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

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.目前已发现多种与CD8阳性T细胞（CD8+ T-cell）介导的HIV复制控制相关的免疫标志物，包括多功能性（polyfunctionality）、增殖能力以及抑制性受体表达等。然而，目前尚未发现能够利用离体（ex vivo）细胞实现直接清除HIV感染细胞的功能性相关标志物。我们近期发现，人类CD8阳性T细胞在经抗原特异性刺激后，可快速上调穿孔素（perforin）的表达——穿孔素是细胞介导细胞毒性（cell-mediated cytotoxicity）的关键效应分子。本研究针对病毒载量水平各异的慢性HIV感染者组成的大型横断面队列，检测了其穿孔素表达能力，该队列包含以下组别：精英控制者（n=35）、病毒血症控制者（n=29）、慢性进展者（n=27）以及病毒血症非进展者（n=6）。本研究采用多色流式细胞术（polychromatic flow cytometry）与标准细胞内细胞因子染色（intracellular cytokine staining）实验方法，对经覆盖HIV全部蛋白的重叠肽库刺激后的样本，检测了穿孔素上调、细胞因子产生以及脱颗粒情况。研究观察到，与其他所有组别相比，精英控制者体内的HIV特异性CD8阳性T细胞在直接离体状态下，始终表现出更强的穿孔素表达能力。该能力并不局限于具有保护作用的HLA-B单倍型（HLA-B haplotypes），无需依赖细胞增殖或添加外源性因子（exogenous factors），且无法通过高效抗逆转录病毒治疗（HAART）恢复，其主要来源于功能能力本就有限的效应CD8阳性T细胞。值得注意的是，本研究发现HIV特异性穿孔素表达与病毒载量呈负相关关系。因此，HIV特异性CD8阳性T细胞快速表达穿孔素的能力，可作为HIV感染控制中的新型相关标志物。