Multi-HLA class II tetramer analyses of citrulline-reactive T cells and early treatment response in rheumatoid arthritis

Abstract

Background: HLA class II tetramers can be used for ex vivo enumeration and phenotypic characterisation of antigen-specific CD4+ T cells. They are increasingly applied in settings like allergy, vaccination and autoimmune diseases. Rheumatoid arthritis (RA) is a chronic autoimmune disorder for which many autoantigens have been described.

Results: Using multi-parameter flow cytometry, we developed a multi-HLA class II tetramer approach to simultaneously study several antigen specificities in RA patient samples. We focused on previously described citrullinated HLA-DRB1*04:01-restricted T cell epitopes from α-enolase, fibrinogen-β, vimentin as well as cartilage intermediate layer protein (CILP). First, we examined inter-assay variability and the sensitivity of the assay in peripheral blood from healthy donors (n = 7). Next, we confirmed the robustness and sensitivity in a cohort of RA patients with repeat blood draws (n = 14). We then applied our method in two different settings. We assessed lymphoid tissue from seropositive arthralgia (n = 5) and early RA patients (n = 5) and could demonstrate autoreactive T cells in individuals at risk of developing RA. Lastly, we studied peripheral blood from early RA patients (n = 10) and found that the group of patients achieving minimum disease activity (DAS28 < 2.6) at 6 months follow-up displayed a decrease in the frequency of citrulline-specific T cells.

Conclusions: Our study demonstrates the development of a sensitive tetramer panel allowing simultaneous characterisation of antigen-specific T cells in ex vivo patient samples including RA 'at risk' subjects. This multi-tetramer approach can be useful for longitudinal immune-monitoring in any disease with known HLA-restriction element and several candidate antigens.

Keywords: Autoimmune disease; Autoreactive CD4+ T lymphocytes; Citrullination; Multi-MHC class II tetramer assay.

Fig. 1

The multi-tetramer approach is sensitive enough to detect antigen-specific CD4+ T cells in healthy controls. a Representative flow plots depicting the gating strategy for CD4+ T cells reactive to influenza (left) and citrullinated CILP/FGB peptides (right). b Frequency of antigen-specific CD4+ T cells is shown for seven healthy controls (different symbols and shades of grey for each buffy coat). Plotted are tetramer-positive cells per million CD4+ T cells from all fourteen experiments (one technical replicate per healthy control) for influenza, citrullinated CILP/FGB and citrullinated α-enolase. Cut-off for positivity is one tetramer-positive cell per million CD4+ T cells, marked with a dotted line. c + d Characterisation of antigen-specific CD4+ T cells by differentiation status, determined by simultaneous or singular expression of CD45RA and CCR7 according to Sallusto et al [22] in naïve (Tnaïve), central memory (Tcm, coloured in red), effector memory (Tem, coloured in salmon) and CD45RA+ effector memory (Temra) T cells. We plotted the proportion of influenza- and citrulline-specific T cells among the four different phenotypes in (c) box plots showing the mean distribution and (d) scatter plots showing the detailed proportion and distribution of influenza- (left, open symbols) and citrulline-specific (right, closed symbols) T cells among the different phenotypes

Fig. 2

Autoreactive T cells in a cross-sectional cohort of RA patients. a Frequency of antigen-specific CD4+ T cells, recognising influenza, citrullinated CILP/FGB or citrullinated α-enolase peptides, is shown for all 14 patients longitudinally. Plotted are tetramer-positive cells per million CD4+ T cells. Cut-off for positivity is one tetramer-positive cell per million CD4+ T cells and marked with a dotted line. The frequencies detected at the three time points (t1, t2, t3) are connected with a continuous line for each patient and with a dotted line in case there were no cells detected at one or two out of the three time points. b + c) Phenotypic characterisation of antigen-specific CD4+ T cells according to (b) simultaneous or singular expression of CD45RA and CCR7 and (c) expression of CD25, CXCR3, CXCR5 and CCR6. Plotted are frequencies from all 42 experiments (three per patient) for influenza- (open circles) and citrulline-specific (closed circles, not included in c) as well as for CILP/FGB- (squares) and α-enolase- (triangles) specific T cells and the general CD4+ population (light grey diamonds). Due to the setup of the experiment which resulted in there being up to three individual data points per patient plotted for each of the different antigen-specific T cell populations, we did not perform any specific statistical analysis

Fig. 3

Citrulline-specific CD4+ T cells in LN biopsies from arthralgia and early RA patients. Lymph node biopsies were stained for citrullinated vimentin-, citrullinated α-enolase- and citrullinated CILP/FGB-reactive T cells after in vitro propagation using the tetramers according to Table 2, i.e. two cit vim-, three cit α-eno-, three cit CILP/FGB-peptide-loaded tetramers. Displayed are plots from both RA and arthralgia patients (upper panel) and one patient with undifferentiated arthritis (UA) as well as one healthy control (HC, lower panel) showing tetramer-positive cells against CD4. Numbers in the upper right quadrant present the mean fluorescence intensity (MFI) of the tetramer-positive staining, which can help in assessing positive staining. Cut-off for positivity for the different tetramer-cocktails are MFI > 400 for cit vim, MFI > 600 for cit α-eno and MFI > 320 for cit CILP/FGB. Plots marked with a red asterisk show positive staining. The numbers of positive tested individuals/tested individuals in total is depicted below the plots for each specificity for both RA and arthralgia patients

Fig. 4

Early RA patients achieving drug-induced remission show a reduction in citrulline-specific T cells. a + b) Frequency of (a) influenza- and citrulline-specific T cells in patients achieving remission (DAS28 at 6 m < 2.6) versus patients not achieving remission (DAS28 at 6 m > 2.6) and (b) of T cells reactive to citrullinated α-enolase, citrullinated CILP/FGB and citrullinated vimentin is depicted at baseline (0 m) and follow-up visit (6 m) for 10 early RA patients. Plotted are tetramer-positive cells per million CD4+ T cells. Cut-off for positivity is one tetramer-positive cell per million CD4+ T cells and depicted by a dotted line. Green symbols and lines depict the patients that achieved clinical remission at follow-up. The patient with missing DAS28-value at 6 m is coloured in grey. The numbers of positive tested individuals/totally tested individuals for each time point is depicted below the graphs showing α-enolase-, CILP/FGB- and vimentin-specific T cells. (c) Phenotypic characterisation of antigen-specific CD4+ T cells according to simultaneous or singular expression of CD45RA and CCR7 in central memory (Tcm) and effector memory (Tem) T cells. Plotted are frequencies and the mean for citrullinated α-enolase-, citrullinated CILP/FGB-, citrullinated vimentin- and influenza-specific T cells as well as the general CD4+ population at 0 m (squares) and 6 m follow-up (triangles). Green symbols depict the patients that achieved clinical remission at follow-up. Statistical analysis was performed using Mann-Whitney test when comparing the frequency of central memory T cells at baseline with the follow-up time point. p-values less than 0.05 were considered significant and marked with an asterisk