Comparison between a deep-learning and a pixel-based approach for the automated quantification of HIV target cells in foreskin tissue.

The availability of target cells expressing the HIV receptors CD4 and CCR5 in genital tissue is a critical determinant of HIV susceptibility during sexual transmission. Quantification of immune cells in genital tissue is therefore an important outcome for studies on HIV susceptibility and prevention. Immunofluorescence microscopy allows for precise visualization of immune cells in mucosal tissues; however, this technique is limited in clinical studies by the lack of an accurate, unbiased, high-throughput image analysis method. Current pixel-based thresholding methods for cell counting struggle in tissue regions with high cell density and autofluorescence, both of which are common features in genital tissue. We describe a deep-learning approach using the publicly available StarDist method to count cells in immunofluorescence microscopy images of foreskin stained for nuclei, CD3, CD4, and CCR5. The accuracy of the model was comparable to manual counting (gold standard) and surpassed the capability of a previously described pixel-based cell counting method. We show that the performance of our deep-learning model is robust in tissue regions with high cell density and high autofluorescence. Moreover, we show that this deep-learning analysis method is both easy to implement and to adapt for the identification of other cell types in genital mucosal tissue.

Effect of cryopreservation on CD4+ T cell subsets in foreskin tissue.

Voluntary medical male circumcision (VMMC) reduces HIV acquisition by at least 60%, but the determinants of HIV susceptibility in foreskin tissues are incompletely understood. Flow cytometry is a powerful tool that helps us understand tissue immune defenses in mucosal tissue like the inner foreskin, but foreskin flow cytometry has only been validated using fresh tissue samples. This restricts immune analyses to timepoints immediately after surgical acquisition and hinders research in this area. We compared fresh analysis with whole tissue cryopreservation and later thawing and digestion to analyze CD4+ T cell populations relevant to HIV susceptibility (CCR5, CD25, CD127, CCR4, CXCR3, CCR6, CCR10, HLA-DR, and CD38). Eight foreskin samples from HIV-negative males aged >18 years were collected after VMMC. For each sample, half the foreskin was immediately cryopreserved for later digestion and flow cytometry analysis, while the remaining tissues were analyzed fresh. We demonstrate no significant impact of cryopreservation on CD4+ T cell expression of CD25, CCR4, CCR6, HLA-DR, CCR10, or CD127. Although expression levels of CCR5, CD38, and CXCR3 were increased after cryopreservation, the relative ranking of participants was retained. In conclusion, cryopreserved foreskin tissues may be suitable for subsequent digestion and flow cytometry phenotyping of HIV-susceptible T cell populations.