Positive regulation of CXCR4 expression and signaling by interleukin-7 in CD4+ mature thymocytes correlates with their capacity to favor human immunodeficiency X4 virus replication

Abstract

The emergence of X4 human immunodeficiency virus type 1 (HIV-1) variants in infected individuals is associated with poor prognosis. One of the possible causes of this emergence might be the selection of X4 variants in some specific tissue compartment. We demonstrate that the thymic microenvironment favors the replication of X4 variants by positively modulating the expression and signaling of CXCR4 in mature CD4(+) CD8(-) CD3(+) thymocytes. Here, we show that the interaction of thymic epithelial cells (TEC) with these thymocytes in culture induces an upregulation of CXCR4 expression. The cytokine secreted by TEC, interleukin-7 (IL-7), increases cell surface expression of CXCR4 and efficiently overcomes the downregulation induced by SDF-1 alpha, also produced by TEC. IL-7 also potentiates CXCR4 signaling, leading to actin polymerization, a process necessary for virus entry. In contrast, in intermediate CD4(+) CD8(-) CD3(-) thymocytes, the other subpopulation known to allow virus replication, TEC or IL-7 has little or no effect on CXCR4 expression and signaling. CCR5 is expressed at similarly low levels in the two thymocyte subpopulations, and neither its expression nor its signaling was modified by the cytokines tested. This positive regulation of CXCR4 by IL-7 in mature CD4(+) thymocytes correlates with their high capacity to favor X4 virus replication compared with intermediate thymocytes or peripheral blood mononuclear cells. Indeed, we observed an enrichment of X4 viruses after replication in thymocytes initially infected with a mixture of X4 (NL4-3) and R5 (NLAD8) HIV strains and after the emergence of X4 variants from an R5 primary isolate during culture in mature thymocytes.

FIG. 1.

Comparison of the levels of expression of CXCR4 and CCR5 in mature SP CD4⁺ and immature CD4^−/+ CD8⁻ CD3⁻ thymocytes. Immunostainings of CXCR4 and CCR5 were performed on freshly isolated thymocytes and analyzed by flow cytometry. White area indicates isotypic control. Percentages of labeled cells are given. The results are representative of independent experiments carried out with thymuses from 13 donors.

FIG. 2.

Influence of TEC on CXCR4 expression in mature SP CD4⁺ and immature CD4^−/+ CD8⁻ CD3⁻ thymocytes. Cell surface expression of CXCR4 was analyzed on immature (A) and mature (B) thymocytes by flow cytometry at the time of isolation or after 24 h in culture alone or with TEC. Immunostaining of CXCR4 was determined in immature and mature thymocytes just after isolation or after 1, 4, or 7 days of culture with or without TEC. (C and D) Results are shown as MFI of CXCR4-labeled cells. The experiment presented here is representative of independent experiments carried out with thymuses from five donors.

FIG. 3.

Effects of SDF-1α and IL-7 on CXCR4 expression in mature SP CD4⁺ and immature CD4^−/+ CD8⁻ CD3⁻ thymocytes. Cell surface expression CXCR4 was analyzed by flow cytometry on immature or mature thymocytes, freshly isolated and after 24 h in culture in the presence of various concentrations of SDF-1α (A), after 24 h in culture in the presence of various cytokines (10 ng/ml each or the indicated concentrations of IL-7) (B), or freshly isolated and after 12, 36, and 84 h in the presence of SDF-1α (0.1 μg/ml) or a combination of SDF-1α (0.1 μg/ml) and IL-7 (1.25 ng/ml) (C). The results are shown as MFI of CXCR4-labeled cells and are expressed as means plus standard deviations of triplicate values for each thymus. The results are representative of independent experiments performed with thymuses from three donors.

FIG. 4.

Upregulation of CXCR4 protein and mRNA levels in mature SP CD4⁺ and immature CD4^−/+ CD8⁻ CD3⁻ thymocytes by IL-7. CXCR4 expression was monitored at the protein (A) and mRNA (B) levels in mature and immature thymocytes after 12, 24, 40, and 60 h in culture in the presence or absence of IL-7 (10 ng/ml). (A) CXCR4 protein expression level was determined by CXCR4 cell surface immunostaining and analysis of labeled cells by flow cytometry. The results are shown as increase in MFI induced by IL-7 compared to untreated cells. (B) CXCR4 mRNA levels were determined by using a real-time kinetic quantitative reverse transcription-PCR. To normalize for differences in the amounts of total RNA, 18S rRNA was amplified as a control. The results are shown as increase of the normalized amount of CXCR4 mRNA induced by IL-7 compared to the amount in untreated cells. The data are representative of independent experiments carried out with thymuses from four donors.

FIG. 5.

Influence of IL-7 on CXCR4 signaling leading to actin polymerization in mature SP CD4⁺ and immature CD4^−/+ CD8⁻ CD3⁻ thymocytes. Thymocytes were cultured for 40 h in the presence (solid lines) or absence (dashed lines) of 10 ng of IL-7/ml. Immunostaining of intracellular F actin was performed with fluorescein isothiocyanate-labeled phalloidin and was analyzed by flow cytometry after the addition of 1 μg of SDF-1α/ml at time zero, in immature (triangles) and mature (circles) thymocytes. Results are shown as percent intracellular F-actin (MFI) relative to the value before addition of SDF-1α. The data are representative of independent experiments carried out with thymuses from five donors.

FIG. 6.

Comparison of mature SP CD4⁺ thymocytes, immature CD4^−/+ CD8⁻ CD3⁻ thymocytes, and PBMC in their capacity to select X4 (NL4-3) versus R5 (NLAD8) viruses. (A, C, and E) Kinetics of HIV replication in mature (A) and immature (C) thymocytes and in PBMC (E). Cells were infected at MOIs of 1.6 × 10⁻⁴ for NL4-3 (filled circle), 2.8 × 10⁻³ for NLAD8 (filled triangle), or 2.8 × 10⁻³ for a mixture of both at TCID₅₀ ratios (NL4-3/NLAD8) of 1/10⁵ (open triangles), 1/10⁴ (open squares), and 1/10³ (open circles). Infected thymocytes were cultured with IL-7 (10 ng/ml) and TNF-α (10 ng/ml). PHA-IL-2-activated PBMC were infected and maintained with IL-2 (540 IU/ml). Freshly isolated thymocytes or activated PBMC were added to the cultures of infected thymocytes or PBMC every 12 days in order to provide new targets for the virus. HIV replication was determined by measuring the p24^gag concentration in the culture supernatants every 3 or 4 days. (B, D, and F) Tropism of viruses recovered from the supernatants of infected mature (B) and immature (D) thymocytes and in PBMC (F). Supernatants of thymocytes or PBMC infected with virus mixtures at ratios of 1/10⁵, 1/10⁴, and 1/10³ were harvested at day 21 for thymocytes and day 24 for PBMC. These supernatants and the initial mixtures used for infection (initial inoculum) were serially diluted and used to infect CD4-CXCR4 and CD4-CCR5 U87 cells. Replication of X4 and R5 viruses was determined by measuring the p24^gag concentrations in culture supernatants at day 7. X4 and R5 TCID₅₀ were then calculated by the method of Kärber (27). The ratios of TCID₅₀ per milliliter for X4 versus R5 are given above the bars. ns, no significant ratio (<0.02) due to a too-low value for CD4-CXCR4 U87 cells.

FIG. 7.

Kinetics of replication of the primary isolates described in Table 1 in PBMC or mature thymocytes. PBMC (A) and mature thymocytes (B) were infected with various primary HIV-1 isolates described in Table 1. Freshly isolated thymocytes or PHA-IL-2-activated PBMC were infected with the primary isolates at MOI of 1.6 × 10⁻³ for V.CT8 (open circles), 9.5 × 10⁻⁴ for 11111D (filled triangles), 3.5 × 10⁻⁴ for J2758 (open squares), and 2.25 × 10⁻³ for 9614C (filled squares). Infected thymocytes were cultured with IL-7 (10 ng/ml) and TNF-α (10 ng/ml). Infected PBMC were maintained with IL-2 (540 IU/ml). Freshly isolated thymocytes or activated PBMC were added to the cultures every 10 days in order to provide new targets for the virus. HIV replication was determined by measuring the p24^gag concentration in the culture supernatants.