Scientists at The Wistar Institute may have discovered a new way of identifying and targeting hidden HIV viral reservoirs during treatment with antiretroviral therapy (ART).
These findings were published today in Cell Reports and may have translational implications for improving the long-term care of HIV positive people.
ART has dramatically increased the health and life expectancy of HIV-infected individuals, suppressing virus replication in the host immune cells and stopping disease progression; however, low yet persistent amounts of virus remain in the blood and tissues despite therapy.
Virus persistency limits immune recovery and is associated with chronic levels of inflammation so that treated HIV-infected individuals have higher risk of developing a number of diseases.
This persistent infection stems from the ability of HIV to hide in a rare population of CD4 T cells. Finding new markers to identify the virus reservoir is of paramount importance to achieve HIV eradication.
“With recent advances that we are making in the fields of glycobiology and glycoimmunology, it has become clear that the sugar molecules present on the surface of immune cells play a critical role in regulating their functions and fate,” said corresponding author Mohamed Abdel-Mohsen, Ph.D., assistant professor in The Wistar Institute Vaccine &
Immunotherapy Center. “However, the relevance of host cell-surface glycosylation in HIV persistence remained largely unexplored, making it a ‘dark matter’ in our understanding of HIV latency.
For the first time, we described a cell-surface glycomic signature that can impact HIV persistence.”
Persistently infected cells can be divided into two groups: cells where the virus is completely silent and does not produce any RNA (i.e., silent HIV reservoir); and cells where the virus produces low levels of RNA (i.e., active HIV reservoir).
Targeting and eliminating both types of reservoirs is the focus of the quest for an HIV cure.
A main challenge in this quest is that we do not have a clear understanding of how these two types of infected cells are different from each other and from HIV-uninfected cells. Therefore, identifying markers that can distinguish these cells from each other is critical.
For their studies, Abdel-Mohsen and colleagues used a primary cell model of HIV latency to characterize the cell-surface glycomes of HIV-infected cells. They confirmed their results in CD4 cells directly isolated from HIV-infected individuals on ART.
They identified a process called fucosylation as a feature of persistently infected T cells in which the viral genome is actively being transcribed. Fucosylation is the attachment of a sugar molecule called fucose to proteins present on the cell surface and is critical for T-cell activation.
Researchers also found that the expression of a specific fucosylated antigen called Sialyl-LewisX (SLeX) identifies persistent HIV transcription in vivo and that primary CD4 T cells with high levels of SLeX have higher levels of T-cell pathways and proteins known to drive HIV transcription during ART.
Such glycosylation patterns were not found on HIV-infected cells in which the virus is transcriptionally inactive, providing a distinguishing feature between these two cell compartments.
Interestingly, researchers also found that HIV itself promotes these cell-surface glycomic changes.
Importantly, having a high level of SLeX is a feature of some cancer cells that allow them to metastasize (spread to other sites in the body).
Indeed, researchers found that HIV-infected cells with high levels of SLeX are enriched with molecular pathways involved in trafficking between blood and tissues.
These differential levels of trafficking might play an important role in the persistence of HIV in tissues, which are the main sites where HIV hides during ART.
Based on these findings, the role of fucosylation in HIV persistence warrants further studies to identify how it contributes to HIV persistence and how it could be used to target HIV reservoirs in blood and tissues.
Levels of Fucosylated Carbohydrate Ligands Associate with Persistent HIV Transcription In Vivo
To validate our preliminary in vitro observations, we next assessed whether differential glycosylation levels corresponded with different levels of persistent HIV transcription in vivo. We used fluorescently labeled versions of the lectins identified in Figure S1E (AOL, UEA-I, ABA, and MAL-I) to sort CD4+ T cells from HIV-infected ART-suppressed individuals into groups with low (lowest 5% binding), medium, or high (highest 5% binding) levels of each tested glycan.
Consistent with our in vitro data, we found that cells with low cell-surface total/core fucose, i.e., fucoselow (lower binding to AOL), exhibited lower levels of cell-associated HIV RNA (total elongated transcripts; Yukl et al., 2018) compared with total CD4+ T cells, suggesting that HIV-infected transcriptionally inactive cells in vivo might be depleted of cell-surface fucose.
However, and unlike our in vitro data, in which levels of fucose were not different between activated-uninfected and activated-infected cells, we found that cells with high cell-surface total/core fucose, i.e., fucosehi (higher binding to AOL), exhibited higher levels of cell-associated HIV RNA compared with total CD4+ T cells (Figure 1A).
Comparing fucosehi cells to fucoselow cells, we found that fucosehi cells exhibited higher levels of cell-associated HIV RNA than did fucoselow cells (average of 17.2-fold enrichment of cell-associated HIV RNA in fucosehi cells compared with fucoselow cells) despite less drastic differences in levels of HIV DNA (average of a 6.0-fold difference of HIV DNA) (Figure 1A).
Furthermore, the ratio between cell-associated HIV RNA and HIV DNA (a potential marker of HIV transcriptional activity) was higher in fucosehi cells (average 51.2, median 41.5) than in fucoselow cells (average 20, median 6.5) (Figure 1A).
Next, we examined binding to UEA-I (lectin that binds to branched fucose). We found that cells with high cell-surface branched fucose exhibited higher cell-associated HIV RNA compared with cells with low branched fucose (average of 8.2-fold enrichment) despite little difference in HIV DNA levels (average of 3.0-fold enrichment), but no significant difference in the ratio between cell-associated HIV RNA and HIV DNA was observed (Figure 1B).
However, unlike our in vitro data, we did not observe any difference in cell-associated HIV RNA levels, DNA levels, or the ratio between HIV RNA and DNA in cells binding to MAL-I (Siaα2-3Gal (β-1,4) GlcNAc) or ABA lectin (TF antigen) (Figures S2A and S2B).
The data in Figures 1A and 1B suggest that altered T cell-surface fucosylation patterns associate with persistent HIV transcriptional activity in vivo: low cell-surface fucosylation is associated with transcriptional-inactive HIV during ART, whereas high cell-surface fucosylation is associated with transcriptional-active HIV during ART.
Surface Expression of the Fucosylated Glycoantigen SLeX on CD4+ T Cells Associates with HIV Transcription In Vivo
The oligosaccharide SLeX is a fucosylated carbohydrate that binds the selectin family of lectins and mediates many important cell-cell processes, including extravasation. Given our finding that total fucosylated glycans are enriched on the surface of HIV-infected transcriptionally active cells (Figures 1A and 1B), we hypothesized that the fucosylated glycoantigen SLeX may be one of the fucosylated antigens enriched on these cells.
SLeX is usually attached to O-glycans and exists in two forms:
(1) a non-sulfated form simply called SLeX and
(2) a sulfated form called CLA (cutaneous lymphocyte antigen). We used antibodies specific for SLeX or CLA to sort CD4+ T cells from HIV-infected ART-suppressed individuals.
We found that cells with high cell-surface levels of SLeX had increased cell-associated HIV RNA levels compared with cells with low-SLeX (SLeX-Low) expression (4.9-fold difference for cell-associated HIV RNA) despite similar levels of HIV DNA (0.9-fold difference) (Figure 1C).
The ratio between cell-associated HIV RNA and HIV DNA was higher in SLeX-Hi cells (average 88.0, median 46.6) compared with SLeX-Low cells (average 20.2, median 21.0) (Figure 1C).
Similar, albeit weaker, results were observed for cells sorted using antibodies for CLA (Figure 1D).
To ensure that our observations were not the result of possible latent reactivation induced by these lectins/antibodies, we treated CD4+ T cells isolated from HIV-infected ART-suppressed individuals with AOL, UEA-I, SLeX, or CLA antibodies for the same duration required to perform cell sorting (3 h).
This treatment did not reactivate latent infection or induce cell-associated HIV RNA expression (Figure S2C). Altogether, these data suggest that high levels of the fucosylated glycomic antigen SLeX on the CD4+ T cell-surface associate with persistent HIV transcription in vivo.We also compared levels of SLeX and CLA on CD4+ T cells (total and memory) of HIV-infected (ART-suppressed and viremic) and HIV-negative controls.
We found higher frequencies of SLeX+ cells on total CD4+ T cells of viremic (median 35%, interquartile range [IQR] 11.2) and ART-suppressed (median 26.8%, IQR 5.4) individuals compared with HIV-negative controls (median 15.3%, IQR 2.1).
Similarly, we found a larger proportion of SLeX+ cells on memory CD4+ T cells of viremic (median 29.7%, IQR 11.4) and ART-suppressed (median 22.6%, IQR 8.9) individuals compared with HIV-negative controls (median 15.5%, IQR 4.5) (Figure 1E).
We also found a higher frequency of cells expressing CLA on total CD4+ T cells of viremic (median 71%, IQR 14.2) and ART-suppressed (median 58.3%, IQR 4.4) individuals compared with HIV-negative controls (median 41.2%, IQR 6.2). A larger proportion of memory CD4+ T cells similarly expressed higher levels of CLAhigh in viremic (median 32%, IQR 10.2) and ART-suppressed (median 26.3%, IQR 5.4) individuals compared with HIV-negative controls (median 17.6%, IQR 3) (Figure 1F).
We also found that levels of SLeX+ and CLA+ CD4+ T cells correlate with viral load in HIV-infected individuals (Figure S2D). These results indicate that high cell-surface expression of SLeX is associated with HIV infection, irrespective of viral suppression by ART.
In this study, we examined the relationship between cell-surface glycosylation patterns of HIV-infected cells and persistent HIV transcription. We identified fucosylated carbohydrates to be enriched on the surface of HIV-infected transcriptionally active cells despite ART suppression.
Conversely, the levels of these fucosylated carbohydrates are low on the surface of HIV-infected transcriptionally inactive cells. This glycomic feature of HIV-infected cells actively producing HIV transcripts is possibly a product of viral transcription and potentially has a phenotype significance on the trafficking abilities of these cells.
In particular, the cell extravasation mediator SLeX is one of the fucosylated carbohydrate ligands enriched on the surface of HIV-infected transcriptionally active cells and reduced on the surface of HIV-infected transcriptionally inactive cells.
This observation would suggest potential differential trafficking abilities of these cells. Such differential abilities might affect maintenance of HIV persistence and should be considered when targeting HIV reservoirs in blood and tissues.Identifying host factors enriched in HIV-infected cells (both latent and actively transcribing) during ART could provide the HIV cure field with vital biological clues into the molecular pathways involved in viral persistence.
These host factors may be different between HIV-infected transcriptionally active and HIV-infected transcriptionally inactive cells. For example, it has been observed that persistent HIV proviruses are enriched in several memory CD4+ T cell compartments (Chomont et al., 2009).
Some host factors are also enriched in HIV-infected cells (transcriptionally active or not) during long-term ART. These factors include the immune negative checkpoints PD-1, TIGIT, LAG-3 (Fromentin et al., 2016), and CTLA-4 (McGary et al., 2017), as well as other factors such as CD2 (Iglesias-Ussel et al., 2013), CCR6 (Gosselin et al., 2017), and Survivin (Kuo et al., 2018). Other host factors are shown to be enriched, in particular, in HIV-infected transcriptionally active cells, such as CD30 (Hogan et al., 2018) and CD20 (Serra-Peinado et al., 2019).
These factors, especially those that reside on the cell surface, may be useful for targeting these cells and can provide a better understanding of HIV persistence biology.Recent advances in the cancer field demonstrated that the aberrant glycosylation pattern of cancer cells alters their functions and interaction with the immune system (Pinho and Reis, 2015; RodrÍguez et al., 2018).
Such advances have promoted increasing interest in developing novel tools to target the tumor glycocode (RodrÍguez et al., 2018). However, the relevance of the host glycosylation machinery to HIV persistence has not been evaluated previously.
We found that cell-surface fucosylation is enriched on the surface of HIV-infected transcriptionally active cells and is reduced on the surface of HIV-infected transcriptionally inactive cells in vitro.
Future studies will be needed to identify the potential additive enrichment of persistent HIV-infected cells when combining cell-surface fucosylation with other host factors, mentioned earlier, that are enriched on HIV-infected cells during ART.
Most proviruses persisting in HIV-infected ART-treated individuals harbor mutations and/or deletions, rendering them defective (Bruner et al., 2016; Ho et al., 2013). However, not all intact viruses are inducible, and both intact and defective proviruses can express viral RNA and proteins (Imamichi et al., 2020; Pollack et al., 2017).
It is possible that cells with high cell-surface fucose in general (and SLeX in particular) are enriched with intact HIV genomes compared with cells with low cell-surface fucose, given the higher HIV transcriptional activity of these former cells.
However, this possibility will need to be investigated using single-genome, near-full-length proviral sequencing to determine the genetic makeup of the virus infecting cells with differential levels of fucosylation.Many T cell processes and functions are shaped by cell-surface glycosylation (Pereira et al., 2018).
For example, T cell-surface fucosylation is critical for T cell activation via T cell receptor (TCR) signaling (Liang et al., 2018), and the fucosylation of T cell immune negative checkpoints (such as PD-1) is critical for their function (Okada et al., 2017).
It is unclear how these two published observations may contribute to our findings. Among the total fucosylated carbohydrate ligands, we found that SLeX, which has important implications for T cell trafficking, is enriched on the surface of HIV-infected transcriptionally active cells.
However, this 5-fold enrichment is significantly lower than the enrichment of total/core fucose (17-fold) found when using AOL (which binds total/core fucose). These results suggest that other fucosylated ligands may be enriched on the surface of HIV-infected transcriptionally active cells and depleted on the surface of HIV-infected transcriptionally inactive cells.
Identifying these other glycan structures and their exact protein and lipid backbones should be the subject of future studies and will likely allow us a better understanding of the role of cell-surface fucosylation in modulating T cell functions and HIV persistence.
Further studies will also be needed to examine the potential biological significance of this altered fucosylation on HIV-infected T cell biology and function.The process of leukocyte extravasation is well described and requires binding of selectins (a type of lectins) to fucosylated carbohydrates.
T cells exit the vasculature to reach their target tissue through the leukocyte adhesion cascade, a coordinated series of events involving (1) selectin-mediated rolling, (2) chemokine-triggered activation, and (3) integrin-dependent arrest (Ley et al., 2007). Selectins are a family of receptors comprising platelet (P), endothelial (E), and leukocyte (L) selectin.
To mediate rolling, selectins bind with their fucosylated carbohydrate ligands, notably SLeX (Impellizzeri and Cuzzocrea, 2014). Upon antigen stimulation, some T cells become activated and induce cell-surface expression of SLeX; SLeX binding to P- and E-selectin on vascular endothelium regulates the trafficking of these T cells into various non-lymphoid tissues. Some of these T cells can traffic back to the lymph nodes through the lymph (Haddad et al., 2003; Wolber et al., 1998; Xie et al., 1999).
Our transcriptomic analysis demonstrated enrichment of T cell extravasation and trafficking pathways in CD4+ T cells with high levels of SLeX compared with cells with low levels of SLeX. These data suggest that HIV-infected transcriptionally active CD4+ T cells may have higher trafficking abilities compared with HIV-infected transcriptionally inactive CD4+ T cells.
These differential characteristics of HIV-infected T cells trafficking to lymphoid and non-lymphoid tissues in their HIV transcriptional activity and cell-surface glycomic features could be considered for targeting the tissue-based HIV reservoir. Selectin antagonists such as bimosiamose (Friedrich et al., 2006; Kirsten et al., 2011; Mayr et al., 2008) and rivipansel (Telen et al., 2015) have been used in humans and could be explored in the context of HIV infection to temporarily trap HIV-infected cells in blood for immunotherapeutic clearance.
Our data suggest that HIV infection can directly induce the expression of SLeX on the surface of CD4+ T cells. The mechanism that underlies this induction is not clear. Different viral infections have been described to induce the cell-surface expression of fucosylated carbohydrate ligands, including HSV-1, VZV, and CMV (Nyström et al., 2007, 2009). HTLV1 infection has been shown to induce SLeX through the viral transactivator Tax (Hiraiwa et al., 2003; Kambara et al., 2002; Umehara et al., 1996).
Future studies will be needed to investigate the possibility that HIV induces SLeX through its viral transactivator Tat. However, infection cannot explain the entirety of the difference we found in the levels of SLeX on the surface of CD4+ T cells from viremic and ART-suppressed HIV-infected individuals, because not all T cells from HIV-infected individuals are infected.
There are two possibilities: (1) Although our data show that activation using αCD3/αCD28, TNF-α, or IL-2 does not induce the expression of SLeX, we cannot exclude the possibility that stimulation using other stimulants, in vivo, would induce SLeX expression. (2)
Viral proteins (including tat and nef) can exist in circulation (especially in exosomes) and can be taken by uninfected cells and affect them. HTLV-1 Tax significantly enhances the ability of cell activation to induce FUT7 (Hiraiwa et al., 1997).
Therefore, although our data show that active HIV infection can directly induce SLeX, likely other factors synergize with HIV infection, leading to this enrichment in vivo. Although our data show that HIV infection can directly induce the expression of SLeX, it does not exclude the possibility that cells are preferentially infected with HIV.
This latter possibility is supported by our observation that cells expressing SLeX are enriched with HIV co-receptors and activation markers. Future studies will be needed to determine the contribution of (1) the direct impact of HIV transcription on SLeX expression and (2) the impact of cellular susceptibility to infection on our observed enrichment of SLeX on the surface of HIV-infected transcriptionally active cells during ART.
In addition, several cancers have higher levels of fucose in general (Agrawal et al., 2017; Blanas et al., 2018), and SLeX in particular (Julien et al., 2011; Nakamori et al., 1997; Trinchera et al., 2017), that affect their functions and contribute to their metastasis (through binding to selectins).
Therefore, several mechanisms clearly contribute to SLeX induction. Understanding the upstream modulator or modulators of fucose and SLeX enrichment on HIV-infected transcriptionally active cells, as well as the downstream consequences of this enrichment, should be the subject of future studies to better understand HIV persistence.
Our study provides the first insight into a potential role of circulating CD4+ T cell-surface glycome in persistent HIV transcription activity. However, our findings have limitations, including the following:
(1) Our data were obtained from blood; there is a need to analyze the potential enrichment of fucosylated glycans, including SLeX, in tissues such as lymph nodes and gut-associated lymphoid tissues.
(2) Our in vivo phenotyping analysis was done mainly on cryopreserved PBMCs, and it is possible that freezing/thawing affects these phenotypes.
(3) Our data were obtained using cross-sectional samples from chronically infected adults; there will be a need to analyze longitudinal changes, acute infection, and samples from geographically and/or age-distributed cohorts.
(4) We did not comprehensively validate in vivo all glycomic signatures of HIV-infected cells identified in in vitro. Future studies will need to examine whether additional glycomic signatures of HIV-infected cells can be confirmed in vivo or identified in vitro (using other HIV latency models and/or other glycomic technologies such as mass spectrophotometry).
That said, we think our study is an important step toward elucidating the potential glycomic underpinnings of HIV persistence. In summary, we identified fucosylation to be a feature of persistent HIV transcriptional activity in vivo. The role of cell-surface fucosylation (including SLeX expression) in HIV persistence warrants further investigation to better understand persistent HIV expression during ART and identify glycan-based interactions that can be targeted for novel HIV immunotherapies.
REFERENCE LINK : https://www.cell.com/cell-reports/fulltext/S2211-1247(20)30976-1?_returnURL=https%3A%2F%2Flinkinghub.elsevier.com%2Fretrieve%2Fpii%2FS2211124720309761%3Fshowall%3Dtrue
More information: Florent Colomb et al. Sialyl-LewisX Glycoantigen Is Enriched on Cells with Persistent HIV Transcription during Therapy, Cell Reports (2020). DOI: 10.1016/j.celrep.2020.107991