Increased CD226 Expression on CD8+ T cells in Patients with Systemic Sclerosis
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ABSTRACT: Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular damage, and fibrosis of the skin and internal organs. Because activated and oligoclonally expanded CD8+ T cells can be detected in peripheral blood and lung of SSc patients, effector memory CD8+ T cells may play a critical role for organ involvement in SSc; however, the pathogenic functions of effector memory CD8+ T cells remain incompletely understood. Here we performed DNA microarray analysis of the sort-purified effector memory CD8+ T cells from SSc patients and healthy controls, and showed that the expression of genes related to immune response and cell adhesion, including CD226 (also known as DNAX accessory molecule-1, DNAM-1), was significantly altered. Moreover, detailed analysis of CD226 revealed that CD226highCD8+ T cells were increased in SSc patients (mean, 50.7%) compared with healthy controls (32.9%) and were appreciably associated with the severity of skin sclerosis and interstitial lung disease. Further, CD226highCD8+ T cells from SSc patients produced abundant IL-13 and were positively correlated with the cytotoxic capacity of CD8+ T cells against HUVECs. Finally, the neutralization of CD226 impaired IL-13 production and cytolysis against HUVECs. These findings indicate that upregulated CD226 expression on CD8+ T cells reflects disease severity and are involved in SSc pathogenesis via the production of profibrotic IL-13 and endothelial cell injury, and that CD226 may be a useful target in the treatment of SSc.
Project description:Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular damage, and fibrosis of the skin and internal organs. Because activated and oligoclonally expanded CD8+ T cells can be detected in peripheral blood and lung of SSc patients, effector memory CD8+ T cells may play a critical role for organ involvement in SSc; however, the pathogenic functions of effector memory CD8+ T cells remain incompletely understood. Here we performed DNA microarray analysis of the sort-purified effector memory CD8+ T cells from SSc patients and healthy controls, and showed that the expression of genes related to immune response and cell adhesion, including CD226 (also known as DNAX accessory molecule-1, DNAM-1), was significantly altered. Moreover, detailed analysis of CD226 revealed that CD226highCD8+ T cells were increased in SSc patients (mean, 50.7%) compared with healthy controls (32.9%) and were appreciably associated with the severity of skin sclerosis and interstitial lung disease. Further, CD226highCD8+ T cells from SSc patients produced abundant IL-13 and were positively correlated with the cytotoxic capacity of CD8+ T cells against HUVECs. Finally, the neutralization of CD226 impaired IL-13 production and cytolysis against HUVECs. These findings indicate that upregulated CD226 expression on CD8+ T cells reflects disease severity and are involved in SSc pathogenesis via the production of profibrotic IL-13 and endothelial cell injury, and that CD226 may be a useful target in the treatment of SSc. We first purified effector memory CD8+ T cells (CD3+CD8+CD45RO+CD62L+ cells) from peripheral blood by cell sorting and subsequently performed cDNA microarray analysis of the sort-purified effector memory CD8+ T cells from 9 SSc patients and 5 healthy controls.
Project description:We recently found that loss of the activating receptor CD226 (DNAM-1) was a critical mechanism affecting CD8+ T cell responsiveness to TCR stimulation. To better understand the molecular differences between CD226- and CD226+ CD8+ T cells, we decided to perform a global transcriptional analysis of purified naive (Tn, CD62L+CD45RA+), central memory (Tcm, CD62L+CD45RA-) and terminal effector memory (Temra, CD62L-CD45RA+) CD226+ and CD226- CD8+ T cells using next generation RNA sequencing.
Project description:We recently found that loss of the activating receptor CD226 (DNAM-1) was a critical mechanism affecting CD8+ T cell responsiveness to TCR stimulation. To better understand the molecular differences between CD226- and CD226+ CD8+ T cells, we decided to perform a global transcriptional analysis of purified CD226+ and CD226- CD8+ effector memory T cells before and after TCR activation using next generation RNA sequencing. We report here the total RNA sequencing of healthy donor resting CD226- and CD226+ CD8+ Tem cells (n=6/group) or activated (ACT, n=4/group) by α-CD2/CD3/CD28 for 24 hrs.
Project description:We found a unique subset of effector memory (EM) CD8+ T cells that expressed high levels of IL-6 receptor in human peripheral blood. These cells which also expressed high levels of IL-7Ra (referred to as IL-6R high IL-7Rahigh cells) had the a distinct gene expression profile and cellular characteristics compared to other EM CD8+ T cells. IL-6R high IL-7Ra high cells were early differentiated EM CD8+ T cells with decreased expression of T-bet, KLRG1, perforin and granzyme B. These cells had increased cell proliferation likely secondary to enhanced IL-2 production and high affinity IL-2R expression. IL-6R high IL-7Ra high EM CD8+ T cells exclusively produced high levels of IL-2, IL-5, IL-9 and IL-13 although IFN-r was produced by this cell subset and other EM CD8+ T cells. Of interest, IL-6R high IL-7Ra high EM CD8+ T cells expanded in the peripheral blood of patients with chronic obstructive pulmonary disease (COPD) and asthma where CD8+ T cells, IL-13 and IFN-r are suggested to be involved in the pathogenesis. Being the early-differentiated EM CD8+ T cells with a potent capacity to proliferate, survive and generate multiple cytokines, IL-6R high IL-7Ra high EM CD8+ T cells may serve as a primary reservoir for effector CD8+ T cells which potently expand and produce cytokines upon immune stimulation. Duplicate experiments were performed for each condition. In each condition, we independently prepared total RNA using the RNeasy mini kit (Qiagen) and assessed RNA integrity using Bioanalyzer 2100 (Agilent)- RINs were close to 10 for all samples. RNA was then amplified and hybridized to the Illumina HumanHT-12 v4.0 BeadChip, according to Illumina standard protocols.
Project description:Immunotherapeutics that modulate T cell activation represent a crucial component for inhibiting the autoimmune pathogenesis of type 1 diabetes (T1D) and improving the efficacy of β-cell replacement therapy. Here, we present a novel strategy for reducing diabetes incidence in the NOD mouse using monoclonal antibodies (mAbs) to block the T cell costimulatory receptor, CD226. Notably, female NOD mice treated with 600 µg of ⍺-CD226 mAbs between 7-8 weeks of age showed decreased disease incidence at 30 weeks and reduced insulitis severity compared to mice treated with an isotype control. Ex vivo analysis performed five weeks post-treatment revealed ⍺-CD226 mAbs persist in vivo, reducing the availability of CD226 on CD8+ T cells and Tregs. Flow cytometric analysis demonstrates that ⍺-CD226 mAbs inhibit the proliferation of both CD4+ and CD8+ T cells in vitro. Similarly, ex vivo samples had reduced CD4+ and CD8+ effector memory T cell proliferation following ⍺-CD226 mAb treatment. Splenocytes treated with ⍺-CD226 mAbs exhibited a more immunoregulatory cytokine profile with decreased IFN-γ and increased IL-10 production. This phenotype was further corroborated by 51Cr-release assays demonstrating reduced cell-mediated lympholysis (CML) of murine β-cells by ⍺-CD226 mAb-treated autoreactive cytotoxic lymphocytes. Ex vivo phenotyping of FOXP3+Helios+ Tregs revealed increased CD25 expression following ⍺-CD226 mAb treatment, with Tregs displaying augmented suppressive capacity of CD4+ responders during in vitro suppression assays. These data suggest that ⍺-CD226 mAbs both reduce T cell cytotoxicity and improve Treg function, with important therapeutic implications for the prevention or suspension of T1D.
Project description:At the peak of the CD8 T cell response to acture viral and bacterial infections, expression of the Interleukin-7 Receptor (IL-7R) marks Memory Precursor Effector CD8 T Cells (MPECs) from other Short-Lived Effector CD8 T cells (SLECs), which are IL-7Rlo. This study was designed to determine the gene expression differences between these two subsets of effector CD8 T cells. Experiment Overall Design: This study compared IL-7Rhi and IL-7Rlo LCMV-specific P14 Transgenic CD8 T cells, sorted from LCMV armstrong infected recipient mice 6/7 days after infection. Data includes 3 independent replicates for the IL-7Rhi and IL-7Rlo groups.
Project description:CD25, the high affinity interleukin-2 (IL-2) receptor alpha-chain, is rapidly upregulated by antigen-specific CD8+ T cells after T cell receptor stimulation. We demonstrated that during an acute viral infection, CD25 expression was dynamic, and a subset of virus-specific CD8+ T cells sustained CD25 expression longer than the rest. Examination of the in vivo fate of effector CD8+ T cells exhibiting differential responsiveness to IL-2 revealed that CD25lo cells, which were relatively less sensitive to IL-2, preferentially upregulated CD127 and CD62L and gave rise to the functional long-lived memory pool. In contrast, CD25hi cells that accumulate enhanced IL-2 signals, proliferated more rapidly, were prone to apoptosis, exhibited a more pronounced effector phenotype, and appeared to be terminally differentiated. Sustained IL-2 receptor signaling resulted in increased CD8+ T cell proliferation, higher granzyme B expression and exaggerated contraction after antigen clearance. These data support the hypothesis that prolonged IL-2 signals during priming promote terminal effector differentiation of CD8+ T cells. Experiment Overall Design: An important question in memory development is understanding the differences between effector CD8 T cells that die versus effector cells that survive and give rise to memory cells. In this study we have performed genomic profiling of terminal effectors and memory precursors as defined by CD25 heterogeneity, towards better understanding the generation of these subsets. The two effector subsets were FACS purified based on the amount of cell surface CD25 expression into CD25lo and CD25hi subsets during the early expansion phase (Days 3-4 post-infection) and analyzed for their gene expression profiles (by genome-wide microarray analyses).
Project description:We found a unique subset of effector memory (EM) CD8+ T cells that expressed high levels of IL-6 receptor in human peripheral blood. These cells which also expressed high levels of IL-7Ra (referred to as IL-6R high IL-7Rahigh cells) had the a distinct gene expression profile and cellular characteristics compared to other EM CD8+ T cells. IL-6R high IL-7Ra high cells were early differentiated EM CD8+ T cells with decreased expression of T-bet, KLRG1, perforin and granzyme B. These cells had increased cell proliferation likely secondary to enhanced IL-2 production and high affinity IL-2R expression. IL-6R high IL-7Ra high EM CD8+ T cells exclusively produced high levels of IL-2, IL-5, IL-9 and IL-13 although IFN-r was produced by this cell subset and other EM CD8+ T cells. Of interest, IL-6R high IL-7Ra high EM CD8+ T cells expanded in the peripheral blood of patients with chronic obstructive pulmonary disease (COPD) and asthma where CD8+ T cells, IL-13 and IFN-r are suggested to be involved in the pathogenesis. Being the early-differentiated EM CD8+ T cells with a potent capacity to proliferate, survive and generate multiple cytokines, IL-6R high IL-7Ra high EM CD8+ T cells may serve as a primary reservoir for effector CD8+ T cells which potently expand and produce cytokines upon immune stimulation.
Project description:Background: IL-33, a cytokine with pleiotropic functions, is elevated in serum of patients with hepatocellular carcinoma (HCC). This study investigated effects of local IL-33 expression in resected HCC on patient survival and on immunological and molecular tumor microenvironment. Methods: Tissue of resected HCCs was stained for H&E, masson trichrome, alpha smooth muscle actin, IL-33, CD8 and IL-13 and analysed by flow cytometry. Besides histomorphologic evaluation, the immunohistochemical stainings were analysed for the respective cell numbers separately for tumor area, infiltrative margin and distant liver stroma. These findings were correlated with clinical data and patient outcome. Further, gene expression of different HCC risk groups was compared using Micro Arrays. Results: In multivariable analysis, infiltration of HCCs by IL-33+ cells (P=0.032) and CD8+ cells (P=0.014) both independently were associated with prolonged patient survival. Flow cytometry demonstrated that cytotoxically active CD8+CD62L-KLRG1+CD107a+ effectory-memory cells are the main producers of IL-33 in these HCC patients. Using infiltration by IL-33+ and CD8+ cells as two separate factors, a HCC immune score (HCCIS) was designed and evaluated that stratified patient survival (P=0.0004). This HCCIS identified high and low risk patients who differ in gene expression profiles (P<0.001). Conclusion: Infiltration of HCCs by IL-33+ and CD8+ cells is independently associated with prolonged patient survival. We suggest that this is due to an induction of highly effective cytotoxically active CD8+CD62L-KLRG1+CD107a+ effector-memory cells producing IL-33. Based on these two independent factors we established a HCC immune score that provides risk stratification for HCC patients and can be used in the clinical setting. To investigate if HCCIS 0 high risk and HCCIS 2 low risk tumors exhibit a distinct molecular environment and gene expression pattern, RNA from fresh tumor tissue was isolated and analyzed by whole genome Microarray 4 patients with low risk tumors (HCCIS 2) were compared to 4 patients with high risk tumors (HCCIS 0).
Project description:“Memory-like T cells” are a subset of thymic cells that acquire effector function through the maturation process rather than interaction with specific antigen. Disruption of genes encoding T cell signaling proteins or transcription factors have provided insights into the differentiation of such cells. We show here that in BALB/c but not C57BL/6 mice, a large portion of thymic CD4-CD8+ T cells exhibit a memory-like phenotype. In BALB/c mice, IL-4 secreted by invariant natural killer T (iNKT) cells is both essential and sufficient for the generation of memory-like T cells. In C57BL/6 mice, iNKT cells are less abundant, producing IL-4 that is insufficient to induce thymic memory-like CD8+ T cells. BALB/c mice deficient in the transcription factor Kruppel-like factor (KLF) 13 have comparable numbers of iNKT cells to C57BL/6 mice and extremely low levels of thymic memory-like CD8+ T cells. This work documents the dramatic impact of a small number of KLF13-dependent iNKT cells. FACS-sorted naive splenic CD8 T cells from wild type and KLF13 ko mice were compared.