Memory T cells specific to citrullinated α-enolase are enriched in the rheumatic joint

Abstract

ACPA-positive rheumatoid arthritis (RA) is associated with distinct HLA-DR alleles and immune responses to many citrullinated self-antigens. Herein we investigated the T cell epitope confined within α-enolase_326-340 in the context of HLA-DRB1*04:01 and assessed the corresponding CD4⁺ T cells in both the circulation and in the rheumatic joint. Comparative crystallographic analyses were performed for the native and citrullinated α-enolase_326-340 peptides in complex with HLA-DRB1*04:01. HLA-tetramers assembled with either the native or citrullinated peptide were used for ex vivo and in vitro assessment of α-enolase-specific T cells in peripheral blood, synovial fluid and synovial tissue by flow cytometry. The native and modified peptides take a completely conserved structural conformation within the peptide-binding cleft of HLA-DRB1*04:01. The citrulline residue-327 was located N-terminally, protruding towards TCRs. The frequencies of T cells recognizing native eno_326-340 were similar in synovial fluid and peripheral blood, while in contrast, the frequency of T cells recognizing cit-eno_326-340 was significantly elevated in synovial fluid compared to peripheral blood (3.6-fold, p = 0.0150). Additionally, citrulline-specific T cells with a memory phenotype were also significantly increased (1.6-fold, p = 0.0052) in synovial fluid compared to peripheral blood. The native T cell epitope confined within α-enolase_326-340 does not appear to lead to complete negative selection of cognate CD4⁺ T cells. In RA patient samples, only T cells recognizing the citrullinated version of α-enolase_326-340 were found at elevated frequencies implicating that neo-antigen formation is critical for breach of tolerance.

Keywords: Autoimmunity; Citrullination; Crystal structure; HLA class II tetramers.

Figure 1. Peptide sequence, binding to HLA-DRB1*04:01 and tetramer staining.

(A) Amino acid sequence from α-enolase aa326–340 in the native (eno_326–340) and citrullinated form (cit-eno_326–340). Numbers indicate the tentative binding position for P1, P4, P6, P7 and P9. (B) Binding affinity to HLA-DRB1*04:01, as determined by europium-based peptide competition assay, was done for the native arginine and the citrullinated version of α-enolase. HA: hemagglutinin_306–318 antigen as a positive control. (C-E) PBMC from DRB1*04:01 healthy subjects were stimulated with peptides, cultured for 14 days and stained with tetramers. (C) Negative control, cells were stimulated with eno_326–340 and stained with HA_306–318 tetramer. (D+E) Cells were stimulated with HA_306–318 (D) or eno_326–340 and cit-eno_326–340 peptides (E) and stained with corresponding tetramers.

Figure 2. Frequency and characterization of tetramer-positive cells.

(A) Representative example of ex vivo analysis for flu specific T cells from SFMC of a DRB1*04:01 RA subject after staining with HA_306–318 tetramer along with CD4, CD45RO, CD25 and CD28 antibodies. (B) The frequency of antigen-specific CD4+ T cells in blood from healthy controls (unfilled symbols) and RA-patients (grey symbols) and synovial fluid from RA-patients (black symbols). Plotted are positive cells per 1 x 10⁶ CD4+ T cells. Cut-off for positivity is 1/1 x 10⁶. (C) The frequency of antigen-specific CD4+ T cells in peripheral blood (PB) and synovial fluid (SF) in paired samples from RA patients is shown. Plotted are positive cells per 1 x 10⁶ CD4+ T cells. Black: cells specific for eno_326–340, grey: cit- eno_326–340 specific cells. (D-E) Further characterization of tetramer positive cells from blood and synovial fluid. Depicted is the percentage of Tetramer-positive cells that are positive for CD45RO (D) or CD25 (E).

Figure 3. Functional T cell assay and murine studies.

(A) PBMC were stimulated with eno_326–340 or cit-eno_326–340 peptides for 5 days. -17A and TNF. We detected immunePatients were then divided into 3 groups (tertiles) based on response pattern: generally more stimulation with eno_326–340peptide (upper panel), generally more stimulation with cit-eno_326–340 peptide (centered panel) and patients with same or mixed stimulation (lower panel). Visualized is the % of expression on CD4+ T cells. (B) HLA-DRB1*04:01-IE transgenic mice were immunized with eno_326–340 peptide, restimulated with either eno_326–340 or cit-eno_326–340 peptide and proliferation was measured.

Figure 4. Tetramer stainings in synovial fluid.

Ex vivo staining from a synovial fluid of a HLA-DRB1*04:01 RA patient. Eno_326–340-Tmr was conjugated to APC and cit-eno_326–340-Tmr was conjugated to PE.

Figure 5. Dual-tetramer staining with PE and APC-Tmr in peripheral blood.

PBMC were expanded for 14 days with either HA_306–318, eno_326–340 or cit-eno_326–340 and stained with corresponding tetramers. Two examples from different HLA-DRB1*04:01 RA patients.

Figure 6. Enolase-specific T cells in synovial tissue.

A synovial biopsy was obtained from a DRB1*04:01 RA patient. The tissue was enzymatically digested and the cells were stimulated with eno_326–340 and cit-eno_326–340, cultured for 14 days and stained with the tetramers.