Hsu, Michelle A.

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)

Abstract Although immune checkpoint blockade that targets the PD-L1 has revolutionized the treatment of human cancers, only a small proportion of patients positively respond to this form of treatment. A limitation of these therapies is that they only target and inhibit PD-L1’s extracellular function, which is to bind its receptor on PD-1 on T cells and inhibit T cell cytotoxicity. Recent discoveries have shown that PD-L1 expressed on tumor cells can signal intrinsically and independently of PD-1 ligation to promote tumor cell survival. Besides tumor cells, host myeloid cells have been shown to have high expression of PD-L1, therefore contributing to the immunosuppressive nature of the tumor. However, PD-L1 intrinsic signaling in human myeloid cells has not been studied. Therefore, the purpose of our work is to determine the intrinsic functions of PD-L1 in myeloid cells and how this impacts anti-tumor immunity. To study this, we used a novel PD-L1 antibody (H1A) that does not block PD-L1’s binding with PD-1, but directs PD-L1 for degradation, thereby decreasing total PD-L1 expression. Additionally, we developed a chimeric mouse model in which the murine PD-1/PD-L1 has been replaced with the human version. This allows us to evaluate PD-L1’s intrinsic function in vivo. In our preliminary studies, the degradation of PD-L1 in human monocytes led to increased chemokine release as well as increased conversion of effector T cells. Additionally, H1A treatment of tumor-bearing mice led to lower tumor burden in a CD8 T cell dependent manner. Based on these results, PD-L1 intrinsic signaling has the potential to regulate the immune-stimulatory response of monocytes, thereby limiting effector T cell responses against cancer. Supported by grants from NIH (R0192389) and Mayo Clinic graduate school of biomedical sciences

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

Abstract The intrinsic and acquired resistance to PD-1/PD-L1 immune checkpoint blockade is an important challenge for patients and clinicians because no reliable tool has been developed to predict individualized response to immunotherapy. In this study, we demonstrate the translational relevance of an ex vivo functional assay that measures the tumor cell killing ability of patient-derived CD8 T and NK cells (referred to as “cytotoxic lymphocytes,” or CLs) isolated from the peripheral blood of patients with renal cell carcinoma. Patient-derived PBMCs were isolated before and after nephrectomy from patients with renal cell carcinoma. We compared the efficacy of U.S. Food and Drug Administration (FDA)-approved PD-1/PD-L1 inhibitors (pembrolizumab, nivolumab, atezolizumab) and a newly developed PD-L1 inhibitor (H1A Ab) in eliciting cytotoxic function. CL activity was improved at 3 mo after radical nephrectomy compared with baseline, and it was associated with higher circulating levels of tumor-reactive effector CD8 T cells (CD11ahighCX3CR1+GZMB+). Treatment of PBMCs with FDA-approved PD-1/PD-L1 inhibitors enhanced tumor cell killing activity of CLs, but a differential response was observed at the individual-patient level. H1A demonstrated superior efficacy in promoting CL activity compared with FDA-approved PD-1/PD-L1 inhibitors. PBMC immunophenotyping by mass cytometry revealed enrichment of effector CD8 T and NK cells in H1A-treated PBMCs and immunosuppressive regulatory T cells in atezolizumab-treated samples. Our study lays the ground for future investigation of the therapeutic value of H1A as a next-generation immune checkpoint inhibitor and the potential of measuring CTL activity in PBMCs as a tool to predict individual response to immune checkpoint inhibitors in patients with advanced renal cell carcinoma.

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 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.