A: CD144 and (B) Podoplanin were solely visible in the submucosa when using the Fast Red Chromogen (Biocare Medical) due to the presence of dark melanocytes in the epithelium. However, when fluorescently labeled markers were used, both (C) CD144 and (D) Podoplanin were detected also in the epithelium (minimum distance to tissue surface [MDTS]: 111 μm and 55 μm, respectively). Black and white arrowheads indicate positive staining. Scale bars = 75 μm. Results are from one of at least four tissue samples yielding similar results.

A: CD4⁺ (green) and CD3⁺ (red) frequently co-localized (yellow) within the submucosa of the human foreskin (0.02 cells/100 μm² tissue). B: The CD4⁺ cells were observed both within the epithelium (I: MDTS: 92 μm) and deep in the submucosa (II: MDTS: 609 μm). A vast number of CD4⁺ cells were also detected within lymphoid aggregates, which were present below the basal membrane (III: MDTS: 231 μm; IV: area 10,587 μm²). White arrowheads indicate positive staining. Scale bars = 75 μm. Results are from one of at least four tissue samples yielding similar results. C: Measurements of (I) distance of cell body to tissue surface; (II) epithelial thickness; and (III) thickness of keratin layer were performed on five randomly selected HSV-2 seronegative (HSV-2⁻) individuals and five randomly selected HSV-2 seropositive (HSV-2⁺) individuals. All measurements were performed in approximately 5 × 10⁵ μm² tissue within 700 μm from the tissue surface. Results are presented in D. No differences were detected between HSV-2⁻ and HSV-2⁺ individuals regarding distance of CD3⁺ (red bars) or HLA-DR⁺ (green bars) cells to tissue surface; median (range) in micrometers were as follows: 238 (43 to 683) vs. 238 (65 to 664) and 207 (51 to 508) vs. 189 (59 to 460), respectively. Neither were differences in epithelial thickness (yellow bars) or thickness of keratin layer (blue bars) detected; median (range) in micrometers were as follows: 85 (42 to 232) vs. 88 (38 to 192) and 22 (10 to 42) vs. 23 (13 to 31), respectively. Bars show the medians and error bars the range.

A: LCs were defined here as CD1a⁺ (green) and/or Langerin⁺ (red), and these markers co-localized on the majority of cells (yellow; I: MDTS: 24 μm). B: CD1a⁺ (green) LCs within the epithelium frequently expressed CD4 (red; yellow cells: double positive; I: MDTS: 37 μm). C: CD1a⁺ CD4⁺ (yellow) LCs were also present deep within the submucosa (I: MDTS: 359 μm). D: CD1a⁺ (red) LCs expressed CD11c (green), and double positive (yellow) cells were found both within the epithelium (I: MDTS: 45 μm) and the submucosa (II: MDTS: 226 μm). E: CD1a⁺ (green) LCs expressing DC-LAMP (red) were solely present in the submucosal compartment (I: magnification of double positive cell). F: Macrophages were defined here as CD68⁺ (red) and/or CD14⁺ (green) cells. Double positive cells (yellow) were rare and located further from the tissue surface as compared with single positive CD68 cells (MDTS I: 248 μm and II: 63 μm). CD68⁺ (green) cells expressed (G) MR (red; I: MDTS: 198 μm), and (H) CD68⁺DC-SIGN (yellow) cells were also present, although less frequent (I: MDTS: 54 μm). CD4 (green) was present on submucosal CD11c⁺ (red) cells (I). CD11c⁺CD68⁺ (yellow) cells were frequent in the submucosa (J), whereas CD11c⁺ (green) cells did not express CD14 (red; K). CD11c⁺ (green) cells co-localized (yellow) with the CLRs (red): (L) Langerin, (M) MR, and (N) DC-SIGN. O: The pDCs specific marker BDCA-2 was rarely present in the foreskin tissue, when present BDCA-2 always co-localized with CD123⁺ (red) cells. The pDCs markers used here did not co-express any of the other investigated markers with one exception; (P) CD123⁺CD4⁺ (yellow) cells were detected in the submucosal compartment. White arrowheads indicate positive staining. Scale bars = 75 μm. Results are from one of at least four tissue samples yielding similar results.

Expression of CD3, CD4, HLA-DR, Langerin/CD207, MR/CD206, DC-SIGN/CD209, CD68, CD11c, CD1a, CD123, BDCA-2, and DC-LAMP mRNA was measured by quantitative PCR. Lines and bars depict geometric mean with 95% confidence interval (asterisk) and boxes indicate statistical significant differences in expression between HSV-2 seropositive individuals (HSV-2⁺) and HSV-2 seronegative individuals (HSV-2⁻; statistical test: Mann-Whitney). HSV-2⁺ individuals had significantly higher levels of MR/CD206 (P = 0.046), CD14 (P = 0.046), and CD123 (P = 0.0002) in their foreskin.