Effector Memory Phenotype Research Articles

Cytotoxic activity of anti-mucin 1 chimeric antigen receptor T cells expressing PD-1-CD28 switch receptor against cholangiocarcinoma cells

Background aimsCholangiocarcinoma (CCA) is a lethal bile-duct cancer that is difficult to treat by current standard procedures. This drawback has prompted us to develop adoptive T-cell therapy for CCA, which requires an appropriate target antigen for binding of chimeric antigen receptor (CAR) T cells. Mucin 1 (MUC1), an overexpressed protein in CCA cells, is a potential target antigen for the CAR T-cell development. However, MUC1 overexpression also is associated with the upregulation of programmed death-ligand 1 (PD-L1), an immune checkpoint protein that prohibits anti-tumor functions of T cells, probably causing poor overall survival of patients with CCA. MethodsTo overcome this problem, we developed anti-MUC1-CAR T cells containing PD-1-CD28 switch receptor (SR), namely αM.CAR/SR T cells, to target MUC1 and switch on the inhibitory signal of PD-1/PD-L1 interaction to activate CD28 signaling. Our lentiviral construct contains the sequences that encode anti-MUC1-single chain variable fragment, CD137 and CD3ζ, linked with P2A, PD-1 and CD28. ResultsInitially, the upregulations of MUC1 and PD-L1 proteins were confirmed in CCA cell lines. αM.CAR and SR were co-expressed in 53.53 ± 13.89% of transduced T cells, mainly CD8+ T cells (85.7 ± 0.75%, P<0.0001) with the effector memory phenotype (59.22 ± 16.31%, P < 0.01). αM.CAR/SR T cells produced high levels of intracellular tumor necrosis factor-α and interferon-γ in response to the activation by CCA cells expressing MUC1, including KKU-055 (27.18 ± 4.38% and 27.33 ± 5.55%, respectively, P < 0.05) and KKU-213A (47.37 ± 12.67% and 54.55 ± 8.66%, respectively, P < 0.01). Remarkably, the cytotoxic function of αM.CAR/SR T cells against KKU-213A cells expressing PD-L1 was significantly enhanced compared with the αM.CAR T cells (70.69 ± 14.38% versus 47.15 ± 8.413%, respectively; P = 0.0301), correlated with increased granzyme B production (60.6 ± 9.89% versus 43.2 ± 8.95%, respectively; P = 0.0402). Moreover, the significantly enhanced disruption of KKU-213A spheroids by αM.CAR/SR T cells (P = 0.0027), compared with αM.CAR T cells, was also observed. ConclusionTaken together, the cytotoxic function of αM.CAR/SR T cells was enhanced over the αM.CAR T cells, which are potential to be further tested for CCA treatment.

Abstract 12926: Senescent CD8 T Cells are Enriched in Atherosclerotic Plaques From Patients With Type 2 Diabetes and Their Circulating Levels Are Normalized by Intensive Lipid Lowering

Introduction: Senescence is a key driver of age-related chronic inflammatory diseases including type 2 diabetes (T2D). Senescent T cells lose their ability to control tissue homeostasis and present aberrant cytokine and cytotoxic responses that ultimately cause tissue damage. Atherosclerotic plaques from T2D present more severe pathology than patients with no diabetes (ND) and circulating senescent CD8 T cells are increased in T2D and associated with myocardial infarction. However, whether senescent CD8 T cells accumulate in atherosclerotic plaques of T2D patients is unknown. Hypothesis: Senescent T cells infiltrate atherosclerotic plaques and contribute to the aggravated cardiovascular risk in T2D. Methods: We performed unbiased scRNA-seq analysis (75,049 total cells) of plaques from T2D (n=12) and ND patients (n=10) enrolled in the Athero-In Study. Circulating immune cells from T2D (n=16) and ND (n=6) patients enrolled in the CHORD study were analyzed using mass cytometry (CyTOF) before and after lipid lowering treatment. Results: We found a cluster of CD8 T cells expressing a senescence gene signature ( B3GAT1, KLRG1, KLRF1), as well as proinflammatory ( IFNG, TNF, CCL4, CCL5 ) and cytotoxic ( GZMB, PRF1, GNLY ) genes. Plaque senescent CD8 T cells showed a terminally differentiated effector memory phenotype (TEMRA) with high expression of CX3CR1 and low expression of lymphoid-homing genes ( SELL, CCR7) . CyTOF analysis revealed that circulating senescent CD8+ CD28- CD57+ T cells were increased in T2D compared to ND patients. Treatment with PCSK9 inhibitor plus statin/ezetimibe, which reduced LDL levels by &gt;70% at 4 weeks, normalized senescent CD8 T cell frequency in T2D patients to the levels of ND subjects at baseline. No changes were observed in cells from cholesterol-reduced ND patients. Conclusions: Our results show that senescent CD8+ T cells are enriched in atherosclerotic plaques and blood of T2D patients. The observation that acute lipid lowering normalized circulating senescent CD8 T cells in T2D patients indicates that T cell senescence can be improved by more aggressive lipid control in high risk T2D subjects. Further analyses are needed to understand how T cell senescence contributes to atherosclerosis and impaired regression in T2D.

T-cell receptor αβ+ double-negative T cells in the kidney are predominantly extravascular and increase in abundance in response to ischemia-reperfusion injury.

T-cell receptor+ CD4- CD8- double-negative (DN) T cells are a population of T cells present in low abundance in blood and lymphoid organs, but enriched in various organs including the kidney. Despite burgeoning interest in these cells, studies examining their abundance in the kidney have reported conflicting results. Here we developed a flow cytometry strategy to clearly segregate DN T cells from other immune cells in the mouse kidney and used it to characterize their phenotype and response in renal ischemia-reperfusion injury (IRI). These experiments revealed that in the healthy kidney, most DN T cells are located within the renal parenchyma and exhibit an effector memory phenotype. In response to IRI, the number of renal DN T cells is unaltered after 24 h, but significantly increased by 72 h. This increase is not related to alterations in proliferation or apoptosis. By contrast, adoptive transfer studies indicate that circulating DN T cells undergo preferential recruitment to the postischemic kidney. Furthermore, DN T cells show the capacity to upregulate CD8, both invivo following adoptive transfer and in response to exvivo activation. Together, these findings provide novel insights regarding the phenotype of DN T cells in the kidney, including their predominant extravascular location, and show that increases in their abundance in the kidney following IRI occur in part as a result of increased recruitment from the circulation. Furthermore, the observation that DN T cells can upregulate CD8 invivo has important implications for detection and characterization of DN T cells in future studies.

Generation of good manufacturing practice grade virus-specific T cells for the management of post-transplant CMV infections

Adoptive transfer of antigen-specific T cells has recently emerged as a successful strategy to treat viral infections following hematopoietic cell transplantation (HCT). Ex-vivo expanded donor-derived virus-specific T cells (VSTs) can be safe and effective, devoid of all the drug-related adverse effects. The study aimed to manufacture cGMP grade VSTs from healthy donors, characterize the VST product and demonstrate its safety and efficacy. Peripheral blood mononuclear cells (PBMCs) collected from six healthy donors were stimulated with pepmix that mimics the pp65 antigenic epitope of CMV and cultured for 14 days in G-Rex culture tubes. Post pepmix exposure and expansion the median CD3% was 98.8% (range:95.5% to 99.9%) while the median CD4% and CD8% were 49.1% (range:21.3% to 86.6%) and 43.9% (range:12.7% to 75.5%) respectively. The percentage of IFNγ+ cells was much higher among the CD8+ T cells (median - 18.47%; range 6.50% - 45.82%) when compared to CD4+ T cells (median − 2.74%; range 0.47% - 18.58%) and there was a switch from the CD45RA+ naive phenotype to CD45RA- effector memory phenotype in the 4 samples that achieved a >5 fold expansion. The VSTs were cytotoxic to the pepmix pulsed lymphoblasts (efficacy) while they did not induce cytolysis in the lymphoblasts that were not exposed to the pepmix (safety). This feasibility exercise helped us optimize the starting cell dose for the culture and clinical grade culture strategies, subset characterization and cytotoxicity assays. The approach could be applied to the clinical practice where virus-specific T cell infusions could be given for post-transplant viral infections.

Remodeling the bladder tumor immune microenvironment by mycobacterial species with changes in their cell envelope composition.

Intravesical BCG instillation after bladder tumor resection is the standard treatment for non-muscle invasive bladder cancer; however, it is not always effective and frequently has undesirable side effects. Therefore, new strategies that improve the clinical management of patients are urgently needed. This study aimed to comprehensively evaluate the bladder tumor immune microenvironment profile after intravesical treatment with a panel of mycobacteria with variation in their cell envelope composition and its impact on survival using an orthotopic murine model to identify more effective and safer therapeutic strategies. tumor-bearing mice were intravesically treated with a panel of BCG and M. brumae cultured under different conditions. Untreated tumor-bearing mice and healthy mice were also included as controls. After mycobacterial treatments, the infiltrating immune cell populations in the bladder were analysed by flow cytometry. We provide evidence that mycobacterial treatment triggered a strong immune infiltration into the bladder, with BCG inducing higher global absolute infiltration than M. brumae. The induced global immune microenvironment was strikingly different between the two mycobacterial species, affecting both innate and adaptive immunity. Compared with M. brumae, BCG treated mice exhibited a more robust infiltration of CD4+ and CD8+ T-cells skewed toward an effector memory phenotype, with higher frequencies of NKT cells, neutrophils/gMDSCs and monocytes, especially the inflammatory subset, and higher CD4+ TEM/CD4+ Treg and CD8+ TEM/CD4+ Treg ratios. Conversely, M. brumae treatment triggered higher proportions of total activated immune cells and activated CD4+ and CD8+ TEM cells and lower ratios of CD4+ TEM cells/CD4+ Tregs, CD8+ TEM cells/CD4+ Tregs and inflammatory/reparative monocytes. Notably, the mycobacterial cell envelope composition in M. brumae had a strong impact on the immune microenvironment, shaping the B and myeloid cell compartment and T-cell maturation profile and thus improving survival. Overall, we demonstrate that the bladder immune microenvironment induced by mycobacterial treatment is species specific and shaped by mycobacterial cell envelope composition. Therefore, the global bladder immune microenvironment can be remodelled, improving the quality of infiltrating immune cells, the balance between inflammatory and regulatory/suppressive responses and increasing survival.

INTRAPLEURAL THERAPY TO DISRUPT IL-6/IL-8 JUXTACTINE SIGNALLING TO BLOCK TUMOR EMT AND TO DRIVE SYSTEMIC ANTI-TUMOR IMMUNITY

The goal of this study was to determine whether antiIL-6Rα block (tocilizumab) will alter the pleural secretome and will diminish tumor-specific immune responses. Pleural T cells were isolated from freshly drained pleural effusions (n=6). Autologous pleural tumor was expanded in vitro using the Mammary Epithelial Growth Medium (Lonza). Pleural T cells were stimulated using anti-CD3/CD28 Dynal beads and low dose IL-2 (60 Cetus U/ml) for 2,4,7, 14 or 21 days in the presence of tocilizumab for the last 48h (0, 0.35, 0.72, 1.43, 2.86 and 5.72ug/ml). Pleural T cell effectors were counted and plated on tumor targets at 12.5:1 E:T in the presence of tocilizumab. Ex vivo expanded pleural T cells were effector-memory phenotype (CD45RA-CD27-) and were highly cytotoxic against autologous tumor (89-100%). The majority of CD8+ T cells were central memory (CD45RA-/CD27-) or effector memory (CD45RA+/CD27-); the majority co-expressed granzyme B, perforin, 20-60% expressed PD-1. Most CD4+ co-expressed granzyme B and perforin and were PD-1+, suggesting cytotoxic CD4+ T cells. The presence of tocilizumab reversed tumor EMT but did not alter cytotoxicity. We show that the IL-6/IL-6Rα axis is prominent in MPE, drives tumor growth and inhibits anti-tumor immunity. Pleural T cells are neither exhausted nor dysfunctional but are suppressed by the pleural environment. Ex vivo expanded MPE CD8 and CD4 T cells are highly cytotoxic against autologous tumor. Anti-IL-6Rα block reverses tumor EMT but does not inhibit effector responses.

CT-335 Cytotoxic Rechallenge to Model Active CAR-T and NK Cell Cytotoxic Activity, Exhaustion, and Persistence

We developed a rechallenge cytotoxic assay that mimics repetitive in vivo immune effector cell (IEC; T and NK) interactions with target cells that can be used for the optimization, comparison, and development of CAR-IEC therapies. To evaluate a target cell rechallenge platform that allows rapid and multiplex evaluation of CAR-IEC cytotoxicity, exhaustion, and persistence in vitro. Four separate ROR1 constructs using different costimulatory domains were designed and optimized in silico. We developed a luciferase-based rechallenge cytotoxicity assay. We diluted the effector to target (E/T) ratio to obtain a logarithmic trendline of the cells' cytotoxicity. All were co-cultured with their targets at E/T ratios resulting in 70% cytotoxicity. After 24 hours, we estimated the remaining IEC amount in culture using GFP exclusion by FACS (IEC cells/mL = total cells/mL x GFP%). We repeated the plating of E/T ratio dilutions to obtain daily IC50 curves using this rechallenge platform for 5 days. We examined IEC exhaustion marker expression on days 0 and 5 by FACS. All tested constructs showed specific cytotoxicity against ROR1-expressing cell lines. Third-generation (3G) constructs showed lower cytotoxicity levels after Day 1 of repetitive rechallenge compared with second-generation (2G) CARs. However, the cytotoxic activity of 3G constructs gradually increased during subsequent rechallenges and exceeded the cytotoxicity observed for 2G-CARs on Day 5 of the rechallenge by an order of magnitude. On Day 5, there were no significant differences in IEC subsets among the constructs, with a predominance of effector memory phenotypes (CCR7-, CD45RA-); however, in some cases, we observed the increased expression of PD-1, TIGIT, LAG-3, and TIM3 in the 2G constructs. Our data shows the functionality of a repetitive in vitro target rechallenge platform for evaluating CAR-IECs. By performing repetitive cytotoxic challenges, we aim to mimic the dynamic in vivo interactions in the tumor microenvironment between IECs and target tumor cells. We propose to use the IEC50 calculated from this platform to evaluate the cytotoxic functionality, exhaustion, and persistence of different CAR-IECs in an efficient and cost-effective manner, which is suited for multiplexing and robotic optimization.

Characterization of peripheral T helper 17 (Th17) cells phenotype in postmenopausal women with estrogen insufficiency

Over the past few years, Th17 cells is considered a key player in osteoporosis pathogenesis. Although extensively studied in murine models, comprehensive Th17 cell characterization in osteoporotic women is elusive. We thus aimed to examine peripheral Th17 cells frequency and phenotypes in healthy and osteoporotic women. Our results demonstrated that Th17 cells were primarily CD4+CD45RA−CCR7−HALDR+CCR6lowT-cells. Compared to Pre-N, Post-L showed increased proportion of Th17 with concomitant decrease in Th1 cells. The Th17 cells frequency in effector memory CD4+ T cells was significantly elevated in Post-N with a decrease of Th1 cells in effector memory subsets compared to Pre-N and Post-L. Both Post-N and Post-L had decreased frequency of dual positive Th1-Th17 cells and increased HLA-DR expression on Th17 cells compared to Pre-N. Thus, our study demonstrates increased Th17 cells frequency and reduced Th1 cells frequency with effector memory phenotype in postmenopausal women with estrogen insufficiency and correlates with aging process.

Broader Epstein-Barr virus-specific T cell receptor repertoire in patients with multiple sclerosis.

Epstein-Barr virus (EBV) infection precedes multiple sclerosis (MS) pathology and cross-reactive antibodies might link EBV infection to CNS autoimmunity. As an altered anti-EBV T cell reaction was suggested in MS, we queried peripheral blood T cell receptor β chain (TCRβ) repertoires of 1,395 MS patients, 887 controls, and 35 monozygotic, MS-discordant twin pairs for multimer-confirmed, viral antigen-specific TCRβ sequences. We detected more MHC-I-restricted EBV-specific TCRβ sequences in MS patients. Differences in genetics or upbringing could be excluded by validation in monozygotic twin pairs discordant for MS. Anti-VLA-4 treatment amplified this observation, while interferon β- or anti-CD20 treatment did not modulate EBV-specific T cell occurrence. In healthy individuals, EBV-specific CD8+ T cells were of an effector-memory phenotype in peripheral blood and cerebrospinal fluid. In MS patients, cerebrospinal fluid also contained EBV-specific central-memory CD8+ T cells, suggesting recent priming. Therefore, MS is not only preceded by EBV infection, but also associated with broader EBV-specific TCR repertoires, consistent with an ongoing anti-EBV immune reaction in MS.

Contribution of pathogenic T helper 1 and 17 cells to bursitis and tenosynovitis in polymyalgia rheumatica.

BackgroundAlthough polymyalgia rheumatica (PMR) is a very common rheumatic inflammatory disease, current insight into the pathobiology of PMR is limited and largely based on studies in blood. We investigated T helper 1 (TH1) and T helper 17 (TH17) cell responses in blood, synovial fluid and bursa tissue of patients with PMR.Materials and methodsBlood samples were collected from 18 patients with new-onset PMR and 32 healthy controls. Synovial fluid was aspirated from the inflamed shoulder bursae or biceps tendon sheath of 13 patients. Ultrasound-guided biopsies of the subacromial-subdeltoid (SASD) bursa were obtained from 11 patients. T cells were examined by flow cytometry, immunohistochemistry and immunofluorescence staining.ResultsBesides an increase of TH17 (CD4+IL-17+IFN-γ-) cells and T cytotoxic 17 (TC17; CD8+IL-17+IFN-γ-) cells, no other major changes were noted in the circulating T cell compartment of patients with PMR. Absolute numbers of CD4+ and CD8+ T cells were similar in blood and synovial fluid of patients with PMR. Synovial fluid T cells showed an effector-memory (CD45RO+CCR7-) phenotype. Percentages of TH1 (CD4+IFN-γ+IL-17-) cells and TH1/TH17 (CD4+IFN-γ+IL-17+) cells, but not TH17 or TC17 cells, were increased in the synovial fluid. Bursa tissue biopsies contained a small number of T cells, which were mostly CD8 negative. The majority of bursa tissue T cells produced IFN-γ but not IL-17. For comparison, B cells were scarcely detected in the bursa tissue.ConclusionAlthough the circulating TH17 cell pool is expanded in patients with PMR, our findings indicate that TH1 cells are involved in the inflammation of bursae and tendon sheaths in this condition. Our study points towards the TH1 cell pathway as a potential target for therapy in PMR.

Inducible Costimulator-C-X-C Motif Chemokine Receptor 3 Signaling is Involved in Chronic Obstructive Pulmonary Disease Pathogenesis.

BackgroundThe role of inducible costimulator (ICOS) signaling in chronic obstructive pulmonary disease (COPD) has not been fully elucidated.MethodsWe compared the percentages of ICOS+ T cells and ICOS+ regulatory T (Treg) cells in CD4+ T cells and CD4+CD25+FOXP3+ Tregs, respectively, in the peripheral blood of smokers with or without COPD to those in healthy controls. We further characterized their phenotypes using flow cytometry. To investigate the influence of ICOS signaling on C-X-C motif chemokine receptor 3 (CXCR3) expression in COPD, we evaluated the expression levels of ICOS and CXCR3 in vivo and in vitro.ResultsICOS expression was elevated on peripheral CD4+ T cells and CD4+ Tregs of COPD patients, which positively correlated with the severity of lung function impairment in patients with stable COPD (SCOPD), but not in patients with acute exacerbation of COPD (AECOPD). ICOS+CD4+ Tregs in patients with SCOPD expressed higher levels of coinhibitors, programmed cell death protein 1 (PD-1) and T-cell immunoreceptor with Ig and ITIM domains (TIGIT), than ICOS−CD4+ Tregs, whereas ICOS+CD4+ T cells mostly exhibited a central memory (CD45RA−CCR7+) or effector memory (CD45RA−CCR7−) phenotype, ensuring their superior potential to respond potently and quickly to pathogen invasion. Furthermore, increased percentages of CXCR3+CD4+ T cells and CXCR3+CD4+ Tregs were observed in the peripheral blood of patients with SCOPD, and the expression level of CXCR3 was higher in ICOS+CD4+ T cells than in ICOS−CD4+ T cells. The percentage of CXCR3+CD4+ T cells was even higher in the bronchoalveolar lavage fluid than in matched peripheral blood in SCOPD group. Lastly, in vitro experiments showed that ICOS induced CXCR3 expression on CD4+ T cells.ConclusionsICOS signaling is upregulated in COPD, which induces CXCR3 expression. This may contribute to increased numbers of CXCR3+ Th1 cells in the lungs of patients with COPD, causing inflammation and tissue damage.

Lymphocyte Exhaustion in AML Patients and Impacts of HMA/Venetoclax or Intensive Chemotherapy on Their Biology.

Simple SummaryPatients with acute myeloid leukemia (AML) are routinely treated with either intensive chemotherapy or DNA hypomethylating agents (HMA) in combination with the Bcl-2 inhibitor, venetoclax. While both treatment regimens are highly cytotoxic to the aggressive AML tumor cells, they are also toxic to immune cells. Therefore, we sought to establish the detrimental impacts of these therapies on lymphocytes and their recovery over time in AML patients. Even prior to treatment initiation, the patients were found to have exhausted lymphocytes in peripheral blood, and additional signs of exhaustion were noted after treatment with HMA/venetoclax. In fact, the lymphocytes were still suppressed for two to three months after the initiation of induction therapy. Furthermore, T cells in a subset of patients subsequently found to be resistant to venetoclax therapy exhibited a higher expression of perforin and CD39 and more pronounced IFN-γ production.Acute myeloid leukemia (AML) is an aggressive malignancy that requires rapid treatment with chemotherapies to reduce tumor burden. However, these chemotherapies can compromise lymphocyte function, thereby hindering normal anti-tumor immune responses and likely limiting the efficacy of subsequent immunotherapy. To better understand these negative impacts, we assessed the immunological effects of standard-of-care AML therapies on lymphocyte phenotype and function over time. When compared to healthy donors, untreated AML patients showed evidence of lymphocyte activation and exhaustion and had more prevalent CD57+NKG2C+ adaptive NK cells, which was independent of human cytomegalovirus (HCMV) status. HMA/venetoclax treatment resulted in a greater fraction of T cells with effector memory phenotype, inhibited IFN-γ secretion by CD8+ T cells, upregulated perforin expression in NK cells, downregulated PD-1 and 2B4 expression on CD4+ T cells, and stimulated Treg proliferation and CTLA-4 expression. Additionally, we showed increased expression of perforin and CD39 and enhanced IFN-γ production by T cells from pre-treatment blood samples of venetoclax-resistant AML patients. Our results provide insight into the lymphocyte status in previously untreated AML patients and the effects of standard-of-care treatments on their biology and functions. We also found novel pre-treatment characteristics of T cells that could potentially predict venetoclax resistance.

Viral Delivery of IL-7 Is a Potent Immunotherapy Stimulating Innate and Adaptive Immunity and Confers Survival in Sepsis Models.

Persistence of an immunosuppressive state plays a role in septic patient morbidity and late mortality. Both innate and adaptive pathways are impaired, pointing toward the need for immune interventions targeting both arms of the immune system. We developed a virotherapy using the nonpropagative modified vaccinia virus Ankara (MVA), which harbors the intrinsic capacity to stimulate innate immunity, to deliver IL-7, a potent activator of adaptive immunity. The rMVA-human IL-7 (hIL-7)-Fc encoding the hIL-7 fused to the human IgG2-Fc was engineered and shown to express a dimeric, glycosylated, and biologically active cytokine. Following a single i.v. injection in naive mice, the MVA-hIL-7-Fc increased the number of total and activated B, T, and NK cells but also myeloid subpopulations (Ly6Chigh, Ly6Cint, and Ly6Cneg cells) in both lung and spleen. It triggered differentiation of T cells in central memory, effector memory, and acute effector phenotypes and enhanced polyfunctionality of T cells, notably the number of IFN-γ-producing cells. The MVA vector contributed significantly to immune cell activation, particularly of NK cells. The MVA-hIL-7-Fc conferred a significant survival advantage in the cecal ligation and puncture (CLP) and Candida albicans sepsis models. It significantly increased cell numbers and activation in both spleen and lung of CLP mice. Comparatively, in naive and CLP mice, the rhIL-7-Fc soluble counterpart overall induced less vigorous, shorter lasting, and narrower immune activities than did the MVA-hIL-7-Fc and favored TNF-α-producing cells. The MVA-hIL-7-Fc represents a novel class of immunotherapeutic with clinical potential for treatment of septic patients.

The characterization analysis of pathogenic T cells in immune-mediated aplastic anemia mouse model

目的通过DsRed小鼠淋巴细胞输注加全身辐照（TBI）建立免疫介导的再生障碍性贫血（AA）C.B10-H2b/LilMcd (C.B10)小鼠模型，分析C.B10 AA小鼠模型中病理性T细胞在不同器官的动态归巢过程及特征。方法通过异体淋巴细胞输注加TBI建立AA小鼠模型，以TBI组为对照，分析外周血细胞和骨髓单个核细胞数，验证造模效果。在造模的第3、6、9、12天，处死小鼠并收集骨髓、脾脏、淋巴结和胸腺单个核细胞，流式细胞术分析DsRed+ T细胞的动态变化及效应记忆T细胞（TEM）、中央记忆T细胞（TCM）、初始T细胞（naïve T）和效应T细胞（TEFF）的比例。PCR Array分析AA小鼠不同组织内DsRed+病理性T细胞活化相关基因的表达谱。结果与TBI组相比，AA组小鼠在造模第9、12天出现严重骨髓衰竭，第12天时骨髓有核细胞数、外周血细胞数均显著减少（P值均<0.05）。AA组小鼠骨髓、脾脏、淋巴结内DsRed+ T细胞比例随时间增加而增加。骨髓中的DsRed+ T细胞比例在第3、6天低于脾脏与淋巴结，但在第12天时高于脾脏与淋巴结（P值均<0.05）。整个骨髓衰竭形成过程中，胸腺中DsRed+ T细胞比例均最低。在第12天时，AA小鼠骨髓、淋巴结和脾脏单个核细胞中，DsRed+CD3+CD4+ T细胞比例分别为（91.38±2.10）％、（39.78±6.98）％、（67.87±12.77）％，三者之间两两比较均有统计学意义（P值均<0.05）；DsRed+CD3+CD8+ T细胞比例分别为（98.21±1.49）％、（94.06±4.20）％、（96.29±1.23）％，差异均无统计学意义（P值均>0.05）。第9、12天，AA组小鼠骨髓中几乎所有的DsRed+CD4+ T或DsRed+CD8+ T细胞均为TEM，而淋巴结中包括TEM、TCM以及naïve T。PCR Array结果显示：骨髓中DsRed+CD4+或DsRed+CD8+ T细胞中CD38、IFN-γ、LAG3、CSF1、SPP1及TNFSF13B表达增高，但 DsRed+CD4+ T细胞中FOXP3、CTLA4表达降低。结论DsRed小鼠淋巴细胞输注可导致C.B10小鼠发生骨髓衰竭，并可以在该AA模型小鼠中追踪DsRed+病理性T细胞。在C.B10 AA模型小鼠体内，DsRed+CD8+和DsRed+CD4+ T细胞先归巢到脾脏与淋巴结，在其中扩增分化，最终转运至骨髓并转化为TEM。胸腺对异体T细胞的归巢过程更具抵抗性，提示胸腺在免疫介导AA模型的骨髓衰竭过程中可能是一个免疫豁免部位。在C.B10 AA模型小鼠的骨髓衰竭形成过程中，归巢到骨髓的DsRed+病理性T细胞较迁移到脾脏与淋巴结的细胞具有更强的免疫活性，而免疫抑制活性则减弱。

Abstract 1372: Macrophages impair antigen specific CD8+ T cell response against HCC in NAFLD mice

Abstract Background: Liver cancer is a leading cause of cancer-related deaths. Non-alcoholic fatty liver disease (NAFLD) has become a burgeoning etiology of liver cancer. Emerging data shows that NAFLD alters adaptive T cell immunity and has a profound influence on both liver cancer development and efficacy of immunotherapy. Tumor antigen-specific T cells recognize tumor and are crucial for controlling malignancy. However, their populations are often low, and detection requires special tools. This study utilized a novel mouse model of HCC to investigate how the tumor antigen-specific T cell response is affected in NAFLD. Methods: We generated a doxycycline-inducible MHC-I OVA257-264 and MHC-II OVA323-339 antigen-expressing HCC cell line which allowed us to investigate tumor antigen-specific T cell responses. The cell line was seeded orthotopically into the livers of diet-induced (methionine choline-deficient [MCD]) and genetic (Ob/Ob [Ob]) NAFLD mouse models. OVA257-264 specific CD8+ T cells were detected by flow cytometry using H-2Kb/OVA257-264 tetramer. Pharmacological depletion experiments were used to identify additional effector cells. Results: The creation of an inducible MHC-I and -II OVA antigen-expressing HCC cell line was validated in both in-vitro and in-vivo settings. Tumor expression of highly immunogenic OVA antigens caused a strong tumor suppression which was mostly mediated by CD8+ T cells in mice fed with normal diet. The inverse correlation between percentage of OVA257-264 specific CD8+ T cells with tumor size further supported their critical role in tumor control. In contrast, in NAFLD mice fed MCD diet as well as Ob/Ob mice, tumor OVA antigens induction failed to reduce liver tumor growth. The presence of OVA257-264 specific CD8+ T cells in NAFLD mice suggested that NAFLD did not inhibit the generation of tumor antigen-specific T cells. This was supported by the finding that OVA257-264 specific CD8+ T cells from NAFLD mice had similar cytotoxic potential, activation, degranulation, PD-1 expression, and effector memory phenotype compared to controls, suggesting an indirect mechanism for the impaired anti-tumor immunity. Immunoprofiling revealed a drastic increase of macrophages in tumor-bearing NAFLD livers. Depletion of macrophages with clodronate reversed the NAFLD caused resistance to tumor-antigen dependent tumor suppression. It was observed that there were minimal changes to the amount of TAS CD8+ T cells in doxycycline-treated mice irrespective of clodronate depletion. There was, however, higher markers of activation such as CD69 and TNFα in clodronate-depleted mice compared to controls. Conclusions: NAFLD impairs antigen-specific CD8+ T cell immunity against liver tumor. The effect was not due to reduced generation or intrinsic functional changes of tumor antigen-specific CD8+ T cells but caused by accumulated macrophages in the liver environment. Citation Format: John C. McVey, Benjamin L. Green, Benjamin Ruf, Tim F. Greten, Chi Ma. Macrophages impair antigen specific CD8+ T cell response against HCC in NAFLD mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1372.

Peptide vaccine-treated, long-term surviving cancer patients harbor self-renewing tumor-specific CD8+ T cells

The behaviors and fates of immune cells in cancer patients, such as dysfunction and stem-like states leading to memory formation in T cells, are in intense focus of investigation. Here we show, by post hoc analysis of peripheral blood lymphocytes of hepatocellular carcinoma patients previously undergoing vaccination with tumour-associated antigen-derived peptides in our clinical trials (registration numbers UMIN000003511, UMIN000004540, UMIN000005677, UMIN000003514 and UMIN000005678), that induced peptide-specific T cell responses may persist beyond 10 years following vaccination. Tracking TCR clonotypes at the single cell level reveals in two patients that peptide-specific long-lasting CD8+ T cells acquire an effector memory phenotype that associates with cell cycle-related genes (CCNA2 and CDK1), and are characterized by high expression of IL7R, SELL, and NOSIP along with a later stage promotion of the AP-1 transcription factor network (5 years or more past vaccination). We conclude that effective anti-tumor immunity is governed by potentially proliferative memory T cells, specific to cancer antigens.

A local human Vδ1 T cell population is associated with survival in nonsmall-cell lung cancer

Murine tissues harbor signature γδ T cell compartments with profound yet differential impacts on carcinogenesis. Conversely, human tissue-resident γδ cells are less well defined. In the present study, we show that human lung tissues harbor a resident Vδ1 γδ T cell population. Moreover, we demonstrate that Vδ1 T cells with resident memory and effector memory phenotypes were enriched in lung tumors compared with nontumor lung tissues. Intratumoral Vδ1 T cells possessed stem-like features and were skewed toward cytolysis and helper T cell type 1 function, akin to intratumoral natural killer and CD8+ T cells considered beneficial to the patient. Indeed, ongoing remission post-surgery was significantly associated with the numbers of CD45RA−CD27− effector memory Vδ1 T cells in tumors and, most strikingly, with the numbers of CD103+ tissue-resident Vδ1 T cells in nonmalignant lung tissues. Our findings offer basic insights into human body surface immunology that collectively support integrating Vδ1 T cell biology into immunotherapeutic strategies for nonsmall cell lung cancer.

Broadly recognized, cross-reactive SARS-CoV-2 CD4 T cell epitopes are highly conserved across human coronaviruses and presented by common HLA alleles.

Sequence homology between SARS-CoV-2 and common-cold human coronaviruses (HCoVs) raises the possibility that memory responses to prior HCoV infection can affect T cell response in COVID-19. We studied T cell responses to SARS-CoV-2 and HCoVs in convalescent COVID-19 donors and identified a highly conserved SARS-CoV-2 sequence, S811-831, with overlapping epitopes presented by common MHC class II proteins HLA-DQ5 and HLA-DP4. These epitopes are recognized by low-abundance CD4 T cells from convalescent COVID-19 donors, mRNA vaccine recipients, and uninfected donors. TCR sequencing revealed a diverse repertoire with public TCRs. T cell cross-reactivity is driven by the high conservation across human and animal coronaviruses of T cell contact residues in both HLA-DQ5 and HLA-DP4 binding frames, with distinct patterns of HCoV cross-reactivity explained by MHC class II binding preferences and substitutions at secondary TCR contact sites. These data highlight S811-831 as a highly conserved CD4 T cell epitope broadly recognized across human populations.

Tauroursodeoxycholic acid (TUDCA) disparate pharmacological effects to lung tissue-resident memory T cells contribute to alleviated silicosis

Exposure to crystalline silica (CS) results in a persistent pulmonary inflammatory response, which results in abnormal tissue repair and excessive matrix deposition. Due to vague pathogenesis, there is virtually no practical therapeutic approach. Here we showed the pharmacological effects of TUDCA on CS-induced pulmonary inflammation and fibrosis. It also helped a faster recovery of CS-impaired pulmonary function. Mechanistically, TUDCA suppressed interferon (IFN)-γ and interleukin (IL)-17A productions by pulmonary helper T (Th) cells. We demonstrated that CS-boosted cytokine-producing Th cells were effector memory (TEM) phenotype. TUDCA decreased the pathogenic TEM cells expansion in the lung. Using in vivo labeling method, we discovered the TEM cells were lung tissue residency with CD103 expression. TUDCA’s anti-fibrotic effects were linked to decreasing IFN-γ producing CD103- TEM-like and IL-17A producing CD103+ TRM-like T cells as well as restricting TRM-like Treg cells in the lung. Specifically, TUDCA could restrain CD103+ TRM-like Treg cell proliferation but not limit the CD103- ones. Further characterization study proved that though the Tregs originally came from the thymus, the expressing levels of ST-2 were different, which provides insights into TUDCA’s various effects on cell proliferation. Collectively, our data paved the way to understanding the pathogenesis of silicosis and may provide new treatments for this pulmonary fibrotic disease.

Bispecific Antibody-Bound T Cells as a Novel Anticancer Immunotherapy

Chimeric antigen receptor T (CAR-T) cell therapy is one of the promising anticancer treatments. It shows a high overall response rate with complete response to blood cancer. However, there is a limitation to solid tumor treatment. Additionally, this currently approved therapy exhibits side effects such as cytokine release syndrome and neurotoxicity. Alternatively, bispecific antibody is an innovative therapeutic tool that simultaneously engages specific immune cells to disease-related target cells. Since programmed death ligand 1 (PD-L1) is an immune checkpoint molecule highly expressed in some cancer cells, in the current study, we generated αCD3xαPD-L1 bispecific antibody (BiTE) which can engage T cells to PD-L1+ cancer cells. We observed that the BiTE-bound OT-1 T cells effectively killed cancer cells in vitro and in vivo. They substantially increased the recruitment of effector memory CD8+ T cells having CD8+CD44+CD62Llow phenotype in tumor. Interestingly, we also observed that BiTE-bound polyclonal T cells showed highly efficacious tumor killing activity in vivo in comparison with the direct intravenous treatment of bispecific antibody, suggesting that PD-L1-directed migration and engagement of activated T cells might increase cancer cell killing. Additionally, BiTE-bound CAR-T cells which targets human Her-2/neu exhibited enhanced killing effect on Her-2-expressing cancer cells in vivo, suggesting that this could be a novel therapeutic regimen. Collectively, our results suggested that engaging activated T cells with cancer cells using αCD3xαPD-L1 BiTE could be an innovative next generation anticancer therapy which exerts simultaneous inhibitory functions on PD-L1 as well as increasing the infiltration of activated T cells having effector memory phenotype in tumor site.