CXCR3-Expressing T Cells in Infections and Autoimmunity.

The chemokine receptor CXCR3 and its ligands (MIG/CXCL9, IP-10/CXCL10, and I-TAC/CXCL11) play a central role in the generation of cellular inflammation, both in the protective responses to invading pathogens, and in different pathological conditions associated with autoimmunity. It is worth noting that CXCR3 is highly expressed on innate and adaptive lymphocytes, as well as on various cell subsets that are localized in non-immune organs and tissues. Our review focuses exclusively on CXCR3-expressing T cells, including Th1, Th17.1, Tfh17, Tfh17.1, CXCR3+ Treg cells, and Tc1 CD8+ T cells. Currently, numerous studies have highlighted the role of CXCR3-dependent interactions in the coordination of inflammation in the peripheral tissues, both to increase recruitment of CD4+ and CD8+ T cells that upregulate inflammation, and also for recruitment of CXCR3+ T regulatory cells to dampen overexuberant responses. Understanding the role of CXCR3 and its ligands might help to apply them as new and effective therapeutic targets in a wide range of diseases.

Th17/1 and ex-Th17 cells are detected in patients with polyarticular juvenile arthritis and increase following treatment.

BACKGROUND: A better understanding of the pathogenesis of polyarticular juvenile idiopathic arthritis (polyJIA) is needed to aide in the development of data-driven approaches to guide selection between therapeutic options. One inflammatory pathway of interest is JAK-STAT signaling. STAT3 is a transcription factor critical to the differentiation of inflammatory T helper 17 cells (Th17s). Previous studies have demonstrated increased STAT3 activation in adult patients with rheumatoid arthritis, but less is known about STAT3 activation in polyJIA. We hypothesized that Th17 cells and STAT3 activation would be increased in treatment-naïve polyJIA patients compared to pediatric controls. METHODS: Blood from 17 patients with polyJIA was collected at initial diagnosis and again if remission was achieved (post-treatment). Pediatric healthy controls were also collected. Peripheral blood mononuclear cells were isolated and CD4 + T cell subsets and STAT activation (phosphorylation) were evaluated using flow cytometry. Data were analyzed using Mann-Whitney U and Wilcoxon matched-pairs signed rank tests. RESULTS: Treatment-naïve polyJIA patients had increased Th17 cells (CD3 + CD4 + interleukin(IL)-17 +) compared to controls (0.15% v 0.44%, p < 0.05), but Tregs (CD3 + CD4 + CD25 + FOXP3 +) from patients did not differ from controls. Changes in STAT3 phosphorylation in CD4 + T cells following ex vivo stimulation were not significantly different in patients compared to controls. We identified dual IL-17 + and interferon (IFN)γ + expressing CD4 + T cells in patients, but not controls. Further, both Th17/1 s (CCR6 + CD161 + IFNγ + IL-17 +) and ex-Th17s (CCR6 + CD161 + IFNγ + IL-17neg) were increased in patients' post-treatment (Th17/1: 0.3% v 0.07%, p < 0.05 and ex-Th17s: 2.3% v 1.4%, p < 0.05). The patients with the highest IL-17 expressing cells post-treatment remained therapy-bound. CONCLUSIONS: Patients with polyJIA have increased baseline Th17 cells, potentially reflecting higher tonic STAT3 activation in vivo. These quantifiable immune markers may identify patients that would benefit upfront from pathway-focused biologic therapies. Our data also suggest that inflammatory CD4 + T cell subsets not detected in controls but increased in post-treatment samples should be further evaluated as a tool to stratify patients in remission on medication. Future work will explore these proposed diagnostic and prognostic biomarkers.

Differential Runx3, Eomes, and T-bet expression subdivides MS-associated CD4+ T cells with brain-homing capacity.

Multiple sclerosis (MS) is a common and devastating chronic inflammatory disease of the CNS. CD4+ T cells are assumed to be the first to cross the blood-central nervous system (CNS) barrier and trigger local inflammation. Here, we explored how pathogenicity-associated effector programs define CD4+ T cell subsets with brain-homing ability in MS. Runx3- and Eomes-, but not T-bet-expressing CD4+ memory cells were diminished in the blood of MS patients. This decline reversed following natalizumab treatment and was supported by a Runx3+ Eomes+ T-bet- enrichment in cerebrospinal fluid samples of treatment-naïve MS patients. This transcription factor profile was associated with high granzyme K (GZMK) and CCR5 levels and was most prominent in Th17.1 cells (CCR6+ CXCR3+ CCR4-/dim ). Previously published CD28- CD4 T cells were characterized by a Runx3+ Eomes- T-bet+ phenotype that coincided with intermediate CCR5 and a higher granzyme B (GZMB) and perforin expression, indicating the presence of two separate subsets. Under steady-state conditions, granzyme Khigh Th17.1 cells spontaneously passed the blood-brain barrier in vitro. This was only found for other subsets including CD28- cells when using inflamed barriers. Altogether, CD4+ T cells contain small fractions with separate pathogenic features, of which Th17.1 seems to breach the blood-brain barrier as a possible early event in MS.

T helper (Th) cell profiles and cytokines/chemokines in characterization, treatment, and monitoring of autoimmune diseases.

Autoimmune diseases (AD) consist of a spectrum of disease entities whose etiologies are very complex and still not well understood. Every individual has the potential for developing AD under appropriate conditions because the body contains lymphocytes that are potentially reactive with self-antigens. The aims of this study are to (1) explore the flow cytometry method to identify the frequency of various circulating CD4+ T helper (Th) cell-subsets, including Th1, Th2, Th9, Th17, Th17.1, and Th22; (2) In parallel, to examine multiplex ELISA method for pathogenic inflammatory cytokines/chemokines, and (3) To assess the correlation of expression of T cell-subsets with serum cytokines/chemokines and understand its clinical importance with available AD treatments. We analyzed Th17, Th17.1, Th22, Th2, Th1, and Th9 Th cell populations and compared the concentrations of 67 cytokines/chemokines in healthy as well as AD-diagnosed patients. We observed that patients with autoimmune markers had significantly elevated percentages of naïve (Th17, Th22, and Th9) as well as memory (Th17 and Th22) Th cell-subsets, along with increased concentrations of cytokines/chemokines (Eotaxin, TNFß, and FABP4). The percentage of Th cell-subsets correlated positively or negatively with the production of cytokines/chemokines of patients diagnosed with AD. Our study demonstrates that the naïve and memory Th cell-subsets with positive correlations to cytokines/chemokines show new diagnostic markers to predict the patients' outcome, while the negative correlation of cytokines/chemokines shows the response to autoimmune therapies. Our findings of Th cell-subsets by flow cytometry and cytokines/chemokines by multiplex ELISA suggest that CCR6+ Th cell-subsets (Th17, Th17.1, Th22, and Th9) contribute to our understanding of the pathogenesis of AD and identify the new onset of AD from the autoimmune spectrum. Our findings highlight the importance of CCR6+ as a possible marker in the characterization, treatment, and monitoring of AD.

Pathogenic Th17 cells in autoimmunity with regard to rheumatoid arthritis.

Th17 cells contribute the pathobiology of autoimmune diseases, including rheumatoid arthritis (RA). However, it was shown that differentiated Th17 cells display a high degree of plasticity under the influence of inflammatory conditions. In some autoimmune diseases, the majority of Th17 cells, especially at sites of inflammation, have a phenotype that is intermediate between Th17 and Th1. These cells, which are described as Th17.1 or exTh17 cells, are hypothesized to be more pathogenic than classical Th17 cells. In this review, the involvement of Th17.1 lymphocytes in RA, and potential features that might render these cells to be more pathogenic are discussed.

Imbalanced distribution of regulatory T cells and Th17.1 cells in the peripheral blood and BALF of sarcoidosis patients: relationship to disease activity and the fibrotic radiographic phenotype.

Rationale: Sarcoidosis is a granulomatous interstitial lung disease involving a complex interplay among different cluster of differentiation 4 (CD4+) thymus cell (T-cell) subsets. Originally described as a type 1 T-helper (Th1) inflammatory disease, recent evidence suggests that both effector and regulatory T-cell subgroups play a critical role in sarcoidosis, but this remains controversial. Objectives: We aimed to investigate the distribution of CD4+ T-cell subpopulations in sarcoidosis patients and its potential associations with clinical disease activity and a radiographic fibrotic phenotype. Methods: We measured the frequencies of regulatory T cells (Tregs), Th1, Th17, and Th17.1 cells in the peripheral blood and/or bronchoalveolar lavage fluid (BALF) of 62 sarcoidosis patients, 66 idiopathic pulmonary fibrosis (IPF) patients, and 41 healthy volunteers using flow cytometry. We also measured the changes in these T-cell subpopulations in the blood at the follow-up visits of 11 sarcoidosis patients. Measurements and results: An increased percentage of Tregs was observed in the peripheral blood of sarcoidosis patients, with a positive association to disease activity and a fibrotic radiographic phenotype. We found a higher frequency of Tregs, a lower proportion of Th17.1 cells, and a lower ratio of Th17.1 cells to total Tregs in the peripheral blood of both active and fibrotic sarcoidosis patients, compared with IPF patients or healthy donors. In contrast, a lower frequency of Tregs and a higher proportion of Th17.1 cells was found in the BALF of sarcoidosis patients than in that of IPF patients. There was an imbalance of Tregs and Th17.1 cells between the peripheral blood and BALF in sarcoidosis patients. Following immunoregulatory therapy, the proportion of circulating Tregs in sarcoidosis patients decreased. Conclusion: A higher proportion of Tregs in the peripheral blood of sarcoidosis patients was related to disease activity, fibrotic phenotype, and the need for immunoregulatory therapy. The imbalanced distribution of Tregs and Th17.1 cells in patients' peripheral blood and BALF suggests that the lung microenvironment has an effect on the immunological pathogenesis of sarcoidosis. Therefore, further studies on the functional analysis of Tregs and Th17.1 cells in sarcoidosis patients are warranted.

SMURF1 inhibits the Th17 and Th17.1 polarization and improves the Treg/Th17 imbalance in systemic lupus erythematosus through the ubiquitination of RORγt.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease. This study aimed to investigate the role of SMAD specific E3 ubiquitin protein ligase 1 (SMURF1) in the Th17 and Th17.1 differentiation and Treg/Th17 imbalance, which are major factors contributing to the pathogenesis of SLE. SLE patients and healthy individuals were recruited to detect the SMURF1 levels in naïve CD4+ cells from peripheral blood. Purified and expanded naïve CD4+ T cells were employed to evaluate the effects of SMURF1 on Th17 and Th17.1 polarization in vitro. MRL/lpr lupus model was employed to explore the disease phenotype as well as Treg/Th17 balance in vivo. The results showed that SMURF1 was down-regulated in naïve CD4+ T cells in peripheral blood of patients with SLE and in spleen of MRL/lpr mice. SMURF1 overexpression suppressed the polarization of naïve CD4+ T cells toward Th17 and Th17.1 phenotype and down-regulated the expression of retinoid-related orphan receptor-gammat (RORγt). Subsequently, SMURF1 down-regulation aggravated the disease phenotype, inflammation, and the Treg/Th17 imbalance in MRL/lpr mice. Furthermore, we found that SMURF overexpression promoted the ubiquitination and decreases the stability of RORγt. In conclusion, SMURF1 inhibited the polarization of Th17 and Th17.1 cells and improved the Treg/Th17 imbalance in SLE, which was mediated as least partly by the ubiquitination of RORγt.

Looking Beyond Th17 Cells: A Role for Th17.1 Cells in Thyroid-associated Ophthalmopathy?

Thyroid-associated ophthalmopathy (TAO), an ordinary extrathyroid syndrome of Graves' disease (GD), is closely associated with immunity. T helper (Th) 17, Th1, and Th2 cells in Th lineages are thought to be related to the disease pathogenesis. Recently, there has been growing evidence that Th17.1 cells are involved in the development and progression of TAO. The characteristics of this pathology are similar to those of Th1 and Th17 lymphocytes, which secrete interferon (IFN)-γ and interleukin (IL)-17A. This paper reviews the potential role of the Th17.1 subgroup pathogenesis of TAO. The therapeutic effects of drugs that can modulate Th17.1 cell populations are also highlighted. Rich Th17.1 cells exist in peripheral blood and ocular tissues of patients suffering from thyroid eye disease (TED), especially those with severe or steroid-resistant TAO. The bias of Th17.1 cells to secrete cytokines partly determines the pathological outcome of TAO patients. Th17.1 cells are important in regulating fibrosis, adipocyte differentiation, and hyaluronic acid production. In summary, the Th17.1 subpopulation is essential in the onset and progression of TED, and targeting Th17.1 cell therapy may be a promising therapeutic approach.

From IL-17 to IFN-γ in inflammatory skin disorders: Is transdifferentiation a potential treatment target?

The targeted inhibition of effector cytokines such as interleukin 17 (IL-17) in psoriasis and IL-13 in atopic dermatitis offers impressive efficacy with a favorable side effect profile. In contrast, the downregulation of interferon gamma (IFN-γ) in T helper (Th) 1-dominant skin disorders may lead to more adverse events, given the crucial role of IFN-γ in antiviral and antitumoral immunity. Modulating Th17 and Th2 cell differentiation is performed by blocking IL-23 and IL-4, respectively, whereas anti-IL-12 antibodies are only moderately effective in downregulating Th1 lymphocyte differentiation. Therefore, a targeted approach of IFN-γ-driven disorders remains challenging. Recent literature suggests that certain pathogenic Th17 cell subsets with Th1 characteristics, such as CD4+CD161+CCR6+CXCR3+IL-17+IFN-y+ (Th17.1) and CD4+CD161+CCR6+CXCR3+IL-17-IFN-y+ (exTh17), are important contributors in Th1-mediated autoimmunity. Differentiation to a Th17.1 or exTh17 profile results in the upregulation of IFN-y. Remarkably, these pathogenic Th17 cell subsets are resistant to glucocorticoid therapy and the dampening effect of regulatory T cells (Treg). The identification of Th17.1/exTh17 cells in auto-immune disorders may explain the frequent treatment failure of conventional immunosuppressants. In this review, we summarize the current evidence regarding the cellular plasticity of Th17 cells in inflammatory skin disorders. A deeper understanding of this phenomenon may lead to better insights into the pathogenesis of various skin diseases and the discovery of a potential new treatment target.

Th17.1 lymphocytes: emerging players in the orchestra of immune-mediated inflammatory diseases.

It is now well established that Th17 lymphocytes associate with myriad immune-mediated inflammatory diseases. Over the past one and a half decades, a subset of Th17 lymphocytes viz. Th17.1 lymphocytes has been identified in pre-clinical and clinical models of inflammatory rheumatic diseases. These lymphocytes secrete IL-17A (signature cytokine of Th17 lymphocytes) as well as IFN-γ (the signature cytokine of Th1 lymphocytes). They express the chemokine markers for Th1 (CXCR3) as well as Th17 (CCR6) lymphocytes. Th17.1 lymphocytes also express the drug efflux protein p-glycoprotein, which associates with resistance to corticosteroids and other immunosuppressive drugs. This narrative review overviews the evidence regarding Th17.1 lymphocytes in different inflammatory rheumatic diseases. It is now recognized that Th17.1 lymphocytes are increased in the synovial fluid of affected joints in rheumatoid arthritis (RA) and associate with poor treatment response to abatacept. Th17.1 lymphocytes from synovial fluid of RA are less responsive to immunosuppression than those from the peripheral blood. In sarcoidosis, Th17.1 lymphocytes are concentrated in mediastinal lymph nodes and alveolar lining. Such Th17.1 lymphocytes in sarcoidosis are the predominant source of IFN-γ in the sarcoid lung. Th17.1 lymphocytes are elevated in lupus and Takayasu arteritis and associate with disease activity. Future studies should evaluate isolated Th17.1 lymphocytes from peripheral blood or sites of pathology such as synovial fluid and assess their modulation with immunosuppressive therapy in vitro. The analysis of gene expression signature of isolated Th17.1 lymphocytes might enable the identification of newer therapeutic strategies specifically targeting these cell populations in inflammatory rheumatic diseases. Key Points • Th17.1 lymphocytes are a subset of Th17 lymphocytes secreting both IFN-γ and IL-17 • Th17.1 lymphocytes drive neutrophilic inflammation, granuloma formation, and corticosteroid resistance • Th17.1 lymphocytes are elevated in rheumatoid arthritis and sarcoidosis at sites of inflammation • Increased circulating Th17.1 lymphocytes have been identified in lupus and Takayasu arteritis and associate with active disease.

Autoreactive memory Th17 cells are principally derived from T-bet+RORγt+ Th17/1 effectors.

Effector Th17 cells, including IFN-γ-IL-17+ (eTh17) and IFN-γ+IL-17+ (eTh17/1) subsets, play critical pathogenic functions in the induction of autoimmunity. As acute inflammation subsides, a small proportion of the effectors survive and convert to memory Th17 cells (mTh17), which sustain chronic inflammation in autoimmune diseases. Herein, we investigated the differential contributions of eTh17 versus eTh17/1 to the memory pool using an experimental model of ocular autoimmune disease. Our results show that adoptive transfer of Tbx21-/- CD4+ T cells or conditional deletion of Tbx21 in Th17 cells leads to diminished eTh17/1 in acute phase and functionally compromised mTh17 in chronic phase. Further, adoptive transfer of disease-specific eTh17/1, but not eTh17, leads to generation of mTh17 and sustained ocular inflammation. Collectively, our data demonstrate that T-bet-dependent eTh17/1 cells generated during the acute inflammation are the principal effector precursors of pathogenic mTh17 cells that sustain the chronicity of autoimmune inflammation.

Treatment With Cladribine Selects IFNγ+IL17+ T Cells in RRMS Patients - An In Vitro Study.

Background: Multiple sclerosis (MS) is an incurable autoimmune disease mediated by a heterogeneous T cell population (CD3+CD161+CXCR3-CCR6+IFNγ-IL17+, CD3+CXCR3+CCR6+IFNγ+IL17+, and CD3+CXCR3+IFNγ+IL17- phenotypes) that infiltrates the central nervous system, eliciting local inflammation, demyelination and neurodegeneration. Cladribine is a lymphocyte-depleting deoxyadenosine analogue recently introduced for MS therapy as a Disease Modifying Drug (DMD). Our aim was to establish a method for the early identification and prediction of cladribine responsiveness among MS patients. Methods: An experimental model was designed to study the cytotoxic and immunomodulatory effect of cladribine. T cell subsets of naïve relapsing-remitting MS (RRMS) patients were analyzed ex vivo and in vitro comparatively to healthy controls (HC). Surviving cells were stimulated with rh-interleukin-2 for up to 14days. Cell proliferation and immunophenotype changes were analyzed after maximal (phorbol myristate acetate/ionomycin/monensin) and physiological T-cell receptor (CD3/CD28) activation, using multiparametric flow cytometry and xMAP technology. Results: Ex vivo CD161+Th17 cells were increased in RRMS patients. Ex vivo to in vitro phenotype shifts included: decreased CD3+CCR6+ and CD3+CD161+ in all subjects and increased CD3+CXCR3+ in RRMS patients only; Th17.1 showed increased proliferation vs Th17 in all subjects; CD3+IL17+ and CD3+IFNγ+IL17+ continued to proliferate till day 14, CD3+IFNγ+ only till day 7. Regarding cladribine exposure: RRMS CD3+ cells were more resistant compared to HC; treated CD3+ cells proliferated continuously for up to 14 days, while untreated cells only up to 7 days; both HC/RRMS CD3+CXCR3+ populations increased from baseline till day 14; in RRMS patients vs HC, IL17 secretion from cladribine-treated cells increased significantly, in line with the observed proliferation of CD3+IL17+ and CD3+IFNγ+IL17+ cells; in both HC/RRMS, cladribine led to a significant increase in CD3+IFNγ+ cells at day 7 only, having no further effect at day14. IFNγ and IL17 secreted in culture media decreased significantly from ex vivo to in vitro. Conclusions: CD3+ subtypes showed different responsiveness due to selectivity of cladribine action, in most patients leading to in vitro survival/proliferation of lymphocyte subsets known as pathogenic in MS. This in vitro experimental model is a promising tool for the prediction of individual responsiveness of MS patients to cladribine and other DMDs.

The heterogeneous human memory CCR6+ T helper-17 populations differ in T-bet and cytokine expression but all activate synovial fibroblasts in an IFNγ-independent manner.

BACKGROUND: Chronic synovial inflammation is an important hallmark of inflammatory arthritis, but the cells and mechanisms involved are incompletely understood. Previously, we have shown that CCR6+ memory T-helper (memTh) cells and synovial fibroblasts (SF) activate each other in a pro-inflammatory feedforward loop, which potentially drives persistent synovial inflammation in inflammatory arthritis. However, the CCR6+ memTh cells are a heterogeneous population, containing Th17/Th22 and Th17.1 cells. Currently, it is unclear which of these subpopulations drive SF activation and how they should be targeted. In this study, we examined the individual contribution of these CCR6+ memTh subpopulations to SF activation and examined ways to regulate their function. METHODS: Th17/Th22 (CXCR3-CCR4+), Th17.1 (CXCR3+CCR4-), DP (CXCR3+CCR4+), and DN (CXCR3-CCR4-) CCR6+ memTh, cells sorted from PBMC of healthy donors or treatment-naïve early rheumatoid arthritis (RA) patients, were cocultured with SF from RA patients with or without anti-IL17A, anti-IFNγ, or 1,25(OH)2D3. Cultures were analyzed by RT-PCR, ELISA, or flow cytometry. RESULTS: Th17/Th22, Th17.1, DP, and DN cells equally express RORC but differ in production of TBX21 and cytokines like IL-17A and IFNγ. Despite these differences, all the individual CCR6+ memTh subpopulations, both from healthy individuals and RA patients, were more potent in activating SF than the classical Th1 cells. SF activation was partially inhibited by blocking IL-17A, but not by inhibiting IFNγ or TBX21. However, active vitamin D inhibited the pathogenicity of all subpopulations leading to suppression of SF activation. CONCLUSIONS: Human CCR6+ memTh cells contain several subpopulations that equally express RORC but differ in TBX21, IFNγ, and IL-17A expression. All individual Th17 subpopulations are more potent in activating SF than classical Th1 cells in an IFNγ-independent manner. Furthermore, our data suggest that IL-17A is not dominant in this T cell-SF activation loop but that a multiple T cell cytokine inhibitor, such as 1,25(OH)2D3, is able to suppress CCR6+ memTh subpopulation-driven SF activation.

The role of TH17 cells in multiple sclerosis: Therapeutic implications.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS) where immunopathology is thought to be mediated by myelin-reactive CD4+ T helper (TH) cells. The TH cells most commonly implicated in the pathogenesis of the disease are of TH1 and TH17 lineage, which are defined by the production of interferon-γ and interleukin-17, respectively. Moreover, there is emerging evidence for the involvement of TH17.1 cells, which share the hallmarks of TH1 and TH17 subsets. In this review, we summarise current knowledge about the potential role of TH17 subsets in the initiation and progression of the disease and put a focus on their response to approved immunomodulatory MS drugs. In this regard, TH17 cells are abundant in peripheral blood, cerebrospinal fluid and brain lesions of MS patients, and their counts and inflammatory mediators are further increased during relapses. Fingolimod and alemtuzumab induce a paramount decrease in central memory T cells, which harbour the majority of peripheral TH17 cells, while the efficacy of natalizumab, dimethyl fumarate and importantly hematopoietic stem cell therapy correlates with TH17.1 cell inhibition. Interestingly, also CD20 antibodies target highly inflammatory TH cells and hamper TH17 differentiation by IL-6 reductions. Moreover, recovery rates of TH cells best correlate with long-term efficacy after therapeutical immunodepletion. We conclude that central memory TH17.1 cells play a pivotal role in MS pathogenesis and they represent a major target of MS therapeutics.

T-bet Expression in Peripheral Th17.0 Cells Is Associated With Pulmonary Function Changes in Sarcoidosis.

Background: Interferon-gamma (IFN-γ) is a key mediator of sarcoidosis-related granulomatous inflammation. Previous findings of IFN-γ-producing Th17 cells in bronchoalveolar lavage fluid from sarcoidosis patients invokes the transition of Th17.0 cells to Th17.1 cells in the disease's pathogenesis. Since the T-bet transcription factor is crucial for this transition, the goal of this study was to determine if T-bet expression in Th17.0 cells reflects the extent of granulomatous inflammation in sarcoidosis patients as assessed by clinical outcomes. Methods: Using a case-control study design, we identified two groups of sarcoidosis subjects (total N = 43) with pulmonary function tests (PFTs) that either (1) changed (increased or decreased) longitudinally or (2) were stable. We used flow cytometry to measure the transcription factors T-bet and RORγt in Th1, Th17.0, and Th17.1 cell subsets defined by CCR6, CCR4 and CXCR3 in blood samples. We compared the percentages of T-bet+ cells in RORγt+Th17.0 cells (defined as CCR6+CCR4+CXCR3-) based on subjects' PFT group. We also assessed the relationship between the direction of change in PFTs with the changes in %T-bet+ frequencies using mixed effects modeling. Results: We found that T-bet expression in subjects' RORγt+Th17.0 cells varied based on clinical outcome. The T-bet+ percentage of RORγt+Th17.0 cells was higher in the cases (subject group with PFT changes) as compared to controls (stable group) (27 vs. 16%, p = 0.0040). In comparisons before and after subjects' PFT changes, the T-bet+ frequency of RORγt+Th17.0 cells increased or decreased in the opposite direction of the PFT change. The percentage of these T-bet+ cells was also higher in those with greater numbers of involved organs. Serum levels of interferon-γ-induced chemokines, CXCL9, CXCL10, and CXCL11, and whole blood gene expression of IFN-γ-related genes including GBP1, TAP1, and JAK2 were independently positively associated with the T-bet+ frequencies of RORγt+Th17.0 cells. Conclusions: These data suggest that expression of T-bet in Th17.0 cells could reflect the extent of granulomatous inflammation in sarcoidosis patients because they represent a transition state leading to the Th17.1 cell phenotype. These findings indicate that Th17 plasticity may be part of the disease paradigm.

Dexamethasone suppresses the Th17/1 cell polarization in the CD4+ T cells from patients with primary immune thrombocytopenia.

INTRODUCTION: Primary immune thrombocytopenia (ITP) is an acquired autoimmune disease with increased Th17 cells in peripheral blood. Th17/1 cells, which were recently characterized as a new differentiated Th17 lineage secreting IL-17 and IFN-γ, play an important role in the pathogenesis of multiple autoimmune diseases. In this study, we investigated whether Th17/1 cells are involved in the pathogenesis of ITP. MATERIALS AND METHODS: Peripheral blood was obtained from 44 ITP patients and 50 healthy controls. The percentages of T cell subsets were evaluated. We also detected molecular signature of Th17/1 cells in CD4+ T cells. Besides, CD4+ T cells from ITP patients were treated with dexamethasone, the inhibitor of NF-κB, or rapamycin to evaluate the impact and mechanism of dexamethasone treatment on Th17/1 cells. RESULTS: We found an elevated percentage and an enhanced specific molecular signature of Th17/1 cells in CD4+ T cells in ITP patients. The percentage of Th17/1 cells was correlated positively with Th17 cells in ITP patients and healthy controls. The percentage of Th17/1 cells was correlated with corticosteroid resistance. Dexamethasone reversed the molecular signature of Th17/1 cells and decreased the percentage of Th17/1 cells in vitro. Treatment of dexamethasone and the inhibitor of NF-κB suppressed the phosphorylation of STAT3, while dexamethasone treatment also inhibited the phosphorylation of NF-κB p65. CONCLUSIONS: Our data suggested Th17/1 cells may contribute to the pathogenesis of ITP and dexamethasone could inhibit Th17/1 cells through NF-κB/STAT3 pathway. These results may provide a potential therapeutic strategy of correcting the Th17/1 cell deviation in ITP.

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

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.

Th17 cells increase in RRMS as well as in SPMS, whereas various other phenotypes of Th17 increase in RRMS only.

BACKGROUND: The nature and extent of inflammation seen in multiple sclerosis (MS) varies throughout the course of the disease. Changes seen in CD4+ T-helper cells in relapsing-remitting (RR) MS and secondary progressive (SP) MS might differ qualitatively and/or quantitatively. OBJECTIVE: The objective of this paper is to study the frequencies of all major CD4+ T-helper subtypes - Th17, Th22 and Th1 lineage cells - in relapse, remission and secondary progression alongside CCR6 status, a chemokine receptor involved in migration of these cells into the central nervous system. METHODS: We compared 100 patients (50 RRMS and 50 SPMS) and 50 healthy volunteers and performed flow cytometric analysis of lymphocytes in blood samples. RESULTS: We demonstrated raised frequencies of various cell types along the Th17 axis; Th17, Th17.1 (IL-17+ interferon gamma+) and dual IL-17+ IL-22+ cells in RRMS. Th22 and CCR6+ Th1 cells (nonclassical Th1) were also increased in RRMS. All these cells were CCR6+. Only Th17 frequencies were elevated in SPMS. CONCLUSIONS: Increased frequencies of Th17 cells are implicated both in RRMS and SPMS. The CCR6 pathway includes Th17, Th22 and Th1 nonclassical cells, of which Th22 and Th1 cells represent the greatest subsets in MS.

αEß7 Integrin Identifies Subsets of Pro-Inflammatory Colonic CD4+ T Lymphocytes in Ulcerative Colitis.

BACKGROUND AND AIMS: The αEß7 integrin is crucial for retention of T lymphocytes at mucosal surfaces through its interaction with E-cadherin. Pathogenic or protective functions of these cells during human intestinal inflammation, such as ulcerative colitis [UC], have not previously been defined, with understanding largely derived from animal model data. Defining this phenotype in human samples is important for understanding UC pathogenesis and is of translational importance for therapeutic targeting of αEß7-E-cadherin interactions. METHODS: αEß7+ and αEß7- colonic T cell localization, inflammatory cytokine production and expression of regulatory T cell-associated markers were evaluated in cohorts of control subjects and patients with active UC by immunohistochemistry, flow cytometry and real-time PCR of FACS-purified cell populations. RESULTS: CD4+αEß7+ T lymphocytes from both healthy controls and UC patients had lower expression of regulatory T cell-associated genes, including FOXP3, IL-10, CTLA-4 and ICOS in comparison with CD4+αEß7- T lymphocytes. In UC, CD4+αEß7+ lymphocytes expressed higher levels of IFNγ and TNFα in comparison with CD4+αEß7- lymphocytes. Additionally the CD4+αEß7+ subset was enriched for Th17 cells and the recently described Th17/Th1 subset co-expressing both IL-17A and IFNγ, both of which were found at higher frequencies in UC compared to control. CONCLUSION: αEß7 integrin expression on human colonic CD4+ T cells was associated with increased production of pro-inflammatory Th1, Th17 and Th17/Th1 cytokines, with reduced expression of regulatory T cell-associated markers. These data suggest colonic CD4+αEß7+ T cells are pro-inflammatory and may play a role in UC pathobiology.

Follicular T Cells from smB- Common Variable Immunodeficiency Patients Are Skewed Toward a Th1 Phenotype.

Germinal center follicular T helper (GCTfh) cells are essential players in the differentiation of B cells. Circulating follicular T helper (cTfh) cells share phenotypic and functional properties with GCTfh cells. Distinct subpopulations of cTfh with different helper capabilities toward B cells can be identified: cTfh1 (CXCR3+CCR6-), cTfh2 (CXCR3-CCR6-), and cTfh17 (CXCR3-CCR6+). Alterations in cTfh function and/or distribution have been associated with autoimmunity, infectious diseases, and more recently, with several monogenic immunodeficiencies. Common variable immunodeficiency (CVID) disease is the commonest symptomatic primary immunodeficiency with a genetic cause identified in only 2-10% of patients. Although a heterogeneous disease, most patients show a characteristic defective B cell differentiation into memory B cells or antibody-secreting cells. We investigated if alterations in CVID cTfh cells frequency or distribution into cTfh1, cTfh2, and cTfh17 subpopulations and regulatory follicular T (Tfr) cells could be related to defects in CVID B cells. We found increased percentages of cTfh exhibiting higher programmed death-1 expression and altered subpopulations distribution in smB- CVID patients. In contrast to smB+ patients and controls, cTfh from smB- CVID patients show increased cTfh1 and decreased cTfh17 subpopulation percentages and increased CXCR3+CCR6+ cTfh, a population analogous to the recently described pathogenic Th17.1. Moreover, Tfr cells are remarkably decreased only in smB- CVID patients. In conclusion, increased cTfh17.1 and cTfh1/cTfh17 ratio in CVID patients could influence B cell fate in smB- CVID patients, with a more compromised B cell compartment, and the decrease in Tfr cells may lead to high risk of autoimmune conditions in CVID patients.