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.
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