Local and Systemic Induction of an Abundant CD4+CD25+ Regulatory T Cell Population by Non-Hodgkin’s Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 357-357
Author(s):  
S. Mittal ◽  
N.A. Marshall ◽  
L. Duncan ◽  
D.J. Culligan ◽  
R.N. Barker ◽  
...  
Keyword(s):  
T Cells ◽  
Tumor Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Cell Contact ◽  
Healthy Controls ◽  
Soluble Factors ◽  
Tissue Samples ◽  
Ann Arbor

Abstract Regulatory T (Treg) cells contribute to immune evasion by malignancies. To investigate their importance in non-Hodgkin’s lymphoma (NHL), we enumerated Treg cells in peripheral blood mononuclear cells (PBMC) and involved tissues from 30 newly diagnosed patients. CD25+FoxP3+CD127lowCD4+ Treg cells were increased markedly in PBMC (median=20.4% CD4 T cells, n=20) versus healthy controls (median=3.2%, n=13; p<0. 001, rank sum test) and correlated with serum lactate dehydrogenase (n=14; Rs=0.79, p <0.0001) and disease stage. The median Treg percentage of CD4 T cells from early stages (Ann Arbor stage I and II, n=4) was 12.2%, whereas it was 25.4% in advanced disease (Ann Arbor stages III, IV or bulky stage II, ≥5cm, n=10; p =0.013). We also enumerated Tr1 cells, both in peripheral blood and involved tissue samples, and again compared with healthy controls but no significant differences were noted. We documented poor proliferation of T cells with mitogen ConA and almost none with recall antigens PPD and DPT in both PBMC and involved tissue samples (n=9). T cell hyporesponsiveness was reversed by depleting CD25+ cells (n=4), or by adding anti-CTLA-4 (n=3), supporting the view that Treg cells explain the systemic immunosuppression seen in NHL. A high proportion of Treg cells was also present in involved tissues (median=38.8% CD4 T cells, n=15) versus reactive nodes (median=11.6%, n=2, p=0.02). Therefore, we tested the hypothesis that a regulatory phenotype is induced from conventional T cells within the tumor microenvironment. When autologous CD25- PBMC fractions were incubated with tumor cells from patients (n=6) in vitro, there was consistent strong induction and then expansion of cells with the CD4+CD25+FoxP3+ phenotype of classic ‘natural’ Treg cells as indicated by CFSE dilution. This induction was dependent on tumor dose and was seen when we depleted lymphoid dendritic cells from the involved tissue cell suspension using anti-CD304, or enriched the tumor cells by positive selection of CD20+ cells. This population was confirmed to be suppressive in function (n=3). We also investigated the mechanisms of this induction. Both cell-cell contact and soluble factors appeared important. In two of four cases, some induction was also noted with transwell experiments or with tumor cell conditioned supernatant, indicating that in these cases soluble factors are also involved apart from direct cell-cell contact mechanism. Reports elsewhere suggest roles for prostaglandin E2, tryptophan catabolism, IL-9 and PD-1 interaction with its ligands in inducing a Treg phenotype. Thus, we used cyclooxygenase inhibitors aspirin and sulindac, the indoleamine 2, 3-dioxygenase (IDO) inhibitor 1-methyl tryptophan (1MT), anti-IL-9 receptor antibody and blocking anti-PDL-1 or anti-PDL-2 antibodies in four samples. None of these reagents inhibited Treg induction apart from one case where both anti-PDL-1 and anti-PDL-2 blocking antibodies inhibited Treg induction. We conclude that NHL cells are powerful inducers of Treg cells, which may represent a new therapeutic target.

mTOR pathway regulates the differentiation of peripheral blood Th2/Treg cell subsets in patients with pemphigus vulgaris

2021 ◽  
Author(s):  
Kuan Lai ◽  
Wenjing Zhang ◽  
Songshan Li ◽  
Zhiwen Zhang ◽  
Shuangde Xie ◽  
...  
Keyword(s):  
T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Pemphigus Vulgaris ◽  
Treg Cells ◽  
Mtor Pathway ◽  
Cd4 T Cell ◽  
Cell Ratio ◽  
Loss Of Balance

Abstract Pemphigus vulgaris (PV) is a chronic and potentially life-threatening autoimmune blistering disease. Aberrant mTOR pathway activity is involved in many autoimmune diseases. This study investigated the correlation of mTOR pathway (PI3K/AKT/mTOR/p70S6K) activity with the loss of balance in T helper 2/regulatory T (Th2/Treg) cells in the peripheral blood of PV patients. CD4+ T cells were isolated from 15 PV patients and 15 healthy controls (HCs), the ratios of Th2/CD4+ T cells and Treg/CD4+ T cells, the activity of the mTOR pathway (PI3K/AKT/mTOR/p70S6K), the transcription factors and cytokines of Th2 and Treg cells were detected. Primary CD4+ T cells from PV patients were cultured under Th2- or Treg-polarizing conditions with or without rapamycin in vitro. We found that PV patients showed significantly elevated serum IL-4 when compared with HCs, and serum IL-4 level was positively correlated with the titer of anti-Dsg1/3 antibody and disease severity, while the serum TGF-β level was negatively correlated with the titer of anti-Dsg3 antibody and disease severity. Meanwhile, PV patients showed increased Th2/CD4+ T cell ratio; decreased Treg/CD4+ T cell ratio; elevated mRNA of PI3K, AKT, mTOR and protein of PI3K (P85), AKT, p-AKT (Ser473), mTOR, p-mTOR (Ser2448), p-p70S6K (Thr389), GATA3; reduced protein of forkhead box protein 3. Rapamycin inhibited Th2 cell differentiation and promoted Treg cell differentiation in vitro. These data suggest a close association between mTOR pathway activation and the loss of balance in Th2/Treg cells in peripheral blood of PV patients. Inhibiting mTORC1 can help restore the Th2/Treg balance.

Analysis of regulatory T cells in patients of NSCLC with malignant pleural effusion.

2012 ◽  
Vol 30 (15_suppl) ◽  
pp. e17511-e17511
Author(s):  
Prabhat Singh Malik ◽  
Vinod Raina ◽  
Amar Singh ◽  
Dipendrea Kumar Mitra
Keyword(s):  
T Cells ◽  
Pleural Effusion ◽  
Peripheral Blood ◽  
Chemokine Receptors ◽  
Malignant Pleural Effusion ◽  
Advanced Nsclc ◽  
Treg Cells ◽  
Healthy Controls ◽  
Anatomic Site ◽  
Treg Frequency

e17511 Background: Enrichment of regulatory T (Treg) cells at the affected anatomic site in cancer may suppress the anti-tumor immune response influencing the cancer progression. Understanding of the clinical relevance of Treg mediated suppression of anti-tumor immune response and mechanisms underlying their preferential trafficking to the affected anatomic site is still limited. The aim of this study was to enumerate the frequencies of Treg cells in malignant pleural effusion and peripheral blood of patients with advanced NSCLC and it’s trend after treatment. Methods: Treg frequencies were evaluated in pleural effusion and peripheral blood of the patients with advanced NSCLC (n=27) using flowcytometry and compared with peripheral blood of age and sex matched healthy controls (n=15) and tubercular pleural effusions (n=10). The Treg cells were characterized as CD4+CD25+Foxp3+ T cells gated on CD4+CD25+ T cells. We assessed the effect of treatment response on Treg frequency. We have also looked for the expression of chemokine receptors CCR4 and CCR6 on the Tregs in pleural effusion and peripheral blood of the patients. Results: Compared to healthy controls, frequency of CD4+CD25+Foxp3+ Tregs was significantly increased in peripheral blood of patients with NSCLC (p=0.0036). In pleural effusion of patients, Treg frequency was higher than their corresponding peripheral blood (p=0.025). As compared to tubercular pleural effusion Treg frequency was higher in malignant effusion (p<0.0001). We had 12 patients who completed treatment and in whom response evaluation was available. Treg frequency reduced at the time of response (PR or SD) and increased again at disease progression. Surface expression of CCR4 and CCR6 was higher on Treg cells as compared to non Treg CD4 cells among the patients (p=0.0001; p=0.001 respectively). However, there was no difference in expression of these chemokine receptors on Tregs in pleural fluid and peripheral blood. Conclusions: Tregs are increased in patients of NSCLC, both at disease site and in systemic circulation. This increase may be chemokine receptors mediated. Treg frequency changes with treatment and response. Modulation of Tregs may have therapeutic implication in the management of advanced NSCLC.

scholarly journals Infectious Tolerance

2002 ◽  
Vol 196 (2) ◽  
pp. 255-260 ◽  
Author(s):  
Helmut Jonuleit ◽  
Edgar Schmitt ◽  
Hacer Kakirman ◽  
Michael Stassen ◽  
Jürgen Knop ◽  
...  
Keyword(s):  
T Cells ◽  
Treg Cell ◽  
Cd4 T Cells ◽  
Transforming Growth Factor ◽  
Treg Cells ◽  
Cell Contact ◽  
Th Cells ◽  
Self Tolerance ◽  
Infectious Tolerance ◽  
Membrane Bound

Regulatory CD4+CD25+ T cells (Treg) are mandatory for maintaining immunologic self-tolerance. We demonstrate that the cell-cell contact–mediated suppression of conventional CD4+ T cells by human CD25+ Treg cells is fixation resistant, independent from membrane-bound TGF-β but requires activation and protein synthesis of CD25+ Treg cells. Coactivation of CD25+ Treg cells with Treg cell–depleted CD4+ T cells results in anergized CD4+ T cells that in turn inhibit the activation of conventional, freshly isolated CD4+ T helper (Th) cells. This infectious suppressive activity, transferred from CD25+ Treg cells via cell contact, is cell contact–independent and partially mediated by soluble transforming growth factor (TGF)-β. The induction of suppressive properties in conventional CD4+ Th cells represents a mechanism underlying the phenomenon of infectious tolerance. This explains previously published conflicting data on the role of TGF-β in CD25+ Treg cell–induced immunosuppression.

Relation of CD4+CD25+ Regulatory T Cells to Immune Status in Patients with HIV Infection.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3106-3106
Author(s):  
Sachi Tsunemi ◽  
Tsuyoshi Iwasaki ◽  
Takehito Imado ◽  
Satoshi Higasa ◽  
Eizo Kakishita ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Immune Responses ◽  
Hiv Infection ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Cd4 T Cell ◽  
Healthy Controls ◽  
Cell Counts ◽  
Hiv 1

Abstract Human immunodeficiency virus (HIV) infection is characterized by marked defects in CD4+ helper T cell (Th) functions that commonly progress to a substantial decline in peripheral CD4+ T cell counts. However, the mechanisms responsible for the loss of Th functions in HIV-infected patients independent of CD4+ T cell counts remains unclear. CD4+CD25+ regulatory T cells (T Reg) are essential for down-regulation of both autoreactive and alloreactive T cells. Therefore, we decided to investigate the role of T Reg in immune status of HIV-infected patients. We examined the expression of cell surface CD25, cytoplasmic IL-4 and cytoplasmic IFN-gamma in peripheral blood CD4+ T cells from both healthy controls (n=9) and HIV-infected patients (n=43). We also compared T Reg functions between the 2 groups. CD4+CD25+ T Reg isolated from both HIV-infected patients and healthy controls strongly expressed CD45RO, HLA-DR, and FoxP3, and suppressed the proliferation of CD4+CD25− T cells, suggesting that CD4+CD25+ T cells from both healthy controls and HIV-infected patients possess phenotypic and functional characteristics of Treg. CD4+CD25high T cells are a subset of circulating CD4+CD25+ T cells in normal humans and exhibit strong in vitro regulatory functions similar to those reported for murine CD4+CD25+ T Reg. We measured the frequency of CD4+CD25high T Reg by analysis of surface CD25 on CD4+ T cells in peripheral blood samples. We also examined Th1 and Th2 frequencies by analysis of cytoplasmic IFN-gamma and IL-4 levels in CD4+ T cells. T Reg from HIV-infected patients with detectable plasma HIV-1 RNA showed a statistically significant increase in CD4+CD25high cell frequency (p<0.05) compared to healthy controls, with T Reg frequencies inversely proportional to CD4+ T cell numbers (p<0.01). However, in HIV-infected patients with undetectable plasma HIV-RNA, frequencies of CD4+CD25high T Reg were not increased and not related to CD4+ T cell numbers. In both HIV-infected patient groups, T Reg frequency was inversely related to Th1 frequency (detectable: p<0.05, undetectable: p<0.001), but positively related to Th2 frequency (detectable: p<0.01, undetectable: p<0.001). Our results indicate that increased frequencies of peripheral blood T Reg were related to disease progression as measured by detectable plasma HIV-1 RNA, decreased peripheral blood CD4+ T cell counts, and polarization toward Th2 immune responses in HIV-infected patients. HIV infection may lead to induction of T reg that inhibit antiviral immune responses, resulting in the progression of the disease. Manipulation of T Reg could help restore antiviral immune responses in HIV infection, and prevent the progression of HIV infection.

Elevated Expression of T-Cell Immunoglobulin and Mucin Domain 3 on T Cells from Peripheral Blood in Patients with Cervical Carcinoma

2020 ◽  
pp. 1-8
Author(s):  
Juan Li ◽  
Xiao-fei Sun ◽  
Ying Shen ◽  
Qing Yang ◽  
Shu-yan Dai
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cervical Carcinoma ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Control Group ◽  
Healthy Controls ◽  
Novel Therapy ◽  
Domain 3

<b><i>Objective:</i></b> To investigate the expression of T-cell immunoglobulin and mucin domain 3 (TIM-3) on peripheral T cells of cervical carcinoma patients. <b><i>Methods:</i></b> Peripheral blood samples from 15 high-grade cervical squamous intraepithelial lesion (HSIL) patients, 24 cervical carcinoma patients, and 21 healthy controls were collected. TIM-3 expressions on the surface of peripheral CD4+ T cells and CD8+ T cells were analyzed with flow cytometry. <b><i>Results:</i></b> There was significantly lower expression of CD4+ T cells and CD8+ T cells in HSIL patients and cervical carcinoma patients compared with healthy controls. We also found that TIM-3 expression on peripheral CD4+ T and CD8+ T cells of both HSIL patients and cervical carcinoma patients was significantly increased compared to the control group. Further analyses revealed that the expression of TIM-3 on peripheral CD4+ T and CD8+ T cells significantly increased in stage III–IV cervical carcinoma patients compared to stages I–II. <b><i>Conclusion:</i></b> The increased expression of TIM-3 on CD4+ T cells and CD8+ T cells of patients with cervical carcinoma and HSIL suggests the potential role of TIM-3 in the development and progression of cervical carcinoma, which may be a novel therapy target for cervical carcinoma.

Local and systemic induction of CD4+CD25+ regulatory T-cell population by non-Hodgkin lymphoma

Blood ◽  
2008 ◽  
Vol 111 (11) ◽  
pp. 5359-5370 ◽  
Author(s):  
Sajjan Mittal ◽  
Neil A. Marshall ◽  
Linda Duncan ◽  
Dominic J. Culligan ◽  
Robert N. Barker ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Hodgkin Lymphoma ◽  
Tumor Cells ◽  
Cd4 T Cells ◽  
Mononuclear Cells ◽  
Treg Cells ◽  
Disease Stage ◽  
Serum Lactate Dehydrogenase ◽  
Non Hodgkin Lymphoma

Abstract Regulatory T (Treg) cells contribute to immune evasion by malignancies. To investigate their importance in non-Hodgkin lymphoma (NHL), we enumerated Treg cells in peripheral blood mononuclear cells (PBMCs) and involved tissues from 30 patients. CD25+FoxP3+CD127lowCD4+ Treg cells were increased markedly in PBMCs (median = 20.4% CD4 T cells, n = 20) versus healthy controls (median = 3.2%, n = 13, P < .001) regardless of lymphoma subtype, and correlated with disease stage and serum lactate dehydrogenase (Rs = 0.79, P < .001). T-cell hyporesponsiveness was reversed by depleting CD25+ cells, or by adding anti–CTLA-4, supporting the view that Treg cells explain the systemic immunosuppression seen in NHL. A high proportion of Treg cells was also present in involved tissues (median = 38.8% CD4 T cells, n = 15) versus reactive nodes (median = 11.6%, n = 2, P = .02). When autologous CD25− PBMC fractions were incubated with tumor cells from patients (n = 6) in vitro, there was consistent strong induction and then expansion of cells with the CD4+CD25+FoxP3+ phenotype of classic “natural” Treg cells. This population was confirmed to be suppressive in function. Direct cell-cell interaction of tumor cells with CD25− PBMCs was important in Treg induction, although there was heterogeneity in the mechanisms responsible. We conclude that NHL cells are powerful inducers of Treg cells, which may represent a new therapeutic target.

Determination of CD4+ and CD8+ T cells in the peripheral blood of dogs with demodicosis

Parasitology ◽  
2010 ◽  
Vol 137 (13) ◽  
pp. 1921-1924 ◽  
Author(s):  
S. K. SINGH ◽  
U. DIMRI ◽  
M. C. SHARMA ◽  
B. SHARMA ◽  
M. SAXENA
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Cd8 T Cells ◽  
Peripheral Blood ◽  
Cd4 T Cells ◽  
Significant Alteration ◽  
Healthy Controls ◽  
Healthy Dogs

SUMMARYThe aim of this study was to evaluate the CD4+/CD8+ ratio in peripheral blood of dogs with localized and generalized demodicosis. Sixteen dogs were examined, 8 with localized and 8 with generalized demodicosis, while 8 healthy dogs were used as controls. Peripheral blood was obtained and CD4+ and CD8+ T cells were determined by flow cytometry. Significantly higher numbers of CD8+ T cells and lower numbers of CD4+ T cells were found in dogs with generalized demodicosis compared to dogs with localized demodicosis and healthy controls. Significantly higher numbers of CD8+ T cells and lower numbers of CD4+ T cells were also found in dogs with localized demodicosis compared to healthy controls. The CD4+/CD8+ ratio was also found to be significantly lower in dogs with generalized demodicosis in comparison with dogs with localized demodicosis and healthy controls. It is concluded that significant alteration in the CD4+/CD8+ ratio may be implicated in the pathogenesis of generalized canine demodicosis.

scholarly journals Study on Quantity and Function of Regulatory T Cell Subsets in Multiple Myeloma and MGUS

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3154-3154
Author(s):  
Jinuo Wang ◽  
Jian Li ◽  
Xinxin Cao ◽  
Hao Cai ◽  
Ai-lin Zhao ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Treg Cell ◽  
Cd4 T Cells ◽  
Treg Cells ◽  
Healthy Controls ◽  
Newly Diagnosed ◽  
Inhibition Rate ◽  
Significant Difference ◽  
Normal Controls

Abstract Introduction Almost all multiple myeloma (MM) cases were progressed from a premalignant condition called monoclonal gammopathy of undetermined significance (MGUS). So far, the pathogenesis of myeloma is not yet clear. The immune cells in the tumor microenvironment, such as regulatory T (Treg) cells with a unique immunosuppressive function, play an important role in myelomagenesis. Although there have been reports on Treg cells in MM patients, the results were still in debate. In this study, we performed a comprehensive analysis of peripheral blood (PB) and bone marrow (BM) Treg subsets and aging Treg-like cells in untreated MM patients and individuals with MGUS, which might help further elucidate mechanisms of immune dysfunction during myelomagenesis. Methods Our study included 20 MGUS patients and 26 newly diagnosed MM patients. Flow cytometry was applied to determine the proportion of Treg cell subsets and aging Treg-like cells in PB and BM. Flow sorting technology was used to separate Treg cell subsets and effector T cells in the bone marrow of newly diagnosed MM patients. The inhibitory function was indirectly calculated by detecting proliferation rate of CFSE-labelled effective T cells which were cocultured with different Treg cell subsets. Concentration of IL-10 from the culture supernatants of proliferation assay was measured using ELISA. Results In PB, the proportion of activated Tregs (aTregs, CD4+CD45RA-FoxP3hi) in CD4+ T cells was significantly higher in MGUS and untreated MM patients than healthy controls (P=0.01, P<0.001); there was no difference in the proportion of resting Tregs (rTregs, CD4+CD45RA+FoxP3lo) between MGUS and untreated MM patients compared with healthy adults (P=0.72, P=0.07). There was also no significant difference in the frequencies of non-Tregs (CD4+CD45RA-FoxP3lo) from MGUS and MM patients with normal controls (P=0.22, P=0.67). The proportion of CD4+CD28-FoxP3+ Treg-like cells in CD4+ T cells was gradually increased in MGUS, untreated MM patients than healthy controls (P<0.01, P<0.01); Treg-like cells in newly diagnosed MM patients were significantly higher than those in MGUS patients (P=0.01). In BM, the proportion of aTregs was significantly higher in MGUS, untreated MM patients compared with healthy controls (P<0.01); the proportion of rTregs in MGUS, untreated MM patients was significantly lower than that of controls (P=0.02, P<0.01). However, there was no significant difference in the frequencies of non-Tregs in BM from MGUS and MM patients with normal controls (P=0.14, P=0.88). The proportion of Treg-like cells in CD4+ T cells was significantly higher in MGUS, untreated MM patients compared with healthy controls (P<0.01, P<0.01). Treg-like cells in untreated MM patients were significantly higher than those in MGUS patients (P<0.01). The inhibition rate of aTreg in bone marrow of newly diagnosed MM patients was significantly higher than that of rTreg (P<0.01), while the inhibition rate of non-Treg was significantly lower than that of rTreg cells (P<0.01). The inhibition rates of aTreg (P=0.21), rTreg (P=0.08) and non-Treg (P=0.09) in healthy controls were no difference from those in MM patients. The level of IL-10 secreted by non-Treg in untreated MM patients was notably higher than that of aTreg and rTreg; the ability of cytokine secretion of Treg subsets in MM patients was similar with that of healthy controls. Conclusions There were significant changes in the frequencies of Treg cell subsets and Treg-like cells in peripheral blood and bone marrow of MGUS and MM patients, suggesting that immunomodulatory abnormality has existed in patients at premalignant stage. The immunosuppressive and cytokine secretory functions of Treg subsets in bone marrow of untreated MM patients were intact compared with that in healthy adults. Disclosures No relevant conflicts of interest to declare.

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.

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