This indicated the fact that inner foreskin can allow for increased penetration of Ad5 IgG antibodies. DISCUSSION We investigated the antibody isotypes present in different areas of the human foreskin CITED2 and compared them to colonic mucosa and blood. whereas foreskin Ad5 IgG was mainly derived from blood, and reached the inner epidermis at higher ratios than the outer (p 0.002). In summary, the foreskin antibody response combines local and systemic sources and there is selective isotype accumulation in the epidermis. INTRODUCTION Substantial evidence has emerged over the years in non-human primate (NHP) models that potent antibodies can mediate protective effects against SIV and SHIV infection1. In humans, the RV144 clinical trial demonstrated 31% protection among Thai volunteers2 where infection risk was directly associated with blood IgA titers against the C1 region of HIV Env, and inversely associated with high titers of anti-Env V1V2 IgG33C5. However, it remains to be determined whether these vaccine correlates can protect at the rectal, vaginal, and penile surfaces, where HIV is predominantly transmitted6,7. Whether passively infused antibodies or HIV vaccine candidates can reach human genital and rectal sites at sufficient concentrations is also unclear. Quantitative methods to measure vaginal and rectal antibody responses have been developed8, but penile Ig assessments are limited. In uncircumcised heterosexual men14C16 and men who have insertive sex with men17,18, the foreskin is an important site of HIV exposure9, as three independent randomized controlled trials showed that circumcision reduced HIV infection risk by 51C60%14C16. However, 70% of the worlds men remain uncircumcised10 and the uptake of circumcision faces logistical11,12 and cultural barriers13, so it remains important to investigate whether the foreskin can be armed with humoral responses that can prevent HIV infection. In fact a recent study demonstrated that SIV can preferentially persist in proximity to target cells at the macaque inner foreskin and glands, but not the outer foreskin,9 indicating that immune control at these sites may be most useful Ouabain for protection. Distinct Ig isotypes predominate in various mucosal surfaces, suggesting that antibody restrictions are present at the sites of host-pathogen interactions. IgM is first induced during the immune response to a new antigen and has C1q and complement activation functions; however, it is present in low concentrations in female genital and intestinal surfaces, and is undetectable in seminal fluid of most healthy men19C21. IgA isotypes dominate in the intestine and can inactivate pathogens by neutralization and exclusion21C23. Both intestinal and genital IgA rely on local production, with minor components transudating from blood24C28. Compared to other isotypes, IgG is most abundant in blood, semen, cervical, and vaginal compartments29. There are four subclasses arranged by their abundance in serum: IgG1, IgG2, IgG3, and IgG4. They have remarkable differences in complement activation, phagocytosis, antibody dependent cell mediated cytotoxicity (ADCC), and Fc-Receptor binding, with a general order of activating capacity being IgG3 IgG1 ? IgG2 IgG430. In addition to the isotypes, the specificity of the antibody response can also be compartmentalized. Exposure to oral or intranasal adenovirus can lead to IgG antibody responses that concentrate in the Ouabain nose and mouth, as well as vaginal IgA31,32; whereas rectal exposure can lead to antigen-specific IgG in tears and IgA that dominates in rectal secretions31. Deltoid delivery of a canarypox HIV Ouabain vaccine can generate both IgG and IgA in rectal secretions, but this is limited after inguinal immunizations, which drain the genitals33. Thus, the immunization strategies and natural infections that trigger penile antibody responses may not match those that successfully generate responses at mucosal surfaces. To better understand the antibody profile that may play a role in controlling infections at the foreskin, we evaluated the humoral responses in the foreskin of sexually active young men who have sex with men (MSM) at high risk of HIV infection, and compared these with colonic and systemic B-cell responses. Our results indicate that some foreskin Ig isotypes transudate directly from blood, whereas others are locally produced. These findings have important implications for the development of strategies to induce relevant Ig responses against sexually transmitted infections (STI) so that immune responses reach this important site of pathogen exposure. METHODS Tissue and Blood Donation We evaluated foreskin and colon biopsy samples collected in Lima, Peru during HVTN 914. They correspond to 20 HIV Ouabain negative, 21C30 year-old MSM at high risk of HIV acquisition34. All study participants provided written informed consent prior to HVTN 914 participation, and met safety criteria to undergo.