Over the course of the past year, we have investigated novel potential reservoirs for CXCR4 (X4)-utilizing strains of HIV-1, including B lymphocytes and macrophages. We demonstrated a mechanism by which B lymphocytes become susceptible to X4-mediated infection in vitro. Highly purified B lymphocytes, cultured in the presence of CD40L and cytokines, expressed high levels of CXCR4 and detectable levels of CD4. The appearance of these receptors coincided with susceptibility of the cells to X4 strains of HIV-1; infection was blocked with inhibitors of either CD4 or CXCR4, directly demonstrating the route of entry. Furthermore, in contrast to the syncytia typically observed when X4 HIV-1 strains infect CD4-positive T cells, B lymphocytes replicated these same viruses in the absence of detectable cytopathic effects. Recent in vitro studies have also established that X4 primary strains of HIV-1 can replicate in macrophages.In light of our in vitro observations, we have pursued the issue of in vivo reservoirs for X4 strains of HIV-1 through the analysis of pure B cell and macrophage fractions isolated from lymph node biopsies of HIV-infected individuals harboring X4 strains. Preliminary findings indicate that tissue macrophages may be a source of X4 strains of HIV-1 in vivo. B cells purified from HIV-infected individuals and initially propagated in the mono-culture system described above, failed to show evidence of harboring endogenous virus. However, we recently discovered that substantial levels of HIV-1 RNA were associated with freshly isolated B cell preparations from several individuals with high viral loads and low CD4 counts. Furthermore, the RNA signal was reduced to background levels by treatments designed to strip cells of surface-associated virus, suggesting that HIV-1 may bind B cells in vivo, yet may not get internalized efficiently. We also established that the surface-bound virus was replication competent as it led to a spreading infection in vitro by co-cultivating the B cells isolated from HIV-infected individuals with HIV-negative activated peripheral blood lymphocytes. These findings indicate that B cells may serve as a source of virus that can efficiently be passed to activated CD4-positive T cells. - AIDS pathogenesis; HIV-1 tissue reservoirs; B lymphocytes; macrophages; HIV-1 tropism; cytopathicity - Human Subjects