Integrative molecular profiling of autoreactive CD4 T cells in autoimmune hepatitis

Abstract

Background & Aims: In most autoimmune disorders, crosstalk of B cells and CD4 T cells resultsin the accumulation of autoantibodies. In autoimmune hepatitis (AIH), the presence of anti-Soluble Liver Antigen (SLA or SepSecs) autoantibodies is associated with significantly reduced overall survival, but the associated autoreactive CD4 T cells have not been characterized yet.Here we isolated and deeply characterized SLA-specific CD4 T cells in AIH patients. Methods: We used brief ex vivo restimulation with overlapping SLA-derived peptides to isolate and phenotype circulating SLA-specific CD4 T cells, and integrative single-cell RNA-seq (scRNAseq) to characterize their transcriptome and TCR repertoire in n=5 AIH patients. SLA-specific CD4 T cells were tracked in peripheral blood through TCR sequencing, to identify their phenotypic niche. We further characterized disease-associated peripheral blood T cells by high content flow cytometry in an additional cohort of n=46 AIH patients and n=18 non-alcoholic steatohepatitis (NASH) controls. Results: Autoreactive SLA-specific CD4 T cells were only detected in patients with anti-SLA autoantibodies and had a memory PD-1 + CXCR5 -CCR6 -CD27 + phenotype. ScRNA-seq revealed their pro-inflammatory/B-Helper profile ( IL21, IFNG, TIGIT, CTLA4, NR3C1, CD109, KLRB1 and CLEC2D). Autoreactive TCR clonotypes were restricted to the memory PD-1 + CXCR5 -CD4 T cells. This subset was significantly increased in the blood of AIH patients and supportedB cell differentiation through IL-21. Finally, we identified a specific phenotype (PD-1 + CD38 + CD27 + CD127 -CXCR5 -) of CD4 T cells linked to disease activity and IgG response during AIH. Conclusions: This work provides for the first time a deep characterization of rare circulating autoreactive CD4 T cells and the identification of their peripheral reservoir in AIH. We also propose a generic phenotype of pathogenic CD4 T cells related to AIH disease activity.

Autoimmmune hepatitis

“…Three groups have recently published observations relevant to the pathogenesis of AIH. Renand et al describe a rare population of CD4 cells, with a memory PD-1 + CXCR5 − CCR6 − CD27 + phenotype, reactive with SLA and only present in patients positive for anti-SLA autoantibodies [ 84 ]. They also describe another phenotype, CD45RA- PD1 + CD38 + -CXCR5-CD127-CD27 + , that when present on CD8 T cells is associated with transaminase levels, while when present on CD4 cells is linked to IgG levels [ 84 ].…”

“…Renand et al describe a rare population of CD4 cells, with a memory PD-1 + CXCR5 − CCR6 − CD27 + phenotype, reactive with SLA and only present in patients positive for anti-SLA autoantibodies [ 84 ]. They also describe another phenotype, CD45RA- PD1 + CD38 + -CXCR5-CD127-CD27 + , that when present on CD8 T cells is associated with transaminase levels, while when present on CD4 cells is linked to IgG levels [ 84 ]. You et al reported that tissue resident CD8 T (CD8TRM) cells are elevated in the liver of patients with AIH compared to CHB, non-alcoholic fatty liver disease and healthy control tissues [ 85 ].…”

The Role of B Cells and B Cell Therapies in Immune-Mediated Liver Diseases

“…These cells are similar in profile to so called T peripheral helper (Tph) cells that have been identified as drivers of B cell inflammation in other autoimmune conditions such as Rheumatoid Arthritis and IgG4related disease (24). The population of PD1 positive activated Tph cells was expanded amongst all CD4 T cells in AIH, produced IL-21 and interferon gamma (IFNg) on ex-vivo restimulation and drove B cell differentiation in co-cultures (23).…”

“…T cell expression of activation markers PD1 and CD38 and the magnitude of ex-vivo cytokine responses towards autoantigenic peptides correlates with AIH disease activity (20)(21)(22). Autoreactive T cell responses specific to autoantigen SLA have been investigated by flow cytometry and single cell RNA sequencing (23), showing SLA CD4 T cells transcriptionally upregulate expression of genes associated with B cell help and inflammation. These cells are similar in profile to so called T peripheral helper (Tph) cells that have been identified as drivers of B cell inflammation in other autoimmune conditions such as Rheumatoid Arthritis and IgG4related disease (24).…”

T peripheral helper cells in autoimmune diseases*

“…In this work, T cells from autoimmune hepatitis patients were stimulated in vitro with peptides derived from soluble liver antigen, an autoantibody target in autoimmune hepatitis, and the phenotype and repertoire of responsive T cells were characterized. 51 Remarkably, the T cells that responded to soluble liver antigen peptides were highly enriched in a Tph cell phenotype, with expression of PD-1 but not CXCR5, and single-cell RNA-seq analyses revealed a typical Tph signature, with expression of IL21, ICOS, MAF, TIGIT, and SLAMF6. Peripheral blood cell analyses confirmed an increased frequency of PD-1 + CXCR5 -Tph cells in the circulation of patients with autoimmune hepatitis; these cells could stimulate B cell responses in vitro, consistent with a B cell-helper function.…”