T-cell Accumulation Research Articles

Tumor-Infiltrating Regulatory T-cell Accumulation in the Tumor Microenvironment Is Mediated by IL33/ST2 Signaling.

Regulatory T cells (Treg) are enriched in the tumor microenvironment (TME) and suppress antitumor immunity; however, the molecular mechanism underlying the accumulation of Tregs in the TME is poorly understood. In various tumor models, tumor-infiltrating Tregs were highly enriched in the TME and had significantly higher expression of immune checkpoint molecules. To characterize tumor-infiltrating Tregs, we performed bulk RNA sequencing (RNA-seq) and found that proliferation-related genes, immune suppression-related genes, and cytokine/chemokine receptor genes were upregulated in tumor-infiltrating Tregs compared with tumor-infiltrating CD4+Foxp3- conventional T cells or splenic Tregs from the same tumor-bearing mice. Single-cell RNA-seq and T-cell receptor sequencing also revealed active proliferation of tumor infiltrating Tregs by clonal expansion. One of these genes, ST2, an IL33 receptor, was identified as a potential factor driving Treg accumulation in the TME. Indeed, IL33-directed ST2 signaling induced the preferential proliferation of tumor-infiltrating Tregs and enhanced tumor progression, whereas genetic deletion of ST2 in Tregs limited their TME accumulation and delayed tumor growth. These data demonstrated the IL33/ST2 axis in Tregs as one of the critical pathways for the preferential accumulation of Tregs in the TME and suggests that the IL33/ST2 axis may be a potential therapeutic target for cancer immunotherapy.

Newly characterized decidual Tim-3+ Treg cells are abundant during early pregnancy and driven by IL-27 coordinately with Gal-9 from trophoblasts.

What is the mechanism of Tim-3+ regulatory T (Treg)-cell accumulation in the decidua during early pregnancy and is its disruption associated with recurrent pregnancy loss (RPL)? IL-27 and Gal-9 secreted by trophoblasts activate the Tim-3 signaling pathway in CD4+ T cells and Treg cells and so promote accumulation of Tim-3+ Treg cells, the abnormal expression of IL-27 and Gal-9 is associated with impaired immunologic tolerance in RPL patients. Tim-3+ Treg cells are better suppressors of Teff cell proliferation, and display higher proliferative activity than Tim-3- Treg cells. Tim-3+ Treg cells are tissue-specific promoters of T-cell dysfunction in many tumors. These cells express a unique factor that influences and shapes the tumor microenvironment. The animal study included 80 normal pregnant mice. In human study, decidua tissues in the first trimester for flow cytometry analysis were collected from 32 normal pregnant women and 23 RPL patients. Placenta tissues for immunohistochemistry analysis were collected from 15 normal pregnant women. Placenta tissues for western blot analysis were collected from 5 normal pregnant women, 5 RPL patients and 5 women who have experienced one miscarriage. Blood samples for in vitro experiments were collected from 30 normal pregnant women. This study was performed between January 2017 and March 2019. In this study, we investigated the kinetics of Tim-3+ CD4+ T-cell accumulation, and the proportions of Tim-3+ Treg cells throughout murine pregnancies using flow cytometry. We compared Tim-3 expression on decidual CD4+ T cells and Treg cells during normal pregnancies with expression on the same cell populations in women suffering from RPL. IL-27 and Gal-9 transcription and protein expression in the placenta were determined by RT-PCR and western blot, respectively. An in vitro co-culture model consisting of peripheral CD4+ T cells and primary trophoblasts from early pregnancy was used to mimic the maternal-fetal environment. The percentage of Tim-3+ Treg cells present in mouse uteri fluctuates as gestation proceeds but does not change in the spleen. Levels of Tim3+ Treg cells in uteri peaked at pregnancy Day 6.5 (E 6.5), then progressively diminished, and fell to non-pregnant levels by E18.5. In pregnant mice, Tim-3+ Treg cells constituted 40-70% of Treg cells in uteri but were present at much lower abundance in spleens. About 60% of decidual Treg cells were Tim-3 positive at E6.5. Of these decidual Tim3+ Treg cells, nearly 90% were PD-1 positive. However, only about 16% of Tim3- Treg cells expressed PD-1. Blocking the Tim-3 signaling pathway decreased the proportion of Treg cells and led to embryo resorption. Moreover, much lower Tim-3 expression was observed on CD4+ T cells and Treg cells in women who had suffered from RPL at 6-9 gestational weeks compared with those who had normal pregnancies at matched gestations. In a normal pregnancy, Tim-3 expression on decidual CD4+ T cells is induced initially by IL-27. Then Gal-9-Tim-3 interaction promotes differentiation of decidual Tim-3+ CD4+ T cells into Treg cells. IL-27 and Gal-9 cooperatively induced Tim-3+ Treg cells in vitro. N/A. We did not investigate the kinetics of human decidual Tim-3+ CD4+ T and Tim-3+ Treg cell populations throughout pregnancy due to limited availability of second and third trimester decidua. In addition, functional suppressive data on the decidual Tim-3+ Treg cells are lacking due to limited and low quantities of these cells in decidua. These findings might have therapeutic clinical implications in RPL. This study was supported by research grants from the National Natural Science Foundation of China (No. 81871186) and National Key Research & Developmental Program of China (2018YFC1003900, 2018YFC1003904). The authors declare no conflict of interest.

A Genomic Approach to Integration of T Category and the Tumor Immune Microenvironment As Part of A Decision Algorithm for Radiation in Prostate Cancer

The nature of the tumor immune microenvironment (TIME) has been shown to have prognostic significance and implications for therapeutic response in many different cancers. Emerging data suggests that radiation may alter the TIME by promoting accumulation of immunosuppressive regulatory T-cells (Treg). Radiation is a mainstay of treatment for prostate cancer (PCa), but the importance of TIME composition with respect to radiation response in PCa has not been fully elucidated. Here, we leverage clinical and genomic data from The Cancer Genome Atlas (TCGA) to further characterize the TIME in a cohort of irradiated T2-T3 PCa patients, and assess relationships between stage, TIME composition, and recurrence free survival (RFS) in the setting of radiation therapy. Tumors treated with radiation were stratified by T2 vs. T3 disease, as well as “High” or “Low” values for measurement of T-regulatory (Treg) cell-specific markers, calculated relative to the mean values of 490 tissue samples. Normalized RNA Seq by expectation maximization (RSEM) values from the TCGA were collected along with expression data for Treg specific markers (CD25, FOXP3, and CTLA4). Clinical data was obtained from the Broad Institute’s Firebrowse tool. RFS was defined as length of time from end of treatment to biochemical recurrence and/or locoregional recurrence and was assessed using Kaplan-Meier estimates. Survival curves were compared using the log-rank test. 490 PCa specimens were retrieved from the TCGA Pan-Cancer Atlas. Of these, 106 tumors were treated with radiation. Patients with T3 disease (n = 67) on initial pathologic diagnosis and high intratumoral co-expression of CD25, FOXP3, and CTLA4 treated with radiation demonstrated significantly decreased estimated 8-year RFS compared to those patients with T3 disease lacking high co-expression of all three markers (p = 0.0084). There was no significant difference in estimated 8-year RFS after radiation in T2 patients (n = 39) with high co-expression of all Treg specific markers compared to those lacking high co-expression of these markers (p = 0.40). Various studies have attempted to decipher the interplay of radiation and Treg cells, the majority having been conducted on mouse models. Our analysis of human specimens revealed that radiation in PCa with extracapsular extension (T3 disease) and Treg specific marker elevation portends significantly worse prognosis than irradiated T3 disease without Treg specific marker elevations - this relationship was not seen in T2 disease. These results suggest a prognostic benefit from risk stratification of PCa patients by both T category and Treg specific activation markers, and warrant investigation into immune checkpoint inhibitors such as anti-CTLA4 antibody as part of the treatment paradigm in this patient subset to enhance anti-tumor effects by inhibiting Treg activity.

Особенности регуляции и функции инфламмасом при воспалительных заболеваниях органов женской репродуктивной системы

Chronic inflammation underlies the progression of many human systemic disorders, including a number of gynecological diseases such as endometriosis and chronic endometritis. Inflammasomes play a special role in the regulation of intercellular interactions under physiological and pathological conditions. The pathogenesis of chronic inflammation assumes a gradual course, accompanied by prolonged maintenance active state of inflammasomes due to the excessive accumulation of intracellular and extracellular endogenous molecules released from destroyed cells – DAMPs (damage-associated molecular patterns), their ineffective utilization by autophagy and stress-reducing cell systems, local production of pro-inflammatory cytokines (IL-1β, IL-18), as well as ROS (reactive oxygen species), accumulation of macrophages and T-lymphocytes, tissue damage and the development of fibrosis. The proteins of the inflammasome complex can serve as targets for drugs, which will increase the efficiency of therapy for inflammation-associated diseases. This review presents the current data on the role of NLRP3-inflammasomes in immune regulation and the molecular mechanisms underlying the pathogenesis of endometriosis and chronic endometritis. Understanding the molecular mechanisms of inflammasome activation and the discovery of their effective inhibitors, as well as the assessment of the therapeutic potential in these pathologies, is an important direction of future research. Keywords: inflammasome, inflammation, innate immunity, endometriosis, chronic endometritis

A Genetic Screen to Identify Gain- and Loss-of-Function Modifications that Enhance T-cell Infiltration into Tumors.

T-cell-mediated cancer immunotherapies, including anti-PD-1 and T cells expressing chimeric antigen receptors (CAR-T cells), are becoming standard treatments for many cancer types. CAR-T therapy, in particular, has been successful in treating circulating, but not solid, tumors. One challenge limiting immunotherapy success is that tumors lacking T-cell infiltration do not respond to treatment. Therefore, one potential strategy to overcome resistance is to enhance the ability of T cells to traffic into tumors. Here, we describe an unbiased in vivo genetic screen approach utilizing the Sleeping Beauty mutagenesis system to identify candidate genes in T cells that might be modified to drive intratumoral T-cell accumulation. This screen identified over 400 candidate genes in three tumor models. These results indicated substantial variation in gene candidate selection, depending on the tumor model and whether or not mice were treated with anti-PD-1, yet some candidate genes were identified in all tumor models and with anti-PD-1 therapy. Inhibition of the most frequently mutated gene, Aak1, affected chemokine receptor expression and enhanced T-cell trafficking in vitro and in vivo Screen candidates should be further validated as therapeutic targets, with particular relevance to enhancing infiltration of adoptively transferred T cells into solid tumors.

THE IMPACT OF OXYGEN FREE RADICAL SCAVENGER ON ACUTE REJECTION IN A HISTOCOMPATIBLE MINIATURE SWINE MODEL

Introduction: Vascularized Composite Allotransplantations (VCAs), like solid organ transplants, are very susceptible to ischemia-reperfusion injury (IRI). IRI leads to tissue hypoxia and the formation of oxygen free radicals which triggers T-cell accumulation and complement activation, causing increased antigenicity of the transplanted tissue. In this study we demonstrate the effect of two oxygen free radical scavengers on acute rejection in a histocompatible miniature swine model. Materials and Methods: To conduct this study, we used our well-established myocutaneous flap model in a major histocompatibility complex (MHC)-defined miniature swine. MHC class I and II antigens were matched between donors and recipients. Myocutaneous allografts consisted of an isolated skin pad with the underlying gracilis muscle. All flaps were treated with similar concentrations of either Saline (9mg/ml, n=2), Sodium Iodide (10mg/ml, n=3) or Hydrogen Sulfide (H2S) (11.6mg/ml). Flaps were subjected to an intermediate ischemia time of 3 hours at 4°C immediately after harvest. End of ischemia time was marked by unclamping the anastomosis. Daily blood draws were performed on postoperative day (POD) 0 to 7, 14 and end of study. Biopsies for histological analysis were collected on POD 4, 10, 14 and end of study. Rejection was assessed by an independent pathologist following the Banff criteria (Grade 0-IV) for skin containing VCA. Results: On POD 4, flaps treated with H2S showed Grade 0 of rejection according to the Banff classification, compared to Grade I in the Sodium Iodide group and Grade II in the Saline. There was no significant difference in grade of rejection between these three groups on or after POD 10.Discussion: Treatment of ischemic myocutaneous flaps with oxygen free radical scavengers delayed acute rejection when compared to saline treated controls. Currently, standard immunosuppression regimens and techniques for graft preservation of VCA do not enable preconditioning of the donor or ischemic-free storage. Therefore, IRI is unavoidable in VCA so far. Diminishing the impact of IRI could reduce local secretion of pro-inflammatory cytokines and sequester recruitment of alloreactive immune cells to the transplant, delaying acute rejection episodes during the immediate post-operative period. Conclusion: With this study we demonstrate the impact of oxygen free radical scavengers on IRI in an immunocompetent and genetically well-defined large animal model. Treating myocutaneous flaps accordingly abrogates early rejection histopathologically at POD 4. Further experiments are ongoing to titrate the impact of these scavengers across later timepoints.

Molecular Pathways of Colon Inflammation Induced by Cancer Immunotherapy

Checkpoint blockade with antibodies specific for the PD-1 and CTLA-4 inhibitory receptors can induce durable responses in a wide range of human cancers. However, the immunological mechanisms responsible for severe inflammatory side effects remain poorly understood. Here we report a comprehensive single-cell analysis of immune cell populations in colitis, a common and severe side effect of checkpoint blockade. We observed a striking accumulation of CD8 Tcells with highly cytotoxic and proliferative states and no evidence of regulatory Tcell depletion. Tcell receptor (TCR) sequence analysis demonstrated that a substantial fraction of colitis-associated CD8 Tcells originated from tissue-resident populations, explaining the frequently early onset of colitis symptoms following treatment initiation. Our analysis also identified cytokines, chemokines, and surface receptors that could serve as therapeutic targets for colitis and potentially other inflammatory side effects of checkpoint blockade.

Reduced Nhe1 (Na+-H+ Exchanger-1) Function Protects ApoE-Deficient Mice From Ang II (Angiotensin II)-Induced Abdominal Aortic Aneurysms.

IgE-mediated activation of Nhe1 (Na+-H+ exchanger-1) induces aortic cell extracellular acidification and promotes cell apoptosis. A pH-sensitive probe pHrodo identified acidic regions at positions of macrophage accumulation, IgE expression, and cell apoptosis in human and mouse abdominal aortic aneurysm (AAA) lesions. Ang II (angiotensin II)-induced AAA in Nhe1-insufficient Apoe-/-Nhe1+/- mice and Apoe-/-Nhe1+/+ littermates tested Nhe1 activity in experimental AAA, because Nhe1-/- mice develop ataxia and epileptic-like seizures and die early. Nhe1 insufficiency reduced AAA incidence and size, lesion macrophage and T-cell accumulation, collagen deposition, elastin fragmentation, cell apoptosis, smooth muscle cell loss, and MMP (matrix metalloproteinase) activity. Nhe1 insufficiency also reduced blood pressure and the plasma apoptosis marker TCTP (translationally controlled tumor protein) but did not affect plasma IgE. While pHrodo localized the acidic regions to macrophage clusters, IgE expression, and cell apoptosis in AAA lesions from Apoe-/-Nhe1+/+ mice, such acidic areas were much smaller in lesions from Apoe-/-Nhe1+/- mice. Nhe1-FcεR1 colocalization in macrophages from AAA lesions support a role of IgE-mediated Nhe1 activation. Gelatin zymography, immunoblot, and real-time polymerase chain reaction analyses demonstrated that Nhe1 insufficiency reduced the MMP activity, cysteinyl cathepsin expression, IgE-induced apoptosis, and NF-κB activation in macrophages and blocked IgE-induced adhesion molecule expression in endothelial cells. A near-infrared fluorescent probe (LS662) together with fluorescence reflectance imaging of intact aortas showed reduced acidity in AAA lesions from Nhe-1-insufficient mice. This study revealed extracellular acidity at regions rich in macrophages, IgE expression, and cell apoptosis in human and mouse AAA lesions and established a direct role of Nhe1 in AAA pathogenesis.

Jak Inhibitors Reverse Vitiligo in Mice but Do Not Deplete Skin Resident Memory T Cells

Jak Inhibitors Reverse Vitiligo in Mice but Do Not Deplete Skin Resident Memory T Cells

Abstract A78: IFNg-activated lymphatic vessels express PD-L1 and suppress effector T-cell function in melanoma

Abstract Peripheral tissues adopt mechanisms of immune suppression that counteract protective immunity to prevent immunopathology and are co-opted by tumors for immune escape. In tumors, cytotoxic, antigen-specific T cells produce IFNg and activate multiple mechanisms of adaptive immune resistance, including upregulation of the T-cell inhibitory molecule programed death ligand 1 (PD-L1). Tumor cell PD-L1 expression does not predict response to anti-PD-L1 immunotherapy, indicating, however, that other sources of PD-L1 in tumor microenvironments may contribute to PD-L1-dependent immune suppression. Lymphatic vessels facilitate antigen and dendritic cell transport to draining lymph nodes and are required for antitumor immunity. Interestingly, lymphatic endothelial cells in lymph nodes are tolerogenic at steady-state and constitutively express PD-L1 to delete self-reactive CD8+ T cells. However, whether lymphatic endothelial cells in tumor microenvironments are also directly immunosuppressive remains unclear. Here we test the hypothesis that peripheral, tumor-associated lymphatic vessels acquire immunosuppressive mechanisms, e.g., PD-L1 expression, that limit effector CD8+ T-cell accumulation and function in melanoma. We demonstrate that host nonhematopoietic, nontumor, PD-L1 limits CD8+ T-cell accumulation in murine melanoma. Further, we show that tumor-associated lymphatic endothelial cells upregulate PD-L1 in response to IFNg produced by tumor-infiltrating, antigen-specific CD8+ T cells. As such, loss of IFNgR by lymphatic endothelial cells improves CD8+ T-cell accumulation in murine melanomas, thereby enhancing tumor control and improved overall survival. Importantly, this robust lymphatic vessel-dependent tumor control was revealed in the context of murine melanomas with significant UVB-induced somatic mutation burden, consistent with a cytotoxic T cell-mediated negative feedback mechanism. Consequently, we present IFNg-mediated activation of tumor-associated lymphatic vessels as a new component of adaptive immune resistance, suggesting that context-dependent lymphatic vessel function may provide novel, targetable mechanisms of immune control in melanoma. Citation Format: Ryan S. Lane, Alec P. Breazeale, Amanda W. Lund. IFNg-activated lymphatic vessels express PD-L1 and suppress effector T-cell function in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A78.

Abstract IA19: Lymphatic vessels: Barriers to adaptive immunity in melanoma

Abstract Mechanisms of immune suppression in peripheral tissues counteract protective immunity to prevent immunopathology and are co-opted by tumors for immune escape. Nonhematopoietic cells, as the resident tissue scaffold, play critical roles in regulating the infiltration, retention, function, and exit of tissue- and tumor-infiltrating leukocytes. While lymphatic vessels facilitate T-cell priming through dendritic cell and antigen delivery to lymph nodes, they also facilitate regional metastasis and exert multiple immune-suppressive mechanisms that maintain peripheral tolerance in steady state lymph nodes. We hypothesized that in tumors peripheral lymphatic vessels exhibit context-dependent function that limits local effector CD8+ T-cell accumulation in melanoma. Our work demonstrates that lymphatic vessels both maintain homeostatic mechanisms of peripheral tolerance in poorly inflamed tumors to limit immune surveillance and activate mechanisms of adaptive immune resistance in the context of potent cytotoxic T-cell activity. Consequently, we present tumor-associated lymphatic vessels as a new, anatomic immune checkpoint in melanoma. Our work suggests that understanding context-dependent lymphatic vessel function will continue to reveal novel mechanisms of immune control and metastasis that may be targeted for therapeutic response. Citation Format: Amanda W. Lund. Lymphatic vessels: Barriers to adaptive immunity in melanoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr IA19.

Abstract A51: Rationally improving T cell-mediated cancer immunotherapies using Sleeping Beauty mutagenesis to identify novel therapeutic targets

Abstract Expanding immunotherapy success to a broader patient population is a top priority in the field, and T-cell infiltration into tumors appears to be an important prerequisite for the success of both immune checkpoint blockade and chimeric antigen receptor T cell (CAR-T) therapies. The data presented here are results from multiple genetic screens designed to identify T-cell genes that promote their intratumoral infiltration. The gene candidates we discovered may be leveraged to enhance T cell-mediated cancer immunotherapies, with particular translational potential for CAR-T therapies. We designed a unique forward genetic screen approach to identify T-cell genes that contribute to intratumoral T-cell accumulation using Sleeping Beauty mutagenesis in T cells. Sleeping Beauty has a unique advantage over CRISPR and shRNA screens because it allows us to induce gain-of-function mutations, in addition to loss-of-function mutations, across the entire genome. Our systematic screen approach was performed in immunocompetent murine models of melanoma and lymphoma that preserve the complexity of the tumor microenvironment. Further, we carried out additional screens where mutagenized mice were treated with immune checkpoint inhibitor anti-PD-1 in order to assess how the selective pressures of this therapy influence the mutation spectra. Our screens identified 368 genes expected to impact intratumoral T-cell infiltration. Some of the candidate genes we identified were previously found to directly regulate intratumoral CD8+ T cell infiltration (e.g., IL-23R), and the candidate gene list was significantly enriched for T cell anergy genes (P = 0.0074). Fifty-one genes were mutated in more than one mouse and represent strong gene candidates for functional validation. Importantly, none of the classical immune checkpoint molecules were identified in our screens, demonstrating the novelty of our results since these genes likely are not currently being investigated as drug targets to enhance immunotherapy. We hypothesize that the biologic functions of these genes have the potential to modify important T-cell functions including trafficking to the tumor, clonal expansion, and sustained viability once inside the tumor. Interestingly, anti-PD-1 treatment had a profound effect on candidate gene selection. One gene in particular was much more frequently mutated in T cells from mice treated with anti-PD-1 therapy than untreated mice. In a series of validation experiments, we demonstrate that modulating one of these gene candidates with a small-molecule inhibitor impacts tumor growth and CD8+ T-cell infiltration in mice when combined with anti-PD-1 therapy. From this, we conclude that this screen approach is a viable way to discover novel immunotherapy targets. Current studies are exploring potential mechanisms by which this candidate gene impacts T-cell function and assessing therapeutic impact of overexpression in a CAR-T system. Citation Format: Laura M. Rogers, Adam J. Dupuy, George J. Weiner. Rationally improving T cell-mediated cancer immunotherapies using Sleeping Beauty mutagenesis to identify novel therapeutic targets [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A51.

Diagnosis-independent loss of T-cell costimulatory molecules in individuals with cytomegalovirus infection

Major depressive disorder (MDD) is associated with physiological changes commonly observed with increasing age, such as inflammation and impaired immune function. Age-related impaired adaptive immunity is characterized by the loss of naive T-cells and the reciprocal accumulation of memory T-cells together with the loss of T-cell co-stimulatory molecules. Additionally, the presence and activity of cytomegalovirus (CMV) alters the architecture of the T-cell compartment in a manner consistent with premature aging. Because CMV is also thought to reactivate with psychological stress, this study tested whether MDD influences age-related phenotypes of T-cell populations in the context of CMV infection in young and middle-aged adults. Morning blood samples from volunteers with a DSM-IV diagnosis of MDD (n = 98, mean age(SD) = 36(10) years, 74.5% female, 57.1% CMV+) and comparison controls (n = 98, mean age(SD) = 34(10) years, 68.4% female, 51.0% CMV+) were evaluated for CMV IgG antibody status and the distribution of late differentiated (CD27−CD28−) cells within CD4+ and CD8+ T-cell subsets, i.e. naive (CCR7+CD45RA+), effector memory (EM, CCR7−CD45RA−), central memory (CM, CCR7+CD45RA−) and effector memory cells re-expressing CD45RA (EMRA, CCR7−CD45RA+). Mixed linear regression models controlling for age, sex, ethnicity and flow cytometry batch showed that CMV seropositivity was associated with a reduction in naive T-cells, expansion of EMRA T-cells, and a greater percent distribution of CD27−CD28− cells within CD4+ and CD8+ memory T-cell subsets (p’s < 0.004), but there was no significant effect of MDD, nor any significant interaction between CMV and diagnosis. Unexpectedly, depressed men were less likely to be CMV+ and depressed women were more likely to be CMV+ than sex-matched controls suggesting a possible interaction between sex and MDD on CMV susceptibility, but this three-way interaction did not significantly affect the T-cell subtypes. Our findings suggest that depression in young and middle-aged adults does not prematurely advance aging of the T-cell compartment independently of CMV, but there may be significant sex-specific effects on adaptive immunity that warrant further investigation.

Abstract IA19: Myeloid cell control of tumor immunity

Abstract While the success of T-cell checkpoint-targeted immunotherapies is predicated on the induction of an effector T-cell response against tumor antigens by potent antigen-presenting cells (APC), very little is known about the APC composition in human tumors during immunotherapy. APC form a diverse population of dendritic cells (DCs) and macrophages (macs) that share the capacity to capture and present cell-associated antigens to T cells and instruct the repertoire and effector versus regulatory function of T lymphocytes. Despite the key role of APC in tumor immunotherapy, targeting the APC compartment has proved difficult. This is because little is known about the APC molecular changes during tumor response or resistance to immunotherapy. In order to develop ways to modulate tumor APC and promote cancer immunity, we sought to gain a deeper understanding of the molecular and functional diversity of APC in human NSCLC. Using paired mass cytometry analysis of tumor/adjacent tissues, we recently identified major changes in APC and T-cell composition in early non-small lung cancer (NSCLC) lesions (Lavin et al., Cell 2017). More recently, we used cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) to further measure APC defects in the tumor lesions. CITE-seq analysis of the tumor lesions revealed profound alterations in tumor APC composition including a reduction of cross-presenting DC at the expense of monocyte-derived cells, which correlated with the presence of dysfunctional T cells compared to the adjacent tissue. Accordingly, we found that DC composition and molecular state controlled CD8+ T-cell accumulation at the tumor site and controlled tumor response to checkpoint blockade in preclinical models. Here, I will discuss the strategies we have developed to enhance DC functionality at the tumor site and the challenges that remain. Citation Format: Miriam Merad. Myeloid cell control of tumor immunity [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr IA19.

Comprehensive TCR repertoire analysis of CD4+ T-cell subsets in rheumatoid arthritis

The pathogenesis of rheumatoid arthritis (RA), a systemic autoimmune disease characterized by autoreactive T-cell accumulation and pro-inflammatory cytokine overproduction, is unclear. Systematically addressing T-cell receptor (TCR) repertoires of different CD4+ T-cell subsets could help understand RA pathogenesis. Here, peripheral CD4+ T cells from treatment-naïve RA patients and healthy controls were sorted into seven subsets including naïve, effector, central memory, effector memory (EMT), Th1, Th17, and regulatory T cells. T-cell receptor β chain repertoires were then analyzed by next-generation sequencing. We identified T-cell clonal expansion in EMT and Th17 cells of RA patients, with highly similar TCR repertoires. Ex vivo experiments demonstrated the preferred differentiation from EMT to Th17 cells in RA. Notably, we showed that TCR diversity and abundance of differentiated T cells of Th17 were significantly correlated with RA disease activity. Based on these observations, we propose that abnormal differentiation from EMT to Th17 and expansion of Th17 play pivotal role in RA pathogenesis.

Abstract WMP76: Interleukin-33 Protects Against Transient Ischemic Stroke by Enhancing Regulatory T-Cell Expansion and Accumulation

Background: The interleukin (IL)-33 could promote proliferation of regulatory T lymphocytes (Tregs) which are negatively related with brain damage after ischemic stroke. How IL-33 works on Tregs after stroke is unclear. The purpose of this study was to investigate the role of IL-33 for Tregs-mediated neuroprotection and further expounded the mechanisms of protection in mice. Methods: In vitro study, primary mice neuronal cells were subjected to 3h oxygen-glucose deprivation (OGD). The vehicle or drug conditioned Tregs were applied to neurons at the time of induction of hypoxia respectively. Neuronal apoptosis, Tregs related cytokines were measured by MTT assay, Western blotting and enzyme-linked immune-sorbent assay (ELISA). In vivo study, Tregs were depleted by intraperitoneal administration of anti-CD25Ab. Intraperitoneal injection of IL-33 immediately post 60 min transient middle cerebral artery occlusion (tMCAO) modeling. The neurological function test at days 1, 3, 5, 7 and 14 after tMCAO. Infarct volume, Brain edema, cell death, percentage of Tregs and related cytokines were respectively measured by 2,3,5-triphenyltetrazolium chloride or MAP2 staining, dry-wet method, TUNEL staining, flow cytometry and immunofluorescence, Western blotting and ELISA. Results: The supernatant of IL-33-treated Tregs reduced neuronal apoptosis in the OGD model meanwhile elevated the production of Tregs related cytokines IL-10, IL-35 and TGF- β in vitro. Intraperitoneal administration of IL-33 significantly reduced infarct volume and stroke-induced cell death and improved sensorimotor functions. Notably, the protective effect of IL-33 was abolished in mice depleted of Tregs. IL-33 increased CD4+CD25+Foxp3+ Tregs in spleens, blood, and brain in vivo. Yet, ST2 blocking muted these IL-33 activities. Mechanistically, the protection of IL-33 was associated with reduced apoptosis protein and production of Tregs related cytokine. Conclusions: This study elucidated that IL-33 afforded neuroprotection against ischemic brain injury by enhancing ST2-dependent regulatory T-cell expansion and activation, which suggested a promising immune modulatory target for the treatment of stroke.

A Comparative Evaluation on the Effect of Oral Zinc 50 mg with or without 0.1% Triamcinolone Orabase on Oral Lichen Planus.

Background:Lichen planus is a T-cell-mediated autoimmune disease, in which CD8+ T-cells releases the cytokines such as tumor necrosis factor-alpha and interleukin-12 disrupting basement membrane integrity. Treatment modalities were directed toward the relief in signs and symptoms and preventing recurrences. Zinc activates caspase-3 and DNA fragmentation, resulting in the apoptosis of keratinocytes. Prevention of matrix metalloproteinases1 (MMP1) activation, inhibits the Tcell accumulation in oral lichen planus (OLP) and by inhibiting MMP9, prevents the cleavage of collagen resulting in maintaining the integrity of the basement membrane.Objectives:The main objective of the study is to compare the efficacy of oral zinc 50 mg and 0.1% triamcinolone Orabase with 0.1% triamcinolone Orabase alone on the healing process of symptomatic OLP.Materials and Methods:A total of forty participants were randomly categorized into two groups: Group A and Group B with 20 patients with OLP and having symptoms of burning sensation. Group A patients had received 0.1% triamcinolone Orabase twice daily application. Group B patients had provided with oral zinc 50 mg and 0.1% triamcinolone Orabase twice daily for 8 weeks. The follow-up period for both the groups was 6 months. Lesional size was measured by Thongprasom scale and burning sensation was assessed by visual analog scale at each visit till the cessation of treatment.Results:There was decrease in the burning sensation and lesional size from the first visit to follow-up period which was statistically significant in both groups (P = 0.000).Conclusion:Oral zinc therapy was adjunctive in reducing the burning sensation and lesional size in the symptomatic OLP.

Signaling through the Inhibitory Fc Receptor FcγRIIB Induces CD8+ T Cell Apoptosis to Limit T Cell Immunity

Effector CD8+ Tcells are important mediators of adaptive immunity, and receptor-ligand interactions that regulate their survival may have therapeutic potential. Here, we identified a subset of effector CD8+ Tcells that expressed the inhibitory fragment crystallizable (Fc) receptor FcγRIIB following activation and multiple rounds of division. CD8+ Tcell-intrinsic genetic deletion of Fcgr2b increased CD8+ effector Tcell accumulation, resulting in accelerated graft rejection and decreased tumor volume in mouse models. Immunoglobulin G (IgG) antibody was not required for FcγRIIB-mediated control of CD8+ Tcell immunity, and instead, the immunosuppressive cytokine fibrinogen-like 2 (Fgl2) was a functional ligand for FcγRIIB on CD8+ Tcells. Fgl2 induced caspase-3/7-mediated apoptosis in Fcgr2b+, but not Fcgr2b-/-, CD8+ Tcells. Increased expression of FcγRIIB correlated with freedom from rejection following withdrawal from immunosuppression in a clinical trial of kidney transplant recipients. Together, these findings demonstrate a cell-intrinsic coinhibitory function of FcγRIIB in regulating CD8+ Tcell immunity.

Accumulation of Skeletal Muscle T Cells and the Repeated Bout Effect in Rats.

The purpose of this investigation was to characterize skeletal muscle T-cell accumulation after contraction-induced muscle damage and test the hypothesis that T cells contribute to postdamage muscle protection (i.e., the repeated bout effect) in a way reminiscent of their role in adaptive immunity. In vivo lengthening contractions were used to model the repeated bout effect and contralateral repeated bout effect in rats. Intramuscular T-cell subsets were characterized by flow cytometry after single and repeated bouts of lengthening contractions, and an adoptive T-cell transfer experiment was done to test whether T cells from muscle damage-experienced rats can confer protection from injury to damage-naive rats. Electrically stimulated lengthening contractions elicited the repeated bout effect, but not the contralateral repeated bout effect. Although leukocytes (CD45+) were scarce in undamaged muscle (2.1% of all cells), substantially more (63% of all cells) were observed after a single bout of lengthening contractions. Within the leukocyte population were several subsets of T cells, including conventional CD4+, CD8+, memory, and regulatory T cells. In contrast, a minimal increase in T cells was observed after a second bout of lengthening contractions. Conventional CD4+ T cells (FoxP3-) were the most abundant subset in muscle after lengthening contractions. Adoptive T-cell transfer from damage-experienced rats did not confer protection to damage-naive recipient rats. The robust T-cell accumulation, particularly the CD4 subset, after contraction-induced damage suggests a role for these cells in muscle repair and adaptation to muscle damaging contractions. Moreover, T cells are unlikely to mediate the protective adaptations of the repeated bout effect in a manner similar to their role in adaptive immunity.

Integrin αDβ2 influences cerebral edema, leukocyte accumulation and neurologic outcomes in experimental severe malaria.

Malaria is an infectious disease of major worldwide clinical importance that causes a variety of severe, or complicated, syndromes including cerebral malaria, which is often fatal. Leukocyte integrins are essential for host defense but also mediate physiologic responses of the innate and adaptive immune systems. We previously showed that targeted deletion of the αD subunit (αD-/-) of the αDβ2 integrin, which is expressed on key leukocyte subsets in mice and humans, leads to absent expression of the integrin heterodimer on murine macrophages and reduces mortality in mice infected with Plasmodium berghei ANKA (P. berghei ANKA). To further identify mechanisms involved in the protective effect of αD deletion in this model of severe malaria we examined wild type C57BL/6 (WT) and αD-/- mice after P. berghei ANKA infection and found that vessel plugging and leukocyte infiltration were significantly decreased in the brains of αD-/- animals. Intravital microscopy demonstrated decreased rolling and adhesion of leukocytes in cerebral vessels of αD-/- mice. Flow cytometry analysis showed decreased T-lymphocyte accumulation in the brains of infected αD-/- animals. Evans blue dye exclusion assays demonstrated significantly less dye extravasation in the brains of αD-/- mice, indicating preserved blood-brain barrier integrity. WT mice that were salvaged from P. berghei ANKA infection by treatment with chloroquine had impaired aversive memory, which was not observed in αD-/- mice. We conclude that deletion of integrin αDβ2 alters the natural course of experimental severe malaria, demonstrating previously unrecognized activities of a key leukocyte integrin in immune-inflammatory responses that mediate cerebral involvement.