Promote Tumor Rejection Research Articles

Abstract PR009: SMARCAL1 is a dual regulator of innate immune signaling and PD-L1 expression that promotes tumor immune evasion

Abstract Genomic instability can trigger cancer-intrinsic innate immune responses that promote tumor rejection. However, cancer cells often evade these responses by overexpressing immune checkpoint regulators, such as PD-L1. Here, we identify the SNF2-family DNA translocase SMARCAL1 as a factor that favors tumor immune evasion by a dual mechanism involving both the suppression of innate immune signaling and the induction of PD-L1-mediated immune checkpoint responses. Mechanistically, SMARCAL1 relieves endogenous DNA damage and suppresses cGAS-STING-dependent immune signaling during cancer cell growth. Simultaneously, it cooperates with the AP-1 family member JUN to maintain chromatin accessibility at a transcriptional regulatory element in the PD-L1 gene, thereby promoting PD-L1 expression in cancer cells. Loss of SMARCAL1 enhances anti-tumor immune responses and sensitizes tumors to immune checkpoint blockade in a mouse melanoma model. Collectively, these studies uncover SMARCAL1 as a valuable target for cancer immunotherapy. Citation Format: Giuseppe Leuzzi, Alessandro Vasciaveo, Angelo Taglialatela, Xiao Chen, Tessa M. Firestone, Allison R. Hickman, Wendy Mao, Tanay Thakar, Alina Vaitsiankova, Jen-Wei Huang, Raquel Cuella-Martin, Samuel B. Hayward, Jordan S. Kesner, Ali Ghasemzadeh, Tarun S. Nambiar, Patricia Ho, Alexander Rialdi, Maxime Hebrard, Yinglu Li, Jinmei Gao, Saarang Gopinath, Oluwatobi A. Adeleke, Bryan Venters, Charles G. Drake, Richard Baer, Benjamin Izar, Ernesto Guccione, Michael-Christopher Keogh, Raphael Guerois, Lu Sun, Chao Lu, Andrea Califano, Alberto Ciccia. SMARCAL1 is a dual regulator of innate immune signaling and PD-L1 expression that promotes tumor immune evasion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR009.

IMMU-07. THE CXCL16/CXCR6 AXIS REGULATES CD8+ T-CELL RECRUITMENT AND FUNCTION IN GLIOBLASTOMA

Abstract INTRODUCTION Glioblastoma (GBM) is the most common and aggressive primary brain tumor with a poor prognosis and no curative treatment. Immunotherapy, revolutionizing oncology, is currently ineffective for GBM due to the immunosuppressive tumor microenvironment. A critical driver is tumor-associated macrophages (TAMs) infiltration, which is correlated to T-cell deficiency and tumor progression. While the interactions between TAMs and T-cells are known to inhibit the antitumor functions, the mechanisms driving these interactions in GBM are still not established. Our data shows that the CXCL16/CXCR6 axis is likely the predominant chemokine-receptor interaction among TAMs and CD8+ T-cells population in GBM. Understanding the role of this axis is crucial in modulating antitumor immune functions in GBM. METHODS We designed several migration and adhesion assays to determine how the CXCL16/CXCR6 axis influences: CD8+ T-cell migration, TAM: CD8+ T-cell interactions, and CD8+ T-cell dysfunction in GBM. In addition, long-term survival with immune checkpoint treatment was done to understand the influence of the axis on murine tumor growth. RESULTS While CXCR6 deficiency prevented T-cell infiltration into tumors, the CD8+ T-cells cells within the tumors expressed more markers associated with cytotoxicity and exhaustion. Furthermore, CXCR6 deficient CD8+ T cells were less adherent to immunosuppressive TAMs. Importantly, CXCR6 deficiency worked in concert with anti-PD1 to promote tumor rejection in the CT2A tumor-bearing model of GBM. CONCLUSION Our data show that while CXCR6 is crucial in trafficking CD8+ T-cells to tumors from the periphery, it promotes immunosuppressive interactions between TAMs/CD8+ T-cells within tumors. Thus, the CXCR6 axis exerts opposing roles in anti-tumor immunity in GBM.

Role of the E3 ubiquitin ligase Peli1 in regulating antitumor T cell responses

Abstract Metabolic fitness of T cells is crucial for immune responses against infections and tumorigenesis. Both the T cell receptor signal (TCR) and environmental cues contribute to the induction of T cell metabolic reprogramming, but the underlying mechanism is incompletely understood. Here, we identified the E3 ubiquitin ligase Peli1 as an important regulator of T cell metabolism and antitumor immunity. Peli1 ablation profoundly promotes tumor rejection, associated with increased effector function and the number of tumor-infiltrating CD4 and CD8 T cells. Moreover, Peli1 deficiency inhibited the expression of PD-1 and TIM-3 and reduced the exhaustion of tumor-infiltrating CD8 T cells. The Peli1-deficient T cells display markedly stronger metabolic activities, particularly glycolysis, than wild-type T cells. Peli1 controls the activation of mTOR signaling, stimulated by both the TCR signal and growth factors, and this function of Peli1 is mediated through the regulation of the mTORC1-inhibitory proteins, TSC1 and TSC2. Peli1 mediates non-degradative ubiquitination of TSC1, thereby promoting the stabilization of the TSC complex. These results establish Peli1 as a novel regulator of mTORC1 and downstream mTORC1-mediated actions on T cell metabolism and antitumor immunity.

Abstract 5073: NT219 induces tumor PD-L1 expression and potentiates anti-PD-1 efficacy

Abstract While the advent of immune checkpoint blockade (ICB) has dramatically improved the prognosis of many immune-infiltrated cancers, for others, unfortunately, these benefits have yet to be realized. The major challenge before the field, then, is to identify combination therapies that act both to combat evolved resistance. NT219 is a novel dual inhibitor of insulin receptor substrates 1 and 2 (IRS) and STAT3. NT219 demonstrated antitumor effects against both in situ and metastatic human melanoma models in mice as a stand-alone treatment and in combination with mutated BRAF and MEK inhibitors. The potential of NT219 to overcome resistance and increase efficacy was demonstrated in PDX models with multiple drug classes. Collectively, these findings provided preclinical proof-of-concept NT219 as a promising novel cancer therapy. Given this data, the goal of our study was to assess the efficacy of combining NT219 with anti-PD-1 and anti-CTLA-4 ICB, and test capacity of the combination to overcome immune resistance. To that end, we have examined PD-L1 expression levels in vitro following NT219 treatment in two different strains of melanoma cells, B16-tdTomato (TMT) and B16 3I-F4, ICB sensitive and ICB resistant derivative lines, respectively. In addition, to evaluate the combination effect of NT219 with ICB therapy in ICB-resistant PDX model, we used a humanized PDX model of pembrolizumab-resistant gastroesophageal tumor (GEJ). These mice were injected with PBMCs from the same patient. Using multiple syngeneic immunocompetent models of melanoma (TMT and 3I-F4 cells) and the humanized PDX model, we investigated the potential of each ICB with NT219. Our findings showed that NT219 increased PD-L1 expression levels on both melanoma cell lines in vitro, however, the PD-L1 expression levels were much more elevated 3I-F4 than TMT cells. Interestingly, the levels of both IRS and activated pSTAT3, the targets of NT219, which consist well known resistance mechanisms, were higher in the resistant cells as compared to the sensitive line. In accordance we found that NT219 was able to restore anti-PD-1 sensitivity in 3I-F4 model (TGI = 58%) in the syngeneic model, promoting tumor rejection and increasing survival rates. The combined approach between NT219 and anti-CTLA-4 displayed a moderate effect (TGI = 37%). In the TMT, NT219 combined with anti-PD-1 showed greater efficacy compared to anti-PD-1 alone (TGI = 63% and TGI = 47%, respectively). These results were confirmed using the humanized ICB-resistant PDX model, where impressive synergy between NT219 and the anti-PD-1 (TGI = 98%) was demonstrated. To summarize, we found a significant synergistic effect of NT219 combined with anti-PD-1 therapy, supported by a mechanism of PDL-1 induction making ICB resistant tumors amenable to ICB treatment. Collectively, these findings demonstrated that NT219 has the potential to reverse ICB resistance in both human PDX and murine syngeneic tumor model systems Citation Format: Ricardo Alexandre de Azevedo, Hadas Reuveni, Menashe Bar-Eli, Michael Curran. NT219 induces tumor PD-L1 expression and potentiates anti-PD-1 efficacy. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5073.

Evaluation of nanobody-based biologics targeting purinergic checkpoints in tumor models in vivo.

Adenosine triphosphate (ATP) represents a danger signal that accumulates in injured tissues, in inflammatory sites, and in the tumor microenvironment. ATP promotes tumor growth but also anti-tumor immune responses notably via the P2X7 receptor. ATP can also be catabolized by CD39 and CD73 ecto-enzymes into immunosuppressive adenosine. P2X7, CD39 and CD73 have attracted much interest in cancer as targets offering the potential to unleash anti-tumor immune responses. These membrane proteins represent novel purinergic checkpoints that can be targeted by small drugs or biologics. Here, we investigated nanobody-based biologics targeting mainly P2X7, but also CD73, alone or in combination therapies. Blocking P2X7 inhibited tumor growth and improved survival of mice in cancer models that express P2X7. P2X7-potentiation by a nanobody-based biologic was not effective alone to control tumor growth but enhanced tumor control and immune responses when used in combination with oxaliplatin chemotherapy. We also evaluated a bi-specific nanobody-based biologic that targets PD-L1 and CD73. This novel nanobody-based biologic exerted a potent anti-tumor effect, promoting tumor rejection and improving survival of mice in two tumor models. Hence, this study highlights the importance of purinergic checkpoints in tumor control and open new avenues for nanobody-based biologics that may be further exploited in the treatment of cancer.

Hydroxychloroquine (HCQ) decreases the benefit of anti-PD-1 immune checkpoint blockade in tumor immunotherapy.

Immunotherapy using checkpoint blockade (ICB) with antibodies such as anti-PD-1 has revolutionised the treatment of many cancers. Despite its use to treat COVID-19 patients and autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis, the effect of hydroxychloroquine (HCQ) on cancer immunotherapy has not been examined. In this study, remarkably, we find that HCQ alone, or in combination with azithromycin (AZ), at doses used to treat patients, decreased the therapeutic benefit of anti-PD-1 in cancer immunotherapy. No deleterious effect was seen on untreated tumors. Mechanistically, HCQ and HCQ/AZ inhibited PD-L1 expression on tumor cells, while specifically targeting the anti-PD-1 induced increase in progenitor CD8+CD44+PD-1+TCF1+ tumor infiltrating T cells (TILs) and the generation of CD8+CD44+PD-1+ effectors. Surprisingly, it also impaired the appearance of a subset of terminally exhausted CD8+ TILs. No effect was seen on the presence of CD4+ T cells, FoxP3+ regulatory T cells (Tregs), thymic subsets, B cells, antibody production, myeloid cells, or the vasculature of mice. This study indicates for the first time that HCQ and HCQ/AZ negatively impact the ability of anti-PD-1 checkpoint blockade to promote tumor rejection.

The E3 ubiquitin ligase Peli1 regulates the metabolic actions of mTORC1 to suppress antitumor T cell responses.

Metabolic fitness of T cells is crucial for immune responses against infections and tumorigenesis. Both the T cell receptor (TCR) signal and environmental cues contribute to the induction of T cell metabolic reprogramming, but the underlying mechanism is incompletely understood. Here, we identified the E3 ubiquitin ligase Peli1 as an important regulator of T cell metabolism and antitumor immunity. Peli1 ablation profoundly promotes tumor rejection, associated with increased tumor-infiltrating CD4 and CD8 T cells. The Peli1-deficient T cells display markedly stronger metabolic activities, particularly glycolysis, than wild-type T cells. Peli1 controls the activation of a metabolic kinase, mTORC1, stimulated by both the TCR signal and growth factors, and this function of Peli1 is mediated through regulation of the mTORC1-inhibitory proteins, TSC1 and TSC2. Peli1 mediates non-degradative ubiquitination of TSC1, thereby promoting TSC1-TSC2 dimerization and TSC2 stabilization. These results establish Peli1 as a novel regulator of mTORC1 and downstream mTORC1-mediated actions on T cell metabolism and antitumor immunity.

Abstract IA02: Next targets for immune checkpoint blockade in melanoma

Abstract Multiple mechanisms of melanoma-induced immune escape contribute to the failure of T-cell responses to control tumor growth in humans. Many inhibitory pathways play a critical role in impeding T-cell responses to tumor antigens (TAs), including PD-1, the T-cell immunoglobulin and mucin-domain containing-protein 3 (Tim-3), and the T-cell immunoreceptor with Ig and ITIM domains (TIGIT). These inhibitory receptors (IRs) are upregulated by TA-specific CD8+T cells in the tumor microenvironment (TME) while their respective ligands are expressed by antigen-presenting cells (APCs) and tumor cells. Circulating TA-specific CD8+ T cells and CD8+ tumor-infiltrating lymphocytes (TILs) that coexpress PD-1, TIGIT and Tim-3 exhibit different levels of T-cell dysfunction. Dysfunctional/exhausted CD8+ TILs are not totally inert and can exhibit cytolytic functions but are likely kept in check by multiple inhibitory pathways that can be targeted by immune checkpoint blockade. TIGIT is an attractive target for the next-generation immune checkpoint blockade for several reasons. First, it is highly expressed by the majority of CD8+ TILs together with PD-1. Second, the TIGIT ligands, CD155/PVR, and CD112, are highly expressed in the TME by melanoma cells and APCs. Third, dual PD-1/TIGIT blockade augments the expansion and function of human TA-specific CD8+ T cells in vitro and promotes tumor rejection in animal models. Fourth, TIGIT also acts in Tregs and NK cells to regulate antitumor immune responses. CD8+ TILs in melanoma and other solid tumors downregulate the costimulatory molecule DNAM-1/CD226, which competes with TIGIT for binding to the same ligands CD155 and CD112. Therefore, in addition to the TIGIT-mediated T-cell intrinsic inhibitory effects, the downregulation of CD226 expression by CD8+TILs in the TME also contributes to impede T-cell responses to melanoma. Collectively, our findings provide the rationale for dual PD-1/TIGIT blockade together with therapeutic strategies to counteract CD226 downregulation in the TME to improve the clinical benefits of single PD-1 blockade in patients with melanoma and other solid tumors. Citation Format: Joe-Marc Chauvin, Mignane Ka, Ornella Pagliano, Julien Foucade, Carmine Menna, Diwakar Davar, John Kirkwood, Vijay Kuchroo, Ana Anderson, Alan Korman, Hassane Zarour. Next targets for immune checkpoint blockade in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr IA02.

Tumor necrosis factor overcomes immune evasion in p53-mutant medulloblastoma.

Many immunotherapies act by enhancing the ability of cytotoxic T cells to kill tumor cells. Killing depends on T cell recognition of antigens presented by class I major histocompatibility complex (MHC-I) proteins on tumor cells. In this study, we showed that medulloblastomas lacking the p53 tumor suppressor do not express surface MHC-I and are therefore resistant to immune rejection. Mechanistically, this is because p53 regulates expression of the peptide transporter Tap1 and the aminopeptidase Erap1, which are required for MHC-I trafficking to the cell surface. In vitro, tumor necrosis factor (TNF) or lymphotoxin-β receptor agonist can rescue expression of Erap1, Tap1 and MHC-I on p53-mutant tumor cells. In vivo, low doses of TNF prolong survival and synergize with immune checkpoint inhibitors to promote tumor rejection. These studies identified p53 as a key regulator of immune evasion and suggest that TNF could be used to enhance sensitivity of tumors to immunotherapy.

Structure Based Prediction of Neoantigen Immunogenicity.

The development of immunological therapies that incorporate peptide antigens presented to T cells by MHC proteins is a long sought-after goal, particularly for cancer, where mutated neoantigens are being explored as personalized cancer vaccines. Although neoantigens can be identified through sequencing, bioinformatics and mass spectrometry, identifying those which are immunogenic and able to promote tumor rejection remains a significant challenge. Here we examined the potential of high-resolution structural modeling followed by energetic scoring of structural features for predicting neoantigen immunogenicity. After developing a strategy to rapidly and accurately model nonameric peptides bound to the common class I MHC protein HLA-A2, we trained a neural network on structural features that influence T cell receptor (TCR) and peptide binding energies. The resulting structurally-parameterized neural network outperformed methods that do not incorporate explicit structural or energetic properties in predicting CD8+ T cell responses of HLA-A2 presented nonameric peptides, while also providing insight into the underlying structural and biophysical mechanisms governing immunogenicity. Our proof-of-concept study demonstrates the potential for structure-based immunogenicity predictions in the development of personalized peptide-based vaccines.

Batf3-Dependent Genes Control Tumor Rejection Induced by Dendritic Cells Independently of Cross-Presentation.

The BATF3-dependent cDC1 lineage of conventional dendritic cells (cDC) is required for rejection of immunogenic sarcomas and for rejection of progressive sarcomas during checkpoint blockade therapy. One unique function of the cDC1 lineage is the efficient cross-presentation of tumor-derived neoantigens to CD8+ T cells, but it is not clear that this is the only unique function of cDC1 required for tumor rejection. We previously showed that BATF3 functions during cDC1 lineage commitment to maintain IRF8 expression in the specified cDC1 progenitor. However, since cDC1 progenitors do not develop into mature cDC1s in Batf3 -/- mice, it is still unclear whether BATF3 has additional functions in mature cDC1 cells. A transgenic Irf8-Venus reporter allele increases IRF8 protein concentration sufficiently to allow autonomous cDC1 development in spleens of Batf3 -/- mice. These restored Batf3 -/- cDC1s are transcriptionally similar to control wild-type cDC1s but have reduced expression of a restricted set of cDC1-specific genes. Restored Batf3 -/- cDC1s are able to cross-present cell-associated antigens both in vitro and in vivo However, Batf3 -/- cDC1 exhibit altered characteristics in vivo and are unable to mediate tumor rejection. These results show that BATF3, in addition to regulating Irf8 expression to stabilize cDC1 lineage commitment, also controls expression of a small set of genes required for cDC1-mediated tumor rejection. These BATF3-regulated genes may be useful targets in immunotherapies aimed at promoting tumor rejection.

Antibodies to Costimulatory Receptor 4-1BB Enhance Anti-tumor Immunity via T Regulatory Cell Depletion and Promotion of CD8 T Cell Effector Function

The costimulatory receptor 4-1BB is expressed on activated immune cells, including activated Tcells. Antibodies targeting 4-1BB enhance the proliferation and survival of antigen-stimulated Tcells invitro and promote CD8 Tcell-dependent anti-tumor immunity in pre-clinical cancer models. We found that T regulatory (Treg) cells infiltrating human or murine tumors expressed high amounts of 4-1BB. Intra-tumoral Treg cells were preferentially depleted by anti-4-1BB mAbs invivo. Anti-4-1BB mAbs also promoted effector Tcell agonism to promote tumor rejection. These distinct mechanisms were competitive and dependent on antibody isotype and FcγR availability. Administration of anti-4-1BB IgG2a, which preferentially depletes Treg cells, followed by eitheragonistic anti-4-1BB IgG1 or anti-PD-1 mAb augmented anti-tumor responses in multiple solid tumor models. An antibody engineered to optimize both FcγR-dependent Treg cell depleting capacity and FcγR-independent agonism delivered enhanced anti-tumor therapy. These insights into the effector mechanisms of anti-4-1BB mAbs lay the groundwork for translation into the clinic.

Abstract IA36: Regulatory T cells, polymorphisms, and response to checkpoint blockade: From mechanisms to potential biomarkers

Abstract In mice, anti-CTLA-4 monoclonal antibodies (mAbs) require Fc-gamma receptor (FcγR)-mediated depletion of intratumoral regulatory T (Treg) cells to promote tumor rejection. However, the relevance of this mechanism in the human setting remains controversial. Using a mouse model expressing human FcγRs, and analysis of clinical samples from ipilimumab-treated melanoma patients, we investigate the potential role of Treg depletion in the activity of antibodies targeting human CTLA-4. Citation Format: Sergio Quezada. Regulatory T cells, polymorphisms, and response to checkpoint blockade: From mechanisms to potential biomarkers [abstract]. In: Proceedings of the Fifth AACR-IASLC International Joint Conference: Lung Cancer Translational Science from the Bench to the Clinic; Jan 8-11, 2018; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(17_Suppl):Abstract nr IA36.

Recombinant Listeria promotes tumor rejection by CD8+ T cell-dependent remodeling of the tumor microenvironment

Agents that remodel the tumor microenvironment (TME), prime functional tumor-specific T cells, and block inhibitory signaling pathways are essential components of effective immunotherapy. We are evaluating live-attenuated, double-deleted Listeria monocytogenes expressing tumor antigens (LADD-Ag) in the clinic. Here we show in numerous mouse models that while treatment with nonrecombinant LADD induced some changes in the TME, no antitumor efficacy was observed, even when combined with immune checkpoint blockade. In contrast, LADD-Ag promoted tumor rejection by priming tumor-specific KLRG1+PD1loCD62L- CD8+ T cells. These IFNγ-producing effector CD8+ T cells infiltrated the tumor and converted the tumor from an immunosuppressive to an inflamed microenvironment that was characterized by a decrease in regulatory T cells (Treg) levels, a proinflammatory cytokine milieu, and the shift of M2 macrophages to an inducible nitric oxide synthase (iNOS)+CD206- M1 phenotype. Remarkably, these LADD-Ag-induced tumor-specific T cells persisted for more than 2 months after primary tumor challenge and rapidly controlled secondary tumor challenge. Our results indicate that the striking antitumor efficacy observed in mice with LADD-based immunotherapy stems from TME remodeling which is a direct consequence of eliciting potent, systemic tumor-specific CD8+ T cells.

Increasing FcγR Binding Activity May Enhance Anti-CTLA4 Efficacy

Abstract Anti-CTLA4 antibodies induce an FcγR-dependent depletion of Tregs to promote tumor rejection.

Fc Effector Function Contributes to the Activity of Human Anti-CTLA-4 Antibodies

SummaryWith the use of a mouse model expressing human Fc-gamma receptors (FcγRs), we demonstrated that antibodies with isotypes equivalent to ipilimumab and tremelimumab mediate intra-tumoral regulatory T (Treg) cell depletion in vivo, increasing the CD8+ to Treg cell ratio and promoting tumor rejection. Antibodies with improved FcγR binding profiles drove superior anti-tumor responses and survival. In patients with advanced melanoma, response to ipilimumab was associated with the CD16a-V158F high affinity polymorphism. Such activity only appeared relevant in the context of inflamed tumors, explaining the modest response rates observed in the clinical setting. Our data suggest that the activity of anti-CTLA-4 in inflamed tumors may be improved through enhancement of FcγR binding, whereas poorly infiltrated tumors will likely require combination approaches.

Fluorinated Adenosine A2A Receptor Antagonists Inspired by Preladenant as Potential Cancer Immunotherapeutics.

Antagonism of the adenosine A2A receptor on T cells blocks the hypoxia-adenosinergic pathway to promote tumor rejection. Using an in vivo immunoassay based on the Concanavalin A mouse model, a series of A2A antagonists were studied and identified preladenant as a potent lead compound for development. Molecular modeling was employed to assist drug design and subsequent synthesis of analogs and those of tozadenant, including fluorinated polyethylene glycol PEGylated derivatives. The efficacy of the analogs was evaluated using two in vitro functional bioassays, and compound 29, a fluorinated triethylene glycol derivative of preladenant, was confirmed as a potential immunotherapeutic agent.

Transcriptional and epigenetic regulation of T cell hyporesponsiveness.

Naive CD8+ T cells differentiate into effector and memory cytolytic T cells (CTLs) during an acute infection. In contrast, in scenarios of persistent antigen stimulation, such as chronic infections and cancer, antigen-specific CTLs show a gradual decrease in effector function, a phenomenon that has been termed CD8+ T cell "exhaustion" or "dysfunction." Another hyporesponsive state, termed "anergy", is observed when T cells are activated in the absence of positive costimulatory signals. Among the many negative regulators induced in hyporesponsive T cells are inhibitory cell-surface receptors, such as PD-1, LAG-3, CTLA-4, and TIM-3; "checkpoint blockade" therapies that involve treatment of patients with cancer with blocking antibodies to those receptors show considerable promise in the clinic because the blocking antibodies can mitigate hyporesponsiveness and promote tumor rejection. In this review, we describe recent advances in our molecular understanding of these hyporesponsive states. We review evidence for the involvement of diverse transcription factors, metabolic programs, and chromatin accessibility changes in hyporesponsive T cells, and we discuss how checkpoint blockade therapies affect the molecular program of CD8+ T cell exhaustion.

A Unique Cellular and Molecular Microenvironment Is Present in Tertiary Lymphoid Organs of Patients with Spontaneous Prostate Cancer Regression.

Multiple solid cancers contain tertiary lymphoid organs (TLO). However, it is unclear whether they promote tumor rejection, facilitate tumor evasion, or simply whether they are a byproduct of chronic inflammation. We hypothesize that although chronic inflammation induces TLO formation, the tumor milieu can modulate TLO organization and functions in prostate cancer. Therefore, our study seeks to elucidate the cellular and molecular signatures in unique prostatectomy specimens from evanescent carcinoma patients to identify markers of cancer regression, which could be harnessed to modulate local immunosuppression or potentially enhance TLO function. We used multicolor immunofluorescence to stain prostate tissues, collected at different stages of cancer progression (prostatic intraepithelial neoplasia, intermediate and advanced cancer) or from patients with evanescent prostate carcinoma. Tissues were stained with antibodies specific for pro-inflammatory molecules (cyclooxygenase 2, CXCL10, IL17), tumor-infiltrating immune cells (mature DC-LAMP+ dendritic cells, CD3+ T cells, CD3+Foxp3+ regulatory T cells (Treg), T bet+ Th1 cells, granzyme B+ cytotoxic cells), and stromal cell populations (lymphatic vessels, tumor neovessels, high endothelial venules (HEV), stromal cells), which promote prostate tumor growth or are critical components of tumor-associated TLO. Generally, inflammatory cells are located at the margins of tumors. Unexpectedly, we found TLO within prostate tumors from patients at different stages of cancer and in unique samples from patients with spontaneous cancer remission. In evanescent prostate carcinomas, accumulation of Treg was compromised, while Tbet+ T cells and CD8 T cells were abundant in tumor-associated TLO. In addition, we found a global decrease in tumor neovascularization and the coverage by cells positive for cyclooxygenase 2 (COX2). Finally, consistent with tumor regression, prostate stem cell antigen was considerably reduced in TLO and tumor areas from evanescent carcinoma patients. Collectively, our results suggest that COX2 and Treg are attractive therapeutic targets that can be harnessed to enhance TLO-driven tumor immunity against prostate cancer. Specially, the presence of HEV and lymphatics indicate that TLO can be used as a platform for delivery of cell-based and/or COX2 blocking therapies to improve control of tumor growth in prostate cancer.

Basophils Promote Tumor Rejection via Chemotaxis and Infiltration of CD8+ T Cells.

Elevated numbers of regulatory T cells (Treg) in patient tumors are known to inhibit efficient antitumor T-cell responses. To study the mechanisms controlling tumor rejection, we assessed different mouse models for Treg depletion. In Foxp3DTR knock-in mice, about 99% Treg depletion was achieved, resulting in complete rejection of transplanted HCmel12 melanomas in a CD8+ T-cell-dependent way. In contrast, about 90% Treg depletion obtained in BAC transgenic Foxp3.LuciDTR4 mice failed to induce complete rejection of HCmel12 melanomas, demonstrating that residual Tregs were able to control CD8+ T-cell responses against the tumor. Ninety-nine percent of Treg depletion provoked drastic changes in the tumor microenvironment, such as strong infiltration of CD8+ T cells and basophils. Intratumoral basophils enhanced CD8+ T-cell infiltration via production of chemokines CCL3 and CCL4; antibody-based blocking of these chemokines inhibited CD8+ T-cell infiltration. Therapeutic induction of basophilia by IL3/anti-IL3 antibody complexes, combined with transfer of CD8+ T cells, resulted in enhanced T-cell infiltration and tumor rejection. Our study identifies a critical role basophils play in tumor rejection and that this role can be exploited for therapeutic intervention. Cancer Res; 77(2); 291-302. ©2016 AACR.