Identification and functional characterization of T cells reactive to citrullinated vimentin in HLA-DRB1*0401-positive humanized mice and rheumatoid arthritis patients

Abstract

Objective: Antibodies toward the citrullinated form of the synovial antigen vimentin are specific for rheumatoid arthritis (RA) and are associated with HLA-DRB1*0401. This suggests that T cells specific for peptides derived from citrullinated vimentin presented in the context of HLA-DRB1*0401 may contribute to the etiopathogenesis of RA. The aim of this study was to identify immunodominant epitopes from citrullinated vimentin presented by HLA-DRB1*0401 and to characterize the resulting T cell responses.

Methods: We first predicted an HLA-binding T cell epitope from citrullinated vimentin based on the binding motif of HLA-DRB1*0401 and then confirmed its affinity. A class II major histocompatibility complex (MHC) tetramer loaded with the citrullinated form of vimentin aa 59-78 (cit-vimentin aa 59-78) was constructed and used to screen for specific T cells in HLA-DRB1*0401-transgenic mice, patients with RA, and healthy control subjects. Additionally, the cytokine output following cit-vimentin aa 59-78 challenge was analyzed in patients and healthy control subjects by multicolor flow cytometry and Luminex-based analysis.

Results: The citrullinated form of vimentin aa 59-78 bound to HLA-DRB1*0401, but the native form could not. Subsequently, cit-vimentin aa 59-78-specific T cells were detected in immunized mice and in the periphery of both HLA-DR*0401-positive healthy control subjects and HLA-DR*0401-positive patients with RA, using class II MHC tetramers, CD154 up-regulation, and intracellular cytokine measurements. As demonstrated in cell culture supernatants, the production of cytokines (predominantly interferon-γ) in response to cit-vimentin aa 59-78 was significantly higher in patients compared with controls.

Conclusion: Here, we describe a posttranslational modification of an RA candidate autoantigen toward which HLA-DRB1*0401-restricted T cells can be detected in both patients with RA and healthy controls but for which a proinflammatory response is observed uniquely in patients with RA.

Figure 1. Vimentin peptides and relative binding affinity to HLA-DRB1*0401

(A) Peptides synthesized from vimentin in their native and citrullinated forms. Residues in bold represent proposed P1 anchors. Binding affinity to DRB1*0401 was determined for either the long version of the peptides (B) or shorter versions, limited to one positional binding frame (C), by europium based peptide competition assay. Non-citrullinated vim peptides are shown in light gray and citrullinated in black. For comparison, the high affinity HA_306-318 peptide is shown in dark gray.

Figure 2. Cit- vim_59-78, but not vim_59-78, is antigenic in DR0401-IE mice

³H-thymidine incorporation from splenocytes of mice immunized with either (A) cit-vim_59-78 or (B) vim_59-78 after in vitro restimulation of vim_59-78, cit- vim_59-78, and HA_306-318 peptides at 4, 20, & 100 μg/ml. Splenocytes alone served as a negative control while anti-CD3/anti-CD28 stimulation served as a positive control. Results are representative of n= 20 and n= 8, respectively. (C) Splenocytes from mice immunized with cit- vim_59-78 were challenged with the different versions of the epitope (Fig 1A) (n=13). Conversely, mice were also immunized with either cit-vim_59-71 (D)(n=7) or cit-vim_66-78 (E)(n=5), and challenged with long and short peptides. In C, D, and E peptide challenge was done at 5 μg/ml and figures display one representative mouse out of each experiment. (F) Cit-vim specific CD4+ T cells were detected in cit- vim_59-78 immunized mice by tetramer staining. Here a representative panel of mice harvested at 13-weeks is shown, where 4/5 cit-vim_59-78 immunized mice are tetramer positive. Samples had to have > 4-fold background to be considered positive. 0401-HA tetramer is used as a negative control for 0401-cit-vim_59-78 tetramer. * designates p values of < 0.05.

Figure 3. Cit-vim_59-78 specific CD4 T cells from both RA subjects and healthy controls can be detected by tetramer

A representative cit-vim_59-78 tetramer positive sample from one HLA-DRB1*0401-positive RA patient (A) and one HLA-DRB1*0401-positive healthy control subject (B). PBMCs were stimulated with either vim_59-78, cit-vim_59-78, or HA_306-318 in the presence of IL-2. Samples were then stained for tetramer on day 14. 0401-CII is shown as a universal negative control.

Figure 4. Reactivity to cit-vim_59-78 in DRB1*0401 RA patients and healthy controls

PBMCs from RA patients (n=22) and control (n=9) were stimulated with either vim_59-78 or cit-vim_59-78 before being stained for expression of (A) CD154 and intracellular content of (B) TNFα, (C) IFNγ and (D) IL-17A in CD4+ T cells as measured by flow cytometry. Percentages of reactive-CD4 T cells are shown following subtraction of background levels (from unstimulated cultures). RA patients’ responses are shown in solid lines, whereas dotted lines represent healthy controls.

Figure 5. Cytokine milieu following in vitro stimulation of PBMCs from RA patients and healthy controls

The levels of 16 different cytokines (IFNγ, TNFα, IL-1b, IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-10, IL-13, IL-17A, IL-17F, IL-21, IL-22 and IL-23) were measured by Luminex Multiplex array following peptide stimulation. (A) Production of inflammatory cytokines by an RA patient in response to cit-vim_59-78 but not vim_59-78. (B) Average cytokine response following stimulation with cit-vim_59-78 or vim_59-78 (in 19 RA patient and 9 healthy control cultures). (C) Cytokine production in response to HA_306-318 stimulation in RA patients and healthy controls, where donors are the same as seen in 5B. Stimulation index (S.I.) was calculated as: pg of cytokine from peptide stimulated culture/pg of cytokine from an unstimulated culture.