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ObjectivesStudies in axial spondyloarthritis (axSpA) have yielded conflicting results regarding the identity of the major IL-17A-producing lymphocyte populations. The goal of this study was to comprehensively assess the production of IL-17A and related cytokines by peripheral blood lymphocytes in axSpA.MethodsPeripheral blood mononuclear cells were isolated from patients with axSpA and healthy controls matched for age, sex and HLA-B27 status. Unstimulated cells and cells activated with PMA/Ionomycin were analyzed by 25-parameter fluorescent flow cytometry. Data were analyzed by hierarchical gating, UMAP and SPICE.ResultsExcept for a reduced frequency of mucosal-associated invariant T (MAIT) cells and natural killer (NK) cells, there were no other significant differences in abundance of major lymphocyte populations in axSpA patients compared with controls. Increased IL-17A production in axSpA was observed in total non-B lymphocytes and in MAIT cells. The fraction of MAIT cells expressing the tissue residency markers CD69 and CD103 was increased in axSpA. CD103 positive MAIT cells were enriched for IL-17A producers. axSpA patients demonstrated an expansion of MAIT cell subsets producing IL-17A, IL-17F, GM-CSF and TNF. This expansion was only observed in HLA-B27+ patients.ConclusionsWe document an expansion of polyfunctional IL-17A+ MAIT cells in the peripheral blood of HLA-B27+ patients with axSpA. These results are consistent with the implied role of intestinal dysbiosis or inflammation in axSpA pathogenesis.Key messagesWhat is already known about this subject? Various IL-17A-producing lymphocyte populations have been implicated in the pathogenesis of axSpA.What does this study add? Polyfunctional MAIT cells capable of producing IL-17A, IL-17F, GM-CSF and TNF are expanded in the peripheral blood of HLA-B27+ patients with axSpA.How might this impact on clinical practice or future developments? Overproduction of IL-17A by MAIT cells is the most consistent finding of peripheral blood lymphocyte studies in axSpA.Our data support the pathogenetic link between intestinal and axial inflammation in axSpA.

BackgroundOverexpression of IL-23 in adult mice by means of hydrodynamic tail vein injection of IL-23 minicircles has been reported to result in spondyloarthritis-like disease. The impact of genetic background and sex on the disease phenotype in this model has not been investigated.MethodsWe compared male B10.RIII mice with male C57BL/6 mice, and male with female B10.RIII mice after hydrodynamic injection of IL-23 enhanced episomal vector (EEV) at 8-12 weeks of age. We monitored clinical arthritis scores, paw swelling, and body weight. Animals were euthanized after two weeks and tissues were harvested for histology, flow cytometry and gene expression analysis. Serum cytokine levels were determined by ELISA.FindingsMale B10.RIII mice developed arthritis in the forepaws and feet within 6 days after IL-23 EEV injection; they also exhibited psoriasis-like skin disease, colitis, weight loss, and osteopenia. In contrast to previous reports, we did not observe spondylitis or uveitis. Male C57BL/6 mice injected with IL-23 EEV had serum IL-23 levels comparable with B10.RIII mice and developed skin inflammation, colitis, weight loss, and osteopenia but failed to develop arthritis. Female B10.RIII mice had more severe arthritis than male B10.RIII mice but did not lose weight.ConclusionsSystemic IL-23 overexpression results in spondyloarthritis-like disease in B10.RIII mice. The development of extra-articular manifestations but absence of arthritis in C57BL/6 mice suggests organ-specific genetic control mechanisms of IL-23 driven inflammation. Discrepancies regarding the phenotype of IL-23 induced disease in different labs and the sexual dimorphism observed in this study warrant further exploration.

Understanding how genetic regulatory variation affects gene expression in different T cell states is essential to deciphering autoimmunity. We conducted a high-resolution RNA-seq time course analysis of stimulated memory CD4+T cells from 24 healthy individuals. We identified 186 genes with dynamic allele-specific expression, where the balance of alleles changes over time. These genes were four fold enriched in autoimmune loci. We found pervasive dynamic regulatory effects within six HLA genes, particularly for a major autoimmune risk gene,HLA-DQB1. EachHLA-DQB1allele had one of three distinct transcriptional regulatory programs. Using CRISPR/Cas9 genomic editing we demonstrated that a single nucleotide variant at the promoter is causal for T cell-specific control ofHLA-DQB1expression. Our study in CD4+T cells shows that genetic variation incisregulatory elements may affect gene expression in a lymphocyte activation status-dependent manner contributing to the inter-individual complexity of immune responses.

High dimensional single-cell analyses have dramatically improved the ability to resolve complex mixtures of cells from human disease samples; however, identifying disease-associated cell types or cell states in patient samples remains challenging due to technical and inter-individual variation. Here we present Mixed effects modeling of Associations of Single Cells (MASC), a novel reverse single cell association strategy for testing whether case-control status influences the membership of single cells in any of multiple cellular subsets while accounting for technical confounds and biological variation. Applying MASC to mass cytometry analyses of CD4+ T cells from blood of rheumatoid arthritis (RA) patients and controls revealed a significantly expanded population of CD4+ T cells, identified as CD27- HLA-DR+ effector memory cells, in RA patients (OR = 1.7; p = 1.1 × 10−3). The frequency of CD27- HLA-DR+ cells was similarly elevated in blood samples from a second RA patient cohort, and CD27- HLA-DR+ cell frequency decreased in RA patients who respond to immunosuppressive therapy. Compared to peripheral blood, synovial fluid and synovial tissue samples from RA patients contained ∼5-fold higher frequencies of CD27- HLA-DR+ cells, which comprised ∼10% of synovial CD4+ T cells. We find that CD27- HLA-DR+ cells are abundant producers of IFN-γ and also express perforin and granzyme A at elevated levels. Thus MASC identified the expansion of a unique Th1 skewed effector T cell population with cytotoxic capacity in RA. We propose that MASC is a broadly applicable method to identify disease-associated cell populations in high-dimensional single cell data.One Sentence SummaryMixed-effects regression of single cells identifies a cytotoxic Th1-like CD4+ T cell subset while accounting for inter-individual and technical variation.