Expression profile of human Fc receptors in mucosal tissue: implications for antibody-dependent cellular effector functions targeting HIV-1 transmission

The majority of new Human Immunodeficiency Virus (HIV)-1 infections are acquired via sexual transmission at mucosal surfaces. Partial efficacy (31.2%) of the Thai RV144 HIV-1 vaccine trial has been correlated with Antibody-dependent Cellular Cytotoxicity (ADCC) mediated by non-neutralizing antibodies targeting the V1V2 region of the HIV-1 envelope. This has led to speculation that ADCC and other antibody-dependent cellular effector functions might provide an important defense against mucosal acquisition of HIV-1 infection. However, the ability of antibody-dependent cellular effector mechanisms to impact on early mucosal transmission events will depend on a variety of parameters including effector cell type, frequency, the class of Fc-Receptor (FcR) expressed, the number of FcR per cell and the glycoslyation pattern of the induced antibodies. In this study, we characterize and compare the frequency and phenotype of IgG (CD16 [FcγRIII], CD32 [FcγRII] and CD64 [FcγRI]) and IgA (CD89 [FcαR]) receptor expression on effector cells within male and female genital mucosal tissue, colorectal tissue and red blood cell-lysed whole blood. The frequency of FcR expression on CD14+ monocytic cells, myeloid dendritic cells and natural killer cells were similar across the three mucosal tissue compartments, but significantly lower when compared to the FcR expression profile of effector cells isolated from whole blood, with many cells negative for all FcRs. Of the three tissues tested, penile tissue had the highest percentage of FcR positive effector cells. Immunofluorescent staining was used to determine the location of CD14+, CD11c+ and CD56+ cells within the three mucosal tissues. We show that the majority of effector cells across the different mucosal locations reside within the subepithelial lamina propria. The potential implication of the observed FcR expression patterns on the effectiveness of FcR-dependent cellular effector functions to impact on the initial events in mucosal transmission and dissemination warrants further mechanistic studies

Defining the interaction of HIV-1 with the mucosal barriers of the female reproductive tract

Worldwide, HIV-1 infects millions of people annually, the majority of whom are women. To establish infection in the female reproductive tract (FRT), HIV-1 in male ejaculate must overcome numerous innate and adaptive immune factors, traverse the genital epithelium, and establish infection in underlying CD4(+) target cells. How the virus achieves this remains poorly defined. By utilizing a new technique, we define how HIV-1 interacts with different tissues of the FRT using human cervical explants and in vivo exposure in the rhesus macaque vaginal transmission model. Despite previous claims of the squamous epithelium being an efficient barrier to virus entry, we reveal that HIV-1 can penetrate both intact columnar and squamous epithelial barriers to depths where the virus can encounter potential target cells. In the squamous epithelium, we identify virus entry occurring through diffusive percolation, penetrating areas where cell junctions are absent. In the columnar epithelium, we illustrate that virus does not transverse barriers as well as previously thought due to mucus impediment. We also show a statistically significant correlation between the viral load of inocula and the ability of HIV-1 to pervade the squamous barrier. Overall, our results suggest a diffusive percolation mechanism for the initial events of HIV-1 entry. With these data, we also mathematically extrapolate the number of HIV-1 particles that penetrate the mucosa per coital act, providing a biological description of the mechanism for HIV-1 transmission during the acute and chronic stages of infection

Localization of CD14+ cells within three different mucosal tissue types.

<p>Deconvolution microscopy images showing location of CD14+ cells within Penile Glans (A), Ectocervical (B) and Colorectal tissue (C) and relative co-expression of FcR CD16 (i), CD32 (ii), CD64 (iii) and CD89 (iv). CD14 is shown in green, the FcR are shown in red and DAPI is shown in blue. Images taken at 40x; Scale bar set to 40 microns.</p

Flow cytometry analysis gating strategies in FlowJo.

<p>A time gate was initially applied to exclude any electronic noise followed by a singlet gate excluded any doublets, then a gate was applied to include the cells of interest, followed by a viability gate to exclude any dead cells. (A) FcR analysis for both CD14+ and mDC; CD3-negative cells were included and split into CD14+ or CD14-CD19- cells. CD14-CD19- were further categorized based on their CD11c expression. Finally, CD14+ cells and mDC were assessed for their FcR expression (only CD32 shown here). (B) NK FcR phenotypic analysis was assess by investigating the CD56+ cells for their FcR expression (only CD16 shown here). Representative plots for cells isolated from penile glans tissue.</p

Characterization of immune cell phenotypes within mucosal tissue and RBC-lysed whole blood.

<p>The percentage of CD3+ T-Cells, CD14+ monocytic cells, CD19+ B cells, myeloid dendritic cells (mDC), NK cells, NKT cells and neutrophils in total viable cells isolated from (A) penile glans (n = 9), ectocervical (n = 5) and colorectal (n = 6) tissue and Whole Blood (n = 6). (B) Relative proportions of CD14+ monocytic cells, mDC and NK cells in penile, cervical and colorectal tissue and Whole Blood (mean and SD values shown).</p

Localization of CD14+ cells within three different mucosal tissue types.

<p>Deconvolution microscopy images showing location of CD14+ cells within Penile Glans (A), Ectocervical (B) and Colorectal tissue (C) and relative co-expression of FcR CD16 (i), CD32 (ii), CD64 (iii) and CD89 (iv). CD14 is shown in green, the FcR are shown in red and DAPI is shown in blue. Images taken at 40x; Scale bar set to 40 microns.</p

Localization of CD11c+ cells within three different mucosal tissue types.

<p>Deconvolution microscopy images showing location of CD11c+ cells within Penile Glans (A), Ectocervical (B) and Colorectal tissue (C) and relative co-expression of FcR CD16 (i), CD32 (ii), CD64 (iii) and CD89 (iv). CD11c is shown in green, the FcR are shown in red and DAPI is shown in blue. Images taken at 40x; Scale bar set to 40 microns.</p

Myeloid Dendritic Cell Fc Receptor (FcR) expression in RBC-lysed whole blood and mucosal tissue compartments.

<p>Percentage expression of CD16, CD32, CD64 and CD89 on viable mDC (A) isolated from penile glans (n = 9); ectocervical (n = 4) and colorectal (n = 4) tissue and RBC-lysed whole blood (n = 6) [Mean/SD values shown; comparisons of the FcR in the different tissue were made using Kruskal-Wallis with Dunn’s multiple comparison test]. (B) and (C) Boolean gating of FcR-positive cells to demonstrate the combinatorial nature of FcR expression in viable mDC across the three tissues and RBC-lysed whole blood (mean values shown).</p

Localization of CD56+ cells within three different mucosal tissue types.

<p>Deconvolution microscopy images showing location of CD56+ cells within Penile Glans (A), Ectocervical (B) and Colorectal tissue (C) and relative co-expression of FcR CD16 (i), CD32 (ii), CD64 (iii) and CD89 (iv). CD56 is shown in green, the FcR are shown in red and DAPI is shown in blue. Images taken at 40x; Scale bar set to 40 microns.</p

Deep Gene Sequence Cluster Analyses of Multi-Virus-Infected Mucosal Tissue Reveal Enhanced Transmission of Acute HIV-1

Exposure of the genital mucosa to a genetically diverse viral swarm from the donor HIV-1 can result in breakthrough and systemic infection by a single transmitted/founder (TF) virus in the recipient. The highly diverse HIV-1 envelope (Env) in this inoculating viral swarm may have a critical role in transmission and subsequent immune response. Thus, chronic (Envchronic) and acute (Envacute) Env chimeric HIV-1 were tested using multivirus competition assays in human mucosal penile and cervical tissues. Viral competition analysis revealed that Envchronic viruses resided and replicated mainly in the tissue, while Envacute viruses penetrated the human tissue and established infection of CD4+ T cells more efficiently. Analysis of the replication fitness, as tested in peripheral blood mononuclear cells (PBMCs), showed similar replication fitness of Envacute and Envchronic viruses, which did not correlate with transmission fitness in penile tissue. Further, we observed that chimeric Env viruses with higher replication in genital mucosal tissue (chronic Env viruses) had higher binding affinity to C-type lectins. Data presented herein suggest that the inoculating HIV-1 may be sequestered in the genital mucosal tissue (represented by chronic Env HIV-1) but that a single HIV-1 clone (e.g., acute Env HIV-1) can escape this trapped replication for systemic infection.IMPORTANCE During heterosexual HIV-1 transmission, a genetic bottleneck occurs in the newly infected individual as the virus passes from the mucosa, leading to systemic infection with a single transmitted HIV-1 clone in the recipient. This bottleneck in the recipient has just been described (K. Klein et al., PLoS Pathog 14:e1006754, https://doi.org/10.1371/journal.ppat.1006754), and the mechanisms involved in this selection process have not been elucidated. However, understanding mucosal restriction is of the utmost importance for understanding dynamics of infections and for designing focused vaccines. Using our human penile and cervical mucosal tissue models for mixed HIV infections, we provide evidence that HIV-1 from acute/early infection, compared to that from chronic infection, can more efficiently traverse the mucosal epithelium and be transmitted to T cells, suggesting higher transmission fitness. This study focused on the role of the HIV-1 envelope in transmission and provides strong evidence that HIV transmission may involve breaking the mucosal lectin trap.</p