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The oral mucosa is the first line of defense against pathogenic bacteria and plays a vital role in maintaining tolerance to food antigens and commensal bacteria. We used CD11c reporter mice to visualize dendritic cells (DCs), a key immune cell population, in the oral cavity. We identified differences in DC density in each oral tissue region. Sublingual immune cell clusters (SLICs) extended from the lamina propria to the epithelium, where DCs and T cells resided in close contact with each other and innate lymphoid cells. Targeted in situ photolabeling revealed that the SLICs comprised mostly CD11c+CD11b+ DCs and were enriched for cDC1s and Langerhans cells. Although the frequency of T cell subsets was similar within and outside the SLICs, tissue-resident memory T cells were significantly enriched within the clusters and cluster size increased in response to inflammation. Collectively, we found that SLICs form a unique microenvironment that facilitates T cell-DC interactions in the steady state and during inflammation. Since the oral mucosa is an important target for needle-free vaccination and sublingual immunotherapy to induce tolerogenic responses, the insight into the localized immunoregulation provided in this study may accelerate the development of these approaches.

Extracellular vesicles (EVs) are mediators of intercellular communication and a promising class of biomarkers. Surface proteins of EVs play decisive roles in establishing a connection with recipient cells, and they are putative targets for diagnostic assays. Analysis of the surface proteins can thus both illuminate the biological functions of EVs and help identify potential biomarkers. We developed a strategy combining high-resolution mass spectrometry (HRMS) and  proximity ligation assays (PLA) to first identify and then validate surface proteins discovered on EVs. We applied our workflow to investigate surface proteins of small EVs found in seminal fluid (SF-sEV). We identified 1,014 surface proteins and verified the presence of a subset of these on the surface of SF-sEVs. Our work demonstrates a general strategy for deep analysis of EVs' surface proteins across patients and pathological conditions, proceeding from unbiased screening by HRMS to ultra-sensitive targeted analyses via PLA., (© 2022. The Author(s).)