Medical Research Council/Uganda Virus Research Institute and London School of Hygiene and Tropical Medicine, Uganda Research Unit (MRC/UVRI & LSHTM Uganda Research Unit), Entebbe, Uganda.
Department of Epidemiology & Biostatistics, and Global Health Sciences, University of California, San Francisco (UCSF), United States.
Vaccine. 2019 Jan 3;37(1):113-122. doi: 10.1016/j.vaccine.2018.11.024. Epub 2018 Nov 17.
Evaluation of antigen-specific T-cell responses to viral antigens is frequently performed on IFN-γ secreting cells. However, T-cells are capable of producing many more functions than just IFN-γ, some of which, like Perforin, are associated with immune protection in HIV-1 disease elite controllers. We evaluated the extent of missed T-cell functions when IFN-γ secretion is used as a surrogate marker for further evaluation of T-cell functions. Intracellular cytokine staining assay and flow cytometry were used to assess peripheral blood mononuclear cells (PBMCs) from 31 HIV-infected ART-naive individuals for the extent to which gated CD4+ and CD8+ IFN-γ producing and non-producing T-cells also secreted IL-2, Perforin, and TNF-α functions. Similarly, the extent of missed virus-specific responses in IFN-γ ELISpot assay negative T-cells from 5 HIV-1 uninfected individuals was evaluated. Cells from HIV-infected individuals were stimulated with pooled consensus group M (Con M) peptides; and those from healthy individuals were stimulated with pooled adenovirus (Ad) peptides. Overall, frequencies of virus-specific IFN-γ secreting CD4+ and CD8+ cells were low. Proportions of IFN-γ negative CD4+ expressing IL-2, Perforin, or TNF-α to Con M were significantly higher (5 of 7 functional profiles) than the corresponding IFN-γ positive CD4+ (0 of 7) T-cell phenotype, p = 0.02; Fisher's Exact test. Likewise, proportions of CD8+ T-cells expressing other functions were significantly higher in 4 of the 7 IFN-γ negative CD8+ T-cells. Notably, newly stimulated Perforin, identified as Perforin co-expression with IL-2 or TNF-α, was significantly higher in IFN-γ negative CD8+ T-cell than in the positive CD8+ T-cells. Using SEB, lower responses in IFN-γ positive cells were most associated with CD4+ than CD8+ T-cells. These findings suggest that studies evaluating immunogenicity in response to HIV and Adenovirus viral antigens should not only evaluate T-cell responsiveness among IFN-γ producing cells but also among those T-cells that do not express IFN-γ.
评估针对病毒抗原的抗原特异性 T 细胞反应通常是通过 IFN-γ 分泌细胞进行的。然而，T 细胞能够产生的功能远不止 IFN-γ，其中一些功能，如穿孔素，与 HIV-1 疾病精英控制器中的免疫保护有关。我们评估了在将 IFN-γ 分泌用作替代标志物来进一步评估 T 细胞功能时，错过 T 细胞功能的程度。我们使用细胞内细胞因子染色测定和流式细胞术评估了来自 31 名未接受 ART 治疗的 HIV 感染个体的外周血单核细胞 (PBMC)，以评估门控 CD4+和 CD8+IFN-γ 产生和非产生 T 细胞是否也分泌 IL-2、穿孔素和 TNF-α 功能。同样，还评估了来自 5 名 HIV-1 未感染个体的 IFN-γ ELISpot 测定阴性 T 细胞中错过的病毒特异性反应的程度。用混合群 M（Con M）肽刺激 HIV 感染个体的细胞；用混合腺病毒（Ad）肽刺激健康个体的细胞。总体而言，特异性针对病毒的 IFN-γ 分泌 CD4+和 CD8+细胞的频率较低。IFN-γ 阴性 CD4+表达 IL-2、穿孔素或 TNF-α与 Con M 的比例（7 种功能谱中的 5 种）明显高于相应的 IFN-γ 阳性 CD4+（7 种中的 0 种）T 细胞表型，p=0.02；Fisher 精确检验。同样，4 种 IFN-γ 阴性 CD8+T 细胞中表达其他功能的比例明显更高。值得注意的是，新刺激的穿孔素，被鉴定为与 IL-2 或 TNF-α共表达的穿孔素，在 IFN-γ 阴性 CD8+T 细胞中明显高于阳性 CD8+T 细胞。使用 SEB，IFN-γ 阳性细胞中的较低反应最与 CD4+T 细胞有关，而不是 CD8+T 细胞。这些发现表明，评估针对 HIV 和腺病毒病毒抗原的免疫原性的研究不仅应评估 IFN-γ 产生细胞中的 T 细胞反应性，还应评估不表达 IFN-γ 的 T 细胞。
