Background/Purpose: Anti-citrullinated protein antibody (ACPA) is highly specific to rheumatoid arthritis (RA). Beyond citrullination, other post-translational protein modifications including malondialdehyde-acetaldehyde (MAA) are targeted by T cells and autoantibodies in RA. MAA is abundant in RA synovial tissues and strongly co-localizes with both citrulline and B cells, suggesting that MAA expression could modulate adaptive immune responses to citrullinated proteins. In this study, we quantified changes T cell responses and autoantibody reactivity resulting from co-modification of a citrullinated protein with MAA.

Methods: Peripheral blood mononuclear cells (PBMCs; n=10) and serum (n=2484) were collected from RA participants in the Nebraska Rheumatology Biobank (NRB) and Veterans Affairs Rheumatoid Arthritis Registry (VARA), respectively. PBMCs (NRB) were isolated and stimulated with citrullinated fibrinogen (FIB-CIT), MAA-modified FIB (FIB-MAA), or co-modified FIB (FIB-MAA-CIT) for 48 hrs, followed by incubation with autologous T cells and evaluated for: 1) T cell proliferation using IFN-γ ELISpot kits; or 2) Th1, Th2, T-Reg, and Th17 cell types by flow cytometry. ELISpot and flow cytometry data were analyzed using one-way ANOVA with Tukey’s multiple comparison test. Serum levels (relative units) of IgG antibody to FIB-CIT, FIB-MAA, and FIB-MAA-CIT were quantified via ELISA (VARA). Antibody positivity was established as >2 SD above mean levels quantified in a separate cohort of 103 healthy controls. Seropositivity to modified and co-modified fibrinogen was compared by chi-square tests.

Results: Participant characteristics were similar in the 2 cohorts (90-91% male; mean age 63-73 years; 78-100% anti-CCP positive). CD4 cells proliferated significantly more to FIB-MAA-CIT than either FIB-CIT or FIB-MAA (p< 0.01) (Fig. 1A). This proliferation represented a predominant Th2 response based on increased GATA3 %-positivity in response to FIB-MAA-CIT vs. FIB-CIT or FIB-MAA (p< 0.0001) (Fig. 1B). In contrast, Th1 (CD183) (Fig. 1C), T-Reg (CD25) (Fig.1D) and Th17 (STAT3) %-positivity (Fig. 1E) showed no differences between FIB-CIT, FIB-MAA, and FIB-MAA-CIT. In VARA, the proportion of participants positive for serum IgG antibodies to FIB-CIT (68%) was significantly greater than positivity for either FIB-MAA-CIT (43%) or FIB-MAA (0.6%) (p˂0.0001) (Fig. 2).  

Conclusion: These results demonstrate that co-modification of CIT-protein with MAA significantly amplifies CD4 T cell proliferation and promotes a Th2 response to antigen stimulation. Given the known role of Th2 cells in promoting autoantibody expression, our data further suggest that resulting T cell responses promote the generation of ACPA but not anti-MAA antibody. Figure 3 provides a summary of the hypothesis for the mechanism behind these results.  Additional investigation is needed to understand whether these observations are driven by linked recognition (binding and uptake of co-modified antigen followed by preferential processing and presentation of CIT antigen) and whether interventions targeting MAA could reduce pathologic immune responses to CIT-protein in RA.

« Back to ACR Convergence 2024

ACR Meeting Abstracts - https://acrabstracts.org/abstract/co-modification-of-citrullinated-proteins-with-malondialdehyde-acetaldehyde-leads-to-amplified-t-cell-responses-and-increased-disease-specific-autoantibody-concentrations/