Bhardwaj
Bhardwaj. representative cell line, HB-2, that expressed DC markers including CD83, CD80 and CD86 could be induced to produce IL-12 through CD40 stimulation. After human immunodeficiency virus (HIV) infection, there was impairment of antigen-presenting cell (APC) function, which was manifested by an inability to stimulate allogeneic T-cell responses. There was no change in expression of major histocompatibility complex class I and class II antigens, CD83, CD40, CD4, CD11c, CD80, CD86, CD54, and CD58, or IL-12 production in the HIV-infected HB-2 cells. The HIV-infected HB-2 cells induced T-cell apoptosis in the cocultures. T-cell proliferation could be partially restored by using ddI, indinivir, and blocking anti-gp120 and anti-IL-10 antibodies. Our data suggest that there are multiple mechanisms that DCs use to inhibit T-cell responses in HIV-infected patients. The HB-2 cell line could be a useful model system to study APC function in HIV-infected DCs. Dendritic cells (DCs) are potent antigen-presenting cells (APCs) that have the unique property of being able to generate primary T-cell responses (3, 4, 60). The roles that DCs play in the immunopathogenesis of human immunodeficiency virus (HIV) disease have been extensively studied, but some questions still remain unanswered. It was not clear whether DCs can be directly infected with HIV or if the virus is only present on the cell surface or if both processes occur simultaneously (37, 46, 47). However, recent work has demonstrated that DCs can take up HIV into endosome-like compartments, where it can be transferred to T cells (35). Turnville et al. have demonstrated that DCs transfer HIV type 1 (HIV-1) to CD4+ T cells in two distinct phases (63). In the first phase DCs divert the virus from the endolysosomal pathway to the DC-T-cell synapse, while in the second phase the transmission occurs by de novo infection of the DCs. Study of the immunologic function of HIV-infected DCs, as well as DCs isolated from HIV-infected individuals, has yielded ambiguous results (7, 8, 10, 11, 12, 13, 19, 24, 25, 44, 44, 45, 53). Some reports have identified APC defects with DCs, whereas others have not. This could be partially explained by different effects of HIV infection on DC subpopulations, the purity of the DC population studied, and different percentages of infected DCs. A cloned stable cell line that can be infected with HIV may be useful for studying the effect of HIV infection on DC APC function, although it may not be reflective of the different types of DCs, including Langerhans cells, monocyte-derived, immature, mature, and myeloid and plasmacytoid DCs. There are currently no available cloned and stable human DC lines. However, it is possible to fuse DCs with tumor cells by using either either polyethylene glycol Cetrorelix Acetate or electrofusion to create a tumor-DC hybrid (54). We have previously demonstrated that both human peripheral blood monocytes and macrophages are able to fuse with the U937 promonocytic cell line to form stable hybrids (56). Using these cell lines, we demonstrated different monocyte defects after HIV infection (57, 58, 65). These studies have been extended here; Rabbit Polyclonal to PAK5/6 we have made human DC hybridomas by fusing immature DCs obtained from peripheral blood monocytes with HGPRT-deficient U937 cells, followed by aminopterin selection. The hybridomas express DC markers and donor class I and II antigens and have functional properties not found in the U937 fusion partner, including interleukin-12 (IL-12) production and the ability to stimulate allogeneic T-cell responses. In addition, these hybridomas can be Cetrorelix Acetate uniformly infected with HIV. In the present study we have used one of these cell lines (HB-2) to investigate DC function after HIV infection and have found that infection leads to defective accessory Cetrorelix Acetate cell function manifested by an inability to stimulate allogeneic T-cell proliferative responses. This is due to multiple factors including gp120, IL-10, and the induction of apoptosis. MATERIALS AND METHODS DC generation. Peripheral blood mononuclear cells were isolated from buffy coats obtained from healthy blood donors by using Ficoll-Paque (Amersham Pharmacia Biotech, Piscataway, N.J.) density gradient centrifugation (65). Cells were washed three times with sterile phosphate-buffered saline (PBS) and resuspended in RPMI 1640 (Life Technologies, Grand Island, N.Y.) supplemented with 10% male AB human serum (Life Technologies) and 1% penicillin-streptomycin-glutamine (Life Technologies). The freshly isolated peripheral blood mononuclear cells were incubated at 37C in 5% CO2 in culture flasks and allowed to adhere for 45 min. The nonadherent cells were removed by several washes with sterile PBS. The adherent monocytes were then cultured in RPMI 1640 (Life Technologies) supplemented with 1% male AB human serum (Life Technologies) and 1% penicillin-streptomycin-glutamine (Life Technologies) and 100 U of granulocyte-macrophage colony-stimulating factor (BD Pharmingen, San Diego, Calif.)/ml and 1,000 U of IL-4 (BD Pharmingen)/ml. The medium.