Background/Purpose: Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a rare and severe autoimmune disease, characterized by a pauci-immune necrotizing vasculitis leading to inflammation and damage of major organs. ANCAs targeting proteinase-3 (PR3) or myeloperoxidase (MPO), and ANCA-producing B cells play a central role in the pathogenesis of AAV. Autoreactive T cells and their features are less well-studied, but recent studies emphasize their importance in AAV. In this study we aimed to investigate autoreactive T cells and their cytotoxic responses in AAV.

Methods: Single cell RNA and TCR sequencing combined with CITE-seq staining was performed of isolated T cells from healthy controls (HC) (n=4), anti-MPO⁺ AAV (n=5) and anti-PR3⁺ AAV (n=4) patients. Secondly, PBMCs of these patients were stimulated in vitro with anti-CD3, Np/pp65, MPO or PR3 autoantigens in combination with anti-CD28 and IL-2 for 72h. After culturing, T cells were analyzed with flow cytometry for activation, proliferation and cytotoxic responses.

Results: We identified 15 clusters of CD4+ and CD8+ T cells based upon differential gene expression in AAV patients (n=9) and HCs (n=4) (Fig 1A-C). Anti-PR3⁺ AAV patients exhibited increased effector memory cells re-expressing CD45RA⁺ (EMRA) GZMB⁺ CD8⁺ T cells (20.8%) versus HC (6.7%) (p=0.03) and anti-MPO⁺ AAV patients (7.2%) (p=0.04) (Fig 1B,D). Both anti-MPO⁺ and anti-PR3⁺ AAV patients had large populations of clonally hyperexpanded T cells, which were found mostly in the GZMB⁺ but also in the GZMK⁺ CD8⁺ T cell subsets, which was not observed in ­­­HCs (Fig 1E-F). Expanded T cells in AAV patients had increased expression of cytotoxic and tissue homing genes, such as GZMB, NKG7, CST7, CCL5, PRF1, GPR56, CX3CR1 and TBX21 (Fig 2A). Gene ontology (GO) pathway analysis demonstrated that the significantly upregulated genes of expanded T cells in AAV were involved in cell activation and positive regulation of the immune response (Fig 2B). Specifically, CD8 EMRA GZMB⁺ T cells of AAV patients had significantly increased expression of highlighted cytotoxic and tissue homing genes as compared to HCs (Fig 2C). Secondly, flow cytometry confirmed the increased proportion of CD8 EMRA and GZMB⁺ CD8⁺ T cells in anti-PR3 AAV patients (Fig 3A-C), whereas anti-MPO⁺ AAV patients had increased levels of GZMB⁺IFNy⁺ CD8⁺ T cells as compared to HCs (Fig 3C). 24h of anti-CD3/anti-CD28 stimulation, used as a positive control in the culture assay, induced GZMB⁺IFNy⁺ responses in CD8⁺ T cells in all samples, which was most pronounced in HCs, and not observed after MPO or PR3 stimulation in AAV patients (Fig 3D). On the other hand, MPO specifically induced expansion and proliferation of GZMK⁺IFNy⁺ CD8⁺ T cells in anti-MPO⁺ AAV patients, in contrast to anti-PR3⁺ AAV patients and HCs (Fig 3E).

Conclusion: This study demonstrated that clonally expanded cytotoxic CD8⁺ T cells are present in both anti-MPO⁺ and anti-PR3⁺ AAV patients, and we identified a specific autoreactive GZMK⁺IFNy⁺ CD8⁺ T cell subpopulation in anti-MPO⁺ AAV patients. The identification and characterization of pathogenic autoreactive T cells in AAV could provide novel insights into next-generation therapeutic targets for this autoimmune disease.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/identification-of-autoreactive-cytotoxic-t-cells-in-anca-associated-vasculitis/