Comprehensive phenotyping of circulating immune cell subsets in people living with HIV

Systemic chronic inflammation and immune dysfunction are recognized as drivers of the development of non-AIDS related comorbidities (NARCs) in people living with HIV (PLHIV). In order to lower the risk of NARCs, it is critical to elucidate what is the contribution of alterations in the composition and function of circulating immune cells to NARCs-related pathogenesis. Findings from previous immunophenotyping studies in PLHIV are highly heterogeneous and it is not fully understood to what extent phenotypic changes on immune cells play a role in the dysregulated inflammatory response observed. In this study, three flow cytometry panels were designed and standardized to phenotypically and functionally identify the main circulating immune cell subsets in PLHIV. To reduce variability, up to 10 markers out of the approximately 20 markers in each panel were used in a custom dry format DURA Innovations (LUCID product line). Intra-assay precision tests performed for the selected cell subsets showed that the three panels had a %CV below 18% for percent of positive cells and the MFI (mean fluorescent intensity) of lineage markers. Our reported pipeline for immunophenotypic analysis facilitated the discrimination of 1153 cell populations, providing an integrated overview of circulating innate and adaptative immune cells as well as the cells' functional status in terms of activation, exhaustion, and maturation. When combined with unsupervised computational techniques, this standardized immunophenotyping approach may support the discovery of novel phenotypes with clinical relevance in NARCs and demonstrate future utility in other immune-mediated diseases.