Larsson M, Jin X, Ramratnam B, Ogg G S, Engelmayer J, Demoitie M A, McMichael A J, Cox W I, Steinman R M, Nixon D, Bhardwaj N
Laboratory of Cellular Physiology and Immunology, The Rockefeller University, New York, NY 10021-6399, USA.
AIDS. 1999 May 7;13(7):767-77. doi: 10.1097/00002030-199905070-00005.
HIV-1-specific CD8 T cells are considered to be critical in anti-HIV responses. It is important to quantify these cells and to determine their antigenic targets. Here quantification of interferon (IFN)-gamma secreting, virus-specific cells was achieved with an enzyme linked immuno spot (ELISPOT) assay.
Peripheral blood mononuclear cells (PBMC) were infected with recombinant vaccinia vectors expressing HIV-1 genes (gag, pol, env or nef) and added to wells precoated with anti-IFN-gamma monoclonal antibodies. Spot forming cells (SFC), i.e. antigen-specific T cells were detected 24 h later by the addition of biotinylated anti-IFN-gamma monoclonal antibodies, followed by avidin-bound biotinylated horseradish peroxidase.
In a cohort of 19 patients, of whom 15 were on highly active antiretroviral therapy, 18 had primed T cells directed against one or more HIV-1 antigens (P < 0.0001). Pol-specific T cells routinely dominated the CD8 response with frequencies up to 2000 SFC per 10(6) PBMC. In HLA A*0201-positive patients, the vaccinia vectors detected much higher frequencies of SFC than haplotype-restricted peptides. Elimination of CD8 T cells resulted in > 90% loss of antigen-specific SFC when vaccinia virus was used as a vector. The number of CD8 SFC exceeded the number of memory cells detected in limiting dilution assays by > 1 log10, whereas a correlation was found between the frequency of effector cells detected by both ELISPOT and MHC class I peptide tetramer assays.
Vaccinia virus vectors used in ELISPOT assays are useful for determining the frequency and specificity of CD8 T cells for individual HIV-1 gene products. The dominance of cytolytic T lymphocytes (CTL) recognizing pol proteins suggests that this antigen should be considered in vaccine strategies.
HIV-1特异性CD8 T细胞被认为在抗HIV反应中起关键作用。对这些细胞进行定量并确定其抗原靶点很重要。在此，通过酶联免疫斑点（ELISPOT）测定实现了对分泌干扰素（IFN）-γ的病毒特异性细胞的定量。
外周血单个核细胞（PBMC）用表达HIV-1基因（gag、pol、env或nef）的重组痘苗病毒载体感染，并添加到预包被抗IFN-γ单克隆抗体的孔中。24小时后，通过添加生物素化抗IFN-γ单克隆抗体，随后加入与抗生物素蛋白结合的生物素化辣根过氧化物酶，检测斑点形成细胞（SFC），即抗原特异性T细胞。
在19名患者队列中，其中15名接受高效抗逆转录病毒治疗，18名有针对一种或多种HIV-1抗原的初始T细胞（P<0.0001）。Pol特异性T细胞通常在CD8反应中占主导，频率高达每10⁶ PBMC中有2000个SFC。在HLA A*0201阳性患者中，痘苗病毒载体检测到的SFC频率比单倍型限制肽高得多。当使用痘苗病毒作为载体时，消除CD8 T细胞导致抗原特异性SFC损失>90%。CD8 SFC的数量比在有限稀释试验中检测到的记忆细胞数量超过>1 log10，而在ELISPOT和MHC I类肽四聚体试验检测到的效应细胞频率之间发现了相关性。
ELISPOT测定中使用的痘苗病毒载体可用于确定针对个体HIV-1基因产物的CD8 T细胞的频率和特异性。识别pol蛋白的细胞毒性T淋巴细胞（CTL）占主导，表明该抗原应在疫苗策略中予以考虑。
