key: cord-0331269-6e7t6lp6 authors: Ma, Tongcui; McGregor, Matthew; Giron, Leila B.; Xie, Guorui; George, Ashley F.; Abdel-Mohsen, Mohamed; Roan, Nadia R. title: CyTOF-Lec: Single-cell Glycomics Analysis Reveals Glycan Features Defining Cells Differentially Susceptible to HIV date: 2022-04-08 journal: bioRxiv DOI: 10.1101/2022.04.06.487312 sha: ee6bc40c9b06d62385d1d1f53b60b22f5288f8de doc_id: 331269 cord_uid: 6e7t6lp6 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. To identify specific glycans that were remodeled, we implemented PP-SLIDE to identify In contrast to fucose and sialic acid, we did not observe any marked upregulation of T antigen To examine whether HIV alters glycan expression in other bystander cellular subsets, we compared glycan levels on multiple subsets of B and T cells from the uninfected vs. infected 216 cultures ( Figure S6 ). Only tonsils exhibited significant differences between uninfected vs. 217 bystander cells, and these differences were observed among all subsets. For example, sialic 218 acid levels (as assessed by both WGA and MAL-1 binding) were significantly higher in all the 219 analyzed subsets of bystander B, CD8+, and CD4+ T cells, relative to their counterparts from 220 uninfected cultures. Fucose expression was also uniformly higher among bystander cells, although the difference among CD8+ Tm cells did not reach statistical significance. Differences The data presented thus far suggest that although there are differences between blood 230 vs. the tissue sites examined, fucose and sialic acid are upregulated on HIV productively-231 infected cells, and CD4+ T cells expressing high levels of fucose or sialic are preferentially 232 targeted for infection. We next conducted manual gating to assess whether the HIV-susceptible 233 cells express high levels of both fucose or sialic acid, or whether they belong to distinct subsets 234 of fucose+ vs. sialic acid+ cells. We focused on the AOL and WGA datasets, as they cover total 235 fucose and different forms of sialic acid, respectively. We first examined, within the HIV-infected 236 cultures, the infection rates among CD4+ Tm cells expressing high vs. low levels of AOL or 237 WGA. In both blood and both tissue compartments, AOL High and WGA High cells exhibited 238 significantly higher HIV infection rates than did AOL Low and WGA Low cells, respectively ( Figure 239 4A). To assess the extent to which this high level of infection was due to preferential infection of 240 the AOL High and WGA High CD4+ Tm cells, we next compared, among the uninfected CD4+ Tm 241 cells and PRE cells, the percentages of cells that were AOL High , WGA High , or AOL High WGA High . Consistent with the MSI data, the percentages of cells expressing high levels of AOL or WGA The results presented thus far suggest that CD4+ Tm cells from endometrium, tonsils, 285 and blood are preferentially susceptible to HIV infection compared to their naïve counterparts, 286 but only in tonsils and blood can high levels of fucose and sialic acid further distinguish HIV-287 susceptible CD4+ Tm cells from non-susceptible CD4+ Tm cells. To experimentally validate these findings, we conducted sorting experiments. As endometrial and tonsillar T cells do not 289 maintain good viability after sorting, we limited these studies to blood specimens. CD4+ Tm 290 cells from blood expressing low (WGA Low ), medium (WGA Medium ), or high (WGA High ) levels of 291 sialic acid were isolated through sorting ( Figure 6A ). These sorted populations (along with total 292 CD4+ Tm cells as a comparison control) were then exposed to HIV-F4.HSA for 3 days and then To better understand the mechanism behind the differential susceptibility of cells Figure 6F ). To further validate the notion that the high susceptibility of WGA High 310 CD4+ Tm cells is closely associated with the activation status of these cells, we phenotyped 311 CD4+ Tm cells from resting vs. PHA-stimulated PBMCs. As expected, the stimulated CD4+ Tm 312 cells expressed higher levels of multiple activation markers ( Figure S8A ). Importantly, the 313 stimulated CD4+ Tm cells also bound higher levels of WGA, consistent with upregulation of sialic acid upon T cell activation ( Figure S8B ). Furthermore, activated cells, as defined as those 315 expressing high levels HLADR, CD69, CD38, CD25, CD28, or ICOS, all expressed higher levels 316 of sialic acid as compared to cells with low levels of these activation markers ( Figure S8C ). These data together strongly support the notion that high sialic expression identifies the most 318 activated subsets of CD4+ Tm cells. We also considered the possibility that WGA High cells may support higher levels of 320 productive infection because these cells better survive the cytopathic effects of HIV replication. Consistent with this hypothesis, we found that WGA High cells expressed higher levels of CD127, Although these results suggest that high levels of sialic acid may simply be a biomarker 329 of the most HIV-susceptible CD4+ Tm cells, it did not rule out the possibility that sialic acid plays 330 a direct role in HIV susceptibility. To test this, we tried two approaches to diminish cell-surface 331 sialic acid expression: transient treatment with either sialic acid synthease inhibitor P-3FAX-332 Neu5Ac or sialidase. Inhibitor treatment did not decrease cell-surface sialic acid levels ( Fig. 333 S8D), but sialidase did ( Figure 6I ). We therefore exposed mock-or sialidase-treated cells to 334 HIV-F4.HSA for 3 days and then assessed infection rates by FACS. We found that infection 335 rates were decreased in a sialidase dose-dependent manner ( Figure 6J (Table S2 ). X8 538 antibody-labeling kits (Fluidigm) were used to label antibodies that required in-house 539 conjugation. The conjugated antibodies were quantitated for protein content by Nanodrop 540 (Thermofisher). Prior to storage at 4ºC, specimens were diluted 1:1 using a PBS-based 541 Antibody Stabilizer (Boca Scientific) supplemented with 0.05% sodium azide. 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