Frequency and Function of HIV-specific T-cells in GALT

描述(申请人提供)：本提案响应PA-02-073，“人类免疫学研究创新基金”。这些研究的目的是为了更好地了解肠道相关淋巴组织(GALT)中CD8+T细胞应答、病毒复制和CD4+T细胞耗竭之间的关系，并阐明GALT T细胞执行抗病毒免疫效应功能的能力。我们已经开发了评估新鲜从人类直肠组织中分离的抗原特异性T细胞的表型和功能的方法。对于特定的AIM 1，我们将评估GALT和血液中HIV特异性CD8+T细胞的频率、广度和记忆/效应表型。血液和直肠活检样本将来自：(I)对30名艾滋病毒感染者(包括15名“接受”HAART和15名“关闭”HAART)和10名对照组的横断面研究；(Ii)对20名艾滋病毒感染者(10名“接受”HAART，10名“关闭”)的纵向研究，每隔6个月采集一次样本。我们推测，在没有接受HAART治疗的患者中，GALT中CDS+T细胞反应的频率和广度可能超过血液中的CDS+T细胞反应。在接受HAART的患者中，我们预测在组织病毒载量下降的同时，这些反应的幅度和广度会下降。我们还预测，病毒载量的减少和CD4+T细胞的重新繁殖将使GALT中的CD4+T细胞对非HIV抗原的反应部分重建。对于特异的AIM 2，我们将评估GALT中HIV特异性CDS+T细胞的效应功能。初步结果表明：(I)血液中HIV特异性CTL表达低水平的穿孔素，(Ii)GALT中所有CD8+T细胞的穿孔素表达均低于血液，以及(Iii)颗粒酶A和穿孔素的表达存在差异。我们推测GALT中的CD8+T细胞可能存在功能损伤。我们将通过检查30名HIV感染男性和女性(15名HAART患者和15名HAART患者)和10名对照组的直肠活检标本，来评估GALT中HIV和CMV特异性CTL的效应功能。将评估GalT CD8+T细胞产生穿孔素、颗粒酶A和B、MIP-1β、IL-2、肿瘤坏死因子-α和干扰素-γ的能力，以及它们以MHC I类限制性方式杀伤靶细胞的能力。我们还将测试不同的假说来解释GALT中穿孔素表达的明显低水平。

DESCRIPTION (provided by applicant): This proposal responds to PA-02-073, "Innovation Grants for Research in Human Immunology." The purpose of these studies is to better understand the relationship between CD8+ T-cell responses, viral replication and CD4+ T-cell depletion in gut-associated lymphoid tissue (GALT), and to clarify the ability of GALT T-cells to perform antiviral immune effector functions. We have developed methods to assess the phenotype and function of antigen-specific T-cells freshly isolated from human rectal tissue. For SPECIFIC AIM 1, we will assess the frequency, breadth and memory/effector phenotype of HIV-specific CD8+ T-cells in GALT and blood. Blood and rectal biopsy samples will be obtained from (i) a cross-sectional study of 30 HIV-infected men and women (including 15 "on" and 15 "off" HAART) and 10 controls; (ii) a longitudinal study of 20 HIV-infected individuals (10 "on" HAART, 10 "off'), with samples taken at 6-month intervals. We hypothesize that in patients not on HAART the frequency and breadth of CDS+ T-cell responses in GALT may exceed that in blood. In patients on HAART, we predict a decline in the magnitude and breadth of these responses concurrent with decreased tissue viral load. We also predict that reduced viral load and CD4+ T-cell repopulation will provide partial reconstitution of CD4+ T-cell responses to non-HIV antigens in GALT. For SPECIFIC AIM 2, we will assess the effector functions of HIV-specific CDS+ T-cells in GALT. Preliminary results suggest that (i) HIV-specific CTL in blood express low levels of perforin, (ii) perforin expression by all CD8+ T-cells is decreased in GALT relative to blood, and (iii) granzyme A and perforin are differentially expressed. We hypothesize that CD8+ T-cells in GALT may be functionally impaired. We will assess effector functions of HIV- and CMV- specific CTL in GALT by examining rectal biopsy specimens obtained in a cross-sectional study of 30 HIV-infected men and women (15 "on" and 15 "off' HAART) and 10 controls. GALT CD8+ T-cells will be assessed for production of perforin, granzymes A and B, MIP-1beta, IL-2, TNF-alpha and IFN-gamma, and for their ability to kill target cells in an MHC class I-restricted manner. We will also test alternate hypotheses to explain the apparent low levels of perforin expression in GALT.