Expression Of Inhibitory Receptors Research Articles

Tumor-associated NK cells facilitate tumor growth via NKp46 in immunocompetent murine hepatocellular carcinoma

Natural killer(NK) cells comprise one subset of the innate lymphoid cells family. Despite reported anti-tumor activity of NK cells, their tangible contribution to tumor control remains controversial. This is due to the incomplete understanding of NK alterations within tumor microenvironment(TME). Here we showed, using murine hepatocellular carcinoma(HCC) model, that early NK cells deletion markedly attenuated tumor growth in a CD8+T cells dependent manner. This effect was accompanied by an enhanced CD8+T cells effector function in tumor rather than circulating blood. Then, we demonstrated that abundant NKp46+ NK subset, but not NKp46− NK, were recruited towards tumor microenvironment during tumor progression. Frequency of intratumor NKP46+ NK cells were inversely related to CD8+T cells activation, and positively correlated with tumor growth. Intratumor NKp46+ NK cells exhibited dysfunction and increased expression of inhibitory receptors, when compared with NKp46− NK cells. Blockade of NK cells-associated NKp46 effectively attenuated HCC growth. Infusion of tumor-derived NKp46+ NK cells markedly enhanced HCC growth in vivo, in contrast to tumor cells inoculation alone. The further mechanistic investigations unveiled that NK cells boosted tumor growth by NKp46-mediated impairment of CD8+T cells effector function.Overall, this work supported a previously unappreciated regulatory property of tumor-associated NK cells in HCC, and NKp46 as a potential target against HCC in clinical setting.

Characterization of the input material quality for the production of tisagenlecleucel by multiparameter flow cytometry and its relation to the clinical outcome.

Tisagenlecleucel (tisa-cel) is a CD19-specific CAR-T cell product approved for the treatment of relapsed/refractory (r/r) DLBCL or B-ALL. We have followed a group of patients diagnosed with childhood B-ALL (n = 5), adult B-ALL (n = 2), and DLBCL (n = 25) who were treated with tisa-cel under non-clinical trial conditions. The goal was to determine how the intensive pretreatment of patients affects the produced CAR-T cells, their in vivo expansion, and the outcome of the therapy. Multiparametric flow cytometry was used to analyze the material used for manufacturing CAR-T cells (apheresis), the CAR-T cell product itself, and blood samples obtained at three timepoints after administration. We present the analysis of memory phenotype of CD4/CD8 CAR-T lymphocytes (CD45RA, CD62L, CD27, CD28) and the expression of inhibitory receptors (PD-1, TIGIT). In addition, we show its relation to the patients' clinical characteristics, such as tumor burden and sensitivity to prior therapies. Patients who responded to therapy had a higher percentage of CD8+CD45RA+CD27+ T cells in the apheresis, although not in the produced CAR-Ts. Patients with primary refractory aggressive B-cell lymphomas had the poorest outcomes which was characterized by undetectable CAR-T cell expansion in vivo. No clear correlation of the outcome with the immunophenotypes of CAR-Ts was observed. Our results suggest that an important parameter predicting therapy efficacy is CAR-Ts' level of expansion in vivo but not the immunophenotype. After CAR-T cells' administration, measurements at several timepoints accurately detect their proliferation intensity in vivo. The outcome of CAR-T cell therapy largely depends on biological characteristics of the tumors rather than on the immunophenotype of produced CAR-Ts.

Abstract 6378: An in vitro human CD8 T cell exhaustion model for the functional screening of immune checkpoint inhibitors

Abstract T cell exhaustion is a state of dysfunction that frequently occurs during chronic infections and cancer due to persistent antigen stimulation and inflammation. It is defined by poor effector function, sustained expression of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. In this state, cells remain in the local environment but lack effector functions necessary to combat the tumor or infection. In the current study, we developed an in vitro human antigen-specific CD8 T cell exhaustion model to evaluate the ability of immune checkpoint inhibitors to restore functionality to exhausted T cells. Using the fully human, autologous MIMIC (Modular IMmune In vitro Construct) effector CD8 T cell assay as a framework, we assessed the effect of antigen dose, timing, and frequency of repeated antigen stimulation with antiviral HLA Class I-restricted peptides to drive the development of Ag-specific CD8 T cells toward an exhausted state. Epitope-specific CD8 T cell responses were monitored by HLA class I-restricted pentamers and establishment of an exhausted profile was evaluated by increased expression of inhibitory co-stimulatory receptors and decreased intracellular expression of antiviral effector cytokine via flow cytometry relative to functional Ag-specific CD8 T cells from the same donor. Repeated antigen exposure led to decreased production of IFN-γ and TNF-α and increased expression of inhibitory co-stimulation markers on antigen-specific CD8 T cells. After establishment of an exhausted phenotype, treatment with various immune checkpoint inhibitor molecules restored functionality to Ag-specific CD8 T cells upon an additional antigen re-stimulation. Moreover, here using this model we characterized distinct transcriptomic signatures of functional and exhausted MIMIC CD8 T cells. Development of an in vitro human viral antigen-specific CD8 T cell exhaustion model with characteristic features of dysfunctional CD8 T cells as observed in vivo and the ability to restore effector function by treatment with immune checkpoint inhibitors, provides a platform to assess the effectiveness of therapeutic mono-specific or bi-specific “checkpoint blockade” antibody candidates and, in general, the potential to evaluate a myriad of immunotherapeutic approaches for cancer treatment. Citation Format: Roberto Carrio, Margot Cucchetti, Mikielia Devonish, Louis Poisson, Mihaela Babiceanu, Andrew Kettring, Yingchun Liu, Donald Jackson, Evan Gomes, Jeremy Baudhuin, Donald R. Drake, Valeria Fantin, Anthony Byers, Ingrid Sassoon. An in vitro human CD8 T cell exhaustion model for the functional screening of immune checkpoint inhibitors [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 6378.

Abstract 5877: Intratumoral lymphocyte networks harbor TCF1+ PD1+ progenitor CD8 T cells in lung cancer

Abstract Cancer immunotherapies that activate cytotoxic T cell responses against tumors have been remarkably effective in some patients, but the determinants of response remain unclear. To investigate how spatial organization of T cells might influence immunotherapy response, we developed a tissue-based cyclic immunofluorescence (t-CyCIF) platform to characterize preclinical genetically engineered mouse models (GEMMs) of cancer. We optimized a 21-antibody panel delineating major myeloid and lymphoid cell subsets, that included key markers for characterizing T cell phenotypes and function. The panel was used to characterize the Kras/p53-mutant GEMM of lung adenocarcinoma, treated with anti-PD-1/anti-CTLA-4 immune checkpoint blockade (ICB) therapy or a neoantigen-targeted long-peptide vaccine. Spatial profiling by t-CyCIF identified intratumoral lymphocyte networks (“lymphonets”) as a key feature associated with a productive immune response against lung tumors. Lymphonets were predominantly composed of B cells and conventional CD4 T cells, but harbored tumor-reactive CD8 T cell populations. TCF1+ PD1+ progenitor CD8 T cells, a subset previously correlated with patient response to ICB, were almost exclusively localized to lymphonets inside tumors. Lymphonets of similar composition harboring TCF1+ PD1+ cells were also found in early-stage human lung adenocarcinomas. In response to ICB and vaccine therapies in mice, cytotoxic CD8 T cells (Granzyme B+/Perforin+) colocalized with TCF1+ PD1+ cells in lymphonets, likely having differentiated from this progenitor pool. Notably, vaccination further induced a highly proliferative (Ki67hi) and cytotoxic (GranzymeBhiPerforinhi) population of CD8 T cells localized outside of tumors that we posit enter tumors without passing through the intratumoral progenitor cell state. Upon becoming dysfunctional, marked by loss of Granzyme B/Perforin expression and upregulation of inhibitory receptors, CD8 T cells were excluded from lymphonets and localized predominantly to the tumor margin. Altogether, these results shed light on the spatial dynamics associated with a productive response to immunotherapies and open avenues for exploration into the function of lymphonets in supporting cytotoxic CD8 T cell responses in tumors. Citation Format: Megan L. Burger, Giorgio Gaglia, Claire C. Ritch, Danae Rammos, Yang Dai, Grace E. Crossland, Sara Z. Tavana, Simon Warchol, Alex M. Jaeger, Santiago Naranjo, Shannon Coy, Ajit J. Nirmal, Robert Krueger, Jia-Ren Lin, Hanspeter Pfister, Peter K. Sorger, Tyler Jacks, Sandro Santagata. Intratumoral lymphocyte networks harbor TCF1+ PD1+ progenitor CD8 T cells in lung cancer [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 5877.

Abstract 2909: Genetically engineered Natural Killer cells as an immunotherapeutic approach for the treatment of neuroblastoma

Abstract In this study we test if knockout of DNAM-1 competing inhibitory receptors on Natural Killer (NK) cells improves cytotoxic response against neuroblastoma cell lines. Neuroblastoma and other peripheral nervous cell tumors account for 6% of all pediatric cancer. While the 5-year survival rate has greatly improved, the prognosis of children with high-risk neuroblastoma at diagnosis is less than 50%. Immune checkpoint therapy is a promising new application for pediatric oncology; however, neuroblastoma is often immunologically “cold” and current immune checkpoint targets are not abundant in pediatric solid tumors. To improve response to checkpoint blockade and directly target tumor cells for elimination, this study evaluated the use of NK cells. NK cells prime the immune system for successful response to cancer immunotherapy. NK cells activated with particle technology (PM21-NK cells) are highly cytotoxic, produce IFNγ and induce PD-L1 on cancer cells. Although highly cytotoxic, PM21-NK cells express inhibitory receptors that compete for ligands that trigger NK cell lysis, such as PVR, a ligand highly expressed on neuroblastoma cells. We knocked out expression of multiple PVR inhibitory receptors in PM21-NK cells to further enhance cytotoxicity and prevent exhaustion. Three inhibitory receptors on NK cells that bind PVR - TIGIT, CD96, and PVRIG were knocked out alone or in combination and effector functions of these edited NK cells tested. Cytotoxicity against 3D spheroids of the neuroblastoma cell line SK-NA-S cells was assessed and compared to wild type PM21-NK cells. Knockout (KO) of TIGIT enhanced NK cell killing compared to wildtype NK cells (72%±10 vs 55%±3% killing) as well as PVIRG KO (84±3% vs 46±6% killing) and CD96 KO (62%±9% vs 46±6% killing) at a 1:3 E:T ratio at 72 h. Double or triple KO of these receptors resulted in further increased killing of cancer cells. The knockouts of each of the receptors could affect cytokine production and cytokine responsiveness and this will be examined. Overall, PM21-NK cells show promise for therapeutic use against neuroblastoma and preventing the PVR-mediated inhibitory axis signaling enhances their function. Citation Format: Tayler J. Croom-Perez, Jeremiah L. Oyer, Md Faqrul Hasan, Liza D. Robles-Carrillo, Brian P. Tullius, Alicja J. Copik. Genetically engineered Natural Killer cells as an immunotherapeutic approach for the treatment of neuroblastoma [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 2909.

Abstract 2283: An immunosuppressive ectoenzyme-expressing T cell population is associated with non-responsiveness to immune checkpoint blockade in metastatic melanoma patients

Abstract The development of antibodies to block immune checkpoints like PD-1, CTLA-4, PD-L1 or LAG-3 has been a major advance in cancer therapy. This has led to the approval of multiple antibodies for metastatic melanoma and other malignancies. A large proportion of the patients do not benefit from CTLA-4 or PD-1 antagonistic antibodies. Understanding the mechanisms of resistance to immunotherapy is thus critical to improve its effectiveness. Previously we identified and validated a novel population of cells characterized by phenotype CD3+CD4+CD127-GARP-CD38+CD39+ that was associated with non-responsiveness to immune checkpoint blockade (ICB) in metastatic melanoma patients (Woods et al, 2020). CD38 and CD39 are ectonucleotidases, that convert intracellular ATP or NADPH to extracellular adenosine which has suppressive effect on T cell functions. We used high-dimension flow cytometry and bulk RNA seq to characterize the phenotype of these cells and evaluated the functions by T cell suppression assays. All characterizations and functional experiments were performed on peripheral blood samples from metastatic melanoma patients undergoing anti-PD1 therapy. The characterization of this population by flow cytomtery shows an enhanced expression of activation markers (4-1BB, HLA-DR, OX-40, GITR), inhibitory receptors (Tim3, CTLA-4, PD-1) and ligands (PD-L1, CD244) and an exhausted phenotype (TOXhiTCF1low). Further functional studies performed on these cells showed a suppressive phenotype, as observed by a significant decrease in the proliferation of target autologous CD4 (p=0.001) and CD8 (p=0.0012) T cells when in co-culture with these cells, compared to the controls. The transcriptomic features of these cells showed an upregulation of genes associated with proliferation (e.g. CDC20, TK1, DHFR, MYL6B), as well as exhaustion (e.g. upregulated TOX), consistent with the flow cytometry data. We also evaluated the presence of these cells and their correlation with the outcome in other melanoma datasets. By performing gene set variation analysis (GSVA) on existing single-cell RNA seq datasets of tumor derived immune cells in melanoma, we have found that an intra-tumoral CD3+CD4+CD127-GARP-CD38+CD39+ signature is associated with resistance to immunotherapy (p= 0.000003). We identified a novel population of ectonucleotidase expressing T cells in peripheral blood and tumor to be associated with immunotherapy resistance in metastatic melanoma patients. This population showed an exhausted phenotype and suppressed autologous T-cells in vitro. Our data identifies CD3+CD4+CD127-GARP-CD38+CD39+ cells as a potential target to enhance the efficacy of immunotherapy in advanced melanoma patients. Citation Format: Ankita Mitra, Brian Thompson, David M. Woods, Jeffrey S. Weber. An immunosuppressive ectoenzyme-expressing T cell population is associated with non-responsiveness to immune checkpoint blockade in metastatic melanoma patients [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 2283.

Abstract 6396: BET inhibition alleviates T-cell dysfunction in chronic lymphocytic leukemia

Abstract Background: The chronic lymphocytic leukemia (CLL) tumor microenvironment (TME) is laden with hyporesponsive T-cells that permit disease persistence. Yet, redundant TME immunosuppressive mechanisms and epigenetic maintenance of T-cell exhaustion limit the efficacy of T-cell targeted therapies in CLL. Bromodomain and extra-terminal (BET) proteins regulate key pathways contributing to CLL pathogenesis and TME interactions, including T-cell function and differentiation. We hypothesize that blocking BET protein function can reverse T-cell exhaustion to yield durable tumor elimination in CLL. Methods: WT C57BL/6 mice were engrafted with Eμ-TCL1 spleen-derived lymphocytes, then treated daily with the novel pan-BET inhibitor, OPN-51107 (OPN5; 20mg/kg PO) for up to 4 weeks. Splenic gene expression was evaluated with the NanoString PanCancer iO360 panel. T-cell differentiation status, immune inhibitory receptor (IR) expression, proliferation (72 h ex vivo α-CD3/α-CD28 stimulation), and cytokine production (6 h ex vivo PMA/ionomycin stimulation) was measured via flow cytometry. CLL patient and healthy donor PBMCs were used for validation studies and to assess T-cell transcription factor (TF) expression via flow cytometry. Evaluation of BRD4 occupancy at select T-cell TFs via ChIP qPCR is ongoing. Results: OPN5 significantly increased cytotoxic cell signatures and reduced exhaustion-associated cell signatures in leukemic mice through inhibition of T-cell exhaustion signaling, as well as activation of Th1, natural killer cell, and IL-7 signaling pathways. Correspondingly, T-cells from OPN5-treated mice demonstrated greater ex vivo proliferative capacity and effector response to stimuli. A greater proportion of CD8+ T-cells from OPN5-treated mice were classified as naïve, and OPN5 significantly reduced KLRG1 expression on antigen-experienced CD8+ T-cells. Importantly, OPN5 curtailed IR co-expression (PD-1, PD-L1, VISTA, CD244, CD160, and LAG3) on splenic T-cells. These findings were confirmed with primary CLL cells ex vivo. OPN5 also impaired expression of terminal differentiation-associated TFs in CLL patient-derived T-cells, enriching for a TCF1+ progenitor T-cell population. While BTK inhibitors are known to similarly improve T-cell function in CLL, ibrutinib treatment was inadequate to revert CLL T-cell terminal differentiation. Future ATAC-sequencing analysis will inform how BET inhibition alleviates exhaustion-associated chromatin organization in CLL T-cells. Conclusion: BET inhibition dismantles immunosuppressive mechanisms in the CLL TME, alleviating CLL-induced T-cell dysfunction and terminal differentiation. These findings suggest that BET inhibition may be a useful component of combination strategies for the treatment of CLL to yield lasting anti-cancer immune memory and prevent relapsed/refractory disease. Citation Format: Audrey L. Smith, Alexandria P. Eiken, Sydney A. Skupa, Christopher R. D'Angelo, Avyakta Kallam, Matthew A. Lunning, Gregory Bociek, Julie M. Vose, Ben Powell, Gideon Bollag, Dalia El-Gamal. BET inhibition alleviates T-cell dysfunction in chronic lymphocytic leukemia [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 6396.

Abstract 1152: NKTR-255, a polymer-conjugated IL-15, dramatically improves ROR1 CAR-T cell persistence and anti-tumor efficacy in an autochthonous model of ROR1+ lung cancer

Abstract CAR-T cells have mediated remarkable clinical responses against hematological malignancies, but this success has yet to extend to solid tumors, where many factors in the tumor microenvironment (TME) inhibit their persistence and function. In a phase 1 trial at our Center, CAR-T cells targeting the tumor-associated antigen ROR1 in patients with non-small cell lung cancer (NSCLC) and triple negative breast cancer rapidly became dysfunctional and persisted poorly at tumor sites, ultimately failing to mediate objective responses. To evaluate strategies to improve ROR1 CAR-T efficacy, we adapted the KrasLSL-G12D/+;p53fl/fl (KP) autochthonous model of lung adenocarcinoma to express ROR1 (KPROR1). This aggressive model mimics the initiation, progression, and suppressive TME of human NSCLC. ROR1 CAR-T cells in KPROR1 mice rapidly declined in number, lost the ability to produce pro-inflammatory cytokines, and failed to significantly enhance tumor control, recapitulating the major barriers to CAR-T efficacy we observed in patients. NKTR-255 is a novel immunotherapeutic consisting of a polymer conjugated recombinant human interleukin-15 (rhIL-15) that could enhance the activity of CAR-T cells. NKTR-255 binds to the IL-15 receptor and IL-2/IL-15 receptor, thus maintaining the full potential spectrum of IL-15 biology while exerting a sustained effect on CD8+ memory T cell and NK cell expansion and persistence. We hypothesized that the combination of CAR-T cells and NKTR-255 would be feasible and safe and may extend the duration of efficacy of ROR1 CAR-T cells in ROR1+ lung tumors by improving their expansion and persistence. To test this, we treated tumor-bearing KPROR1 mice with control or ROR1 CAR-T cells with or without weekly injections of NKTR-255. NKTR-255 treatment resulted in a robust expansion of ROR1 CAR-T cells in the peripheral blood relative to vehicle-treated mice, which correlated with increased CAR-T cell infiltration into lung tumors. CAR-T cells in NKTR-255-treated mice also showed reduced signs of T cell exhaustion, including reduced expression of inhibitory receptors and enhanced ability to produce effector cytokines upon ex vivo re-stimulation. Importantly, combination therapy with both NKTR-255 and ROR1 CAR-T cells resulted in significantly improved tumor control relative to either treatment alone. Together, our work shows that NKTR-255 dramatically improves the persistence, function, and anti-tumor activity of ROR1 CAR-T cells in an aggressive autochthonous model of ROR1+ lung cancer. Our findings provide rationale to combine NKTR-255 with CAR-T cell therapy as a strategy to enhance the anti-tumor efficacy of CAR-T cells in patients with solid tumors. Citation Format: W. Sam Nutt, Takahiro Miyazaki, Mario Marcondes, Stanley R. Riddell, Shivani Srivastava. NKTR-255, a polymer-conjugated IL-15, dramatically improves ROR1 CAR-T cell persistence and anti-tumor efficacy in an autochthonous model of ROR1+ lung cancer [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 1152.

Abstract 3281: GEN1042 (DuoBody-CD40x4-1BB)®in combination with PD-1 blockade reverses T-cell exhaustion in vitro

Abstract GEN1042 (DuoBody®-CD40x4-1BB) is an investigational, novel, agonistic, bispecific antibody that combines targeting and conditional activation of CD40 and 4-1BB on immune cells for priming and (re-) activation of tumor-specific immune responses. We previously showed that GEN1042 induces conditional CD40 and 4-1BB agonist activity, leading to dendritic-cell maturation and enhanced T-cell activation and effector functions in vitro. However, dysfunction of exhausted T cells in the solid tumor microenvironment may represent a potential resistance mechanism to checkpoint inhibitors/agonists. Here we utilized a multi-omics approach to evaluate whether GEN1042 could reverse T-cell exhaustion in vitro. Publicly available single cell RNA sequencing datasets were harmonized across multiple solid-tumor indications (including treatment-naïve and anti-PD-1 and/or anti-CTLA-4 pretreated samples) and analyzed for expression of CD40, 4-1BB and PD-1 on various immune-cell subsets based on their transcriptomic signatures to characterize target prevalence. In vitro mixed lymphocyte reaction (MLR) functional assays were performed where unstimulated healthy donor CD3+ T cells or CD3+ T cells exhausted by repeated stimulation with anti-CD3/CD28 beads were co-cultured with allogeneic lipopolysaccharide (LPS)-matured dendritic cells. Cultures were analyzed for cytokine secretion and expression of cell surface proteins. In the unstimulated T cell/mDC MLR assay, concurrent treatment with GEN1042 and PD-1 blockade potentiated IFNγ, TNFα and IL-2 production compared with either compound alone. In the T-cell exhaustion MLR assay, T cells showed increased expression of inhibitory receptors (e.g., TIM-3, LAG-3) and exhibited hyporesponsiveness for both proliferation and cytokine secretion upon restimulation with anti-CD3/CD28 beads. Here, the combined functional effect of GEN1042 plus PD-1 blockade further potentiated IFNγ secretion compared to single agent activity and induced IL-2 at similar levels to that observed for GEN1042 alone. These data suggest that, in addition to enhancing T-cell effector functions during T cell priming, GEN1042 can amplify the magnitude of the immune response in combination with PD-1 blockade by re-establishing the functional activity of dysfunctional T cells. GEN1042 is currently being evaluated in patients with advanced solid tumors in a phase 1/2 clinical trial (NCT04083599). Citation Format: Vanessa M. Spires, Gregg Masters, Christina Yu, Matt Hancock, Monique N. Luijten, Alexander Muik, Kristina Nuermberger, Friederike Gieseke, Tahamtan Ahmadi, Özlem Türeci, Maria Jure-Kunkel, Ugur Sahin, Mark Fereshteh, Homer C. Adams, Jordan M. Blum. GEN1042 (DuoBody-CD40x4-1BB)®in combination with PD-1 blockade reverses T-cell exhaustion in vitro [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 3281.

Abstract PL04-05: LAG3: The third checkpoint inhibitor

Abstract Immunotherapies targeting the PD1/PDL1 pathway have had a major impact on cancer treatment. However, only a proportion of patients respond, and an even smaller proportion exhibit a long-term, durable cure. Several mechanisms of resistance and potential combinatorial approaches will be discussed. Lack of response to inhibitory receptor (IR) blockade therapy and increased disease burden has been associated with circulating, peripheral CD8+ T cell exhaustion, which is defined by poor T cell function linked to increased IR expression (eg: PD1, LAG3, neuropilin-1 [NRP1]). LAG3 is the third IR to be targeted in the clinic, consequently garnering considerable interest and scrutiny. However, persistent antigen exposure in the tumor microenvironment results in sustained PD1/LAG3 expression, contributing to a state of exhaustion manifest in impaired proliferation and cytokine production. This presentation will include a brief introduction LAG3, discussion of the signaling mechanism used by LAG3, and assessment of new insight in to the function of LAG3, alone and in combination with PD1, from mouse models of cancer and analysis of samples from patients treated with LAG3/PD1 therapeutics. Citation Format: Dario A. Vignali. LAG3: The third checkpoint inhibitor. [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 PL04-05.

Establishment and validation of exhausted CD8+ T cell feature as a prognostic model of HCC.

The exhausted CD8+T (Tex) cells are a unique cell population of activated T cells that emerges in response to persistent viral infection or tumor antigens. Tex cells showed the characteristics of aging cells, including weakened self-renewal ability, effector function inhibition, sustained high expression of inhibitory receptors including PD-1, TIGIT, TIM-3, and LAG-3, and always accompanied by metabolic and epigenetic reprogramming. Tex cells are getting more and more attention in researching immune-related diseases and tumor immunotherapy. However, studies on Tex-related models for tumor prognosis are still lacking. We hope to establish a risk model based on Tex-related genes for HCC prognosis. Tex-related GEO datasets from different pathologic factors (chronic HBV, chronic HCV, and telomere shortening) were analyzed respectively to acquire differentially expressed genes (DEGs) by the 'limma' package of R. Genes with at least one intersection were incorporated into Tex-related gene set. GO, KEGG, and GSEA enrichment analyses were produced. Hub genes and the PPI network were established and visualized by the STRING website and Cytoscape software. Transcription factors and targeting small molecules were predicted by the TRUST and CLUE websites. The Tex-related HCC prognostic model was built by Cox regression and verified based on different datasets. Tumor immune dysfunction and exclusion (TIDE) and SubMap algorithms tested immunotherapy sensitivity. Finally, qRT-PCR and Flow Cytometry was used to confirm the bioinformatic results. Hub genes such as AKT1, CDC6, TNF and their upstream transcription factor ILF3, Regulatory factor X-associated protein, STAT3, JUN, and RELA/NFKB1 were identified as potential motivators for Tex. Tex-related genes SLC16A11, CACYBP, HSF2, and ATG10 built the HCC prognostic model and helped with Immunotherapy sensitivity prediction. Our study demonstrated that Tex-related genes might provide accurate prediction for HCC patients in clinical decision-making, prognostic assessment, and immunotherapy. In addition, targeting the hub genes or transcription factors may help to reverse T cell function and enhance the effect of tumor immunotherapy.

Peripheral immune characteristics of hepatitis B virus-related hepatocellular carcinoma.

Liver cancer is the sixth most common cancer worldwide and the third leading cause of cancer-related death. As a chronic liver disease, many studies have shown that the immune response plays a key role in the progression of liver cancer. Chronic hepatitis B virus (HBV) infection is one of the high-risk factors for HCC, accounting for 50%-80% of HCC cases worldwide, and little is known about the immune status of HBV associated hepatocellular carcinoma (HBV-HCC), therefore, we aimed to explore the changes in peripheral immunity in patients with HBV-HCC. In this study, patients with HBV-HCC (n=26), patients with hepatitis B-related cirrhosis (HBV-LC) (n=31) and healthy volunteers (n=49) were included. The lymphocytes and their subpopulation phenotypes in peripheral blood were characterized. In addition, we explored the effect of viral replication on peripheral immunity in patients with HCC and analyzed the circulating immunophenotypic characteristics at different stages of HCC with flow cytometry. Firstly, our results showed that the percentages of total αβ T cells in the peripheral blood of HBV-HCC patients was significantly decreased compared to healthy subjects. Secondly, we found that naïve CD4+ T cells in HBV-HCC patients were significantly reduced, terminally differentiated CD8+ T cells, homing memory CD8+ T cells and Th2 cells were increased in peripheral circulation in HBV-HCC patients. Moreover, in the peripheral blood of HBV-HCC patients, expression of TIGIT on CD4+ T cells and PD-1 on the surface of Vδ 1 T cells was increased. In addition, we found that sustained viral replication resulted in up-regulation of TIM3 expression on CD4+ T cells, and TIM3+ γδ T cells increased in peripheral circulation in patients with advanced HBV-HCC. Our study showed that circulating lymphocytes in HBV-HCC patients exhibited features of immune exhaustion, especially in HCC patients with persistent viral replication and in patients with intermediate and advanced HBV-HCC, including decreased frequency of T cells and elevated expression of inhibitory receptors including TIGIT and TIM3 on CD4+ T cells and γδ T cells. Meanwhile, our research suggests that the combination of CD3+ T cell and CD8+HLADR+CD38+ T cell may be a potential diagnostic indicator for HBV-HCC. These findings could help us to better understand the immune characteristics of HBV-HCC and explore the immune mechanisms and immunotherapy strategies for HBV-HCC.

MP04-18 LEVERAGING PATIENT-DERIVED RENAL CELL CARCINOMA ORGANOIDS TO ENRICH TUMOR-REACTIVE T CELLS AND IDENTIFY NOVEL IMMUNE CHECKPOINTS

You have accessJournal of UrologyCME1 Apr 2023MP04-18 LEVERAGING PATIENT-DERIVED RENAL CELL CARCINOMA ORGANOIDS TO ENRICH TUMOR-REACTIVE T CELLS AND IDENTIFY NOVEL IMMUNE CHECKPOINTS Elliot Merritt, Julie-Ann Cavallo, John Sfakianos, Amir Horowitz, Laura Zuluaga, Jordan Rich, Indu Saini, Kennedy E. Okhawere, Burak Upcinar, Ketan Badani, Benjamin Hopkins, and Anna Tocheva Elliot MerrittElliot Merritt More articles by this author , Julie-Ann CavalloJulie-Ann Cavallo More articles by this author , John SfakianosJohn Sfakianos More articles by this author , Amir HorowitzAmir Horowitz More articles by this author , Laura ZuluagaLaura Zuluaga More articles by this author , Jordan RichJordan Rich More articles by this author , Indu SainiIndu Saini More articles by this author , Kennedy E. OkhawereKennedy E. Okhawere More articles by this author , Burak UpcinarBurak Upcinar More articles by this author , Ketan BadaniKetan Badani More articles by this author , Benjamin HopkinsBenjamin Hopkins More articles by this author , and Anna TochevaAnna Tocheva More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.18AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Despite that renal cell carcinomas (RCC) are heavily infiltrated by intratumoral T cells, >70% of RCC patients fail to respond to immune checkpoint inhibitors (ICIs) immunotherapy. Our knowledge of the cellular and molecular mechanism underlying ICI efficacy in RCC is limited. Tumor cell-intrinsic IFNg responsivity has vital yet opposing functions in ICI efficacy by inducing the expression of antigen presenting molecules and T cell recruiting chemokine ligands from tumor cells, while in parallel increasing the expression of therapeutically relevant immune checkpoint ligands (IC-L) such as PD-L1. Major obstacles to improving ICI efficacy in RCC are the lack of (1) knowledge as it pertains to tumor-intrinsic IFNg responsivity; and (2) human models that recapitulate tumor cell heterogeneity and ICI responses. METHODS: We leverage patient-derived organoids (PDO) to determine IFNg-responsivity by analyzing the expression of antigen presenting molecule and IC-Ls. For each PDO, we generated blood and tumor derived PDO-reactive T cell lines that were phenotyped to determine T cell subset composition and inhibitory receptor expression. We performed PDO/T cell co-culture assays (PDOTs) and measured intracellular cytokine production to determine the increase in anti-tumor T cell reactivity in response to PD1 ICI and in combination with PDO relevant IC-Ls. RESULTS: We identified patient-specific intratumoral heterogeneity in IFNg responsivity to reveal IFNg-dependent and independent IC-L expression and soluble mediator secretion. Paired PDO IC-L expression and TIL checkpoint receptor expression data were used to identify ICI strategies in patient-specific manner. PDOTs were used to identify patient-specific therapeutically relevant IC-Ls, which in combination with PD-1 ICI improved anti-tumor T cell responses in autologous PDOT co-cultures. Functional immunophenotyping showed that in addition to CD8 T cells, intratumoral double negative T cells play a key role in ICI responses in RCC. CONCLUSIONS: PDO model intra-tumoral and inter-patient heterogeneity in the tumor-immune phenome and IFNg responsivity. Our paired autologous PDOT biobank enables patient-specific identification of targetable immune checkpoints and PDOT co-cultures enable characterization of patient-specific anti-tumor response to rationally design ICI combination therapies that augment anti-tumor T cell responses in RCC. Source of Funding: none © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e40 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Elliot Merritt More articles by this author Julie-Ann Cavallo More articles by this author John Sfakianos More articles by this author Amir Horowitz More articles by this author Laura Zuluaga More articles by this author Jordan Rich More articles by this author Indu Saini More articles by this author Kennedy E. Okhawere More articles by this author Burak Upcinar More articles by this author Ketan Badani More articles by this author Benjamin Hopkins More articles by this author Anna Tocheva More articles by this author Expand All Advertisement PDF downloadLoading ...

Regulatory Mechanisms and Reversal of CD8+T Cell Exhaustion: A Literature Review.

CD8+T cell exhaustion is a state of T cell dysfunction during chronic infection and tumor progression. Exhausted CD8+T cells are characterized by low effector function, high expression of inhibitory receptors, unique metabolic patterns, and altered transcriptional profiles. Recently, advances in understanding and interfering with the regulatory mechanisms associated with T cell exhaustion in tumor immunotherapy have brought greater attention to the field. Therefore, we emphasize the typical features and related mechanisms of CD8+T cell exhaustion and particularly the potential for its reversal, which has clinical implications for immunotherapy.

IL-12 and IL-27 Promote CD39 Expression on CD8+ T Cells and Differentially Regulate the CD39+CD8+ T Cell Phenotype.

Tumor-specific CD8+ T cells are critical components of antitumor immunity; however, factors that modulate their phenotype and function have not been completely elucidated. Cytokines IL-12 and IL-27 have recognized roles in promoting CD8+ T cells' effector function and mediated antitumor responses. Tumor-specific CD8+ tumor-infiltrating lymphocytes (TILs) can be identified based on surface expression of CD39, whereas bystander CD8+ TILs do not express this enzyme. It is currently unclear how and why tumor-specific CD8+ T cells uniquely express CD39. Given the important roles of IL-12 and IL-27 in promoting CD8+ T cell functionality, we investigated whether these cytokines could modulate CD39 expression on these cells. Using invitro stimulation assays, we identified that murine splenic CD8+ T cells differentially upregulate CD39 in the presence of IL-12 and IL-27. Subsequently, we assessed the exhaustion profile of IL-12- and IL-27-induced CD39+CD8+ T cells. Despite the greatest frequency of exhausted CD39+CD8+ T cells after activation with IL-12, as demonstrated by the coexpression of TIM-3+PD-1+LAG-3+ and reduced degranulation capacity, these cells retained the ability to produce IFN-γ. IL-27-induced CD39+CD8+ T cells expressed PD-1 but did not exhibit a terminally exhausted phenotype. IL-27 was able to attenuate IL-12-mediated inhibitory receptor expression on CD39+CD8+ T cells but did not rescue degranulation ability. Using an immunogenic neuro-2a mouse model, inhibiting IL-12 activity reduced CD39+CD8+ TIL frequency compared with controls without changing the overall CD8+ TIL frequency. These results provide insight into immune regulators of CD39 expression on CD8+ T cells and further highlight the differential impact of CD39-inducing factors on the phenotype and effector functions of CD8+ T cells.

Fingolimod Administration Following Hypoxia Induced Neonatal Seizure Can Restore Impaired Long-term Potentiation and Memory Performance in Adult Rats

Neonatal seizures commonly caused by hypoxia can lead to long-term neurological outcomes. Early inflammation plays an important role in the pathology of these outcomes. Therefore, in the current study, we explored the long-term effects of Fingolimod (FTY720), an analog of sphingosine and potent sphingosine 1-phosphate (S1P) receptors modulator, as an anti-inflammatory and neuroprotective agent in attenuating anxiety, memory impairment, and possible alterations in gene expression of hippocampal inhibitory and excitatory receptors following hypoxia-induced neonatal seizure (HINS). Seizure was induced in 24 male and female pups (6 in each experimental group) at postnatal day 10 (P10) by premixed gas (5% oxygen/ 95% nitrogen) in a hypoxic chamber for 15 minutes. Sixty minutes after the onset of hypoxia, FTY720 (0.3 mg/kg) or saline (100 µl) was administered for 12 days (from P10 up to P21). Anxiety-like behavior and hippocampal memory function were assessed at P90 by elevated plus maze (EPM) and novel object recognition (NOR), respectively. Long-term potentiation (LTP) was recorded from hippocampal dentate gyrus region (DG) following stimulation of perforant pathway (PP). In addition, the hippocampal concentration of superoxide dismutase activity (SOD), malondialdehyde (MDA), and thiol as indices of oxidative stress were evaluated. Finally, the gene expression of NR2A subunit of N-Methyl-D-aspartic acid (NMDA) receptor, GluR2 subunit of (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) AMPA receptor and γ2 subunit of γ-Aminobutyric acid (GABAA) receptor were assessed at P90 by the quantitative real-time PCR. FTY720 significantly reduced later-life anxiety-like behavior, ameliorated object recognition memory and increased the amplitude and slope of the field excitatory postsynaptic potential (fEPSP) in the rats following HINS. These effects were associated with restoration of the hippocampal thiol content to the normal values and the regulatory role of FTY720 in the expression of hippocampal GABA and glutamate receptors subunits. In conclusion, FTY720 could restore the dysregulated gene expression of excitatory and inhibitory receptors. It also increased the reduced hippocampal thiol content, which was accompanied with attenuation of HINS-induced anxiety, reduced the impaired hippocampal related memory, and prevented hippocampal LTP deficits in later life following HINS.

Tonic-signaling chimeric antigen receptors drive human regulatory T cell exhaustion

Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor invivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. To "benchmark" exhaustion in human Tregs, we used a method known to induce exhaustion in conventional T cells: expression of a tonic-signaling chimeric antigen receptor (TS-CAR). We found that TS-CAR-expressing Tregs rapidly acquired a phenotype that resembled exhaustion and had major changes in their transcriptome, metabolism, and epigenome. Similar to conventional T cells, TS-CAR Tregs upregulated expression of inhibitory receptors and transcription factors such as PD-1, TIM3, TOX and BLIMP1, and displayed a global increase in chromatin accessibility-enriched AP-1 family transcription factor binding sites. However, they also displayed Treg-specific changes such as high expression of 4-1BB, LAP, and GARP. DNA methylation analysis and comparison to a CD8+ T cell-based multipotency index showed that Tregs naturally exist in a relatively differentiated state, with further TS-CAR-induced changes. Functionally, TS-CAR Tregs remained stable and suppressive invitro but were nonfunctional invivo, as tested in a model of xenogeneic graft-versus-host disease. These data are the first comprehensive investigation of exhaustion in Tregs and reveal key similarities and differences with exhausted conventional T cells. The finding that human Tregs are susceptible to chronic stimulation-driven dysfunction has important implications for the design of CAR Treg adoptive immunotherapy strategies.

Increased co-expression of TIM-3 with TIGIT or 2B4 on CD8+ T cells is associated with poor prognosis in locally advanced nasopharyngeal carcinoma.

The use of immune checkpoint inhibitors in malignant tumors improves patient outcomes. Because single-agent immune checkpoint blockade has a low objective response rate, it is meaningful to explore combined blockade of immune checkpoint receptors. We aimed to investigate the co-expression of TIM-3 with TIGIT or 2B4 on peripheral blood CD8+ T cells from patients with locally advanced nasopharyngeal carcinoma. The correlation between co-expression level and clinical characteristics and prognosis was studied to provide a basis for immunotherapy for nasopharyngeal carcinoma. Flow cytometry was used to detect TIM-3/TIGIT and TIM-3/2B4 co-expression on CD8+ T cells. The differences in co-expression between patients and healthy controls were analyzed. The correlation between co-expression of TIM-3/TIGIT or TIM-3/2B4 and the patient clinical characteristics and prognosis was examined. Also, the correlation between the TIM-3/TIGIT or 2B4 co-expression and other common inhibitory receptors was analyzed. We further validated our results using mRNA data from the Gene Expression Omnibus (GEO) database. TIM-3/TIGIT and TIM-3/2B4 co-expression was upregulated on peripheral blood CD8+ T cells from patients with nasopharyngeal carcinoma. They were both correlated with poor prognosis. There was a correlation between TIM-3/TIGIT co-expression and patient age and pathological stage, whereas TIM-3/2B4 co-expression correlated with age and sex. CD8+ T cells with elevated mRNA levels of TIM3/TIGIT and TIM3/2B4 also showed increased expression of multiple inhibitory receptors, indicating T cell exhaustion in locally advanced nasopharyngeal carcinoma. TIM-3/TIGIT or TIM-3/2B4 can be used as potential targets for combination immunotherapy in locally advanced nasopharyngeal carcinoma.

Bispecific GPC3/PD‑1 CAR‑T cells for the treatment of HCC.

Constantly stimulated by the tumor microenvironment (TME), programmed death1 (PD‑1) is elevated, and it interacts with PD ligand1 (PD‑L1), rendering chimeric antigen receptor (CAR)‑Tcells dysfunctional. Hence, CAR‑Tcells immune to PD‑1‑induced immunosuppression were constructed to improve the function of CAR‑Tcells in hepatocellular carcinoma (HCC). Double‑target CAR‑Tcells, targeting glypican‑3 (GPC3) [a tumour-associated antigen (TAA)] and hindering PD‑1‑PD‑L1 binding, were established. The expression of GPC3, PD‑L1, and inhibitory receptors was measured using flow cytometry. The cytotoxicity, cytokine release, and differentiation level of CAR‑Tcells were determined using lactate dehydrogenase release assay, enzyme‑linked immunosorbent assay, and flow cytometry, respectively. HCC cells were targeted and eliminated by double‑target CAR‑Tcells. These double‑target CAR‑Tcells limit PD‑1‑PD‑L1 binding and sustain cytotoxicity to PD‑L1+ HCC cells. The relatively low IR expression and differentiation level in double‑target CAR‑Tcells in tumour tissues induced tumour‑suppression and extended survival in PD‑L1+ HCC TX models, as opposed to their single‑target counterparts. The results of the present study suggested that the newly constructed double‑target CAR‑T cells exhibit stronger tumour‑suppressing effects in HCC than their single‑target counterparts, which are common, suggesting the potential of strengthening CAR‑T cell activity in HCC treatment.

Combined flow cytometry natural killer immunophenotyping and KIR/HLA-C genotyping reveal remarkable differences in acute myeloid leukemia patients, but suggest an overall impairment of the natural killer response.

Natural killer (NK) cells are key anti-tumor effectors of the innate immunity. Phenotypic differences allow us to discriminate in between three functional stages of maturation, named immature, mature and hypermature that are distinctive in terms of receptor expression, cytokine secretion, cytotoxic properties and organ trafficking. NKs display an impressive repertoire of highly polymorphic germline encoded receptors that can be either activating, triggering the effector's function, or inhibitory, limiting the immune response. In our study, we have investigated peripheral blood NK cells of acute myeloid leukemia (AML) patients. The Killer Immunoglobulin-like receptors (KIRs) and the HLA-C genotypes were assessed, as HLA-C molecules are cognate antigens for inhibitory KIRs. The AA mainly inhibitory KIR haplotype was found in a higher proportion in AML, while a striking low frequency of the 2DS3 characterized the mainly activating Bx haplotype. Flow cytometry immunophenotyping evidenced a lower overall count of NK cells in AML versus healthy controls, with lower percentages of the immature and mature subpopulations, but with a markedly increase of the hypermature NKs. The analysis of the KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL1, and NKG2A inhibitory receptors surface expression revealed a remarkable heterogeneity. However, an overall trend for a higher expression in AML patients could be noticed in all maturation subpopulations. Some of the AML patients with complex karyotypes or displaying a FLT3 gene mutation proved to be extreme outliers in terms of NK cells percentages or inhibitory receptors expression. We conclude that while the genetic background investigation in AML offers important pieces of information regarding susceptibility to disease or prognosis, it is flow cytometry that is able to offer details of finesse in terms of NK numbers and phenotypes, necessary for an adequate individual evaluation of these patients.