Anti-citrulline immunity in rheumatoid arthritis : characterization of peptide-HLA interactions and CD4+ T cell responses

Abstract: Rheumatoid Arthritis (RA) is a complex, systemic autoimmune disorder characterized by chronic inflammation in small joints in hands and feet. It is a common disease that if untreated leads to joint destruction, disability, comorbidities and a reduced lifespan. Both genetic and environmental factors contribute to disease pathogenesis that in the majority of patients is characterized by the occurrence of antibodies against citrullinated proteins (ACPA). The strong genetic association with certain MHC class II alleles led to autoreactive T cells being assigned a major role in the course of disease. Today, citrulline-specific CD4+ T cells are regarded the perfect target for antigen-specific immunotherapy and have so far been investigated in a handful of studies. The focus of this thesis was therefore to expand our knowledge of citrulline-reactive T cells with a specific emphasis on their phenotype as well as the interaction with their cognate peptide-HLA complexes on antigen-presenting cells. We have examined functional T cell responses to native and citrullinated epitopes derived from the candidate antigen α-enolase and compared their relevance in peripheral blood of patients with different RA-associated HLA-DR alleles. While HLA-DRB1'04:01 and '04:04 presented a similar set of peptides, a clear bias in functional responses towards citrullinated epitopes was observed in HLA-DRB1'04:01 patients. When analysing crystal structures of both native and citrullinated versions of two α-enolase epitopes in complex with HLADRB1' 04:01, we found the citrulline residues at peptide positions p-1 and p2, respectively. In both cases the citrulline was not involved in binding to the HLA molecule but instead pointed upwards readily available for interaction with the TCR. Particular recognition of these citrullinated epitopes by the T cells is thus based on the creation of neoantigens. Using HLA class II tetramer technology, we even detected cross-reactive T cells in some patients recognizing both native and citrullinated version of the epitope with citrulline at position p-1 implicating the existence of TCRs with different docking patterns towards these pHLA complexes. When comparing frequencies of T cells reactive to either of the two versions, we found memory T cells specific for the citrullinated version enriched in the synovial fluid compared to peripheral blood. Additionally, by successfully combining HLA class II tetramer sorting with single cell RNAsequencing, we could compare the transcriptional profile of citrulline- versus virus-specific CD4+ T cells in peripheral blood and synovial fluid of RA patients. Here, we repeatedly found genes associated with cytolytic and cytotoxic features upregulated in the citrulline-specific T cells both in blood and synovial fluid. Furthermore, we developed a multi-tetramer staining panel that allows the simultaneous assessment of multiple specificities making it applicable for longitudinal monitoring of T cells in clinical samples. Using this tool to examine the frequency and phenotype of T cells specific for eight citrullinated peptides from four RA candidate antigens, we could show that the frequencies of citrulline-specific CD4+ T cells in early RA patients decline upon disease improvement. In summary, our data demonstrate that autoreactive citrulline-specific T cells are present in RA patients both early and late in the disease course and that the breach of tolerance includes several non-related autoantigens.