Quarter in review – Basic science

Chengappa Kavadichanda MD (General Medicine), DM (Clinical Immunology)
Associate Professor, Department of Clinical Immunology, Jawaharlal Institute of Postgraduate medical education and Research, Puducherry, India

Study in focus 1: Citrullination modulates antigen processing and presentation by revealing cryptic epitopes in rheumatoid arthritis1

One of the mechanisms of loss of immune tolerance resulting in autoimmune diseases like Rheumatoid arthritis (RA) is the presentation of cryptic peptides/ epitopes which are otherwise hidden from the immune system. It is well known that individuals with RA mount immune responses to citrullinated self-antigens and form anti citrullinated peptide antibodies (ACPA). The authors of this study tested their hypothesis stating that citrullination exposes cryptic peptides by modifying protein structure and proteolytic cleavage and thus results in RA.

To test this hypothesis, they used a variety of advanced techniques including proteolytic mapping by cathepsins B, S, and H of native and citrullinated fibrinogen and vimentin. This was followed by analysis using mass spectroscopy (MS). Using SEQUEST or MSFragger algorithms, the authors then mapped the citrullination sites on fibrinogen and vimentin. After citrullination mapping, they identified the changed regions and peptides in the amino acid sequence followed by synthesizing the predicted native and citrullinated protein structures for vimentin, fibrinogen (β and γ), and hn-RNP A2/B1. Monocyte derived dendritic cells were derived from cryopreserved PBMCs from an HLA-DRB1*04:01/16:01-positive samples and natural antigen processing assay was performed. Following this, T cell stimulation, peptide binding assay and T cell cytokine release assays were performed.

The authors first found that the process of citrullination altered antigen processing, resulting in the generation of cryptic peptides and the destruction of previously dominant peptides. They recognised changes in antigen processing both proximal and distal to the citrullinated sites in the linear amino acid sequence and further promoted destabilization in protein folding. They concluded that, the generation of such unique repertoire was achieved through altered protease cleavage and protein destabilization, and not because of direct presentation of citrulline-containing epitopes. Following this, the authors found that RA-associated HLA-DR peptide-binding cores primarily consisted of native non citrullinated sequences (88%). But the presence of these native sequences was enriched by the process of citrullination in the other 12% of the peptides. While co cultured with dendritic cells they found that peptide repertoire presented from the citrullinated antigen bound with a higher median relative affinity than the repertoire presented from the native antigen. They also found that citrullination-dependent epitopes stimulated ACPA+SE+ RA CD4+ T cells and the memory T cells more robustly than native antigen–derived epitopes.

All put together, these findings supported the hypothesis that cryptic peptides revealed by citrullination (away from the citrullination site) may play a role in the development of immune responses and loss of immune tolerance to citrullinated antigens in RA.

Study in focus 2: TAp63, a methotrexate target in CD4+ T cells, suppresses Foxp3 expression and exacerbates autoimmune arthritis2

Methotrexate (MTX) is an anchor drug used in the treatment of rheumatoid arthritis (RA) and many other autoimmune diseases. There is limited understanding about mechanism of drug action and our understanding is predominantly based on the drugs’ antifolate activity. Although it has been reported that MTX suppresses T cell activity and proliferation and increases Treg cells, the precise mechanisms of action are not known. This study aimed to identify candidate genes that are regulated by MTX treatment in CD4+ T cells in patients with RA and its functional implication.

The authors recruited patients classified as RA (ACR 1987 criteria) and took their blood samples before starting MTX and after 3 months of treatment. They first isolated the CD4+T cells and performed DNA microarray analyses from the before and after samples. The authors found that TP63 was the most significantly downregulated gene following treatment with MTX. This finding was followed up with in-vitro experiments that showed that TAp63, an isoform of TP63, was highly expressed in human Th17 cells. These experiments also showed that coculturing these cells with MTX in vitro significantly downregulated TAp63. On adoptive transfer arthritis model experiments, knocking out (KO) TAp63 in murine Th17 cells ameliorated onset of arthritis. By combining RNA-Seq analyses of human Th17 cells overexpressing TAp63 and those with TAp63 knockout in the murine model, the authors identified FOXP3 as a possible TAp63 target gene. They further tested this by co-culturing TAp63 KO out CD4+ T cells under conditions that would polarise them into Th17. This resulted in increased Foxp3 expression, suggesting that TAp63 balances Th17 cells and Treg cells. Upon evaluating the mechanistic link between TAp63 and FOXP3, they found that knocking out TAp63 in murine induced Treg (iTreg) cells increased hypomethylation of conserved noncoding sequence 2 (CNS2) of the Foxp3 gene and enhanced the suppressive function of i-Treg cells. Collectively, the authors concluded that Tap63 suppressed FOXP3 expression and methotrexate downregulates TAp63 resulting in increased regulatory capacity of i-Tregs in RA.


  1. Curran AM, Girgis AA, Jang Y, Crawford JD, Thomas MA, Kawalerski R, et al. Citrullination modulates antigen processing and presentation by revealing cryptic epitopes in rheumatoid arthritis. Nat Commun. 2023 Feb 24;14(1):1061.
  2. Suga K, Suto A, Tanaka S, Sugawara Y, Kageyama T, Ishikawa J, et al. TAp63, a methotrexate target in CD4+ T cells, suppresses Foxp3 expression and exacerbates autoimmune arthritis. JCI Insight. 2023 May 22;8(10):e164778.s1