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.