Defining the human mucosal CD4+ T cell and Mononuclear Phagocyte landscape by high parameter technologies

Abstract

Tissue resident mononuclear phagocytes (MNP) are among the first immune cells that sexually transmitted pathogens encounter during transmission. These antigen presenting cells rapidly endocytose pathogens and then interact with CD4+ T cells to initiate adaptive immunity. In the case of HIV, the virus can be transferred from MNP to infect CD4+ T cells which are the primary target cell in which HIV undergoes replication. These MNP:CD4+ T cell interaction were originally believed to occur in lymph nodes but there is now an increasing body of evidence that this occurs within tissue at the site of transmission.

There has been recent interest in defining the phenotype and the role of CD4+ TRMs in diseases. In HIV, CD4+ TRM have been shown to be a major target for productive and latent infection in the cervix. However, there are limited investigations into the CD4+ T cell landscape in other genital tissues. In this thesis, we optimised the enzymatic digestion process to maintain the cells in their physiological status. We have designed, optimised and implemented a 24-color flow cytometry panel to characterise T cells by memory, residency, activation.

CD127 is known to be enriched on TRM. It is also a marker used in blood to identify regulatory T cells by surface expression. By preventing the enzymatic cleavage of surface markers during tissue digestion, we were able to identify a CD127+FOXP3+ T cell subset. We find its expression is dichotomous with TIGIT expression. The CD127+ Tregs express markers that are associated with TRM.

Lastly, we examined the mononuclear phagocyte and CD4+ T cell landscape in human anogenital mucosa by single cell RNA sequencing (sc-RNAseq). We apply scRNA-Seq to conclude that Langerhans cells are not the only cells within the stratified epidermal layer of tissues, as the latter also contain conventional DC2 (cDC2). Further, we examine tissue pDCs and Axl+Siglec6+ DCs (ASDCs) by RNA sequencing and compare these to their counterparts in blood.