LOSS OF TH1 IMMUNE FUNCTION IN FIV-INFECTED CATS

此申请是竞争续订的重新提交 免疫缺陷病毒免疫致病机制的应用(S) Thl免疫功能，这是艾滋病毒和FIV艾滋病的标志。CD8， L-选择素-阴性，整合素-Hi效应/记忆细胞迅速取代幼稚的CD8 感染FIV的猫血液中的细胞，因此90%的CD8细胞 在无症状晚期，血液中可能存在效应/记忆表型。这 观察与HIV感染患者血液中的这些变化非常相似。 此外，在FIV和HIV感染中，CD4显著增加， L-选择素-阴性细胞在感染晚期出现在循环中 淋巴结中高比例的T细胞表达效应/记忆 表型。最近，CD4T细胞丢失与细胞凋亡率增加有关 HIV感染者的LN和免疫激活状态增加以及 在非人类灵长类艾滋病模型中。CD4和CD8 T细胞均有所增加 B7分子在FIV感染猫淋巴组织中的表达 在疾病的整个过程中，细胞类型逐渐增加 它们代表了75%-100%的LN-T细胞。然而，上调 表达B7的T细胞在血液中含量极低。在许多细胞和动物中 免疫调节模型，B7-1(CD80)与CD28相互作用导致 共刺激激活信号，而B7-2(CD86)与CTLA4相互作用 对这种激活信号的下调或无能。基于以上几点 观察，申请人提出艾滋病患者的CD4T细胞丢失是 由B7-CTLA4相互作用引起的无能和细胞凋亡 CD8、B7 T细胞和活化的CD4、CTLA4 T细胞。特定目标的研究1 将验证他们的假设，即激活淋巴组织中的CD8B7细胞 感染FIV的猫具有表型和功能特征 FIV-抑制细胞。将对激活的CD8B7细胞进行T细胞分析 流式细胞仪检测活化标志物、RT-qcPCR、FIV检测细胞因子和趋化因子 抑制子活性和CTL活性。在具体目标2中，申请人将 验证他们的假设，即人的大脑中存在高水平的淋巴细胞凋亡。 无症状FIV感染猫的淋巴结和LN CD8 B7细胞 体外诱导活化的LN CD4T细胞无能和凋亡。Ln细胞 将对无症状的FIV感染猫的病毒进行评估 Annexin试剂盒流式细胞术和免疫组织化学检测细胞凋亡 采用隧道法。凋亡细胞的表型将通过以下方式确定 用适当的荧光标记单抗标记猫的CD_4、CD_8和B_(21) 流式细胞仪检测系统或免疫组织化学染色检测系统。具体而言 目的3、体外克隆FIV LN CD8 B7细胞，以检测其克隆性 扩增的细胞可以保留特定的表型和功能。以特定的目标 申请者将检验他们的中心假设，即相互作用 活化CD8T细胞表面B7分子与活化CD4T细胞表面CTLA4分子间的关系 细胞诱导抗FIV抑制活性和CD4细胞无能。 CTLA4-Ig融合蛋白将用于封闭CD8 B7上的B7分子 细胞及其对CD4细胞无能和FIV抑制活性的影响将 被监视。此外，IL-2基因和FIV gag基因在CD4细胞中的表达 反应细胞也将在CTLA4-Ig阻断研究中进行检查。这个 以后的研究将确定B7-CTLA4信号是否介导其活性 通过影响基因转录。

This application is a resubmission of a competitive renewal application on the mechanism(s) of FIV immunopathogenesis leading to the loss of Thl immune functions which is the hallmark of both HIV and FIV AIDS. CD8+, L-selectin-neg, integrin-hi effector/memory cells rapidly replace naive CD8+ cells in the blood of FIV-infected cats, such that 90 percent of the CD8+ cells in blood may be of effector/memory phenotype by late asymptomatic stage. This observation closely mimics those changes in blood of HIV-infected patients. Furthermore, in both FIV and HIV infection, a marked increase in CD4+, L-selectin-neg cells occurs in the circulation during late stages of infection and high proportion of the T cells in the lymph node express effector/memory phenotype. Recently, CD4 T cell loss has been correlated to increased apoptosis of LN and increased state of immune activation in HIV-infected individuals and in nonhuman primate AIDS models. Both CD4+ and CD8+ T cells had increased expression of B7 molecules in the lymph nodes of FIV-infected cats and these cell types increase progressively throughout the course of the disease such that they represent 75-100 percent ofthe LN T cells. However, the upregulation of B7 expressing T cells was minimal in the blood. In many cellular and animal models of immune modulation, B7- 1 (CD80) interaction with CD28 leads to costimulatory activation signal, while B7-2 (CD86) interaction with CTLA4 leads to down-regulation or anergy of such activation signal. Based on above observations, the applicant proposes that the CD4+ T cell loss in AIDS is caused by anergy and apoptosis that develops upon B7-CTLA4 interaction between CD8+B7+ T cells and activated CD4+, CTLA4+ T cells. Studies in specific aim 1 will test their hypothesis, that activated CD8+B7+ cells in lymph node of FIVinfected cats have phenotype and functional characteristics of FIV-suppressor cells. The activated CD8+B7+ cells will be analyzed for T-cell activation markers by FACS, cytokine and chemokine profile by RT-qcPCR, FIV suppressor activity, and CTL activity. In specific aim 2, the applicant will test their hypothesis that there is a high level of lymphocyte apoptosis in the lymph nodes of asymptomatic FIV-infected cats and that the LN CD8+B7+ cells induce anergy and apoptosis of activated LN CD4+ T cells in vitro. LN cells from asymptomatic FIV-infected cats will be evaluated for the presence of apoptotic cells by flow cytometry using annexin kit and by immunohistochemistry using TUNNEL assay. The phenotype of the apoptotic cells will be determined by using mAb to feline CD4, CD8, and B21 with appropriate flourochrome labeled system for FACS or counter stain system for immunohistochemistry. In specific aim 3, FIV+ LN CD8+B7+ cells will be cloned in vitro to test whether clonally expanded cells can retain the specific phenotype and function. In specific aim 4, the applicant will test their central hypothesis, that the interaction between the B7 on the activated CD8+T cells and CTLA4 on the activated CD4+ T cells induces the anti-FIV suppressor activity and the CD4+ cell anergy. CTLA4-Ig fusion protein will be used to block the B7 molecules on the CD8+B7+ cells and the effect on the CD4+ cell anergy and FIV suppressor activity will be monitored. Furthermore, both IL2 gene and FIV gag mRNA expression in CD4+ responder cells will also be examined in the CTLA4-Ig blocking studies. The latter studies will determine whether B7-CTLA4 signaling mediates its activity by affecting gene transcription.