Michael Mingueneau

Identification of Novel CD4+ T Cell Subsets in the Target Tissue of Sjögren’s Syndrome and Their Differential Regulation by the Lymphotoxin/LIGHT Signaling Axis

Despite being one of the most common rheumatologic diseases, there is still no disease-modifying drug for primary Sjögren’s syndrome (pSS). Advancing our knowledge of the target tissue has been limited by the low dimensionality of histology techniques and the small size of human salivary gland biopsies. In this study, we took advantage of a molecularly validated mouse model of pSS to characterize tissue-infiltrating CD4+ T cells and their regulation by the lymphotoxin/LIGHT signaling axis. Novel cell subsets were identified by combining highly dimensional flow and mass cytometry with transcriptomic analyses. Pharmacologic modulation of the LTβR signaling pathway was achieved by treating mice with LTβR-Ig, a therapeutic intervention currently being tested in pSS patients (Baminercept trial NCT01552681). Using these approaches, we identified two novel CD4+ T cell subsets characterized by high levels of PD1: Prdm1+ effector regulatory T cells expressing immunoregulatory factors, such as Il10, Areg, Fgl2, and Itgb8, and Il21+ effector conventional T cells expressing a pathogenic transcriptional signature. Mirroring these observations in mice, large numbers of CD4+PD1+ T cells were detected in salivary glands from Sjögren’s patients but not in normal salivary glands or kidney biopsies from lupus nephritis patients. Unexpectedly, LTβR-Ig selectively halted the recruitment of PD1− naive, but not PD1+, effector T cells to the target tissue, leaving the cells with pathogenic potential unaffected. Altogether, this study revealed new cellular players in pSS pathogenesis, their transcriptional signatures, and differential dependency on the lymphotoxin/LIGHT signaling axis that help to interpret the negative results of the Baminercept trial and will guide future therapeutic interventions.

Clinical relevance of RORγ positive and negative subsets of CD161+CD4+ T cells in primary Sjögren’s syndrome

Objective. The relevance of the Th17 pathway in primary SS (pSS) is unclear. Published studies have relied on restimulating circulating CD161+ T cells in vitro for quantitation of IL-17–producing cells. While CD161 marks all IL-17+ T cells, it is also expressed by other Th subsets. The aim of this study was to directly analyse retinoic acid receptor–related orphan nuclear receptor (ROR)−&ggr; expressing and non-expressing subsets of CD161+ T cells to determine the relevance of the Th17 pathway in pSS. Methods. We quantitated the frequencies of both CD161- and ROR&ggr;-expressing T cells by comparative flow cytometry in peripheral blood mononuclear cells from a well-stratified cohort of pSS patients and control subjects. We also analysed the expression of antigen D–related HLA (HLA-DR) and CD161 in labial salivary glands from nine subjects undergoing a diagnostic biopsy. Results. While the frequencies of both ROR&ggr;+ and ROR&ggr;− subsets of CD161+ CD4+ T cells were increased in peripheral blood from pSS patients, the increase in the ROR&ggr;+ subset positively correlated with humoral manifestations of the disease (anti-SSA/SSB autoantibodies and hypergammaglobulinaemia), but not with disease activity, and vice versa for the ROR&ggr;− subset. An increased frequency of HLA-DR+ CD161+CD4+ T cells was observed in labial salivary gland biopsies from pSS patients, suggesting chronic activation of CD161+CD4+ T cells in the target tissue of the disease. Conclusion. In addition to pointing to CD161 as a marker of a pathogenic subset of CD4+ T cells in pSS patients, our data indicate that even though the ROR&ggr;+ (Th17) CD161+ subset might contribute to humoral manifestations of the disease, the ROR&ggr;− (non-Th17) CD161+ subset is the one associated with disease activity in pSS patients.

BAFF overexpression increases lymphocytic infiltration in Sjögren's target tissue, but only inefficiently promotes ectopic B-cell differentiation

B-cell activating factor (BAFF) levels are increased in rheumatoid arthritis, lupus and primary Sjögrens syndrome (pSS). However, BAFF contribution to pathogenesis is not completely understood. In pSS, immune infiltration of the salivary and lacrimal glands leads to xerostomia and xerophtalmia. Glandular B cell hyperactivation, differentiation into germinal center (GC)-like structures and plasma cell accumulation are histopathological hallmarks that were attributed to increased BAFF. Here, we experimentally tested this hypothesis by overexpressing BAFF in a mouse model of pSS. BAFF overexpression enhanced lymphocytic infiltration and MHCII expression on B cells. Increased BAFF also induced B cell differentiation into GC B cells within the autoimmune target tissue. However, even in these conditions, GC B cells only accounted for <1% of glandular B cells, demonstrating that BAFF is not efficiently promoting ectopic GC formation in pSS and warranting further investigation of therapeutics targeting both BAFF and the related TNF-family member APRIL.

Role of the IL-12/IL-35 balance in patients with Sjögren syndrome

Background: An interferon signature is involved in the pathogenesis of primary Sjögren syndrome (pSS), but whether the signature is type 1 or type 2 remains controversial. Mouse models and genetic studies suggest the involvement of TH1 and type 2 interferon pathways. Likewise, polymorphisms of the IL‐12A gene (IL12A), which encodes for IL‐12p35, have been associated with pSS. The IL‐12p35 subunit is shared by 2 heterodimers: IL‐12 and IL‐35. Objective: We sought to confirm genetic association of the IL12A polymorphism and pSS and elucidate involvement of the IL‐12/IL‐35 balance in patients with pSS by using functional studies. Methods: The genetic study involved 673 patients with pSS from 2 French pSS cohorts and 585 healthy French control subjects. Functional studies were performed on sorted monocytes, irrespective of whether they were stimulated. IL12A mRNA expression and IL‐12 and IL‐35 protein levels were assessed by using quantitative RT‐PCR and ELISA and a multiplex kit for IL‐35 and IL‐12, respectively. Results: We confirmed association of the IL12A rs485497 polymorphism and pSS and found an increased serum protein level of IL‐12p70 in patients with pSS carrying the risk allele (P = .016). Serum levels of IL‐12p70 were greater in patients than control subjects (P = .0001), especially in patients with more active disease (P = .05); conversely, IL‐35 levels were decreased in patients (P = .0001), especially in patients with more active disease (P = .05). In blood cellular subsets both IL12p35 and EBV‐induced gene protein 3 (EBI3) mRNAs were detected only in B cells, with a trend toward a lower level among patients with pSS. Conclusion: Our findings emphasize involvement of the IL‐12/IL‐35 balance in the pathogenesis of pSS. Serum IL‐35 levels were associated with low disease activity, in contrast with serum IL‐12p70 levels, which were associated with more active disease.

RNA-Seq and CyTOF immuno-profiling of regenerating lacrimal glands identifies a novel subset of cells expressing muscle-related proteins

The purpose of the present studies was to use CyTOF and RNA-Seq technologies to identify cells and genes involved in lacrimal gland repair that could be targeted to treat diseases of lacrimal gland dysfunction. Lacrimal glands of female BALB/c mice were experimentally injured by intra-glandular injection of interleukin 1 alpha (IL-1α). The lacrimal glands were harvested at various time points following injury (1 to 14 days) and used to either prepare single cell suspensions for CyTOF immuno-phenotyping analyses or to extract RNA for gene expression studies using RNA-Seq. CyTOF immuno-phenotyping identified monocytes and neutrophils as the major infiltrating populations 1 and 2 days post injury. Clustering of significantly differentially expressed genes identified 13 distinct molecular signatures: 3 associated with immune/inflammatory processes included genes up-regulated at days 1–2 and 3 associated with reparative processes with genes up-regulated primarily between days 4 and 5. Finally, clustering identified 65 genes which were specifically up-regulated 2 days post injury which was enriched for muscle specific genes. The expression of select muscle-related proteins was confirmed by immunohistochemistry which identified a subset of cells expressing these proteins. Double staining experiments showed that these cells are distinct from the myoepithelial cells. We conclude that experimentally induced injury to the lacrimal gland leads to massive infiltration by neutrophils and monocytes which resolved after 3 days. RNAseq and immunohistochemistry identified a group of cells, other than myoepithelial cells, that express muscle-related proteins that could play an important role in lacrimal gland repair.

The Hippo pathway effector YAP is an essential regulator of ductal progenitor patterning in the mouse submandibular gland

Salivary glands, such as submandibular glands (SMGs), are composed of branched epithelial ductal networks that terminate in acini that together produce, transport and secrete saliva. Here, we show that the transcriptional regulator Yap, a key effector of the Hippo pathway, is required for the proper patterning and morphogenesis of SMG epithelium. Epithelial deletion of Yap in developing SMGs results in the loss of ductal structures, arising from reduced expression of the EGF family member Epiregulin, which we show is required for the expansion of Krt5/Krt14-positive ductal progenitors. We further show that epithelial deletion of the Lats1 and Lats2 genes, which encode kinases that restrict nuclear Yap localization, results in morphogenesis defects accompanied by an expansion of Krt5/Krt14-positive cells. Collectively, our data indicate that Yap-induced Epiregulin signaling promotes the identity of SMG ductal progenitors and that removal of nuclear Yap by Lats1/2-mediated signaling is critical for proper ductal maturation. DOI: http://dx.doi.org/10.7554/eLife.23499.001

AB0170 Cytof Analysis of Lip Biopsies from SjÖgren's Subjects Identifies Dysregulated Immune and Non-Immune Cell Subsets

Background The cellular characterization of the immune infiltrate in exocrine glands from patients with primary Sjögrens syndrome (pSS) has relied so far on methods with low dimensionality. As a result, our knowledge of cellular events taking place in the exocrine glands of these patients is limited. Objectives In order to quantify cellular variations associated with pathogenesis, we used here highly multiparametric cytometry by time-of-flight (CyTOF) to analyze diagnostic biopsies from patients with pSS and non-pSS sicca controls. Methods The CyTOF instrument combines single cell analysis used in flow cytometry with mass spectrometry detection of metal-conjugated antibodies. Using a panel of 36 antibodies, cell populations present in heterogeneous cell suspensions were characterized and correlated with clinical parameters. In parallel, paired formalin-fixed, paraffin embedded sections of labial glands from 15 individuals from this cohort were used to examine anatomic relationships between cell subsets identified by CyTOF. Results We studied 16 pSS patients and 13 non-pSS sicca controls, among which 81% were anti-SSA+, 44% were anti-SSB+, and 63% had a focus score of 1 or more. In addition to confirming the well-characterized presence of CD4+ T cells and B cells in Sjögrens biopsies, this experimental approach revealed several additional pathologic features, among which are: (a) a dramatic accumulation of CD38++CD27+HLA-DRlow cells (up to 50% of glandular B cells); (b) an unexpected abundance of CD8+ T cells showing marks of activation; (c) the upregulation of HLA-DR on epithelial cells in pSS patients with grades ≥3, which constitutes the first direct evidence of a potential pathological contribution of the epithelium using un-manipulated, ex vivo, primary human cells. Conclusions The CyTOF instrument is a new tool allowing precise determination of salivary gland subsets, which may generate new mechanistic hypotheses on pathogenesis of the disease. Disclosure of Interest None declared