nature publishing group ARTICLES See COMMENTARY page XX Foreskin T-cell subsets differ substantially from blood with respect to HIV co-receptor expression, inflammatory profile, and memory status J L P r o d g e r 1 , R G r a y 2 , G K i g o z i 3 , F N a l u g o d a 3 , R G a l i w a n g o 3 , T H i r b o d 4 , M Wa w e r 2 , S O P H o f e r 5 , N S e w a n k a m b o 6 , D S e r w a d d a 7 a n d R K a u l 1 The foreskin is the main site of heterosexual human immunodeficiency virus (HIV) acquisition in uncircumcised men, but functional data regarding T-cell subsets present at this site are lacking. Foreskin tissue and blood were obtained from Ugandan men undergoing elective adult circumcision. Tissue was treated by mechanical and enzymatic digestion followed by T-cell subset identification and assessment of cytokine production using flow cytometry. Foreskin CD4 + T cells were predominantly an effector memory phenotype, and compared with blood they displayed a higher frequency of CCR5 expression (42.0 % vs. 9.9 % ) and interleukin-17 production. There was no difference in T-regulatory cell frequency, but interferon- and tumor necrosis factor- production were increased in foreskin CD8 + T cells. These novel techniques demonstrate that the foreskin represents a proinflammatory milieu that is enriched for HIV-susceptible T-cell subsets. Further characterization of foreskin T-cell subsets may help to define the correlates of HIV susceptibility in the foreskin. INTRODUCTION the efficacy of male circumcision, as evidenced by the fact As of 2009 there were 33.3 million people infected with that only a third of eligible men opted to avail themselves of human immunodeficiency virus (HIV)-1, and only a third of free male circumcision during a recent HIV vaccine trial in those requiring treatment were receiving it. 1 In addition, there South Africa. 8 were an estimated 2.6 million new infections in that year, the In the cervix, HIV and SIV infection is initiated by a small majority transmitted through heterosexual sex, emphasizing founder population of infected CD4 + T cells that expands the urgent need for better HIV prevention strategies. Clinical through the local production of chemoattractant cytokines, fol- trials have demonstrated that circumcision reduces HIV lowed by subsequent recruitment of activated memory CD4 + acquisition by 50 – 60 % in heterosexual men, proving that the T cells. 9,10 It is likely that the efficiency with which this founder foreskin is the site of most acquisition in uncircumcised men virus population expands depends on the immune milieu in the exposed to HIV during insertive vaginal sex. 2 – 4 Although genital mucosa at the time of exposure to HIV.11 While resting other penile sites such as the urethra may also have a role, 5 CD4 + T cells can be infected, viral replication within such cells the central role of the foreskin in HIV acquisition was fur- is less efficient, and HIV propagation and dissemination from ther supported by the observation that an increased foreskin the site of initial infection are driven by the rapid recruitment surface area correlated with increased risk of HIV acquisi- of activated CD4 + T cells, in which the virus can more read- tion. 6 However, the immune events that surround acquisition ily replicate.9,12 Recruitment of these activated CD4 + T cells and establishment of productive infection in the foreskin are to the initial site of exposure may be assisted by HIV-induced poorly defined. 7 Understanding the immunopathogenesis of changes in the local immune milieu, including the expression HIV acquisition in the foreskin remains an important priority of chemokines such as MIP-3 and MIP-1 by epithelial and for the development of new prevention modalities, despite plasmacytoid dendritic cells.9,13 1 Clinical Science Division, Department of Medicine, University of Toronto, Toronto , Canada . 2 Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USA . 3 Rakai Health Sciences Program, Kalisizo , Uganda . 4 Department of Medicine, Solna, Center for Molecular Medicine, Karolinska Institutet , Stockholm , Sweden . 5 Department of Surgery, University of Toronto, Toronto , Canada . 6 College of Health Sciences, Makerere University, Kampala , Uganda . 7 Institute of Public Health, Makerere University, Kampala , Uganda . Correspondence: R Kaul ([email protected] ) Received 5 September 2011; accepted 12 October 2011; published online 16 November 2011. doi: 10.1038/mi.2011.56 MucosalImmunology | VOLUME 5 NUMBER 2 | MARCH 2012 121 ARTICLES The presence or absence of certain T-cell subsets at the T-cell proportions in the blood and foreskin mucosal site of HIV exposure may be an important determi- Foreskin T cells were identified based on the expression of nant of HIV susceptibility. Genital herpes is associated with CD3, and comprised between 0.1 and 0.6 % of total recorded an increase in activated CD4 + T cells within the foreskin and events from digested, filtered foreskin tissue (Supplementary female genital tract, 14 – 16 perhaps contributing to the three- Material online). Upon permeabilization, many contaminating fold increase in HIV susceptibility associated with this infec- events (non-CD3 + ) were removed from the cell solution, so tion. 17 The proinflammatory Th17 cells that normally protect that CD3 + events constituted 10 – 15 % of total recorded events, skin and mucosal sites against bacterial and fungal infection allowing for easier identification of lymphocytes based on for- are present at high frequency in the female genital mucosa ward and side scatter alone. Owing to differences in the size of and display enhanced HIV susceptibility.18,19 Interleukin (IL)- foreskin samples and to variation in tissue physical properties 22 is an important effector molecule of Th17 cells, having a leading to differential cell loss during the digestion procedure, role in epithelial integrity and repair. IL-22 is also produced the absolute number of CD3 cells per foreskin was not defined. by proinflammatory Th22 cells, which may be preferentially Rather, we report proportions of cells, standardized to CD3 + , infected by HIV. 20 Conversely, CD25 + / FoxP3 + (Forkhead CD3 / 4 + , or CD3 / 8 + . The majority of foreskin CD3 + cells were box P3) T-regulatory cells (Tregs) have an important role in found to express either CD4 (mean, 51.4 % of CD3 + cells) or controlling inflammation, and higher Treg frequencies in CD8 (mean, 35.1 % ). Peripheral blood cells isolated from the the blood have been linked to reduced HIV susceptibility.21 same participants in parallel contained a higher proportion Furthermore, individuals who are HIV exposed but seronega- of CD4 + cells (63.4 % , P = 0.0001) and slightly lower propor- tive show a quiescent immune phenotype with reduced basal tion of CD8 + cells (31.8 % , P = 0.005; Figure 1a ), resulting in T-cell cytokine production and lower proportions of activated a substantially reduced CD4 / CD8 ratio in the foreskin com- T cells.21 – 24 pared with blood (1.53 vs. 2.27; P < 0.0001). A small propor- Although immunohistochemistry is able to demonstrate tion of CD3 + cells in both the foreskin and peripheral blood the tissue position of specific cells in three dimensions, the were found to express both CD8 and CD4 (0.41 % and 0.84 % , ability of this technique to define cellular immune function respectively, not significantly different). Of note, the foreskin is very limited. Therefore, we have developed techniques to contained more than twice as many CD4 − / CD8 − (double isolate a single-cell suspension from fresh foreskin tissues, negative) CD3 + cells as the blood (12.4 % vs. 5.1 % , P < 0.0001; and to characterize the frequency and function of foreskin Figure 1b ). T-cell subsets using multiparameter flow cytometry. Our results indicate that the foreskin constitutes a proinflamma- CCR5 expression and CD4 + Th17 and T-regulatory subsets tory immune environment that is enriched for HIV-suscep- in the foreskin tible T-cell subsets. The great majority of sexually transmitted viruses use CCR5 as a co-receptor. 26 Therefore, we examined the expression of RESULTS CCR5 on CD3 / CD4 + T cells isolated from the foreskin and Study population the blood of study participants ( Figure 2a ). The proportion Participants were recruited from a longstanding community of foreskin CD4 + T cells expressing CCR5 was over four-fold cohort in Rakai, Uganda. 25 Foreskin and whole blood were higher than that in blood (41.7 % in the foreskin vs. 9.9 % in collected from 46 men between the ages 15 and 49 years, peripheral blood mononuclear cells (PBMCs), P < 0.0001; who had requested elective circumcision at the Rakai Health Figure 2b ). Sciences Program clinic in Kalisizo, Uganda, and who had Th17 cells may be preferentially infected by HIV 18 and the provided written informed consent. All men were free of symp- ratio of mucosal Th17 / Treg cells is important in HIV immuno- tomatic sexually transmitted diseases at the time of surgery. pathogenesis.27 Tregs were defined as CD3 + / CD4 + cells that P < 0.0001 100 P = 0.0001 P = 0.005 40 80 30 60 20 40 10 % Of CD3+ cells 20 % DN of CD3+ cells 0 0 PBMC Foreskin PBMC Foreskin PBMC Foreskin CD4+ CD8+ Figure 1 CD4 + and CD8 + T-cell subsets within the foreskin and peripheral blood. Peripheral blood mononuclear cells (PBMCs) and foreskin cells from 46 men were stained with CD3-FITC, CD4-PE, and CD8-PerCP. Graphs show percentages of CD3 + cells within PBMCs or foreskin cells that co-express (a ) either CD4 or CD8, or ( b ) expressed neither CD4 nor CD8 (double-negative, DN, T cells).