HIV-1 Efficient Entry in Inner Foreskin Is Mediated by Elevated CCL5/RANTES that Recruits T Cells and Fuels Conjugate Formation with Langerhans Cells

Male circumcision reduces acquisition of HIV-1 by 60%. Hence, the foreskin is an HIV-1 entry portal during sexual transmission. We recently reported that efficient HIV-1 transmission occurs following 1 h of polarized exposure of the inner, but not outer, foreskin to HIV-1-infected cells, but not to cell-free virus. At this early time point, Langerhans cells (LCs) and T-cells within the inner foreskin epidermis are the first cells targeted by the virus. To gain in-depth insight into the molecular mechanisms governing inner foreskin HIV-1 entry, foreskin explants were inoculated with HIV-1-infeceted cells for 4 h. The chemokine/cytokine milieu secreted by the foreskin tissue, and resulting modifications in density and spatial distribution of T-cells and LCs, were then investigated. Our studies show that in the inner foreskin, inoculation with HIV-1-infected cells induces increased CCL5/RANTES (1.63-fold) and decreased CCL20/MIP-3-alpha (0.62-fold) secretion. Elevated CCL5/RANTES mediates recruitment of T-cells from the dermis into the epidermis, which is blocked by a neutralizing CCL5/RANTES Ab. In parallel, HIV-1-infected cells mediate a bi-phasic modification in the spatial distribution of epidermal LCs: attraction to the apical surface at 1 h, followed by migration back towards the basement membrane later on at 4 h, in correlation with reduced CCL20/MIP-3-alpha at this time point. T-cell recruitment fuels the continuous formation of LC-T-cell conjugates, permitting the transfer of HIV-1 captured by LCs. Together, these results reveal that HIV-1 induces a dynamic process of immune cells relocation in the inner foreskin that is associated with specific chemokines secretion, which favors efficient HIV-1 entry at this site

HIV-1-infected cells affect secretion of chemokines by the inner foreskin.

<p>Inner foreskin explants were inoculated in a polarized manner for 4 h with either non-infected or HIV-1-infected PBMCs, washed, and further incubated in medium in a non-polarized manner. The levels of twelve chemokines and cytokines were measured the next day in the culture supernatants, using a custom multiplex bead immunoassay and quantified by flow cytometry or specific ELISA. Shown are mean folds±SEM secretion of each analyte (derived from two independent explants), calculated as [secretion after exposure to HIV-1-infected PBMCs/secretion after exposure to non-infected PBMCs], for either chemokines/cytokines not altered (A) or altered (B) following exposure to HIV-1-infected cells. In (B), p = 0.0105, 0.0308, 0.0166, 0.0045 for INF gamma, CCL3/MIP-1 alpha, CCL20/MIP-3 alpha and CCL5/RANTES, respectively, Student's t-test, n = 2.</p

HIV-1-infected cells modify the spatial distribution of LCs in inner foreskin.

<p>(A) Representative images of normal inner foreskin (top), and inner foreskin explants exposed to either non-infected (left) or HIV-1-infected (right) PBMCs for 1 h (middle) or 4 h (bottom). Explants were then stained with anti-langerin Ab and visualized with AEC (normal foreskin) or DAB (foreskin explants) peroxidase substrates. The black double-headed arrows denote individual distances of LCs from the apical surface for each condition and are marked as [a_i]-[e_i] respectively. Images are representative of three independent experiments. Scale bars = 20 µm. (B) Calculated mean±SEM distances covered by LCs, following exposure of inner foreskin explants to either non-infected (white bars) or HIV-1-infected (grey bars) PBMCs. For exposure to non-infected PBMCs, the distances after 1 h and 4 h were calculated as [b]-[a] and [c]-[b], respectively, where a, b, c are means of the individual [a_i], [b_i], [c_i] distances (measured for 106, 193 and 148 different cells in three independent explants). For exposure to HIV-1-infected PBMCs, the distances after 1 h and 4 h were calculated as [a]-[d] and [e]-[d], respectively, where d and e are mean of the individual [d_i] and [e_i] distances (measured for 186 and 108 different cells in three independent explants). *p<0.0001 infected vs. non-infected at 4 h; Student's t-test. (C) shows the actual distances from the apical surface measured for each experimental condition. *p<0.0001 vs. normal foreskin; Student's t-test.</p

(A, B) HIV-1-infected cells induce retention of LCs within the epidermis.

<p>Parallel inner (A) and outer (B) foreskin explants were exposed for 4 h to either HIV-1-infected (solid lines) or non-infected (broken lines) PBMCs. Explants were then stained with anti-langerin Ab and visualized with DAB peroxidase substrate. Shown are means±SEM langerin+ cell densities (cells/mm²) in epidermis derived from three independent explants. Cells were counted in a minimum of 10 fields/section in each experiment. In (A): p = 0.0322 non-infected epidermis 4 h vs. 0 h and p = 0.0415 HIV-1-infected vs. non-infected at 4 h; Student's t-test. (C, D) HIV-1 entry and capture by LCs in inner, but not outer, foreskin explants. Single sections observed by confocal microscopy of parallel inner (C) and outer (D) foreskin explants, following 4 h exposure to HIV-1-infected PBMCs. Horizontal and vertical lines represent the localization of the sectioned stacks along the z axis (25 sections, 0.2 µm apart) that are shown below and to the right of each image. Sections were double-stained with goat-anti-human langerin Ab followed by TRITC-conjugated anti-goat IgG Ab and a mixture of several human/mouse anti-HIV-1 mAbs followed by FITC-conjugated anti-human/mouse IgG Abs. In (C), langerin is detected around cell bodies and dendrites reaching the apical surface (red arrowheads); HIV-1 virions are detected as dots in the epidermis associated with epithelial cells or in dermis (green arrowheads), as well as co-localized with LCs (green arrows). Higher magnification image with the xyz planes rotated shows viral particles internalized into LCs. Cell nuclei were counterstained with DAPI. White dotted lines denote the epidermis/dermis interface. Scale bars = 10 µm. Representative images of n = 3 experiments.</p

CCL5/RANTES mediates T-cell recruitment into the epidermis.

<p>(A) Representative FACS profiles out of two independent experiments, showing epidermal single-cell suspensions derived from inner foreskin explants exposed for 4 h to either medium alone (left), non-infected PBMCs (middle), or HIV-1-infected PBMCs (right). Cells were stained with PE-conjugated anti-human CD3 mAb and numbers represent the percentages of CD3+ cells in the framed windows (determined based on the non-specific staining of a matched isotype control Ab). (B) Inner foreskin explants were exposed for 4 h to either non-infected PBMCs (white bar) or HIV-1-infected PBMCs: alone (black bar); in the presence of 40 µg/ml control goat IgG Ab (dark grey bar); in the presence of 20 or 80 µg/ml neutralizing goat Ab to CCL5/RANTES (light grey bars). Following infection, epidermal single-cell suspensions were prepared and stained for CD3 expression as described above. Shown are mean folds increase±SEM of the percentages of CD3+ cells, normalized against that following exposure to non-infected PBMCs and derived from two independent explants. p = 0.0129 infected vs. non-infected and p = 0.0128 infected+CCL5 Ab 80 µg/ml vs. infected; Student's t-test.</p

Inhibition of PI3K pathway increases immune infiltrate in muscle-invasive bladder cancer

International audienceAlthough immune checkpoint inhibitors have shown improvement in survival in comparison to chemotherapy in urothelial bladder cancer, many patients still fail to respond to these treatments and actual efforts are made to identify predictive factors of response to immunotherapy. Understanding the tumor-intrinsic molecular basis, like oncogenic pathways conditioning the presence or absence of tumor-infiltrating T cells (TILs), should provide a new rationale for improved anti-tumor immune therapies. In this study, we found that urothelial bladder cancer from human samples bearing PIK3CA gene mutations was significantly associated with lower expression of a defined immune gene signature, compared to unmutated ones. We identified a reduced 10-gene immune gene signature that discriminates muscle-invasive bladder cancer (MIBC) samples according to immune infiltration and PIK3CA mutation. Using a humanized mouse model, we observed that BKM120, a pan-PI3K inhibitor, significantly inhibited the growth of a human bladder cancer cell line bearing a PIK3CA mutation, associated to increased immune cell infiltration (hCD45+). Using qRT-PCR, we also found an increase in the expression of chemokines and immune genes in PIK3CA-mutated tumors from mice treated with BKM120, reflecting an active immune infiltrate in comparison to untreated ones. Moreover, the addition of BKM120 rendered PIK3CA-mutated tumors sensitive to PD-1 blockade. Our results provide a relevant rationale for combination strategies of PI3K inhibitors with immune checkpoint inhibitors to overcome resistance to immune checkpoint inhibitors