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

Unutmaz D, KewalRamani VN, Marmon S, Littman DR - J. Exp. Med. (1999)

Bottom Line: Lentiviral vectors have been advocated to be effective vehicles for the delivery and stable expression of genes in nondividing primary cells.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, USA. unutmaz@saturn.med.nyu.edu

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.

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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.
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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.

Mentions: Finally, we asked whether cytokine-activated T cells can also be infected with replication-competent HIV-1, since this may be an important mode of infection of T cells in vivo and in the establishment of latency in resting cells. We used a CCR5-tropic GFP-expressing replication-competent HIV-1 to infect cytokine-stimulated resting T cells under conditions similar to those used for the HDV transduction. Infection of cytokine-stimulated resting T cells was clearly observed 4 d after challenge (Fig. 7). The GFP expression in the cytokine-treated cells remained stable for >2 wk (data not shown). However, the percentage of cells expressing GFP was lower as compared with VSV-G–pseudotyped viruses. This could be due to the relatively low titers of the R5 virus, which limit the infection to an MOI of 1–2 compared with 10–20 for VSV-G viruses. It is also possible that the infection is influenced by the expression of CCR5 on cytokine-stimulated versus TCR-activated T cells. Thus we examined CCR5 expression on resting T cells after culture in the presence of cytokines. The expression of CCR5 was modestly upregulated on IL-2 or IL-7 cultured resting T cells and more strikingly in the presence of IL-15 (Fig. 8). The TCR- mediated stimulation induced CCR5 expression in nearly half of the cells (Fig. 8). The expression of CCR5 was exclusively induced on the memory (CD45RO+) subset of T cells (Fig. 8), consistent with the expression pattern of CCR5 on PBMCs (52). The T-tropic HIV coreceptor CXCR4 is expressed on all the naive T cells and on most of the memory T cells (data not shown and reference 52); however, we also observed an approximately fivefold increase in CXCR4 expression levels in the presence of IL-4 (data not shown), similar to recent reports (53–55).


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

Unutmaz D, KewalRamani VN, Marmon S, Littman DR - J. Exp. Med. (1999)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2193071&req=5

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.
Mentions: Finally, we asked whether cytokine-activated T cells can also be infected with replication-competent HIV-1, since this may be an important mode of infection of T cells in vivo and in the establishment of latency in resting cells. We used a CCR5-tropic GFP-expressing replication-competent HIV-1 to infect cytokine-stimulated resting T cells under conditions similar to those used for the HDV transduction. Infection of cytokine-stimulated resting T cells was clearly observed 4 d after challenge (Fig. 7). The GFP expression in the cytokine-treated cells remained stable for >2 wk (data not shown). However, the percentage of cells expressing GFP was lower as compared with VSV-G–pseudotyped viruses. This could be due to the relatively low titers of the R5 virus, which limit the infection to an MOI of 1–2 compared with 10–20 for VSV-G viruses. It is also possible that the infection is influenced by the expression of CCR5 on cytokine-stimulated versus TCR-activated T cells. Thus we examined CCR5 expression on resting T cells after culture in the presence of cytokines. The expression of CCR5 was modestly upregulated on IL-2 or IL-7 cultured resting T cells and more strikingly in the presence of IL-15 (Fig. 8). The TCR- mediated stimulation induced CCR5 expression in nearly half of the cells (Fig. 8). The expression of CCR5 was exclusively induced on the memory (CD45RO+) subset of T cells (Fig. 8), consistent with the expression pattern of CCR5 on PBMCs (52). The T-tropic HIV coreceptor CXCR4 is expressed on all the naive T cells and on most of the memory T cells (data not shown and reference 52); however, we also observed an approximately fivefold increase in CXCR4 expression levels in the presence of IL-4 (data not shown), similar to recent reports (53–55).

Bottom Line: Lentiviral vectors have been advocated to be effective vehicles for the delivery and stable expression of genes in nondividing primary cells.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pathogenesis Program, Skirball Institute of Biomolecular Medicine, New York University Medical Center, New York, USA. unutmaz@saturn.med.nyu.edu

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.

Show MeSH
Related in: MedlinePlus