Highly purified CD25⁻resting T cells cannot be infectedde novowith HIV-1

Abstract

Previous studies have demonstrated that the expression of CD25 can distinguish CD25 ؊ latently infected cells from CD25 ؉ cells actively producing virus. Our studies were designed to characterize the nature and stability of the viral genome in CD25 ؊ quiescent HIV-1-infected cells and to determine whether these cells could be infected Using a model of acute in vitro infection, we have previously demonstrated that the expression of CD25 can distinguish latently infected cells (CD25 Ϫ ) from cells actively producing virus (CD25 ϩ ) (1) and that these two populations display significant differences with regard to the nature of the proviral genome. Thus, by combining the use of a potent anti-CD25 immunotoxin and sensitive immunofluorescence staining we have obtained highly purified resting cells. This is important because interpretations of viral latency have been confounded, in part, by the degree of purity of the resting cells. Indeed, partially reverse-transcribed DNAs (2, 3), full-length extrachromosomal DNA (4, 5), and stable integrated viral genomes (6) have been reported to occur in resting peripheral blood mononuclear cells (PBMCs). Although these differences can be explained in part by the different methodologies employed, they could also suggest the existence of several subsets of latently infected cells and possibly the contamination of resting cells with activated ones.Using our in vitro model of acute HIV-1 infection, the present studies were designed to characterize the nature and stability of the viral genome in quiescent HIV-1-infected CD25 Ϫ cells and to determine whether CD25 Ϫ cells can be infected de novo. The results of these analyses have implications regarding the origin of latently infected cells. MATERIALS AND METHODSVirus Strain and Standard Inoculum. HIV-1 JR-CSF is a clinical strain provided by the National Institutes of Health AIDS research and reference reagent program (7). The virus was cultured with phytohemagglutinin (PHA)-activated PBMCs for 6 days. The culture supernatant was purified on an Amicon concentrator (Centriprep 100) by centrifugation at 500 ϫ g at 4ЊC for 1 hr to remove cytokines and mitogens (M r Ͻ100 kD). This standard inoculum was adjusted to 10 times its original concentration, aliquoted, and stored at Ϫ80ЊC.Purification of CD4 ؉ CD25 ؊ Human Quiescent T Lymphocytes. Fresh PBMCs were obtained from normal healthy donors by centrifugation over Ficoll͞Hypaque and were cultured in RPMI 1640 supplemented with 5% autologous plasma [complete-medium (CM)] for 18 hr to remove peripheral blood macrophages by plastic adhesion. Adherent cells were used for additional experiments. The nonadherent cells were further cultured under the same conditions for 2 hr, and were treated with 5 mM leucine methyl ester in RPMI 1640 for 1 hr at 25ЊC to further deplete the macrophages and natural killer cells (8). CD4 ϩ T cells were purified by positive selection on anti-CD4-coated magnetic beads (Dynal, Great Neck, NY) according to the instructions of the manufacturer. By trypan blue...

Distinct gene-expression profiles associated with the susceptibility of pathogen-specific CD4 T cells to HIV-1 infection

“…18,[26][27][28] We observed that, compared with TT-and C albicans-specific CD4 T cells, in which CD25 expression was evident, CMV-specific CD4 T cells expressed little CD25 18,25 (supplemental Figure 2A). This low CD25 expression on CMV-specific cells may be because CMV-specific CD4 T cells produced very little endogenous IL-2 in response to recall antigen stimulation 18 (data not shown).…”

Preferential Feline Immunodeficiency Virus (FIV) Infection of CD4+CD25+T-Regulatory Cells Correlates both with Surface Expression of CXCR4 and Activation of FIV Long Terminal Repeat Binding Cellular Transcriptional Factors

“…Further, it has been demonstrated that HIV and FIV replicate efficiently in CD4 ϩ CD25 ϩ T cells that are partially activated but not in naive CD4 ϩ CD25 Ϫ resting T cells (7,9,24,43). To date there have been limited reports regarding FIV replication in activated versus resting T cells.…”

“…While HIV infection has also been demonstrated in naive T cells in vivo (33), establishment of a stable HIV infection in resting T cells in vitro remains controversial. Some studies suggest that HIV can efficiently bind and enter resting T cells (42,46), whereas others have shown that naive T cells are resistant to de novo HIV infection (9,19). In this regard, the data suggest that susceptibility of different lymphocyte subsets to HIV infection is regulated, to some extent, by differential expression of viral coreceptors.…”

Interleukin-7–treated naive T cells can be productively infected by T-cell–adapted and primary isolates of human immunodeficiency virus 1

“…[1][2][3] Whether HIV productively infects naive T cells in vivo is still controversial. [4][5][6][7][8][9] Naive T cells were shown to harbor replicationcompetent HIV, 7,10 but are not an active site of HIV replication in the absence of mitogen stimulation. 11 Even in studies demonstrating HIV infection of naive T cells, further analysis of integrated HIV indicated that HIV DNA did not always integrate into the genome.…”