¹Institut für Immunologie, Universitätsmedizin Johannes Gutenberg-Universität, Mainz
²Gene Transfer Department, Translationale Onkologie an der Universitätsmedizin der Johannes-Gutenberg-Universität, Mainz
³IDC Immunomonitoring, Translationale Onkologie an der Universitätsmedizin der Johannes-Gutenberg-Universität, Mainz

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Introduction: Increasing the number and function of regulatory T cells (Tregs) may provide an effective strategy for treating rheumatoid arthritis (RA) by maintaining immune tolerance [1]. The chimeric antigen receptor (CAR) approach is an important factor that could enable Tregs to exert targeted effects. However, peripheral CD4 T cells used for this method lose their ability to induce tolerance over time due to plasticity. In this study, we aim to design a CAR that is resistant to inflammatory signals and maintains FoxP3 stability [2].

Methods: STAT5 is a crucial transcription factor for FoxP3 stability in Tregs and is primarily activated in response to IL-2 stimulation. In contrast, cytokines such as IL-6 and IL-8 antagonize the effects of STAT5 and reduce FoxP3 activity through STAT3 activation [3]. Therefore, we designed a CAR containing the cytoplasmic domains of IL2RB to promote STAT5 activation. By adding the scFv portion of the E44 antibody specific to citrullinated proteins isolated from an RA patient to the design, we aimed to ensure targeted efficacy. We compared the phosphorylation states of STAT5 and STAT3 in CD4 T cells expressing classic and IL2RB-containing CAR (CARB) molecules when exposed to the target antigen using FACS method.

Results: When cultured with citrullinated collagen-II483-498, both classic and CARB T cells reached maximum pSTAT5 and pSTAT3 MFI values by the 4^th hour. It was observed that the additional IL2RB domain in the CARB structure did not create a significant difference in pSTAT5 levels. However, it did result in a meaningful increase in pSTAT3 levels. These data suggest that CARB may have triggered STAT3 phosphorylation through an alternative JAK/STAT activation pathway.

Conclusion: The finding that the IL2RB domain increases STAT3 phosphorylation instead of STAT5, as we expected, indicates that different kinases may play a role in chimeric receptor signal activation. In the subsequent phases of the study, we will aim to identify the regions responsible for inducing STAT3 phosphorylation through mutation analyses.

Literatur

[1] Zhang J, Liu H, Chen Y, Liu H, Zhang S, Yin G, Xie Q. Augmenting regulatory T cells: new therapeutic strategy for rheumatoid arthritis. Front Immunol. 2024 Jan 23;15:1312919. DOI: 10.3389/fimmu.2024.1312919
[2] Lee GR. The Balance of Th17 versus Treg Cells in Autoimmunity. Int J Mol Sci. 2018 Mar 3;19(3):730. DOI: 10.3390/ijms19030730
[3] Contreras-Castillo E, García-Rasilla VY, García-Patiño MG, Licona-Limón P. Stability and plasticity of regulatory T cells in health and disease. J Leukoc Biol. 2024 Jun 28;116(1):33-53. DOI: 10.1093/jleuko/qiae049
[4] van de Stadt LA, van Schouwenburg PA, Bryde S, Kruithof S, van Schaardenburg D, Hamann D, Wolbink G, Rispens T. Monoclonal anti-citrullinated protein antibodies selected on citrullinated fibrinogen have distinct targets with different cross-reactivity patterns. Rheumatology (Oxford). 2013 Apr;52(4):631-5. DOI: 10.1093/rheumatology/kes371