Resident CD8 Research Articles

CD103 promotes the proinflammatory response of gastric resident CD4+ T cell in Helicobacter pylori- positive gastritis

Abstract As an integrin found on T cell residence, CD103 is associated with the CD8+ T cell response in allogeneic transplant rejection, autoimmune diseases and cancer, but its role in infection and inflammation remains unclear. In this study, biopsy specimens of the gastric mucosa were obtained from patients with H. pylori-positive gastritis. We found that CD103 was highly expressed in CD4+T cells of the gastric mucosa from patients with H. pylori-positive gastritis. Mucosal resident CD103+CD4+T cells exhibited an increase in the CD45RO+CCR7− effector memory phenotype and high expression of the chemokines CXCR3 and CCR9 compared with those in CD103−CD4+ T cells. An In vitro coculture study demonstrated that H. pylori-specific antigen CagA/ VacA-primed dendritic cells (DCs) induced proliferation as well as IFN-γ, TNF and IL-17 production by CD103+CD4+ T cells from patients with H. pylori-positive gastritis. Furthermore, blocking CD103 with a neutralizing antibody reduced proliferation and IFN-g, TNF and IL-17 production by CD103+CD4+ T cells cocultured with DCs. Moreover, immunoprecipitation revealed that CD103 interacted with TCR α/β and CD3ζ, and activating CD103 enhanced the phosphorylation of ZAP70 induced by the TCR signal. Finally, increased T-bet and Blimp1 levels were also observed in CD103+CD4+ T cells, while activating CD103 increased T-bet and Blimp1 expression in CD4+ T cells. Our results explored the function of CD103 in gastric T cells from patients with H. pylori-positive gastritis, which may provide a therapeutic target for the treatment of gastritis

The human IL-15 superagonist N-803 promotes migration of virus-specific CD8+ T and NK cells to B cell follicles but does not reverse latency in ART-suppressed, SHIV-infected macaques.

Despite the success of antiretroviral therapy (ART) to halt viral replication and slow disease progression, this treatment is not curative and there remains an urgent need to develop approaches to clear the latent HIV reservoir. The human IL-15 superagonist N-803 (formerly ALT-803) is a promising anti-cancer biologic with potent immunostimulatory properties that has been extended into the field of HIV as a potential “shock and kill” therapeutic for HIV cure. However, the ability of N-803 to reactivate latent virus and modulate anti-viral immunity in vivo under the cover of ART remains undefined. Here, we show that in ART-suppressed, simian-human immunodeficiency virus (SHIV)SF162P3-infected rhesus macaques, subcutaneous administration of N-803 activates and mobilizes both NK cells and SHIV-specific CD8+ T cells from the peripheral blood to lymph node B cell follicles, a sanctuary site for latent virus that normally excludes such effector cells. We observed minimal activation of memory CD4+ T cells and no increase in viral RNA content in lymph node resident CD4+ T cells post N-803 administration. Accordingly, we found no difference in the number or magnitude of plasma viremia timepoints between treated and untreated animals during the N-803 administration period, and no difference in the size of the viral DNA cell-associated reservoir post N-803 treatment. These results substantiate N-803 as a potent immunotherapeutic candidate capable of activating and directing effector CD8+ T and NK cells to the B cell follicle during full ART suppression, and suggest N-803 must be paired with a bona fide latency reversing agent in vivo to facilitate immune-mediated modulation of the latent viral reservoir.

DOP34 Transplantation of human intestine into the mouse: A novel platform for study of inflammatory enterocutaneous fistulas

Abstract Background In Crohn’s disease patients, enteric and perianal fistulas represent a severe and medically challenging comorbidity that affects a broad number of patients. Enteric fistulas do not develop in animal models of colitis what prevents in vivo experiments so far. Based on our preliminary data, we proposed transplantation of the human foetal gut into mice as a novel platform for studying inflammatory enterocutaneous fistulas. Methods Human foetal gut segments were transplanted subcutaneously into mature SCID mice, where they grew and fully developed over the course of several months. We first analysed the resident immune cells and inflammatory response elicited by systemic lipopolysaccharide in normal, fully developed human gut xenografts. Thereafter, we used immunostaining to analyse fully developed xenografts that spontaneously developed entero-cutaneous fistulas. Results We found a broad number of resident human innate and adaptive immune cells in gut xenografts during steady-state and inflammation. The expression of human IL-8, IL-1β, IL-6, TNF-α, A20, and IkBα was significantly elevated in response to LPS, with no change in IL-10 gene expression. Approximately 17% [19/110] of fully developed subcutaneous human gut xenografts spontaneously developed enterocutaneous fistulas, revealing striking histopathological similarities with CD fistula specimens. Immunohistochemical analyses of fistulating xenografts revealed transmural lymphocytic enteritis associated with a massive expansion of resident human CD4+ lymphocytes and their migration into the intraepithelial compartment. Regionally, mucosal epithelial cells assumed spindle-shaped mesenchymal morphology and formed fistulous tracts towards chronic non-healing wounds in the host mouse skin overlying the transplants. Conclusions Inflammation and fistulas developed in human gut xenografts lacking IL-10 gene response. This novel model system will enable systematic studies of the inflamed and fistulating human gut in live animals.

Generation of highly activated, antigen-specific tumor-infiltrating CD8+ T cells induced by a novel T cell-targeted immunotherapy

ABSTRACT The induction of tumor-targeted, cytotoxic T lymphocytes has been recognized as a key component to successful immunotherapy. DPX-based treatment was previously shown to effectively recruit activated CD8+ T cells to the tumor. Herein, we analyze the unique phenotype of the CD8+ T cells recruited into the tumor in response to DPX-based therapy, and how combination with checkpoint inhibitors impacts T cell response. C3-tumor-bearing mice were treated with cyclophosphamide (CPA) for seven continuous days every other week, followed by DPX treatment along with anti-CTLA-4 and/or anti-PD-1. Efficacy, immunogenicity, and CD8+ T cells tumor infiltration were assessed. The expression of various markers, including checkpoint markers, peptide specificity, and proliferation and activation markers, was determined by flow cytometry. tSNE analysis of the flow data revealed a resident phenotype of CD8+ T cells (PD-1+TIM-3+CTLA-4+) within untreated tumors, whereas DPX/CPA treatment induced recruitment of a novel population of CD8+ T cells (PD-1+TIM-3+CTLA-4−) within tumors. Combination of anti-CTLA-4 (ipilimumab) with DPX/CPA versus DPX/CPA alone significantly increased survival and inhibition of tumor growth, without changing overall systemic immunogenicity. Addition of checkpoint inhibitors did not significantly change the phenotype of the newly recruited cells induced by DPX/CPA. Yet, anti-CTLA-4 treatment in combination with DPX/CPA enhanced a non-antigen specific response within the tumor. Finally, the tumor-recruited CD8+ T cells induced by DPX/CPA were highly activated, antigen-specific, and proliferative, while resident phenotype CD8+ T cells, seemingly initially exhausted, were reactivated with combination treatment. This study supports the potential of combining DPX/CPA with ipilimumab to further enhance survival clinically.

BRAF V600E and Pten deletion in mice produces a histiocytic disorder with features of Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is characterized by the accumulation of Langerin (CD207)-expressing histiocytes. Mutational activation of mitogen-activated protein kinase pathway genes, in particular BRAF, drives most cases. To test whether activated BRAF is sufficient for the development of LCH, we engineered mice to express BRAF V600E under the control of the human Langerin promoter. These mice have shortened survivals, smaller lymphoid organs, absent Leydig cells, and fewer epidermal LCs than controls, but do not accumulate histiocytes. To test whether the absence of histiocyte proliferation could be due to oncogene-induced senescence, we engineered homozygous Pten loss in the same cells that expressed BRAF V600E. Like mice with intact Pten, these mice have shortened survivals, smaller thymi, and absent Leydig cells. However, loss of Pten also leads to the accumulation of CD207+ histiocytes in spleen, thymus, and some lymph nodes. While many CD207+ histiocytes in the thymus are CD8-, reminiscent of LCH cells, the CD207+ histiocytes in the spleen and lymph nodes are CD8+. These mice also accumulate large numbers of CD207- cells in the lamina propria (LP) of the small intestine. Both the lymphoid and LP phenotypes are likely due to human Langerin promoter-driven BRAF V600E expression in resident CD8+ dendritic cells in the former and LP dendritic cells in the latter and confirm that Pten loss is required to overcome inhibitory pathways induced by BRAF V600E expression. The complex phenotype of these mice is a consequence of the multiple murine cell types in which the human Langerin promoter is active.

T Cell Metabolism Is Dependent on Anatomical Location within the Lung.

The metabolic shift from oxidative phosphorylation to glycolysis is universally accepted as a necessary step for immune cells to mount effector functions. However, it is unknown if this paradigm holds true for T cells regardless of anatomical location. In this study, we compared metabolic responses among distinct mouse pulmonary CD4+ effector T cell (Teff) pools following intranasal vaccination with either Francisella tularensis or Bordetella pertussis Surprisingly, in contrast to circulating CD4+ Teff, upon ex vivo stimulation, resident CD4+ Teff did not shift to glycolysis. This impairment in the resident pool was modestly overcome following in vivo infection. However, consistent with an ex vivo triggered shift toward glycolysis, circulating CD4+ Teff remained superior compared with resident CD4+ Teff after in vivo infection. These data indicate differences in lung T cell metabolism is associated with anatomic location, a feature which may be exploited to enhance or dampen pulmonary T cell responses.

Robust Iterative Stimulation with Self-Antigens Overcomes CD8+ T Cell Tolerance to Self- and Tumor Antigens

SUMMARYThe immune system adapts to constitutive antigens to preserve self-tolerance, which is a major barrier for anti-tumor immunity. Antigen-specific reversal of tolerance constitutes a major goal to spur therapeutic applications. Here, we show that robust, iterative, systemic stimulation targeting tissue-specific antigens in the context of acute infections reverses established CD8+ T cell tolerance to self, including in T cells that survive negative selection. This strategy results in large numbers of circulating and resident memory self-specific CD8+ T cells that are widely distributed and can be co-opted to control established malignancies bearing self-antigen without concomitant autoimmunity. Targeted expansion of both self- and tumor neoantigen-specific T cells acts synergistically to boost anti-tumor immunity and elicits protection against aggressive melanoma. Our findings demonstrate that T cell tolerance can be re-adapted to responsiveness through robust antigenic exposure, generating self-specific CD8+ T cells that can be used for cancer treatment.

LB1143 New insights into the role of T cells in psoriasis: Identification of multi-cytokine producing T cell subsets and a skewed relative contribution of IL17A production by tissue resident CD103+CD8+ T cells

LB1143 New insights into the role of T cells in psoriasis: Identification of multi-cytokine producing T cell subsets and a skewed relative contribution of IL17A production by tissue resident CD103+CD8+ T cells

Dynamic Metabolic State of Tissue Resident CD8 T Cells.

In the past years, there have been significant advances in the understanding of how environmental conditions alone or in conjunction with pathogen invasion affect the metabolism of T cells, thereby influencing their activation, differentiation, and longevity. Detailed insights of the interlinked processes of activation and metabolism can contribute to major advances in immunotherapies. Naive and memory T cells circulate the body. In a quiescent state with low metabolic demands, they predominantly use oxidative phosphorylation for their energy needs. Recognition of cognate antigen combined with costimulatory signals results in a proliferative burst and effector molecule production, requiring rapid release of energy, achieved via dynamically reprogramming metabolic pathways. After activation, most T cells succumb to activation induced cell death, but few differentiate into memory T cells. Of note, some memory T cells permanently occupy tissues without circulating. These, tissue resident T cells are predominantly CD8 T cells, maintained in a metabolic state distinct from naïve and circulating memory CD8 T cells with elements similar to effector CD8 T cells but without undergoing proliferative burst or secreting immune mediators. They continually interact with tissue cells as part of an immune surveillance network, are well-adapted to the tissues they have made their home and where they may encounter different metabolic environments. In this review, we will discuss recent insights in metabolic characteristics of CD8 T cell biology, with emphasis on tissue resident CD8 T cells at the epithelial barriers.

Abstract 4989: T-distributed stochastic neighbor embedding (t-SNE) analysis of tumor infiltrating lymphocytes after treatment with a T cell activating therapy identifies a unique population of recruited CD8+ T cells and novel options for combination immunotherapy

Abstract Immune therapies for cancer seek to alter the immune profile of the tumor microenvironment (TME). A suppressive TME can counteract active immune responses by CD8+ T cells by multiple mechanisms, including induction of checkpoint inhibitor (CPI) expression. DPX™ is a lipid nanoparticle-based platform that can be formulated with cancer targets to induce potent and sustained antigen-specific T cell responses. In preclinical and clinical testing previously published, combination of DPX with low dose intermittent cyclophosphamide (ldCPA) resulted in increased tumor infiltration of antigen-specific CD8+ T cells. These T cells express PD-1, and combination treatment with PD-1 blocking antibody was shown previously to result in better tumor control in preclinical tumor models. In this study, we used tSNE analysis to understand changes to the immune profile induced by DPX/ldCPA treatment in the C3 model. We prioritized the expression of CPIs on tumor infiltrating CD8+T cells to identify potential combinations with blocking monoclonal antibodies to enhance tumor CD8+ T cell activity. Mice (C57BL/6) bearing HPV16 E7-transformed C3 tumor cells were treated with ldCPA (20 mg/kg/day PO, 7 days), followed by subcutaneous injection with HPV16 E749-57 peptide in a DPX formulation (DPX-R9F). Tumors were collected for analysis 10 days after DPX-R9F treatment and a single cell suspension prepared by digestion. Immune profiling was performed by 12-color flow cytometry, to observe differences in infiltrating immune cells and CPIs. tSNE analysis of flow cytometry data was performed to analyze differences in cellular infiltrates and phenotype with treatment. We found CD8+ T cells that infiltrated tumors in mice treated with DPX-R9F/ldCPA expressed most CPIs tested (including PD-1, Tim-3, Lag-3, ICOS), and fewer expressed CTLA-4. Treatment induced infiltration of a unique population of CD8+ T cells that was PD-1+Tim-3+CTLA-4-, whereas CD8+ T cells present in untreated tumors were primarily PD-1+Tim-3+CTLA-4+. Tumor challenge was performed to evaluate combination of DPX-R9F/ldCPA treatment with anti-CTLA-4 (Clone 9D9) to potentially impact these resident CD8+ T cells. Mice treated with the triple combination had better control of C3 tumor growth compared to mice treated with DPX-R9F/ldCPA or anti-CTLA-4 separately. With tSNE analysis we were able to have a better model to observe differences in the CD8 population with DPX/ldCPA treatment and determine the mechanism for how certain checkpoint targets are impacting tumor infiltrating cells to further sculpt the anti-tumor response to increase efficacy of treatment. Citation Format: Ava Vila-Leahey, Alecia MacKay, Genevieve Weir, Marianne Stanford. T-distributed stochastic neighbor embedding (t-SNE) analysis of tumor infiltrating lymphocytes after treatment with a T cell activating therapy identifies a unique population of recruited CD8+ T cells and novel options for combination immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4989.

Life-long control of cytomegalovirus (CMV) by T resident memory cells in the adipose tissue results in inflammation and hyperglycemia.

Cytomegalovirus (CMV) is a ubiquitous herpesvirus infecting most of the world’s population. CMV has been rigorously investigated for its impact on lifelong immunity and potential complications arising from lifelong infection. A rigorous adaptive immune response mounts during progression of CMV infection from acute to latent states. CD8 T cells, in large part, drive this response and have very clearly been demonstrated to take up residence in the salivary gland and lungs of infected mice during latency. However, the role of tissue resident CD8 T cells as an ongoing defense mechanism against CMV has not been studied in other anatomical locations. Therefore, we sought to identify additional locations of anti-CMV T cell residency and the physiological consequences of such a response. Through RT-qPCR we found that mouse CMV (mCMV) infected the visceral adipose tissue and that this resulted in an expansion of leukocytes in situ. We further found, through flow cytometry, that adipose tissue became enriched in cytotoxic CD8 T cells that are specific for mCMV antigens from day 7 post infection through the lifespan of an infected animal (> 450 days post infection) and that carry markers of tissue residence. Furthermore, we found that inflammatory cytokines are elevated alongside the expansion of CD8 T cells. Finally, we show a correlation between the inflammatory state of adipose tissue in response to mCMV infection and the development of hyperglycemia in mice. Overall, this study identifies adipose tissue as a location of viral infection leading to a sustained and lifelong adaptive immune response mediated by CD8 T cells that correlates with hyperglycemia. These data potentially provide a mechanistic link between metabolic syndrome and chronic infection.

PF755 PD‐1 PATHWAY IS A KEY IMMUNOSUPPRESSIVE MECHANISM IN PATIENTS AFTER HAPLOIDENTICAL STEM CELL TRANSPLANTATION WITH TCR AB–DEPLETION

Background:Programmed cell death protein 1 (PD‐1) is a surface receptor expressed normally on distinct immune cells. It's well known PD‐1 pathway blockade is a negative immune regulatory mechanism that has detrimental effects on anti‐tumor immunity. Currently some studies have revealed different immune cells overexpressed PD‐1 as a predictable marker of leukemia relapse. On the other hand its assay after allo‐HSCT could be controversial due to alternative GVHD prophylaxis regimens that might affect PD‐1 expression, such as post‐transplant high dose Cyclophosphamide (PT‐Cy) or graft‐manipulated TCR αβ‐depletion are taken place if mismatched or haploidentical donors are used.Aims:To evaluate PD‐1 expression on bone marrow (BM) resident CD8+ memory T‐cell subsets in leukemia patients after allo‐HSCT and its input on immunosuppressive mechanisms of different GVHD prophylaxis regimens after allo‐HSCT.Methods:BM samples were collected from 56 leukemia patients with a median age of 33 years on day +30, +60 and +90 after allo‐HSCT. Detailed patients characteristics are presented in Table 1. Flow cytometry analysis was performed on BD FACS Canto II (Becton Dickinson, USA) to define CD8+ T‐memory subsets with PD‐1 expression: T‐naive and T‐stem cell memory (Tnv+Tscm) –CD45R0‐CCR7+CD28+; T‐central memory (Tcm) ‐ CD45R0+CCR7+CD28+; T‐transitional memory (Ttm) ‐ CD45R0+CCR7‐CD28+; T‐effector memory (Tem) ‐ CD45R0+CCR7‐CD28‐; T‐terminal effector (Tte) ‐ CD45R0‐CCR7‐CD28‐. Sysmex XE‐2100 was used to calculate absolute count of different CD8+ T‐ memory cell subsets with PD‐1. Mann–Whitney U test was used for nonparametric data analysis. A p‐value less than 0.05 was considered as significant.Table 1. Patients characteristics.Results:During all follow‐up period PD‐1 expression remains increased on all CD8+ T‐memory cell subsets after TCR αβ‐depletion compared to PT‐Cy–based and classical immunosuppressive regimen based on Cyclosporine+MMF. On day +30 we observe significantly higher expression of PD‐1 on Tnv+scm, Tcm, Ttm after TCR αβ‐depletion compared to PT‐Cy or classical regimen (p < 0.05). On day +60 PD‐1 is still overexpressed on Tnv+scm after TCR αβ‐depletion compared to PT‐Cy or classical regimen (p = 0.008). Moreover on day +60 and +90 PD‐1 expression is elevated on terminally differentiated CD8+ T cells (Ttm, Tem) after TCR αβ‐depletion comparing PT‐Cy or classical regimen (р<0.05). (Figure 1).imageSummary/Conclusion:According to our data PD‐1 is overexpressed on distinct subsets of BM resident CD8+ T‐memory cells after TCR αβ‐depletion during follow‐up period in first 3 months after allo‐HSCT. Since other immune signaling pathways are inhibited due to the other immunosupressive agents had been used, PD‐1 pathway seems to be a key mechanism of immunological tolerance after haploidentacal allo‐HSCT with TCR αβ‐depletion.image

Prognostic significance of resident CD103+CD8+T cells in human colorectal cancer tissues

The prognostic significance and clinical implications of resident CD103+CD8+T cells in human colorectal cancer tissues still remains largely unexplored. In our present study, we aimed to characterize the resident CD8+T cells in human colorectal cancer tissues by using double staining of CD103 and CD8, and further evaluated the prognostic significance of resident CD8+T cells in colorectal cancer. We found that the OS rate of the colorectal cancer patients with higher infiltration of CD8+T cells, or with higher numbers of resident CD103+CD8+T cells, or with higher ratio of CD103+CD8+T cells over total CD8+T cells in cancer tissues was significantly better than that of the patients with lower infiltration of CD8+T cells, or with lower numbers of resident CD103+CD8+T cells, or with higher ratio of CD103+CD8+T cells over total CD8+T cells in cancer tissues, respectively. Moreover, higher infiltration of CD8+T cells in colorectal cancer tissues was significantly and inversely correlated with advanced TNM stage. Higher numbers of resident CD103+CD8+T cells in colorectal cancer tissues were significantly and inversely correlated with distant metastasis status. Higher ratio of CD103+CD8+T cells over total CD8+T cells in colorectal cancer tissues was significantly and inversely correlated with age status. The COX model analysis demonstrated that higher infiltration of CD8+T cells, higher numbers of resident CD103+CD8+T cells, or higher ratio of CD103+CD8+T cells over total CD8+T cells in colorectal cancer tissues, could serve as independent prognostic predictors for colorectal cancer patients. Taken together, our present study demonstrated the density of tumor infiltrating CD8+T cells or the numbers of resident CD103+CD8+T cells in colorectal tissues could be used as an important prognostic predictor for this malignancy.

Dynamic changes in the immune infiltrate within hepatocellular carcinoma tumor correlate with response to PD-1 blockade.

e15644 Background: Hepatocellular carcinoma (HCC) is the second leading cause of cancer deaths in men worldwide, and is the most rapidly rising cause of cancer mortality in the US. Patients with resectable disease experience high rates of locoregional recurrence, and there are few effective systemic treatment options available. PD-1 blockade was recently approved for 2nd line therapy, and there are promising response rates seen in the 1st-line setting. However, only 20% of patients achieve significant response, thus we must investigate further the tumor-immune microenvironment to understand how to better modulate the immune system in this highly immunosuppressive organ. Methods: We used mass cytometry (CyTOF) and cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to study protein and transcriptomics of untreated tumor and tumor adjacent tissue, on a single-cell level. Results: Paired CyTOF analysis of 10 tumor lesions/adjacent tissues identified a significant increase in frequency of CD4+ T cells and T regulatory cells, a significant decrease in NK cells and neutrophils, and no difference in CD8+ T cells, macrophages, or dendritic cells (DCs) in the tumor microenvironment compared to the adjacent liver tissue. We also analyzed four patients who received nivolumab and subsequently went on to have their liver tumors resected; two out of the four patients treated had near-complete necrosis of their tumors (radiographically and histologically). Of note, the tumors of the two patients with significant pathologic response contained a significantly higher number of CD103+ tissue resident CD8+ and CD4+ T cells, compared to adjacent tissue. Moreover, activated CD8+ T cells (TIGIT+, PD-1+, TIM3+, and/or CD38+) were significantly higher in the tumor of nivolumab responders, indicating a tumor-specific response. We will also present single cell transcriptomic analyses of T cell and myeloid populations in responders and non-responders. Interestingly, high levels of nivolumab were detected on PD-1+ T cells in the tumor in all 4 patients; we will discuss transcriptional differences between nivolumab-targeted T cells in responders and non-responder patients. Conclusions: Based on this preliminary data we will be opening a clinical trial of neoadjuvant PD-1 blockade in HCC (NCT# pending) in March 2019.

Differentiation and dynamics of circulating and tissue-resident memory CD8 T cells in the lung

Abstract Memory CD8 T cells are capable of providing robust heterosubtypic immunity to Influenza. In contrast to other tissues, relatively little is known about the differentiation and localization of the memory CD8 T cell subsets within the lung. We utilized intravital 2-photon microscopy of live mice to examine the real-time dynamics of circulating and/or resident memory (Trms) CD8 T cells in the lung. LCMV-induced transgenic P14 CD8 memory T cells, which are exclusively circulating, were highly motile, while Influenza-experienced P14s, which are comprised of both circulating and resident memory CD8 T cells, exhibited heterogenous motility patterns including both highly motile cells and those exhibiting a slow to moderate motility pattern. Selective depletion of circulating memory CD8 T cells resulted in the loss of highly motile cells, suggesting that lung CD8 Trms exhibit slow to moderate motility. Intranasal administration of cognate peptide or recombinant Influenza virus infection induced the arrest of a large number of LCMV-experienced circulating P14s in the lung shortly after administration in an antigen-specific manner. Interestingly, within 12–48 hours post stimulation, the majority of lung-residing LCMV-experienced circulating P14s became un-arrested and began a moderate scanning motility phenotype that persisted in a substantial fraction of P14s and correlated with the generation of secondary memory CD103+ Trms in the lung. Overall, these data shed light into the differentiation and dynamics of circulating and resident CD8 memory T cells in the lung.

Pulmonary resident effector CD4+ T cells fail to effectively utilize glycolysis

Abstract The transition of T cells from naïve to effector cells has been reported to require a metabolic shift from oxidative phosphorylation to glycolysis for the production of effector molecules such as IFN-γ. This phenomenon is primarily restricted to study of splenic T cells and it is largely unknown if T cells in other organs undergo the same metabolic shift. Intranasal vaccination establishes a pool of lung tissue-resident, effector memory T cells (trTem) that are typically sufficient to control a secondary infection via production of cytokines. Therefore, we hypothesized trTem would be highly glycolytic upon activation. To test this hypothesis, we compared the metabolic activity of both trTem and circulating Tem (cTem) isolated from mice vaccinated with either Bordetella pertussis or Francisella tularensis. As expected, regardless of the vaccinating pathogen, CD4+ cTem readily shifted to glycolysis following activation with anti-CD3/CD28 beads. In contrast, CD4+ trTem cells did not shift to glycolysis following the same activation scheme, nor did they increase mitochondrial respiration as a mechanism to compensate for the absence of glycolysis. Consistent with an inability to shift to glycolysis, ex vivo stimulation of trTem with antigen also resulted in limited uptake of glucose as compared to cTem. These data suggested that, unlike previous reports, pulmonary CD4+ trTem do not rapidly shift to glycolysis even in the presence of antigen. Together these data highlight the unique environment of the pulmonary compartment and present critical information that will influence our understanding of pulmonary T cell responsiveness during infection which may shape future vaccination strategies.

Indiscriminate nature of lung resident CD8+ TRM cells reactivation and their varied reactivation profiles

Abstract CD8+ tissue resident memory (TRM) cells are poised at the portals of infection and confer immediate protection upon reinfection. Despite the critical role of CD8+ TRM cells in long-term protective immunity, the precise mechanics governing their reactivation in-situ are unclear. Seminal work by Leo Lefrançois' group showed that circulating (TCIRC) memory CD8+ T cell depends on CD11c+ antigen-presenting cells (APCs) for reactivation, however, TRM cells are reactivated within peripheral tissues, raising the question of whether the same principle is conserved. We examined this question, focusing on influenza infection, because APCs in the lung are well-defined and lung-resident TRM cells are critical for heterologous immunity to influenza. Using a TCR transgenic Nur77 reporter to delineate TCR-specific from bystander activation in CD8+ TRM cells, we characterized the superior reactivation kinetics in CD8+ TRM cell compared to CD8+ TCIRC. We also identified unique TCR-specific signatures and bystander-specific signatures in the kinetics of CD8+ TRM cells and TCIRC cells reactivation respectively. While we observed the strict requirement for CD11c+APCs in TCR-driven CD8+ TCIRC reactivation, CD8+ TRM cells exhibit promiscuity in their interactions with antigen-presenting partners in that they can be reactivated by both hematopoetic and non-hematopoetic cells. This study explains how the anatomical location of CD8+ TRM cells can confer unique advantage by demonstrating their superior reactivation kinetics and the indiscriminate nature of CD8+ TRM cell reactivation as well as showcasing the transcription profiles of TRM and TCIRC cells following different modes of activation.

BHLHE40 programs mitochondrial regulation of resident CD8+ T cell fitness and functionality

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.

Endometrial Tumor Microenvironment Alters Human NK Cell Recruitment, and Resident NK Cell Phenotype and Function.

Endometrial Cancer is the most common cancer in the female genital tract in developed countries, and with its increasing incidence due to risk factors such as aging and obesity tends to become a public health issue. However, its immune environment has been less characterized than in other tumors such as breast cancers. NK cells are cytotoxic innate lymphoid cells that are considered as a major anti-tumoral effector cell type which function is drastically altered in tumors which participates to tumor progression. Here we characterize tumor NK cells both phenotypically and functionally in the tumor microenvironment of endometrial cancer. For that, we gathered endometrial tumors, tumor adjacent healthy tissue, blood from matching patients and healthy donor blood to perform comparative analysis of NK cells. First we found that NK cells were impoverished in the tumor infiltrate. We then compared the phenotype of NK cells in the tumor and found that tumor resident CD103+ NK cells exhibited more co-inhibitory molecules such as Tigit, and TIM-3 compared to recruited CD103− NK cells and that the expression of these molecules increased with the severity of the disease. We showed that both chemokines (CXCL12, IP-10, and CCL27) and cytokines profiles (IL-1β and IL-6) were altered in the tumor microenvironment and might reduce NK cell function and recruitment to the tumor site. This led to hypothesize that the tumor microenvironment reduces resident NK cells cytotoxicity which we confirmed by measuring cytotoxic effector production and degranulation. Taken together, our results show that the tumor microenvironment reshapes NK cell phenotype and function to promote tumor progression.

SUN-174 Severe Insulin Resistance and Diabetic Ketoacidosis after Brentuximab Vendotin and Cyclosporin Treatment

Introduction: Brentuximab Vendotin(BV) is antibody-drug conjugate that selectively delivers cytotoxic agent, monomethyl auristatin E, targeting cells expressing surface CD30, treatment approved for refractory Hodgkin’s lymphoma. Previous research suggested MDR1 inhibition (cyclosporine) can overcome BV resistance. Case: 23 year old male with relapsed and refractory classical Hodgkin lymphoma underwent clinical trial with BV and Cyclosporin( CyA). He was given one dose of BV( 1.8mg/kg) and CyA ( 725mg 2 times a day for 5 days). A week after BV and 3 days after the last dose of CyA, patient had acute onset of hyperglycemia and ketoacidosis; glucose 435 mg/dL, bicarbonate 14 mmol/L, anion gap 14( 8-14), beta hydroxybutyrate 40.3 mg/dL (0.0-3.0 mg/dL), urine ketone +3, C-peptide 9.0 ng/ml (0.8-3.5 ng/ml). Serum cyclosporine level was high as 453 ng/ml (150-300ng/ml) at the time of presentation, which was reduced to 116 ng/ml within a week. He had no previous diabetes history. His severe hyperglycemia and metabolic acidosis persisted after aggressive hydration and multiple daily insulin injections. He was started on modified intravenous insulin infusion protocol. Within 24 hours, his glycemic control was improved at goal requiring average 34 units/h of regular insulin with NPO status. This was transitioned to U500 regular insulin divided dose injection every 8 hours with holding criteria. 10 days after initial clinical presentation, patient's insulin requirement was abruptly reduced and eventually discontinued. Patient was discharged and glucose level remained within reference range (80-128 mg/dl) without additional medical management from follow-up. Patient had completed given clinical trial with reduced dose of BV and CyA, and there was no further similar episode. During the hospital course, underlying endocrine disease related to insulin resistance was ruled out including thyroid, glucagon, cortisol, catecholamine, or growth hormone excess. Autoimmune workup was negative except elevated ESR. Insulin autoantibody panels were negative. Pancreatitis and infections were also ruled out. Lymphocytes were reduced and monocytes were increased in CBC during the episode. Peripheral blood mononuclear cell (PBMC) analysis by flow cytometry showed that absolute B and T cell counts were low but in the residual CD4+ and CD8+ T cells, expression of CD25 and PD-1 was increased. Major portion of the PBMC did not stain with our standard immune phenotype panel, which may include plasma cells, NKT, or dendritic cells. Discussion: Adverse events ≥ Grade 3 hyperglycemia (12%) has been reported with BV treatment. On the other hand, CyA is known to be related to insulin resistance as well. This is the first case report of a patient who developed rare condition of severe insulin resistance and diabetic ketoacidosis in the context of hyperinsulinemia likely due to impaired insulin signaling induced by BV and CyA.