The integrin alpha4beta7 forms a complex with cell-surface CD4 and defines a T-cell subset that is highly susceptible to infection by HIV-1

Abstract

Both activated and resting CD4(+) T cells in mucosal tissues play important roles in the earliest phases of infection after sexual transmission of HIV-1, a process that is inefficient. HIV-1 gp120 binds to integrin alpha(4)beta(7) (alpha(4)beta(7)), the gut mucosal homing receptor. We find that alpha(4)beta(7)(high) CD4(+) T cells are more susceptible to productive infection than are alpha(4)beta(7)(low-neg) CD4(+) T cells in part because this cellular subset is enriched with metabolically active CD4(+) T cells. alpha(4)beta(7)(high) CD4(+) T cells are CCR5(high) and CXCR4(low); on these cells, alpha(4)beta(7) appears in a complex with CD4. The specific affinity of gp120 for alpha(4)beta(7) provides a mechanism for HIV-1 to target activated cells that are critical for efficient virus propagation and dissemination following sexual transmission.

Fig. 1.

α₄β₇^high CD4⁺ T cells are a preferential target of productive infection. In vitro infection of CD4⁺ T cells after 6 days of culture in the presence of retinoic acid. (A) Flow cytometric analysis of cell surface integrin β₇ and intracellular Gag p24 expression on and in CD4⁺ T cells 3, 6, and 8 days after inoculation with the R5 HIV-1 SF162. (B) Percent intracellular Gag p24 expression in α₄β₇^high and α₄β₇^low-neg CD4⁺ T cells 3 days post-inoculation using three different amounts of SF162. Values represent the % p24⁺ cells within each subset. (C) Comparison of integrin β₇ expression on CD4⁺ T cells 8 days post-inoculation with the same cells left uninfected. (D) p24 antigen ELISA of culture supernatants harvested from α₄β₇^high and α₄β₇^low-neg CD4⁺ T-cell subsets 3, 6, and 8 days post-inoculation with SF162. Levels of integrin β₇ expression on both subsets on the day of inoculation are included (Inset). These results are representative of three independent experiments using different donor CD4⁺ T cells.

Fig. 2.

α₄β₇^high CD4⁺ T cells express high levels of CCR5 and are metabolically active. Flow cytometric analysis of peripheral CD4⁺ T cells cultured in retinoic acid for 6 days followed by staining with fluoresceinated mAbs specific for (A) β₇ and CD45RO. (B) β₇ and CCR5. (C) β₇ and Ki-67. This is a representative analysis of receptor expression on more than three independent donor CD4⁺ T cells.

Fig. 3.

Activated CD4⁺ T cells in the gut and rectum are enriched in the α₄β₇^high subset. Cells freshly isolated from rectal and colon biopsies were analyzed by flow cytometry. (A) A representative analysis of CCR5 and Ki-67 expression on β₇^high and β₇^low-negCD4⁺ T cells. (B) Summary of Ki-67 and CCR5 expression on β₇^high and β₇^low-negCD4⁺ T cells isolated from the colon and rectum of three healthy donors. Values are reported as % within each population. Average % expression of CCR5 and Ki-67 expression in all β₇^high and β₇^low-negCD4⁺ T-cell samples analyzed is presented along with a significance value (nonparametric Wilcoxon signed-rank test for paired samples). (C) α₄β₇⁺/CD4⁺ T cells are detected in female genital mucosa (Mean 17.5, S.D. 13.4).

Fig. 4.

Delay in HIV-1 replication by an α₄ mAb. (A–C) Purified CD4⁺ T cells cultured in retinoic acid and inoculated with a low amount of HIV-1 SF162 in the presence of IgG1 or the α₄ mAb 2B4 (as indicated). Viral replication was determined by measuring by p24 Gag levels in culture supernatants on days 3, 6, and 9 post-infection. Three representative replication time-courses are presented. (D) Cumulative inhibition of viral replication (percent reduction in p24 Gag) from six independent infections mediated by mAb 2B4 relative to an IgG1 control on days 6 and 9 is presented. 2B4 significantly inhibited viral replication on day 6 (P < 0.001), but not on day 9 (P < 0.139). On average there is an 80% (95% CI: 71–89%) reduction in the level of p24 on day 6 in the infection in presence of 2B4. Error bars represent standard deviation from mean inhibition. Inhibition on day 6 was significantly greater than on day 9 (Wilcoxon signed-rank test).

Fig. 5.

On gut α₄β₇^high CD4⁺ T cells α₄β₇ colocalizes with CD4 and CCR5. Freshly isolated gut cells obtained from biopsies taken from healthy donors were stained with the α₄β₇ mAb Act-1 (red), the CD4 mAb OKT4 (purple), and the CCR5 mAb 2D7 (green) and viewed under a confocal microscope. Unstained and individual stains of a representative cell are presented along with digitally defined regions of colocalization between α₄β₇ and CD4 (yellow) and α₄β₇, CD4 and CCR5 (blue). This cell is representative of greater that 60 cells analyzed from four donors.

Fig. 6.

Integrin β₇ coprecipitates with the CD4 receptor. (A) A Western blot stained with a an integrin β₇ polyclonal antisera of cell lysates derived from α₄β₇^high CD4⁺ T cells treated or not with the crosslinking reagent DTSSP followed by coprecipitation with protein A agarose beads and the CD4 mAb OKT4 (lane 1), IgG1+DTSSP (lane 2), and OKT4+DTSSP (lane 3). A recombinant soluble α₄β₇ was run directly as a positive control (lane 4). These results are representative of three independent experiments. (B) A schematic depicting approximate sizes of α₄β₇, CD4, and a gp120 trimer.