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.

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 CD4^LowCD28^Low 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 CD127^High 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 CD127^High 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?

We describe here the development and implementation of CyTOF-Lec, a high-parameter single-cell method to simultaneously quantitate multiple glycans and proteins on the surface of human cells. We used CyTOF-Lec to identify glycomic and phenotypic differences between immune subsets from blood and multiple tissues, and between uninfected and HIV-infected CD4+ T cells. Moreover, by performing PP-SLIDE bioinformatics on our datasets of HIV-infected blood and tissue cells, we identified unique glycan features characteristic of HIV-susceptible CD4+ T cells, and identified glycan structures that were remodeled as a result of cell-intrinsic HIV replication.

Multiple studies have demonstrated remodeling of host cells upon HIV infection. HIV infection of cell lines (Matheson et al., 2015) as well as primary cells (Cavrois et al., 2017; Ma et al., 2020; Xie et al., 2021) leads to up- and downregulation of a variety of host proteins, reflecting the ability of the virus to hijack host processes to complete its replication cycle. We demonstrate in the current study that HIV also remodels the surface glycome of infected cells. By implementing PP-SLIDE to discern antigens differentially expressed as a result of remodeling from those differentially expressed as a reflection of HIV selection, we found that both fucose and sialic acid were upregulated on CD4+ T cells after infection. As core fucosylation on the cell surface is critical for CD4+ T cell activation (Fujii et al., 2016; Liang et al., 2018) and activated cells are more permissive to HIV infection (Stevenson et al., 1990), HIV may upregulate fucosylation to maintain an elevated state of activation facilitating the completion of its replication. We cannot rule out, however, that upregulation of fucose is a byproduct of T cell activation, and not a direct consequence of HIV infection. Of note, fucosylation is also important for lymphocyte trafficking (Colomb et al., 2020), and the upregulation of fucose on infected cells, even if not directly promoting viral replication, may facilitate the circulation of infected cells from blood into tissues where ample populations of HIV-permissive CD4+ T cells reside.

Sialic acid, like fucose, was also upregulated as a result of HIV infection. In general, sialylated glycans on the surface of cells can elicit an immunosuppressive response by effector cells of the immune system. For example, binding of sialic acid on the surface of cancer cells to Siglec-7 and Siglec-9 proteins on the surface of NK cells can diminish NKG2D-mediated activation of the NK cells (Xiao et al., 2016). Moreover, NKG2D may play an important role in NK cell-mediated killing of HIV-infected cells and NKG2D ligands are actively downregulated by HIV Nef to escape immune recognition (Jost and Altfeld, 2012). We postulate that through a combination of upregulating sialic acid and downregulating NKG2D ligands, HIV may evade NKG2D-mediated killing by NK cells.

Interestingly, sialic acid was also preferentially expressed on the memory CD4+ T cells that were preferentially targeted for infection by HIV. These results suggest that the high levels of sialic acid on HIV-infected cells results from preferential infection of cellular subsets with high levels of cell-surface sialic acid, followed by further upregulation of this glycan. Interestingly, however, the preferential selection of WGA^High memory CD4+ T cells for HIV infection was only observed in tonsils and PBMCs, but not in the endometrium (Figure 4C). Because memory CD4+ T cells from the endometrium express higher levels of cell-surface sialic acid than those from tonsils or PBMCs (Figure 4E), most endometrial memory CD4+ T cells may already have the WGA^High HIV-permissive phenotype, resulting in minimal selection of these cells by HIV. Indeed, this would be consistent with the many subsets of endometrial memory CD4+ T cells that are susceptible to HIV infection, as compared to a markedly lower number of HIV-susceptible memory CD4+ T cell subsets from the blood compartment (Ma et al., 2020). It is also worthwhile to note that the endometrium is a unique tissue in that it serves as the site of blastocyst implantation and development of a semi-allogenic fetus. The unusually high levels of sialic acid on immune cells from this tissue may be important to prevent immune-mediated rejection of spermatozoa or the developing fetus.

Importantly, HIV’s preference for cells expressing high levels of sialic acid was validated by demonstrating via sorting experiments that blood-derived memory CD4+ T cells expressing high levels of WGA were significantly more susceptible to infection than those expressing low levels of WGA. The WGA^High population exhibits many features that could explain its high susceptibility: higher expression levels of HIV receptor/co-receptor, higher activation status, and higher expression of markers associated with survival of HIV-infected cells. However, our somewhat surprising finding that sialidase-treated cells are more poorly susceptible to HIV additionally suggest that sialic acid may exert a direct role in promoting HIV infection; the precise step in the viral replication cycle affected by sialic acid should be interrogated in future studies. Regardless, since thus far markers that distinguish memory CD4+ T cells with differing susceptibilities to HIV have been difficult to identify, our findings that WGA may be useful as a tool to isolate a highly susceptible population of CD4+ T cells could be of practical use for the field. On a more fundamental level, our results suggest that host glycan expression can very much influence HIV replication dynamics.

Of note, we cannot rule out the possibility that the high levels of fucose and sialic acid on HIV-infected cells may also result in part from binding of HIV virions to the surface of the infected CD4+ T cells. HIV particles contain cell-derived glycolipids, including the sialic acid-containing GM3 ganglioside (Puryear et al., 2012). Furthermore, HIV gp120 is heavily N-glycosylated, with a majority of high-mannose N-glycan structures and a lower proportion of complex N-glycans carrying lactosamine residues and terminal sialic acid. Sialic acid on gp120 is recognized by multiple members of the Siglec family of lectins, and these lectins can facilitate trans- or cell-to-cell infection of CD4+ T cells (Izquierdo-Useros et al., 2012; Varchetta et al., 2013; Zou et al., 2011). However, we note that our studies characterizing infected cells defined these cells as those that express HSA – which is not on the surface of input virions – and that have downregulated cell-surface CD4, a hallmark of productive HIV infection. The fact that we found unique glycan features of these cells relative to bystander CD4+ T cells suggests that productively infected cells have distinct cell-surface glycan features in a manner not attributable to just surface virion sticking.

Our study has limitations. First, since cells from the different compartments (blood, tonsils, and endometrium) were run in separate batches, it is difficult to compare expression levels between cells from these compartments, which is why our analysis focused on relative glycan expression levels among cells within each compartment. Second, while WGA is commonly used as a reagent to monitor cell-surface sialic acid levels, it also binds GlcNAc, so we cannot rule out that some of the WGA effects we observed were due to GlcNAc and not sialic acid. Notably, a recent study reported that cell-surface GlcNAc is important for HIV binding and infection (Spillings et al., 2022), which is in line with our observation that WGA^High cells are highly susceptible to HIV infection. Third, a limited number of donors were analyzed, particularly in the sialidase treatment experiments, as those experiments were technically challenging due to the toxic effects of sialidase treatment on primary CD4+ T cells from most tested donors.

While our study focused on characterizing cells productively infected with HIV-1, identified as those expressing on their cell surface the LTR-driven HSA reporter protein, we envision that CyTOF-Lec will also be useful for studying the HIV reservoir that persists despite antiretroviral therapy (ART). Recent observations suggest that a significant portion of the reservoir in ART-suppressed people living with HIV (PLWH) is transcriptionally active, expressing short and incomplete HIV transcripts in the absence of ex vivo stimulation (Yukl et al., 2018). Interestingly, we recently demonstrated using cells from ART-suppressed PLWH that CD4+ T cells expressing high levels of fucose contain significantly more transcriptionally active HIV reservoir cells than those expressing low levels of fucose (Colomb et al., 2020). With the development of CyTOF-Lec as a single-cell analysis tool, we will now be able to interrogate glycan expression patterns on transcriptionally active, as well as transcriptionally silent HIV reservoir cells. Specifically, we envision combining CyTOF-Lec with PP-SLIDE analysis of reservoir cells from PLWH. This will entail using bioinformatics approaches to trace ex vivo reactivated cells to their original pre-stimulation states by PP-SLIDE, an approach we previously described and validated experimentally (Neidleman et al., 2020b), but under conditions where we incorporate assessment of the glycan features of the reservoir cells with the CyTOF-Lec panel.

Beyond HIV, CyTOF-Lec has potentially wide-ranging applications. Glycosylated host receptors play important roles in enabling the attachment of virus. For example, influenza virus enters cells by binding of its hemagglutinin (HA) protein to terminal sialic acid moieties attached to surface proteins of host cells (Li et al., 2021). The novel beta-coronavirus SARS-CoV-2 may also exploit host cell glycosylation for infection, as its primary receptor ACE2 is heavily glycosylated. Indeed, blockage of N- and O-glycan synthesis on the host cell can inhibit SARS-CoV-2 entry by diminishing the ability of the viral spike protein to bind ACE2 (Yang et al., 2020). Better understanding the surface glycans of virally susceptible host cells through single-cell glycomic analysis by CyTOF-Lec, in conjunction with identifying the protein backbones harboring the glycans, will improve our understanding of viral pathogenesis and can potentially lead to novel prophylactic or therapeutic agents for clinically relevant viral pathogens.

Further development of CyTOF-Lec in conjunction with next-generation sequencing approaches will also be valuable for developing the tools to better understand how glycan expression associates with or directly regulates host cell processes. A recent report combined glycomic analysis with single-cell RNAseq (Kearney et al., 2021) by incubating cells with a biotinylated version of the lectin L-Pha followed by a DNA-barcoded anti-biotin antibody. Subsequent droplet encapsulation and sequencing revealed the transcriptomes of cells with different levels of L-Pha binding. We envision that by directly conjugating lectins to barcoded DNA oligos, we can expand the numbers of lectins we can simultaneously monitor. Furthermore, additional inclusion of DNA-barcoded antibodies (Peterson et al., 2017; Stoeckius et al., 2017) will enable simultaneous surface proteome analysis. Such a technology, while not as high-throughput, scalable, and cost-effective as CyTOF-Lec, will enable a genome-wide analysis of cellular processes associated with differential surface glycosylation.

Raw CyTOF data have been deposited in Dryad (https://doi.org/10.7272/dryad.Q6FT8J92).

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Author details

1. Tongcui Ma

   1. Department of Urology, University of California, San Francisco, San Francisco, United States
   2. Gladstone Institutes, San Francisco, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Validation, Visualization, Writing - original draft

   Competing interests

   No competing interests declared

2. Matthew McGregor

   1. Department of Urology, University of California, San Francisco, San Francisco, United States
   2. Gladstone Institutes, San Francisco, United States

   Contribution

   Methodology

   Competing interests

   No competing interests declared

3. Leila Giron

   The Wistar Institute, Philadelphia, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

4. Guorui Xie

   1. Department of Urology, University of California, San Francisco, San Francisco, United States
   2. Gladstone Institutes, San Francisco, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Ashley F George

   1. Department of Urology, University of California, San Francisco, San Francisco, United States
   2. Gladstone Institutes, San Francisco, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. Mohamed Abdel-Mohsen

   The Wistar Institute, Philadelphia, United States

   Contribution

   Conceptualization, Funding acquisition, Methodology, Resources, Supervision, Writing – review and editing

   For correspondence

   mmohsen@Wistar.org

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9945-4314

7. Nadia R Roan

   1. Department of Urology, University of California, San Francisco, San Francisco, United States
   2. Gladstone Institutes, San Francisco, United States

   Contribution

   Conceptualization, Data curation, Funding acquisition, Methodology, Project administration, Resources, Supervision, Validation, Writing - original draft

   For correspondence

   nadia.roan@gladstone.ucsf.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5464-1976

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