Background: CD4+ T cells reacting to post-translationally modified, citrullinated self-antigens are thought to play a central role in the pathogenesis of rheumatoid arthritis (RA) ¹ . This is evidenced by a strong HLA class II association, the success of therapeutic co-stimulation blockade and the detection of autoantigen specific T-cells using HLA class II multimers ² . These cells may represent a target for antigen-specific, tolerogenic therapies and their in-depth phenotyping may provide the means by which to monitor such treatment.

Objectives: To identify the citrullinated-peptide (cit-peptide) induced cytokine repertoire of antigen-specific memory CD4+ T cells in both healthy controls (HCs) and ACPA positive RA patients using intracellular cytokine staining and flow cytometry. Of note, the HLA-types of both HCs and RA patients were not known.

Methods: Cryopreserved peripheral blood mononuclear cells (PBMC) from both HCs (n = 8) and RA patients (n = 13) with both early (untreated) and established disease were thawed and labelled with a proliferation tracking dye (PTD). Labelled PBMC were then either incubated alone or with a pool of cit-peptides for 9-days, followed by a 5-hour restimulation with PMA and ionomycin, where cytokine secretion was blocked for the final 4-hours using brefeldin-A. Cells were then harvested, permeabilised and stained for T cell surface markers and intracellular cytokines including IFN-γ, IL-4, IL-21 and IL-17. Stained cells were immediately acquired using a BD Fortessa X20, where antigen-specific CD4+ T cells were identified as the viable CD45RO+ (memory) CD4+ T cell population that had proliferated (PTDlow) in response to the cit-peptides. Stimulation indices (SI) were calculated as the percentage of proliferated memory CD4+ T cells in the stimulated wells divided by the percentage in the unstimulated conditions, and cit-peptide responders were defined as those with an SI > 2.0. Net cytokine production was measured by subtracting the percentage cytokine production from unstimulated CD4+ CD45RO+ PTDlow cells, from those stimulated with the cit-peptides.

Results: Comparable proliferative responses were observed in both donor groups in response to stimulation with the cit-peptide pool, where 37 % of HCs and 31 % of RA patients responded with an SI > 2.0 ( Fig. 1A ). While little cytokine production was observed in the cit-peptide responding HC T cells, for responding RA donors, cit-peptide responsive CD4+ memory T cells were predominantly IFN-γ and IL-21 producing ( Fig. 1B and 1C ). In contrast, these donors did not produce significant levels of either IL-17 or IL-4 ( Fig. 1D and 1E ).

Conclusion: Cit-peptides were able to induce proliferation in both HCs and RA memory CD4+ T cells which, amongst the RA donors only, were of a Th1/Tfh subtype. In contrast, and while based only on a small sample, cit-peptides did not induce either IL-17 or IL-4 production in either donor group, suggesting a lack of Th17/Th2 responses. Not all donors responded to the peptide pool, possibly reflecting the limited number of pooled cit-peptides or to a lack of confirmed HLA-DRB1*04:01 positive donors, as peptides were selected for their specificity on this basis. Future work will therefore include HLA-typing, as well as the inclusion of additional citrullinated-epitopes to demonstrate autoreactivity in a wider cross-section of patients. Further phenotyping of the cit-peptide specific T cells will be performed, and future plans will be to study the assay data alongside clinical outcomes to assess its value for immune monitoring.

REFERENCES:

[1]Malmström, V et al Nat Rev Immunol. 2017; 17(1):60-75.

[2]Gerstner, C et al BMC Immunol. 2020; 21(27):1-14.

Disclosure of Interests: Jessica Swift: None declared, James Stanway: None declared, Ioana Nicorescu: None declared, Catharien Hilkens: None declared, Frederik Stevenaert Employee of: Janssen, Amy Anderson Grant/research support from: Pfizer, GSK and Janssen, Arthur Pratt Grant/research support from: Pfizer, GSK and Janssen, John D Isaacs Speakers bureau: Abbvie, Gilead, Roche, UC, Consultant of: Abbvie, Gilead, Roche, UC, Grant/research support from: Pfizer, GSK and Janssen

Figure 1.

Citrullinated-peptide specific memory CD4+ T cell proliferation (A) and net % cytokine production of IFN-γ (B), IL-21 (C), IL-17 (D) and IL-4 (E) positive cells.