McElrath M J, Hoffman M, Kluckling S, Corey L, Greenberg P D
Department of Medicine, University of Washington, Seattle 98195.
AIDS Res Hum Retroviruses. 1994 May;10(5):541-9. doi: 10.1089/aid.1994.10.541.
The induction of CD8+ CTL responses is a goal of most HIV-1 vaccine trials, but such potentially protective effector responses have been difficult to evaluate, particularly in these vaccine prevention trials, due to technical obstacles. We report a method to evaluate CTL responses based on the ability to infect autologous macrophages with a monocytotropic strain of HIV-1, and to use these cells as efficient stimulators. This approach does not require the addition of exogenous cytokines, allows detection of class I-restricted CTLs against multiple HIV-1 gene products, and circumvents the problem, often detected using other stimulator cells, of high levels of lytic activity against target cells expressing vaccinia and/or EBV antigens. Adherent monocyte-derived macrophages were infected with HIV-1 Ba-L, and used within 2-3 weeks as autologous stimulators. Fresh PBMCs were cultured with the infected macrophages, harvested after 1 week, replated with fresh infected macrophages and filler cells, and tested after 5-7 days for cytolytic activity. CD8+ CTL responses specific for HIV-1 envelope were detected at an E:T ratio as low as 5:1 in two of four HIV-1-uninfected recipients of an HIV vaccine regimen that included a recombinant live vaccinia virus. Cytotoxic T lymphocyte activity could be detected > 1 year following vaccination. Similar lytic activity was detected with cryopreserved responder cells. In two HIV-1-infected individuals participating in a blinded therapeutic vaccination trial, the use of infected macrophages as in vitro stimulators permitted detection of the presence of envelope and gag-specific CTLs. No responses were observed in nonimmunized, uninfected controls. Thus, HIV-1-infected macrophages can stimulate in vitro the repertoire of primed HIV-reactive CD8+ precursors from seronegative and seropositive participants in HIV-1 vaccine trials, and should facilitate the identification of potentially effective candidate HIV vaccines.
诱导CD8 +细胞毒性T淋巴细胞（CTL）反应是大多数HIV-1疫苗试验的目标，但由于技术障碍，这种潜在的保护性效应反应一直难以评估，尤其是在这些疫苗预防试验中。我们报告了一种基于用嗜单核细胞株HIV-1感染自体巨噬细胞并将这些细胞用作高效刺激剂的能力来评估CTL反应的方法。这种方法不需要添加外源性细胞因子，能够检测针对多种HIV-1基因产物的I类限制性CTL，并规避了通常使用其他刺激细胞检测到的针对表达痘苗和/或EBV抗原的靶细胞的高水平裂解活性问题。用HIV-1 Ba-L感染贴壁的单核细胞衍生巨噬细胞，并在2至3周内用作自体刺激剂。将新鲜的外周血单核细胞（PBMC）与感染的巨噬细胞一起培养，1周后收获，重新接种新鲜的感染巨噬细胞和填充细胞，并在5至7天后检测细胞溶解活性。在接受包括重组活痘苗病毒的HIV疫苗方案的四名未感染HIV-1的受试者中的两名中，以低至5：1的效靶比检测到了针对HIV-1包膜的特异性CD8 + CTL反应。接种疫苗1年多后仍可检测到细胞毒性T淋巴细胞活性。用冷冻保存的反应细胞检测到类似的裂解活性。在参与一项盲法治疗性疫苗试验的两名HIV-1感染者中，使用感染的巨噬细胞作为体外刺激剂能够检测到包膜和gag特异性CTL的存在。在未免疫、未感染的对照中未观察到反应。因此，HIV-1感染的巨噬细胞可以在体外刺激HIV-1疫苗试验中血清阴性和血清阳性参与者中已致敏的HIV反应性CD8 +前体细胞库，并且应有助于鉴定潜在有效的候选HIV疫苗。
