Background/Purpose: Granzyme K (GZMK)-expressing CD8 T cells have recently been detected in tissues from an increasing number of autoimmune conditions, but their role in disease pathogenesis is unclear. We have identified and characterized these cells in rheumatoid arthritis (RA), where they represent up to 30% of cells in inflamed synovium (joint lining) and express a gene program different from granzyme B (GZMB)+ cytotoxic CD8 T cells. We recently showed that Gzmk knock-out mice exhibit a milder course antigen-induced arthritis, a mouse model of inflammatory arthritis, compared to wildtype mice, supporting a direct role for GZMK in the pathogenesis of inflammatory arthritis such as RA. GZMK+ CD8 T cells have many potential effector functions. Resting GZMK+ CD8 T cells continuously release GzmK, even in the absence of stimulation. They can also release cytokines such as IFNγ upon T-cell receptor (TCR)- or cytokine-mediated stimulation. Lastly, GzmK can initiate complement cascades, generating anaphylatoxins C3a and C5a. Our goal with this project is to localize GZMK+ CD8 T cells in RA synovial tissue and characterize their molecular interactions with neighboring cells.

Methods: Spatial transcriptomic data were collected from three samples of human synovial tissue from patients with RA using a custom 377-probe panel on the Xenium platform (10x Genomics). For in vitro experiments, CD14+ myeloid cells isolated from human peripheral blood mononuclear cells by magnetic-activated cell sorting and stimulated with recombinant or serum-purified GZMK, IFNγ, C3a, and/or C5a, for 24 hours and then analyzed by ELISA or RT-PCR.

Results: In spatial transcriptomic and immunofluorescence microscopy data, GZMK+ CD8 T cells were preferentially located among myeloid cells in perivascular areas of the sublining in human RA synovial tissue (Figure 1). To investigate and compare molecular interactions between GZMK+ CD8 T cells and myeloid cells, we stimulated CD14+ blood monocytes in vitro with soluble factors produced by GZMK+ CD8 T cells directly (GZMK, IFNγ) or via activation of complement cascades (C3a, C5a). We paid special attention to chemokines CCL2, CCL4, CXCL9, and CXCL10 since both monocytes and GZMK+ CD8 T cells express the cognate receptors for these chemokines, CCR2, CCR5, and CXCR3. We found that recombinant GZMK induced monocytes to produce CCL2, CCL4, and IL-6 (Figure 2). GZMK did not induce CXCL9, CXCL10, or STAT1 on its own, but it amplified the ability of IFNγ to induce these classic interferon signature markers. The combination of GZMK and IFNγ also had modestly augmented effects on IL-1β and IL-6, while they dampened CCL2 production. C5a displayed more potent effects than C3a in terms of CCL2, CCL4, IL-1β, and IL-6 production.

Conclusion: GZMK+ CD8 T cells localize near myeloid cells in the sublining of inflamed RA synovium. GZMK can directly induce monocytes to secrete CCL2, CCL4, and IL-6, and it can act together with IFNγ or through complement cascades to induce IL-1β and interferon signature genes. Together, these molecular interactions form a strong positive feedback loop, recruiting and activating additional GZMK+ CD8 T cells and blood monocytes in inflamed RA synovium.

Figure 1. GZMK+ CD8A+ cells are located near myeloid cells in the sublining of RA synovial tissue. FFPE RA synovial tissue was submitted for spatial transcriptomic analysis on the Xenium platform. The panels on the left show density gradients of transcripts encoding CD68 (myeloid marker) and GZMK, respectively, superimposed on the tissue. Red arrows point to areas enriched in both CD68 and GZMK. The panel on the right shows a close-up view with cells shaded by cluster, showing intermingling of GZMK+ T cells (teal) and myeloid cells (red).

Figure 2. GZMK directly stimulates blood monocytes and also amplifies the effects of IFNγ. Human CD14+ monocytes were isolated from blood and stimulated with recombinant or serum-purified GZMK, IFNγ, C3a, or C5a, or a combination of GZMK and IFNγ for 24 hours. The supernatants were collected for analysis by ELISA, while the cells were lysed for analysis by quantitative RT-PCR. The lines connect samples from the same blood donor (N&#3f6).

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/granzyme-k-cd8-t-cells-colocalize-with-myeloid-cells-and-activate-them-through-several-pathways/