Background: The male genital tract is of major importance in the transmission and acquisition of HIV-1. Studying cellular immunity in the male genital tract is important in development of HIV-1 vaccines protective at mucosal sites. Semen is the primary HIV-1 containing fluid released from the male genital tract and reducing virus levels in semen would also reduce HIV-1 spread. Characterizing lymphocytes from semen requires the isolation of viable T cells that can be analyzed by downstream applications such as flow cytometry. The aims of this study were to investigate the influence of various parameters on CD3(+) T cell yields from semen and to compare isolation methods to maximize CD3(+) T cell yields for the purpose of functional characterization by flow cytometry.
Methods: The influence of abstinence, storage temperature and time till processing on semen CD3(+) T cell yields was investigated. Seminal CD3(+) T cell yields were evaluated by comparing gradient separation, enzymatic digestion, filtration and magnetic bead capture. The function and viability of seminal CD4(+) and CD8(+) T cells were assayed by flow cytometry.
Results: We found that the use of pronase and cell strainers resulted in significantly higher CD3(+) T cell yields when compared to gradient separation alone. Positive selection of CD3(+) cells using magnetic bead purification resulted in significantly higher yields and improved resolution of lymphocyte subsets by flow cytometry. Processing of samples should occur as expediently as possible to maximize CD3(+) T cell yields. However, if this is not possible, loss of CD3(+) T cells can be minimized by storing samples at 37°C for up to one day post ejaculation.
Conclusions: We describe a simple method for the isolation of functional T cells from semen. Developing standardized methods for processing samples and measuring immunity in the male genital tract may be important in clinical trials of not only candidate HIV-1 vaccines, but in better understanding cellular immunity to a range of sexually transmitted infections of global significance.
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