MAdCAM costimulation through Integrin-α4β7 promotes HIV replication

Abstract

Human gut-associated lymphoid tissues (GALT) play a key role in the acute phase of HIV infection. The propensity of HIV to replicate in these tissues, however, is not fully understood. Access and migration of naive and memory CD4⁺ T cells to these sites is mediated by interactions between integrin α₄β₇, expressed on CD4⁺ T cells, and MAdCAM, expressed on high endothelial venules. We report here that MAdCAM delivers a potent costimulatory signal to naive and memory CD4⁺ T cells following ligation with α₄β₇. Such costimulation promotes high levels of HIV replication. An anti-α₄β₇ mAb that prevents mucosal transmission of SIV blocks MAdCAM signaling through α₄β₇ and MAdCAM-dependent viral replication. MAdCAM costimulation of memory CD4⁺ T cells is sufficient to drive cellular proliferation and the upregulation of CCR5, while naive CD4⁺ T cells require both MAdCAM and retinoic acid to achieve the same response. The pairing of MAdCAM and retinoic acid is unique to the GALT, leading us to propose that HIV replication in these sites is facilitated by MAdCAM-α₄β₇ interactions. Moreover, complete inhibition of MAdCAM signaling by an anti-α₄β₇ mAb, an analog of the clinically approved therapeutic vedolizumab, highlights the potential of such agents to control acute HIV infection.

Figure 1

MAdCAM stimulation promotes HIV replication. (a) Primary CD4⁺ T cells cultured for three days in the presence of stimulatory ligands – either anti CD3 alone or anti CD3 + MAdCAM, in the presence of anti-α4β7 mAb or a control mAb (Synagis). Three days post-stimulation, cells were infected with the HIV isolate SF162 and viral replication was assessed by measuring the concentration of p24 antigen in cell supernatants 4 and 6 days post-infection. (b) Replication of HIV-SF162 in CD4⁺ T cells isolated from 6 independent donors, in the presence of stimulatory ligands as described in (a). The concentration of viral p24 antigen in culture supernatants on day 6 post-infection is shown. *P < 0.05 (two-tailed parametric paired t-test).

Figure 2

The anti-α4β7 mAb inhibits MAdCAM-mediated CD4⁺ T cell proliferation. (a) Cellular proliferation in primary CD4⁺ T cells cultured for 5 days in the presence of anti CD3 alone, anti CD3 + MAdCAM, anti CD3 + MAdCAM + a control mAb (Synagis), or anti CD3 + MAdCAM + anti-α4β7 mAb. Proliferation was measured by CFSE dye dilution: red, draw model sum; blue, draw model components; black, total division profile. The percent of diluted CFSE labeled cells (divided cells) is shown in blue, above the blue bar, and undiluted (undivided) cells is shown in red. (b) Division of primary CD4⁺ T cells isolated from 5 independent donors and stimulated with ligands as in (a), represented by a division index (measure of the average number of divisions). * P < 0.0001 (two tailed parametric paired t test). (c) The expression of Integrin-β7 (y-axis, top row) and CD45RO (y-axis, bottom row) on CD4⁺ T cells stimulated for 5 days with anti CD3 or anti CD3 + MAdCAM is shown by CFSE dye (x-axis).

Figure 3

MAdCAM stimulation promotes loss of CD62L expression. (a) Expression of CD62L (y-axis) and CD45RO (x-axis) on purified, primary, CD4⁺ T cells 5 hours post-stimulation with anti CD3 alone, anti CD3 + MAdCAM, anti CD3 + MAdCAM + anti-α4β7 mAb, or anti CD3 + MAdCAM + control mAb (Synagis). Cells below the black dashed line reflect loss of CD62L expression. In CD45RO⁻ of CD45RO⁺ cells, the percent of CD62L expressing cells is labeled in red or blue respectively. (b) The percent loss of CD62L expression (y-axis) in either CD45RO⁻ or CD45RO⁺ subsets of CD4⁺ T cells from four independent donors, treated as in (a). (c) A representative dot plot illustrating the expression of Integrin-β7 (y-axis) by CD45RO (x-axis) on unstimulated CD4⁺ T cells, with red boxes around the β7⁺/CD45RO⁺ and β7⁻/CD45RO⁺ subsets. (d) The percent loss of CD62L expression on purified CD4⁺ T cells subdivided into the CD45RO⁺/β7⁺ and CD45RO⁺/β7⁻ populations as in (c) and treated as in (a). *P < 0.05 (two-tailed parametric paired t-test).

Figure 4

RA combined with MAdCAM drives costimulation of naïve CD4⁺ T cells. (a) Purified naïve CD45RO⁻/CD4⁺ T cells stained with CFSE dye (x-axis), then stimulated for 5 days with anti CD3 alone or anti CD3 + MADCAM, with the addition of either RA or anti CD28. The blue bar marks cellular proliferation in CFSE diluted cells with the percent of either divided or undivided cells indicated in blue or red, respectively, as in (Figure 2a). (b) Division index of purified naïve CD45RO⁻/CD4⁺ T cells isolated from 7 independent donors and stimulated as in (a). * P < 0.008 (two-tailed parametric paired t-test). (c) The expression of Integrin-β₇ (y-axis) by CFSE (x-axis), on purified naïve CD45RO⁻/CD4⁺ T cells, stimulated for 5 days with anti CD3 + MAdCAM + RA or anti CD3 + MAdCAM + anti CD28.

Figure 5

Naïve CD4⁺ T cells stimulated with RA and MAdCAM support viral replication. Replication of HIV SF162 in purified CD45RO⁻/CD4⁺ T cells from 4 independent donors, where cells were stimulated with anti CD3 alone or in combination with MAdCAM, RA or anti CD28 as designated, for 3 days prior to infection. Viral replication was quantified by p24 AlphaLISA (y-axis) on days 4 (purple) and 7 (red) post-infection (x-axis). The purity of CD45RO⁻/CD4⁺ T cell cultures assessed on day 0, is shown.

Figure 6

MAdCAM stimulation of PBMCs from HIV infected subjects promotes viral replication. PBMCs isolated from six HIV infected donors were CD8⁺ T cell depleted, and cultured over ligand coated plates. The stimulatory ligands include anti CD3, anti CD28, MAdCAM and RA in various combinations as designated. Viral p24 was measured by AlphaLISA (y-axis) from culture supernatants at day 7, 11, 14, and 18 (x- axis).