Background: The autoimmune disease rheumatoid arthritis (RA) is distinguished by the pivotal involvement of autoreactive CD4 ⁺ T helper cells [1, 2]. Our research group investigated the clonality and expression of these CD4 ⁺ T helper cells using single-cell RNA sequencing (scRNA-seq) in healthy donors (HD) [3]. Clonality assessment demonstrated that CD4 ⁺ CD8ααl ^ow T cells constitute substantial T cell clonal populations, which distinctly segregate from CD4 ⁺ CD8 ^- T cells [3]. In this process, we identified the SLAMF7 receptor as a characteristic marker on CD4 ⁺ CD8ααl ^ow T cells. SLAMF7 (CD319, CS-1, CRACC), is a transmembrane glycoprotein, that has been discovered on several immune cell populations, especially cytotoxic immune cells [4, 5]. Functionally, SLAMF7 can induce activating and inhibiting signals and plays a regulatory role [5, 6].

Objectives: The aim was to investigate SLAMF7 expression on CD4 ⁺ CD8αα ^low T cells. Furthermore, we were interested in the expression of cytotoxic markers, like granzymes or perforin and the proliferation rate.

Methods: For the study, peripheral blood mononuclear cells (PBMCs) from HD (n=53) were isolated by density gradient centrifugation. Surface and intracellular T cell markers were analyzed by flow cytometry. Using an in-vitro assay, CD4 ⁺ T cells from peripheral blood of HD (n=4) were cultured for 48h unstimulated or with anti-CD3/CD28, and IL-2. CD107a expression was assessed over the last 24-hour period with a fluorophore-conjugated monoclonal antibody. An additional in-vitro assay for examining the proliferation rate of SLAMF7 ^± CD4 ⁺ T cells was conducted using CFSE proliferation assay after 3 days stimulation (n=3). The measurement was performed by flow cytometry.

Results: First results showed an increased expression of SLAMF7 on CD4 ⁺ CD8αα ^low T cells (MFI=741.3) in comparison to CD4 ⁺ CD8 ^- T cells (MFI=220.9) in HD (n=53). Specifically, CD4 ⁺ CD8αα ^low T cells showed an increased production of granzym A/B (27%/29.2%) and perforin (4.6%) that was similar to the percentage of SLAMF7 ⁺ CD4 ⁺ T cells (n=21). In contrast to SLAMF7 ^- CD4 ⁺ T cells, the expression of granzyme A/B (3%/4.6%) and perforin (2.4%) was significantly downregulated, as evidenced by markedly reduced production levels. Further analysis of the data revealed a higher expression of CD107a on SLAMF7 ⁺ CD4 ⁺ compared to SLAMF7 ^- CD4 ⁺ T cells after 24h stimulation. Moreover, comparison of the SLAMF7 ⁺ CD4 ⁺ and SLAMF7 ^- CD4 ⁺ T cells yielded statistically significant differences in the proliferation rate. Flow cytometric analysis revealed that SLAMF7 ⁺ CD4 ⁺ T lymphocytes exhibited a significantly higher proliferation rate (*p < 0.04).

Conclusion: In summary, our findings demonstrate that elevated SLAMF7 expression on peripheral CD4 ⁺ CD8αα ^low T cells is associated with increased expression of cytotoxic molecules and an enhanced proliferation rate. However, precise functional role of SLAMF7 in this context remains to be elucidated. Further investigations are needed to clarify the mechanisms and potential therapeutic implications of SLAMF7 in CD4 ⁺ CD8αα ^low T cell clones. These studies will be crucial in unraveling the complex interplay between SLAMF7 expression and the observed phenotypic and functional characteristics of CD4 ⁺ CD8αα ^low T cells.

REFERENCES: [1] Weyand CM, Zeisbrich M, Goronzy JJ. Metabolic signatures of T-cells and macrophages in rheumatoid arthritis. Curr Opin Immunol. 2017;46:112-120. doi:10.1016/j.coi.2017.04.010.

[2] Nguyen P, Melzer M, Beck F, et al. Expansion of CD4+CD8+ double-positive T cells in rheumatoid arthritis patients is associated with erosive disease. Rheumatology (Oxford). 2022;61(3):1282-1287. doi:10.1093/rheumatology/keab551.

[3] Beck F, Nguyen P, Hoffmann A, et al. CD4+CD8αlow T Cell Clonal Expansion Dependent on Costimulation in Patients With Rheumatoid Arthritis. Arthritis Rheumatol. 2024;76(12):1719-1729. doi:10.1002/art.42960.

[4] Boles KS, Stepp SE, Bennett M, Kumar V, Mathew PA. 2B4 (CD244) and CS1: novel members of the CD2 subset of the immunoglobulin superfamily molecules expressed on natural killer cells and other leukocytes. Immunol Rev. 2001;181:234-249. doi:10.1034/j.1600-065x.2001.1810120.x.

[5] Loyal L, Warth S, Jürchott K, et al. SLAMF7 and IL-6R define distinct cytotoxic versus helper memory CD8+ T cells. Nat Commun. 2020;11(1):6357. Published 2020 Dec 11. doi:10.1038/s41467-020-19002-6.

[6] Guo H, Cruz-Munoz ME, Wu N, Robbins M, Veillette A. Immune cell inhibition by SLAMF7 is mediated by a mechanism requiring src kinases, CD45, and SHIP-1 that is defective in multiple myeloma cells. Mol Cell Biol. 2015;35(1):41-51. doi:10.1128/MCB.01107-14.

Acknowledgements: We thank Cornelia Arnold, Erik Schilling, Maximilian Neuenfeld, Annika Müller, Philipp Engelhard, Caroline Schmidt for discussions and/or technical assistance.

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.