Background/Purpose: The “Targeting Immune Responses for Prevention of RA” (TIP-RA) collaboration studies individuals at high risk for developing rheumatoid arthritis (RA) because of serum anti-citrullinated protein antibody (ACPA) positivity in absence of arthritis at baseline, and is focused on defining how they transition from at-risk to classifiable disease. One potential mechanism is the expansion of antigen specific T cells that recognize self-antigens and acquisition of disease associated T cell phenotypes. ACPA emerge years prior to clinically apparent disease and subsequently increase in their titer and breadth of specificity.  However, few studies have characterized T cells during this transition.  Therefore, individuals enrolled in the TIP-RA cohort were analyzed by multi-parameter flow cytometry to identify features associated with progression to RA.

Methods: HLA class II tetramer staining and flow cytometry were performed on peripheral blood samples from a baseline visit from CCP3- controls (n=34), CCP3+ at-risk (n=26), CCP3+ positive individuals who transitioned in the near-term to RA (called “RA converters”, n=4), and seropositive early-RA (n=21).  Our staining panel allowed us to assess the frequencies of T cells specific for citrullinated alpha-enolase, aggrecan, cartilage intermediate layer protein, fibrinogen and vimentin.  We then applied both supervised phenotyping and a cluster-based computational approach to compare the phenotypic landscape and specificity of CD4+ T cells in each cohort.

Results: In comparison with controls, we observed higher frequencies of T cells that recognize citrullinated epitopes in CCP3+ at-risk subjects (p< 0.05), suggesting that these T cells expand prior to disease onset.  Supervised phenotypic analysis revealed an increase in CCR4+ CD4+ T cells in CCP3+ at risk subjects (p< 0.001) and a corresponding decrease in CXCR3+ CD4+ T cells that was most pronounced in RA converters and seropositive early-RA (p< 0.05).  Cluster-based phenotypic analysis revealed a landscape of ten distinct immunophenotypes present within all subjects.  Among these, a CCR4 immunotype was progressively enriched in ACPA+ at risk subjects, RA converters, and seropositive early-RA (p=0.007).  Correspondingly, a CXCR3/CCR4/CCR6 immunotype was progressively depleted in ACPA+ at risk subjects, RA converters, and seropositive early-RA (p=0.05).  Each of these ten immunotypes was shown to contain tetramer positive T cells that recognize citrullinated epitopes.  However, the predominant immunotype varied for different antigens and unique phenotypic patterns were observed for different subject groups.

Conclusion: Our data suggest a progressive change in T cell phenotypes during disease development and subsequent transition to classified RA.  Disease associated changes in the antigen specificity and phenotype of CD4+ T cells are present in CCP3+ at-risk subjects before the onset of symptoms and development of classified RA.  This finding suggests that there is a continuum of immunologic changes that drive disease evolution and that exploring emerging antigen specificities and immunophenotypes will aid our understanding of risk and shape approaches for intervention.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/differences-in-the-phenotypic-landscape-and-antigen-specificity-of-cd4-t-cells-are-present-in-ccp-subjects-before-the-onset-of-rheumatoid-arthritis/