scholarly journals SLAM/SAP Decreased Follicular Regulatory T Cells in Patients With Graves’ Disease 

2021 ◽  
Author(s):  
Lina Geng ◽  
Jun Yang ◽  
Xinyi Tang ◽  
Huiyong Peng ◽  
Jie Tian ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Regulatory T Cells ◽  
Negative Correlation ◽  
Real Time Pcr ◽  
Graves Disease ◽  
Healthy Controls ◽  
Significant Difference ◽  
Ifn Γ ◽  
Signaling Lymphocytic Activation Molecule

Abstract Background: Signaling lymphocytic activation molecule (SLAM) and SLAM-associated protein (SAP) play important role in inflammatory and autoimmune diseases. Our study aimed to detect the expression of SLAM and SAP in patients with Graves’ disease (GD) and analyze the effect of SLAM/SAP on circulating blood CD4+CXCR5+ Foxp3+ follicular regulatory T (Tfr) cells.Methods: The expression of SLAM and SAP was assessed by flow cytometry and real-time PCR. The percentages of IFN-γ+ cells, IL-4+ cells, IL-17+ cells and Foxp3+ cells in CD4+CXCR5+ T cells and circulating CD4+CXCR5+ Foxp3+ Tfr cells after treatment with anti-SLAM and anti-CD3 antibodies were also assessed by flow cytometry. The correlations between the percentages of Tfr cells and the levels of autoantibodies as well as SAP were analyzed.Results: The level of SAP in CD4+CXCR5+ T cells and the level of SLAM on CD19+ B cells were significantly increased in the patients with GD, but no significant difference in the level of SLAM on CD4+CXCR5+ T cells was observed between the patients with GD and the healthy controls. A decrease in the percentage of Foxp3+ cells in CD4+CXCR5+ T cells was observed following anti-SLAM treatment, but the percentages of IFN-γ+ cells, IL-4+ cells and IL-17+ cells showed no obvious differences. The proportion of circulating Tfr cells was decreased in the patients with GD, and the proportion of circulating Tfr cells had a negative correlation with the level of SAP in CD4+CXCR5+ T cells and the levels of autoantibodies in the serum of the patients with GD.Conclusions: Our results indicate that the SLAM/SAP signaling pathway regulates Tfr cells, which may be involved in the pathogenesis of Graves’ disease.

scholarly journals SLAM/SAP Decreased Follicular Regulatory T Cells in Patients with Graves’ Disease

2021 ◽  
Vol 2021 ◽  
pp. 1-11
Author(s):  
Lina Geng ◽  
Jun Yang ◽  
Xinyi Tang ◽  
Huiyong Peng ◽  
Jie Tian ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Regulatory T Cells ◽  
Autoimmune Diseases ◽  
Negative Correlation ◽  
Graves Disease ◽  
Healthy Controls ◽  
Significant Difference ◽  
Ifn Γ ◽  
Signaling Lymphocytic Activation Molecule

Signaling lymphocytic activation molecule (SLAM) and SLAM-associated protein (SAP) play important role in inflammatory and autoimmune diseases. Our study is aimed at detecting the expression of SLAM and SAP in patients with Graves’ disease (GD) and analyzing the effect of SLAM/SAP on circulating blood CD4+CXCR5+Foxp3+ follicular regulatory T (Tfr) cells. The level of SAP in CD4+CXCR5+ T cells and the level of SLAM on CD19+ B cells were significantly increased in the patients with GD, but no significant difference in the level of SLAM on CD4+CXCR5+ T cells was observed between the patients with GD and the healthy controls. A decrease in the percentage of Foxp3+ cells in CD4+CXCR5+ T cells was observed following anti-SLAM treatment, but the percentages of IFN-γ+ cells, IL-4+ cells, and IL-17+ cells showed no obvious differences. The proportion of circulating Tfr cells was decreased in the patients with GD, and the proportion of circulating Tfr cells had a negative correlation with the level of SAP in CD4+CXCR5+ T cells and the levels of autoantibodies in the serum of the patients with GD. Our results suggested that the SLAM/SAP signaling pathway is involved in the decrease of circulating Tfr cells in Graves’ disease.

Reduction of Tregs with Expansion of Th1 Cells and Lack of IFN-γ Secretion by GPI Negative T-Cells In PNH

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2240-2240
Author(s):  
Shahram Kordasti ◽  
Modupe Elebute ◽  
Pilar Perez Abellan ◽  
Austin G Kulasekararaj ◽  
Janet Hayden ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
High Resolution ◽  
Peripheral Blood ◽  
Clonal Expansion ◽  
T Cell Subsets ◽  
Th1 Cells ◽  
Healthy Controls ◽  
Significant Difference ◽  
Ifn Γ

Abstract Abstract 2240 Introduction and Aim: Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haematopoietic stem cell disorder characterized by intravascular haemolysis and thrombosis. The pathogenetic link with bone marrow failure syndromes is well recognized, however the process of clonal expansion of the glycosylphosphatidylinositol (GPI)-deficient cells over normal haemotopoiesis remains unclear. To further elucidate mechanisms leading to clonal expansion in PNH, we investigated the immunological profile and performed high-resolution genome-wide karyotyping using Affymetrix SNP6 microarrays. Patients and Methods: The percentage and absolute numbers of CD4+ and CD8+ T-cell subsets, NK cells and B cells in peripheral blood were assessed in 8 patients with PNH prior to any therapy and 8 healthy age matched controls. High resolution SNP6 karyotyping was performed on bone marrow (n=15) and peripheral blood (n=12) of these patients. Bone marrow from an additional 8 patients was enriched for CD34+59- and CD34+59+ cell fractions for SNP array karyotyping. Abberations that overlapped by >50% with variations found in the Database of Genomic Variants, as well as an internal series of 91 normal subjects were excluded from further analysis. T-cells were stimulated and then stained intracellularly for TNF-α and IFN-γ (Th1), IL-4 (Th2) and IL-17 (Th17). NK cells were defined as CD3– CD56+. B cells were defined as CD3-CD19+. CD3+ CD4+ T-cell subsets were defined as CD45RO–CD27+ naïve, CD45RO+ CD27+ CD62L+ central memory, CD45RO+ CD27+ CD62L– effector memory, CD45RO+CD27– effectors and CD45RO–CD27– terminal effectors. CD4+ Tregs were defined as CD3+CD4+ CD25high CD27+Foxp3+. Results: There were no significant differences in the number or percentage of different CD8+ and CD4+ T-cells compared to healthy controls except for the number of Tregs and Th1 cells. In our cohort of patients, the number of Th1 cells was significantly higher than healthy controls (4.1×107/L v 0.93 × 107/L, p=0.039), whereas the number of Tregs cells was lower (0.75 × 107/L v 1.36 × 107/L, p=0.028). There was no significant difference in the number of Th2 and Th17 cells between patient and healthy subjects. Within CD4+ T-cells two distinct CD59+ and CD59- populations were identified, of which the CD59- cells were unable to secrete IFN-γ in response to stimulation compared to CD59+ population. On average 48% of CD4+ CD59+ T-cells secrete IFN-γ compared to 2% in the CD59- population. There was no significant difference in IL17 and IL4 secretion between CD59+ and CD59- T-cells. SNP karyotyping revealed three regions of uniparental disomy (UPD); UPD1p26.11-p34.3, UPD1p13.3-p13.1in one peripheral blood sample and UPD7q32.1-q34 in one bone marrow sample. There were no additional somatic genomic aberrations detected in any of the samples. Of note, purified CD34+59- cells did not reveal any clonal copy number changes or regions of UPD. Conclusion: Specific analysis of Xp22.1 did not reveal any aberrations of the PIGA gene, suggesting aberrations of the PIGA gene may be restricted to mutations or epigenetic abnormalities. Our immunological profiling revealed an expansion of Th1 cells and diminished Tregs in the peripheral blood, which is in contrast to our published data from both MDS and AA patients. The lack of IFN-γ secretion by GPI deficient T-cells also suggests an additional immunological defect in these patients, which may contribute in disease pathogenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Imbalance between IL-17A-Producing Cells and Regulatory T Cells during Ischemic Stroke

2014 ◽  
Vol 2014 ◽  
pp. 1-8 ◽  
Author(s):  
Yuehua Hu ◽  
Yanhua Zheng ◽  
Ya Wu ◽  
Bing Ni ◽  
Shugui Shi
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Ischemic Stroke ◽  
T Cell ◽  
Animal Models ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Real Time Pcr ◽  
Treg Cells ◽  
T Cell Subsets

Immune responses and inflammation are key elements in the pathogenesis of ischemic stroke (IS). Although the involvement of IL-17A in IS has been demonstrated using animal models, the involvement of IL-17A and IL-17-secreting T cell subsets in IS patients has not been verified, and whether the balance of Treg/IL-17-secreting T cells is altered in IS patients remains unknown. In the present study, we demonstrated that the proportion of peripheral Tregs and the levels of IL-10 and TGF-βwere reduced in patients with IS compared with controls using flow cytometry (FCM), real-time PCR, and ELISA assays. However, the proportions of Th17 andγδT cells, the primary IL-17A-secreting cells, increased dramatically, and these effects were accompanied by increases in the levels of IL-17A, IL-23, IL-6, and IL-1βin IS patients. These studies suggest that the increase in IL-17A-producing cells and decrease in Treg cells might contribute to the pathogenesis of IS. Manipulating the balance between Tregs and IL-17A-producing cells might be helpful for the treatment of IS.

scholarly journals Quantification of Regulatory T Cells in Septic Patients by Real-Time PCR–Based Methylation Assay and Flow Cytometry

PLoS ONE ◽  
2012 ◽  
Vol 7 (11) ◽  
pp. e49962 ◽  
Author(s):  
Roman Tatura ◽  
Michael Zeschnigk ◽  
Michael Adamzik ◽  
Michael Probst-Kepper ◽  
Jan Buer ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Regulatory T Cells ◽  
Real Time ◽  
Real Time Pcr ◽  
Methylation Assay

scholarly journals Altered Frequencies of CD4+ CD25+ Foxp3+ and CD8+ CD25+ Foxp3+ Regulatory T Cells in Pre-eclampsia

2019 ◽  
Author(s):  
Maryam Zare ◽  
Mehrnoosh Doroudchi ◽  
Behrouz Gharesi-Fard
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Regulatory T Cells ◽  
Negative Correlation ◽  
Peripheral Blood ◽  
Pregnancy Complications ◽  
Treg Cells ◽  
Positive Correlation ◽  
Blood Pressures ◽  
In Pregnancy

Regulatory T cells are of utmost importance for tolerating the fetus. In some pregnancy complications such as pre-eclampsia, the frequency of CD4+CD25+Foxp3+ regulatory T cells is altered, but there is no consistency regarding the results. Besides, little is known about the frequency of CD8+CD25+Foxp3+ Treg cells in pregnancy complications. Therefore, we aimed to investigate the frequency of both CD4+ and CD8+ regulatory T cells in the peripheral blood of women afflicted by preeclampsia. Ten non-pregnant, ten healthy pregnant, and ten preeclamptic women participated in this study. Four colors flow cytometry method was used to identify the frequency of the CD4+ and CD8+ regulatory T cells in the peripheral blood. Results indicated that the frequencies of CD4+CD25+Foxp3+ and CD8+CD25+Foxp3+ cells were significantly lower in preeclamptic women compared to healthy pregnant and non-pregnant ones (p<0.05). A positive correlation was also observed between CD4+ and CD8+ regulatory T cells (R=0.532, p=0.002). Moreover, CD4+ regulatory T cells negatively correlated with systolic and diastolic blood pressures (R=-0.760 and -0.753, respectively; p<0.001). CD8+ regulatory T cells also had a negative correlation with systolic (R=-0.503, p=0.001) and diastolic (R=-0.590, p=0.005) blood pressures. In conclusion, a reduction in the frequencies of both CD4+ CD25+ Foxp3+ and CD8+CD25+Foxp3+ regulatory T cells might be important in the pathogenesis of pre-eclampsia.

scholarly journals Insufficient Phosphorylation of STAT5 in Tregs Inhibits the Expression of BLIMP-1, Leading to a Lack of Tregs in Pediatric Aplastic Anemia

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2183-2183
Author(s):  
Lifen Huang ◽  
Junbin Huang ◽  
Chengming Zhu ◽  
Hongman Xue ◽  
Chi Kong Li ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Aplastic Anemia ◽  
Inflammatory Cytokines ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Healthy Controls ◽  
Healthy Donors ◽  
Ifn Γ ◽  
Low Expression

Abstract Aplastic anemia (AA) is a group of bone marrow failure diseases characterized by three-line blood cell reduction and decreased myeloproliferation. It is believed that T cell immune disorder is the leading cause of the disease, especially the number and functional damage of regulatory T cells (Tregs). BLIMP-1 is a transcription factor encoded by PRDM1 gene, which is indispensable for Tregs. The expression of BLIMP-1 is mainly induced by the IL-2/STAT5 signaling pathway. However, the level of phosphorylation of STAT5 and the expression of BLIMP-1 in Tregs from patients with AA has not been revealed, and the mechanism of Tregs damage in AA has not yet been clarified. In the present study, we collected peripheral blood from 10 newly diagnosed AA children and 10 age-matched healthy donors. We observed that the ratio of Tregs/lymphocytes and Tregs/CD4 + T cells decreased significantly in AA patients, compared with healthy controls by flow cytometry. In addition, we found significantly elevated levels of inflammatory cytokines IL-2, IL-6, and IFN-γ, but decreased levels of anti-inflammatory cytokines IL-10 and TGF-β in plasma of children with AA, compared with healthy controls. Quantitative real-time PCR showed decreased transcriptional level of BLIMP-1 in peripheral blood mononuclear cells (PBMC) from children with AA, compared with healthy donors. We used flow cytometry to detect the protein level of BLIMP-1 in Tregs and found that the level of BLIMP-1 in Tregs in the peripheral blood of children with AA was significantly lower than that of healthy donors. The correlation analysis showed that the percentage of BLIMP-1 + Tregs was positively correlated with the ratio of Tregs/CD4 + T cells (r=0.829, p＜0.001), the plasma level of IL-10 (r=0.492, p=0.027), and TGF-β (r=0.482, p=0.030), suggesting that low expression level of BLIMP-1 in Tregs may lead to decreased number of Tregs in peripheral blood and declined levels of IL-10 and TGF-β in children with AA. When stimulated IL-2, the level of pSTAT5 in CD4 + T cells of children with AA was significantly reduced compared with that of healthy donors. The level of pSTAT5 in CD4 + T cells was also positively correlated with the ratio of Tregs/CD4 + T cells (r= 0.575, p= 0.008) and the expression of BLIMP-1 in Tregs (r=0.693, p＜0.0001)，suggesting that STAT5 signal is poorly activated in pediatric AA, and it may be an important cause for the low expression of BLIMP-1 in Tregs and the decrease in the number of Tregs in children with AA. Furthermore, we constructed an AA mouse model by co-administering IFN-γ and busulfan for 10 consecutive days. These mice exhibited decreased ratio of Tregs/CD4 +T cells in the spleen and lower BLIMP-1 in Tregs, compared with controls. Also, we isolated Tregs with immunomagnetic beads from spleens of mice and observed that the level of IL-2-stimulated pSTAT5 was lower in isolated Tregs from AA mice than controls. These phenotypes were partially rescued by intervention of IL-2-JES6-1, an antibody complex tends to promote the proliferation of Tregs in mice, while inhibiting the proliferation of effector T cells. IL-2-JES6-1 increased the level of pSTAT5 and the expression of BLIMP-1 in Tregs from spleen of the AA mice, and elevated the ratio of Tregs/CD4 + T cells in the spleen. In conclusion, we found that Tregs from AA patients have impaired phosphorylation of STAT5 and insufficient expression of BLIMP-1, which may contribute to the pathogenesis of AA. Disclosures No relevant conflicts of interest to declare.

scholarly journals CXCR6 within T-helper (Th) and T-cytotoxic (Tc) type 1 lymphocytes in Graves’ disease (GD)

2005 ◽  
Vol 152 (4) ◽  
pp. 635-643 ◽  
Author(s):  
G Aust ◽  
M Kamprad ◽  
P Lamesch ◽  
E Schmücking
Keyword(s):  
T Cells ◽  
Normal Subjects ◽  
Peripheral Blood Lymphocytes ◽  
Th2 Cells ◽  
Thyroid Peroxidase ◽  
Graves Disease ◽  
Mrna Levels ◽  
T Helper ◽  
Significant Difference ◽  
Ifn Γ

Objective: In Graves’ disease (GD), stimulating anti-TSH receptor antibodies are responsible for hyperthyroidism. T-helper 2 (Th2) cells were expected to be involved in the underlying immune mechanism, although this is still controversial. The aim of this study was to examine the expression of CXCR6, a chemokine receptor that marks functionally specialized T-cells within the Th1 and T-cytotoxic 1 (Tc1) cell pool, to gain new insights into the running immune processes. Methods: CXCR6 expression was examined on peripheral blood lymphocytes (PBLs) and thyroid-derived lymphocytes (TLs) of GD patients in flow cytometry. CXCR6 cDNA was quantified in thyroid tissues affected by GD (n = 16), Hashimoto’s thyroiditis (HT; n = 2) and thyroid autonomy (TA; n = 11) using real-time reverse transcriptase PCR. Results: The percentages of peripheral CXCR6+ PBLs did not differ between GD and normal subjects. CXCR6 was expressed by small subsets of circulating T-cells and natural killer (NK) cells. CXCR6+ cells were enriched in thyroid-derived T-cells compared with peripheral CD4+ and CD8+ T-cells in GD. The increase was evident within the Th1 (CD4+ interferon-γ+ (IFN-γ+)) and Tc1 (CD8+IFN-γ+) subpopulation and CD8+ granzyme A+ T-cells (cytotoxic effector type). Thyroid-derived fibro-blasts and thyrocytes were CXCR6−. There was no significant difference between the CXCR6 mRNA levels in GD compared with HT and normal TA tissues. The lowest CXCR6 mRNA levels were obtained from thyroid nodules from TA patients and GD patients with low thyroid peroxidase autoantibody levels. Conclusions: CXCR6 was overexpressed in Th1 and Tc1 TLs compared with PBLs in GD. CXCR6 could be a marker for lymphocytes that have migrated into the thyroid and assist in the thyroid, independently of the bias of the underlying disease.

scholarly journals Endometrial mesenchymal stem/stromal cells: The Enigma to code messages for generation of functionally active regulatory T cells

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Mehdi Aleahmad ◽  
Mahmood Bozorgmehr ◽  
Shohreh Nikoo ◽  
Alireza Ghanavatinejad ◽  
Mohammad-Reza Shokri ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
T Cells ◽  
Mesenchymal Stem Cells ◽  
Regulatory T Cells ◽  
Immune Responses ◽  
Cd4 Cells ◽  
Ifn Γ ◽  
Pre Treatment

Abstract Background Regulatory T cells (Tregs) play an important role in fine-tuning of immune responses and are pivotal for a successful pregnancy. Recently, the importance of mesenchymal stem cells in regulation of immune responses in general and Tregs in particular has been highlighted. Here, we hypothesized that menstrual stromal/stem cells (MenSCs) contribute to uterine immune system regulation through induction of functionally active Tregs. Methods MenSCs were collected from 18 apparently healthy women and characterized. Bone marrow mesenchymal stem cells (BMSCs) served as a control. The effect of MenSCs on proliferation of anti-CD3/CD28-stimulated T CD4 + cells and generation of Tregs with or without pre-treatment with mitomycin C, IFN-γ and IL-1β was evaluated by flow cytometry. The potential role of IDO, PGE2, IL-6, IL-10, and TGF-β on proliferation of T CD4 + cells and generation of Tregs was assessed using blocking antibodies or agents. IDO activity was evaluated in MenSCs and BMSCs culture supernatants by a colorimetric assay. IL-10 and IFN-γ production in MenSCs-primed T CD4 + was measured using intracellular staining. To investigate the functional properties of Tregs induced by MenSCs, Treg cells were isolated and their functional property to inhibit proliferation of anti-CD3/CD28-stimulated PBMCs was assessed by flow cytometry. Results According to the results, proliferation of T CD4 + lymphocytes was enhanced in the presence of MenSCs, while pre-treatment of MenSCs with pro-inflammatory cytokines reversed this effect. PGE2 and IDO were the major players in MenSCs-induced T cell proliferation. Non-treated MenSCs decreased the frequency of Tregs, whereas after pre-treatment with IFN-γ and IL-1β, they induced functional Tregs with ability to inhibit the proliferation of anti-CD3/CD28-stimulated PBMCs. This effect was mediated through IL-6, IL-10, TGF-β and IDO. IFN-γ/IL-1β-treated MenSCs induced IL-10 and IFN-γ production in CD4 + T cells. Conclusion Collectively, these findings indicate that immunomodulatory impact of menstrual blood stem cells (MenSCs) on generation of Tregs and inhibition of T cells proliferation is largely dependent on pre-treatment with IFN-γ and IL-1β. This is the first report on immunomodulatory impact of MenSCs on Tregs and highlights the pivotal role of endometrial stem cells in regulation of local endometrial immune responses.

PD-1/PD-L Pathway Potentially Involved in ITP Immunopathogenesis

2019 ◽  
Vol 119 (05) ◽  
pp. 758-765 ◽  
Author(s):  
Mu Nie ◽  
Yang Liu ◽  
Xiu-xiu Li ◽  
Ya-nan Min ◽  
Dan-dan Yang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Cell Apoptosis ◽  
Cd4 T Cells ◽  
Peripheral Tolerance ◽  
Healthy Controls ◽  
T Cell Apoptosis ◽  
Ifn Γ

AbstractThe binding of programmed death 1 (PD-1) to its ligands PD-L1 and PD-L2 on antigen-presenting cells turns off autoreactive T cells and induces peripheral tolerance. Aberrant PD-1/PD-L signalling could result in a breakdown of peripheral tolerance and lead to autoimmune diseases. In this study, we detected PD-1 and PD-L expression on T cells and dendritic cells (DCs) in immune thrombocytopenia (ITP) patients with active disease by flow cytometry. The effects of PD-L1-Fc fusion protein (PD-L1-Fc) on T cells and on secretion of interferon-γ (IFN-γ) and interleukin-2 (IL-2) were detected by flow cytometry and enzyme-linked immunosorbent assay, respectively. Compared with healthy controls, PD-1 expression was significantly increased in CD4+ T cells and CD8+ T cells from patients with active ITP. However, PD-L1 expression on monocyte-derived DCs was lower in patients with active ITP than in healthy controls. In vitro assays revealed that PD-L1-Fc increased T cell apoptosis, inhibited activation and proliferation of CD4+ T cells and CD8+ T cells and decreased IFN-γ and IL-2 secretion in patients with active ITP. These results suggest that the aberrant PD-1/PD-L negative co-stimulatory pathway may play a role in ITP. Enhancing PD-1/PD-L signalling might be a promising therapeutic approach for ITP patients by enhancing T cell apoptosis, inhibiting T cell activation and proliferation and reducing secretion of inflammatory factors.

scholarly journals PD-1/PD-L Pathway Potentially Involved in ITP Immunopathogenesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3700-3700
Author(s):  
Mu Nie ◽  
Qi Feng ◽  
Yu Hou ◽  
Miao Xu ◽  
Jun Peng ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
T Cell Activation ◽  
Cd8 T Cells ◽  
Cell Apoptosis ◽  
Cell Activation ◽  
Healthy Controls ◽  
T Cell Apoptosis ◽  
Significant Difference ◽  
Ifn Γ

Abstract Introduction Immune thrombocytopenia (ITP) is an autoimmune disease in which autoreactive T and B cells are activated by platelet autoantigens resulting in immune-mediated platelet destruction and/or suppression of platelet production. The binding of programmed death 1 (PD-1) to its ligands PD-L1 and PD-L2 on antigen-presenting cells turns off autoreactive T cells and induces peripheral tolerance. Aberrant PD-1/PD-L signalling could result in a breakdown of peripheral tolerance and lead to autoimmune diseases. Methods Thirty-four patients with primary active ITP who were diagnosed and/or followed up and 26 healthy controls were enrolled in this study. Platelet counts in all ITP patients were less than 30×109 /L at sampling. They had not been treated with any immunosuppressive agents for at least one week prior to sampling for this study. To determine the role of the PD-1/PD-L signalling pathway in ITP, we detected PD-1 expression on T cells and PD-L expression on dendritic cells (DCs) in both ITP patients with active disease and healthy controls by flow cytometry. To investigate the effects of PD-L1-Fc fusion protein (PD-L1-Fc) on T cells, PBMCs from ITP patients and healthy controls with autologous platelets were cultured with soluble anti-CD3 monoclonal antibody (mAb) and anti-CD28 mAb in the presence (PD-L1-Fc+ group) or absence (PD-L1-Fc- group) of the PD-L1-Fc (0.5 μg/mL) at 37 ˚C with 5% CO2 for 4 days. The cells were harvested and stained for flow cytometry to detect the apoptosis, activation and proliferation of T cells. IL-2 and IFN-γ levels in the co-culture supernatant were assayed by ELISA. Results Enhanced PD-1 expression on T cells and decreased PD-L1 expression on mDCs in ITP patients We found that PD-1 mean fluorescence intensities (MFIs) increased in CD4+ cells (P < 0.01) and in CD8+ cells (P < 0.01) from ITP patients compared with those from healthy controls. However, PD-L1 expression on monocyte-derived DCs was lower in patients with active ITP than in healthy controls (P < 0.01, Figure 1). PD-L1-Fc promoted T cell apoptosis Annexin V-FITC and propidium iodide (PI) were used to detect T cell apoptosis. The percentage of apoptotic cells and dead cells were analysed to determine T cell apoptosis levels. In ITP patients, the percentage of apoptotic cells and dead cells were higher in PD-L1-Fc+ group than in the PD-L1-Fc- group (P < 0.01 for both CD4+ and CD8+ T cells). However, we found no significant difference in apoptosis between PD-L1-Fc- and PD-L1-Fc+ groups in healthy controls (Figure 2). PD-L1-Fc inhibited T cell activation and proliferation CD25 MFIs were analysed to determine the activation level of cocultured T lymphocytes. Compared with healthy controls, CD25 expression on CD4+ and CD8+ T cells was significantly increased in ITP patients (P < 0.05 for both CD4+ and CD8+ T cells). These results suggest that ITP patients had more activated CD4+ and CD8+ T cells than in healthy controls. PD-L1-Fc significantly inhibited T cell activation in ITP patients (P < 0.01, n=34) but not in healthy controls (P = 0.0834 in CD4+ T cells, P = 0.6834 in CD8+ T cells, n=26, Figure 3). To analyse proliferation, the frequency of divided cells was calculated according to the loss of CFSE fluorescence intensity. PD-L1-Fc inhibited proliferation of T cells from ITP patients (P < 0.01 in both CD4+ T and CD8+ T cells, n=34, Figure 4). We found no significant difference in proliferation in T cells from healthy controls (CD4+ T cells P = 0.9758, CD8+ T cells P = 0.5658, n=26). PD-L1-Fc inhibited IL-2 and IFN-γ production IL-2 secretion was higher in ITP than in healthy controls in the absence of PD-L1-Fc (P = 0.0308, Figure 5). PD-L1-Fc significantly inhibited IL-2 and IFN-γ production in ITP patients. IL-2 and IFN-γ levels were lower in the PD-L1-Fc+ group than in the PD-L1-Fc- group in ITP (P < 0.01 for both IL-2 and IFN-γ, n=34). However, we did not detect a significant difference in secretion of IL-2 (P = 0.2016, n=26) or IFN-γ (P = 0.2989, n=26) in healthy controls. Conclusions In summary, our study suggests that the aberrant PD-1/PD-L negative costimulatory pathway may play a role in ITP. Enhancing PD-1/PD-L signalling might be a promising therapeutic approach for ITP by promoting T cell apoptosis, inhibiting T cell activation and proliferation, and reducing secretion of inflammatory factors. Disclosures No relevant conflicts of interest to declare.

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