Absolute Numbers Of Th17 Cells Research Articles

Decreased absolute number of peripheral regulatory T cells in patients with idiopathic retroperitoneal fibrosis.

In order to determine whether the immune balance of T helper 17(Th17)/regulatory T(Treg) is related to the pathogenesis of idiopathic retroperitoneal fibrosis (IRPF), we analyzed the differences in peripheral blood lymphocytes, CD4+T cell subsets and cytokines between patients with IRPF and healthy people to clarify the CD4+T cell subsets, especially Treg cell subsets, and the role of cytokines in the pathogenesis of IRPF. This study included 22 patients with IRPF, 36 patients with IgG4-related diseases (IgG4-RD) without retroperitoneal fibrosis (RPF), and 28 healthy controls. The absolute numbers and percentage of peripheral blood lymphocyte subsets and CD4+T cell subsets in each group were detected by flow cytometry, and the serum cytokine level was detected by flow cytometric bead array (CBA). Compared with the healthy group, the absolute value of B cells in peripheral blood of IRPF patients was significantly decreased, and T, natural killer (NK), CD4+ and CD8+ were not significantly abnormal. The absolute numbers of Th2 cells were lower than healthy group(p=0.043). In particular, the absolute numbers of Treg cells were significantly lower than healthy group(p<0.001), while the absolute numbers of Th17 cells increased(p=0.682). Th17/Treg was significantly higher than healthy group (p< 0.001). Cytokine analysis showed that the level of interleukin (IL)-4 in IRPF patients was higher than healthy group(p=0.011), IL-6, IL-10, IL-17, TNF-α and IFN-γ were significantly higher than healthy group (all p<0.001). Receiver operating characteristic (ROC) curves showed that IL-10 and TNF-α could distinguish bilateral ureteral dilatation in IRPF patients, with areas under the ROC curve (AUCs) of 0.813 (95% CI:0.607-1.000, p=0.026) and 0.950 (95% CI:0.856-1.000, p=0.001), respectively. IL-6 could distinguish bilateral ureteral obstruction, with an AUC of 0.861 (95% CI: 0.682-1.000, p=0.015). Our study showed that IRPF patients had reduced Treg cells and indeed had Th17/Treg imbalance, which may be related to the pathogenesis of the disease. The levels of IL-6, IL-10 and TNF-α appear to be associated with the progression of IRPF.

Elevated Th17 cells are associated with cardiovascular complications in ankylosing spondylitis.

Patients with ankylosing spondylitis (AS) carry an increased burden of cardiovascular diseases (CVD), but features denoting the development of CVD in AS are unclear. This study aimed to evaluate the percentage and absolute number of lymphocytes and CD4+T cells in AS patients complicated with CVD (AS-CVD) and determine whether circulating T helper 17 (Th17) cells are associated with the development of CVD in AS. A total of 117 AS patients (46 had CVD and 71 had no CVD) were enrolled in this retrospective study. The percentage and absolute number of lymphocytes and CD4+T cells were determined by flow cytometry. Associations between CVD and clinical markers were analysed using logistic regression. The ratio of Th17/Treg cells (0.30 vs 0.19, P = 0.014) and the absolute number of Th17 cells (7.27 cells/μL vs 4.34 cells/μL, P < 0.001) was significantly elevated in AS-CVD group compared with AS-no-CVD group. Multivariate logistic regression revealed that elevated Th17 cells (OR = 1.20, P = 0.016) were associated with CVD complications in AS. Receiver operating characteristic (ROC) curves showed a contribution of Th17 cell for distinguishing AS patients with CVD, with the areas under the ROC curve (AUCs) of 0.729 (95% CI: 0.632, 0.825, P < 0.001). Our findings provide evidence for the association between Th17 cells and increased cardiovascular risk in AS. Th17 cells may contribute to accelerated atherogenesis and increased cardiovascular burden in AS and be valuable for early assessment and management of AS-CVD.

Elevated Peripheral T Helper Cells Are Associated With Atrial Fibrillation in Patients With Rheumatoid Arthritis.

Patients with rheumatoid arthritis (RA) have a significantly high risk of atrial fibrillation (AF). This study aimed to compare the absolute and relative changes in peripheral T cells in patients with RA who were also affected with and without AF. To help make an early diagnosis and prevent the initiation and progression of AF, the changes in the lymphocyte subsets were assessed in RA patients with and without AF. A propensity score matching (PSM) system (1:3) was used to perform a matched case-control study with 40 RA-AF cases and 120 RA controls. Changes in the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), anti-citrullinated peptide antibody (ACPA), and rheumatoid factor (RF) were examined. The percentage and absolute number of T, B, natural killer (NK), T helper (Th)1, Th2, Th17, and T-regulatory (Treg) cells in the peripheral blood of patients with and without RA-AF were determined using flow cytometry. Univariate and multivariate analyses were performed to determine the association between peripheral lymphocytes and RA-AF. Demographic data, ESR, CRP, ACPA, and the percentage, as well as the absolute value of B, NK, Th2, and Treg cells, showed no significant differences between the propensity score-matched groups of RA and RA-AF. Meanwhile, the absolute number and percentage of Th1 cells, the absolute number of Th17 cells, the ratio of Th1/Treg, Th17/Treg, and RF were significantly higher in patients with RA-AF than those in the control groups (P < 0.05). Univariate and multivariate logistic regression analyses also revealed that the percentage of Th1 cells, the absolute number of Th17 cells, and the ratio of Th1/Treg were associated with a significantly higher risk of AF. This PSM study demonstrated that the incidence of AF was higher in RA patients with Th cell immunological derangements.

SAT0278 LOW-DOSE IL-2 RESTORES TREG-MEDIATED IMMUNE TOLERANCE IN PATIENTS WITH ANCA-ASSOCIATED VASCULITIS

Background:Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of autoimmune disease that can cause systemic organ damage, including granulomatosis with polyangiitis(GPA), microscopic polyangiitis (MPA), and eosinophilic granulomatosis with polyangiitis(EGPA)[1]. Several observations have showed that the breakdown of immune tolerance was involved in the pathogenesis of AAV [2], furthermore, a single, open and clinical trial demonstrates that IL-2 can be used to treat patients with GPA [3]. But there is still a lack of understanding of the relationship between Th17 / Treg and AAV and evidence for the therapeutic effect of IL-2 on AAV, which needs further exploration.Objectives:We first measured the absolute number of CD4+T subsets in peripheral blood of patients to explore the pathogenesis of AAV, and then investigated the effects of short-term and low-dose recombinant human IL-2 (rhIL-2) on CD4+T subsets of patients to analyze the regulatory effect of IL-2 on AAV.Methods:49 patients with AAV, hospitalized at the Second Hospital of Shanxi Medical University from the May 2016 to the November 2019 were enrolled, including 36 patients who were only received conventional glucocorticoids and DMARDs, and other 13 patients who were not only received these treatments but were also injected subcutaneously rhIL-2(50WIU/day for a 5-day course). 31 age and gender-matched healthy adults were selected as controls. The absolute number of Th17 and Treg cells in peripheral blood of health controls and the patients before and after treatment was detected by flow cytometry.Results:There was significant decreased level of Treg cells in the patients with AAV compared with healthy controls (P&lt;0.001) leading to a higher Th17/Treg ratio in the patients with AAV, but there was no statistically significant in the absolute number of Th17 cells between the patients and healthy controls. After the treatment of short-term and low-dose IL-2, there was a significant increase in the absolute number of Treg cells (P&lt;0.01) leading to a decrease in the ratio of Th17 and Treg (p&lt;0.05).The absolute number of Th17 had a trend towards higher values but was not statistical significance.Conclusion:The difference of Treg cells between the patients and healthy controls suggested that the decreased number of Treg cells failed to control autoimmune inflammatory response contributing to the pathogenesis of AAV. After the treatment of short-term and low-dose rhIL-2, there was a more significant increase in the absolute number of Treg cells showing that IL-2 could selectively stimulate the growth of Treg cells and restore the Treg-mediated immune tolerance in patients with AAV to achieve disease remission.

Exogenous IL-2 Induces FoxP3+ Th17 Cells In Vivo in Melanoma Patients.

Th17 cells represent a distinct subset of CD4 effector T cells with potent pathogenic qualities, capable of directly mediating tumor cell destruction. IL-2 has frequently been shown to have a negative effect on Th17 differentiation while supporting regulatory T-cell (FoxP3CD4, TREG) growth and development in both in vitro models and in vivo animal models. We investigated the effect of in vivo IL-2 on both the Th17 and FoxP3CD4 T-cell compartments in a human model of cancer. High-dose IL-2 (HDIL-2) was administered at a dose of 720,000 IU/kg to patients with melanoma (n=7) and peripheral blood was collected at baseline and at 24, 48, 72, and 96 hours posttreatment. Peripheral blood mononuclear cells (PBMCs) were isolated and subjected to intracellular cytokine and extracellular receptor staining for flow cytometry. We report that HDIL-2 increased both frequencies and absolute numbers of Th17 cells on day 4 of treatment. The administration of HDIL-2 to patients with melanoma increased IL-6 production by peripheral immune cells, a cytokine vital in the downregulation of FoxP3 expression and expansion of the Th17-cell population. Furthermore, we demonstrated that FoxP3CD4 T cells express IL-17 in patients with melanoma undergoing HDIL-2 therapy. Taken together, our findings indicate that HDIL-2 combined with the conditions of malignancy create an immune environment supportive of Th17 differentiation and that expansion of this compartment may occur through the transdifferentiation of IL-17-secreting FoxP3CD4 T cells.

Parallel assessment of Th17 cell frequencies by surface marker co-expression versus ex vivo IL-17 production in HIV-1 infection.

Th17 cells can either be identified by co-staining of surface markers or by intracellular cytokine staining (ICS) for IL-17 production. Discrepancies regarding the published frequencies of Th17 cells in peripheral blood mononuclear cells (PBMC) of HIV patients may partly be due to the different methodologies used. Cryopreserved PBMC from healthy controls and HIV-infected subjects, including treated (cART) and viremic patients, were split and analyzed side-by-side by flow cytometry for expression of surface markers CCR6, CXCR3, CCR4, and CD161, or for intracellular expression of IL-17A and IFNγ after stimulation. The characterization of Th17 cells as CXCR3 - CCR6 + CCR4 + CD161+ yielded considerably higher frequencies than the corresponding frequencies obtained by characterization via cytokines (IL-17 + IFNγ-), regardless of the HIV status. However, the overall frequencies delivered by the two methods significantly correlated. The relative frequency of Th17 cells within the CD4+ T cell compartment was preserved in HIV infection but there was a significant decrease in the absolute Th17 number, which was restored after initiation of cART, paralleling CD4+ T cell recovery. Absolute Th17 numbers inversely correlated with HIV viral load. The definition of Th17 cells by surface markers might overestimate their frequency in comparison to functional assessment of IL-17 production by ICS, regardless of the HIV infection status. However, both methods yield proportionate results with reduced absolute numbers of Th17 cells in untreated HIV disease, reflecting the depletion of total CD4+ T cells in viremic HIV patients, and restoration with cART. © 2016 International Clinical Cytometry Society.

Redox regulation of a new autoimmune mouse model, glucose-6-phosphate isomerase peptide induced arthritis in mice (BA12P.108)

Abstract Human glucose-6-phosphate isomerase (hG6PI) peptide-induced arthritis is established by immunizing C57Bl/6N.Q (B6N.Q) mice with single hGPI325-339 peptide emulsified in complete freund’s adjuvant. Immunized B6N.Q mice start to develop arthritis on day 10-12, reach to the peak value of severity on day 14-20 and then recover during the next 7-10 days. This peptide-induced arthritis mouse model represents an acute arthritic disease progression. By taking advantage of μMT mice (B cell deficient) and TCRβ-/- (T cell deficient) mice, we have demonstrated that this peptide-induced arthritis is T cell- and B cell- dependent. In addition, by using Ncf1 mutant mice, which are lack of oxidative burst, we have found that reduced reactive oxygen specifics aggravate and prolong the peptide-induced arthritis. Phenotypic analysis results show that increased frequency and absolute numbers of Th17 cells, accompanied by significant increased CD11b+cells, are observed in Ncf1 mutant mice comparing with wild-type mice; moreover, IL-17 levels were dramatically increased in isolated splenic cells or lymph nodes cells from immunized Ncf1 mutant mice when restimulated with hGPI325-339 peptide. Moreover, in vitro experiments suggest that APCs from Ncf1 mutant mice show higher capability to present hGPI325-339 peptide to stimulate hGPI325-339 peptide specific T cell hybridoma, which suggest that enhanced antigen-presentation capability may aggravate the arthritic disease in Ncf1 mutant mice.

Blockade of Notch Signaling Ameliorates Murine Collagen-Induced Arthritis via Suppressing Th1 and Th17 Cell Responses

Recent studies have demonstrated that Notch signaling is critically involved in the regulation of immune response and contributes to autoimmune pathogenesis. Here, Notch signaling was found to be activated in CD4(+) T cells and synovial tissue from collagen-induced arthritis mice. In vivo administration of the γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl-l-alanyl)]-S-phenylglycine t-butyl ester (DAPT) substantially reduced the severity of arthritic symptoms and joint damage in collagen-induced arthritis mice. Notably, DAPT treatment significantly suppressed Th1- and Th17-cell responses in spleen and lymph nodes and reduced IFN-γ and IL-17 levels in plasma. In polarization culture, DAPT treatment markedly reduced Th17 cell expansion from naïve T cells, whereas fusion protein of the Notch receptor ligand delta-like 3 significantly increased the frequency and absolute number of Th17 cells. These results suggest a novel therapeutic strategy for treatment of human rheumatoid arthritis by targeting Notch signaling using γ-secretase inhibitors.

Transcriptional factor E2A regulates the differentiation of Th17 cells (69.10)

Abstract The molecular mechanisms that direct transcription of the gene encoding the transcription factor RORγt, a master regulator of Th17 cells, remain ill-defined. We previously showed that transcription factor E2A is enriched at the Foxp3 promoter and regulates the differentiation of regulatory T cells in response to TGF-β. Since TGF-β is also required to generate Th17 cells, we hypothesized that E2A might also be involved in the differentiation of Th17 cells. We utilized Id3 deficient mice, as Id3 is reported to inhibit E2A binding to DNA, and found that Id3 deficient mice had a much greater frequency and absolute number of Th17 cells in the lamina propria and Peyer’s patches. In vitro study showed that Id3 deficient CD4(+)CD25(-) naïve T cells showed greater differentiation into Th17 cells in response to TGF-β1 plus IL-6. Treatment with TGF-β1 substantially enriched E2A at the Rorc promoter in naïve CD4(+) T cells. In addition, E2A knockdown by siRNA resulted much less Rorc transcription and IL-17 production in CD4(+) T cells stimulated with TGF-β1 plus IL-6. These data suggest that E2A plays an important role in promoting the RORγt expression through enrichment to Rorc promoter and the consequent Th17 differentiation. We plan to investigate how IL-6-mediated signaling pathway is integrated with E2A-mediated Rorc activation in directing IL-17 production in CD4(+) T cells.

Plasmacytoid dendritic cells and Th17 immune response contribution in gastrointestinal acute graft-versus-host disease

The contribution of Th17 cells in acute graft-versus-host disease (aGVHD) has been demonstrated in aGVHD mouse models. However, their contribution in human gastrointestinal aGVHD remains unclear. We evaluated Th17 cells in a cohort of 23 patients at diagnosis of aGVHD. In this study, we have shown that the absolute number of Th17 cells using the CCR6 and CD161 markers were significantly higher in the intestinal mucosa of patients with aGVHD compared with intestinal mucosa of patients without aGVHD. Moreover, in keeping with the increase of CCR6+ and CD161+ T cells, RORγt the key transcription factor that orchestrates the differentiation of Th17 cells, was significantly increased in the intestinal mucosa of patients with aGVHD compared with intestinal mucosa of patients without aGVHD (P=0.01). Since plasmacytoid dendritic cells (PDCs) have been reported to drive the differentiation of the Th17 subset, we quantified PDCs in these patients. PDC CD123+ cells were increased in the intestinal mucosa of patients with aGVHD. Furthermore, the number of CD123+ PDCs paralleled the histological grade of aGVHD, providing evidence for a role of Th17-mediated responses and a potential new pathophysiological link between PDCs and Th17 in human aGVHD.

Th17 and non-Th17 interleukin-17-expressing cells in chronic lymphocytic leukemia: delineation, distribution, and clinical relevance

The levels and clinical relevance of Th17 cells and other interleukin-17-producing cells have not been analyzed in chronic lymphocytic leukemia. The objective of this study was to quantify blood and tissue levels of Th17 and other interleukin-17-producing cells in patients with this disease and correlate blood levels with clinical outcome. Intracellular interleukin-17A was assessed in blood and splenic mononuclear cells from patients with chronic lymphocytic leukemia and healthy subjects using flow cytometry. Interleukin-17A-producing cells were analyzed in formalin-fixed, paraffin-embedded spleen and lymph node sections using immunohistochemistry and immunofluorescence. The absolute numbers of Th17 cells in peripheral blood mononuclear cells and the percentages of Th17 cells in spleen cell suspensions were higher in patients with chronic lymphocytic leukemia than in healthy subjects; in six out of eight paired chronic lymphocytic leukemia blood and spleen sample comparisons, Th17 cells were enriched in spleen suspensions. Circulating Th17 levels correlated with better prognostic markers and longer overall survival of the patients. Two "non-Th17" interleukin-17-expressing cells were identified in chronic lymphocytic leukemia spleens: proliferating cells of the granulocytic lineage and mature mast cells. Granulocytes and mast cells in normal spleens did not express interleukin-17. Conversely, both chronic lymphocytic leukemia and healthy lymph nodes contained similar numbers of interleukin-17+ mast cells as well as Th17 cells. Th17 cells are elevated in chronic lymphocytic leukemia patients with better prognostic markers and correlate with longer survival. Furthermore, non-Th17 interleukin-17A-expressing cells exist in chronic lymphocytic leukemia spleens as maturing granulocytes and mature mast cells, suggesting that the microenvironmental milieu in leukemic spleens promotes the recruitment and/or expansion of Th17 and other IL-17-expressing cells. The pathophysiology of Th17 and non-Th17-interleukin-producing cells in chronic lymphocytic leukemia and their distributions and roles in this disease merit further study.

Increased Number of IL-17 Producing CD4+ T Cells in Low Risk Myelodysplastic Syndrome (MDS)

Immunological responses are increasingly recognized as important in the initiation and progression of MDS. Indeed, autoimmune diseases commonly occur in association with low risk MDS. Different studies, including our own, have shown that Foxp3+ Tregs play a role in the inhibition of tumour immune surveillance in high risk MDS; however, less clear are the potential factors which are inducing inflammatory response in low risk MDS that may also predispose to autoimmunity. CD4+IL 17 producing T cells (Th17) are considered as important pro-inflammatory T cells and contribute to the pathogenesis of a number of autoimmune diseases as well as some malignancies. The aim of this study was to investigate the potential role of Th17 cells in low risk MDS. We show for the first time that Th17 cells are markedly increased in low risk MDS in comparison to high risk disease and healthy age matched controls. We also describe an inverse relationship between numbers of Th17 cells and naturally occurring Tregs in MDS.88 patients with newly diagnosed MDS, and 15 age matched healthy donors, were recruited. Analysis of Th17 cells was performed in a subset of 43 patients. WHO classifications were as follows: 19% had RA, 33% had RCMD, 32% had RAEB I & II and 16% had 5q- syndrome. A clear difference was observed in the median percentage and absolute number of Th17 cells between patients with low risk (based on IPSS) compared to high risk MDS (p<0.01). In low risk, but not high risk MDS, CD4+ cells had greater median number of committed Th17 cells than healthy donors (p<0.01). The absolute number of polyclonal Foxp3+ Tregs inversely correlated with Th17 cells, with higher Tregs in the high risk MDS when compared to low risk MDS or healthy donors (p<0.001), consistent with our previous report. Analysis according to IPSS, BM blast percentage or WHO classification revealed the same patterns of Th17 and Tregs expression, with the higher risk phenotypes being associated with significantly higher Treg number and lower risk phenotypes with greater Th17 cells. This finding was specific for Th17 as there was no significant difference in the number of circulating Th1 and Th2 cells between MDS subtypes.Co-culture of Tregs from patients with MDS with their own T effectors lead to suppression of supernatant IFNγ levels. Importantly, Tregs did not have an inhibitory effect on IL17 production and indicates that reduced Th17 number in the presence of high Tregs does not indicate active suppression of the Th17 phenotype.To investigate differences in serum cytokines between low and high risk MDS and healthy donors, serum samples from patients were analyzed by Luminex for multiple cytokines. The cytokines IL12(p<0.01) IL7(p<0.005) IFNγ(p<0.01) and RANTES (p<0.005) were significantly higher in patients with low risk compared to high risk MDS and healthy controls. The inhibitory cytokine IL10 and soluble IL2 receptor were present at higher levels in the serum of patients with high risk MDS (p<0.01 and p<0.005 respectively). To confirm that the peripheral blood cytokines are a true reflection of cytokine concentration in the bone marrow, the concentration of 30 different cytokines was measured by Luminex in 10 matched bone marrow and peripheral blood samples from these patients and there was no significant difference between these two sets of samples. In addition, the level of apoptosis, assessed by TUNEL assay, was also higher in the bone marrow of low risk patients.In conclusion, the “unfavourable” Th17 to Tregs ratio in low risk MDS may explain the higher risk of autoimmunity and bone marrow apoptosis. These findings may help identify subtype of low risk MDS patients likely to respond to immunosuppressive therapy.