Characteristics and functions of an atypical inflammation-associated GZMK+GZMB+CD8+ T subset in people living with HIV-1

Abstract

HIV-1 chronically infects host CD4⁺ T lymphocytes and further affects a variety of immune cells, including CD8⁺ T cells. In our previous study, by analyzing unbiased high-dimensional single-cell RNA-seq data (scRNA-seq), we found that the frequency of GZMK⁺CD8⁺ T cells expressing granzyme K (GZMK) was increased in people living with HIV-1 (PLWHs). However, the phenotypic and functional characteristics of these cells in chronic HIV-1 infection and their correlation with disease are not well understood. In this study, we conducted a comprehensive analysis of scRNA-seq and matched T-cell receptor repertoire (TCR) sequencing data to delve into the characterizations of GZMK⁺CD8⁺ T cells, which was further validated by flow cytometry. We observed heterogeneity within the GZMK⁺CD8⁺ T cells, which could be further subdivided into a GZMK⁺GZMB^- subset and a GZMK⁺GZMB⁺ subset, with the latter being significantly enriched in PLWHs. The GZMK⁺GZMB⁺ cells are a unique subset within CD8⁺ T cells, characterized by high proliferation, activation, inflammatory response, clone transition, etc., and are one of the differentiation endpoints by pseudotemporal analysis of CD8+αβ T cells. Despite being predominantly composed of effector memory T cells (Tem), similar to the GZMK⁺GZMB^- subset, the GZMK⁺GZMB⁺ subset exhibits differentiation at a later stage than the GZMK⁺GZMB^- subset. We also observed that the frequency/count of GZMK⁺GZMB⁺CD8⁺ T cells was negatively correlated with CD4/CD8 ratio, and positively correlated with HIV DNA, IP-10, and MIG levels in PLWHs. In vitro experiments demonstrate that GZMK can potentiate the stimulatory effects of lipopolysaccharide (LPS) on THP-1 macrophages via the TLR-4 pathway, significantly enhancing the secretion of IP-10, MIG, and MCP-1, as well as increasing the proportion of TNF-α⁺ cells. In conclusion, in PLWHs, GZMK⁺GZMB⁺CD8⁺ T cells are a highly reactive and inflammatory-inducing subset that may be associated with systemic inflammation.