Expression Profile of Human Fc Receptors in Mucosal Tissue: Implications for Antibody-Dependent Cellular Effector Functions Targeting HIV-1 Transmission

Abstract

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.

Fig 1. Characterization of immune cell phenotypes within mucosal tissue and RBC-lysed whole blood.

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).

Fig 2. Flow cytometry analysis gating strategies in FlowJo.

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.

Fig 3. CD14+ Fc Receptor (FcR) expression in RBC-lysed whole blood and mucosal tissue compartments.

Percentage expression of CD16, CD32, CD64 and CD89 on viable CD14+ cells (A) isolated from penile glans (n = 9); ectocervical (n = 4) and colorectal (n = 6) tissue and 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 CD14+ cells across the three tissues and RBC-lysed whole blood (mean values shown).

Fig 4. Myeloid Dendritic Cell Fc Receptor (FcR) expression in RBC-lysed whole blood and mucosal tissue compartments.

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).

Fig 5. NK Cell Fc Receptor (FcR) expression in RBC-lysed whole blood and mucosal tissue compartments.

Percentage expression of CD16, CD32, CD64 and CD89 on viable NK (A) cells isolated from penile glans (n = 5); ectocervical (n = 4) and colorectal (n = 3) 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 NK cells across the three tissues and RBC-lysed whole blood (mean values shown).

Fig 6. Localization of CD14+ cells within three different mucosal tissue types.

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.

Fig 7. Localization of CD11c+ cells within three different mucosal tissue types.

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.

Fig 8. Localization of CD56+ cells within three different mucosal tissue types.

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.