Small CD4 Mimetics Prevent HIV-1 Uninfected Bystander CD4 + T Cell Killing Mediated by Antibody-dependent Cell-mediated Cytotoxicity

Abstract

Human immunodeficiency virus type 1 (HIV-1) infection causes a progressive depletion of CD4 + T cells. Despite its importance for HIV-1 pathogenesis, the precise mechanisms underlying CD4 + T-cell depletion remain incompletely understood. Here we make the surprising observation that antibody-dependent cell-mediated cytotoxicity (ADCC) mediates the death of uninfected bystander CD4 + T cells in cultures of HIV-1-infected cells. While HIV-1-infected cells are protected from ADCC by the action of the viral Vpu and Nef proteins, uninfected bystander CD4 + T cells bind gp120 shed from productively infected cells and are efficiently recognized by ADCC-mediating antibodies. Thus, gp120 shedding represents a viral mechanism to divert ADCC responses towards uninfected bystander CD4 + T cells. Importantly, CD4-mimetic molecules redirect ADCC responses from uninfected bystander cells to HIV-1-infected cells; therefore, CD4-mimetic compounds might have therapeutic utility in new strategies aimed at specifically eliminating HIV-1-infected cells.

Keywords: ADCC; Bystander killing; CD4; CD4-bound conformation; CD4-mimetics; Envelope glycoproteins; HIV-1; Non-neutralizing antibodies; gp120.

Graphical abstract

Fig. 1

ADCC-mediating antibodies and sera from HIV-1-infected individuals efficiently recognize uninfected bystander CD4 + T cells. Cell-surface staining of primary CD4 + T cells either mock-infected or infected with NL4-3 GFP ADA based virus, either wild-type (wt) or expressing D368R Env with (a,b) A32 mAbs or (c) sera from 10 HIV-1-infected individuals and sera from 5 uninfected individuals. Shown in (a,c) are (left) dot blots depicting representative staining and (right) mean fluorescence intensities (MFI) obtained for multiple stainings. (b) Correlation between the levels of infection and recognition of uninfected bystander (GFP −) (depicted in gray) and infected (GFP +) CD4 + T cells (depicted in green) by the A32 mAb. (d) Cell-surface staining of primary CD4 + T cells infected with an X4-tropic virus (NL4-3 GFP) and a primary HIV-1 isolate (CH77) with sera from 10 HIV-1-infected individuals. Shown in (left) are dot blots depicting representative staining and (right) MFI for all tested sera. (e) Cell-surface staining of primary CD4 + T cells isolated from 6 HIV-1-infected individuals after activation with autologous sera. Shown in (left) and (center) are dot blots depicting representative staining obtained for two different HIV-1-infected individuals, and (right) Median Fluorescence Intensity (MFI) values obtained with all individuals. Error bars indicate the mean ± SEM. Statistical significance was tested using (a,c) an ordinary one-way ANOVA test with a Holm–Sidak post-test, (d) an unpaired t test, (e) a Kruskal–Wallis with a Dunn's post-test and (b) a Spearman rank correlation (**p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant).

Fig. 2

Shed gp120 represents the major source of Env present on the surface of uninfected bystander cells. (a,b) Cell-surface staining of primary CD4 + T cells infected with the NL4-3 GFP ADA wt virus with anti-Env Abs (A32, C11, 2G12, F240 and PGT151). Shown in (a) are dot blots depicting a representative staining obtained 72 h post-infection. (b) Quantification of data presented in A as fold binding over mock. (c) Quantification of cell-surface staining of primary CD4 + T cells infected with CH77 with anti-Env Abs (A32, C11, F240 and PGT151), as fold binding over mock. Statistical significance was tested using the Mann–Whitney t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns: not significant).

Fig. 3

Shed gp120 from productively infected cells is the major source of gp120 present on the surface of uninfected bystander CD4 + T cells. Detection of uninfected bystander primary CD4 + T cells infected with (a) NL4-3 GFP ADA wt virus or (b) CH77, by anti-gp120 Abs (A32, C11, 2G12; depicted in red) and anti-gp41 (F240) and anti-gp120-gp41 Abs (PGT151; depicted in blue) over the course of the infection. The data presented are representative of the results obtained in cells from at least two donors. (c,d) Uninfected cells, stained with the cellular dye eFluor 450, were co-cultured with unstained autologous mock-infected or HIV-1-infected cells. The ability of anti-Env Abs (A32, C11, 2G12, F240 and PGT151) to recognize uninfected bystander cells, designated as eFluor-450 + GFP − cells, was evaluated by FACS at 0, 24 and 48 h of co-culture. Shown in (c) are the gating strategy and a representative staining obtained with the A32 Ab. Shown in (d) are the quantification of the results obtained with at least 4 donors with all tested anti-Env Abs. Statistical significance was tested using the Mann–Whitney t test (*p < 0.05, **p < 0.01, ns: not significant).

Fig. 4

Cell-to-cell transmission is not required for the presence of gp120 on the surface of bystander cells. Uninfected cells, stained with the cellular dye eFluor 450, were co-cultured with unstained autologous mock-infected cells or with HIV-1-infected cells, either in the context or not of a transwell system. The ability of the anti-Env Ab A32 to recognize uninfected bystander cells, designated as eFluor-450 + GFP − cells, was evaluated by FACS after (a) 24 h and (b) 48 h of co-culture. Shown in the top panels are the gating strategy and a representative staining obtained with the A32 Ab. Shown in the bottom panels are the quantification of the results obtained with 5 donors. Error bars indicate the mean ± SEM. Statistical significance was tested using a Kruskal–Wallis with a Dunn's post-test (**p < 0.01, ns: not significant).

Fig. 5

Uninfected bystander cells can be eliminated by ADCC. CEM.NKr cells infected with the NL4-3 ADA GFP wt virus were used as target cells in our FACS-based ADCC assays. Shown in (a) are dot blots depicting the cell count and flow cytometry particle count (bottom left) of a representative ADCC killing experiment with A32 mAb and serum from an HIV-1-infected individual. (b) Percentage of ADCC killing obtained with the A32 mAb in 6 independent experiments. (c) Percentage of ADCC-mediated killing of uninfected bystander (GFP −) cells with the A32 mAb in the presence or absence of the A32 Fab fragment. (d) Percentage of ADCC-mediated killing obtained with sera from 22 HIV-1-infected individuals. Error bars indicate the mean ± SEM. Statistical significance was tested using (b,d) an ordinary one-way ANOVA test with a Holm–Sidak post-test or (c) a Mann–Whitney t test (*p < 0.05, **p < 0.01, ****p < 0.0001).

Fig. 6

CD4-binding site ligands abrogate recognition of bystander CD4 + T cells. Uninfected cells, stained with the cellular dye eFluor 450, were co-cultured with unstained autologous mock-infected cells or with HIV-1-infected cells in the absence or presence of the CD4-mimetic JP-III-48 or VRC01 Fab fragment. The ability of HIV + sera to recognize uninfected bystander cells, designed as eFluor-450 + GFP − cells, was evaluated by FACS after (a) 24 h and (b) 48 h of co-culture. Shown in the top panels are the gating strategy and a representative staining. Shown in the bottom panels are the quantification of the results obtained with 8 HIV + sera using 4 different CD4 + T-cell donors. (c) Staining of primary CD4 T cells infected with the NL4-3 ADA GFP wt virus with sera from HIV-1-infected individuals, in presence of the CD4mc JP-III-48 or VRC01 Fab fragment. Shown on the left are histograms depicting representative staining. Shown in the right panel is the quantification of the results obtained with 6 different HIV + sera. Error bars indicate the mean ± SEM. Statistical significance was tested using a Mann–Whitney t test (**p < 0.01, ***p < 0.001, ns: not significant).