Collagen-specific TCR Repertoire usage in RA and cytokine secretion

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic joint inflammation, associated with HLA-DR4. Collagen II specific T cells repertoire in DR4+ patients is characterized by a limited number of TCR-beta chain during the acute phase, in part enriched in Synovial fluid. The two more used TCR-beta chains are BV11 and BV13. PBMC from 85 RA patients (47 acute presentations and 38 remissions), cultured with or without collagen peptide, were examined by immunoscope. We also studied IL-17 and IL-13 secretion of collagen-specific individual T cells of 3 DR4+ patients after in vitro stimulation with the peptide and with or without bacteria-derived products. At acute presentation 6/15 patients showed BV11+ T cells and 7/15 showed BV13+ T cell, while after remission the number falls to 1/11 and 2/10, respectively. Some DR7+ patients displayed T cells using similar BV11 and BV13 chains. In this latter case, frequency of positive BV13 samples did not decrease with remission. We observed that 1/17 T cell clonotypes secreted IL-17 or IL-13 after stimulation with peptide. However, after stimulation with peptide in the presence of bacterial products, 3 more individual T cells became able to secrete IL-17. Thus, BV11+ and BV13+ cells are bystander of RA at acute presentation in DR4+ patients. The relative high frequency of these TCRs also in DR1+ and DR7+ patients is possibly due to similarities in peptide selection. In RA patients, secretion of the pro-inflammatory cytokine IL- 17 is modulated by exogenous (or endogenous) factors, possibly interacting with PRRs. Background/Purpose: Joint damage in Rheumatoid Arthritis (RA) is likely due to pro-inflammatory T cells specific for collagen and recruited into the synovium, where they are activated and promote acute presentation of the disease. We previously described the T cell repertoire specific for human Collagen II peptide 261-273 (hColl261) in HLA-DRB1*04 Early Rheumatoid Arthritis (ERA) patients, compared to that of DRB1*04 healthy subjects and followed it along disease course, showing that T cells specific for this epitope appear during flares of the disease but tend to disappear during remission1. Here we report preliminary observations regarding the presence of specific clonotypes recognized by the ability of hColl-specific (hColl261) T cells to respond to Collagen II peptide and we describe that they seem to secrete IL-17 more than IL-13. Methods: We enrolled 72 early (disease duration <12 months) RA patients in different disease phases; 44 with an active disease (active ERA) and 28 with non active disease (non active ERA). PBMCs were collected from all patients. All patients were typed for HLA-DRB1: 14 were DRB1*04, 7 were DRB1*01, one was DRB1*04/*01 and 22 were not DRB1*04/*01. Patients were tested for the presence of hColl261 peptide response by immunoscope. In 3 ERA patients synovial fluid mononuclear cells were also examined. In addition, PBMCs were purified from 2 active ERA patients and 1 RA patient at his first remission of disease, stimulated in vitro with hColl261 and IL-17 and IL-13 secreting cells were enriched by MACS® secretion assay. The presence of huColl261-specific TCRs was assessed by immunoscope in samples enriched or depleted for each cytokine, and in samples allowed to proliferate for 3 days in response to the peptide antigen. Results: We examined the usage of two TCR beta chains that we showed to be frequently used in DRB1*04 ERA patients. Collagen-specific T cells carrying TRBV25 (Vb11(139b)) were used by 6 out of 13 ERA DRB1*04 subjects, 2 out of 7 DRB1*01 subjects (that were both also DRB1*15) and 4 out 21 patients negative for both DRB1*04 and DRB1*01. Usage of TRBV6-4 (Vb13b (199)) appears to be more associated with DRB1*04 subjects, since we found collagen specific T cells using this TCR-beta chain in 6 out of 11 DRB1*04 ERA patients versus 4 out of 21 not DRB1*04 ERA subjects. T cells obtained from 3 PB samples were tested for their capacity to secrete specific cytokines (IL-17 or IL-13) in response to stimulation with collagen II. T cells secreting any of the two cytokines represent only a part of the circulating T cell repertoire specific for huColl261, similar to the observation that a minority of the circulating repertoire is actually able to home to the synovial compartment. IL-17-secreting cells were detected in all 3 samples, but it appears that T cells from the patient in disease remission needed additional stimuli that include bacterial derived non antigenic moieties in order to produce IL-17. hColl261-specific IL-13 producing cells were detected only in one of the 2 active ERA patients. Conclusions: Eight rearrangements of the TCR beta-chain co-segregate with the collagen-specific response restricted by DRB1*04 and/or DRB1*01. TRBV25 (vβ11)-Jb 2.2 and TRBV6-4(vb13b)-Jb 2.3 rearrangements are associated with the acute phase of disease in DRB1*04 and *01 RA patients; TRBV25 (vβ11)+IL17+ T cells were detected in three patients affected by RA (2 active and 1 ERA and one remission) while TRBV25 (vβ11)+IL13+ T cells were detected only in one ERA patient. TRBV6-4(vβ13b)T cells seems not to secrete any of the two cytokines. From these data we conclude that T cell precursors driving the proinflammatory response in the synovium by the production of IL-17 seem to represent a limited portion of the entire collagen specific repertoire.