Gicobi, Joanina K.

Abstract Although cancer burden in patients with advanced diseases results in T cell exhaustion and failed prior therapies, some of them achieved durable clinical responses to salvage therapy (chemotherapy or radiation therapy) implying a presence of tumor-reactive resilient T cells in them. However, the phenotype and functional features of resilient T cells have not been clearly defined. We found that CX3CR1+ CD8+ T cells with low mitochondrial membrane potential demonstrated features of resilient T cells: (a) their cytotoxic T cell function (degranulation/CD107a expression) recovered quickly after tumor burden has been reduced by radiation therapy or chemotherapy; (b) they maintain a low profile of glycolysis but produce optimal levels of ATP; (c) they express lower levels of PD-1 and TOX; (4) they share a feature of stem-like T cells (TCF1+PD-1+) that can be responsive to immunotherapy; (5) they avoid accumulation of a harmful level of cytosolic reactive oxygen species (ROS) that may cause T cell exhaustion; (6) they upregulate malic enzyme 1 (ME1) to balance glycolysis and ROS, and to expand cytotoxic effector T cells. These unique features of resilient T cells may not only be used as T cell biomarker to design rational combination therapy to control advanced cancers but can also be used to convey a resilient functional state to T cells in patients who lack them. Citation Format: Joanina K. Gicobi, Yiyi Yan, Hu Zeng, Sean Park, Haidong Dong. Characterization of cytotoxic resilient T cells in patients with advanced cancers in response to salvage radiation therapy or chemotherapy. [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 4160.

Abstract The immune responses to anti-PD-1 therapy in cancer patients are not completely defined due to lack of reliable biomarkers that identifies responsive and functional T cells. Bim, a molecule of the Bcl-2 family, was recently identified as a downstream signaling molecule of PD-1 pathway in CD8+ T cells and has both pro-apoptotic and effective roles in CD8 T cells. Our clinical data shown that high level of Bim in CD8+ T cells (before checkpoint blockade) was predictive of response in metastatic melanoma whereas in colorectal cancer, low level of Bim was associated with treatment response. Using co-immunoprecipitation, high resolution microscopy and FRET we identified ANT2 (Adenine Nucleotide Translocase 2, an inner membrane protein of mitochondria) as a protein interacting with Bim. Accordingly, we found CD8+ T cells with low and high mitochondrial potential demonstrated different ANT2/Bim ratio, and high ANT2/Bim ratio fall in effector T cells with low mitochondrial potential but high cytotoxic activity. Given the pro-survival of ANT2, our results indicate that ANT2 may function to balance the pro-apoptotic and effective role of Bim in mitochondria in order to preserve effector T cells before contraction. Understanding the molecular mechanism of Bim and ANT2 interaction in CD8+ T cells may provide a novel marker to identify resilient CD8+ T cells responsive to anti-PD-1 therapy, and predict clinical response to the therapy.

Abstract Seven different anti–PD-1 and PD-L1 mAbs are now widely used in the United States to treat a variety of cancer types, but no clinical trials have compared them directly. Furthermore, because many of these Abs do not cross-react between mouse and human proteins, no preclinical models exist in which to consider these types of questions. Thus, we produced humanized PD-1 and PD-L1 mice in which the extracellular domains of both mouse PD-1 and PD-L1 were replaced with the corresponding human sequences. Using this new model, we sought to compare the strength of the immune response generated by Food and Drug Administration–approved Abs. To do this, we performed an in vivo T cell priming assay in which anti–PD-1/L1 therapies were given at the time of T cell priming against surrogate tumor Ag (OVA), followed by subsequent B16-OVA tumor challenge. Surprisingly, both control and Ab-treated mice formed an equally robust OVA-specific T cell response at the time of priming. Despite this, anti–PD-1/L1-treated mice exhibited significantly better tumor rejection versus controls, with avelumab generating the best protection. To determine what could be mediating this, we identified the increased production of CX3CR1+PD-1+CD8+ cytotoxic T cells in the avelumab-treated mice, the same phenotype of effector T cells known to increase in clinical responders to PD-1/L1 therapy. Thus, our model permits the direct comparison of Food and Drug Administration–approved anti–PD-1/L1 mAbs and further correlates successful tumor rejection with the level of CX3CR1+PD-1+CD8 + T cells, making this model a critical tool for optimizing and better utilizing anti–PD-1/L1 therapeutics.

Abstract The immune response to anti-PD-1 therapy in cancer patients is not completely defined due to lack of reliable biomarkers that can determine T cell function. Bim, a member of the Bcl-2 family, was recently identified as a downstream signaling molecule in the PD-1 pathway. Within CD8 T cells, Bim appears to have a dual function, and can be pro-apoptotic or promote effector activity. Our clinical data has shown that high levels of Bim in CD8 T cells are predictive of response to anti-PD-1 therapy in metastatic melanoma whereas in colorectal cancer, a low level of Bim is associated with treatment response. Thus, we sought to determine what molecular partners might influence the role of Bim. Using co-immunoprecipitation, high resolution microscopy and FRET we identified ANT2 (Adenine Nucleotide Translocase 2), an inner membrane protein of mitochondria, as a protein that interacts with Bim. Accordingly, we found CD8 T cells with low and high mitochondrial potential demonstrated different ANT2/Bim ratios, and a low ANT2/Bim ratio was present in effector T cells with low mitochondrial potential but high cytotoxic activity. Given the pro-survival function of ANT2, our results indicate that ANT2 may balance the pro-apoptotic and effector roles of Bim in mitochondria in order to preserve effector T cells before contraction. Understanding the molecular mechanism of Bim and ANT2 interactions in CD8 T cells may provide a novel marker to predict clinical response to anti-PD-1 therapy.

Abstract Seven different anti-PD-1 and PD-L1 monoclonal antibodies are now widely used in the US to treat a variety of cancer types, but there have been no clinical trials comparing them directly. Furthermore, because many of these antibodies do not cross react between mouse and human proteins, no preclinical models exist in which to consider these types of questions. Thus, we produced humanized PD-1 and PD-L1 mice in which the extracellular domains of both mouse PD-1 and PD-L1 were replaced with the corresponding human sequences. Using this new model, we sought to compare the strength of the immune response generated by FDA-approved antibodies. To do this, we performed an in vivo T cell priming assay in which anti-PD-1/L1 therapies were given at the time of T cell priming against surrogate tumor antigen (OVA), followed by subsequent B16-OVA tumor challenge. We found that anti-PD-1/L1-treated mice exhibited significantly better tumor rejection than controls, although both the control and antibody-treated mice generated comparable numbers of OVA-specific T cells at the time of priming. To determine what could mediate this strong antitumor immune response, we identified the increased production of CX3CR1+PD-1+CD8+ cytotoxic T cells in the anti-PD-1/L1-treated mice, the same subset of effector T cells known to increase in clinical responders to PD-1/L1 therapy. Thus, our model permits the direct comparison of FDA-approved anti-PD-1/L1 monoclonal antibodies and further correlates successful tumor rejection with the level of CX3CR1+PD-1+CD8 + T cells, making this model a critical tool for future studies to optimize and better utilize anti-PD-1/L1 therapeutics.

Abstract Cytotoxic CD8+ T cells (CTL) are a crucial component of the immune system notable for their ability to eliminate rapidly proliferating malignant cells. However, the T-cell intrinsic factors required for human CTLs to accomplish highly efficient antitumor cytotoxicity are not well defined. By evaluating human CD8+ T cells from responders versus nonresponders to treatment with immune checkpoint inhibitors, we sought to identify key factors associated with effective CTL function. Single-cell RNA-sequencing analysis of peripheral CD8+ T cells from patients treated with anti–PD-1 therapy showed that cells from nonresponders exhibited decreased expression of the cytolytic granule-associated molecule natural killer cell granule protein-7 (NKG7). Functional assays revealed that reduced NKG7 expression altered cytolytic granule number, trafficking, and calcium release, resulting in decreased CD8+ T-cell–mediated killing of tumor cells. Transfection of T cells with NKG7 mRNA was sufficient to improve the tumor-cell killing ability of human T cells isolated from nonresponders and increase their response to anti–PD-1 or anti–PD-L1 therapy in vitro. NKG7 mRNA therapy also improved the antitumor activity of murine tumor antigen–specific CD8+ T cells in an in vivo model of adoptive cell therapy. Finally, we showed that the transcription factor ETS1 played a role in regulating NKG7 expression. Together, our results identify NKG7 as a necessary component for the cytotoxic function of CD8+ T cells and establish NKG7 as a T-cell–intrinsic therapeutic target for enhancing cancer immunotherapy. See related article by Li et al., p. 154.

Abstract Cytotoxic T lymphocytes (CTL's) are capable of killing tumor cells throughout the body, and can be stimulated to do so by a form of immunotherapy known as PD-1 checkpoint blockade. However, the majority of patients given anti-PD-1 therapeutics do not respond to treatment. Thus, we sought to identify key factors associated with failure of PD-1 blockade in order to develop strategies that could significantly improve response rates. We performed single-cell RNA-seq analysis of peripheral CD8+ T cells from responding and non-responding patients and found that the non-responders had less NKG7 expression. Functional assays revealed that knock-down of NKG7 compromised cytolytic granule localization and trafficking within CTL's. Since decreasing NKG7 reduced the cytolytic efficiency of CD8+ T cells, we next hypothesized that by increasing expression of NKG7 we could promote better T cell killing of tumor and enhance the effects of immune checkpoint inhibitors. To test this, we utilized peripheral blood samples from melanoma patients who failed to respond to anti-PD-1 treatment and transfected the PBMC's with NKG7 mRNA. The addition of NKG7 mRNA significantly increased T cell killing of melanoma tumor cells in an in vitro cytotoxicity assay, and this effect was even more robust in combination with anti-PD-1 or anti-PD-L1 pre-treatment. To confirm that over-expression of NKG7 could improve CD8+ T cell killing in an antigen-specific manner, we transfected OT-1 transgenic T cells with murine sequence of NKG7 mRNA or control mRNA. OT-1 CD8+ T cells with increased NKG7 expression exhibited enhanced cytolytic ability when challenged with either EL4 cells pulsed with OVA peptide or B16-OVA tumor cells in vitro. In addition, NKG7 mRNA-transfected OT-1 CD8+ T cells suppressed the aggressive growth of B16-OVA cells in vivo significantly better than control OT-1 CD8+ T cells. These results indicate that deficits in a T cell-intrinsic cytolytic factor can lead to failure of PD-1/L1 blockade and establish NKG7 as a new therapeutic target for enhancing response to cancer immunotherapy. Citation Format: Whitney J. Barham, Ti Wen, Ying Li, Henan Zhang, Cindy Liu, Jacob Hirdler, Joanina Gicobi, Fabrice Lucien-Matteoni, Yiyi Yan, Haidong Dong. NKG7 mRNA therapy increases the anti-tumor cytotoxicity of CD8 T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 504.