LOSS OF TH1 IMMUNE FUNCTION IN FIV-INFECTED CATS

感染 5 种猫的 TH1 免疫功能丧失

• 批准号: 6646453
• 负责人: Mary B Tompkins
• 金额: $ 27.33万
• 依托单位: NORTH CAROLINA STATE UNIVERSITY RALEIGH
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-07-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6646453
• 关键词: B lymphocyte; Retroviridae disease; anergy; apoptosis; cats; cellular immunity; cytokine; cytotoxic T lymphocyte; feline immunodeficiency virus; flow cytometry; helper T lymphocyte; leukocyte activation /transformation; lymph nodes; pathologic process; polymerase chain reaction; terminal nick end labeling; tissue /cell culture

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.

此申请是重新提交的竞争性更新 应用于FIV免疫发病机制导致的损失 Th 1免疫功能的改变，这是HIV和FIV AIDS的标志。CD8+， L-选择素阴性、整联蛋白hi效应/记忆细胞快速取代初始CD 8 + FIV感染猫血液中的CD 8+细胞， 在晚期无症状阶段可能具有效应/记忆表型。这 观察密切模仿艾滋病毒感染者血液中的这些变化。 此外，在FIV和HIV感染中，CD 4+， L-选择素阴性细胞出现在感染后期的循环中 淋巴结中高比例的T细胞表达效应/记忆 表型最近，CD 4 T细胞丢失与细胞凋亡增加相关 LN和HIV感染者的免疫激活状态增加， 在非人类灵长类艾滋病模型中。CD 4+和CD 8 + T细胞均增加， B7分子在FIV感染猫的淋巴结中的表达， 细胞类型在整个疾病过程中逐渐增加，例如 它们代表了75- 100%的淋巴结炎T细胞。然而，上调 表达B7的T细胞在血液中是最小的。在许多细胞和动物 免疫调节模型，B7- 1（CD 80）与CD 28的相互作用导致 共刺激激活信号，而B7-2（CD 86）与CTLA 4的相互作用导致 涉及这种激活信号的下调或无反应性。基于上述 根据观察，申请人提出，艾滋病中的CD 4 + T细胞损失是 由B7-CTLA 4相互作用后发生的无反应性和凋亡引起， CD 8 +B7+ T细胞和活化的CD 4+、CTLA 4 + T细胞。具体目标研究1 将测试他们的假设，即淋巴结中活化的CD 8 +B7+细胞， FIV感染的猫具有以下表型和功能特征： FIV抑制细胞。将分析活化的CD 8 +B7+细胞的T细胞 通过FACS测定活化标志物，通过RT-qcPCR测定细胞因子和趋化因子谱，FIV 抑制子活性和CTL活性。在具体目标2中，申请人将 测试他们的假设，即有一个高水平的淋巴细胞凋亡， 淋巴结的无症状FIV感染猫和LN CD 8 +B7+细胞 体外诱导活化LN CD 4 + T细胞无能和凋亡。LN细胞 将评估无症状FIV感染猫中是否存在 通过使用膜联蛋白试剂盒的流式细胞术和通过免疫组织化学的凋亡细胞 使用隧道测定。凋亡细胞的表型将通过 使用mAb对猫CD 4、CD 8和B21进行标记，并标记适当的荧光染料 流式细胞术系统或免疫组织化学复染系统。在特定 目的3、体外克隆FIV+ LN CD 8 +B7+细胞，检测其克隆性是否 扩增的细胞可以保留特定的表型和功能。具体目标 4、申请人将检验他们的中心假设，即相互作用 活化的CD 8 +T细胞上的B7和活化的CD 4 + T细胞上的CTLA 4之间的差异 细胞诱导抗FIV抑制活性和CD 4+细胞无反应性。 CTLA 4-IG融合蛋白将用于阻断CD 8 +B7+上的B7分子 细胞和对CD 4+细胞无反应性和FIV抑制活性的影响将 被监控。此外，IL 2基因和FIV gag mRNA在CD 4 + T细胞中的表达也与IL 2基因的表达和FIV gag mRNA的表达有关。 在CTLA 4-IG阻断研究中也将检查应答细胞。的 后面的研究将确定B7-CTLA 4信号传导是否介导其活性 通过影响基因转录。