Thanks to antiretroviral therapy, HIV infection is no longer the life sentence it once was. But despite the effectiveness of drugs to manage and treat the virus, it can never be fully eliminated from the human body, lingering in some cells deep in different human tissues where it goes unnoticed by the immune system.
Now, new research by University of Alberta immunologist Shokrollah Elahi reveals a possible answer to the mystery of why infected people can’t get rid of HIV altogether.
Elahi and his team found that in HIV patients, killer T cells – a type of white blood cells responsible for identifying and destroying cells infected with viruses – have very little to none of a protein called CD73.
Because CD73 is responsible for migration and cell movement into the tissue, the lack of the protein compromises the ability of killer T cells to find and eliminate HIV-infected cells, explained Elahi.
“This provides us the opportunity to come up with potential new treatments that would help killer T cells migrate better to gain access to the infected cells in different tissues.”
After identifying the role of CD73 – a three-year project – Elahi turned his focus to understanding potential causes for the drastic reduction. He found it is partly due to the chronic inflammation that is common among people living with HIV.
“Following extensive studies, we discovered that chronic inflammation results in increased levels of a type of RNA found in cells and in blood, called microRNAs,” he explained. “These are very small types of RNA that can bind to messenger RNAs to block them from making CD73 protein. We found this was causing the CD73 gene to be suppressed.”
The team’s discovery also helps explain why people with HIV have a lower risk of developing multiple sclerosis, Elahi noted.
“Our findings suggest that reduced or eliminated CD73 can be beneficial in HIV-infected individuals to protect them against MS. Therefore, targeting CD73 could be a novel potential therapeutic marker for MS patients.”
Elahi said the next steps in his research include identifying ways the CD73 gene can be manipulated to turn on in patients living with HIV and off in those with MS.
The study is published in PLOS Pathogens.
It has been reported that infection of Tregs with HIV-1 impairs their suppressive potential through DNA methylation of the FOXP3 gene,21, 90 but ART reverses this impaired function.91 In contrast, some studies have reported that Treg functions are not altered in the setting of HIV infection.92-94 However, the potential differences in Tregs of different HIV subgroups at the transcriptional level or functionality have not been fully elucidated.
In this report, we show that Tregs from the ART group demonstrate a more suppressive phenotype than Tregs from LTNPs and HCs. Our IPA revealed the upregulation of IL-2 and TCR signalling in Tregs of ARTs compared to HCs. Also, we observed the upregulation of mTOR, hippo and MAPK signalling pathways that are associated with Treg activation in patients on ART.39-42, 52, 54 The mTOR pathway has a key role in the activation and differentiation of cTregs to eTregs and is crucial for the peripheral T-cell tolerance and homeostasis.95 The TCR signalling induces IRF4 and contributes to the acquisition of an effector phenotype in Tregs.96, 97 The mTOR controls the expression of IRF4 at the post-transcriptional level95 and induces the expression of ICOS, which derives eTreg differentiation.98 Therefore, a higher expression of IRF4 and its controlled genes in Tregs of ARTs than those in HCs may support their suppressive effector phenotype. A higher mTOR activity contributes to the higher expression of the large neutral amino acid transporter CD98 (LAT1) gene and the transferrin receptor (CD71) in Tregs of ARTs, which are required for Treg activation.39
We also observed the hippo pathway upregulation in Tregs of patients on ART. The hippo pathway controls the size of animal organs through tight regulation of cell proliferation and apoptosis.53, 99 The core enzyme of the hippo pathway, MST-1, has several key roles in Treg stability. For example, it mediates IL-2-induced STAT5 phosphorylation, resulting in the expression of the genes that are associated with the inhibitory activity of Tregs.54 Also, MST-1 increases FOXP3 acetylation.55 While FOXP3 is expressed transiently in effector T cells, stable expression of FOXP3 is required for the development and establishment of a Treg phenotype.100, 101 Moreover, the interaction of MST-1 with the DOCK8-LRCH complex controls T-cell migration.54 Interestingly, both DOCK8 and LRCH were upregulated in Tregs of ARTs compared to HCs (Supplementary figure 4d). This suggests a higher migratory ability for Tregs in the ART group, which may enhance their access to the effector site.
In contrast, we observed a lower ceramide signalling in Tregs of the ART group. Protein phosphatase 2A (PP2A), the core enzyme of the ceramide pathway, is a highly conserved and ubiquitously expressed serine–threonine phosphatase, which regulates many cellular functions.102, 103 This enzyme has three different subunits, with the Ac subunit being the catalytic and Aa subunit as the scaffold subunit. The abundance of catalytic PP2Ac is similar in different cells. However, its activity is regulated through the phosphorylation of the carboxy-terminal end of PP2Ac at the Tyr307 residue.104 The SET oncoprotein inhibits PP2A activity through its phosphorylation.102, 103 Besides, ceramide interacts with the SET oncoprotein and inhibits its function (Supplementary figure 4e), thus preventing its inhibitory effect on PP2A.47 It has been shown that the inhibition of acid sphingomyelinase increases the suppressive activity of Tregs through the increased CTLA-4 turnover.105 Therefore, decreased ceramide levels and subsequent decline in PP2A activity in Tregs of ART may enhance their suppressive activity through the upregulation of the mTOR pathway.49 However, increased ceramide level in CD4+ T cells and monocyte reduces their infectivity to HIV-1.106 As such, decreased ceramide levels in Tregs of ARTs may enhance their susceptibility to HIV-1 infection and make them a favorite target for the virus.
Our further analysis revealed an increase in MYC, IL-5, TCR and HIF1A but a decrease in IL-10 signalling in LTNP Tregs versus HC Tregs. The upregulation of genes involved in IL-15 and the downregulation of genes involved in IL-10 signalling suggest the decreased suppressive function of Tregs in LTNPs.63, 107 MYC and HIF1A can increase the glycolysis in Tregs,64 which was demonstrated by the upregulation of the glycolysis pathway in Tregs of LTNPs. HIF-1α also reduces the suppression role of Tregs through the degradation and destabilisation of FOXP3 gene.108, 109
Compared to other subsets of T cells such as Th1, Th2 and Th17 cells, Tregs rely less on glycolysis but mainly on mitochondrial metabolism and oxidative phosphorylation (OXPHOS) as their source of energy110 because glycolysis inhibits Treg-suppressive function.65, 66, 111, 112 As such, FOXP3 and CTLA-4 suppress glycolysis and enhance OXPHOS, resulting in reprogramming of Treg metabolism.113
Interestingly, the expression of glucose transporter Glut-1, the rate-limiting step in glycolysis,114 was higher in Tregs of LTNPs than in those of HCs. It has been shown that the overexpression of Glut-1 decreases the expression of FOXP3 in Tregs.66 In contrast to glycolysis, OXPHOS results in the production of reactive oxygen species (ROS) in T cells115 preventing nuclear transport of BTB domain and CNC homolog 2 (Batch2) through Bach2 deSUMOylation.116 DeSUMOylation of BACH2 prevents its nuclear export, which leads to a stable FOXP3 expression and stability of Tregs.116 Another important observation was the upregulation of gluconeogenesis in Tregs of LTNPs. Although gluconeogenesis mainly occurs in the liver and kidney,117 it has been reported to occur in memory T cells.118
The similarity of the metabolic pathway in memory T cells and Tregs110 may account for the observed gluconeogenesis pathway in Tregs. Overall, the metabolic pattern of Tregs in LTNPs and the activation of the IL-6/STAT3 pathway suggest impaired suppressive properties of Tregs in this group. This was further demonstrated when we noted a significant deficiency in Treg-mediated suppression of effector T-cell proliferation by Tregs of LTNPs versus those in the ART group.
We further found that Tregs in ARTs were more apoptotic and showed higher expression of surface co-inhibitory receptors (e.g. CTLA-4 and ICOS), which might contribute to their enhanced suppressive functions. Apoptotic Tregs exhibit more suppressive function through the release of ATP and its conversion to adenosine by the enzymatic activity of CD39 and CD73 ectoenzymes.85 Of note, we observed significant elevation of HLA-F mRNA and HLA-F protein in Tregs from HIV-infected individuals (both ART and LTNP) compared to HCs. Upregulation of HLA-F in Tregs of HIV-infected individuals might be related to their hyper-activation status. HLA-F is largely located in the endoplasmic reticulum, and its expression is tightly regulated.119
This suggests that HLA-F surface expression on Tregs of HIV-infected patients might serve as a marker of cell stress including endoplasmic reticulum stress. Additionally, HIV-1 infection of CD4+ T cells can increase the transcription of HLA-F mRNA but decrease the binding of its ligand (KIR3DS1) as a potential mechanism of avoiding recognition by NK cells.120 Thus, it is possible to speculate that the ability of KIR3DS1 to bind to HLA-F expressed on Tregs in HIV-infected patients provides a mechanistic link between Tregs and NK cells. This hypothesis merits further investigation and might also have relevance in other infectious diseases, autoimmune disorders and cancers.
Taken together, we have provided a novel insight into the differential properties of Tregs in HIV-infected individuals. In agreement with these observations, we have previously reported that CD8+ T cells restricted by HLA-B27 and HLA-B57 upon recognition of their cognate epitopes do not upregulate TIM-3 and therefore evade Treg-mediated suppression.13 In contrast, CD8+ T cells restricted by non-protective HLA alleles upon encounter with their cognate epitopes upregulate TIM-3 and therefore become suppressed by Tregs.13
Thus, T-cell evasion of Treg-mediated immune regulation13 and lower suppressive properties of Tregs in LTNPs may explain the higher propensity of autoimmune diseases in individuals possessing HLA-B27 and HLA-B57.37 In contrast, it is possible to speculate that the robust immune response against HIV infection in LTNPs may in part be associated with impaired Treg functions. More recently, we demonstrated that CD8+ T cells restricted by HLA-B35Px were not suppressed by Tregs in HIV-infected progressors.15 Although because of the cell limitation, we were unable to conduct RNAseq analysis on Tregs from HIV-infected progressors, we have shown that Tregs in these individuals exhibit impaired suppressive properties. This hypothesis can be further supported by the higher susceptibility of individuals restricted by HLA-B35Px to autoimmune diseases such as subacute thyroiditis.121
We are aware of several study limitations. For example, we performed limited functional studies on Tregs in vitro. Isolating a large number of Tregs was not practical for functional assays, mainly because of the cell limitation. Similarly, the discrepancy in the sample size in some experiments was related to the same issue. There were no selection criteria for the samples in each set of experiments, and samples were randomly selected; however, we tried to use the same subset of samples for experiments that were related to each other (e.g. phospho assays).
The period of HIV infection may influence the functionality of Tregs, and we were unable to obtain accurate information for our patients on ART, but they have been on ART beyond 2 years. The sex of HIV-infected individuals is also an important factor to be taken into consideration, but our patients were a combination of men and women.
The activation status of T cells in HCs versus HIV patients may influence the results. As such, higher activated T cells in one group than in the other may influence the transcriptional levels. However, our observations do not support this concept since non-Treg-CD4+ T cells exhibited similar activation status as measured by Ki67 and HLA-DR in our cohort. In this study, we were unable to examine the effects of ART on Treg transcriptional profile/functionality. Nevertheless, future studies comparing Treg transcriptional profiles and effector functions pre- and post-treatment are warranted.
Taken together, we demonstrated differential Treg phenotype and function in different HIV subgroups versus HCs. In particular, by conducting RNAseq analysis, we provided a novel insight into the mechanism underlying differential Treg properties in HIV-infected individuals.
reference link :https://onlinelibrary.wiley.com/doi/full/10.1002/cti2.1289
More information: Shima Shahbaz et al, Elevated ATP via enhanced miRNA-30b, 30c, and 30e downregulates the expression of CD73 in CD8+ T cells of HIV-infected individuals, PLOS Pathogens (2022). DOI: 10.1371/journal.ppat.1010378