Using immunofluorescence techniques to Identify T cells in the foreskin tissue after medical male circumcision

Background: Medical Male Circumcision (MMC) plays an important role in reducing the risk of acquiring sexually transmitted infections (STIs) such as Human papilloma virus (HPV), Herpes simplex type 2 (HSV-2) and HIV-1. The foreskin tissue (FS) is a site abundant in Langerhans cells (LCs), macrophages and T helper cells that express CD4 and CCR5 that are target markers for HIV1 binding and viral infection. The foreskin tissue may also contribute chemokines and cytokines including those that promote inflammation such as IL-17, IL-1β, IL-8, MCP-1 and MIG. The inner foreskin has been shown to contain higher levels of CD4+CCR5+ cells and thus more susceptible to HIV infection compared to the outer foreskin. It was demonstrated that the majority of chemokines measured were highly expressed in the inner foreskin compared to the outer foreskin including CCL27 which was approximately 7-fold higher in the inner foreskin compared to the outer foreskin, in congruent with the higher density of CD4+CCR5+ observed in the epithelium of the inner foreskin. In this study, we hypothesized that CCL27 upregulation in the inner foreskin triggers the recruitment of CD4+ T cells to the epithelium of the foreskin tissue. This could subsequently lead to increased susceptibility to infections in the inner foreskin tissue. The aims of this dissertation were: 1) to measure the impact of CCL27 on the recruitment of CD4+ T cells to the epithelium of the foreskin tissue using immunofluorescence imaging. 2) to compare manual counting and semi-automated method for counting dually positive cells. 3) to use multiparameter flow cytometry to characterize the cells recruited under the influence of CCL27. Methodology: Inner foreskin tissue (n=11) and outer foreskin tissue (n=4) explants were treated with either TNFα or CCL27 and evaluated using immunofluorescence imaging to quantify the levels of CD3 and CD4 expressing cells. Dually positive CD3+CD4+ cells were counted manually using softworx software on the Deltavision microscope and with ...