol, among other small molecules, emerged as possible enhancers of CD8 T cell activity. Conclusions: We have identified several gene candidates as potentially novel and targetable checkpoint-like molecules, as well as small molecule compounds that may be able to enhance the anti-tumor activity of CD8 T cells. Efforts are ongoing in order to validate these targets and to screen larger libraries.

Blocking tumor immunosuppression

Immunology Tumor-derived regulatory T cells (Tregs) suppress the antitumor effects of CD8+ T cells. Budhu et al. investigated the underlying mechanisms with an in vitro system that recreated some of the key features of the in vivo tumor microenvironment. Tregs inhibited the antitumor activity of CD8+ T cells toward cocultured melanoma cells from mouse tumors through the cytokine TGF-β (transforming growth factor-β), which increased the levels of an immune checkpoint protein on CD8+ T cells. A TGF-β-blocking antibody prevented immunosuppression, suggesting a therapeutic strategy to inhibit Treg activity in tumors. Sci. Signal. 10 , eaak9702 (2017).