The discrepant effects of the characteristic secretory cytokines (IL-17/IFN-γ) of different Th17 cells phenotypes in rats with experimental periodontitis

2018 ◽  
Author(s):  
Jingyi Tan
Keyword(s):  
Th17 Cells ◽  
Ifn Γ ◽  
Experimental Periodontitis

Evidence for Associations Between Th1/Th17 “Hybrid” Phenotype and Altered Lipometabolism in Very Severe Graves Orbitopathy

2020 ◽  
Vol 105 (6) ◽  
pp. 1851-1867 ◽  
Author(s):  
Sijie Fang ◽  
Shuo Zhang ◽  
Yazhuo Huang ◽  
Yu Wu ◽  
Yi Lu ◽  
...  
Keyword(s):  
T Cell ◽  
Th17 Cell ◽  
Th17 Cells ◽  
T Cell Subsets ◽  
Th1 Cell ◽  
Effector T Cell ◽  
Cell Lineages ◽  
Graves Orbitopathy ◽  
Ifn Γ

Abstract Purpose The purpose of this article is to investigate the characteristics of Th1-cell and Th17-cell lineages for very severe Graves orbitopathy (GO) development. Methods Flow cytometry was performed with blood samples from GO and Graves disease (GD) patients and healthy controls, to explore effector T-cell phenotypes. Lipidomics was conducted with serum from very severe GO patients before and after glucocorticoid (GC) therapy. Immunohistochemistry and Western blotting were used to examine orbital-infiltrating Th17 cells or in vitro models of Th17 polarization. Results In GD, Th1 cells predominated in peripheral effector T-cell subsets, whereas in GO, Th17-cell lineage predominated. In moderate-to-severe GO, Th17.1 cells expressed retinoic acid receptor-related orphan receptor-γt (RORγt) independently and produced interleukin-17A (IL-17A), whereas in very severe GO, Th17.1 cells co-expressed RORγt and Tbet and produced interferon-γ (IFN-γ). Increased IFN-γ–producing Th17.1 cells positively correlated with GO activity and were associated with the development of very severe GO. Additionally, GC therapy inhibited both Th1-cell and Th17-cell lineages and modulated a lipid panel consisting of 79 serum metabolites. However, in GC-resistant, very severe GO, IFN-γ–producing Th17.1 cells remained at a high level, correlating with increased serum triglycerides. Further, retro-orbital tissues from GC-resistant, very severe GO were shown to be infiltrated by CXCR3+ Th17 cells expressing Tbet and STAT4 and rich in triglycerides that promoted Th1 phenotype in Th17 cells in vitro. Conclusions Our findings address the importance of Th17.1 cells in GO pathogenesis, possibly promoting our understanding of the association between Th17-cell plasticity and disease severity of GO.

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

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

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

scholarly journals T Cell Subsets and Associated Cytokines in Chronic Graft-Versus-Host-Disease Using G-CSF Primed Bone Marrow in Allogeneic Hematopoietic Stem Cell Transplantation

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4580-4580
Author(s):  
Monica M Rivera Franco ◽  
Eucario Leon Rodriguez ◽  
Diana Gomez Martin ◽  
Javier Merayo Chalico ◽  
Jorge Alcocer Varela
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Stem Cell ◽  
Th17 Cells ◽  
Mononuclear Cells ◽  
Chronic Gvhd ◽  
The Other ◽  
Hematopoietic Stem ◽  
Graft Versus Host ◽  
Ifn Γ

Abstract Background Graft versus host disease (GVHD) is the major complication of allogeneic hematopoietic stem cell transplantation. It is characterized by an imbalance between the effector and regulatory arms of the immune system which results in the over production of inflammatory cytokines. Regulatory T (T regs) cells and T helper 17 (Th17) cells are two recently described lymphocyte subsets with opposing actions. Both can develop from naïve CD4+ T cell precursors under the influence of TGFβ1. Th17 lymphocytes, are key effector cells in rodent models of human diseases including GVHD. The other subset, T regs, is essential for dominant immunologic tolerance. At our institution, patients transplanted using G-CSF primed bone marrow (G-BM), have a lower incidence of acute and chronic GVHD when compared to those transplanted with peripheral blood and not primed bone marrow. Some microenvironment characteristics of this hematopoietic stem cells (HSC) source remain unknown, as well as the difference between Tregs, Th17 and cytokine levels in patients who develop GVHD and those who do not. Objective To analyze the characteristics of thirty-eight G-BM donor samples, identifying lymphocytes subsets and associated cytokines, and comparing patients who developed chronic GVHD (cGVHD) and those who did not. Materials and Methods A prospective analysis was performed in 38 G-BM samples from donors from 1999 to 2016. Mononuclear cells were defrosted, counted, and viability was evaluated. A 24 hour resting with RPMI, and posterior activation with PMA (50 ng/ml) for 48 hours was performed. Cells were harvested and cytokines were evaluated by flow cytometry (CBA assay). From each sample, one million mononuclear cells were permeabilized, fixed, and stained with CD4-FITC, IL17A-PE, IFN-γ APC, and IL-4 PECy7, for their posterior phenotipication by flow cytometry. The samples were obtained in a BD LSR Fortessa cytometry, and analyzed with the Flow-Jo software. Patients (recipients) information was analyzed using SPSS v.21. Results GVHD incidence was reported as following: Three (8%) patients developed acute GVHD (2 grade II, and 1 grade IV), 11 patients (29%) developed chronic GVHD (9% extensive, and 91% limited), and 24 patients did not present either. Mononuclear cells from G-BM from donors of patients who developed cGVHD showed a pro inflammatory response, characterized by an increased concentration of IL-17A (15.5 vs 0.71 pg/mL, p=0.013), TNF-α (80.27 vs 0.13 pg/mL, p=0.001), and IL-6 (4953.6 vs 11.75 pg/mL, p=0.025), after a mitogenic stimulation, compared to cells from donors of patients who did not developed GVHD. On the other hand, a decreased IL-10 production (2.62 vs 52.81 pg/mL, p=0.001) was documented in mononuclear cells from donors of patients who developed chronic GVHD, compared to donor cells of patients who did not. No significant difference in the production of IL-2, IL-4, and IFN-γ was observed. There was no difference in Th1 and Th2 between both groups, but mononuclear cells from donors of patients who developed chronic GVHD had a higher percentage of Th17 (1.02% vs 0.46%, p<0.001), and less Tregs (0.88% vs 1.95%, p<0.001), compared to those who did not developed GVHD. Conclusions Patients who develop cGVHD (29%) are characterized by a pro inflammatory response with an increased production of IL-17A, IL-6, and IFN-γ, and also a major percentage of Th17 cells. Also, a decreased suppressive response was documented with reduced IL-10 and Tregs levels. The low incidence of cGVHD show that G-CSF primed bone marrow is an excellent source for allogeneic HSC transplantations, and would be useful to compare these results with other HSC sources. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sarcoidosis Th17 Cells are ESAT-6 Antigen Specific but Demonstrate Reduced IFN-γ Expression

2012 ◽  
Vol 33 (2) ◽  
pp. 446-455 ◽  
Author(s):  
Bradley W. Richmond ◽  
Kristen Ploetze ◽  
Joan Isom ◽  
Isfahan Chambers-Harris ◽  
Nicole A. Braun ◽  
...  
Keyword(s):  
Th17 Cells ◽  
Ifn Γ

scholarly journals IFN-γ–Expressing Th17 Cells Are Required for Development of Severe Ocular Surface Autoimmunity

2017 ◽  
Vol 199 (3) ◽  
pp. 1163-1169 ◽  
Author(s):  
Yihe Chen ◽  
Sunil K. Chauhan ◽  
Chunyi Shao ◽  
Masahiro Omoto ◽  
Takenori Inomata ◽  
...  
Keyword(s):  
Ocular Surface ◽  
Th17 Cells ◽  
Ifn Γ

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

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

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

IL-17A-Expressing CD4+ T Cells Must Acquire the Expression of T-Bet and IFN-γto Destroy Tumor Cells In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 468-468
Author(s):  
Pawel Muranski ◽  
Sid P Kerkar ◽  
Zachary A Borman ◽  
Robert Reger ◽  
Luis Sanchez-Perez ◽  
...  
Keyword(s):  
T Cells ◽  
Treatment Efficacy ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
End Effector ◽  
Polarized Cells ◽  
Ifn Γ

Abstract Abstract 468 We have recently demonstrated that Th17-polarized TCR transgenic CD4+ T cells specific for TRP-1 melanoma antigen are superior to Th1-polarized cells in mediating effective anti-tumor responses against advanced disease after adoptive transfer. The therapeutic activity of Th17-skewed cells is critically dependent on their ability to secrete IFN-γ, suggesting that the Th17 subset might evolve in vivo. However, the developmental program of Th17-polarized cells in vivo remains substantially un- elucidated. We developed a novel TCR-transduction technique that enabled us to rapidly confer specificity for a cognate antigen upon any population of T cells, regardless of its genetic background, its previous polarization history or its state of differentiation. Using adoptive transfers into tumor-bearing hosts, we were able to study the functionality of these genetically-engineered T cells in vivo. In vitro, CD4+ T cells cultured in type 17 conditions acquired end-effector phenotype (CD62Llow, CD45RBlow), but proliferated slower than cells grown in type 1 condition. Thus, we hypothesized that Th17-polarized cells might represent a less mature, more central-memory like subset. This notion was supported by their ability to secrete high quantities of IL-2 and higher expression of IL-7 receptor. In contrast, Th1-polarized cells upon in vitro re-stimulation upregulated PRDM1 that encodes BLIMP1, a molecule associated with the end-effector senescent phenotype. Moreover, Th1-skewed cells overexpressed caspase 3 and were prone to activation-induced cell death as measured by annexin V assay, while type 17 cells were resistant to apoptosis, and robustly expanded in secondary cultures. Using the TCR gene transfer technique we tested the treatment outcomes when Th17-polarized cells deficient for IL-17A were used. In contrast to wild-type (WT)-derived Th17 cells that effectively eradicated established tumors, we observed significant impairment of treatment with IL-17A-deficent cells. Similarly, we observed reduction in treatment efficacy when CCR6-deficient Th17 cells were transferred. CCR6 is a receptor for CCL20, a chemokine highly induced Th17 cells and thought to contribute to the trafficking of those cells to the site of inflammation. In both cases however, the addition of exogenous vaccination and IL-2 significantly improved treatment efficacy. Thus, we concluded that Th17-associated factors play the role in the anti-cancer activity of type 17 cells. To address the question whether plasticity of Th17-skewed effectors is important for their function upon ACT, we treated animals with TCR-transduced Th17-skewed cells derived from IFN-γ-deficient CD4+ cells as well as from t-bet-deficient mice, which are not able to develop type 1 responses. In contrast to WT-derived Th17 effectors, IFN-γ-deficient cells did not show any anti-tumor activity, while t-bet-deficient Th17 cells were able to mediate only minimal delay in tumor growth, suggesting that indeed the capacity to acquire Th1-like properties is essential for the anti-tumor function of Th17-skewed lymphocytes. Overall, here we demonstrate that TCR gene engineered Th17-polarized cells can efficiently treat advanced tumor. The high activity of in vitro-generated anti-tumor Th17 cells relies on the contribution of type 17-associated characteristics, including both the secretion of inflammatory factors IL-17A and CCL20, as well as the superior capacity to survive and expand upon the secondary stimulation. Importantly however, type 1-defining t-bet-mediated plasticity in the lineage commitment is required for the full therapeutic effect, underscoring the dualistic nature of Th17-skewed cells. Disclosures: No relevant conflicts of interest to declare.

Hyperactivable NFAT1 Ameliorates Autoimmune Encephalitis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 711-711
Author(s):  
Srimoyee Ghosh ◽  
Sergei B Koralov ◽  
Irena Stevanovic ◽  
Mark S Sundrud ◽  
Yoshiteru Sasaki ◽  
...  
Keyword(s):  
T Cells ◽  
Transgenic Mice ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Autoimmune Encephalitis ◽  
Cell Types ◽  
Wild Type ◽  
Ifn Γ

Abstract Abstract 711 Naïve CD4 T cells differentiate into diverse effector and regulatory subsets to coordinate the adaptive immune response. TH1 and TH2 effector subsets produce IFN-γ and IL-4, respectively, whereas proinflammatory TH17 cells are key regulators of autoimmune inflammation, characteristically produce IL-17 and IL-22 and differentiate in the presence of inflammatory cytokines like IL-6 and IL-21 together with TGF-β. Naive T cells can also differentiate into tissue-protective induced T regulatory (iTreg) cells. NFAT proteins are highly phosphorylated and reside in the cytoplasm of resting cells. Upon dephosphorylation by the Ca2+/calmodulin-dependent serine phosphatase calcineurin, NFAT proteins translocate to the nucleus, where they orchestrate developmental and activation programs in diverse cell types. In this study, we investigated the role of the Ca/NFAT signaling pathway in regulating T cell differentiation and the development of autoimmune diseases. We generated transgenic mice conditionally expressing a hyperactivable version of NFAT1 (AV-NFAT1) from the ROSA26 locus. To restrict AV-NFAT1 expression to the T cell compartment, ROSA26-AV-NFAT1 transgenic mice were bred to CD4-Cre transgenic mice. Naïve CD4 T cells freshly isolated from AV mice produced significantly less IL-2 but increased amounts of the inhibitory cytokine IL-10. To investigate the role of NFAT1 in the generation of TH1, TH2, Tregand TH17 cells, the respective cell types were generated from CD4 T cells of AV mice by in vitro differentiation. T cells from AV-NFAT1 mice exhibited a dysregulation of cytokine expression, producing more IFN-γ and less IL-4. While the numbers of CD4+CD25+ “natural” Treg cells in peripheral lymphoid organs and their in vitro suppressive functions were slightly decreased in AV mice, iTreg generation from CD4+CD25- T cells of AV mice as compared to wild type cells was markedly enhanced. Moreover, TH17 cells generated in vitro from CD4 T cells of AV mice in the presence of IL-6, IL-21 and TGF-β exhibited dramatically increased expression of both IL-10 and IL-17 as compared to wild type controls. To investigate putative NFAT binding sites in the IL-10 and IL-17 gene loci, we performed chromatin immunoprecipitation experiments. We show that NFAT1 can bind at the IL-17 locus at 3 out of 9 CNS regions which are accessible specifically during TH17 but not during TH1 and TH2 differentiation. Furthermore, we provide evidence that NFAT1 binds one CNS region in the IL10-locus in TH17 cells. To verify our observations in vivo, we induced experimental autoimmune encephalitis (EAE) in AV mice and wild type controls with the immunodominant myelin antigen MOG33-55 emulsified in complete Freund‘s adjuvant. While wild type animals showed a normal course of disease with development of tail and hind limb paralysis after approximately 10 days, AV mice showed a markedly weaker disease phenotype with less severe degrees of paralysis and accelerated kinetics of remission. Moreover at the peak of the response, there were fewer CD4+CD25- but more CD4+CD25+ T cells in the CNS of AV animals compared to wild type controls. Surprisingly, these cells produced significantly more IL-2, IL-17 and IFN-γ upon restimulation, even though they displayed decreased disease. In summary, our data provide strong evidence that NFAT1 contributes to the regulation of IL-10 and IL-17 expression in TH17 cells and show that increasing NFAT1 activity can ameliorate autoimmune encephalitis. This could occur in part through upregulation of IL-10 expression as observed in vitro, but is also likely to reflect increased infiltration of regulatory T cells into the CNS as well as increased conversion of conventional T cells into Foxp3+ regulatory T cells within the CNS. Disclosures: No relevant conflicts of interest to declare.

Functional Characterization of CD4+ T-Cells in Aplastic Anemia (AA)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1340-1340 ◽  
Author(s):  
Shahram Y Kordasti ◽  
Judith C. W. Marsh ◽  
Sufyan Al-Khan ◽  
Jie Jiang ◽  
Alexander E Smith ◽  
...  
Keyword(s):  
T Cells ◽  
Treatment Group ◽  
High Throughput ◽  
Cd4 T Cells ◽  
Th17 Cells ◽  
Absolute Number ◽  
Th2 Cells ◽  
Th1 Cells ◽  
Ifn Γ ◽  
Pre Treatment

Abstract Abstract 1340 We have examined the role of CD4+ T-cells in the pathogenesis of AA in 63 patients, 48 of whom were analyzed at diagnosis and 15 following immunosuppressive therapy (IST). Absolute numbers of CD4+ regulatory T cells (Tregs, defined as CD3+CD4+CD25highCD27+Foxp3+) were lower in pre-treatment AA patients compared to 10 healthy donors (HDs) (5.5 × 106 v 1.4 × 107)(p=0.01). In patients with severe (SAA) and very severe AA (VSAA), the absolute number and frequency of Tregs were lower than non-severe AA (NSAA) (4.4 × 106/L v 1 × 107/L)(p=0.01) and HDs (4.4 × 106/L v 3 × 107/L) (p<0.001). Absolute numbers of Th1 and Th2 cells in all pre-treatment patients were higher compared to HDs (6.4 × 107/L v 1.8 × 107/L)(p=0.03) for Th1 and (2.6 × 107/L v 2.4 × 106/L)(p=0.006) Th2 cells. Although mean percentages of AA Th17 cells were higher than in HDs (1.5% v 0.15%)(p=0.04), differences in absolute numbers were not significant. Absolute numbers of Th2 and Th17 cells were increased in SAA (1.3 × 107/L v 7.4 × 106/L for Th2)(p=0.01) compared to NSAA (5.7 × 106/L v 2.15 × 106/L for Th17)(p=0.02). Ratios of Th1/Tregs (p=0.003), Th2/Tregs (p=0.02), and Th17/Tregs (p=0.001) were higher in SAA and VSAA compared to NSAA. Percentage of both activated (CD4+CD45RA−CD25highFoxp3high) and resting (CD4+CD45RA+ CD25highFoxp3low) Tregs was decreased in AA patients, compared to HDs (p=0.004 and p=0.01), whereas cytokine secreting Tregs (CD4+CD45RA−CD25high Foxp3low) were increased in AA (p<0.003). Sorted Tregs from AA patients did not suppress cytokine secretion by autologous or HD T effectors (Te) cells in 1:1 co-cultures, whereas IL-2 and IFN-γ secretion by AA Te (CD4+CD25lowCD127high) was suppressible by allogeneic Tregs from HDs, confirming Tregs dysfunction. AA Tregs did not inhibit either CD154 or CD69 expression on Te cells. Tregs from AA patients secreted significantly more IFN-γ, TNF-α and IL-17 (p=0.02, p=0.02 and p=0.01, respectively) after 4 hours stimulation with PMA/Ionomycine compared to HDs. Expression levels of FoxP3, ROR□c and T-bet in AA Tregs was normal. IFN-γ secreting cells (Th1) were enriched using enrichment kit then further enriched by FACS sorting. CDR3 region products of TCR Vβ-chain were amplified using Vβ specific forward and Cβ reverse primers. CDR3 PCR products from AA patients and HDs were subjected 454 sequencing (Roche GS FLX titanium). Sequencing was performed to yield an average ‘depth’ in excess of 1000 clonally reads (1000x) for each sample specific CDR3 PCR amp icon. Reads were processed using Roche Amp icon Variant Analyzer software (AVA). Diversity of TCR receptors (measured by spectratyping and confirmed by high throughput deep sequencing) in AA Th1 cells was lower than HDs (p=0.037), as shown by the percentage and number of consensus clusters in total sequence reads. Interestingly, percentages of the most dominant CDR3 clones, revealed by high throughput sequencing, were higher in AA compared to HDs, regardless of spectratyping pattern. Global gene expression of Tregs was compared in 3 pre-IST AA patients and 5 HDs. A unique gene signature consisting of 86 genes that were significant was identified. There were 8 down regulated genes (fold change) in the pre-treatment group; PIN4 (−4.1), OR2T12 (−3.3), AMAC1 (−2.73), PERP (−2.69), UTS2 (−2.27), RNF139 (−2.13), COMMD9 (−2.09) and LOC100128356 (−2.01). The top 10 of 78 up-regulated genes in the pre-treatment group were HBB (19.5), PSME2 (13.8), CSDA (13.07), FAM127A (7.78), EXOSC1 (7.73), BPGM (7.43), CYSLTR1 (7.17), CHPT1 (6.96) and PLAC8 (6.71). qPCR analysis for CSDA, HBB, PSMiE2, PERP, PIN4, and UTS2 confirmed a similar trend to the microarray results. Interestingly absolute number of Tregs, and Th2/Treg ratio were higher in 10 IST responsive patients compared to 5 non-responsive patients (p=0.005 and 0.02, respectively). We show that expansion of Th1, Th2, Th17, and decreased/skewed Tregs immunophenotype and function are a consistent and defining feature of SAA and VSAA. Clonal expansion of Th1 cells is likely to be antigen driven and the presence of dysfunctional Tregs aggravates this autoimmune response. Increases of Tregs, and Th2/Treg ratios following IST predicts a favourable response to this treatment. Disclosures: No relevant conflicts of interest to declare.

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