Anti-PAD4 antibodies link autoimmunity to PAD4 with CTL-associated rheumatoid arthritis.

Abstract

We aimed to study the serologic, genetic, and immunologic underpinnings associated with cytotoxic T lymphocytes (CTLs) in rheumatoid arthritis (RA), using T-large granular lymphocytic leukaemia with comorbid RA (T-LGLL/RA) as a model of CTL-linked RA. We used blood samples and paired clinical data from patients with RA, T-LGLL/RA, T-LGLL and healthy controls. Serological characterisation was performed using anti-cyclic citrullinated peptide 3.1 (anti-CCP3.1) enzyme-linked immunosorbent assay, multiplex RA autoantigen array, and radiolabelled peptidylarginine deiminase type III and IV (PAD4) immunoprecipitations. Genetic characterisation was performed using PADI4 single nucleotide polymorphism TaqMan genotyping assays and droplet digital polymerase chain reaction for signal transducer and activator of transcription 3 (STAT3) mutations. Multiparameter flow cytometry and pentamer binding assays were performed to immunophenotype CTLs and characterise PAD4-specific CTLs, respectively. Patients with T-LGLL/RA had enhanced anticitrullinated protein antibody responses compared with RA, and a strikingly higher frequency of anti-PAD4 antibodies (60% vs 27%, P < .0001). Among patients with T-LGLL, PADI4 single nucleotide polymorphisms were associated with anti-PAD4-positive RA (20% vs 3%, P = .02), and anti-PAD4 antibody levels were inversely correlated with absolute neutrophil count (Spearman's rho = -0.236; P = .02). Activating STAT3 mutations were associated with anti-PAD4 antibodies in both T-LGLL/RA (90% vs 74%, P = .047) and RA (39% vs 9%, P = .002). Flow cytometric characterisation of CTLs showed that anti-PAD4-positive RA was enriched for CD57+, CD7 low, T effector memory cells re-expressing CD45RA (TEMRA) CTLs. Furthermore, PAD4-specific CTLs were detected in anti-PAD4+ patients with RA and expressed elevated levels of CD69, CD57, and KLRG1, and a TEMRA phenotype. These data demonstrate a mechanistic link between autoimmunity to PAD4 and CTL-associated disease in RA.