Granzyme K drives a newly-intentified pathway of complement activation.

Granzymes are a family of serine proteases mainly expressed by CD8+ T cells, natural killer cells, and innate-like lymphocytes1,2. Although their major role is thought to be the induction of cell death in virally infected and tumor cells, accumulating evidence suggests some granzymes can regulate inflammation by acting on extracellular substrates2. Recently, we found that the majority of tissue CD8+ T cells in rheumatoid arthritis (RA) synovium, inflammatory bowel disease and other inflamed organs express granzyme K (GZMK)3, a tryptase-like protease with poorly defined function. Here, we show that GZMK can activate the complement cascade by cleaving C2 and C4. The nascent C4b and C2a fragments form a C3 convertase that cleaves C3, allowing further assembly of a C5 convertase that cleaves C5. The resulting convertases trigger every major event in the complement cascade, generating the anaphylatoxins C3a and C5a, the opsonins C4b and C3b, and the membrane attack complex. In RA synovium, GZMK is enriched in areas with abundant complement activation, and fibroblasts are the major producers of complement C2, C3, and C4 that serve as targets for GZMK-mediated complement activation. Our findings describe a previously unidentified pathway of complement activation that is entirely driven by lymphocyte-derived GZMK and proceeds independently of the classical, lectin, or alternative pathways. Given the widespread abundance of GZMK-expressing T cells in tissues in chronic inflammatory diseases and infection, GZMK-mediated complement activation is likely to be an important contributor to tissue inflammation in multiple disease contexts.

Granzyme K+ CD8 T cells form a core population in inflamed human tissue.

T cell-derived pro-inflammatory cytokines are a major driver of rheumatoid arthritis (RA) pathogenesis. Although these cytokines have traditionally been attributed to CD4 T cells, we have found that CD8 T cells are notably abundant in synovium and make more interferon (IFN)-γ and nearly as much tumor necrosis factor (TNF) as their CD4 T cell counterparts. Furthermore, using unbiased high-dimensional single-cell RNA-seq and flow cytometric data, we found that the vast majority of synovial tissue and synovial fluid CD8 T cells belong to an effector CD8 T cell population characterized by high expression of granzyme K (GzmK) and low expression of granzyme B (GzmB) and perforin. Functional experiments demonstrate that these GzmK+ GzmB+ CD8 T cells are major cytokine producers with low cytotoxic potential. Using T cell receptor repertoire data, we found that CD8 GzmK+ GzmB+ T cells are clonally expanded in synovial tissues and maintain their granzyme expression and overall cell state in blood, suggesting that they are enriched in tissue but also circulate. Using GzmK and GzmB signatures, we found that GzmK-expressing CD8 T cells were also the major CD8 T cell population in the gut, kidney, and coronavirus disease 2019 (COVID-19) bronchoalveolar lavage fluid, suggesting that they form a core population of tissue-associated T cells across diseases and human tissues. We term this population tissue-enriched expressing GzmK or TteK CD8 cells. Armed to produce cytokines in response to both antigen-dependent and antigen-independent stimuli, CD8 TteK cells have the potential to drive inflammation.