Perforin Expression Directly Ex Vivo by HIV-Specific CD8+ T-Cells Is a Correlate of HIV Elite Control

Abstract

Many immune correlates of CD8⁺ T-cell-mediated control of HIV replication, including polyfunctionality, proliferative ability, and inhibitory receptor expression, have been discovered. However, no functional correlates using ex vivo cells have been identified with the known ability to cause the direct elimination of HIV-infected cells. We have recently discovered the ability of human CD8⁺ T-cells to rapidly upregulate perforin—an essential molecule for cell-mediated cytotoxicity—following antigen-specific stimulation. Here, we examined perforin expression capability in a large cross-sectional cohort of chronically HIV-infected individuals with varying levels of viral load: elite controllers (n = 35), viremic controllers (n = 29), chronic progressors (n = 27), and viremic nonprogressors (n = 6). Using polychromatic flow cytometry and standard intracellular cytokine staining assays, we measured perforin upregulation, cytokine production, and degranulation following stimulation with overlapping peptide pools encompassing all proteins of HIV. We observed that HIV-specific CD8⁺ T-cells from elite controllers consistently display an enhanced ability to express perforin directly ex vivo compared to all other groups. This ability is not restricted to protective HLA-B haplotypes, does not require proliferation or the addition of exogenous factors, is not restored by HAART, and primarily originates from effector CD8⁺ T-cells with otherwise limited functional capability. Notably, we found an inverse relationship between HIV-specific perforin expression and viral load. Thus, the capability of HIV-specific CD8⁺ T-cells to rapidly express perforin defines a novel correlate of control in HIV infection. While the majority HIV-infected individuals progress to AIDS, a fraction of these individuals—for reasons not completely understood—do not develop AIDS and also display sustained control over viral replication; these subjects are sometimes referred to as elite controllers (EC). Prior evidence has shown that HIV-specific CD8⁺ T-cells, a component of adaptive immunity against intracellular pathogens, from EC exhibit enhanced functionality compared to individuals with progressive disease. Therefore, HIV-specific CD8⁺ T-cells likely play an important role in the favorable clinical outcomes witnessed in EC. We show in this study that the ability to control HIV replication in EC is associated with the expression of a protein called perforin, a critical molecule that enables CD8⁺ T-cells to directly kill infected cells - thereby preventing the spread of HIV to previously uninfected cells. In infected subjects with nonprogressive disease, we show that HIV-specific CD8⁺ T-cells demonstrate a superior ability to express perforin upon antigen-specific stimulation, whereas in progressors this property is diminished. Thus, we identify a functional capability of CD8⁺ T-cells, readily measured by standard intracellular cytokine staining assays, that potentially has a direct impact on HIV replication in vivo. These findings may, therefore, provide an important qualifier for future HIV vaccine research.