Author response: Single-cell glycomics analysis by CyTOF-Lec reveals glycan features defining cells differentially susceptible to HIV (2022)

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract 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. Editor's evaluation This study applies a new novel method of single cell detection to biologically relevant systems to try to understand whether glycans on the surface of CD4+ T cells impact HIV susceptibility. They find that cells expressing higher levels of fucose and sialic acid are more likely to be infected with HIV than those with low levels. The findings point to glycans as biomarkers and potential determinants for cellular susceptibility to HIV, and open the door to new avenues for studying the interplay between cell surface glycans and viral infections. https://doi.org/10.7554/eLife.78870.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest Living cells have a sugar coating. These sugars include molecules called glycans, which help cells interact with the outside world. The types of sugars on cells can affect their properties, including potentially their susceptibility to infection by viruses, such as the human immunodeficiency virus, HIV. To date, most research examining cells susceptible to HIV has focused on cell surface proteins, not sugars. To study these proteins, researchers had previously covered them in metal-studded antibodies (which stick to proteins) and used a technique called cytometry time of flight, or CyTOF for short, to quantify the levels of these proteins on the surface of cells susceptible to HIV. Adapting this tool to investigate sugars could answer questions about HIV infection. For example, does the virus prefer to infect cells coated in certain sugar molecules? And does it change the pattern of sugars on the surface of the cells it infects? Ma et al. adapted CyTOF to use molecules called lectins (which stick to sugars) in conjunction with the metal-studded antibodies. This made it possible to simultaneously measure the levels of 34 different proteins and 5 different types of sugars on individual cells. The pattern of sugars on the surface of cells from the immune system differed depending on what tissues the cells came from, and what types of cells they were. The results showed that HIV preferred to infect memory CD4 T cells with high levels of two types of sugar: fucose and sialic acid. Furthermore, during infection, the levels of both these sugars increased. Current treatments for HIV keep virus levels low but do not cure the infection. Further research could determine whether sugars have a role to play in HIV persistence. It is possible that the sugar patterns preferred by the virus help it to avoid detection. A clearer understanding of cell surface sugars could lead to sugar-targeting drugs that kill infected cells. Introduction Viruses generally need to hijack multiple host cell processes to complete their replication cycle. This virus-mediated manipulation of host processes is called viral-induced remodeling, and has been studied using a variety of approaches, most entailing virally infected cell lines analyzed in bulk. More recently, viral remodeling of primary cells was studied at the single-cell level by characterizing, via cytometry by time of flight (CyTOF), human tonsillar T cells infected ex vivo by varicella zoster virus (VZV) (Sen et al., 2014). Bioinformatics analyses of high-dimensional CyTOF datasets of VZV-infected cells revealed that VZV infection elicits significant host cell remodeling and alters the skin-trafficking property of subsets of infected cells. We recently implemented a follow-up approach, termed predicted precursor as determined by single-cell linkage using distance estimation (PP-SLIDE), to document HIV-induced remodeling of T cells from blood, lymph node, and genital tract, define the subsets of cells most susceptible to HIV infection, and characterize the phenotypes of HIV-infected cells in viremic and virally suppressed PLWH (Cavrois et al., 2017; Ma et al., 2020; Neidleman et al., 2020b; Xie et al., 2021). One important feature of PP-SLIDE is that it enables assessment of whether a receptor differentially expressed on HIV-infected cells reflects HIV-induced remodeling or preferential infection of cells harboring that pattern of expression of the receptor. For example, PP-SLIDE established that HIV-infected T cells express low levels of CD4 and CD28 not because HIV preferentially infects CD4LowCD28Low T cells, but rather because HIV downregulates these receptors (Cavrois et al., 2017; Ma et al., 2020; Neidleman et al., 2020b; Xie et al., 2021), which were independently shown to be down-modulated by HIV accessory genes (Garcia and Miller, 1991; Swigut et al., 2001). Other HIV-remodeled surface receptors identified by PP-SLIDE include those involved in T cell migration to lymph nodes and markers of Tfh cells (Cavrois et al., 2017; Ma et al., 2020; Neidleman et al., 2020b; Xie et al., 2021). By contrast, the low levels of surface CD127 expression on HIV-infected tonsillar T cells reflected preferential sparing of CD127High T cells from productive infection (Cavrois et al., 2017; Ma et al., 2020; Neidleman et al., 2020b; Xie et al., 2021). Subsequent studies demonstrated that CD127High memory T cells preferentially undergo latent infection by HIV (Hsiao et al., 2020). These and other PP-SLIDE-generated findings of preferential infection of cellular subsets have been experimentally validated through a variety of sorting experiments (Cavrois et al., 2017; Ma et al., 2020; Neidleman et al., 2020b; Xie et al., 2021). Together, these studies suggest that important insights into HIV pathogenesis and persistence can be gained from characterizing HIV-induced remodeling of primary cells at a single-cell level. However, such remodeling studies – and in fact all phenotypic characterizations of virally infected cells to date – have only examined the cells' proteomes. Completely overlooked has been the diverse collection of glycans that are assembled on the surface of all living cells (Williams and Thorson, 2009). Cell-surface glycosylation plays critical roles in regulating multiple cellular processes and immune functions (Barrera et al., 2002), as well as cell-cell (de Freitas Junior et al., 2011) and cell-pathogen (Colomb et al., 2019; Everest-Dass et al., 2012; Giron et al., 2020b) interactions. Furthermore, multiple viruses (e.g., HSV-1, CMV, and HTLV1) have been shown to alter the surface glycosylation of infected cells (Hiraiwa et al., 2003; Kambara et al., 2002; Nyström et al., 2007; Nyström et al., 2009). To date, studies of host glycomes have been limited to analysis of bulk cells, using techniques such as mass spectrometry, liquid chromatography, and lectin microarrays (Chen et al., 2021), although bacteria have been characterized at the single-cell level using lectins (Leipold et al., 2011). A recent study analyzing bulk populations of CD4+ T cells with different glycan features demonstrated that they harbor different levels of HIV transcripts (Colomb et al., 2020), suggesting that the host cell glycome can affect HIV susceptibility and/or replication. However, robust tools to deeply characterize, at the single-cell level, the glycan features of immune cells – including HIV-infected ones – are lacking to date. In this study, we developed a new approach taking advantage of the high-parameter analysis capabilities of CyTOF (Bendall et al., 2011), to phenotype cells simultaneously for protein and glycan features. This was achieved through conjugating a collection of lectins (proteins that specifically bind different types of glycans) to metal lanthanides, an approach that has previously been validated at the bulk level (Leipold et al., 2009). We call our approach CyTOF-Lec – as it combines traditional CyTOF (using lanthanide-conjugated antibodies) with lanthanide-conjugated lectins to characterize surface glycosylation patterns of cells – and applied it on both blood and tissue cells. Taking advantage of the high-dimensional nature of our resulting CyTOF-Lec datasets and our PP-SLIDE analysis pipeline, we set out to address the following two fundamental questions about HIV infection: (1) Does HIV preferentially infect cells exhibiting distinct glycan features, and (2) to what extent does HIV remodel the glycan features of its host cell? Results Development and validation of CyTOF-Lec To establish a methodology that could simultaneously characterize protein and glycan features at the single-cell level, we developed a panel of lanthanide metal-conjugated antibodies and lectins compatible with CyTOF, which we refer to as CyTOF-Lec (Supplementary file 1 A). As tonsils provide an abundant source of both T and B cells, we used these cells for our initial validation of the panel. First, we confirmed that the staining patterns of the lanthanide-conjugated antibodies were consistent with the known differential expression of their target antigens on tonsillar T vs. B cells (Figure 1—figure supplement 1), and with results previously reported using CyTOF without lectin staining (Ma et al., 2020). To ensure that the lectin staining did not displace or alter the antibodies bound to their protein targets, we developed a protocol whereby surface antibody staining was completed prior to lectin staining (see Materials and methods). We confirmed that all five lectins (AOL, MAL-1, WGA, UEA-1, ABA, see Supplementary file 1) conjugated to lanthanides stained both T and B cells (Figure 1—figure supplement 1). Furthermore, we confirmed that antibody binding to CD3, CD4, and CD8 was the same whether or not the specimens were subsequently stained with lectins (Figure 1A). To establish the specificity of lectin binding, we assessed the effect of sialidase, which degrades cell-surface sialic acid. As expected, binding by WGA and MAL-1, which detect different forms of sialic acid (Supplementary file 1), was decreased after sialidase treatment of the cells (Figure 1B). By contrast, binding by AOL and UEA-1, which detect different forms of fucose, and ABA, which detects T antigen (Supplementary file 1), were all increased (Figure 1C–D). This was expected as removal of sialic acid should enable better detection of these other glycan structures, and is consistent with prior reports (Giron et al., 2020b). Figure 1 with 1 supplement see all Download asset Open asset Validation of cytometry by time of flight (CyTOF)-Lec. (A) Antibody staining for protein markers is not altered by lectins. Shown are histograms of tonsil cells expressing CD3, CD8, or CD4, as detected by CyTOF after antibody staining followed or not by staining with metal-conjugated lectins (AOL: Aspergillus oryzae; MAL-1: Maackia amurensis I; WGA: wheat germ agglutinin; UEA-1: Ulex europaeus I; and ABA: Agaricus bisporus agglutinin). Protein expression (y-axis) is represented as the percentage of the maximal expression level detected for each staining. (B–D) Sialidase treatment elicits expected changes in lectin binding. Tonsil cells were treated with sialidase (20 μg/ml) for 1 hr at 37°C, and then stained with the CyTOF-Lec panel. Shown are histograms depicting the extent of interaction with sialic acid-binding (B), fucose-binding, (C) or T antigen-binding (D) lectins. Removal of sialic acid by sialidase decreases binding by sialic acid-binding lectins, while increasing binding by the fucose- and T antigen-binding lectins, as expected. To determine whether the conjugated lectins could detect differences in cell-surface glycans between subsets of immune cells, we compared binding by each of the five lectins to B cells, memory CD8+ T cells (CD8+ Tm cells), naïve CD8+ T cells (CD8+ Tn cells), memory CD4+ T cells (CD4+ Tm cells), and naïve CD4+ T cells (CD4+ Tn cells). We assessed the expression of glycans on these subsets not only among tonsillar cells, but also among PBMCs and endometrial T cells for comparison. For all three sites, the fucose-specific lectins AOL and UEA1 bound more to CD4+ Tm and CD8+ Tm cells than to their respective naïve counterparts, although the difference only reached significance for AOL binding in tonsils (Figure 2A). These results are consistent with the known role of core surface fucosylation for T cell activation, which is more prominent within the memory compartment (Fujii et al., 2016; Liang et al., 2018). The sialic acid-binding lectins WGA and MAL1 also consistently bound to memory T cells more than to their naïve counterparts, although this only reached statistical significance for MAL1 binding of endometrial CD8+ T cells (Figure 2B). Binding by ABA did not show a consistent pattern between memory vs. naïve T cells (Figure 2C). Binding by all five lectins was low on B cells, particularly as compared to memory CD8+ T cells (Figure 2A–C). Figure 2 Download asset Open asset Glycan expression in lymphocytes from human endometrium, tonsils, and blood. (A) Box plots showing staining by fucose-binding lectins on B and T cells from the endometrium, tonsils, and PBMCs, quantified as median signal intensity (MSI). T cells were subdivided into memory CD8+ T cells (CD8+ Tm), naïve CD8+ T cells (CD8+ Tn), memory CD4+ T cells (CD4+ Tm), and naïve CD4+ T cells (CD4+ Tn) based on their expression of Tm- and Tn-specific CyTOF markers. AOL binds to total/core fucose, UEA1 binds to α1–2 branched fucose. Although there were some differences in binding between sites and between the different lectins, in all instances fucose-binding proteins bound CD4+ Tm at higher levels than they did CD4+ Tn. (B) Box plots showing binding by sialic acid-binding lectins WGA and MAL-1. Results are presented as in panel A. WGA binds to total sialylated glycans and MAL-1 binds to α2–3 sialylated glycans. Overall, the sialic acid-binding lectins bound CD8+ T cells at higher levels than they did CD4+ T cells and B cells. (C) Box plots showing binding by the T antigen-binding lectin ABA. Results are presented as in panel A. Overall, ABA bound T cells at higher levels than they did B cells. *p<0.05, **p<0.01, ***p<0.001 as assessed using the Student's paired t test and adjusted for multiple testing using the Holm method. These results establish CyTOF-Lec as a panel that can quantitate glycan and protein expression at the single-cell level, and detect differential glycan expression between different subsets of immune cells. HIV alters expression of fucose and sialic acid in a tissue site-dependent manner We next applied CyTOF-Lec to determine the extent to which glycans are remodeled on the surface of HIV-infected cells. Fresh endometrial biopsies (n=6 donors) and whole tonsils from tonsillectomies (n=4 donors) from HIV seronegative individuals were processed into single-cell suspensions, and then immediately exposed to the HIV-F4.HSA, a replication-competent and Nef-sufficient virus that harbors the CCR5-tropic 109FPB4 transmitted/founder (T/F) Env (Cavrois et al., 2017). In addition, PBMCs were isolated from whole blood of HIV seronegative individuals (n=4 donors) and exposed to HIV-F4.HSA. To limit potential confounding effects of ex vivo-induced T cell activation, infection was carried on unstimulated cells. Infection was allowed to proceed for 3 days, after which the cells, as well as cells from paired uninfected control cultures, were harvested for CyTOF-Lec analysis. CD4+ T cells were identified as intact, live singlet CD3+ CD8- cells, while infected cells were identified as intact, live singlet CD3+ CD8- CD4Low cells (Figure 3—figure supplement 1), to account for the downregulation of cell-surface CD4 by HIV (Doms and Trono, 2000; Garcia and Miller, 1991; Lama, 2003; Piguet et al., 1999). Consistent with our prior studies (Ma et al., 2020), endometrial T cells were the most susceptible to HIV-F4.HSA infection (Figure 3—figure supplement 2A). HIV-infected cells from all three sites were remodeled, as established qualitatively by assessing their locations on a t-SNE (Figure 3—figure supplement 2B) as well as quantitatively using SLIDE (Sen et al., 2014; Figure 3—figure supplement 2C). The remodeling of the infected cells is consistent with prior studies (Cavrois et al., 2017; Ma et al., 2020; Xie et al., 2021), and confirms that CyTOF-Lec is a valid panel for the analysis of remodeling. To identify specific glycans that were remodeled, we implemented PP-SLIDE to identify the predicted precursor (PRE) cells (Cavrois et al., 2017; Ma et al., 2020; Neidleman et al., 2020b; Xie et al., 2021). PRE cells harbor the predicted original (pre-remodeling) features of T cells infected by HIV and are identified using k-nearest neighbor approaches by matching, in the high-parameter CyTOF space, the T cells in the uninfected culture most similar in phenotype to every HIV-infected cell (Figure 3—figure supplement 3A). As expected (Ma et al., 2020), the PRE cells from all three sites were preferentially memory CD4+ T cells (Figure 3—figure supplement 3B, C). Having identified the PRE cells, we then determined which glycans, if any, were remodeled by HIV infection. This was accomplished by assessing for lectins that differentially bound the PRE as compared to the infected cells. Glycans were quantitated by reporting the median signal intensity (MSI) of their corresponding lectins among each population of cells from each donor. Interestingly, we found that both fucose and sialic acid were upregulated during HIV infection. Infected cells from all three sites potently upregulated total fucose as assessed by AOL binding (Figure 3A, Figure 3—figure supplement 4A), although this upregulation did not reach statistical significance (after correcting for multiple comparisons) for the endometrium. Binding by UEA1, however, was not significantly different between infected and PRE cells, and tended to be downregulated in the endometrium and tonsils, and upregulated in PBMCs (Figure 3A). As UEA1 binds α1–2 branched fucose (Supplementary file 1), these results suggest that fucosylation is globally upregulated upon infection of tissue CD4+ T cells with HIV, although not the type of fucosylation that creates α1–2 branched structure. In contrast to the fucose-binding lectins, both sialic acid-binding lectins were increased on infected compared to PRE cells (Figure 3B, Figure 3—figure supplement 4B), and this was observed for all three sites although results did not reach statistical significance for the endometrium. These results are consistent with an upregulation of both total sialic acid (recognized by WGA, which also binds to N-acetylglucosamine [GlcNAc]) (Schwarz et al., 1999) and α2–3 linked sialic acid (recognized by MAL-1) by HIV during infection. In contrast to fucose and sialic acid, we did not observe any marked upregulation of T antigen on infected cells (Figure 3C). Figure 3 with 6 supplements see all Download asset Open asset HIV alters expression of fucose and sialic acid in a tissue site-dependent (A) HIV preferentially infects cells and upregulates fucose expression in a tissue site-dependent Box plots showing binding by fucose-binding proteins on uninfected predicted precursor and infected CD4+ T cells from the endometrium, tonsils, and populations were on singlet CD4+ Tm cells. AOL binds total/core fucose, while UEA1 binds α1–2 branched fucose. (B) HIV preferentially infects sialic cells and upregulates sialic acid in a tissue site-dependent Box plots showing binding by sialic acid-binding lectins. Results are presented as in panel A. WGA binds total sialylated glycans and MAL1 binds α2–3 sialylated glycans. (C) Box plots showing binding by T antigen-binding lectin ABA. Results are presented as in panel A. *p<0.05, as assessed using the Student's paired t test and adjusted for multiple testing using the for HIV preferentially infects memory CD4+ T cells with higher fucose and sialic acid levels In to antigens that have been remodeled by infection, the PP-SLIDE approach can also identify antigens that are differentially expressed on cells infection. In antigens more abundant on PRE than uninfected cells to antigens preferentially expressed on cells, while those abundant on PRE cells to those preferentially expressed on cells. We used as our uninfected population CD4+ Tm cells, CD4+ CD8+ and CD8+ Tn cells because these three populations of cells (Figure 3—figure supplement This was important because antigens differentially expressed between uninfected and PRE cells could phenotypic differences between these differences in lectin binding between PRE and uninfected (CD4+ cells were only observed in PBMCs, with UEA1, WGA, and MAL-1 all binding at significantly higher levels on PRE cells (Figure Figure 3—figure supplement PRE cells also bound these lectins more than their uninfected but these results did not reach statistical PRE cells did not show significant differences in lectin binding to their uninfected These results with the remodeling analysis suggest that in blood and tonsils not HIV preferentially infects memory CD4+ T cells with higher levels of fucose and sialic acid, and then upregulates these cell-surface glycans through viral remodeling. HIV infection alters the surface glycome of immune cells in tonsils of cells can in a cell-intrinsic manner as a of direct infection, but also from For example, the by HIV replication phenotypic changes in cells in the infected We assessed whether HIV infection elicits any glycosylation in cells. To identify memory CD4+ T cells, we the infected culture on CD4+ Tm cells that were binding by all five lectins was observed among tonsillar CD4+ Tm cells to their from uninfected (Figure 3—figure supplement suggesting that in at some tissue sites, remodeling of glycans on CD4+ T cells Interestingly, however, to the CD4+ Tm cells, the infected cells higher levels of total fucose and sialic acid assessed by AOL and WGA binding, suggesting possible cell-intrinsic glycan remodeling by virus (Figure 3—figure supplement To whether HIV alters glycan expression in other cellular we compared glycan levels on multiple subsets of B and T cells from the uninfected vs. infected (Figure 3—figure supplement tonsils significant differences between uninfected vs. cells, and these differences were observed among all For example, sialic acid levels assessed by both WGA and MAL-1 were significantly higher in all the analyzed subsets of and CD4+ T cells, to their from uninfected cultures. expression was also higher among cells, although the difference among CD8+ Tm cells did not reach statistical in levels of T antigen were also observed among B cells, CD8+ Tm cells, and CD4+ Tm cells (Figure 3—figure supplement These glycan changes be by HIV et al., et al., 2003; et al., which can alter cell-surface glycosylation patterns et al., 2016; Giron et al., HIV preferentially infects memory CD4+ T cells from tonsils and PBMCs co-expressing high levels of fucose and sialic acid The data presented suggest that although there are differences between blood vs. the tissue sites fucose and sialic acid are upregulated on HIV infected cells, and CD4+ T cells expressing high levels of fucose or sialic are preferentially for infection. We next to whether the cells express high levels of both fucose or sialic acid, or whether they to distinct subsets of vs. sialic cells. We focused on the AOL and WGA as they total fucose and different forms of sialic acid, We within the HIV-infected cultures, the infection among CD4+ Tm cells expressing high vs. low levels of AOL or In both blood and both tissue compartments, and cells significantly higher HIV infection than did and cells, (Figure To the extent to which this high level of infection was to preferential infection of the and CD4+ Tm cells, we next among the uninfected CD4+ Tm cells and PRE cells, the of cells that were or Consistent with the the of cells expressing high levels of AOL or WGA were higher among PRE cells in both tonsils and PBMCs (Figure 4B), suggesting preferential infection of fucose- and sialic cells by HIV at these Interestingly, cells were also significantly among PRE cells at these sites (Figure 4B), suggesting that the Tm cells fucose and sialic acid. of the PRE cells by t-SNE revealed cells binding high levels of both AOL and WGA among the tonsillar and blood (Figure Although PRE cells from the endometrium did not preferentially express fucose or sialic acid (Figure 4B), PRE cells co-expressing AOL and WGA could be detected from this (Figure suggesting the endometrium, the other two sites, harbors cells co-expressing fucose and sialic acid. To better understand the endometrial CD4+ Tm cells, their tonsillar and blood counterparts, did not preferentially express high levels sialic acid or fucose, we compared the levels of WGA and AOL binding on uninfected CD4+ Tm cells from the three This analysis revealed WGA, but not to be expressed at the levels on the endometrial cells (Figure These results suggest that the cells are not preferentially for infection in the endometrium be that endometrial CD4+ Tm cells all express high levels of sialic acid. however, are higher in PBMCs than endometrium, suggesting that the fact that endometrial CD4+ Tm cells preferentially for infection be by high levels of fucose these results suggest that among blood and tonsillar memory CD4+ T cells, those with the levels of fucose and sialic acid are preferentially for infection by this was not observed among endometrial cells. Figure Download asset Open asset HIV preferentially infects memory CD4+ T cells from tonsils and PBMCs with high levels of fucose and sialic acid. (A) to identify CD4+ Tm populations expressing different levels of fucose and sialic acid detected by AOL and WGA binding, (B) The of CD4+ Tm cells that were infected assessed by are higher among the and cells than among their and for all three *p<0.05, **p<0.01, and ***p<0.001 as assessed using the Student's paired t to a different donor. for the populations are shown in panel A. (C) of uninfected CD4+ Tm and PRE cells expressing high levels of fucose, sialic acid, or both determined by high binding by AOL or WGA, as assessed by In tonsils