Intracellular Expression Of IL-17 Research Articles

Isolation and in vitro Expansion of Regulatory T Cells from β-Thalassemia Major Kids and in vitro Evaluation of Their Plasticity and Inhibitory Effects in the Presence of Pro-Inflammatory Cytokines

Introduction: Graft-versus-host disease (GvHD) is a life-threatening syndrome commonly associated with hematopoietic stem cell transplantation (HSCT). Preventing the incidence of GVHD after HSCT along with minimizing long-term immunosuppression is currently under investigation with regulatory T cells (Tregs). As Tregs are a low-frequency population and the yield of all memory Tregs is not sufficient for clinical application, an initial Treg expansion is essential. Methods: Thirty milliliters of peripheral blood from the β-thalassemia major (beta-TM) patients and healthy controls were obtained and Tregs were isolated using MACS. Isolated cells were cultured in the presence of rapamycin and rIL-2 followed by activated with anti-CD3/CD28-coated beads. To evaluate Treg plasticity, expanded Tregs were cultured in a medium containing IL1β, IL6, TGFβ, and IL2, with or without 500 nM rapamycin for 72 h. To assess the functional properties of Tregs, CFSE dilution assays were performed to evaluate the ability of in vivo expanded Tregs from beta-TM patients. Statistical analysis was performed using paired t-test and independent t-test, with the aid of SPSS version 12.0. p-value ≤0.05 was considered significant. Results: The percentage of Tregs isolated from the control group was significantly higher than the Tregs isolated from patients (p-value = 0.01), which is probably due to the iron overload in beta-TM patients as a result of continuous blood transfusion. Also, the percentage of Tregs after 5 days of expansion had a significant increase in both groups compared to before expansion (p-value = 0.03). Our results also showed that the expansion of Tregs after 72 h in the presence of inflammatory cytokines and in the absence of rapamycin led to the increase in the intracellular expression of IL-17 (p-value = 0.01), while intracellular expression of IL-17 remained low following the addition of 100 nM rapamycin to the culture medium (pvalue = 0.073). The results of the functional evaluation of expanded Tregs showed relatively differences in both patient and control groups. Thus, expanded Tregs inhibited the proliferation of responder T cells in a dose-dependent manner in the control group (p-value = 0.028), while in the patient group this inhibitory effect was not significant (p-value = 0.055). Conclusion: Tregs isolated from beta-TM patients have poorer inhibitory performance than Tregs isolated from healthy individuals. Also, we concluded that rapamycin stabilizes the Treg population by inhibiting the production of IL-17, all necessitating the administration of appropriate immunosuppressive drugs in patients receiving Treg therapy.

Simplified assay for enrichment of primed human Th17 and Tc17 lymphocytes from peripheral blood

BackgroundInterleukin-17A (IL-17A) is a potent pro-inflammatory cytokine that has been implicated in the pathogenesis of various autoimmune diseases. The production of IL-17A is commonly associated with subsets of CD4+ T cells (Th17) and CD8+ T cells (Tc17). Identifying these subsets based on intracellular expression of IL-17 or transcription factor RORC precludes isolation of viable Th17 and Tc17 cells and there by limits studies involving cell-cell interaction or cellular functions. Therefore, identifying surface markers that can help in identifying and enriching these cells is important.ResultsWe used MCAM as a surrogate marker to identify in vivo committed human Th17 and Tc17 subsets. By employing high-speed fluorescence activated cell sorting, we enriched IL-17A-producing subsets from human specimens without the need for in vitro polarization using exogenous cytokines. These subsets can be investigated, following sorting, using a variety of methods such as ELISA, ex vivo functional assays and next generation sequencing to gain insights into the role of human Th17 and Tc17 in health and disease.ConclusionWe here demonstrate that both CD4+ T cells (Th17) and CD8+ T cells (Tc17) cell populations can be identified based on the surface expression of melanoma cell adhesion molecule (MCAM or CD146).

Lamina Propria CD4+LAP+ Regulatory T Cells Are Increased in Active Ulcerative Colitis but Show Increased IL-17 Expression and Reduced Suppressor Activity.

A CD4+CD25- regulatory T cell population expressing the surface TGF-β in its latent form LAP+ [latency associated peptide] cells was proved to be protective in experimental colitis and to be suppressive of human peripheral blood [PB] T proliferation. We investigated the frequency and function of lamina propria [LP] CD4+LAP+ T cells in inflammatory bowel disease [IBD] patients. Specimens from patients undergoing colonoscopy or bowel resection for IBD and colonic cancer were used as source of lamina propria mononuclear cells [LPMC]. The ulcerative colitis [UC] group was divided according to endoscopic activity evaluated with modified Baron Score. IL-17, IFN-γ, IL-10, LAP, and Foxp3 expression in CD3+CD8- [CD4] or CD3+/CD4+ gated cell population was assessed by immunofluorescence. The ability of FACS-sorted LP CD3+CD8-[CD4] LAP+CD25- to inhibit stimulated autologous PB CD3+CD8-[CD4] LAP- CD25- cells proliferation was assessed. LP CD4LAP+ cells were significantly increased, when compared with controls, in active UC patients and not in Crohn's disease patients. The majority of LP CD4+LAP+ cells were Foxp3-. The percentage of IL-17+ cells in LP CD3+CD8-[CD4] LAP+ cells was significantly higher in active UC patients when compared with controls. LP CD3+CD8-[CD4]LAP+CD25- isolated from UC patients showed reduced or no ability to inhibit autologous PB CD3+CD8-[CD4]LAP-CD25- cell proliferation when compared with controls. Removal of IL-17+ cells from LP CD3+CD8-[CD4] LAP+ cells increases their suppressive ability. The percentage of LP CD4LAP+ cells is increased in active UC, showing reduced suppressor activity due to their increased proportion of intracellular IL-17 expression.

Effects of leflunomide on experimental autoimmune uveitis in Lewis rats

Objectives To investigate the protective efficacy of leflunomide on the Lewis rats with experimental autoimmune uveitis (EAU). Methods Complete randomized controlled trials research. Lewis rats were immunized with interphotoreceptor retinoid-binding peptide (IRBP) in order to build the model of EAU. Rats were randomized assigned into four groups, that is control group as A, model group without leflunomide as B, model group with leflunomide administrations as C, and model group with cyclosporine A as D. Rats in group C received intragastric administration of three doses of leflunomide at 3mg/kg/d; 6mg/kg/d; 12mg/kg/d. Rats in group D received 10 mg/kg cyclosporin A were considered as a positive control. Each group has 6 to 8 rats. At the second day of immunization with IRBP, rats were intragastric administrated one time every day till day 13. Rats were investigated for EAU symptoms under slit lamp. Enucleated eyes were collected for sections with HE staining as histopathological evidences at the peak point of disease activity day 14. Treatment effectiveness was evaluated referred by Agarwal standard for clinical EAU and histopathological scoring. The expression of IL-17 in ocular sections was detected by immunohistochemistry (SP method). The expression levels of IL-17 and IFN-γ in the serum were quantified by ELISA. Intracellular expression of IL-17 in the activated CD4 + T cells was assessed by flow cytometry. Ocular of rats were harvested and mRNA expression of IL-17 and IFN-γ were quantified through RT-PCR. Continuous variables were reported as mean ± SD. The comparison among groups was done by using analysis of students't test. Nonparametric test was used in Hierarchical data comparison and multiple comparison method was Bonferroni. Results The model of EAU disease was built successfully in Lewis rats. With giving IRBP for 14 days, the clinic EAU scores were lower in model rats than those without leflunomide. Moreover, the effects of leflunomide on the clinic EAU scores was dose-dependent. Comparing to vehicle-treated eyes, treatment with leflunomide significantly prevented the onset of EAU-induced ocular inflammation [1.5 (1,2) vs. 3 (3,4), P=0.0006, P<α', α'=0.05/15]. The pathological examination showed model rats eye characterized by severe inflammatory cells infiltration and all layers of retina damaged. The pathologic grade was significant higher in model group than in medium dose leflunomide. [3(3, 4) vs. 2(1,3), P=0.0014, P<α', α'= 0.05/15]. IL-17 was positively expressed in iris, ciliary and retina in model group. While, it was markedly reduced in leflunomide-treated eyes. Flow cytometry detection found that compared with normal group, Th17 cells rates in rats' spleen of model group also increased significantly (8.5%±1.3% vs. 0.5%±0.2%; t=8.057, P=0.000, P<α', α'=0.05/15). Compared with model group, Th17 cells in spleen of rats in leflunomide groups showed a decreased number by flow cytometry. And it showed dosage dependent. It was significant different between different doses leflunomide treated group compared with control group. The results showed as below, in low dose group (4.1%±0.6% vs. 8.5%±1.3%; t=6.372, P=0.01, P<α', α'=0.05/15), in medium dose group (2.8%±0.2% vs. 8.5%±1.3%; t=4.49, P=0.002, P<α', α'=0.05/15) and in high dose group (1.8%±0.2% vs. 8.5%±1.3%; t=5.743, P=0.000, P<α', α'=0.05/15). Gene expression of IL-17 and IFN-γ were markedly reduced in leflunomide-treated eyes. Leflunomide significantly decreased the serum levels of IL-17 and IFN-γ. Compared with model group, in leflunomide-treated low dosage group (0.603±0.03 vs. 0.787±0.104; t= 0.183, P=0.002, P<α', α'=0.05/15), medium dosage group (0.535±0.048 vs. 0.787±0.104; t=0.252, P=0.000, P<α', α'=0.05/15) and high dosage group (0.374±0.051 vs. 0.787±0.104; t=0.412, P=0.000, P<α', α'=0.05/15), IL-17 mRNA showed lower expression. Moreover, IFN-γ mRNA in the tissue of EAU eyes were suppressed by medium dosage leflunomide group (0.375±0.018 vs. 0.427±0.056;t=0.69, P=0.001, P<α', α'= 0.05/15) and high leflunomide dosage group respectively (0.367±0.018 vs. 0.427±0.056; t=0.077, P=0.000, P<α', α'=0.05/15) . The difference was statistically significant. All the results suggested that IL-17, which was secreted by Th17 cell, a subtype of T lymphocytes, might play an important role in the pathogenesis of uveitis. Conclusions Oral administration of leflunomide effectively suppressed IRBP-induced uveitis in rats, not only reduced exudation in iris but also alleviated the infiltration damage of inflammation cells in fundus. It might be ascribed to the effect that leflunomide could treat inflammation by down-regulating the expressions of IL-17 and IFN-γ. Therefore, it suggested that leflunomide had protective effects against EAU in Lewis rats. (Chin J Ophthalmol, 2015, 51:754-761) Key words: Uveitis; Autoimmune diseases; Isoxazoles; Th17 cells; Rats

The Clinical Relevance of IL-17-Producing CD4+CD161+ Cell and Its Subpopulations in Primary Sjögren's Syndrome.

Objective. Th17 cells have been demonstrated to play an important role in the onset and development of primary Sjögren's syndrome (pSS). In this study, we evaluated the expansion and clinical significance of circulating CD4+CD161+ T cell and its “effector” (CD4+CD25−CD161+ T cell) and “regulatory” (CD4+CD25+CD161+ T cell) subpopulations. Methods. Fifty-eight pSS patients and 16 healthy controls (HCs) were recruited in our study. The cell populations and intracellular IL-17 expression were analyzed by flow cytometry. The disease activity was evaluated by the EULAR-SS Disease Activity Index (ESSDAI). Autoantibodies were measured by ELISA or indirect immunofluorescence assay. Results. The CD161+ T cell fractions showed higher proportions of IL-17-producing cells. The frequencies of the overall CD4+CD161+ T cell population and its effector subset were positively correlated with disease activity parameters and more severe disease manifestations. A significant elevation of the CD4+CD25+CD161+ T cell subpopulation was observed in the peripheral blood of pSS patients compared to HCs and this subset showed decreased regulatory functions compared with the CD4+CD25+CD161− population. Conclusion. Circulating CD4+CD161+ T cell populations associated with pSS disease activity and severity. These cells might be involved in the development of pSS and could be potential therapeutic targets in the treatment of pSS.

Cytokines Elicited by HSP60 in Periodontitis with and without Coronary Heart Disease

The human 60 kDa and microbial 65 kDa heat shock proteins (HSP) have been implicated in the pathogenesis of chronic periodontitis (CP) and coronary heart disease (CHD). We have studied 100 subjects: Group (a) consisted of patients with gingivitis (n = 25), group (b) were patients with CP (n = 25), group (c) patients with CHD and gingivitis (n = 25) and group (d) patients with CHD and CP (n = 25). PBMCs separated from peripheral blood were stimulated with medium, PMA/ionomycin, human HSP60, microbial HSP65, or no stimulus for 18 hours before intracellular IL-2, IFN-γ, TNF-α, IL-4, IL-5, or IL-17 were detected by flow cytometry. The mean fluorescence intensity (MFLI) for intracellular TNF-α was significantly increased when PBMC were stimulated with human HSP60 amongst the four groups (p = 0.001, ANOVA); pairwise comparisons revealed significant differences in MFLI between the gingivitis group and the CP (p = 0.017); between gingivitis and ging/CHD (p = 0.001) as well; but no significant difference between the CP and CP/CHD (p = 0.442). There was no significant difference in intracellular expression of IL-17, or any of the other cytokines tested; and the MFLI for HSP-stimulated were comparable to unstimulated cultures. When heat-labile human HSP60 was heated, intracellular cellular TNF-α expression was abrogated. In contrast, heat-stable LPS elicited TNF-α expression from monocytes in bulk cultures in all groups. These results suggest that the cytokine expression was dependent on human HSP60 and not LPS. Serum CRP was significantly associated with MFLI of intracellular TNF-α in CP patients (rs = 0.665, p = 0.026) and CP/CHD (rs = 0.699, p = 0.011). We conclude that human HSP60 elicits increased monocytic expression of TNF-α in patients with CP, CP/CHD or ging/CHD compared to patients with gingivitis. Since the marker of inflammation, namely CRP correlates with CP with or without CHD and not with mild chronic gingivitis or ging/CHD, this suggests that human HSP60-induced production of TNF-α is associated with CP and not CHD. There was no significant difference in intracellular expression of IL-17.

Decreased microRNA-155 Expression in Ocular Behcet's Disease but Not in Vogt Koyanagi Harada Syndrome

MicroRNAs (miRNAs) have emerged as a class of gene expression regulators involved in immune regulation. In the present study, we investigated the role of miRNA in two uveitis entities: Behcet's disease (BD) and Vogt Koyanagi Harada syndrome (VKH). The expression of five miRNAs was studied in PBMCs, DCs, and CD4(+) T cells from BD patients with active and inactive uveitis, VKH patients with active uveitis, and healthy controls using real-time PCR. MiR-155 mimics and inhibitor were transfected to DCs to evaluate the effect on DC maturation and cytokine production by these cells and CD4(+) T cells. Luciferase reporter assays and Western blotting were performed to identify the target gene of miR-155. Only miR-155 expression was significantly decreased in PBMCs and DCs from BD patients with active uveitis and no differences were observed in the miRNA expression in cells from patients with VKH as compared with controls. Overexpression of miR-155 in DCs was shown to inhibit the production of IL-6 and IL-1β, and to promote the expression of IL-10 by these cells. MiR-155 transfected DCs significantly inhibited intracellular IL-17 expression in allogeneic CD4(+) T cells; however, it did not influence the expression of cell surface markers CD80, CD40, CD83, CD86, and HLA-DR. Luciferase reporter assays revealed that TAB2 was a target gene of miR-155, which was confirmed by Western blotting. The present results suggest that miR-155 expression is decreased in active BD but not in VKH patients. Downregulated miR-155 may be involved in BD pathogenesis by targeting TAB2.

Activity of Childhood Lupus Nephritis is Linked to Altered T Cell and Cytokine Homeostasis

Standard therapy for lupus nephritis is based on non-specific immunosuppression. We aimed to identify specific alterations in T cell and cytokine homeostasis and possible associations with disease activity in children with lupus nephritis (LN). The phenotype of circulating T cells from children with LN and healthy controls (HC) was analyzed by flow cytometry. Intracellular expression of IL-17 and INF-γ was assessed after stimulation with anti-CD3 and anti-CD28. Serum concentrations of IP10, CCL2, TGF-β, IL-17, and IL-23 were measured by ELISA. Disease activity was determined using the Systemic Lupus Erythematosus Disease Activity Index 2000 update (SLEDAI-2K). Children with active LN displayed increased frequencies of effector memory CD4(+)CD45RO(+)CCR7(-) and terminal differentiated CD4(+)CD45RA(+)CCR7(-) T cells and reduced naive CD4(+)CD45RA(+)CCR7(+) T cells compared to those with inactive LN or HC. Circulating CD4(+)CXCR3(+) and CD4(+)CCR2(+) T cells correlated inversely with the renal SLEDAI-2K, whereas IP10 and CCL2 showed a positive correlation. Reduced CD4(+)Foxp3(+) T cells and serum TFG-β levels in active LN were associated with high serum IL-17 and IL-23 levels and correlated inversely with the renal SLEDAI-2K (r = -0.5855, p = 0.0013 and r = -0.6246, p = 0.0005, respectively), whereas IL-17 and IL-23 correlated positively (r = 0.5516, p = 0.0029 and r = 0.6116, p = 0.0007, respectively). Expansion of Th17 and Th1/Th17 cells in children with LN was significantly greater than in HC (p = 0.0304 and p = 0.0067, respectively). Children with active LN display high levels of pro-inflammatory cytokines associated with an increase in effector T cells and reduction of regulatory T cells. Therapeutic regulation of the aberrant cytokine profile might specifically interrupt pathogenic mechanisms.

Th17/IL-17 Might Play a Protective Role in CLL Immunity

Abstract 2843 Background:Immune system disorders play an important role in the development and progression of CLL. Many deficiencies, contributing to disease progression, were found in T cell populations. Th17 cells represent a recently-discovered distinct lineage of helper T-cells, characterized by IL-17 secretion. IL-17 play an active role in inflammation and autoimmune diseases, however the role of IL-17 and Th17 cells in CLL immunopathogenesis remains undefined. We investigated IL-17A mRNA and IL-17A protein expression in peripheral blood (PB) CD4+ T cells and the plasma levels of IL-17 in 70 untreated patients with CLL in the context of clinical and immunological parameters. Th17 cell percentage was measured also in the bone marrow of CLL patients. The control group consisted of 20 healthy age-matched subjects. Materials and methods:Quantitative ‘real-time' reverse transcription-polymerase chain reaction (qRT-PCR) was used for the analysis of IL-17A mRNA in PB CD4+ T-cells. Intracellular IL-17A expression in CD3+/CD4+cells was analyzed using a three-color flow cytometry technique. Plasma levels of IL-17A, IL-4 and IL-10 were measured by ELISA. Results:None of the CD4+ T cells in the samples obtained from healthy volunteers (HV) contained detectable amounts of IL-17 mRNA, whereas it could be found in 5 out of 70 CLL samples. All CLL patients with detectable IL-17 mRNA in T-cells were in 0 Rai stage and negative both for ZAP-70 and CD38 expression. It is possible that IL-17 mRNA in these cells is expressed only for a short time thus evading detection by PCR, or that CLL cells contain low amounts of the IL-17 transcript and the RT-PCR method was not sufficiently sensitive to detect these small amounts. Frequently, in HV as well as in CLL patients, the percentage of CD4+ T cells with intracellular IL-17 expression in non-activation assays was lower than 1%, comparable to the level of autofluorescence. Despite the fact that IL-17 mRNA was not detected in the T cells of the majority of CLL patients, IL-17 protein was present in T cells after PMA and ionomycin stimulation. We found a significantly higher median percentage of CD4+/CD3+/IL-17+ cells in CLL patients (18.2%) than in HV (2.9%) (p<0.01). Median percentage of Th17 cells was significantly higher in CLL patients in stages 0–1 according to Rai, as compared with stages 2–4 (36.2% vs 4.5%, p<0.05). There were no differences in Th17 percentages between the PB and BM of CLL patients. Accordingly, the IL-17 plasma level was significantly higher in CLL patients than in HV (101.9 pg/ml vs 75.6 pg/ml; p<0.05) and there was a significant correlation between Th17 cell percentage and IL-17 plasma level. There was also significant correlation between the percentages of Th17 and iNKT cells (R=0.56; p<0.05), and inverse correlations between the percentage of Th17 and: Treg cells (R=−0.28; p<0.05), CD4+IL-4+ (R=−0.55; p<0.05) and CD4+TNF+ T cells (R=−0.59; p<0.01). The percentage of Th17 inversely correlated with β2-microglobulin serum level (R=−0.29; p<0.05) and the expression of ZAP-70 (R=−0.244; p<0.05) and CD38 (R=−0.33; p<0.01). The plasma level of IL-17 inversely correlated with the stage of disease (R=−0.23; p<0.05), IL-10 (R=−0.56; p<0.01) and TNF (R=−0.66; p<0.01) plasma levels, CD38 antigen (R=−0.25; p<0.05) and ZAP-70 expression (R=−0.28; p<0.05). In patients requiring therapy during the observation period, the median percentage of Th17 cells and median plasma IL-17 level were significantly lower comparing to untreated ones (5.7% vs 23.1%, p<0.05; 53.8 vs 95.6 pg/ml, p<0.05; respectively). Moreover, median plasma IL-17A level correlated with the time from CLL diagnosis to the start of therapy (R=0.49; p<0.01). Conclusions: both the Th17 cell percentage, and the IL-17 plasma level are significantly higher in the PB of untreated CLL patients compared with the HV, and IL-17 mRNA expression was found only in the T cell of CLL patients, but not in the control group. Correlations found with disease activity parameters, the subsets of T cells involved in tumor surveillance, and also with the length of time from diagnosis to the start of therapy, suggest an important protective role for Th17 cells in CLL immunity. Disclosures:No relevant conflicts of interest to declare.

Lack of Th17 Cell Generation in Patients with Severe Burn Injuries

Immunodeficient patients with severe burn injuries are extremely susceptible to infection with Candida albicans. In addition to Th1 cells, IL-17-producing CD4(+) T cells (Th17 cells) have recently been described as an important effector cell in host anti-Candida resistance. In this study, therefore, we tried to induce Th17 cells in cultures of severely burned patient PBMC by stimulation with the C. albicans Ag (CAg). In the results, the biomarkers for Th17 cells (IL-17 production and intracellular expression of IL-17 and retinoic acid receptor-related orphan receptor γt) were not displayed by burn patient PBMC stimulated with CAg, whereas these biomarkers of Th17 cells were detected in cultures of healthy donor PBMC stimulated with CAg. Burn patient sera were shown to be inhibitory on CAg-stimulated Th17 cell generation in healthy donor PBMC cultures; however, Th17 cells were induced by CAg in healthy donor PBMC cultures supplemented with burn patient sera that were previously treated with anti-IL-10 mAb. Also, the biomarkers of Th17 cells were not induced by CAg in healthy donor PBMC cultures supplemented with rIL-10. IL-10 was detected in serum specimens derived from severely burned patients. These results indicate that Th17 cells are not generated in burn patient PBMC cultures supplemented with CAg. IL-10, produced in response to burn injuries, is shown to be inhibitory on Th17 cell generation. The high susceptibility of severely burned patients to C. albicans infection might be influenced if burn-associated IL-10 production is intervened.

Modulatory effect of triptolide on differentiation of human Th17 cells

To investigate the effect of triptolide on the differentiation of human Th17 cells. Human peripheral blood mononuclear cells, purified CD4+ T cells and CD4+CD45RA- memory T cells were treated with various concentrations of triptolide in vitro. Cell proliferation was determined by MTT assay. Flow cytometry was used to analyze the intracellular expression of IL-17 and IFN-gamma. Cytokine production of IL-17 and IFN-gamma was measured by ELISA. Cell proliferation, intracellular expression of IL-17 and IL-17 secretion were inhibited by triptolide in a dose-dependent manner. IFN-gamma expression and production were also inhibited by triptolide. Triptolide inhibits the differentiation of human Th17 cell. The observation may indicate at least one of the mechanisms of the immunosuppressive and anti-inflammatory effects of triptolide.

Endogenous IL-β Selectively Converts T Regulatory Cells Into IL-17 Producers During Antigen Stimulation

AbstractAbstract 586A major challenge of the immune system is to fight pathogens and tumor antigens while preserving tolerance to self-antigen. T regulatory cells (Treg) are critical extrinsic regulators of immune tolerance and maintenance of lymphoid homeostasis. Recently it was determined that, when used as cell-based immunosuppressive therapy, Treg have a potent effect in preventing GvHD in patients undergoing allogeneic stem cell transplantation. However, several studies suggest that the Treg phenotype is not at end stage of differentiation. Treg can express and produce effector cytokines including IFN-γ and IL-17 under certain conditions, particularly in the context of inflammatory milieu, suggesting that Treg may convert into inflammatory mediators. IL-1β and TNF-α are critical inflammatory cytokines that have been implicated in GvHD. The precise role and the mechanism(s) via which these cytokines may affect development of GvHD remain unclear. In the presence study, we sought to determine whether IL-1β and TNF-α regulate the properties of Treg and specifically whether these cytokines affect Treg expansion and/or conversion into IL-17 producing cells. CD4+CD25+Treg cells were isolated from B6 mice and were stimulated with anti-CD3-plus-anti-CD28 mAbs in the presence of either media, IL-1β or TNF-α. Addition of either cytokine induced Treg proliferation as determined by CFSE. Assessment of intracellular IL-17 expression by flow cytometry and IL-17 production by ELISA revealed that IL-1β but not TNF-α induced conversion of Treg into IL-17 producing cells, suggesting that conversion was mediated via pathways distinct from those that regulate cell cycle progression. To evaluate conversion of Treg to IL-17 producers during antigen stimulation and to determine the role of IL-1β in this process, we used neutral culture conditions in which no exogenous cytokines were supplied. Treg cells isolated from Foxp3GFP-KI mouse were added to cultures of naive conventional CD4+ T cells (Tc) in the presence of APC and anti-CD3 mAb. We found that these conditions preferentially induced conversion of Treg to IL-17 producing cells. To determine the role of IL-1β in this conversion process, we used IL-1β neutralizing antibody. Addition of anti-IL-1β neutralizing antibody reduced IL-17 production to almost undetectable levels. Because it has exogenous IL-6 can induce IL-17 production by both Treg and Tc, we evaluated whether endogenous IL-6 was involved in the conversion of Treg into IL-17 producing cells in our system. Addition of a combination of IL-6 neutralizing and IL-6 receptor blocking antibodies did not affect IL-17 production, suggesting that the conversion process of Treg into IL-17 producing cells was dependent on endogenous IL-1β rather than IL-6. To determine whether IL-1β was mandatory for this process, we used T cells from IL-1R deficient mice. Individual culture of IL−1R−/− Tc or IL-1R−/− Treg with wild type (wt) APC and co-culture of IL-1R−/− Tc and IL-1R−/− Treg with wt APC did not result in detectable IL-17 production. Similarly, no IL-17 production was observed when wt instead of IL-1R−/− Tc were used. In contrast, substitution of IL-1R−/− Treg with wt Treg resulted in abundant IL-17 production. To investigate the in vivo biological relevance of our findings we adoptively transferred Treg cells from either congenic B6.PL mice or IL-1R1−/− mice into IL-1R1−/− recipients, which were then immunized with KLH in IFA. Three days after immunization both IL-1R−/− Treg and IL-1R−/− Tc cells were incapable of producing detectable levels of IL-17 or expressing RORγt, the key transcriptional factor of IL-17. In contrast, a significant percentage of IL-17 and RORγt positive cells were detected within the adoptively transferred Thy1.1+ Treg population. Mechanistic analysis revealed that IL-1β induced activation of p38 and JNK in Treg and addition of pharmacologic inhibitors specific for these MAPKs abrogated IL-17 production. Our studies reveal that although Treg have primarily immunosuppressive functions they may also facilitate pro-inflammatory responses as they can be converted into IL-17 producing cells by IL-1β. These observations may have significant implications on clinical strategies that employ Treg for control of GvHD and suggest that further intervention might be required to prevent attainment of pro-inflammatory properties by Treg while maintaining their suppressive function. Disclosures:No relevant conflicts of interest to declare.

Linking Power Doppler Ultrasound to the Presence of Th17 Cells in the Rheumatoid Arthritis Joint

BackgroundPower Doppler ultrasound (PDUS) is increasingly used to assess synovitis in Rheumatoid Arthritis (RA). Prior studies have shown correlations between PDUS scores and vessel counts, but relationships with T cell immunopathology have not been described.Methodology/Principal FindingsPBMC were isolated from healthy controls (HC) or RA patients and stimulated ex vivo with PMA and ionomycin for 3 hours in the presence of Golgistop. Paired synovial fluid (SF) or synovial tissue (ST) were analysed where available. Intracellular expression of IL-17, IFNγ, and TNFα by CD4+ T cells was determined by flow cytometry. Synovial blood flow was evaluated by PDUS signal at the knees, wrists and metacarpophalangeal joints of RA patients. Serum, SF and fibroblast culture supernatant levels of vascular endothelial growth factor-A (VEGF-A) were measured by ELISA. The frequency of IL17+IFNγ-CD4+ T cells (Th17 cells) was significantly elevated in peripheral blood (PB) from RA patients vs. HC (median (IQR) 0.5 (0.28–1.59)% vs. 0.32 (0.21–0.54)%, p = 0.005). Th17 cells were further enriched (mean 6.6-fold increase) in RA SF relative to RA PB. Patients with active disease had a higher percentage of IL-17+ T cells in ST than patients in remission, suggesting a possible role for Th17 cells in active synovitis in RA. Indeed, the percentage of Th17 cells, but not Th1, in SF positively correlated with CRP (r = 0.51, p = 0.04) and local PDUS-defined synovitis (r = 0.61, p = 0.002). Furthermore, patients with high levels of IL-17+CD4+ T cells in SF had increased levels of the angiogenic factor VEGF-A in SF. Finally, IL-17, but not IFNγ, increased VEGF-A production by RA synovial fibroblasts in vitro.Conclusions/SignificanceOur data demonstrate a link between the presence of pro-inflammatory Th17 cells in SF and local PDUS scores, and offer a novel immunological explanation for the observation that rapid joint damage progression occurs in patients with persistent positive PDUS signal.

Th-17/IL-17 Axis in Chronic Lymphocytic Leukemia.

Abstract 2357Poster Board II-334 Introduction:Maintenance of chronic lymphocytic leukemia (CLL) clones is dependent on complex interactions between leukemic cells and soluble factors released by cells within the microenvironment of the blood, bone marrow, lymph node and spleen. Studies in our laboratory demonstrated elevated levels of IL-17, a pro-inflammatory cytokine secreted by a subset of CD4+ effector T cells (Th17 cells), in the sera of CLL patients as compared to those of healthy age-matched subjects. We hypothesized that the Th17/IL-17 axis is active in CLL, creating a proinflammatory microenvironment that promotes growth and angiogenesis. To test this hypothesis, we analyzed the expression of both IL-17 and IL-17 receptors in blood, spleen, and lymph nodes of CLL patients and normal control subjects. Methods:All studies involved patients with CLL and healthy subjects matched for age. Constitutive and inducible intracellular IL-17 expression was assessed in cryopreserved peripheral blood mononuclear cells (PBMCs) isolated from untreated CLL patients (n = 27) and healthy individuals (n = 8). Flow cytometric analyses were performed at baseline (Day 0) and after 7-day culture with or without the Th17-polarizing cytokines, IL-1b and IL-23. Surface expression of IL-17 receptors A (IL-17RA) and B (IL-17RB) was analyzed on PBMCs of CLL patients (n = 25) and healthy controls (n = 8) using flow cytometry. Lastly, IL-17 expression was assessed by immunohistochemistry in spleen and lymph nodes from CLL patients and healthy subjects. Results:Intracellular analysis of IL-17 expression in PBMCs revealed a higher median percentage of CD4+ Th17 cells in CLL patients (Median=0.73; range: 0.0-3.6%) compared to healthy subjects (Median=0.13; range: 0.0-0.8%) at baseline; this was a statistically significant difference (P<0.05). While there was no significant difference between the two major patient subgroups, i.e., (Mutated IGHV >2% and CD38low <30% and unmutated IGHV <2% and CD38high, >30%). CLL patients in the good prognosis group had significantly higher median percentage of CD4+ IL-17+ cells compared to healthy controls at baseline (Median=0.73 vs 0.13, P<0.05). Complementary studies also revealed that expression of IL-17 in the CD4+ T cell compartment after culturing total PBMC's for 7 days in the absence of Th17-polarizing cytokines was significantly higher in CLL patients than healthy subjects (Median =1.34, P<0.05). When PBMCs were cultured for 7 days in the presence of Th17-polarizing cytokines, the percentage of IL-17-producing CD4+ T cells increased in cells from both patients and healthy subjects, however again the percentage of IL-17 expressing CD4+cells was higher in cultures of PBMC's from CLL patients as compared to those from healthy subjects (Median= 1.99 vs 0.84). Surface expression of IL-17RA and IL-17RB was observed on monocytes from both patients and healthy subjects. The density of expression of IL-17RA on monocytes was more intense relative to IL-17RB in both patients and healthy subjects; however IL-17RA and IL-17RB expression was not significantly different between patients and healthy subjects with respect to percent of positive cells, mean fluorescence intensity or median fluorescence intensity. Of note, IL-17RA and IL-17RB were expressed on a small fraction of B lymphocytes from patients (Mean for RA and RB= 6.59 and 3.99, respectively) and normal individuals (Mean for RA and RB= 4.31 and 3.43, respectively). For CLL patients, these receptors were detected on CD19+CD5+ cells, whereas for healthy subjects they were found on CD19+CD5− cells. Preliminary immunohistochemical analyses of lymphoid tissues from CLL patients and healthy subjects revealed strong IL-17 expression in tissues from CLL patients but not in healthy age-matched subjects. Conclusions:Patients with CLL have higher baseline levels of circulating IL-17, as well as increased expression of Th17 cells in the blood and lymphoid tissues, as compared to healthy age-matched subjects. Furthermore, IL-17RA and IL-17RB are expressed on monocytes and on a small population of B cells in CLL patients enabling them to respond to the elevated levels of IL-17 in the microenvironment. Such interactions may stimulate release of pro-survival factors and promote growth and angiogenesis, contributing to the development and evolution of CLL. Further studies are in progress to demonstrate the functional role of IL-17 in CLL. Disclosures:No relevant conflicts of interest to declare.

T helper17 Cells Are Sufficient But Not Necessary to Induce Acute Graft-Versus-Host Disease

T helper (Th)1 cells were considered responsible for the induction of graft-versus-host disease (GVHD), but recently the concept has been challenged. Th17 cells play a critical role in mediating autoimmune diseases, but their role in the pathogenesis of GVHD remains unclear. Herein we compare the ability of in vitro generated Th1 and Th17 cells from C57BL/6 mice to induce GVHD in lethally irradiated BALB/c recipients. Allogeneic Th17 cells had superior expansion and infiltration capabilities in GVHD target organs, which correlated with their increased pathogenicity when compared with naïve or Th1 controls. Th17 cells caused no pathology in the syngeneic recipients, indicating that antigen-activation was required for their pathogenicity. Polarized Th17 cells could not maintain their phenotype in vivo as they produced a significant amount of interferon (IFN)-gamma after being transplanted into allogeneic recipients; however, IFN-gamma was not required for Th17 cell-induced GVHD. Further, we evaluated the pathogenesis of Th17 cells in GVHD by using polyclonal nonprimed CD4T cells in a clinically relevant allogeneic bone marrow transplantation (BMT) setting. We found that disruption of Th17-differentiation alone by targeting RORgammat (Th17-specific transcription factor) had no significant effect on GVHD development. We conclude that Th17 cells are sufficient but not necessary to induce GVHD.

The Pronounced Th17 Profile in Systemic Sclerosis (SSc) Together with Intracellular Expression of TGFβ and IFNγ Distinguishes SSc Phenotypes

BackgroundSystemic sclerosis (SSc) is an autoimmune disease where controversy on Th1/Th2 balance dominates. We investigated whether the recently discovered Th17 pattern was present in SSc.Methodology and Principal FindingsPatients were subdivided as having limited cutaneous SSc (lcSSc, n = 12) or diffuse cutaneous SSc (dcSSc, n = 24). A further arbitrary subdivision was made between early dcSSc (n = 11) and late dcSSc (n = 13) based upon the duration of disease. As a comparator group 14 healthy controls were studied. CD3+ cells were isolated using FACS and subsequently studied for the expression of CD4, CD8, CD25, CD45Ro, CD45Ra, IL-23, GITR, CD69 and intracellular expression of IL-17, TGFβ and IFNγ using flow cytometry. Levels of IL-17, IL-6, IL-1α and IL-23 were measured using Bioplex assays. SSc patients had more and more activated CD4+ cells. In addition, CD4, CD45Ro and CD45Ra cells from all SSc patients highly expressed the IL23R, which was associated with a higher IL-17 expression as well. In contrast, IFNγ and TGFβ were selectively up regulated in SSc subsets. In line with these observation, circulating levels of IL-17 inducing cytokines IL-6, IL-23 and IL-1α were increased in all or subsets of SSc patients.Conclusion and SignificanceThe combination of IL-17, IFNγ and TGFβ levels in CD45Ro and CD45Ra cells from SSc patients is useful to distinguish between lSSc, ldSSc or edSSc. Blocking Th17 inducing cytokines such as IL-6 and IL-23 may provide a useful tool to intervene in the progression of SSc.

In vivo activated monocytes from the site of inflammation in humans specifically promote Th17 responses.

Th17 cells are a recently defined subset of proinflammatory T cells that contribute to pathogen clearance and tissue inflammation by means of the production of their signature cytokine IL-17A (henceforth termed IL-17). Although the in vitro requirements for human Th17 development are reasonably well established, it is less clear what their in vivo requirements are. Here, we show that the production of IL-17 by human Th17 cells critically depends on both the activation status and the anatomical location of accessory cells. In vivo activated CD14+ monocytes were derived from the inflamed joints of patients with active rheumatoid arthritis (RA). These cells were found to spontaneously and specifically promote Th17, but not Th1 or Th2 responses, compared with resting CD14+ monocytes from the blood. Surprisingly, unlike Th17 stimulation by monocytes that were in vitro activated with lipopolysaccharide, intracellular IL-17 expression was induced by in vivo activated monocytes in a TNF-alpha- and IL-1beta-independent fashion. No role for IL-6 or IL-23 production by either in vitro or in vivo activated monocytes was found. Instead, in vivo activated monocytes promoted Th17 responses in a cell-contact dependent manner. We propose that, in humans, newly recruited memory CD4(+) T cells can be induced to produce IL-17 in nonlymphoid inflamed tissue after cell-cell interactions with activated monocytes. Our data also suggest that different pathways may be utilized for the generation of Th17 responses in situ depending on the site or route of accessory cell activation.

Intracellular IL-17 Expression in Peripheral Blood CD4+ and CD4- T Cells of HIV-Infected Individuals

Intracellular IL-17 Expression in Peripheral Blood CD4+ and CD4- T Cells of HIV-Infected Individuals