Increased tumor-infiltrating CD45RA-CCR7- regulatory T-cell subset with immunosuppressive properties foster gastric cancer progress.
ABSTRACT: Regulatory T cells (Tregs) are major components of tumor-infiltrating immune cells with potent immunosuppressive properties in gastric cancer (GC) microenvironment. However, different subsets of the Tregs and their relevance to GC are unknown. Here, we found that patients with GC showed a significantly higher Tregs infiltration in tumors, and CD45RA-CCR7- Treg subset constituted most tumor-infiltrating Tregs. Tumor-infiltrating CD45RA-CCR7- Treg subset with an effector/memory phenotype accumulated in tumors and expressed low level of HLA-DR. Gastric tumor-derived TNF-? induced CD45RA-CCR7- Treg subset with similar phenotype to their status in tumors and inhibited their HLA-DR expression via activating STAT3 phosphorylation. These tumor-associated CD45RA-CCR7- Treg subset exerted superior immunosuppressive properties to effectively suppress CD8+ T cells' anti-tumor function including CD8+ T-cell IFN-? and granzyme B (GrB) production as well as CD8+ T-cell proliferation in vitro, and also contributed to the growth and progression of human gastric tumors in vivo, via IL-10 secretion and cell-cell contact mechanisms. Moreover, increased tumor-infiltrating CD45RA-CCR7- Treg subset as well as higher intratumoral CD45RA-CCR7- Treg/CD8+ T-cell ratio was associated with advanced disease progression and reduced GC patient survival. This study therefore identifies a novel immunosuppressive pathway involving CD45RA-CCR7- Treg subset development within the GC microenvironment. Efforts to inhibit this pathway may therefore prove a valuable strategy to prevent, and to treat this immune suppressive of GC.
Project description:CD8(+) T cells play important roles in anti-tumor immunity but distribution profile or functional characteristics of effector memory subsets during tumor progression are unclear. We found that, in oral squamous carcinoma patients, circulating CD8(+) T cell pools skewed toward effector memory subsets with the distribution frequency of CCR7(-)CD45RA(-)CD8(+) T cells and CCR7(-) CD45RA(+)CD8(+) T cells negatively correlated with each other. A significantly higher frequency of CD127(lo) CCR7(-)CD45RA(-)CD8(+) T cells or CCR7(-)CD45RA(+)CD8(+) T cells among total CD8(+) T cells was found in peripheral blood or tumor infiltrating lymphocytes, but not in regional lymph nodes. The CD127(hi) CCR7(-)CD45RA(-)CD8(+) T cells or CCR7(-)CD45RA(+)CD8(+) T cells maintained significantly higher IFN-?, IL-2 productivity and ex vivo proliferative capacity, while the CD127(lo) CCR7(-)CD45RA(-)CD8(+) T cells or CCR7(-)CD45RA(+)CD8(+) T cells exhibited higher granzyme B productivity and susceptibility to activation induced cell death. A higher ratio of CCR7(-)CD45RA(+)CD8(+) T cells to CCR7(-)CD45RA(-)CD8(+) T cells was associated with advanced cancer staging and poor differentiation of tumor cells. Therefore, the CD127(lo) CCR7(-)CD45RA(-)CD8(+) T cells and CCR7(-)CD45RA(+)CD8(+) T cells are functionally similar CD8(+) T cell subsets which exhibit late differentiated effector phenotypes and the shift of peripheral CD8(+) effector memory balance toward CCR7(-)CD45RA(+)CD8(+) T cells is associated with OSCC progression.
Project description:CD8 T cells stimulated with a suboptimal dose of anti-CD3 Abs (100 pg/ml) in the presence of IL-15 retain a naive phenotype with expression of CD45RA, CD28, CD27, and CCR7 but acquire new functions and differentiate into immunosuppressive T cells. CD8+CCR7+ regulatory T cells (Tregs) express FOXP3 and prevent CD4 T cells from responding to TCR stimulation and entering the cell cycle. Naive CD4 T cells are more susceptible to inhibition than memory cells. The suppressive activity of CD8+CCR7+ Tregs is not mediated by IL-10, TGF-?, CTLA-4, CCL4, or adenosine and relies on interference with very early steps of the TCR signaling cascade. Specifically, CD8+CCR7+ Tregs prevent TCR-induced phosphorylation of ZAP70 and dampen the rise of intracellular calcium in CD4 T cells. The inducibility of CD8+CCR7+ Tregs is correlated with the age of the individual with PBLs of donors older than 60 y yielding low numbers of FOXP3(low) CD8 Tregs. Loss of CD8+CCR7+ Tregs in the elderly host may be of relevance in the aging immune system as immunosenescence is associated with a state of chronic smoldering inflammation.
Project description:BACKGROUND:CD4+ T cells are of great importance in the pathogenesis of systemic lupus erythematosus (SLE), as an imbalance between CD4+ regulatory T cells (Tregs) and CD4+ responder T cells (Tresps) causes flares of active disease in SLE patients. In this study, we aimed to find the role of aberrant Treg/Tresp cell differentiation for maintaining Treg/Tresp cell balance and Treg functionality. METHODS:To determine differences in the differentiation of Tregs/Tresps we calculated the percentages of CD45RA+CD31+ recent thymic emigrant (RTE) Tregs/Tresps and CD45RA+CD31- mature naive (MN) Tregs/Tresps, as well as CD45RA-CD31+ and CD45RA-CD31- memory Tregs/Tresps (CD31+ and CD31- memory Tregs/Tresps) within the total Treg/Tresp pool of 78 SLE remission patients compared with 94 healthy controls of different ages. The proliferation capacity of each Treg/Tresp subset was determined by staining the cells with anti-Ki67 monoclonal antibodies. Differences in the autologous or allogeneic Treg function between SLE remission patients and healthy controls were determined using suppression assays. RESULTS:With age, we found an increased differentiation of RTE Tregs via CD31+ memory Tregs and of RTE Tresps via MN Tresps into CD31- memory Tregs/Tresp in healthy volunteers. This opposite differentiation of RTE Tregs and Tresps was associated with an age-dependent increase in the suppressive activity of both naive and memory Tregs. SLE patients showed similar age-dependent Treg cell differentiation. However, in these patients RTE Tresps differentiated increasingly via CD31+ memory Tresps, whereby CD31- memory Tresps arose that were much more difficult to inhibit for Tregs than those that emerged through differentiation via MN Tresps. Consequently, the increase in the suppressive activity of Tregs with age could not be maintained in SLE patients. Testing the Tregs of healthy volunteers and SLE patients with autologous and nonautologous Tresps revealed that the significantly decreased Treg function in SLE patients was not exclusively attributed to an age-dependent diminished sensitivity of the Tresps for Treg suppression. The immunosuppressive therapy reduced the accelerated age-dependent Tresp cell proliferation to normal levels, but simultaneously inhibited Treg cell proliferation below normal levels. CONCLUSIONS:Our data reveal that the currently used immunosuppressive therapy has a favorable effect on the differentiation and proliferation of Tresps but has a rather unfavorable effect on the proliferation of Tregs. Newer substances with more specific effects on the immune system would be desirable.
Project description:Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) is characterized by a low percentage of neoplastic lymphocyte predominant (LP) cells in a background of lymphocytes. The goal of this study is to characterize the microenvironment in NLPHL. Ten NLPHL cases and seven reactive lymph nodes (RLN) were analyzed by flow cytometry for the main immune cells and multiple specific subpopulations. To discriminate between cells in or outside the tumor cell area, we used CD26. We observed significantly lower levels of CD20+ B-cells and CD56+ NK cells and higher levels of CD4+ T-cells in NLPHL in comparison to RLN. In the subpopulations, we observed increased numbers of PD-1+CD4+ T follicular helper cells (TFH), CD69+CD4+ and CD69+CD8+ T-cells and CCR7-CD45RA-CD4+ effector memory T-cells, while FoxP3+CD4+ T regulatory cells (Tregs) and CCR7-CD45RA+ terminally differentiated CD4+ T-cells were decreased in NLPHL compared to RLN. CD69+ cells were increased in the tumor cell area in CD4+ and CD8+ T-cells, while FoxP3+CD25+CD4+ Tregs and CD25+CD8+ T-cells were significantly increased outside the tumor area. Thus, we show a markedly altered microenvironment in NLPHL, with lower numbers of NK cells and Tregs. PD-1+CD4+ and CD69+ T-cells were located inside, and Tregs and CD25+CD8+ cells outside the tumor cell area.
Project description:BACKGROUND AND AIM:Thymus-derived regulatory T cells (Tregs) mediate dominant peripheral tolerance and treat experimental colitis. Tregs can be expanded from patient blood and were safely used in recent phase 1 studies in graft versus host disease and type 1 diabetes. Treg cell therapy is also conceptually attractive for Crohn's disease (CD). However, barriers exist to this approach. The stability of Tregs expanded from Crohn's blood is unknown. The potential for adoptively transferred Tregs to express interleukin-17 and exacerbate Crohn's lesions is of concern. Mucosal T cells are resistant to Treg-mediated suppression in active CD. The capacity for expanded Tregs to home to gut and lymphoid tissue is unknown. METHODS:To define the optimum population for Treg cell therapy in CD, CD4(+)CD25(+)CD127(lo)CD45RA(+) and CD4(+)CD25(+)CD127(lo)CD45RA(-) Treg subsets were isolated from patients' blood and expanded in vitro using a workflow that can be readily transferred to a good manufacturing practice background. RESULTS:Tregs can be expanded from the blood of patients with CD to potential target dose within 22-24?days. Expanded CD45RA(+) Tregs have an epigenetically stable FOXP3 locus and do not convert to a Th17 phenotype in vitro, in contrast to CD45RA(-) Tregs. CD45RA(+) Tregs highly express ?4?7 integrin, CD62L and CC motif receptor 7 (CCR7). CD45RA(+) Tregs also home to human small bowel in a C.B-17 severe combined immune deficiency (SCID) xenotransplant model. Importantly, in vitro expansion enhances the suppressive ability of CD45RA(+) Tregs. These cells also suppress activation of lamina propria and mesenteric lymph node lymphocytes isolated from inflamed Crohn's mucosa. CONCLUSIONS:CD4(+)CD25(+)CD127(lo)CD45RA(+) Tregs may be the most appropriate population from which to expand Tregs for autologous Treg therapy for CD, paving the way for future clinical trials.
Project description:Regulatory T cells (Tregs) play an important role in controlling alloreactivity after solid organ transplantation, but they may also impair antiviral immunity. We hypothesized that the Treg frequency and the Treg phenotype are altered in hepatitis C virus (HCV)-infected recipients of liver transplantation (LT) with possible prognostic implications. Tregs from 141 individuals, including healthy individuals, LT recipients with or without persistent HCV infections, and nontransplant patients with chronic HCV, were studied. A comprehensive phenotypic analysis was performed with multicolor flow cytometry, which included standard Treg markers [CD4(+), CD25(hi), CD127(-), and FoxP3(+) in addition to HLA DR, CCR7, CD45RA, CD62L, CD49d, CD39, ICOS and LAP-TGFβ stainings. Healthy individuals and LT patients displayed similar Treg frequencies and largely comparable Treg phenotypes, which were stable over time after transplantation. In contrast, Tregs with a CD45RA(-) CCR7(-) effector phenotype were enriched in LT recipients with chronic HCV versus HCV-negative transplant patients. HCV infection, rather than LT, altered the expression of functional markers on Tregs. A principal component analysis revealed distinct Treg phenotypes in HCV-infected LT recipients with rejection and patients with recurrent graft HCV. In conclusion, Treg phenotypes are altered in HCV-infected LT patients. An investigation of Tregs may possibly help to distinguish recurrent HCV from graft rejection. Further functional studies are needed to define the role of Tregs in determining the balance between antiviral and allogenic immunity.
Project description:BackgroundAutologous tumor-infiltrating lymphocytes (Tils) immunotherapy is a promising treatment in patients with advanced hepatocellular cancer. Although Tils treatment has shown great promise, their persistence and the efficacy after adoptive-transfer are insufficient and remain a challenge. Studies have demonstrated that IL-15 and Akt inhibitor can regulate T cell differentiation and memory. Here, we constructed S-15 (Super human IL-15), a fusion protein consisting of human IL-15, the sushi domain of the IL-15 receptor ? chain and human IgG-Fc. Herein we compared the effects of S-15 with IL-2 or in combination with Akti on the expansion and activation of Tils.MethodsHepatocellular cancer tissues were obtained from 6 patients, Tils were expanded using IL-2, IL-2/S-15, IL-2/Akti or in combination IL-2/S-15/Akti. At day 10, anti-CD3 antibody was added to the culture media and expanded to day 25. The composition, exhaustion and T-cell differentiation markers (CD45RA/CCR7) were analyzed by flow cytometry.ResultsWe found that IL-2/S-15/Akti expanded Tils and showed the highest percentage of central memory CD45RA?CCR7+ phenotype prior to anti-CD3 antibody activation and after anti-CD3 antibody activation. T cells cultured with IL-2/S-15/Akti exhibited a mixture of CD4+, CD8+, and CD3+CD4?CD8? T cells; S-15 in combination with Akt inhibitor downregulated the expression of PD-1+Tim-3+ on Tils and decreased the Tregs in Tils. Additionally, the Tils expanded in the presence of the Akt inhibitor and S-15 showed enhanced antitumor activity as indicated by the increase in IFN-? producing tumor infiltrating CD8+ T cells and without comprising the Tils expansion.ConclusionOur study elucidates that IL-2/S-15/Akti expanded Tils and represent a viable source for the cellular therapy for patients with hepatocellular cancer.
Project description:Immune aging results in progressive loss of both protective immunity and T cell-mediated suppression, thereby conferring susceptibility to a combination of immunodeficiency and chronic inflammatory disease. Here, we determined that older individuals fail to generate immunosuppressive CD8+CCR7+ Tregs, a defect that is even more pronounced in the age-related vasculitic syndrome giant cell arteritis. In young, healthy individuals, CD8+CCR7+ Tregs are localized in T cell zones of secondary lymphoid organs, suppress activation and expansion of CD4 T cells by inhibiting the phosphorylation of membrane-proximal signaling molecules, and effectively inhibit proliferative expansion of CD4 T cells in vitro and in vivo. We identified deficiency of NADPH oxidase 2 (NOX2) as the molecular underpinning of CD8 Treg failure in the older individuals and in patients with giant cell arteritis. CD8 Tregs suppress by releasing exosomes that carry preassembled NOX2 membrane clusters and are taken up by CD4 T cells. Overexpression of NOX2 in aged CD8 Tregs promptly restored suppressive function. Together, our data support NOX2 as a critical component of the suppressive machinery of CD8 Tregs and suggest that repairing NOX2 deficiency in these cells may protect older individuals from tissue-destructive inflammatory disease, such as large-vessel vasculitis.
Project description:Fas ligand expression in certain tumors has been proposed to contribute to immunosuppression and poor prognosis. However, immunotherapeutic approaches may elicit the Fas-mediated elimination of immunosuppressive regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) within tumors that represent major obstacles for cancer immunotherapy. Previously, we showed that IL-2 and agonistic CD40 Ab (?CD40) elicited synergistic antitumor responses coincident with the efficient removal of Tregs and MDSCs. We demonstrate in this study in two murine tumor models that Treg and MDSC loss within the tumor microenvironment after IL-2/?CD40 occurs through a Fas-dependent cell death pathway. Among tumor-infiltrating leukocytes, CD8(+) T cells, neutrophils, and immature myeloid cells expressed Fas ligand after treatment. Fas was expressed by tumor-associated Tregs and immature myeloid cells, including MDSCs. Tregs and MDSCs in the tumor microenvironment expressed active caspases after IL-2/?CD40 therapy and, in contrast with effector T cells, Tregs significantly downregulated Bcl-2 expression. In contrast, Tregs and MDSCs proliferated and expanded in the spleen after treatment. Adoptive transfer of Fas-deficient Tregs or MDSCs into wild-type, Treg-, or MDSC-depleted hosts resulted in the persistence of Tregs or MDSCs and the loss of antitumor efficacy in response to IL-2/?CD40. These results demonstrate the importance of Fas-mediated Treg/MDSC removal for successful antitumor immunotherapy. Our results suggest that immunotherapeutic strategies that include exploiting Treg and MDSC susceptibility to Fas-mediated apoptosis hold promise for treatment of cancer.
Project description:Regulatory T cells (Tregs) are physiologically designed to prevent autoimmune disease and maintain self-tolerance. In tumour microenvironments, their presence is related to a poor prognosis, and they influence the therapeutic outcome due to their capacity to suppress the immune response by cell-cell contact and to release immunosuppressive cytokines. In this study, we demonstrate that Treg immunosuppressive activity can be modulated by the cross-linking between the CD45RA expressed by Tregs and the C-type lectin MGL. This specific interaction strongly decreases the immunosuppressive activity of Tregs, restoring the proliferative capacity of co-cultured T lymphocytes. This effect can be attributed to changes in CD45RA and TCR signalling through the inhibition of Lck and inactivation of Zap-70, an increase in the Foxp3 methylation status and, ultimately, the reduced production of suppressive cytokines. These results indicate a role of MGL as an immunomodulator within the tumour microenvironment interfering with Treg functions, suggesting its possible use in the design of anticancer vaccines.