Zhang, Wenjing

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)

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 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 Though anti-PD-1/PD-L1 immune checkpoint blockade therapy has demonstrated robust anti-tumor immunity in human patients with advanced cancers such as melanoma, only a fraction of patients achieved such durable anti-tumor responses. Among all the non-responders, some patients responded initially but developed resistance to the therapy in the later treatments. This reflects an urgent need to understand the role of PD-1 signals in CD8+ T cells at priming and at effector stages during tumor development for the benefit of immunotherapy. The controversy of rapid tumor progression in PD-1 conditional knockout in regulatory T cells and tumor regression in PD-1 targeted deletion in regulatory T cells indicated germline PD-1 knockout mice might not be sophisticated enough to study the role of PD-1 in CD8+ T cells during tumor development. Thus, we produced a CD8 T cell-specific PD-1 knockout mouse model (CD8 E8ICre-PD-1fl/fl, also called CD8-PD-1 cKO mice) in which PD-1 is deleted in CD8+ T cells in the peripheral tissues. When CD8-PD-1 cKO mice were challenged with B16-OVA mouse melanoma cells, we found a delayed tumor growth and prolonged survival of CD8-PD-1 cKO compared to control mice. However, after 20 days of tumor challenge, the delayed tumor growth rebound in CD8-PD-1 cKO mice. In an OVA antigen immunization model, we found increased effector cells and tissue resident cells in the spleen of CD8-PD-1 cKO mice compared with control mice on day 7 after immunization. Taken together, our results suggest that PD-1 may restrain the expansion of effector T cells that contribute to the early phase of tumor control, but the augmented effector T cell response may cause an attrition of memory T cell pool that will be needed for long term protection. R01CA256927