Mao, Zhiming

Abstract Description Therapies targeting the PD-1/PD-L1 axis are among the leading treatments of cancer, but only a minority of patients benefit. Many therapies focus on preventing PD-L1 from binding to its receptor, PD-1, but do not address PD-L1’s ability to be recycled to the cell surface and any intrinsic functions it may possess. Our lab has developed an antibody (H1A) that binds to PD-L1 and prevents its association with a stabilizing protein, CMTM6, therefore targeting it for lysosomal degradation. Consequently, H1A limits any PD-L1 intrinsic function while also decreasing interactions with PD-1. Here, we use human-derived immune cells to test H1A’s effect on immune responses through flow cytometry, multiplex screening, and cytotoxicity assays. Additionally, we have developed a humanized PD-1/PD-L1 mouse model that allows us to test H1A’s effect on tumor growth inhibition in an immunocompetent host. Our results show that H1A specifically targets human myeloid cells, resulting in increased co-stimulation, antigen presentation, and cytokine secretion. H1A demonstrated enhanced T cell cytotoxicity in a whole PBMC culture. Finally, H1A resulted in greater tumor growth inhibition and survival in our in vivo tumor models. This work highlights the importance of targeting beyond PD-1/PD-L1 ligation and that myeloid cells are instrumental to enhancing the immune response against cancer. H1A is emerging as a new immunotherapy tool to target PD-L1 and CMTM6 in myeloid cells for treatment of cancers. Funding Sources NIH R01 92389022; NIH T32 AI170478; Mayo Clinic Graduate School of Biomedical Sciences Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description A durable T cell immunity against cancer is dependent on a continually refilling of effector CD8+ T cells. Thymic output has been correlated with favorable prognosis in cancer patients of advanced ages, suggesting thymus is an important source for refilling T cells that are capable of controlling cancer progression in the periphery. However, it remains unclear how PD-1 signals regulate the cytotoxic potential of CD8+ T cells in thymus before they egress to the periphery to establish durable anti-tumor immunity. Here we report PD-1 signaling prelimits thymic single positive (SP) CD8+ T cells to acquire cytotoxic potential by downregulating Gzmb and Nkg7, which are required for CD8+ T cell-mediated cytotoxicity. Unexpectedly, we found the absence of Pdcd1 also led to upregulation of Tox and Tim3 in thymic SP CD8+ T cells, thereby pre-programming these cells for exhaustion within the tumor. Thus, the absence of PD-1 signals in CD8+ T cells only partially delayed tumor growth in peripheral tissues. To improve therapeutic effects of PD-1 inhibition, we observed that tumor growth in CD8-specific Pdcd1 conditional knockout mice was further suppressed by treatment with a PD-L1 antibody that blocks PD-L1 and CD80 interaction. Our results imply that PD-1 inhibition promotes the expansion of effector cytotoxic CD8+ T cells but also retains T cell exhaustion potential, which can be mitigated by PD-L1 blockade that allows more CD80 to provide costimulatory signals to T cells via CD28. Funding Sources NCI R01CA256927 NCI R01CA261932 NCI P30CA15083 Topic Categories Tumor Immunology: Cellular Responses and Tumor Microevironment (TIME)

Abstract Description The efficacy of ICI and T cell-based therapies is dependent on sufficient killing of tumor cells by T cells. Thus, it’s critical for effector T cells to maintain their function and overcome metabolic stress within the tumor microenvironment. Our previous work in human resilient CD8+ T cells identified malic enzyme 1 (ME1) as a key enzyme that augments effector CD8+ T cells’ metabolic and functional fitness via increased ATP production and curbed ROS production. To further study ME1’s role in promoting CD8+ T cell resilience in vivo, we produced CD8-specific ME1 conditional knockout and transgenic (ME1 Tg) mouse models. In MC38, ME1 was upregulated in CD8+ T cells in TILs compared to those in spleen and ME1 loss in CD8+ T cells abrogated tumor responses to ICI. In ME1 Tg mice, MC38 had reduced tumor growth, while B16F10 tumors were sensitized to ICI. We observed lower ROS and increased lipid content in CD8+ T cells in the TILs of ME1 Tg mice, underscoring ME1’s role in activating alternative metabolic pathways to fuel mitochondrial respiration while keeping ROS in check. The association of ME1 with 6PGD, a key enzyme in the pentose phosphate pathway (PPP), implies its regulation of redox homeostasis in CD8+ T cells. Importantly, ME1 overexpression in TILs dramatically improved the efficacy of TIL therapy in treatment of established tumors. Overall, ME1 upholds T cell resilience in tumor tissues and provides a new therapeutic target to improve the efficacy of TIL therapy. Funding Sources This work was supported by the Mayo Clinic Center for Biomedical Discovery (to H.D.); the Mayo Clinic Cancer Center’s David F. and Margaret T. Grohne Cancer Immunology and Immunotherapy program (to H.D.); R01 AI095239 (to H.D.), R01 CA200551 (to H.D. and S.S.P.), and R01 CA256927 (to H.D.). Topic Categories Tumor Immunology: Checkpoints, Prevention, and Treatment (TIPT)

Background Immune checkpoint therapies targeting programmed cell death protein 1 (PD-1) and programmed death ligand 1 (PD-L1) have been met with limited clinical responses in the treatment of patients of varying cancer types. Current US Food and Drug Administration (FDA)-approved therapies function to prevent PD-1 binding to PD-L1, thereby preventing T-cell suppression. However, these therapies fail to prevent PD-L1 intrinsic signaling and recycling, which can render these treatments ineffective. Moreover, most research primarily focuses on how these antibodies affect tumor cells and their interactions with T cells. Myeloid cells, which also express PD-L1 and influence T-cell responses, are integral to the efficacy of these therapies. However, strategies that can target these cells to boost antitumor responses have not been effective. Methods Our group has developed a new anti-PD-L1 antibody (H1A), which disrupts PD-L1 recycling and redirects it towards degradation. Due to the loss of available PD-L1 protein, H1A effectively prevents PD-L1 intrinsic signaling. Using in vivo humanized PD-1/PD-L1 mouse tumor models, we evaluated the therapeutic efficacy of H1A against current FDA-approved PD-L1 blocking antibodies. Using human-derived immune cell in vitro assays, we pinpoint the biological consequences of H1A-induced PD-L1 degradation in myeloid cells. Results H1A demonstrated improved tumor control and established immunological memory responses in vivo in the treatment of tumors with moderate immunogenicity that are less responsive to current immunotherapies. This was supported by enhanced activation of myeloid cells (major histocompatibility complex (MHC)-II and CD80) and frequencies of effector T-cell populations found intratumorally. Human myeloid cells treated with H1A were also observed to have increased activation (MHC-II and CD80) and cytokine secretion. Similar to our in vivo findings, human peripheral blood lymphocyte cultures demonstrated increased frequencies of effector T-cell populations and greater tumor cell killing. Conclusions This work highlights the importance of targeting PD-L1 recycling and intrinsic signaling, specifically in myeloid cells, to boost antitumor responses. Our studies suggest that H1A can provide a solution to the lack of responses seen with current therapeutics, while also revealing previously unknown intrinsic functions of PD-L1 in myeloid cells.

Abstract The effectiveness of T cell–based immunotherapy depends on durable T-cell responses that can efficiently eliminate tumor cells. NKG7 was discovered three decades ago as a protein associated with lytic granules. However, only studies published over the past 5 years have contributed substantially to our understanding of NKG7 in T-cell biology. NKG7 has been recognized as an important T-cell functional marker in responses to immune checkpoint inhibitor therapy and in the prognosis of certain cancers. Besides its role in the generation, trafficking, and release of lytic granules, which is critical for efficient T-cell cytotoxicity against tumor cells, NKG7 has been identified as a key negative regulator of mTORC1 activity. By restraining mTORC1 activity, NKG7 promotes T-cell longevity and memory generation after infection. Importantly, NKG7 upregulation has demonstrated therapeutic potential in preclinical T-cell therapy for cancer. Collectively, NKG7 is emerging as a promising biomarker and therapeutic addition to T cell–based immunotherapies.

Abstract Durable T cell immunity against cancer depends on the continual replenishment of effector CD8+ T cells. Thymic output has been correlated with favorable prognosis in cancer patients across a range of ages, suggesting that the thymus is an important source for replenishing T cells capable of controlling cancer progression. However, the effector potential of thymic mature CD8+ T cells and their regulation have not been clearly defined. In this study, we identified the ability of thymic single positive CD8+ T cells to gain effector potential after thymic selection, but they are subject to the regulation of PD-1. We found a previously undisclosed role of PD-1 in limiting both the cytotoxic and exhaustion potential of thymic and peripheral CD8+ T cells. Our results show that although PD-1 inhibition facilitates the expansion of effector CD8+ T cells, effector CD8+ cells gradually lose their antitumor activity within tumor tissues due to advanced exhaustion in the absence of PD-1. Thus, although the preset effector potential in thymic mature CD8+ T cells allows them to rapidly respond to malignant cells in the periphery, PD-1, as a checkpoint, is embedded in the thymic mature CD8+ T cells after positive selection to balance their effector function from exaggeration and exhaustion. Therefore, we propose that a strategy capable of upholding the cytotoxic capacity and avoiding exhaustion of CD8+ T cells during the early stages of PD-1 inhibition therapy is needed to achieve durable antitumor immunity.

Abstract Cytotoxic CD8+ T cells play a key role in response to anti-PD-1/PD-L1 immune checkpoint inhibitors (ICI) therapy. However, the exact effector molecules that are regulated by PD-1 signals in cytotoxic CD8+ T cells have not been completely defined. Lack of this knowledge is a crucial problem in addressing how to overcome the low clinical response rate to ICI therapy. When exploring why certain patients not responding to ICI therapy, we found non-responders did not have an elevated NKG7 expression in CD8+ T cells as responders to ICI therapy. The difference in NKG7 expression in CD8+ T cells between non-responders and responders to anti-PD-1 therapy prompted us to investigate the potential role of PD-1 signaling in regulating NKG7 expression. To that end, we generated a CD8+ T cell-specific Pdcd1 conditional knockout mice (CD8 E8ICre-Pdcd1fl/fl, also known as CD8-PD-1 cKO) where Pdcd1 is deleted after thymic selection of single positive CD8+ T cells. We observed a delayed tumor growth and prolonged survival in CD8-PD-1 cKO mice compared to control mice after B16-OVA melanoma tumor challenge. On Day 12, when the CD8-PD-1 cKO mice demonstrated their ability to control tumor growth, we conducted single cell RNA sequencing on isolated tumor infiltrating CD8+ T cells, revealing a significant increase of Nkg7 expression in effector CD8+ T cells in the Pdcd1 deficient CD8+ T cells compared to the control wild type T cells. Pdcd1-deficient effector CD8+ T cells also had increased expression of genes linked to NFAT/Calcineurin signaling pathway including Nfatc1, S100a4, Tbet and Tox, suggesting Nkg7 could be regulated by Pdcd1 via the NFAT pathway. To determine the impact of increased NKG7 expression in CD8+ T cells, we used OVA protein as a surrogate tumor antigen in vaccination. We found a complete B16-OVA tumor rejection in CD8-PD-1 cKO mice compared to control mice when tumor cells were injected on day 7 after vaccination with OVA protein and poly (I:C) (as an adjuvant), but only a partial rejection on day 21. The CD8-PD-1 cKO mice that were tumor-free for 30-40 days rejected 2nd tumor challenge with B16-OVA tumors. This long-term protection is tumor antigen (OVA) specific because these mice did not reject B16F10 tumor cells that do not express OVA antigen. We also found effector memory CD8+ T cells with high cytotoxicity increased in the spleen of CD8-PD-1 cKO mice after OVA antigen immunization compared to control. In conclusion, we found NKG7 is one of the effector molecules in cytotoxic CD8+ T cells, which can be regulated by PD-1 signals through NFAT signaling. Targeting the PD-1-NFAT pathway may be a new option to overcome low responses of T cells to ICI therapy in treatment of refractory cancer. Citation Format: Zhiming Mao, Joanina Gicobi, Jacob Hirdler, Xin Liu, Michelle Hsu, Emilia Dellacecca, Wenjing Zhang, Fabrice Lucien-Matteoni, Hai dong. PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses [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 3979.

Patients with advanced cancers who either do not experience initial response to or progress while on immune checkpoint inhibitors (ICIs) receive salvage radiotherapy to reduce tumor burden and tumor-related symptoms. Occasionally, some patients experience substantial global tumor regression with a rebound of cytotoxic CD8 + T cells. We have termed the rebound of cytotoxic CD8 + T cells in response to salvage therapy as T cell resilience and examined the underlying mechanisms of resilience. Resilient T cells are enriched for CX3CR1 + CD8 + T cells with low mitochondrial membrane potential, accumulate less reactive oxygen species (ROS), and express more malic enzyme 1 (ME1). ME1 overexpression enhanced the cytotoxicity and expansion of effector CD8 + T cells partially via the type I interferon pathway. ME1 also increased mitochondrial respiration while maintaining the redox state balance. ME1 increased the cytotoxicity of peripheral lymphocytes from patients with advanced cancers. Thus, preserved resilient T cells in patients rebound after salvage therapy and ME1 enhances their resiliency.

Abstract Cytotoxic T lymphocytes (CTL) are indispensable in anti-tumor immunity. Although CTLs are prone to exhaustion in patients with advanced cancer, T cell resiliency explains the presence of tumor-reactive CTLs that are less exhausted, capable of cytolytic function, expansion, and rebound in response to immunotherapy to reject metastatic malignancies. However, the features of resilient T cells have not been clearly defined. In this report, we demonstrate that peripheral CX3CR1+ CD8+ T cells with low mitochondrial membrane potential rebounded CTL function quickly after radiation therapy in patients with large tumor burden portraying their functional resiliency. Furthermore, CX3CR1+ CD8+ T cell with low, but not high, mitochondrial membrane potential are highly cytotoxic, accumulate less reactive oxygen species (ROS), and express more Malic enzyme 1 (ME1). ME1 overexpression increases ATP production in a glycolysis-independent manner while concurrently curtailing excessive ROS in activated CD8+ T cells; and expands CX3CR1+NKG7+ effector CD8+ T cells with enhanced cytotoxicity. Importantly, transfection of ME1 mRNA promotes tumoricidal activity in CD8+ T cells from patients with advanced cancers. Our study reveals a mechanism used by CTLs to balance excessive ROS via ME1 to maintain a metabolic and functional resiliency. Modification of ME1 expression in CTLs may be a novel method to improve the efficacy of cancer immunotherapy by preventing T cell exhaustion. Supported by grants from Mayo Clinic Foundation and NIH (R01CA 256927-2)