HIV-Specific Cd8+ T Cells Produce Antiviral Cytokines but Are Impaired in Cytolytic Function

TLDR

Tetramer staining has enabled identification of antigen‑specific CD8⁺ T cells, yet their precise functional roles, especially in HIV, remain unclear, as cytotoxic lymphocytes largely act through cytokine and chemokine secretion. The study presents a combined tetramer and intracellular cytokine staining approach to assess functional heterogeneity of antigen‑specific CD8⁺ T cells ex vivo. By stimulating peripheral blood cells with peptide antigens, the method quantifies IFN‑γ, TNF‑α, MIP‑1β, and perforin production in tetramer‑positive cells via flow cytometry, allowing comparison of HIV‑specific and CMV‑specific CD8⁺ T‑cell phenotypes. While most CMV‑ and HIV‑specific CD8⁺ T cells produce antiviral cytokines, HIV‑specific cells exhibit markedly reduced perforin, sustained CD27 expression, and diminished ex vivo lysis, indicating impaired cytolytic function.

Abstract

The use of peptide–human histocompatibility leukocyte antigen (HLA) class I tetrameric complexes to identify antigen-specific CD8+ T cells has provided a major development in our understanding of their role in controlling viral infections. However, questions remain about the exact function of these cells, particularly in HIV infection. Virus-specific cytotoxic T lymphocytes exert much of their activity by secreting soluble factors such as cytokines and chemokines. We describe here a method that combines the use of tetramers and intracellular staining to examine the functional heterogeneity of antigen-specific CD8+ T cells ex vivo. After stimulation by specific peptide antigen, secretion of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, macrophage inflammatory protein (MIP)-1β, and perforin is analyzed by FACS® within the tetramer-positive population in peripheral blood. Using this method, we have assessed the functional phenotype of HIV-specific CD8+ T cells compared with cytomegalovirus (CMV)-specific CD8+ T cells in HIV chronic infection. We show that the majority of circulating CD8+ T cells specific for CMV and HIV antigens are functionally active with regards to the secretion of antiviral cytokines in response to antigen, although a subset of tetramer-staining cells was identified that secretes IFN-γ and MIP-1β but not TNF-α. However, a striking finding is that HIV-specific CD8+ T cells express significantly lower levels of perforin than CMV-specific CD8+ T cells. This lack of perforin is linked with persistent CD27 expression on HIV-specific cells, suggesting impaired maturation, and specific lysis ex vivo is lower for HIV-specific compared with CMV-specific cells from the same donor. Thus, HIV-specific CD8+ T cells are impaired in cytolytic activity.