Background/Purpose: Recognition of self-peptides by autoreactive CD4+ T-cells is central to the disruption of immune tolerance. Although systemic autoimmune diseases such as Sjogren’s syndrome (SjS) and systemic sclerosis (SSc) are both immune responsive to anti-nuclear antigens (ANA), the mechanisms underlying their distinct clinical manifestations are largely unknown. Our aim was to address these mechanisms by identifying auto-reactive T and B cells in multiple tissues and holistically analyzing their properties.

Methods: To identify reactive lymph nodes containing auto-reactive T and B cells, we performed ¹⁸F-fluorolevothymidine PET scans in patients with SjS (n=10) and SSc (n=10). Ultrasound-guided biopsies were performed to subsequently obtain  PET-avid and PET-negative LNs. In addition matched affected tissues (salivary gland and skin, respectively) were also sampled. To obtain auto-reactive T cells for detailed analysis, ex vivo T-cell stimulation assays (n=40 SSc and 33 SjS) were combined with paired blood, LN and affected tissue multi-modal single-cell techniques (transcriptome, proteome, and TCR/BCR sequencing) (n=8 SSc and 8 SjS) to identify SSA/SSB and scl70 reactive CD4+ T-cells and B-cells. Flow cytometry and multiplex immunofluorescence staining were used for validation and detailed analysis of spatial localization of the autoreactive cells.

Results: In both SjS and SSc,  memory ANA-reactive T-cell clones were expanded in the blood compared to healthy individuals and their numbers were associated with disease severity. A proportion of ANA-reactive clones was shared in  blood, LNs, and affected tissues. In comparison to cold LNs, reactive LNs and blood in SjS patients were characterized by increased SSA/B-reactive Th2, Th17, follicular, and peripheral T-helper populations. Additionally, a naïve-like CD4+TRAIL+ T cell population uniquely occurred in reactive LNs and patients’ blood. In contrast, in SSc patients, reactive LNs and blood contained predominantly scl70-reactive CD4+ TRAIL+ T cells and no CD4+ effector T cells. This coincided with an increased presence of expanded autoreactive memory B and plasma cell clones in SjS compared to SSc reactive LNs and affected tissues. Strikingly, in vitro blockade of the TRAIL-DR5 axis increased ANA-reactive T-cell responses and plasma cell differentiation in both diseases.

Conclusion: Our data provide evidence of differential and disease specific autoreactive T cell immunity. Reactive LNs of SjS patients are characterized by increased ANA-reactive effector T cells and B cells compared to those in SSc patients. Naive-like CD4+ TRAIL+ ANA-reactive T-cells may differentially regulate the ANA immune response in SjS and SSc. Targeting this potentially immune regulatory axis will assist in the rational development of tolerogenic treatments for systemic autoimmune diseases.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/single-cell-multi-omics-analysis-of-reactive-lymph-nodes-affected-tissues-and-blood-reveals-a-naive-like-cd4trail-t-cell-population-that-differentially-directs-effector-anti-nuclear-antigen-reacti/