Migration of antigen-specific T cells away from CXCR4-binding human immunodeficiency virus type 1 gp120

Abstract

Cell-mediated immunity depends in part on appropriate migration and localization of cytotoxic T lymphocytes (CTL), a process regulated by chemokines and adhesion molecules. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode chemotactically active proteins, suggesting that dysregulation of immune cell trafficking may be a strategy for immune evasion. HIV-1 gp120, a retroviral envelope protein, has been shown to act as a T-cell chemoattractant via binding to the chemokine receptor and HIV-1 coreceptor CXCR4. We have previously shown that T cells move away from the chemokine stromal cell-derived factor 1 (SDF-1) in a concentration-dependent and CXCR4 receptor-mediated manner. Here, we demonstrate that CXCR4-binding HIV-1 X4 gp120 causes the movement of T cells, including HIV-specific CTL, away from high concentrations of the viral protein. This migratory response is CD4 independent and inhibited by anti-CXCR4 antibodies and pertussis toxin. Additionally, the expression of X4 gp120 by target cells reduces CTL efficacy in an in vitro system designed to account for the effect of cell migration on the ability of CTL to kill their target cells. Recombinant X4 gp120 also significantly reduced antigen-specific T-cell infiltration at a site of antigen challenge in vivo. The repellant activity of HIV-1 gp120 on immune cells in vitro and in vivo was shown to be dependent on the V2 and V3 loops of HIV-1 gp120. These data suggest that the active movement of T cells away from CXCR4-binding HIV-1 gp120, which we previously termed fugetaxis, may provide a novel mechanism by which HIV-1 evades challenge by immune effector cells in vivo.

FIG. 1.

Checkerboard transmigration analysis of CD8⁺ CD45RO⁺ T cells in response to recombinant HIV-1_IIIB gp120. T cells (5 × 10⁴) were placed in the upper chamber, and recombinant gp120 was added to the upper and/or lower chamber at the indicated concentrations, creating a negative gradient (above the diagonal line), a positive gradient (below the diagonal line), or equal concentrations in both chambers (along the diagonal line). After 3 h of incubation, cells were counted in the lower chamber. Results represent the mean percentage of cells placed in the top chamber that migrated to the lower chamber ± the standard error of the mean from six independent experiments.

FIG. 2.

Transmigration responses of a representative HIV-specific CTL clone (161JD27) with recombinant HIV-1_IIIB gp120 used at concentrations of 20 and 200 ng/ml. Media alone (M/M) was used as a control. Movement away (fugetaxis), movement toward (chemotaxis), and random movement (chemokinesis) were quantitated in a standard transmigration assay and are expressed along the y axis as the percentage of cells placed in the upper chamber of the transwell that migrated to the lower chamber after 3 h of incubation. Results represent the mean and standard error from three independent experiments.

FIG. 3.

Pertussis toxin and anti-CXCR4 antibodies inhibit active movement towards (A) and away from (B) X4 gp120. CD8⁺ CD45RO⁺ T cells were incubated with pertussis toxin (PTX; striped bars) or 12G5 (anti-CXCR4; open bars) prior to their addition to the transmigration assay. Control cells (No Tx; filled bars) received no pretreatment. The percentage of cells that had migrated to the lower transwell is shown for gp120 concentrations of 20 and 200 ng/ml. Results represent the mean and standard error from three independent experiments.

FIG. 4.

Migration of CD8⁺ T cells in response to HIV-1_IIIB gp120 containing variable loop deletions. Freshly isolated peripheral blood CD8⁺ T cells were exposed to concentrations of gp120 at 20 and 200 ng/ml in the lower or upper chambers of a transwell assay (A), HIV-1_IIIB ΔV1,V2 (B), and HIV-1_IIIB ΔV1,V2,V3 (C). The percentage of cells moving away from (fugetaxis; striped bars), moving towards (chemotaxis; filled bars), and moving in the absence of (chemokinesis; open bars) a gradient of the different recombinant gp120 molecules was quantitated by using the standard transmigration assay. Cell migration in medium alone (M) served as a control. Results represent the mean of three independent experiments.

FIG. 5.

Modifications to the standard ⁵¹Cr release assay demonstrate that CTL migration influences killing efficacy. (A) CTL killing in the standard ⁵¹Cr assay in round-bottom 96-well plates (⧫) was compared to experiments done in a flat-bottom plate (▪). (B) Standard assay in the flat-bottom well plate (▪) performed in parallel with a modified ⁵¹Cr assay in which the total number of cells was kept constant at 110,000 cells per well and only the E:T ratio was changed (⧫). Results represent the mean and standard error from three independent experiments with a representative Nef-specific CTL clone. (C) Correlation of the mathematical model to experimental data. The modified ⁵¹Cr release assay was performed in flat-bottom wells with decreasing numbers of cells per well (cell density). The percent specific lysis observed at each cell density is plotted against the calculated mean distance from effector to target cell obtained from the mathematical model for three E:T ratios (equation 1). Results represent the mean from three independent experiments with a representative HIV Nef-specific clone. R² values represent correlation coefficients and are highly significant at all three E:T ratios.

FIG. 6.

Effects of X4 gp120 expression by target cells on CTL lysis. Two HIV-1 Nef-specific CTL clones, DMD (A) and ND-25 (B), were tested for lytic activity against peptide-pulsed, autologous BLCL (⧫), BLCL transduced with an rAAV vector encoding RFP (▪), or X4 gp120 (▴). Lysis was measured by using the standard ⁵¹Cr release assay in flat-bottom 96-well plates at the indicated E:T ratios. Results represent the mean and standard error from three independent experiments.

FIG. 7.

HIV-1 X4 gp120 inhibits T-cell infiltration into a site of antigen challenge in vivo. C57BL/6 (A) and OT-1 (B) mice were immunized with OVA subcutaneously. Three days later, mice were challenged with IP OVA (time zero). Twenty-four hours after IP OVA injection, one of several forms of recombinant HIV-1 X4 gp120, HIV-1_IIIB gp120 (▪), HIV-1_IIIB gp120 ΔV1/V2 (ΔV1V2) (▴ [A]), or HIV-1_IIIB gp120 ΔV1/V2/V3 (ΔV1V2V3) (×), was administered. Control mice received an IP injection of normal saline (⧫) or heat-inactivated (HI) HIV-1_IIIB gp120 (▴ [B]). T-cell infiltration following the IP injection of recombinant gp120 or the control was then quantitated 3 h later and after 24 h by cell counting, immunostaining, and flow cytometry. The results represent the mean and standard error of three independent experiments.