SOCS2-deficient T cells more readily produce Th2 cytokines, and SOCS2-deficient mice exhibit exacerbated atopic dermatitis and allergic airway inflammation.

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 multimers2. 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 JanssenFigure 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.

Background:Self-reactive CD4+ T cells are thought to play a key role in the pathogenesis of rheumatoid arthritis (RA) and represent a target for antigen-specific, tolerogenic therapies1. Phenotyping such cells could provide the means with which to monitor these treatments. Here, we aimed to demonstrate antigen-induced T cell responses to RA relevant epitopes using peptide stimulation of peripheral blood mononuclear cells (PBMC). Several autoantigens have been described in RA, including various citrullinated peptide (cit-peptide) epitopes2; here we have used a combination of cit-peptides that will be used to load tolerogenic dendritic cells in a forthcoming experimental medicine study (AuToDeCRA II).Objectives:To detect peripheral blood T cell proliferation in response to candidate autoantigens in RA patients and healthy controls (HCs) using flow cytometry.Methods:PBMC from RA patients and HCs were obtained following informed consent and were cryopreserved. Prior to use, cells were thawed in medium supplemented with human serum and were labelled with a proliferation tracking dye (PTD). 2x105labelled cells were plated into 96 well plates with a minimum of 7 replicates per condition and were either cultured alone or stimulated with individual peptides at varying concentrations or a peptide pool. On day 9, cells from replicate wells were harvested, pooled and stained for surface markers and viability. Samples were acquired on a BD Fortessa X20 and analysis was performed in FlowJo version 10. A manual gating strategy was used to identify CD45RO+ (memory) CD4+ T cells and amongst this population, activated/proliferated cells were defined as CD25+ PTD low. Stimulation indices (SI) were calculated as the percentage of activated/proliferated cells from stimulated wells divided by the percentage from unstimulated wells.Results:Ten RA patients (including early and established disease) were recruited alongside seven HCs. Cit-peptides were initially titrated on an individual basis to determine the optimum concentration for use (n = 2). We then compared stimulation with either single peptides or a pool (n = 2), and found that PBMC stimulated with the pooled cit-peptides had a higher proliferative response. Both single and pooled cit-peptide induced T cell proliferation was observed in both RA and HC samples (Figure 1); the largest responses were seen amongst RA patients, with a maximum stimulation index of 52.4 compared to a maximum of 6.75 in the HC group. Only 1 HC sample responded with an SI greater than 3.0, whereas 50 % of RA patients elicited responses above this. Two of the RA patients failed to respond to the peptide pool (SI < 1.0). Of note, RA patients were not selected on the basis of tissue type whereas selected peptides bind preferentially to class II HLA containing the shared epitope (SE).Figure 1Antigen-specific CD4+ CD45RO+ responses of citrullinated peptide stimulated PBMC from HCs and RA patients.Conclusion:In non-HLA typed individuals, cit-peptide induced proliferative T cell responses were detectable in both RA patients and HCs, and although SIs overall were higher amongst RA patients this did not reach statistical significance in this small sample. Not all RA patients responded to the peptide pool which may be due to the limited number of citrullinated epitopes used, or to RA patients with a non-SE HLA type. Additional work should establish the need for HLA typing in this assay; around half of seropositive RA patients would be expected to be SE positive3. Furthermore, a wider array of cit-peptides may be needed to demonstrate autoreactivity in a broader cross-section of RA patients. Our future plans are to further phenotype the cellular subsets responding to the peptide pool and to study assay data in the context of clinical outcomes, to assess its utility for immune monitoring.References:[1]McInnes, I. B. & Schett, G. N Engl J Med. 2011; 365(23):2205-2219[2]James, E. Aet al. Arthritis Rheum. 2014; 66(7):1712-1722[3]Thomson, Wet al. Arthritis Rheum. 1999; 42(4):757-762Disclosure of Interests:Jessica Swift: None declared, James Stanway: None declared, Catharien Hilkens: None declared, Amy Anderson: None declared, Arthur Pratt Grant/research support from: Pfizer, GlaxoSmithKlein, John Isaacs Consultant of: AbbVie, Bristol-Myers Squibb, Eli Lilly, Gilead, Janssen, Merck, Pfizer, Roche