Anti-tumor Effector Functions Research Articles

The ω-3 polyunsaturated fatty acid docosahexaenoic acid enhances NK-cell antitumor effector functions.

ω-3 polyunsaturated fatty acids (PUFAs) are known to directly repress tumour development and progression. In this study, we explored whether docosahexaenoic acid (DHA), a type of ω-3 PUFA, had an immunomodulatory role in promoting tumour growth in immunocompetent mice. The number of natural killer (NK) cells but not the number of T or B cells was decreased by DHA supplementation in various tissues under physiological conditions. Although the frequency and number of NK cells were comparable, IFN-γ production by NK cells in both the spleen and lung was increased in DHA-supplemented mice in the mouse B16F10 melanoma tumour model. Single-cell RNA sequencing (scRNA-seq) revealed that DHA promoted effector function and oxidative phosphorylation in NK cells but had no obvious effects on other immune cells. Using Rag2-/- mice and NK-cell depletion by PK136 antibody injection, we demonstrated that the suppression of B16F10 melanoma tumour growth in the lung by DHA supplementation was dependent mainly on NK cells. In vitro experiments showed that DHA directly enhanced IFN-γ production, CD107a expression and mitochondrial oxidative phosphorylation (OXPHOS) activity, and slightly increased proliferator-activated receptor gamma coactivator-1α (PGC-1α) protein expression in NK cells. The PGC-1α inhibitor SR-18292 in vitro and NK cell-specific knockout of PGC-1α in mice reversed the antitumour effects of DHA. In summary, our findings broaden the current knowledge on how DHA supplementation protects against cancer growth from the perspective of immunomodulation by upregulating PGC-1α signalling-mediated mitochondrial OXPHOS activity in NK cells.

Abstract 3191: Chk1/2 inhibition enhances response to lurbinectedin treatment in small cell lung cancer

Abstract Small cell lung cancer (SCLC) remains a significant clinical concern, with only a 7% 5 year survival rate; therefore, there remains an unmet need for efficacious treatments. Lurbinectedin was FDA approved in 2019 to treat platinum-resistant SCLC. Preliminary experiments in SCLC cell lines indicated an increase in intracellular pChk1 when treated with lurbinectedin. Based on these data and our understanding of the role of checkpoint kinases in the DNA damage response (DDR), we hypothesize that the activity of Chk1 is indicative of upregulated DDR activity as a result of double stranded breaks and replicative stress induced by lurbinectedin. In subsequent experiments, we sought to identify the role of Chk1 and Chk2 in the tumor cell response to lurbinectedin by inhibiting their activity using prexasertib (LY2606368, Acrivon Therapeutics). Cell viability experiments indicate decreased tumor cell viability when three SCLC cell lines (H1048, H1105, H1417) are treated with prexasertib as a single agent and in combination with lurbinectedin. Western blot analysis of intracellular proteins from the same SCLC cell lines treated with both drugs demonstrate dose-dependent effects on cell death marker cPARP, DNA damage marker γH2AX, and DDR/cell cycle pathway kinases cdk1 and 2. Ongoing experiments seek to replicate results using alternative Chk1/2 inhibitors, and to explore the effect of lurbinectedin-Chk1/2 inhibitor combination treatment on antitumor immune effector function by in vitro co-culture. Citation Format: Ashley Sanchez Sevilla Uruchurtu, Tyler Roady, Agenxnie Anderson, Wafik S. El-Deiry. Chk1/2 inhibition enhances response to lurbinectedin treatment in small cell lung 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 3191.

Abstract 2359: New insights into targeting the CD200R1 pathway in T and NK cells using 23ME-00610 as a single agent or in combination

Abstract 23ME-00610 is a monoclonal antibody that is being evaluated as a single agent in a Phase 1/2a clinical trial for advanced solid malignancies (NCT05199272). It binds with high affinity (KD < 0.1 nM) to CD200R1, a promising immune-oncology (IO) target identified by surveying the 23andMe genetic and health survey database. CD200R1 is an inhibitory receptor that when bound to its ligand CD200, reduces pro-inflammatory cytokine production, and leads to an immunosuppressive tumor environment. To gain further insight into the mechanism of action of 23ME-00610 and its potential for combination therapy, we profiled CD200R1 and other immune inhibitory receptors in human dissociated solid tumor samples using spectral flow cytometry. CD200R1 was broadly expressed across tumor-infiltrating immune cells, including T cells and NK cells, cytotoxic cell types with known antitumor functions. Notably, CD200R1 expression pattern was differentiated from PD-1, which is mostly restricted to T cells, indicating differences in patient populations that could potentially benefit from anti-CD200R1 and anti-PD-1 treatments. The ligand, CD200 was broadly expressed, including on tumor cells and endothelial cells. The differential expression profiles of CD200R1 and PD-1 on immune cells, along with CD200 expression on endothelial cells, suggest that combining 23ME-00610 with other clinically validated immunomodulatory agents could enhance antitumor responses. Follow-up experiments were conducted to validate and extend these findings. In an in vitro assay with cancer patient-derived PBMCs, 23ME-00610 promoted higher production of IFNγ, a key functional antitumor cytokine, compared to anti-PD-1 in 9 of 10 donors. Subsequent attempts were made to pinpoint immune cells responsible for 23ME-00610's anti-tumor activity. 23ME-00610 reversed CD200-mediated suppression of chronically stimulated primary T cells and CD200-mediated inhibition of NK cell degranulation, suggesting that 23ME-00610 can mediate tumor cell killing through at least two different cell types. To test the potential of 23ME-00610 to combine with anti-PD-1 to enhance T cell function, an artificial antigen-presenting cell (aAPC) line overexpressing PD-L1 and CD200 was co-cultured with chronically stimulated primary human T cells. Anti-PD-1 and 23ME-00610 each independently enhanced IFNy secretion levels compared to isotype control, yet their combination resulted in a more than additive effect. Taken together, these data demonstrate that 23ME-00610 can enhance both NK and T cell antitumor effector functions, and 23ME-00610 could synergize with anti-PD-1 to further increase the activity of T cells against cancer cells. The results of this study provide evidence that 23ME-00610 could expand IO treatment options for patients with advanced solid malignancies as a single agent or in combination with other anti-tumor therapeutics. Citation Format: Mithra R. Kumar, Shiteng Duan, Shruti Yadav, Abigail Jarret, Zhonghao Yan, Jill Fenaux, Martin Kinisu, Zuoan Yi, Mauro Poggio, Patrick Collins, Antony Symons, Lorena Riol Blanco, Maike Schmidt, Sophia Majeed, Anh Diep, Clarissa Lee. New insights into targeting the CD200R1 pathway in T and NK cells using 23ME-00610 as a single agent or in combination [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 2359.

Phenotypic and functional exhaustion of circulating CD3+ CD56+ NKT-like cells in colorectal cancer patients.

CD3+ CD56+ NKT-like cells are crucial to antitumor immune surveillance and defense. However, research on circulating NKT-like cells in colorectal cancer (CRC) patients is limited. This investigation selected 113 patients diagnosed with primary CRC for preoperative peripheral blood collection. The blood from 106 healthy donors at the physical examination center was acquired as a healthy control (HC). The distribution of lymphocyte subsets, immunophenotype, and functional characteristics of NKT-like cells was comprehensively evaluated. Compared to HC, primary CRC patients had considerably fewer peripheral NKT-like cells in frequency and absolute quantity, and the fraction of NKT-like cells was further reduced in patients with vascular invasion compared to those without. The NKT-like cells in CRC patients had a reduced fraction of the activating receptor CD16, up-regulated expression of inhibitory receptors LAG-3 and NKG2A, impaired production of TNF-α and IFN-γ, as well as degranulation capacity. Moreover, the increased frequency of NKG2A+ NKT-like cells and the decreased expression of activation-related molecules were significantly correlated with tumor progression. In detail, NKG2A+ NKT-like cells indicated increased PD-1 and Tim-3 and reduced TNF-α than NKG2A- subgroup. Blocking NKG2A invitro restored cytokine secretion capacity in NKT-like cells from CRC patients. Altogether, this research revealed that circulating NKT-like cells in CRC patients exhibited suppressive phenotype and functional impairment, which was more pronounced in NKG2A+ NKT-like cells. These findings suggest that NKG2A blockade may restore anti-tumor effector function in NKT-like cells, which provides a potential target for immunotherapy in CRC patients.

The highly selective and oral phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib induces apoptosis in mesothelioma cells and increases immune effector cell composition

Targeting aberrantly expressed kinases in malignant pleural mesothelioma (MPM) is a promising therapeutic strategy. We here investigated the effect of the novel and highly selective Phosphoinositide 3-kinase delta (PI3K-δ) inhibitor roginolisib (IOA-244) on MPM cells and on the immune cells in MPM microenvironment.To this aim, we analyzed the expression of PI3K-δ by immunohistochemistry in specimens from primary MPM, cell viability and death in three different MPM cell lines treated with roginolisib alone and in combination with ipatasertib (AKT inhibitor) and sapanisertib (mTOR inhibitor). In a co-culture model of patient-derived MPM cells, autologous peripheral blood mononuclear cells and fibroblasts, the tumor cell viability and changes in immune cell composition were investigated after treatment of roginolisib with nivolumab and cisplatin. PI3K-δ was detected in 66/89 (74%) MPM tumors and was associated with reduced overall survival (12 vs. 25 months, P=0.0452). Roginolisib induced apoptosis in MPM cells and enhanced the anti-tumor efficacy of AKT and mTOR kinase inhibitors by suppressing PI3K-δ/AKT/mTOR and ERK1/2 signaling. Furthermore, the combination of roginolisib with chemotherapy and immunotherapy re-balanced the immune cell composition, increasing effector T-cells and reducing immune suppressive cells. Overall, roginolisib induces apoptosis in MPM cells and increases the antitumor immune cell effector function when combined with nivolumab and cisplatin.These results provide first insights on the potential of roginolisib as a therapeutic agent in patients with MPM and its potential in combination with established immunotherapy regimen.

Isolation and expansion of pure and functional γδ T cells.

γδ T cells are important components of the immune system due to their ability to elicit a fast and strong response against infected and transformed cells. Because they can specifically and effectively kill target cells in an MHC independent fashion, there is great interest to utilize these cells in anti-tumor therapies where antigen presentation may be hampered. Since only a small fraction of T cells in the blood or tumor tissue are γδ T cells, they require extensive expansion to allow for fundamental, preclinical and ex vivo research. Although expansion protocols can be successful, most are based on depletion of other cell types rather than γδ T cell specific isolation, resulting in unpredictable purity of the isolated fraction. Moreover, the primary focus only lies with expansion of Vδ2+ T cells, while Vδ1+ T cells likewise have anti-tumor potential. Here, we investigated whether γδ T cells directly isolated from blood could be efficiently expanded while maintaining function. γδ T cell subsets were isolated using MACS separation, followed by FACS sorting, yielding >99% pure γδ T cells. Isolated Vδ1+ and Vδ2+ T cells could effectively expand immediately after isolation or upon freeze/thawing and reached expansion ratios between 200 to 2000-fold starting from varying numbers using cytokine supported feeder stimulations. MACS/FACS isolated and PHA stimulated γδ T cells expanded as good as immobilized antibody mediated stimulated cells in PBMCs, but delivered purer cells. After expansion, potential effector functions of γδ T cells were demonstrated by IFN-γ, TNF-α and granzyme B production upon PMA/ionomycin stimulation and effective killing capacity of multiple tumor cell lines was confirmed in killing assays. In conclusion, pure γδ T cells can productively be expanded while maintaining their anti-tumor effector functions against tumor cells. Moreover, γδ T cells could be expanded from low starting numbers suggesting that this protocol may even allow for expansion of cells extracted from tumor biopsies.

CD8+ T cell metabolic flexibility elicited by CD28-ARS2 axis-driven alternative splicing of PKM supports antitumor immunity

Metabolic flexibility has emerged as a critical determinant of CD8+ T-cell antitumor activity, yet the mechanisms driving the metabolic flexibility of T cells have not been determined. In this study, we investigated the influence of the nuclear cap-binding complex (CBC) adaptor protein ARS2 on mature T cells. In doing so, we discovered a novel signaling axis that endows activated CD8+ T cells with flexibility of glucose catabolism. ARS2 upregulation driven by CD28 signaling reinforced splicing factor recruitment to pre-mRNAs and affected approximately one-third of T-cell activation-induced alternative splicing events. Among these effects, the CD28-ARS2 axis suppressed the expression of the M1 isoform of pyruvate kinase in favor of PKM2, a key determinant of CD8+ T-cell glucose utilization, interferon gamma production, and antitumor effector function. Importantly, PKM alternative splicing occurred independently of CD28-driven PI3K pathway activation, revealing a novel means by which costimulation reprograms glucose metabolism in CD8+ T cells.

Abstract A048: Enhancing the immune response in locally advanced pancreatic cancer (LAPC) with intratumoral endoscopic ultrasound-guided fine needle injection of large surface area microparticle paclitaxel (LSAM-PTX)

Abstract Background: The complex tumor microenvironment (TME) of locally advanced pancreatic cancer (LAPC) is resistant to immune effector cell infiltration and drug bioavailability, limiting the effectiveness of current systemic therapies. Large Surface Area Microparticle Paclitaxel (LSAM-PTX) was developed for intratumoral (IT) injection to solid tumors to provide a paclitaxel depot at therapeutic levels with a prolonged residence time. We previously reported enhanced tumor kill in LAPC subjects receiving two monthly IT LSAM-PTX injections and demonstrated immunomodulation in the TME. Multiplex immunofluorescence (mIF) performed on pre-treatment biopsy tissue and post-LSAM-PTX resection showed increases in densities of effector T cells and NK cells and decreases in myeloid derived suppressor cells (MDSC). To further evaluate immunomodulation following IT LSAM-PTX therapy, blood samples were obtained for immunophenotyping using flow cytometry (NCT03077685). Methods: Pretreatment samples were obtained from subjects administered four injections (n=14) and at visits throughout the treatment period and up to six months post first LSAM-PTX injection. Flow cytometric analysis was performed using three panels to evaluate absolute counts and percentages of up to 45 immunophenotypes. Results: Treatment with four IT injections of LSAM-PTX (ongoing median overall survival = 12.6 months; follow-up = 12.2 months) was associated with increases in peripheral lymphocytes during the treatment period. An enhancement of immune subsets associated with anti-tumor effector functions was found, including significant increases in circulating CD3+ and CD4+ cells (p=0.0012 and 0.002 respectively) which is consistent with the 4-fold increase in CD4+helper T cells detected in the TME of resected tissue. A significant increase in LAG3+CD8+ T cells was seen following treatment initiation (p=0.048) marking early T cell activation. Increases in circulating CD8+ T cells, B cells, and NK cells were seen during the treatment period corresponding with effector T cells and NK cells increases (4.8-fold increase in density) within the TME. Six months following treatment initiation there was a significant decrease in circulating Treg cells (p=0.0183) and a decrease in peripheral immunosuppressive G-MDSC, consistent with the reduction in MDSC measured in the TME. Notably, systemic taxane toxicities, including immunosuppression, were not reported following IT LSAM-PTX and negligible amounts of paclitaxel were found in blood samples. Conclusion: Immunophenotyping of blood from LAPC subjects treated with IT LSAM-PTX demonstrates immunomodulation to a phenotype associated with anti-tumor immune effects, including favorable immunosurveillance, and is consistent with changes found in the TME in resected tissues. Immunosuppressive cell types typically associated with poor outcomes were reduced at six months. Anti-tumor immunomodulation without immunosuppression suggests that IT LSAM-PTX may be amenable to combination with immunotherapy. Citation Format: Andrew E. Hendifar, Max M. Wattenberg, Holly A. Maulhardt, Alyson M. Marin, Alison E. Wendt, Shelagh J. Verco, Gere S. diZerega. Enhancing the immune response in locally advanced pancreatic cancer (LAPC) with intratumoral endoscopic ultrasound-guided fine needle injection of large surface area microparticle paclitaxel (LSAM-PTX) [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A048.

Optimisation of a primary human CAR-NK cell manufacturing pipeline.

Autologous chimeric antigen receptor (CAR) T-cell therapy of B-cell malignancies achieves long-term disease remission in a high fraction of patients and has triggered intense research into translating this successful approach into additional cancer types. However, the complex logistics involved in autologous CAR-T manufacturing, the compromised fitness of patient-derived Tcells, the high rates of serious toxicities and the overall cost involved with product manufacturing and hospitalisation have driven innovation to overcome such hurdles. One alternative approach is the use of allogeneic natural killer (NK) cells as a source for CAR-NK cell therapy. However, this source has traditionally faced numerous manufacturing challenges. To address this, we have developed an optimised expansion and transduction protocol for primary human NK cells primed for manufacturing scaling and clinical evaluation. We have performed an in-depth comparison of primary human NK cell sources as a starting material by characterising their phenotype, functionality, expansion potential and transduction efficiency at crucial timepoints of our CAR-NK manufacturing pipeline. We identified adult peripheral blood-derived NK cells to be the superior source for generating a CAR-NK cell product because of a higher maximum yield of CAR-expressing NK cells combined with potent natural, as well as CAR-mediated anti-tumor effector functions. Our optimised manufacturing pipeline dramatically improves lentiviral transduction efficiency of primary human NK cells. We conclude that the exponential expansion pre- and post-transduction and high on-target cytotoxicity make peripheral blood-derived NK cells a feasible and attractive CAR-NK cell product for clinical utility.

Dual-Armed Oncolytic Myxoma Virus Encoding IFN-γ and CD47 Promotes Lymphocyte Infiltration and Tumor Suppression of Syngeneic Murine Melanoma.

Myxoma virus (MyxV) is a rabbit-specific poxvirus. However, its ability to selectively target tumor cells has established it as a safe and effective anticancer therapy. To strengthen its preclinical efficacy, transgenes that can prolong cancer cell infection and enhance anti-tumor effector functions are currently being investigated. We engineered MyxV armed with CD47, to turn on a 'do not eat me' signal within infected cells with actively replicating viruses, and with IFN-γ to further activate host immune anticancer responses. Tumor suppressive activities were significantly enhanced by the dual-armed MyxV_CD47/IFN-γ compared to parental MyxV or single-armed MyxV_CD47 or MyxV_IFN-γ. In addition, significant increases in IFN-γ+ CD8+T-cells and CD4+ T-cells populations within tumor-infiltrating lymphocytes (TIL) were observed after MyxV_CD47/IFN-γ treatment. Notably, all groups treated with MyxV showed a marked reduction in Foxp3+ CD4+ regulatory T-cells (Tregs) within TIL. We also show that MyxV infection induces PD-L1 up-regulation in cancer cells, and combinational treatment of MyxV with anti-mouse PD-L1 antibodies (αPD-L1) further controlled tumor burden and increased survival in the syngeneic melanoma model B16F10. Our data demonstrate that a CD47 and IFNγ dual-armed MyxV is an effective oncolytic viral immunotherapeutic. These findings strongly support further preclinical investigations to develop next-generation MyxV-based immunotherapy approaches.

SOCS3 deletion in effector T cells confers an anti-tumorigenic role of IL-6 to the pro-tumorigenic cytokine

Interleukin (IL)-6 is abundantly expressed in the tumor microenvironment and is associated with poor patient outcomes. Here, we demonstrate that the deletion of the suppressor of cytokine signaling 3 (SOCS3) in Tcells potentiates anti-tumor immune responses by conferring the anti-tumorigenic function of IL-6 in mouse and human models. In Socs3-deficient CD8+ Tcells, IL-6 upregulates the expression of type I interferon (IFN)-regulated genes and enhances the anti-tumor effector function of Tcells, while also modifying mitochondrial fitness to increase mitochondrial membrane potential and reactive oxygen species (ROS) levels and to promote metabolic glycolysis in the energy state. Furthermore, Socs3 deficiency reduces regulatory Tcells and increases T helper 1 (Th1) cells. SOCS3 knockdown in human chimeric antigen receptor T (CAR-T) cells exhibits a strong anti-tumor response in humanized mice. Thus, genetic disruption of SOCS3 offers an avenue to improve the therapeutic efficacy of adoptive Tcell therapy.

Antitumor T-cell function requires CPEB4-mediated adaptation to chronic endoplasmic reticulum stress.

Tumor growth is influenced by a complex network of interactions between multiple cell types in the tumor microenvironment (TME). These constrained conditions trigger the endoplasmic reticulum (ER) stress response, which extensively reprograms mRNA translation. When uncontrolled over time, chronic ER stress impairs the antitumor effector function of CD8 T lymphocytes. How cells promote adaptation to chronic stress in the TME without the detrimental effects of the terminal unfolded protein response (UPR) is unknown. Here, we find that, in effector CD8 T lymphocytes, RNA-binding protein CPEB4 constitutes a new branch of the UPR that allows cells to adapt to sustained ER stress, yet remains decoupled from the terminal UPR. ER stress, induced during CD8 T-cell activation and effector function, triggers CPEB4 expression. CPEB4 then mediates chronic stress adaptation to maintain cellular fitness, allowing effector molecule production and cytotoxic activity. Accordingly, this branch of the UPR is required for the antitumor effector function of T lymphocytes, and its disruption in these cells exacerbates tumor growth.

Exploring the potential of combining IL-2-activated NK cells with an anti-PDL1 monoclonal antibody to target multiple myeloma-associated macrophages

Multiple myeloma (MM) is an incurable disease, characterized by malignant plasma cells in the bone marrow. MM growth is largely dependent on the tumor microenvironment (TME), consisting of complex cellular networks that shape a tumor-permissive environment. Within the TME, tumor-associated cells (TAC) comprise heterogeneous cell populations that collectively support immunosuppression. Reshaping the TME toward an immunostimulatory environment may enhance effectiveness of immunotherapies. Here, we investigated interactions between donor-derived natural killer (NK) cells and TAC, like tumor-associated macrophages (TAM) and M1 macrophages, and assessed whether anti-tumor effector functions of NK cells could be enhanced by an ADCC-triggering antibody targeting macrophages. Monocytes were polarized in vitro toward either M1 or TAM before co-culture with high-dose IL-2-activated NK cells. NK cell responses were assessed by measuring degranulation (CD107a) and IFN-γ production. We found that NK cells degranulated and produced IFN-γ upon interaction with both macrophage types. NK cell responses against PD-L1+ M1 macrophages could be further enhanced by Avelumab, an anti-PD-L1- and ADCC-inducing antibody. Additionally, NK cell responses were influenced by HLA class I, shown by stronger degranulation in NK cell subsets for which the corresponding HLA ligand was absent on the macrophage target cells (KIR-ligand mismatch) compared to degranulation in the presence of the HLA ligand (KIR-ligand match). Our results suggest that NK cells could, next to killing tumor cells, get activated upon interaction with TAC, like M1 macrophages and TAMs, and that NK cells combined with PD-L1 blocking antibodies with ADCC potential could, through IFN-γ secretion, promote a more immune-favorable TME.

CD200+ cytotoxic T lymphocytes in the tumor microenvironment are crucial for efficacious anti-PD-1/PD-L1 therapy.

Anti-PD-1/PD-L1 therapy, either by anti-PD-1 antibody or anti-PD-L1 antibody, has efficacy by reinvigorating tumor-infiltrating CD8+ T cells in a subset of patients with cancer, but it has unequal effects on heterogeneous CD8+ T cell populations. Hence, the subset crucial to efficacious PD-1 blockade therapy remains elusive. Here, we found an increase in tumor-infiltrating CD200+ cytotoxic T lymphocytes (CTLs) upon PD-1/PD-L1 blockade, with higher proportions of CD200+ T cells positively related to a favorable clinical outcome to anti-PD-1/PD-L1 therapy in three independent cohorts of patients with cancer. Using multiple mouse tumor models, we demonstrated that CD200+ CTLs are essential for efficacious anti-PD-L1 therapy. Mechanistically, we observed a unique chromatin landscape in CD200+ CTLs and found that these cells are enriched for tumor antigen-specific CTLs and have antitumor effector functions. Coinoculation of CD200+ CTLs with tumor cells led to robust tumor regression in two transplanted mouse models. Clinically, we found that infiltration of CD200+ CTLs into tumors could predict immunotherapy efficacy in six patient cohorts. Together, our findings reveal that CD200+ CTLs in the tumor microenvironment are crucial for efficacious anti-PD-1/PD-L1 therapy and could serve as a predictor of successful immunotherapy in the clinic.

Immune Cell Metabolism and Immuno-Oncology

With the significant successes of immune checkpoint blockade and adoptive cellular therapy, immunotherapy has now become an established treatment option to effectively treat cancer. However, the full potential of this treatment modality has yet to be realized, as there are many additional mechanisms whereby tumors continue to evade immune destruction. To this end, metabolic reprogramming by cancer cells serves not only to promote their own growth but also to create an immunosuppressive tumor microenvironment. The tumor metabolic microenvironment not only inhibits antitumor effector function but also supports the differentiation and function of suppressive immune cells. In this review, we delineate the major metabolic programs of cancer cells and immune cells. Furthermore, we discuss the role of so-called metabolic checkpoints that promote immune evasion and tumor growth. Finally, we review current and potential future strategies to target metabolism in order to not simply inhibit tumor growth but also enhance antitumor immune responses. Such strategies have the great potential to enhance the breadth and depth of immunotherapy for cancer by targeting metabolic checkpoints.

Perioperative corticosteroid treatment impairs tumor-infiltrating dendritic cells in patients with newly diagnosed adult-type diffuse gliomas.

Adult-type diffuse gliomas are malignant primary brain tumors characterized by very poor prognosis. Dendritic cells (DCs) are key in priming antitumor effector functions in cancer, but their role in gliomas remains poorly understood. In this study, we characterized tumor-infiltrating DCs (TIDCs) in adult patients with newly diagnosed diffuse gliomas by using multi-parametric flow cytometry and single-cell RNA sequencing. We demonstrated that different subsets of DCs are present in the glioma microenvironment, whereas they are absent in cancer-free brain parenchyma. The largest cluster of TIDCs was characterized by a transcriptomic profile suggestive of severe functional impairment. Patients undergoing perioperative corticosteroid treatment showed a significant reduction of conventional DC1s, the DC subset with key functions in antitumor immunity. They also showed phenotypic and transcriptional evidence of a more severe functional impairment of TIDCs. Overall, the results of this study indicate that functionally impaired DCs are recruited in the glioma microenvironment. They are severely affected by dexamethasone administration, suggesting that the detrimental effects of corticosteroids on DCs may represent one of the mechanisms contributing to the already reported negative prognostic impact of steroids on glioma patient survival.

Dual Immunostimulatory Pathway Agonism through a Synthetic Nanocarrier Triggers Robust Anti-Tumor Immunity in Murine Glioblastoma.

Myeloid cells are abundant, create a highly immunosuppressive environment in glioblastoma (GBM), and thus contribute to poor immunotherapy responses. Based on the hypothesis that small molecules can be used to stimulate myeloid cells to elicit anti-tumor effector functions, a synthetic nanoparticle approach is developed to deliver dual NF-kB pathway-inducing agents into these cells via systemic administration. Synthetic, cyclodextrin-adjuvant nanoconstructs (CANDI) with high affinity for tumor-associated myeloid cells are dually loaded with a TLR7 and 8 (Toll-like receptor, 7 and 8) agonist (R848) and a cIAP (cellular inhibitor of apoptosis protein) inhibitor (LCL-161) to dually activate these myeloid cells. Here CANDI is shown to: i) readily enter the GBM tumor microenvironment (TME) and accumulate at high concentrations, ii) is taken up by tumor-associated myeloid cells, iii) potently synergize payloads compared to monotherapy, iv) activate myeloid cells, v) fosters a "hot" TME with high levels of T effector cells, and vi) controls the growth of murine GBM as mono- and combination therapies with anti-PD1. Multi-pathway targeted myeloid stimulation via the CANDI platform can efficiently drive anti-tumor immunity in GBM.

Metabolic regulation of immune responses to cancer.

The tumor microenvironment is an ecosystem composed of multiple types of cells, such as tumor cells, immune cells, and cancer-associated fibroblasts. Cancer cells grow faster than non-cancerous cells and consume larger amounts of nutrients. The rapid growth characteristic of cancer cells fundamentally alters nutrient availability in the tumor microenvironment and results in reprogramming of immune cell metabolic pathways. Accumulating evidence suggests that cellular metabolism of nutrients, such as lipids and amino acids, beyond being essential to meet the bioenergetic and biosynthetic demands of immune cells, also regulates a broad spectrum of cellular signal transduction, and influences immune cell survival, differentiation, and anti-tumor effector function. The cancer immunometabolism research field is rapidly evolving, and exciting new discoveries are reported in high-profile journals nearly weekly. Therefore, all new findings in this field cannot be summarized within this short review. Instead, this review is intended to provide a brief introduction to this rapidly developing research field, with a focus on the metabolism of two classes of important nutrients-lipids and amino acids-in immune cells. We highlight recent research on the roles of lipids and amino acids in regulating the metabolic fitness and immunological functions of T cells, macrophages, and natural killer cells in the tumor microenvironment. Furthermore, we discuss the possibility of "editing" metabolic pathways in immune cells to act synergistically with currently available immunotherapies in enhancing anti-tumor immune responses.

Low-Dose JAK3 Inhibition Improves Antitumor T-Cell Immunity and Immunotherapy Efficacy.

Terminal T-cell exhaustion poses a significant barrier to effective anticancer immunotherapy efficacy, with current drugs aimed at reversing exhaustion being limited. Recent investigations into the molecular drivers of T-cell exhaustion have led to the identification of chronic IL2 receptor (IL2R)-STAT5 pathway signaling in mediating T-cell exhaustion. We targeted the key downstream IL2R-intermediate JAK 3 using a clinically relevant highly specific JAK3-inhibitor (JAK3i; PF-06651600) that potently inhibited STAT5-phosphorylation in vitro. Whereas pulsed high-dose JAK3i administration inhibited antitumor T-cell effector function, low-dose chronic JAK3i significantly improved T-cell responses and decreased tumor load in mouse models of solid cancer. Low-dose JAK3i combined with cellular and peptide vaccine strategies further decreased tumor load compared with both monotherapies alone. Collectively, these results identify JAK3 as a novel and promising target for combination immunotherapy.

Abstract 4188: CD4+ T cells mediate non-canonical rejection of major histocompatibility class-I deficient pancreatic tumors independently of CD8+ T cells

Abstract Identifying and interrogating novel immune-mediated mechanisms of tumor rejection is a major clinical opportunity. Immune checkpoint blockade (ICB) induces tumor rejection primarily via cytolytic CD8+ T cells, but many patients have primary or acquired resistance to CD8+ T cell immunity. Reduced major histocompatibility complex I (MHC I) expression is a contributing factor to resistance, and pancreatic ductal adenocarcinoma (PDAC) is historically treatment-refractory due to, among other features, low MHC expression and a dearth of T cells in the tumor. Interrogating interventions in the context of the PDAC tumor microenvironment will reveal novel mechanisms regulating sensitivity to immune-mediated destruction. Using a genetically engineered mouse model of PDAC, we previously reported that combination immunotherapy (agonistic anti-CD40 and dual ICB, anti-PD-1/CTLA-4) induced both a CD8+ and a CD4+ T cell response sufficient to mediate tumor rejection and long-lived protection. Here, we utilized CRISPR-Cas9 to disrupt the dominant MHC I (H-2Kb allele) expression in PDAC cell lines, precluding direct antigen presentation to CD8+ T cells via this molecule. Subcutaneous injection of MHC I-deficient tumor clones into wild-type mice revealed normal tumor growth at baseline, and significantly delayed tumor growth kinetics in response to combination immunotherapy (CD40/ICB) in a CD4+ T cell-dependent manner (p < 0.0001 vs. vehicle-treated mice). These findings were observed in parental PDAC clones lacking H-2Kb expression, as well as in an ovalbumin (OVA)-expressing PDAC clone. CD4+ T cells mediated tumor rejection including in OVA-tolerized mice, excluding the role of tumor-neoantigens as a major determinant of response. There was no direct contribution of tumor rejection by CD8+ T cells, despite the generation of an activated, tumor-specific CD8+ T cell response in the tumor site (24.5% vs. 1.98% in vehicle-treated mice, p < 0.003). CD4+ T cells upregulated IFN-γ production (52.3% vs. 11.9% in vehicle-treated mice, p < 0.0001), and host IFN-γ and IFN-γ receptor expression were required for response. No changes were observed in the expression of cytotoxic molecules (Granzyme A or B) by CD4+ T cells after CD40/ICB, and perforin was not required for tumor rejection. Furthermore, depletion of macrophage and myeloid cells or genetic knockout of inducible nitric oxide synthase did not dampen CD4+ T cell immunity against established PDAC tumors. Thus, CD4+ T cells mediated PDAC tumor rejection via non-canonical mechanisms, excluding cytotoxic molecules and re-direction of anti-tumor macrophage effector functions after treatment with anti-CD40/ICB. This study provides critical insight into novel mechanisms of tumor clearance beyond direct CD8+ T cell:tumor cell interactions, and opportunities to exploit CD4 T+ cells as regulators of tumor immunity. Citation Format: Samuel I. Kim, Charu Arora, Ioannis I. Verginadis, Christopher R. Cassella, Nune Markosyan, Constantinos Koumenis, Robert H. Vonderheide, Katelyn T. Byrne. CD4+ T cells mediate non-canonical rejection of major histocompatibility class-I deficient pancreatic tumors independently of CD8+ T cells [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 4188.