The Effect of Cytokines on Granzyme K and Granzyme B Expression in CD8+ T Cells

Abstract

CD8+ T cells make up nearly half of all T cells in the synovium of patients with rheumatoid arthritis (RA). To date, most studies have focused on the role of CD4+ T cells in RA. Recent unbiased studies of T cell subsets in RA synovium using single cell RNA sequencing revealed that the majority of CD8+ T cells expressed granzyme K (GzmK), either alone or in combination with granzyme B (GzmB). GzmK has pro-inflammatory effects on synovial fibroblasts, inducing them to produce IL-6, CCL2, and reactive oxidative species (ROS), all of which are up regulated in RA synovium. In this project, we studied whether cytokines regulated GzmK and GzmB expression. To test the effects of selected candidate cytokines on GzmK and GzmB expression in CD8+ T cells, we designed an experimental system. CD8+ T cells from healthy control blood were cultured for up to 4 days in the presence of selected cytokines, either alone or in combination. The cytokines were tested with and without concurrent TCR stimulation, using plate-bound anti-CD3 antibodies and soluble anti-CD28 antibodies. On days 0, 1, 2, 3, and 4, the harvested cells were stained for surface activation markers (CD69 and HLA DR) and intracellular markers (GzmK, GzmB, Nur77, and Ki67). Data were collected on a BD Fortessa flow cytometer and analyzed using FlowJo 10.5 software. Stimulating peripheral blood CD8+ T cells via their TCR led to GzmB expression. Furthermore, CD8+ T cells showed no increase in GzmK and GzmB co-expression following individual cytokine stimulation. However, stimulation with IL-12 in combination with IL-15 (in the absence of TCR stimulation) led to the maintenance of GzmK and GzmB expression, and an up regulation of the activation markers CD69, HLA DR, and Ki67. Studying the role of cytokines in the differentiation and activation of CD8+ T cells can help us better understand the factors that drive RA pathogenesis.