Similar levels of human immunodeficiency virus type 1 replication in human TH1 and TH2 clones

Abstract

Studies on the development and function of CD4+ TH1 and TH2 cells during the progression to AIDS may increase the understanding of AIDS pathogenesis. The preferential replication of human immunodeficiency virus (HIV) in either TH1 or TH2 cells could alter the delicate balance of the immune response. TH1 (gamma interferon [IFN-gamma] positive, interleukin-4 [IL-4] and IL-5 negative) and TH2 (IFN-gamma negative, IL-4 and IL-5 positive) clones, developed from several healthy donors, pedigreed by reverse transcriptase PCR (RT-PCR) and enzyme linked immunosorbent assay have similar levels of cell surface expression of CD4 and several chemokine receptor cofactors necessary for viral entry. After activation by specific antigens and infection with T-cell-tropic strains of HIV type 1 (HIV-1), TH1 and TH2 clones showed similar levels of viral entry and reverse transcription. At days 3 through 14 postinfection, HIV replicated to similar levels in several TH1 and TH2 clones as measured by release of HIV p24 and total number of copies of gag RNA/total cell RNA as measured by RT-PCR. When values were normalized for viable cell number in three clones of each type, there was up to twofold more HIV RNA in TH1 than TH2 cells. In addition, several primary monocytotropic HIV-1 strains were able to replicate to similar levels in TH1 and TH2 cells. These studies suggest that the importance of TH1 and TH2 subsets in AIDS pathogenesis transcends clonal differences in their ability to support HIV replication.

FIG. 1

Characterization of human T_H clones by RT-PCR. Single-cell cloning of human peripheral blood was performed as described in Materials and Methods. Presence of mRNA for IFN-γ and IL-4 in these clones was measured 7 days after antigen activation, using RT-PCR as described in Materials and Methods. (A) Clones isolated under T_H1 conditions. Lane 1, IFN-γ; lane 2, IL-4; lane 3, GAPDH. Arrows indicate positions of cytokine standards. (B) Clones isolated under T_H2 conditions. Lane 1, standard (STD) for IL-4 (427 bp); lane 2, standard for IFN-γ (459 bp) (Clontech). GAPDH (not shown) was used as a loading control.

FIG. 2

Flow cytometric analysis of a human T_H1 clone and a human T_H2 clone for cell surface expression of chemokine receptors. A total of 10⁶ cells of each of the T-cell clones H1.5 and H2.25 were suspended in PBS containing 1% heat-inactivated human AB serum and 0.5% sodium azide and stained with phycoerythrin (PE)-labeled anti-CXCR2, -CXCR4, -CD4, or -CD44 or an isotype control labeled immunoglobulin G antibody. After staining, the cells were extensively washed and then fixed with 1% paraformaldehyde. The clones were analyzed on a FACStar Plus flow cytometer.

FIG. 3

HIV-1 DNA detection in T_H1 and T_H2 clones. Lysates were prepared 24 h p.i. A primer pair was used to detect the earliest region of DNA formed by reverse transcription (141 bp of the minus strong-stop strand). Another primer pair was used to detect full-length HIV-1 DNA (200 bp of LTR-gag). The sense primers of each pair were radiolabeled. Three different T_H1 (H1.12, H1.15, and H1.18) and T_H2 (H2.25, H2.29, and H2.33) clones shown for each infection are representative of three separate experiments. Tenfold serial dilutions of the ACH-2 cell line, containing one integrated copy of HIV-1 DNA per cell, were made in a background of uninfected T cells and shown for estimation of copy number.

FIG. 4

Analysis of HIV gag mRNA in HIV-infected T_H1 and T_H2 clones. HIV infection of antigen-activated T-cell clones, RT-PCR analysis, and use of HIV gag RNA standards were performed as described in Materials and Methods. Cells were infected with HIV_MN. Lanes 1 and 2, T_H1 H1.15 with and without HIV, day 2; lanes 3 and 4, mitogen-stimulated antigen-presenting cells (APC) with and without HIV, day 2; lanes 5 and 6, T_H1 H1.15 with and without HIV, day 7; lane 7, T_H1 H1.12 with HIV, day 7; lanes 8 and 9, T_H2 H2.29 with and without HIV, day 7; lane 10, T_H2 H2.29; lane 11, T_H2 H2.29 with HIV, day 14; lanes 12 to 16, RNA standard curve.

FIG. 5

HIV-1 DNA detection in T_H1 and T_H2 clones. Lysates were prepared at 10 and 24 h p.i. A primer pair was used to detect the earliest region of DNA formed by reverse transcription (141 bp of the minus strong-stop strand). Another primer pair was used to detect full-length HIV-1 DNA (200 bp of LTR-gag) and human β-globin. The sense primers of each pair were radiolabeled. Two different T_H1 (H1.20 and H1.25) and T_H2 (H2.10 and H2.25) clones are shown. Lanes: A, control; B, SF162 infection; C, US657; D, US714; E, US727. Tenfold serial dilutions of the ACH-2 cell line, containing one integrated copy of HIV-1 DNA per cell, were made in a background of uninfected T cells and shown for estimation of copy number.