High-parameter single-cell phenotyping has enabled in-depth classification and interrogation of immune cells, but to date has not allowed for glycan characterization. Here, we develop CyTOF-Lec as an approach to simultaneously characterize many protein and glycan features of human immune cells at the single-cell level. We implemented CyTOF-Lec to compare glycan features between different immune subsets from blood and multiple tissue compartments, and to characterize HIV-infected cell cultures. Using bioinformatics approaches to distinguish preferential infection of cellular subsets from viral-induced remodeling, we demonstrate that HIV upregulates the levels of cell-surface fucose and sialic acid in a cell-intrinsic manner, and that memory CD4+ T cells co-expressing high levels of fucose and sialic acid are highly susceptible to HIV infection. Sialic acid levels were found to distinguish memory CD4+ T cell subsets expressing different amounts of viral entry receptors, pro-survival factors, homing receptors, and activation markers, and to play a direct role in memory CD4+ T cells' susceptibility to HIV infection. The ability of sialic acid to distinguish memory CD4+ T cells with different susceptibilities to HIV infection was experimentally validated through sorting experiments. Together, these results suggest that HIV remodels not only cellular proteins but also glycans, and that glycan expression can differentiate memory CD4+ T cells with vastly different susceptibility to HIV infection.
© 2022, Ma et al.

Relatively little is known about features of T cells targeted by HIV in vivo. By applying bioinformatics analysis to mass cytometry (CyTOF)-phenotyped specimens from individuals with viremia and in-vitro-infected cells from uninfected donors, we provide an atlas of the phenotypes of in vivo and in vitro HIV-susceptible cells. T helper 17 (Th17) and α4β1+ cells are preferentially targeted in vivo, whereas T effector memory (Tem), T transitional memory (Ttm), Th1, and Th1/Th17 subsets are targeted in vitro. Multiple proteins-including chemokine and cytokine receptors-are remodeled by HIV in vivo, and these changes are mostly recapitulated in vitro. HIV remodels cells to a T follicular helper (Tfh) phenotype. Using clustering, we uncover a subset of CD29-expressing, Tem-like cells that are highly susceptible to infection in vivo and in vitro and experimentally confirm that susceptibility. These studies provide an in-depth look at features of HIV-susceptible cells in individuals with viremia and demonstrate that some-but not all-HIV-susceptible cells identified in vitro effectively model in vivo susceptibility.
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.

The latent reservoir is a major barrier to HIV cure. As latently infected cells cannot be phenotyped directly, the features of the in vivo reservoir have remained elusive. Here, we describe a method that leverages high-dimensional phenotyping using CyTOF to trace latently infected cells reactivated ex vivo to their original pre-activation states. Our results suggest that, contrary to common assumptions, the reservoir is not randomly distributed among cell subsets, and is remarkably conserved between individuals. However, reservoir composition differs between tissues and blood, as do cells successfully reactivated by different latency reversing agents. By selecting 8-10 of our 39 original CyTOF markers, we were able to isolate highly purified populations of unstimulated in vivo latent cells. These purified populations were highly enriched for replication-competent and intact provirus, transcribed HIV, and displayed clonal expansion. The ability to isolate unstimulated latent cells from infected individuals enables previously impossible studies on HIV persistence.
© 2020, Neidleman et al.

Asthma is one of the most common chronic diseases affecting both children and adults. We report a genome-wide association meta-analysis of 69,189 cases and 702,199 controls from Iceland and UK biobank. We find 88 asthma risk variants at 56 loci, 19 previously unreported, and evaluate their effect on other asthma and allergic phenotypes. Of special interest are two low frequency variants associated with protection against asthma; a missense variant in TNFRSF8 and 3' UTR variant in TGFBR1. Functional studies show that the TNFRSF8 variant reduces TNFRSF8 expression both on cell surface and in soluble form, acting as loss of function. eQTL analysis suggests that the TGFBR1 variant acts through gain of function and together with an intronic variant in a downstream gene, SMAD3, points to defective TGFβR1 signaling as one of the biological perturbations increasing asthma risk. Our results increase the number of asthma variants and implicate genes with known role in T cell regulation, inflammation and airway remodeling in asthma pathogenesis.

FCRL4 is an immunoregulatory receptor that belongs to the Fc receptor-like (FCRL) family. In healthy individuals, FCRL4 is specifically expressed by memory B cells (MBCs) localized in sub-epithelial regions of lymphoid tissues. Expansion of FCRL4+ B cells has been observed in blood and other tissues in various infectious and autoimmune disorders. Currently, the mechanisms involved in pathological FCRL4+ B cell generation are actively studied, but they remain elusive. As in vivo FCRL4+ cells are difficult to access and to isolate, here we developed a culture system to generate in vitro FCRL4+ B cells from purified MBCs upon stimulation with soluble CD40 ligand and/or CpG DNA to mimic T-cell dependent and/or T-cell independent activation, respectively. After 4 days of stimulation, FCRL4+ B cells represented 17% of all generated cells. Transcriptomic and phenotypic analyses of in vitro generated FCRL4+ cells demonstrated that they were closely related to FCRL4+ tonsillar MBCs. They strongly expressed inhibitory receptor genes, as observed in exhausted FCRL4+ MBCs from blood samples of HIV-infected individuals with high viremia. In agreement, cell cycle genes were significantly downregulated and the number of cell divisions was two-fold lower in in vitro generated FCRL4+ than FCRL4- cells. Finally, due to their reduced proliferation and differentiation potential, FCRL4+ cells were less prone to differentiate into plasma cells, differently from FCRL4- cells. Our in vitro model could be of major interest for studying the biology of normal and pathological FCRL4+ cells.