Background: Juvenile Idiopathic Arthritis (JIA) is a chronic, T-cell-driven auto-immune disease that causes destructive, erosive joint inflammation. Its treatment involves immunosuppression with disease-modifying anti-rheumatic drugs (DMARDs). Bridling the immune response with DMARDs can potentially dampen the protective response post-vaccination and increase the risk of infections. Hence, there is a critical unmet medical need to delineate the effects of DMARDs on vaccine response and rationalise the vaccination strategy in these vulnerable patients. We hypothesise that treatment with methotrexate (MTX), a commonly used DMARD in JIA, can dampen the antigen-specific T cell response to the Coronavirus Disease of 2019 (COVID-19) mRNA vaccination, despite the effectiveness and strong immunogenicity of this vaccine to generate a strong, robust immune response in healthy adolescents and young adults (AYA).

Objectives: To address this unmet need and our hypothesis, we aim to determine the effects of methotrexate on the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) specific T-cell immune response after the first and second vaccine doses in patients with JIA. Secondly, for those on long-term MTX, we evaluated the effects of withholding MTX for a week before the first and second COVID-19 vaccinations. Translationally, this knowledge will help to refine the vaccine strategy in an evidence-based manner.

Methods: Patients with JIA were recruited in KK Women’s and Children’s Hospital and followed up longitudinally with samples collected before COVID-19 mRNA vaccination, after the first and second vaccine doses. Peripheral blood mononuclear cells (PBMCs) were cryopreserved for experimental batching and were stimulated with SARS-CoV-2 peptide pools before data acquisition using mass cytometry to quantify the antigen-specific responses. Increased expression of activation-induced markers (AIM+) such as CD154 and CD69 were used in tandem to define the antigen-specific CD4+ T cells. Summary statistics with median with interquartile ranges (IQR) and statistical testing with Mann-Whitney U test were used without assumption of the normality of data distribution.

Results: Children and adolescents were recruited (n=79, median age 16.4 (IQR 14.1 - 19.3) years, males=49 (62.0%)). The major subtype is enthesis-related arthritis (n=54, 68.4%), followed by systemic JIA (n=12, 15.2%) and oligo-articular JIA (n=6, 7.6%). Samples were obtained at 2.4 (2.3 - 2.7) weeks and 5.1 (4.2-7.4) weeks after the first and second vaccine dose respectively. We observed a modest increase in the frequency of CD4+ AIM+ SARS-CoV-2 Spike-specific T cells (frequency as % of memory T cells defined as non-CD45RA+CCR7+) with vaccination: pre-vaccine - 0.45 (0.34 - 0.63) %, after dose 1 - 0.45 (0.35 - 0.69) % and after dose 2 - 0.54 (0.36 - 0.87) % (Figure 1A). With MTX treatment, the frequency of CD4+ AIM+ SARS-CoV-2 Spike-specific T cells was significantly reduced only after the second vaccination 0.60 (0.41 - 0.89) % versus 0.41 (0.26 - 0.79) % for no MTX versus MTX respectively (p=0.015) (Figure 1B). Notably for those on long-term MTX therapy, withholding MTX one week before the first and second vaccine doses significantly increased the CD4+ AIM+ SARS-CoV-2 Spike-specific T cells: 0.27 (0.20 - 0.36) % versus 0.49 (0.35 - 1.20) % for MTX versus MTX withheld respectively (p=0.0063) (Figure 1C).

Figure 1.

(A ) Frequency of CD4+ AIM+ T cells at baseline before COVID-19 mRNA vaccination (pre-vac), after doses 1 and 2. (B ) Effect of MTX on CD4+ AIM+ T cell frequency. (C ) Effect on withholding MTX on antigen-specific T cell response. Data presented as median (IQR).

Conclusion: We have determined that although MTX negatively impacts the strength of the antigen-specific CD4+ T cell response after COVID-19 mRNA vaccination in patients with JIA, withholding MTX can ameliorate this and increase the CD4+ AIM+ antigen-specific T cells.

REFERENCES: NIL.

Acknowledgements: This research was supported by the National Research Foundation Singapore under its National Medical Research Council (NMRC) Centre Grant Programme (MOH-000988) and is administered by the Ministry of Health, Singapore’s NMRC. Other NMRC grant support CIRG21nov-0031 and CSAINV22jul-0008 is gratefully acknowledged.

Disclosure of Interests: None declared.

© The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license ( http://creativecommons.org/licenses/by-nc-nd/4.0/ ). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.