Caroline E. Broos

IFN-γ–Producing T-Helper 17.1 Cells Are Increased in Sarcoidosis and Are More Prevalent than T-Helper Type 1 Cells

RATIONALE Pulmonary sarcoidosis is classically defined by T-helper (Th) cell type 1 inflammation (e.g., IFN-γ production by CD4(+) effector T cells). Recently, IL-17A-secreting cells have been found in lung lavage, invoking Th17 immunity in sarcoidosis. Studies also identified IL-17A-secreting cells that expressed IFN-γ, but their abundance as a percentage of total CD4(+) cells was either low or undetermined. OBJECTIVES Based on evidence that Th17 cells can be polarized to Th17.1 cells to produce only IFN-γ, our goal was to determine whether Th17.1 cells are a prominent source of IFN-γ in sarcoidosis. METHODS We developed a single-cell approach to define and isolate major Th-cell subsets using combinations of chemokine receptors and fluorescence-activated cell sorting. We subsequently confirmed the accuracy of subset enrichment by measuring cytokine production. MEASUREMENTS AND MAIN RESULTS Discrimination between Th17 and Th17.1 cells revealed very high percentages of Th17.1 cells in lung lavage in sarcoidosis compared with controls in two separate cohorts. No differences in Th17 or Th1 lavage cells were found compared with controls. Lung lavage Th17.1-cell percentages were also higher than Th1-cell percentages, and approximately 60% of Th17.1-enriched cells produced only IFN-γ. CONCLUSIONS Combined use of surface markers and functional assays to study CD4(+) T cells in sarcoidosis revealed a marked expansion of Th17.1 cells that only produce IFN-γ. These results suggest that Th17.1 cells could be misclassified as Th1 cells and may be the predominant producer of IFN-γ in pulmonary sarcoidosis, challenging the Th1 paradigm of pathogenesis.

Granuloma formation in pulmonary sarcoidosis

Sarcoidosis is a granulomatous disorder of unknown cause, affecting multiple organs, but mainly the lungs. The exact order of immunological events remains obscure. Reviewing current literature, combined with careful clinical observations, we propose a model for granuloma formation in pulmonary sarcoidosis. A tight collaboration between macrophages, dendritic cells, and lymphocyte subsets, initiates the first steps toward granuloma formation, orchestrated by cytokines and chemokines. In a substantial part of pulmonary sarcoidosis patients, granuloma formation becomes an on-going process, leading to debilitating disease, and sometimes death. The immunological response, determining granuloma sustainment is not well understood. An impaired immunosuppressive function of regulatory T cells has been suggested to contribute to the exaggerated response. Interestingly, therapeutical agents commonly used in sarcoidosis, such as glucocorticosteroids and anti-TNF agents, interfere with granuloma integrity and restore the immune homeostasis in autoimmune disorders. Increasing insight into their mechanisms of action may contribute to the search for new therapeutical targets in pulmonary sarcoidosis.

Impaired survival of regulatory T cells in pulmonary sarcoidosis

BackgroundImpaired regulatory T cell (Treg) function is thought to contribute to ongoing inflammatory responses in sarcoidosis, but underlying mechanisms remain unclear. Moreover, it is not known if increased apoptotic susceptibility of Tregs may contribute to an impaired immunosuppressive function in sarcoidosis. Therefore, the aim of this study is to analyze proportions, phenotype, survival, and apoptotic susceptibility of Tregs in sarcoidosis.MethodsPatients with pulmonary sarcoidosis (n = 58) were included at time of diagnosis. Tregs were analyzed in broncho-alveolar lavage fluid and peripheral blood of patients and healthy controls (HC).ResultsIn sarcoidosis patients no evidence was found for a relative deficit of Tregs, neither locally nor systemically. Rather, increased proportions of circulating Tregs were observed, most prominently in patients developing chronic disease. Sarcoidosis circulating Tregs displayed adequate expression of FoxP3, CD25 and CTLA4. Remarkably, in sarcoidosis enhanced CD95 expression on circulating activated CD45RO+ Tregs was observed compared with HC, and proportions of these cells were significantly increased. Specifically sarcoidosis Tregs - but not Th cells - showed impaired survival compared with HC. Finally, CD95L-mediated apoptosis was enhanced in sarcoidosis Tregs.ConclusionIn untreated patients with active pulmonary sarcoidosis, Tregs show impaired survival and enhanced apoptotic susceptibility towards CD95L. Increased apoptosis likely contributes to the insufficient immunosuppressive function of sarcoidosis Tregs. Further research into this field will help determine whether improvement of Treg survival holds a promising new therapeutic approach for chronic sarcoidosis patients.

Decreased Cytotoxic T-Lymphocyte Antigen 4 Expression on Regulatory T Cells and Th17 Cells in Sarcoidosis: Double Trouble?

Cytotoxic T-lymphocyte antigen 4 (CTLA4) is an important suppressor of T-cell–mediated immune responses and has become a major target in tumor therapy (1). Intriguingly, various case reports on initiation or exacerbation of sarcoidosis during anti-CTLA4 treatment of metastatic melanoma have recently emerged. This phenomenon is considered biotherapy-induced sarcoidosis, as in all reported cases, granuloma lesions occurred and progressed during biotherapy employment and disappeared after drug discontinuation (2). In patients with metastatic melanoma, CTLA4 blockade increased circulating proportions and absolute numbers of IL-17–producing CD4 T cells, whereas it only marginally changed Th1 or Th2 cell percentages (3). Although the effect on regulatory T cell (Treg) proportions remains a matter of debate, CTLA4 blockade was found to impair Treg-mediated suppression (1). Moreover, in both mice and humans, CTLA4 deletion, blockade, or dysfunction is strongly associated with the development of autoimmune lesions (1), in which Tregs and Th17 cells are known to play a pivotal role. Because sarcoidosis is characterized by an abnormal Th1/Th17 cell response and impaired Treg function (4), it is conceivable that CTLA4 blockade exacerbates underlying or preexistent disease. These clinical observations provide a rationale to examine CTLA4 expression on CD4 T cells in sarcoidosis in lung-draining mediastinal lymph nodes (MLN), which are an important site of primary and memory T-cell activation by dendritic cells. We hypothesize that altered CTLA4 expression contributes to ongoing Th1/Th17 cell responses in sarcoidosis. Using flow cytometry, we determined CTLA4 expression on CD4 T-cell subsets (for gating strategy, see Figure E1 in the online supplement) in MLN from patients with sarcoidosis (n = 18) and controls (n = 24) (for methods, seeMATERIALS AND METHODS in the online supplement and Table E1). Activated T cells from sarcoidosis MLN showed decreased CTLA4 expression compared with control MLN (Figure 1A). CTLA4 expression was also decreased on nonactivated memory T cells but normal on naive T cells (Figure E2). Evaluating Th cell subsets showed a significant decrease in CTLA4 expression specifically on sarcoidosis Th17 cells (P = 0.004) but not on Th1 or Th2 cells (Figures 1B and 1C). Furthermore, proportions of Th17 cells were significantly increased in sarcoidosis MLN (Figure 1D), consistent with our previous finding of enhanced Th17 cell proportions in peripheral blood and lungs of patients (5). Notably, not only Th17 cells but also activated Tregs showed decreased CTLA4 expression in sarcoidosis MLN compared with controls (P = 0.016) (Figures 1B and 1C). Treg proportions were not increased (Figure 1E). Intriguingly, as CTLA4 protein expression is critical for Treg function (6) and Th17 cells are uniquely sensitive toward CTLA4 co-inhibition (7), impaired CTLA4 expression on these specific subsets contributes to Th17 cell differentiation and activation and to ongoing Th(17) cell proliferation. Although there is evidence that sarcoidosis MLN parallel the lungs in terms of CD4 T-cell (8) and Th17-cell (this manuscript) influx, we also determined CTLA4 expression on T cells in bronchoalveolar lavage fluid. Importantly, similar to in sarcoidosis MLN, sarcoidosis bronchoalveolar lavage fluid memory Th cells showed significantly decreased CTLA4 expression compared with disease controls (P, 0.0001) (Figure E3). CTLA4 expression depends on the strength of T-cell stimulation, mediated by the T-cell receptor and CD28 expression (9). Decreased CTLA4 expression on CD4 T cells in sarcoidosis can therefore be a consequence of ongoing inflammation, leading to loss of CD28 expression, as described in chronic beryllium disease (10). However, we only found a slight, nonsignificant decrease of CD28 expression on sarcoidosis Th17 cells compared with controls (Figure 1F). Beryllium-responsive CD4 T cells in the lung up-regulated programmed death (PD)-1, reflecting T-cell exhaustion in response to chronic antigenic (beryllium) exposure (10). We did not find increased PD-1 expression on sarcoidosis Th17 cells (Figure 1G). Taken together, our findings do not support T-cell differentiation status or exhaustion as an explanation for low CTLA4 levels but do suggest a specific defect in CTLA4 expression instead. It is conceivable that genetic changes could underlie impaired CTLA4 protein expression in sarcoidosis. Genetic variations in the CTLA4 gene are associated with various autoimmune disorders (1), and CTLA4 gene mutations have been identified in families with common variable immunodeficiency, including patients with granulomatous lung disease (6). These mutations led to impaired CTLA4 up-regulation in activated Tregs, indicating that two functional CTLA4 alleles are necessary to drive high protein levels (6). We found up-regulation of CTLA4 on sarcoidosis activated Tregs compared with Th1, Th2, or Th17 cells (Figure 1C). Paired analyses showed that this was significant (P = 0.001). Furthermore, CTLA4 gene polymorphisms in sarcoidosis were not reported to be associated with disease susceptibility but merely with the extent of disease (11). Taken together, it is unlikely that genetic mutations within the CTLA gene impair CTLA4 protein expression in sarcoidosis. The mechanisms that regulate CTLA4 expression on individual T-cell subsets remain largely unclear to date. The cause of impaired CTLA4 expression in sarcoidosis Tregs and Th17 cells may be T-cell nonintrinsic and depend on the local inflammatory microenvironment. Characterizing cytokine profiles in sarcoidosis MLNmight help unravel further causes of diminished CTLA4 expression. In summary, we are the first to demonstrate a significant decrease in CTLA4 expression specifically on Tregs and Th17 cells at an important site of T-cell activation in sarcoidosis. Because Th17 cell, and not Treg, proportions were increased, impaired CTLA4 expression in sarcoidosis could influence the delicate Th cell/Treg M.K. was supported by a FP7 Marie Curie CIG grant.

T-cell immunology in sarcoidosis: Disruption of a delicate balance between helper and regulatory T-cells.

Purpose of review Although the aetiology of sarcoidosis is not yet completely understood, immunological changes within the T-cell compartment are characteristic for an exaggerated antigen-driven immune response. In this review, we describe the most recent findings on T-cell subset responses and regulation in sarcoidosis. We discuss how future immunological research can advance the field to unravel pathobiological mechanisms of this intriguingly complex disease. Recent findings Research into the field of T-cell plasticity has recently challenged the long-held T helper type 1 (Th1) paradigm in sarcoidosis and striking parallels with autoimmune disorders and common variable immunodeficiency were recognized. For instance, it was demonstrated that Th17.1-cells rather than Th1-cells are responsible for the exaggerated IFN-&ggr; production in pulmonary sarcoidosis. Furthermore, impaired regulatory T-cell function and alterations within the expression of co-inhibitory receptors that control T-cell responses, such as PD-1, CTLA-4 and BTNL2, raise new questions regarding T-cell regulation in pulmonary sarcoidosis. Summary It becomes increasingly clear that Th17(.1)-cells and regulatory T-cells are key players in sarcoidosis T-cell immunology. New findings on plasticity and co-inhibitory receptor expression by these subsets help build a more comprehensive model for T-cell regulation in sarcoidosis and will finally shed light on the potential of new treatment modalities.

Increased T-helper 17.1 cells in sarcoidosis mediastinal lymph nodes

The lung-draining mediastinal lymph nodes (MLNs) are currently widely used to diagnose sarcoidosis. We previously reported that T-helper (Th) 17.1 cells are responsible for the exaggerated interferon-γ production in sarcoidosis lungs. In this study, we aimed to investigate 1) whether Th17.1 cells are also increased in the MLNs of sarcoidosis patients and 2) whether frequencies of the Th17.1 cells at diagnosis may correlate with disease progression. MLN cells from treatment-naive pulmonary sarcoidosis patients (n=17) and healthy controls (n=22) and peripheral blood mononuclear cells (n=34) and bronchoalveolar lavage fluid (BALF) (n=36) from sarcoidosis patients were examined for CD4+ T-cell subset proportions using flow cytometry. Higher proportions of Th17.1 cells were detected in sarcoidosis MLNs than in control MLNs. Higher Th17.1 cell proportions were found in sarcoidosis BALF compared with MLNs and peripheral blood. Furthermore, BALF Th17.1 cell proportions were significantly higher in patients developing chronic disease than in patients undergoing resolution within 2 years of clinical follow-up. These data suggest that Th17.1 cell proportions in pulmonary sarcoidosis can be evaluated as a diagnostic and/or prognostic marker in clinical practice and could serve as a new therapeutic target. Th17-lineage cells are increased in sarcoidosis MLNs and BALF Th17.1 cell proportions correlate with disease progression http://ow.ly/hBb730hSEv7

Daily home spirometry to detect early steroid treatment effects in newly treated pulmonary sarcoidosis

Prednisone is the mainstay of sarcoidosis treatment. However, prednisone treatment optimisation is warranted, since prolonged high-dose prednisone therapy is associated with burdensome and harmful side-effects [1, 2]. Early prednisone dose tapering has the potential to reduce side-effects. Gaining insight in the early treatment response can help to determine when tapering could be initiated. To date, there are no prospective studies that look at early treatment response to prednisone in sarcoidosis by monitoring clinical symptoms and daily patient-administered lung function. Therefore, we initiated a multicentre, prospective and observational study with daily home spirometry to detect early steroid treatment effects in newly treated pulmonary sarcoidosis (Dutch National Trial Register NTR4328; www.trialregister.nl/trialreg). The major treatment effect of prednisone on FVC is reached within 2 to 3 weeks in newly treated sarcoidosis patients http://ow.ly/3A3E30h5ZuT

Th17-lineage cells in pulmonary sarcoidosis and Löfgren's syndrome: Friend or foe?

Sarcoidosis, a multisystem granulomatous disorder, has historically been classified as Th1-driven disease. However, increasing data demonstrate a key role of Th17-cell plasticity in granuloma formation and maintenance. In Löfgrens syndrome (LS), an acute and distinct phenotype of sarcoidosis with a favorable outcome, differences in Th17-lineage cell subsets, cytokine expression and T-cell suppressive mechanisms may account for differences in clinical presentation as well as prognosis compared to non-LS sarcoidosis. In contrast with LS, up to 20% of non-LS sarcoidosis patients may progress to irreversible pulmonary fibrosis. In non-LS sarcoidosis patients, IFN-γ-producing Th17.1-cells appear to be more pathogenic and possibly linked to disease progression, while a broader range of cytokines is found in bronchoalveolar lavage fluid (BALF) in LS patients. Differences in Cytotoxic T-lymphocyte antigen 4 (CTLA-4) expression on Th17-cells and regulatory T-cells (Treg) could contribute to Th17-cell pathogenicity and consequently to either disease resolution or ongoing inflammation in sarcoidosis. Furthermore, several genes and SNPs are associated with disease susceptibility and outcome in sarcoidosis, the majority of which are involved in antigen presentation, T-cell activation or regulation of T-cell survival. Novel insights into the role of Th17-cells in the pathogenesis of both LS and non-LS sarcoidosis will unravel pathogenic and benign Th17-lineage cell function and drivers of Th17-cell plasticity. This will also help identify new treatment strategies for LS and non-LS sarcoidosis patients by altering Th17-cell activation, suppress conversion into more pathogenic subtypes, or influence cytokine signaling towards a beneficial signature of Th17-lineage cells. In this review, we summarize new insights into Th17-cell plasticity in the complex pathogenesis of sarcoidosis and connect these cells to the different disease phenotypes, discuss the role of genetic susceptibility and autoimmunity and focus on possible treatment strategies.

Specific activation of non-classical Th1 cells in pulmonary sarcoidosis

Rationale: Sarcoidosis (SRC) is characterized by an exaggerated T helper(Th)1/Th17 response. Increased proportions of IL17A + /IFN-γ + double-positive T cells are present in blood and lungs of active SRC patients. These non-classical (NC) Th1 cells are thought to be highly pathogenic and to derive from Th17 cells, but their contribution to the pathogenesis of SRC remains unclear. Objective: To determine T cell subset activity in SRC-affected organs. Methods: Using flow cytometry, T cell subsets (CCR6 - CXCR3 + Th1, CCR6 + CCR4 + Th17 and CCR6 + CXCR3 + NC Th1) were quantified within bronchoalvolar lavage (BAL) and mediastinal lymph nodes (MLN) of 20 active SRC patients and controls. Healthy control (HC)MLN were collected from 10 transplantation donors. Results: SRC MLN contained increased proportions of Th17 and NC Th1, but not Th1 cells, compared with HC. Both Th17 and NC Th1 cells were increased activated and proliferative in SRC MLN compared with HC. However, only Th17 cells showed decreased expression of the co-inhibitory receptor CTLA4. Proportions NC Th1 cells were increased in SRC BAL compared with MLN, whereas Th17 cells were decreased, and Th1 cells remained stable. In SRC BAL NC Th1 cells were the most prominent population and highly activated compared with Th1 and Th17 cells. Conclusions: These data show that NC Th1, and not 9classical9 Th1 cells, are most prominently present and activated in SRC-affected organs. As specifically Th17 cells showed reduced expression of the co-inhibitory marker CTLA4 and were highly proliferative, it is conceivable that they can shift towards pathological IL17A + /IFN-γ + NC Th1 cells, indicating that SRC is predominantly a Th17-mediated disease.