Elevated Expression of IL-17 and IL-23 in Patients with Immune Thrombocytopenic Purpura

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4537-4537
Author(s):  
Feng Li ◽  
Shanhua Zou ◽  
Yunfeng Cheng
Keyword(s):  
T Cells ◽  
Immune Thrombocytopenic Purpura ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
Statistical Difference ◽  
Plasma Concentrations ◽  
Healthy Controls ◽  
Cd4 Cells ◽  
Thrombocytopenic Purpura ◽  
Ifn Γ

Abstract Introduction: In conjunction with IL-6 and TGF-β, IL-23 stimulates naïve CD4+ T cells to differentiate into Th17 cells. Th17 cells produce IL-17, a pro-inflammatory cytokine that play an important role in the pathogenesis of several autoimmune disorders. However, to date, the role of Th17 cells in immune thrombocytopenic purpura (ITP), a type of autoimmune diseases, has not been clearly established yet. Methods: Peripheral bloods were obtained from 10 patients with ITP at onset, in remission and from 15 healthy control subjects. The frequencies of IL-17 producing T cells in peripheral blood were analyzed by flow cytometry. Peripheral blood Mononuclear cells (PBMCs) were isolated using Ficoll density-gradient centrifugation and the CD4+ cells were separated by immuno-magnetic microbeads selection. Plasma concentrations of Th17 cell-associated cytokines such as IL-12, IL-17, IL-23, IFN-γ, IL-6 and TGF-β were measured using ELISA. The mRNA expression levels of IL-17, IL-12p40, IFN-γ, IL-23p19 in CD4+ cells were determined by Real-Time PCR. Results: The frequencies of IL-17-producing T cells were significantly increased in ITP patients at onset, compared to ITP patients in remission (10.7±5.5 % vs 4.1±3.5 %, p < 0.05) and healthy controls (10.7±5.5 % vs 2.1±1.6 %, p < 0.05), however there was no statistical difference between ITP patients in remission and healthy controls. Comparing to healthy subjects, the plasma concentrations of IL-12 (33.3±15.25 pg/ ml vs 12.8±7.24 pg/ml, p<0.05), IL-17 (37.3±12.1 pg/ml vs 5.1±3.6 pg/ml, p < 0.05), IL-23 (30.01±9.33 pg/ml vs 10.42±13.19 pg/ml, p < 0.05) in patients with ITP at onset were found significantly elevated whereas no statistical difference was observed for the levels of IL-12, IL-17 and IL-23 between ITP patients in remission and healthy controls. Furthermore, the expression levels of IL-23p19 mRNA were significantly increased in ITP patients at onset, compared to healthy controls. Changes in IL-23p19 mRNA expression and IL-17 were strongly correlated (R = 0.66, p < 0.05). Conclusion: Our results support the hypothesis that Th17 cells are involved in the development of ITP and Th17 cells could potentially constitute a novel therapeutic target.

scholarly journals Interleukin-12 Is the Optimum Cytokine To Expand Human Th17 Cells In Vitro

2009 ◽  
Vol 16 (6) ◽  
pp. 798-805 ◽  
Author(s):  
Soad Nady ◽  
James Ignatz-Hoover ◽  
Mohamed T. Shata
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
Interleukin 12 ◽  
Interleukin 17 ◽  
Functional Evaluation ◽  
Optimum Conditions ◽  
T Helper ◽  
Ifn Γ

ABSTRACT Recently, a new lineage of CD4+ T cells in humans and in mice has been reported. This T helper cell secretes interleukin-17 (IL-17) and has been defined as T helper 17 (Th17). Th17 cells express the IL-23 receptor (IL-23R) and play an important pathogenic role in different inflammatory conditions. In this study, our aim was to characterize the optimum conditions for isolation and propagation of human peripheral blood Th17 cells in vitro and the optimum conditions for isolation of Th17 clones. To isolate Th17 cells, two steps were taken. Initially, we negatively isolated CD4+ T cells from peripheral blood mononuclear cells of a normal human blood donor. Then, we isolated the IL-23R+ cells from the CD4+ T cells. Functional studies revealed that CD4+ IL-23R+ cells could be stimulated ex vivo with anti-CD3/CD28 to secrete both IL-17 and gamma interferon (IFN-γ). Furthermore, we expanded the CD4+ IL-23R+ cells for 1 week in the presence of anti-CD3/CD28, irradiated autologous feeder cells, and different cytokines. Our data indicate that cytokine treatment increased the number of propagated cells 14- to 99-fold. Functional evaluation of the expanded number of CD4+ IL-23R+ cells in the presence of different cytokines with anti-CD3/CD28 revealed that all cytokines used (IL-2, IL-7, IL-12, IL-15, and IL-23) increased the amount of IFN-γ secreted by IL-23R+ CD4+ cells at different levels. Our results indicate that IL-7 plus IL-12 was the optimum combination of cytokines for the expansion of IL-23R+ CD4+ cells and the secretion of IFN-γ, while IL-12 preferentially stimulated these cells to secrete predominately IL-17.

Profiles of Th1, Th2, Th17 and Treg Cells in Patients with Chronic Idiopathic Thrombocytopenic Purpura.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3404-3404
Author(s):  
Rong-Fu Zhou ◽  
Jian Ou-yang ◽  
Da-Yu Chang ◽  
Jing-Yan Xu ◽  
Bing Chen ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Idiopathic Thrombocytopenic Purpura ◽  
Peripheral Blood ◽  
Th17 Cells ◽  
Treg Cells ◽  
Chronic Idiopathic Thrombocytopenic Purpura ◽  
Active Stage ◽  
Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Ifn Γ

Abstract Objective: To explore the profiles of Th1,Th2, Th17 and Treg cells in patients with chronic idiopathic thrombocytopenic purpura. Methods: Samples of peripheral blood were collected from 30 chronic ITP patients ( 9 males and 21 females), aged 41, 21 being in active stage, and 9 in remission stage, and 9 healthy persons in control (3 males and 6 females), aged 36. Peripheral blood was cultured, and activated with PMA/ionomycin when Th1, Th2 and Th17 cells were detected. Flow cytometry was used to measure the intracellular cytokines interferon (IFN)-γ, interleukin (IL)-4 and interleukin (IL)-17 so as to identify the Th1 cells (CD3+ CD8− IFN-γ+ IL-4− cells), Th2 cells (CD3+ CD8− IFN-γ − IL-4+ cells) and IL-17 cells (CD3+ CD8− IL-17+ cells); Treg cells were identified to CD4+ CD25+ Foxp3+ cells and uncultured peripheral blood was used to measured the CD4+ CD25+ Foxp3+ cells by flow cytometry. The ratios of Th1/Th2 were calculated. Results: The Th1/Th2 ratio for patients in active stage was 15.04±9.67, significantly higher than those for patients in remission stage (7.17±5.38, P <0.05) and in control (8.47±3.78, P <0.05); the percentage of Treg cells of the patients in active stage was 0.89±0.58%, significantly decreased than those of patients in remission stage (6.41±1.86%, P <0.001) and in control (6.06±0.85%, P <0.001); the percentage of Th17 cells was 1.94±0.77% for patients in active stage, 2.16±0.52% for patients in remission stage and 1.82±0.58% for patients in control, respectively, and there was no statistic significance between them. Conclusion: Chronic ITP is a Th1 predominant disease; decreased number and function of Treg cells might be one of mechanisms that cause immune regulation dysfunction in chronic ITP; Th17 cells might not play a role in the development of chronic ITP.

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 Aberrant Expression of MicroRNAs in CD4+ Cells May Contribute to the Imbalance of Th17/Treg Cells in Primary Immune Thrombocytopenia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1140-1140
Author(s):  
Mingqiang Hua ◽  
Qi Feng ◽  
Ju Li ◽  
Yu Hou ◽  
Shuwen Wang ◽  
...  
Keyword(s):  
Peripheral Blood ◽  
Th17 Cells ◽  
Culture Medium ◽  
Treg Cells ◽  
Healthy Controls ◽  
Cd4 Cells ◽  
Rt Pcr ◽  
Aberrant Expression ◽  
Relative Expression ◽  
Regulated Expression

Abstract Backgrounds:Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease characterized by reduced platelet count and an increased risk of bleeding. The imbalance of Treg/Th17 cells has been demonstrated in ITP, but the mechanism of Th17/Treg cells imbalance is still not clear. In this study, we aimed to investigate whether the expression of helper T (Th) or Treg cell-related microRNAs, such as miR-183-96-182 cluster, miR-17-5p, miR-99a, miR-146-5p, miR-155-5p, miR-181-5p, and miR-326, regulates the ratio of Th17/Treg in CD4+ T cells and could be used to evaluate the clinical implications of ITP patients. Methods: Peripheral blood was obtained from 54 patients with active ITP and 34 healthy controls. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density-gradient centrifugation and the CD4+ cells were separated by immuno-magnetic microbeads selection. Amplification technique of RT-PCR using stem-loop primers was applied to detect the relative expression of microRNAs (miR-17-5p, miR-99a, miR-96-5p, miR-146a-5p, miR-155-5p, miR-181a-5p, miR-182-5p, miR-183-5, miR-326) and U6 was normalized as control for miRNA quantification. The frequencies of Th17 and Treg cells in peripheral blood were analyzed by flow cytometry. The mRNA expression levels of Il-6, Il-10, Il-17, Rorγ-t and Foxp-3 in CD4+ cells were determined by RT-PCR. Platelet autoantibodies specific for GPIIb/IIIaor GPIb/IX were measured using MAIPA method. CD4+ cells were transfected with miRNAs (miR-99a, miR-182-5p, miR-183-5), mimics or inhibitors, which were used to detect the function of miRNAs. Cytokines in culture medium were determined by ELISA. Results: Our results showed that the relative expression of miR-182-5p and miR-183-5p in CD4+ cells was significantly increased in active ITP patients, compared to healthy controls (miR-182-5p, median 9.2678 vs 5.2723, p < 0.05, Fig. 1a; miR-183-5p, median 5.4435 vs 2.009, p < 0.05, Fig. 1b). In addition, the relative expression of miR-99a in ITP patients was lower than that of healthy controls (median 3.4214 vs 7.9648, p < 0.05; Fig. 1c). Moreover, the frequency of Treg cells decreased significantly in ITP patients compared to those in controls (1.89±1.59% vs 4.12±1.42%, p < 0.05; Fig. 2a), and the percentage of Treg cells was positively correlated with the relative expression of miR-99a in ITP patients(r=0.461, p< 0.05; Fig. 2c) and health controls(r=0.729, p< 0.05; Fig. 2d). Though the percentage of Th17 cells increased in ITP patients compared to the health controls (3.51±2.13%vs 1.85±0.63%, p < 0.05; Fig. 2b), there was no correlation between the percentage of Th17 and the relative expression of microRNAs in ITP patients or health controls. Besides, there was no correlation between the expression of mRNAs (Il-10, Il-17, Rorγ-t and Foxp-3) and microRNAs (miR-99a, miRNA-182-5p or miR-183-5p). No significant correlation was found between the microRNAs expression and platelets counts or different autoantibody subsets in ITP patients. The relative expression of other microRNAs (miR-17-5p, miR-96-5p, miR-146a-5p, miR-155-5p, miR-181-5p, miR-326) revealed no difference in CD4+ cells between ITP patients and health controls. Furthermore, the down-regulated expression of miR-183-5p with inhibitors promoted to the differentiation of Th17 cells(Fig. 3a), while up-regulated expression of miR-99a with mimics contributed to Treg cells in CD4+ cells from ITP patients (Fig. 3b). Meanwhile, the IL-17A in culture medium decreased in inhibitor group of miR-183-5p or miR-183-5p. However, miR-182-5p inhibitor had no effect on the differentiation of Th17 cells. Conclusions: Our results show the abnormal expression of microRNAs (miR-99a, miRNA-182-5p and miR-183-5p) in CD4+ cells and the miR-99a was closely correlated with the Treg cells. The aberrant expression of microRNAs may contribute to the imbalance of Th17/Treg cells in the development of ITP patients and potentially constitute a novel therapeutic target. Disclosures No relevant conflicts of interest to declare.

scholarly journals Activated but impaired IFN-γ production of mucosal-associated invariant T cells in patients with hepatocellular carcinoma

2021 ◽  
Vol 9 (11) ◽  
pp. e003685
Author(s):  
Wenyong Huang ◽  
Dongmei Ye ◽  
Wenjing He ◽  
Xiaoshun He ◽  
Xiaomin Shi ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
T Cells ◽  
Peripheral Blood ◽  
Healthy Controls ◽  
Activation Markers ◽  
Mait Cells ◽  
Hla Dr ◽  
Ifn Γ ◽  
And Function

ObjectiveMucosal-associated invariant T (MAIT) cells are innate T cells with immunoregulatory activity and were recently found to be associated with various tumor types. The role of intrasinusoidal MAIT cells in hepatocellular carcinoma (HCC) has not been fully characterized.DesignPeripheral blood samples were obtained from patients with HCC and healthy controls. Liver-associated mononuclear cells (LMCs) were collected from liver perfusions of donors and patients with HCC undergoing liver transplantation. Blood and liver perfusates from patients with HCC were analyzed by flow cytometry for CD3 +CD161+Vα7.2+MAIT cell frequency, phenotype, and function.ResultsThere were fewer MAIT cells in the peripheral blood and liver of patients with HCC than in the healthy controls. Interferon-γ (IFN-γ) production by these cells was also reduced. Peripheral MAIT cells showed upregulation of HLA-DR (Human Leukocyte Antigen DR） and the inhibitory molecule PD-1 (Programmed Cell Death Protein 1), but no significant differences in upregulation were found in intrasinusoidal MAIT cells. MAIT cells were significantly enriched in the liver relative to that in the peripheral blood of patients with HCC. High levels of activation markers and exhaustion markers including HLA-DR, CD69, and PD-1 were observed in LMCs of patients with HCC but not in the peripheral blood. Single-cell RNA sequencing revealed that intrasinusoidal MAIT cells exhibited distinct features in patients with HCC and the controls.ConclusionOur study showed that alterations in MAIT cells are associated with HCC. The distinct activity and function of MAIT cells in the peripheral blood and liver of patients with HCC might suggest a potential role of these cells in disease pathogenesis.

scholarly journals Peritoneal Fluid from Patients with Ovarian Endometriosis Displays Immunosuppressive Potential and Stimulates Th2 Response

2021 ◽  
Vol 22 (15) ◽  
pp. 8134
Author(s):  
Joanna Olkowska-Truchanowicz ◽  
Agata Białoszewska ◽  
Aneta Zwierzchowska ◽  
Alicja Sztokfisz-Ignasiak ◽  
Izabela Janiuk ◽  
...  
Keyword(s):  
T Cells ◽  
Immune Responses ◽  
Cytotoxic Activity ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Peritoneal Fluid ◽  
Th17 Cells ◽  
Th2 Response ◽  
Immunoregulatory Cytokines ◽  
Ifn Γ

Endometriosis is a common gynaecological disorder characterized by the ectopic growth of endometrial tissue outside the uterine cavity. It is associated with chronic pelvic inflammation and autoimmune reactivity manifesting by autoantibody production and abrogated cellular immune responses. Endometriotic peritoneal fluid contains various infiltrating leucocyte populations and a bulk of proinflammatory and immunoregulatory cytokines. However, the nature and significance of the peritoneal milieu in women with endometriosis still remains obscure. Therefore, the aim of the present study was to investigate the immunoregulatory activity of the peritoneal fluid (PF) from women with endometriosis. The peritoneal fluid samples were collected during laparoscopic surgery from 30 women with and without endometriosis. Immunoregulatory cytokines (IL-2, IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF) and chemokines (CCL2, CCL5, CXCL8 and CXCL9) were evaluated in PF and culture supernatants generated by unstimulated and CD3/CD28/IL-2-stimulated CD4+ T cells cultured in the presence of PF. The effect of PF on the generation of Treg and Th17 cells in CD4+ T cell cultures, as well as the natural cytotoxic activity of peripheral blood mononuclear cells, was also investigated. Concentrations of IL-6, IL-10, CCL2, CXCL8 and CXCL9 were significantly upregulated in the PF from women with endometriosis when compared to control women, whereas concentrations of other cytokines and chemokines were unaffected. The culturing of unstimulated and CD3/CD28/IL-2-stimulated CD4+ T cells in the presence of endometriotic PF resulted in the downregulation of their IL-2, IFN-γ, IL-17A and TNF production as compared to culture medium alone. On the other side, endometriotic PF significantly stimulated the production of IL-4 and IL-10. Endometriotic PF also stimulated the release of CCL2 and CXCL8, whereas the production of CCL5 and CXCL9 was downregulated. Endometriotic PF stimulated the generation of Treg cells and had an inhibitory effect on the generation of Th17 cells in cultures of CD4+ T cells. It also inhibited the NK cell cytotoxic activity of the peripheral blood lymphocytes. These results strongly imply that the PF from patients with endometriosis has immunoregulatory/immunosuppressive activity and shifts the Th1/Th2 cytokine balance toward the Th2 response, which may account for deviation of local and systemic immune responses. However, a similar trend, albeit not a statistically significant one, was also observed in case of PF from women without endometriosis, thus suggesting that peritoneal milieu may in general display some immunoregulatory/immunosuppressive properties. It should be stressed, however, that our present observations were made on a relatively small number of PF samples and further studies are needed to reveal possible mechanism(s) responsible for this phenomenon.

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 Defective Regulatory B Cells Are Associated With Thyroid-Associated Ophthalmopathy

2019 ◽  
Vol 104 (9) ◽  
pp. 4067-4077 ◽  
Author(s):  
Guo Chen ◽  
Yungang Ding ◽  
Qian Li ◽  
Yanbing Li ◽  
Xiaofeng Wen ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Healthy Controls ◽  
Regulatory B Cells ◽  
Peripheral Blood Mononuclear ◽  
Thyroid Associated Ophthalmopathy ◽  
Ifn Γ

Abstract Purpose To investigate the change in IL-10–producing regulatory B cells (Breg), which suppress peripheral immune responses, in patients with thyroid-associated ophthalmopathy (TAO). Methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls (n = 54), patients with Graves disease (n = 26), and patients with TAO (N=125), and stimulated with CpG/CD40L. The frequency of IL-10–producing Bregs and the expression of IL-10 in response to TSH stimulation were measured by flow cytometry. CD4+ T cells were cultured with Breg-depleted PBMCs to elucidate the function of Bregs in patients with TAO. The potential immunoregulatory mechanism was also investigated by Western blot and chromatin immunoprecipitation assays. Results Patients with active TAO had higher baseline levels of Bregs in their peripheral blood than both healthy controls and inactive patients. TSH promoted Bregs. Bregs from patients with TAO were defective in suppressing the activation of interferon (IFN)-γ+ and IL-17+ T cells in vitro. Conclusions Regulatory B cells in patients with TAO are functionally defective, suggesting that the defective Bregs might be responsible for the pathogenesis of TAO.

Defective Circulating CD25 Regulatory T Cells in Patients with Chronic Immune Thrombocytopenic Purpura.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 570-570
Author(s):  
Jin Yu ◽  
Vivek Patel ◽  
Susanne Heck ◽  
Jared Levan ◽  
Yu Yu ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Thrombocytopenic Purpura ◽  
Peripheral Tolerance ◽  
Regulatory Function ◽  
Healthy Controls ◽  
Thrombocytopenic Purpura ◽  
Chronic Immune Thrombocytopenic Purpura ◽  
Chronic Itp ◽  
Treg Function

Abstract Chronic Immune thrombocytopenic purpura (ITP) is a bleeding disorder characterized by increased destruction of platelets due to the production of anti-platelet autoantibodies. Regulatory CD4+ T cell (Treg) populations characterized by co-expression of CD25hi and the forkhead/winged helix transcription factor (Foxp3) play an important role in the maintenance of peripheral tolerance. Reduced levels of Foxp3 mRNA in circulating mononuclear cells (Sakakura et al., 2007) and abnormal Treg function in spleen biopsies (Liu et al., 2007) in patients with ITP have been reported, indicating that deficiency in generation and/or defective functions of Tregs contribute to loss of immunologic self-tolerance in patients with ITP. To test the hypothesis that the pathogenesis of chronic ITP may be directly related to the levels or function of circulating Tregs, we first compared the frequency of Tregs in peripheral blood of patients with chronic ITP (n=16) and controls (n=26). We found similar frequency of CD4+CD25bright T cells characteristic of Treg phenotype in patients and controls (6.2% ±0.6% vs. 5.6% ±0.3%, p=0.31). In addition, we found no significant differences in the level of FoxP3-expressing CD4+CD25hi Tregs (patients 78.9% ±2.6% vs. controls 85.7% ±2.4%, p=0.06) and in the frequency of FoxP3+CD25bright population (patients 4.9% ±0.5% vs. controls 5.1% ±0.3%, p=0.7) as measured by intracellular staining of CD4+ T cells. To assess the regulatory function of Tregs in control versus patient samples, sorted CD4+CD25hi T cells were cultured alone or in combination with autologous responder CD4+CD25− T cells at different ratios (responder/suppressor ratios: 1:1, 1:4, and 1:16) using two different T cell receptor signal strengths. We found that when cultured alone, CD4+CD25hi Tregs derived from the circulation of patients with chronic ITP (n=13) had an anergic phenotype characteristic of normal Tregs whereas CD4+CD25− cells from both patients with chronic ITP and healthy individuals responded similarly to stimulation (p≥0.7). In contrast, in cocultures, the patient Tregs had an overall twofold lower ability to suppress the proliferation of responder CD4+CD25− T cells as compared with CD4+CD25hi cells from healthy subjects (p ≤0.02). To determine whether the decrease in Treg function was due to a defect in the CD4+CD25hi Tregs or whether the effector CD4+CD25− T cells from chronic ITP patients were resistant to suppression, we performed co-mixing experiments, in which Tregs from either healthy controls or patients with chronic ITP were cocultured with the autologous and the converse responder patient and control CD4+CD25− cells. We found that patient CD4+CD25hi Tregs (n=5) had a decreased suppressive effect on proliferation of CD4+CD25− cells from either patients or healthy controls (suppression =44%). In contrast, CD4+CD25hi Tregs from controls (n=3) suppressed the proliferation of responder cells from both patients and controls to a similar degree (suppression =74%). We therefore demonstrate that CD4+CD25hi T cells are functionally defective in patients with chronic ITP and that the lack of suppression is not due to increased resistance to suppression by the responder cells. In conclusion, these data support a role for Tregs in loss of tolerance in chronic ITP, opening up the possibility of using Tregs as a therapeutic target in the treatment of chronic ITP by transferring fully functional Tregs or by restoration of Treg function.

Immunodominant epitopes on glycoprotein IIb-IIIa recognized by autoreactive T cells in patients with immune thrombocytopenic purpura

Blood ◽  
2001 ◽  
Vol 98 (1) ◽  
pp. 130-139 ◽  
Author(s):  
Masataka Kuwana ◽  
Junichi Kaburaki ◽  
Hidero Kitasato ◽  
Miyako Kato ◽  
Shinichi Kawai ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Thrombocytopenic Purpura ◽  
Peripheral Blood ◽  
Thrombocytopenic Purpura ◽  
Amino Terminal ◽  
Healthy Donors ◽  
T Cell Lines ◽  
Immunodominant Epitopes

Abstract It was recently reported that autoreactive CD4+ T cells to glycoprotein IIb-IIIa (GPIIb-IIIa) mediate antiplatelet autoantibody production in patients with immune thrombocytopenic purpura (ITP). To further examine the antigenic specificity of the GPIIb-IIIa–reactive T cells, 6 recombinant fragments encoding different portions of GPIIbα or GPIIIa were generated and tested for their ability to stimulate antigen-specific T-cell proliferation and anti–GPIIb-IIIa antibody production in vitro. T cells from the peripheral blood of 25 patients with ITP and 10 healthy donors proliferated in response to recombinant GPIIb-IIIa fragments in various combinations. The amino-terminal portions of both GPIIbα and GPIIIa (IIbα18-259 and IIIa22-262) were frequently recognized (60% and 64%, respectively) compared with other fragments (4%-28%) in patients with ITP, but this tendency was not detected in healthy donors. In subsequent analyses in patients with ITP, T-cell reactivities to IIbα18-259 and IIIa22-262 were consistently detected, whereas those to other fragments were sometimes lost. In vitro antigenic stimulation of peripheral blood mononuclear cells with IIbα18-259 or IIIa22-262 promoted the synthesis of anti–GPIIb-IIIa antibodies in patients with ITP, but not in healthy donors. Of 15 CD4+ T-cell lines specific for platelet-derived GPIIb-IIIa generated from 5 patients with ITP, 13 lines recognized IIbα18-259, IIIa22-262, or both. T-cell lines reactive to IIbα18-259 or IIIa22-262 promoted the production of anti–GPIIb-IIIa antibodies that were capable of binding to normal platelet surfaces. These results indicate that the immunodominant epitopes recognized by pathogenic CD4+ T cells in patients with ITP are located within the amino-terminal portions of both GPIIbα and GPIIIa.

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