Citations

424 Background: Understanding the impact of anticancer therapies on immune system is critical to successful design and incorporation of immunotherapies. We developed a novel immune monitoring platform that measures the relative amount of circulating effector T cells and antitumor cytotoxicity of circulating immune cells. We previously showed that this assay can differentiate patients’ clinical response to anti-PD-1 therapy. Here, we applied this assay to patients with gastric or gastroesophageal junction adenocarcinoma (GGEA) who received chemoradiation or chemotherapy-based therapies. Methods: Patients underwent peripheral blood collection at baseline and during follow-up. Levels of circulating effector T cells (CX3CR1+NKG7+/CD3+CD8+) were quantified by flow cytometry using peripheral blood mononuclear cells (PBMCs). Antitumor cytotoxicity of circulating immune cells was evaluated by co-culturing CD3/CD28 activated PBMCs with calcein-AM labeled GGEA tumor cells (FLO-1). Antitumor cytotoxicity of individual patients’ PBMCs was calculated based on the amount of calcein released by dead tumor cells. Results: In a pilot study of 8 patients with mismatch repair-proficient (pMMR) GGEA, the changes in the frequency of effector T cells appeared to be associated with radiographic response to treatment based on RECIST (3 partial response [PR], 3 stable disease [SD], and 2 progressive disease [PD]). The median interval between sequential blood collections was 64 days and an increase in effector T cells was observed in patients with PD. Antitumor cytotoxicity was evaluated in 4 patients who did not receive targeted therapies or immune checkpoint inhibitors. Two of these patients (a, b) had blood collected at the time of diagnosis (treatment naïve) and after neoadjuvant therapy. One developed pulmonary metastases after chemoradiation while the other had significant tumor shrinkage after chemotherapy. The decrease in antitumor cytotoxicity of PBMCs was less in the patient with PD (-19.4%) than the one with PR (-48.5%). Two patients (c, d) had blood collected twice while receiving the same chemotherapy for metastatic disease (not treatment naïve). The decrease in antitumor cytotoxicity of PBMCs was less in the patient with SD (-0.3%) than the one with PR (-14.5%). Conclusions: We demonstrated the feasibility of using PBMCs to monitor immune response to anticancer therapies. Our data raise the possibility that the amount of circulating effector T cells and antitumor cytotoxicity of PBMCs may be associated with tumor burden in patients with pMMR solid tumors. [Table: see text]

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 Aging has been shown to be one of the major risk factors for host morbidity and mortality following infections with respiratory viruses including influenza virus and SARS-CoV-2. The unique immune properties of the aged hosts not only influence pathogen clearance and tissue damage responses, but also modulate the pathogenesis of the long-term sequalae post acute viral clearance. Currently, the dynamics and features of mucosal immune responses following acute respiratory viral infections in the aged hosts remain to be fully elucidated. Here, we characterized the lung cellular and molecular profiles following influenza virus infection in young (~3-month-old) or aged (~24-month-old) mice with a chronological manner from acute to chronic phase. Using DNA-oligo-conjugated CD45 antibody and i.v. labeling, we were able to distinguish the unique responses of circulating vs lung-resident immune cells to influenza virus infection during aging. These responses were further validated by spectral flow cytometry analysis of the lung innate and adaptive immune cells. Further data analysis and integration are warranted for this unique large data set detailing the single cell molecular profiles of mucosal responses to influenza virus infection comparing young and aged hosts.

Abstract Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells, which recognize specific sialic acid modifications on cell surface glycans. Sialyltransferases are the enzymes that catalyzing the addition of sialic acids onto glycoproteins and glycolipids. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6 on O-linked glycoproteins and functions to inhibit activation of innate immune cells. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered macrophage polarization and increased tumor growth through engagement of ligands generated by ST8Sia6 with Siglec-E. Here, we examine the role of ST8Sia6 on immune cells in regulating anti-tumor immunity. The loss of ST8Sia6 in tumor bearing mice reduced tumor growth, increased survival, and altered the immune response as compared to ST8Sia6 sufficient tumor bearing mice. Using MC38, B16, and B16-OVA flank tumor mouse models, tumor growth was decreased in ST8Sia6 KO mice as compared to C57Bl/6 mice. Within the tumor microenvironment of tumors present in ST8Sia6 KO mice compared to tumors present in C57Bl/6 mice, macrophages and dendritic cells altered their phenotype towards enhanced immune activation. Tregs from tumors in ST8Sia6 KO mice also displayed an increase in RORγt and a reduction in TNFRII, suggesting a possible shift to a less suppressive Treg. These alterations in the immune response to tumors were due to the loss of ST8Sia6 in macrophages and dendritic cells and not T-cells. Thus, ST8Sia6 generates ligands for Siglecs that dampen anti-tumor immunity. Supported by grants from NIH (R01CA243545, 2T32AI007425) and Mayo Clinic Center of Biomedical Discovery

Abstract Though anti-PD-1/L1 antibody therapies have generated unprecedented responses in a limited number of patients, the biological and immunological parameters that define their effective use are still largely unknown. The absence of appropriate pre-clinical models has limited our ability to compare the mechanistic differences between the approved PD-1/L1 antibodies in vivo and to determine the context in which each might be most effective. To address this critical need, we have produced humanized PD-1 and PD-L1 mice in which the extracellular domains (antibody binding sites) of mouse PD-1 and PD-L1 are replaced with the sequences of human PD-1 and PD-L1. The PD-1 and PD-L1 in the resulting “HuPD” mice can be recognized by FDA-approved antibodies, while the intracellular domains are kept intact for normal downstream signals. Following characterization of the new mouse model, in our first in vivo treatment studies, HuPD mice were immunized with OVA/Poly I:C in the context of FDA-approved PD-1/L1 antibodies and antigen-specific T cell production was quantified. Nivolumab, Pembrolizumab, and Atezolizumab all increased effector CD8+T cell production against the OVA antigen as expected given their immune stimulating function. Surprisingly, OVA-tetramer staining was unable to detect antigen-specific CD8+T cells in the spleens of immunized mice treated with Avelumab. This may reflect the unique ability of Avelumab to induce ADCC, and indicates that our model is capable of distinguishing key differences among the antibodies in vivo. Ongoing studies will identify how these differences might be exploited to render the most effective results for patients in a variety of tumor types and immune environments.

Abstract Development of effective vaccination approaches to treat established tumors represents a focus of intensive research because such approaches offer the promise of enhancing immune system priming against tumor antigens via re-stimulation of pre-existing (memory) antitumoral helper and effector immune cells. However, inhibitory mechanisms which function to limit the recall responses of tumor-specific immunity exist remain poorly understood and interfere with therapies anticipated to induce protective immunity. The mouse renal cell carcinoma (RENCA) tumor model was used to investigate variables affecting vaccination outcomes. We demonstrate that although a whole cell irradiated tumor cell vaccine can trigger a functional anti-tumor memory response in the bone marrows of mice with established tumors, these responses do not culminate in the regression of established tumors. In addition, a CD103+ regulatory T (Treg) cell subset accumulates within the draining lymph nodes of tumor-bearing mice. We also show that B7-H1 (CD274, PD-L1), a negative costimulatory ligand, and CD4+ Treg cells collaborate to impair the recall responses of tumor-specific memory T cells. Specifically, mice bearing large established RENCA tumors were treated with tumor cell vaccination in combination with B7-H1 blockade and CD4+ T cell depletion (triple therapy treatment) and monitored for tumor growth and survivability. Triple treatment therapy induced complete regression of large established RENCA tumors and raised long-lasting protective immunity. These results have implications for developing clinical antitumoral vaccination regimens in the setting where tumors express elevated levels of B7-H1 in the presence of abundant Treg cells.

Abstract Checkpoint blockade has improved patient outcome through reinvigorating T cells against cancer. Despite this success, many patients don’t respond, leaving them with few options. Tumors utilize many immunosuppressive mechanisms, including altering the function of innate immune cells. Cancer cells can promote differentiation of macrophages into tolerogenic “M2-like” macrophages, which support tumor growth. Macrophage differentiation by cancer cells is influenced by many receptors, including Siglec-E. Siglec-E binds to α2,8-linked disialic acids, generated by the enzyme ST8Sia6, on O-linked glycoproteins, and functions to inhibit activation of innate immune cells. Cancer patients with high expression of ST8Sia6 exhibit reduced survival. Therefore, ST8Sia6 overexpressing tumors may engage Siglec-E, leading to immune suppression and accelerating tumor growth. Mice injected with ST8Sia6 overexpressing MC38 and B16-F10 tumors had increased tumor burden, reduced survival and an increase M2 macrophage polarization. The acceleration in tumor growth was abrogated when injected into Siglec-E knockout mice. Thus, the primary function of ST8Sia6 in tumors is an extrinsic alteration of the immune response, rather than an intrinsic effect on cell growth. Similarly, in a spontaneous colon cancer model, ST8Sia6 overexpression in a murine colon cancer model accelerated the development of tumors, decreasing survival to 2.5 months instead of 6 months. The overexpression of ST8Sia6 on tumors leads to increased Arg-1 expression by CD11b+ CD11c+Ly6C−Ly6G− cells, which suppresses the immune response. Therefore, ST8Sia6 expression in tumors inhibits the immune response to promote tumor growth in a Siglec-E dependent manner.

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 Tissue-resident memory CD8+ T cells (TRM) share characteristics and core residency gene programs with tumor-infiltrating lymphocytes (TILs). However, the transcriptional, metabolic and epigenetic regulation of TRM and TIL differentiation, maintenance and function are largely undefined. Furthermore, it is unknown whether metabolic and epigenetic manipulations can be employed to promote TRM/TIL functional programs for cancer immunotherapy. Here we report a critical mechanism programming the mitochondrial and epigenetic regulation of TRM/TIL functionality in situ. We find that mouse and human TRM/TIL express the transcription factor BHLHE40 and the BHLHE40-associated gene signature. Bhlhe40 is specifically required for TRM and TIL development and poly-functionality. Local PD-1 signaling inhibits TIL Bhlhe40 expression, and Bhlhe40 is critical for TIL reinvigoration following anti-PD therapy. Mechanistically, Bhlhe40 sustains TRM/TIL mitochondrial fitness for the promotion of a functional epigenetic state. Building on these findings, we also identify an epigenetic and metabolic regimen that promotes TRM/TIL gene signatures associated with tissue residency and poly-functionality through in vitro T cell screening. Strikingly, this regimen empowers the anti-tumor activity of CD8+ T cells and possesses therapeutic potential even at an advanced tumor stage in mouse models. Our results provide mechanistic insights on the local regulation of TRM and TIL function, and offer a viable strategy for developing novel immunotherapeutic means for cancer.

Abstract B7-H1 (also named PD-L1) is up-regulated on activated CD8 T cells at the late stage of antigen stimulation, and is required for T cell survival during contraction phase. Since more anti-B7-H1 (PD-L1) antibody has been used in clinical trials of treatment of human cancers, it is necessary to know whether antibody ligation of B7-H1 expressed by T cells would affect T cell function and survival. After screening several anti-B7-H1 antibodies, we found that some antibody triggered the activation of p38 MAPK in activated CD8 T cells. In addition, the levels of activated p38 MAPK increased in T cells isolated from B7-H1 knockout mice compared to wild type mice. Inhibition of DNA-PKcs activation increased the activation of p38 MAPK in wild type T cells but not in B7-H1 KO T cells, suggesting B7-H1 may negatively regulate p38 MAPK through DNA-PKcs pathway. To that end, we identified an association of B7-H1 with DNA-PKcs in activated human T cells but not in resting T cells. Since p38 MAPK inhibition rescued activated CD8 T cells from apoptosis, the pro-survival function of T cell intrinsic B7-H1 may be mediated by suppression of p38 MAPK. However, ligation of B7-H1 by agonist antibody may disrupt B7-H1’s pro-survival function by increasing p38 MAPK activation. Our results identified a link of B7-H1 with p38 MAPK pathway and may help to define agonist function of anti-B7-H1 antibody prior to clinical application.