Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 1999 Jun 7;189(11):1735-46.
doi: 10.1084/jem.189.11.1735.

Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes

D Unutmaz  1 , V N KewalRamani, S Marmon, D R Littman
Affiliations

Cytokine signals are sufficient for HIV-1 infection of resting human T lymphocytes

D Unutmaz et al. J Exp Med. .

Abstract

Lentiviral vectors have been advocated to be effective vehicles for the delivery and stable expression of genes in nondividing primary cells. However, certain cell types, such as resting T lymphocytes, are resistant to infection with HIV-1. Establishing parameters for stable gene delivery into primary human lymphocytes and approaches to overcome the resistance of resting T cells to HIV infection may permit potential gene therapy applications, genetic studies of primary cells in vitro, and a better understanding of the stages of the lentiviral life cycle. Here we demonstrate that an HIV-1-derived vector can be used for stable delivery of genes into activated human T cells as well as natural killer and dendritic cells. Remarkably, a sizeable fraction of resting T cells was stably transduced with the HIV-1 vector when cultured with the cytokine interleukin (IL)-2, IL-4, IL-7, or IL-15, or, at a lower level, with IL-6, in the absence of any other stimuli. Resting T cells stimulated with these cytokines could also be infected with replication-competent HIV-1. To test the utility of this system for performing structure-function analysis in primary T cells, we introduced wild-type as well as a mutant form of murine CD28 into human T cells and showed a requirement for the CD28 cytoplasmic domain in costimulatory signaling. The ability to stably express genes of interest in primary T cells will be a valuable tool for genetic and structure-function studies that previously have been limited to transformed cell lines. In addition, the finding that cytokine signals are sufficient to permit transduction of resting T cells with HIV may be relevant for understanding mechanism of HIV-1 transmission and pathogenesis.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Stable delivery and expression of genes in activated human T cell subsets through the HDV system. Purified T cells were activated with PHA and allogeneic feeder cells for 4 d and transduced with the HDV system. (A) Activated primary hCD4+ T cells were infected with HDV-EGFP viruses and GFP expression was analyzed by a FACS® 3 d after infection. (B) GFP+ T cells were sorted and expanded in culture with repeated mitogen stimulation and expression was assessed 2 wk and 4 mo after infection. (C) GFP expression in CD4+ and CD8+ T cell lines 5 d after HDV-IRES.EGFP infection. PI, post infection.
Figure 2
Figure 2
Transduction of myeloid cells and lymphocytes other than α/β T cells. To enrich for γ/δ+ T cells, α/β+ T cells were depleted by bead sorting and PHA lines were established as described in Materials and Methods. NK cells and B cells were positively sorted using antibodies to CD16 and CD19 respectively. NK cells were activated with 1,000 U/ml IL-2. B cells were activated as described in Materials and Methods. DC cultures were established as described by Sallusto and Lanzavecchia (44). Lymphocyte infections were done as described in Materials and Methods, whereas macrophages and DCs were infected in the absence of polybrene and at a lower MOI to prevent cytotoxicity induced by the viral supernatants. GFP was analyzed 2 wk after infection for γ/δ T cells and NK cells (gated on CD3−CD56+ cells), and 5 d after infection for B cells. Quadrants were set according to uninfected control stainings. DC infections were assessed 10 d after infection; cells were stained with PE-conjugated anti-CD14 and PercP-conjugated HLA-DR; and GFP expression was determined after gating on CD14− cells.
Figure 3
Figure 3
HDV-mediated transduction of both naive and memory resting T cells cultured in the presence of various cytokines. Purified resting CD4+ T cells were cultured for 4 d in media alone or with the following cytokines: IL-6 (400 U/ml), IL-2 (200 U/ml), IL-4 (20 ng/ml), IL-7 (20 ng/ml), or IL-15 (10 ng/ml). Cells were infected with HDV-IRES.GFP in the presence of cytokines for an additional 5 d and GFP expression was assessed. (A) GFP expression in resting T cells transduced in the presence of cytokines. (B) GFP expression in CD45RA+RO−CD4+ purified naive T cells transduced with HDV-IRES.EGFP.
Figure 4
Figure 4
Transduction efficiency of CD4+ and CD8+ resting T cells stimulated with individual or combinations of cytokines. Resting T cells were cultured and transduced with HDV-IRES.GFP as described in Fig. 3, with individual cytokines or in cytokine combinations (TNF-α was used at 50 ng/ml). Percentage of GFP+ cells was determined by FACS® analysis 5 d after infection.
Figure 5
Figure 5
Cell cycle entry and HDV transduction of cytokine-stimulated resting T cells. Resting T cells were cocultured in the presence of cytokines and 10 μM BrdU for 4 d and infected with HDV-EGFP for an additional 4 d while being maintained in continuous culture with fresh BrdU. T cells stimulated with anti-CD3 plus anti-CD28 antibodies were cultured, 3 d after activation, with 10 μM BrdU for 2 d. Cells were then permeabilized and stained with a PE-conjugated anti-BrdU antibody. (A) GFP expression and BrdU incorporation in cytokine-stimulated T cells. (B) BrdU staining of TCR-activated T cells.
Figure 6
Figure 6
Maintenance of naive phenotype of cytokine-stimulated and HDV-EGFP–transduced resting T cells. HDV-EGFP–infected T cells were cultured for 30 d in the presence of cytokines and stained with PE-conjugated anti-CD45RO antibody. Numbers displayed above FACS® analysis profiles are the percentage of positive cells in each quadrant.
Figure 7
Figure 7
Replication-competent CCR5 tropic HIV-1 can infect cytokine-activated resting T cells. Cytokine stimulation and HIV-1 infection was done as described in Fig. 4. Cells were fixed in 4% paraformaldehyde before FACS® analysis.
Figure 8
Figure 8
Expression of CCR5 on cytokine- or TCR-stimulated T cells. Purified resting CD4+ T cells were cultured with cytokines as described in Fig. 3. Cells were stained with PE-conjugated anti-CCR5 and FITC-conjugated anti-CD45RO antibodies.
Figure 9
Figure 9
Functional assessment of mCD28 expressed on human T cells. (A) Expression of mCD28 in sorted T cells 2 wk after transduction with HIV. (B) CD4+ and CD8+ human T cell lines transduced with wild-type (w+) or cytoplasmic domain–deleted (Δcyto+) mCD28 were stimulated 2 or 3 wk after the last activation with different concentrations of plate-bound anti-CD3 in the presence or absence of 1 μg/ml soluble anti-hCD28 or -mCD28 antibodies. Proliferation was measured after 2 d by [3H]thymidine incorporation.
Figure 10
Figure 10
IL-2 production by costimulation through mCD28 expressed on human T cells. IL-2 production was measured by titrating supernatants from cultures of CD4+ or CD8+ T cell lines that were stimulated with anti-CD3 and anti-CD28. Supernatants were collected after 36 h of stimulation and IL-2 was measured using the CTLL bioassay. 1 U of IL-2 is defined as half-maximal proliferation of CTLL. (A) IL-2 production by CD4+ T cell lines. (B) IL-2 production by CD8+ T cell lines that are negative for hCD28 expression as determined by FACS® analysis.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Mulligan RC. The basic science of gene therapy. Science. 1993;260:926–932. - PubMed
    1. Verma IM. Gene therapy: hopes, hypes, and hurdles. Mol Med. 1994;1:2–3. - PMC - PubMed
    1. Miller AD, Miller DG, Garcia JV, Lynch CM. Use of retroviral vectors for gene transfer and expression. Methods Enzymol. 1993;217:581–599. - PubMed
    1. Humphries EH, Temin HM. Requirement for cell division for initiation of transcription of Rous sarcoma virus RNA. J Virol. 1974;14:531–546. - PMC - PubMed
    1. Miller DG, Adam MA, Miller AD. Gene transfer by retrovirus vectors occurs only in cells that are actively replicating at the time of infection. Mol Cell Biol. 1990;10:4239–4242. - PMC - PubMed

Publication types

MeSH terms