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. 2011;6(8):e23202.
doi: 10.1371/journal.pone.0023202. Epub 2011 Aug 5.

HIV envelope gp120 activates LFA-1 on CD4 T-lymphocytes and increases cell susceptibility to LFA-1-targeting leukotoxin (LtxA)

Catarina E Hioe  1 , Michael Tuen, Gaia Vasiliver-Shamis, Yelina Alvarez, Kathleen C Prins, Sagarika Banerjee, Arthur Nádas, Michael W Cho, Michael L Dustin, Scott C Kachlany
Affiliations

HIV envelope gp120 activates LFA-1 on CD4 T-lymphocytes and increases cell susceptibility to LFA-1-targeting leukotoxin (LtxA)

Catarina E Hioe et al. PLoS One. 2011.

Abstract

The cellular adhesion molecule LFA-1 and its ICAM-1 ligand play an important role in promoting HIV-1 infectivity and transmission. These molecules are present on the envelope of HIV-1 virions and are integral components of the HIV virological synapse. However, cellular activation is required to convert LFA-1 to the active conformation that has high affinity binding for ICAM-1. This study evaluates whether such activation can be induced by HIV itself. The data show that HIV-1 gp120 was sufficient to trigger LFA-1 activation in fully quiescent naïve CD4 T cells in a CD4-dependent manner, and these CD4 T cells became more susceptible to killing by LtxA, a bacterial leukotoxin that preferentially targets leukocytes expressing high levels of the active LFA-1. Moreover, virus p24-expressing CD4 T cells in the peripheral blood of HIV-infected subjects were found to have higher levels of surface LFA-1, and LtxA treatment led to significant reduction of the viral DNA burden. These results demonstrate for the first time the ability of HIV to directly induce LFA-1 activation on CD4 T cells. Although LFA-1 activation may enhance HIV infectivity and transmission, it also renders the cells more susceptible to an LFA-1-targeting bacterial toxin, which may be harnessed as a novel therapeutic strategy to deplete virus reservoir in HIV-infected individuals.

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Conflict of interest statement

Competing Interests: Dr. Scott C. Kachlany is a consultant for Actinobac Biomed, Inc. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. Naïve CD4 T cells do not form stable interaction with the ICAM-1 bilayer.
The naïve CD4 T cells were injected onto a bilayer containing only ICAM-1 and monitored over one hour for the presence of cells (bright-field panels), contact with the bilayer (interference reflection microscopy (IRM) panels) and contact with ICAM-1 (ICAM-1 panel). The same representative field is shown at the indicated time points. Thin black arrows show the cells that interacted transiently with the bilayers at 15 min but had no ICAM-1 accumulation and migrated by 30 min. Three new cells interacting with the bilayer at 50 min were also observed and indicated by thick white arrows; again these cells made no ICAM-1 contact and did not induce any ICAM-1 aggregation.
Figure 2
Figure 2. HIV gp120 interaction with quiescent naïve CD4 T cells triggers LFA-1 activation and supramolecular rearrangement.
(A) Naïve CD4 T cells establish LFA-1-ICAM-1 interaction upon gp120 binding. The naïve CD4 T cells purified by negative selection from ex vivo HIV-seronegative PBMCs were introduced to a bilayer containing Alexa Fluor 488-labeled gp120 and Cy5-labeled ICAM-1, and images were acquired over an hour. Images from one representative cell to show the dynamics of cell interaction with gp120 and ICAM-1 over time are presented. Top panels show gp120 contact the cell made at the indicated time points, middle panels show ICAM-1 contact, and bottom panels display merged images (gp120 in green, ICAM-1 in red). (B) Morphology of ICAM-1 contact areas made upon the interaction of naïve CD4 T cells with gp120 and ICAM-1 on the bilayers. Images of representative cells and the percentages of cells that form symmetrical (top panel) or asymmetrical (bottom panel) ICAM-1 rings are shown. gp120 was added onto the bilayers at a density of 200 to 250 molecules/µm2. (C) Naïve T cells were added to the bilayers in the presence of an anti-gp120 mAb that blocks gp120-CD4 interaction (654), an anti-V3 mAb that interferes with gp120 binding to the chemokine receptor (2219), or a mAb to the N-terminus of gp120 (EH21) that does not affect gp120 interaction with its receptors. The percentages of cells making gp120-positive (left) and ICAM-1-positive (right) contacts out of the total number of cells seen in the fields were calculated. The MAbs were used at 20 µg/ml. The graphs represent the averages +/- SEM of three independent experiments. Statistical analysis was done by one-sided Student's t test. (D and E) Morphology of ICAM-1 contact areas made upon the interaction of naïve CD4 T cells with bilayers containing ICAM-1 and monoclonal antibodies to CD4 (OKT4) or CD3 (OKT3). The percentages of cells that form symmetrical (top panel) or asymmetrical (bottom panel) ICAM-1 rings are shown for comparison with those observed in gp120 + ICAM-1 bilayers (B). The densities of OKT4 and OKT3 on the bilayer were 250 molecules/µm2.
Figure 3
Figure 3. The interaction of quiescent CD4 T cells with surface-bound gp120 renders the cells more susceptible to leukotoxin LtxA.
(A) CD4 T cells purified ex vivo from HIV-seronegative PBMCs were incubated on wells coated with 10 µg/ml wild type gp120 (JRFL), mutant gp120 (JRFL) lacking CD4-binding site and V3, or no gp120 and then treated with titrated amounts of LtxA. (B) For comparison, CD4 T cells were also incubated on wells coated with anti-CD3 and anti-CD28 antibodies (5 µg/ml each) or anti-CD4 antibody (10 µg/ml) and treated with the designated concentrations of LtxA. (C) CD4 T cells were incubated on gp120-coated wells in the presence of monoclonal antibodies (mAb) against the CD4-binding site (anti-CD4bs) or the V3 loop of gp120, soluble CD4 (sCD4), or the chemokine receptor antagonists, and then treated with titrated amounts of LtxA. (D) R5 gp120 (JRFL) or X4 gp120 (HXB2) was used to coat the wells, incubated with CD4 T cells, and tested for induction of LtxA-mediated killing. In A, C, and D, the gp120 proteins were coated on microtiter wells at 10 µg/ml. (E) CD4 T cells were treated with soluble gp120 (10 µg/ml), anti-CD3/anti-CD28 coated on wells, or gp120 coated on wells, prior to addition of LtxA. After incubation with LtxA for 20 hrs, the cell viability was measured based on cellular ATP. Data from one of two or more representative experiments are shown. The mean and standard deviation values from duplicate wells are presented.
Figure 4
Figure 4. (A) CD4 T cells bearing HIV p24 antigen express higher levels of total LFA-1 on the surface.
Ex vivo PBMCs from two viremic HIV-infected subjects (PS05 with 38,165 vRNA copies/ml and PS07 with 35,201 vRNA copies/ml) were stained with mAbs for surface expression of CD3, CD8, and total LFA-1, as well as for intracellular p24 antigen after permeabilization. The cells were subjected to flow cytometric analyses, and the data analyzed by the FlowJo software. The dot plots (top) show the percentages of p24+ cells among CD4 T cells (CD3+ CD8-) from subjects PS05 and PS07. The p24+ gating were determined based on p24 staining of HIV-seronegative PBMCs (Fig. S3). The histograms (bottom) compare LFA-1 expression on p24+ and p24- CD4 T cells; the mean fluorescence intensity (mfi) for p24+ and p24- CD4 T cells are 462 and 376 for PS05, and 311 and 228 for PS07. (B) Expression of active LFA-1 on p24+ versus p24- CD4 T cells. PBMCs from viremic HIV-infected subjects (PS15 with 5,075 vRNA copies/ml and PS16 with 7,077 vRNA copies/ml) were stained with mAb NKI-L16 and secondary fluorescent anti-mouse antibodies, followed with direct staining with fluorescent mAbs to CD3, CD8 and p24. The histograms compare mAb NKI-L16 staining on p24+ and p24- CD4 T cells; the mfi for p24+ and p24- cells are 405 and 95 for PS15 and 835 and 86 for PS16. p24+ CD4 T cells treated with the secondary antibody (no NKI-L16) were also shown for control (mfi = 19 and 18). (C) Reduction of viral DNA in HIV-infected PBMCs due to LtxA cytotoxicity. PBMCs from two viremic HIV-infected subjects (PS05 with 38,165 vRNA copies/ml and CD4 count of 814 and PS14 with 21,815 vRNA copies/ml and CD4 count of 494) were treated with LtxA (7.8 µg/ml) for 20 hrs. The viral DNA were quantified by real time PCR with the specific primers. Averages and standard deviation from epeat experiments are presented. Statistical analysis was done by one-sided Student's t test.
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