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

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

Parasitology ◽

10.1017/s0031182010000879 ◽

2010 ◽

Vol 137 (13) ◽

pp. 1921-1924 ◽

Cited By ~ 20

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.

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

Thrombosis and Haemostasis ◽

10.1055/s-0039-1679909 ◽

2019 ◽

Vol 119 (05) ◽

pp. 758-765 ◽

Cited By ~ 5

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.

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Abstract 001: Axl Expression by CD4+ T Lymphocytes Promotes Salt-Dependent Hypertension

Hypertension ◽

10.1161/hyp.64.suppl_1.001 ◽

2014 ◽

Vol 64 (suppl_1) ◽

Author(s):

Kristine M Wadosky ◽

Sri N Batchu ◽

Angie Hughson ◽

Kathy Donlon ◽

Craig N Morrell ◽

...

Keyword(s):

Flow Cytometry ◽

T Cells ◽

T Cell ◽

T Lymphocytes ◽

Cd4 T Cells ◽

White Blood Cells ◽

Interleukin 4 ◽

Th2 Cells ◽

Salt Hypertension ◽

Ifn Γ

Introduction: Our laboratory has shown that Axl, a receptor tyrosine kinase, is important in both vascular and immune functions during deoxycorticosterone acetate (DOCA)-salt hypertension. We hypothesized that Axl activity specifically in T lymphocytes could explain the dependence of hypertension on Axl. Methods and Results: We did adoptive transfers of either Axl+/+ or Axl-/- CD4+ T cells to RAG1-/- mice that lack mature T cells. Once CD4+ T cell repopulations were confirmed, we induced DOCA-salt hypertension for 6 weeks. Systolic blood pressure (BP, mmHg) increased by 20±5 in Axl+/+RAG-/- mice after DOCA-salt, but Axl-/- RAG-/- mice had increases in BP by only 6+3 after 6 weeks of DOCA-salt. We isolated naïve CD4+ T cells from both Axl+/+ and Axl-/- littermates and primed them under either Th1 or Th2 polarizing conditions in culture. Production of interferon gamma (IFN-γ ng/mL) was significantly decreased (-23%, p<0.05) in Axl-/- (396±23) compared to Axl+/+ (512±42) under Th1-priming. However, Axl had no effect on interleukin 4 (IL-4, ng/mL) production under Th2 polarizing conditions. Intracellular staining of the Th1/Th2 cells with IFN-γ and IL-4 antibodies by flow cytometry confirmed expression of cytokines in culture media. Complete blood counts showed that Axl-/- mice had significantly lower white blood cells due to decreased numbers of lymphocytes (4.5±0.7x10 9 ) compared to Axl+/+ mice (7.8±0.7x10 9 ). We found a higher population of AnnexinV (marker of early apoptosis)-positive peripheral leukocytes in Axl-/- mice (10±1%) compared to Axl+/+ (4±1%) by flow cytometry; while the percentages of dead cells (~10%) were similar between Axl+/+ and Axl-/- mice. Conclusions: Altogether we show that expression of Axl by T cells drives salt-induced hypertension. The mechanism of Axl-dependent effects on T cells occurs via T-cell-dependent expression of the pro-inflammatory cytokine IFN-γ. In addition, Axl plays a role in inhibiting lymphocyte apoptosis in the circulation. Future work will focus on how Axl expression in T cells affects T cell-dependent vascular remodeling during hypertension.

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

Blood ◽

10.1182/blood.v110.11.357.357 ◽

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.

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Study on Mechanism of Human Marrow Mesenchymal Stem Cell in Treating Patients with Aplastic Anemia.

Blood ◽

10.1182/blood.v110.11.4099.4099 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4099-4099

Author(s):

Zhenhua Qiao ◽

Xiujuan Zhao

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Aplastic Anemia ◽

Hematopoietic Cell ◽

Control Group ◽

Rt Pcr ◽

Tnf Α ◽

Ifn Γ ◽

Tgf Β1

Abstract Objective: To explore mechanism of human marrow mesenchymal stem cells (MSCs) in treating patients with aplastic anemia(AA). Methods: MSCs in patients with aplastic anemia(AA) and the control group were separated with Percoll(1.073g/m L) and cultured in low glucose DMEM. Then, observed their morphologies,checked their molecule surface antigen by flow cytometry and examined the process of adipogenic differention. The mononuclear cells (MNC)of marrow in patients with AA were enriched based 1.077g/L density centrifuge and cultured in the 1640 medium. (1)MSC in control group and MNC in AA group were co-cultured with or without cytokines. The function of supporting hematopoiesis for MSC was to be observed in single confluence layer after plating by counting the total cells and the clones in every well every week. Then analyzed the dynamics of proliferation. T cells were harvested by using nylon column. MSC in control group and T cells in AA group were co-cultured. The proliferation of T cell was measured by MTT method. The CD25,CD69,CD4,CD8,Annexin-V expression rates of CD3+T cells were analyzed by flow cytometry .The gene and protein of IL-2, IL-4,IL-10,TNF-α,IFN-γ,TGF-β1 were examined by RT-PCR and ELISA respectively. MSC treated to the model of AA, by the examination of peripheral hemogram, bone marrow biopsy, pathological section of spleen. Results: There was no significant difference between control group MSC and AA-MSC in morphologies but adipogenic differentiation in AA patients is earlier than controls. The clones of CFU-GM in group(MSC)(78.46±3.58)/2×105 cells, after 14 days cultured was significantly higher than(9.21±4.32)/2×105 cells in group(CK + DMEM medium), while lower than (99.32±4.34)/2×105 cells in group(MSC+CK). (1)the Treg cells (TCD4+CD25+) in AA group (2.01±1.21)/ 2×105 was significantly lower than (4.43±1.67)/2×105 cells in control group, while(5.43±2.31) / 2×105 in group (MSC+AAT) was no more than (4.43±1.67)/2×105 cells in control group. (2) MSCs significantly inhibited T cell proliferation (P< 0. O5)by MTT. (3) RT-PCR and ELISA analysis showed that MSCs induced the expression of IL-4, IL-10, TGF-β1 and decreased significantly the expression of IL-2, TNF-α, IFN -γ in T cells of AA. the model of AA treated by MSCs showed improvements in 3 blood components greatly(p<0.05), Bone marrow proliferated and restored to the normal level, hematopoietic cell increased obviously (hematopoietic cell capacity was more than 40%), and atrophied spleen restore to normality. Conclusions: morphologies of AA’MSC had no evident different with the control but was more easy adipogenic differention. aplastic anemia belongs to autoimmune diseases in which T cells effect organ-specific destruction. The fundamental mechanism of MSC in treating AA should be potential to promote hematopoietic cell proliferation by adjusting immunity.

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Functional characterization of CD4+ T cells in aplastic anemia

Blood ◽

10.1182/blood-2011-08-368308 ◽

2012 ◽

Vol 119 (9) ◽

pp. 2033-2043 ◽

Cited By ~ 83

Author(s):

Shahram Kordasti ◽

Judith Marsh ◽

Sufyan Al-Khan ◽

Jie Jiang ◽

Alexander Smith ◽

...

Keyword(s):

T Cells ◽

Aplastic Anemia ◽

Cd4 T Cells ◽

High Throughput Sequencing ◽

Functional Characterization ◽

Effector T Cells ◽

T Cell Subsets ◽

Th2 Cells ◽

Healthy Donors ◽

Th1 And Th2

Abstract The role of CD4+ T cells in the pathogenesis of aplastic anemia (AA) is not well characterized. We investigate CD4+ T-cell subsets in AA. Sixty-three patients with acquired AA were studied. Th1 and Th2 cells were significantly higher in AA patients than in healthy donors (HDs; P = .03 and P = .006). Tregs were significantly lower in patients with severe AA than in HDs (P < .001) and patients with non-severe AA (P = .01). Th17 cells were increased in severe AA (P = .02) but normal in non-severe AA. Activated and resting Tregs were reduced in AA (P = .004; P = .01), whereas cytokine-secreting non-Tregs were increased (P = .003). Tregs from AA patients were unable to suppress normal effector T cells. In contrast, AA effector T cells were suppressible by Tregs from HDs. Th1 clonality in AA, investigated by high-throughput sequencing, was greater than in HDs (P = .03). Our results confirm that Th1 and Th2 cells are expanded and Tregs are functionally abnormal in AA. The clonally restricted expansion of Th1 cells is most likely to be antigen-driven, and induces an inflammatory environment, that exacerbate the functional impairment of Tregs, which are reduced in number.

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Oligoclonal Expansion and T-Cells Subpopulations in Patients with Aplastic Anemia at Different Stages of the Disease

Blood ◽

10.1182/blood-2018-99-119414 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3864-3864

Author(s):

Anastasia V. Abramova ◽

Elena A. Mikhaylova ◽

Zalina T. Fidarova ◽

Yuliya O. Davydova ◽

Nikolay M. Kapranov ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

T Cell ◽

Aplastic Anemia ◽

Memory T Cells ◽

Clonal Expansion ◽

Th Cells ◽

Healthy Donors ◽

Oligoclonal Expansion

Abstract Background. The main mechanism of the bone marrow (BM) failure in idiopathic aplastic anemia (AA) has an immunomediated character. Researching the T-cell clone's effect in the AA pathogenesis is very relevant at the present time. Oligoclonal expansion of T cells is frequent in AA and the identification of immunodominant T-cell clones can correlate with the disease activity and may possibly serve as response predictor to immunosuppressive therapy (IST). The aim. To identify T-cells subpopulations, expression of PD-1 and PD-L1 on T-cells and TCR-Vβ repertoires by flow cytometry in different groups of AA patients. Methods. Thirty AA patients (pts) with median age of 30.5 (19-71), m/f ratio 1:1,3 were divided in 3 groups: pts with newly diagnosed (ND) AA (n=13), pts with overall response to IST (OR) (n=10), non-response pts (NR) for 2 and more lines of IST (n=7). Flow cytometry was performed with BD FACS Canto II. We used commercial kit (IOTest® Beta Mark TCR Vb Repertoire) for evaluation of TCR-Vβ repertoire in the bone marrow (BM) of these patients. We performed analysis of BM samples from healthy donors as a control group (n=8). Due to low amount of donor samples the maximal value each of the 24 subclones (for CD4+ (T-helpers - Th) and CD8+ cells (T-cytotoxic cells - TCL)) was accepted as threshold. We concluded the presence of clonal expansion if TCR subclone exceeded this threshold. We identified different T-cell subpopulations in all 3 groups of AA and healthy donors by flow cytometry: double positive T-cells (CD3+CD4+CD8+), double negative T-cells (CD3+CD4- CD8-), Th (CD3+CD4+), TCL (CD3+CD8+), NK-T-cells (CD3+CD56+) out of CD3+ cells. Among Th and TCL cells was determined naive T-cells (CD28+CD95-), effector T-cells (CD28-CD95+), memory T-cells (CD28+CD95+), regulatory T-cells (CD4+CD127-CD25high) and subpopulations Th and TCL co-expressed PD-1 and PD-L1. Multiple comparisons were assessed by ANOVA or Kruskal Wallis test by GraphPad Prism software. Results. In our study all 30 AA patients had an immunodominant TCR-Vβ clones among Th and/or TCL cells. We identified the most common clonotypes in comparison with healthy donors - Vβ1, Vβ2, Vβ3 among the Th cells and Vβ3, Vβ9, Vβ13.1 among the TCL cells. In ND group Vβ1 was highly expanded in 5 (38.5%), Vβ3 - in 7 (53.8%) pts among Th, and Vβ3 - in 3 (23.1%) and Vβ9 - in 4 (30.8%) out of 13 pts among TCL. In OR group Vβ2 expansion was in 4 (40%) and Vβ3 - in 5 (50%) pts among Th; Vβ3 in 6 (60%) and Vβ9 in 6 (60%) out of 10 pts among TCL. In NR group the most frequent was Vβ13.1 clone in TCL - in 3 (42.9%) out of 7 pts. In NR group in overall clonal expansion was less frequent than in ND and OR groups. We also analyzed the previously mentioned subpopulations of T-cells in patients with AA in three groups (ND, OR, NR) compared to healthy donors (table 1). We obtained significant differences in the count of naive Th and TCL cells, memory T-cells in all three groups of AA patients compared to donors: proportion of naive Th and TCL cells was significantly higher and proportion of memory Th cells was lower in the donor group than in AA pts. The percent of TCL effectors was higher in ND AA pts compare to donors. We also found that cell count of activated Th (CD4+CD25+) was higher in the group of refractory pts. In OR pts proportion of PD-1-positive Th was higher than in donors. In NR pts Th and TCL co-expressed with PD-L1 were lower compare to donors (table 1). Conclusions. In our study we found immunodominant clonotypes in different AA pts and depletion of the pool of naive T cells. Dynamic observation of changes in the most common clonotypes in AA pts during treatment will provide suitable therapy tactics (allogenic bone marrow transplantation or IST). Disclosures No relevant conflicts of interest to declare.

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

10.21203/rs.3.rs-146076/v1 ◽

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.

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CD137 Agonist Induces Gastric Cancer Cell Apoptosis by Enhancing The Functions of CD8+ T Cells via NF-κB Signaling

10.21203/rs.3.rs-40870/v1 ◽

2020 ◽

Author(s):

Ben-Shun Hu ◽

Tian Tang ◽

Tie-Long Wu ◽

Ying-Yue Sheng ◽

Yu-Zheng Xue

Keyword(s):

Gastric Cancer ◽

Flow Cytometry ◽

T Cells ◽

Real Time ◽

Peripheral Blood ◽

Cell Apoptosis ◽

Nuclear Translocation ◽

Granzyme B ◽

Ifn Γ

Abstract Background: CD137 is identified as a target for tumor immunotherapy. However, the role of CD137 in gastric cancer (GC), especially in inducing GC cell apoptosis has not been studied yet. Methods: Foxp3+ and CD8+ T cells in GCs were investigated by immunohistochemistry (IHC). CD137 expression in GCs was detected by flow cytometry, IHC and immunofluorescence (IF). Peripheral blood mononuclear cells (PBMCs) and CD8+ T cells isolated from peripheral blood were stimulated with a CD137 agonist in vitro. CD8+ T cells proliferation and p65 expression were explored by flow cytometry. p65 nuclear translocation was analyzed by IF. IL-10, TGF-β, IFN-γ, Perforin and Granzyme B were detected by real-time quantitative PCR (real-time PCR). PBMCs and primary GC cells were cocultured and stimulated with the CD137 agonist in vitro. Apoptosis of the primary GC cells was detected by flow cytometry. Results: Our data demonstrated that GC tumors show characteristics of an immunosuppressive microenvironment. CD137 was predominantly expressed in CD8+ T cells in GCs and had a positive correlation with tumor cell differentiation. CD137 agonist promoted CD8+ T cells proliferation and increased the secretion of IFN-γ, Perforin and Granzyme B, which induced primary GC cell apoptosis. Mechanistically, this study found that CD137 agonist could induce NF-κB nuclear translocation in CD8+ T cells. Conclusion: Our results demonstrate that CD137 agonist can induce primary GC cell apoptosis by enhancing CD8+ T cells via activating NF-κB signaling.

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Circulating cytokines and lymphocyte subsets in patients who have recovered from COVID-19

10.1101/2020.07.22.20160259 ◽

2020 ◽

Author(s):

Hasi Chaolu ◽

Xinri Zhang ◽

Xin Li ◽

Dongyan Li

Keyword(s):

T Cells ◽

B Cells ◽

Nk Cells ◽

Cd8 T Cells ◽

Cd4 T Cells ◽

Lymphocyte Subsets ◽

Healthy Controls ◽

Tnf Α ◽

Ifn Γ ◽

Circulating Cytokines

To investigate the immune status of people who previously had COVID-19 infections, we recruited patients 2 weeks post-recovery and analyzed circulating cytokines and lymphocyte subsets. We measured levels of total lymphocytes, CD4+ T cells, CD8+ T cells, CD19+ B cells, CD56+ NK cells, and the serum concentrations of interleukin (IL)-1, IL-4, IL-6, IL-8, IL-10, transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ) by flow cytometry. We found that in most post-recovery patients, levels of total lymphocytes (66.67%), CD3+ T cells (54.55%), CD4+ T cells (54.55%), CD8 + T cells (81.82%), CD19+ B cells (69.70%), and CD56+ NK cells(51.52%) remained lower than normal, whereas most patients showed normal levels of IL-2 (100%), IL-4 (80.88%), IL-6 (79.41%), IL-10 (98.53%), TNF-α (89.71%), IFN-γ (100%) and IL-17 (97.06%). Compared to healthy controls, 2-week post-recovery patients had significantly lower absolute numbers of total lymphocytes, CD3+ T cells, CD4+ T cells, CD8+ T cells, CD19+ B cells, and CD56+ NK cells, along with significantly higher levels of IL-2, IL-4, IL-6, IL-10, TNF-α, IFN-γ and IL-17. Among post-recovery patients, T cells, particularly CD4+ T cells, were positively correlated with CD19+ B cell counts. Additionally, CD8+ T cells positively correlated with CD4+ T cells and IL-2 levels, and IL-6 positively correlated with TNF-α and IFN-γ. These correlations were not observed in healthy controls. By ROC curve analysis, post-recovery decreases in lymphocyte subsets and increases in cytokines were identified as independent predictors of rehabilitation efficacy. These findings indicate that the immune system has gradually recovered following COVID-19 infection; however, the sustained hyper-inflammatory response for more than 14 days suggests a need to continue medical observation following discharge from the hospital. Longitudinal studies of a larger cohort of recovered patients are needed to fully understand the consequences of the infection.

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