Metabolomic and immune alterations in long COVID patients with chronic fatigue syndrome.

Introduction: A group of SARS-CoV-2 infected individuals present lingering symptoms, defined as long COVID (LC), that may last months or years post the onset of acute disease. A portion of LC patients have symptoms similar to myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS), which results in a substantial reduction in their quality of life. A better understanding of the pathophysiology of LC, in particular, ME/CFS is urgently needed. Methods: We identified and studied metabolites and soluble biomarkers in plasma from LC individuals mainly exhibiting ME/CFS compared to age-sex-matched recovered individuals (R) without LC, acute COVID-19 patients (A), and to SARS-CoV-2 unexposed healthy individuals (HC). Results: Through these analyses, we identified alterations in several metabolomic pathways in LC vs other groups. Plasma metabolomics analysis showed that LC differed from the R and HC groups. Of note, the R group also exhibited a different metabolomic profile than HC. Moreover, we observed a significant elevation in the plasma pro-inflammatory biomarkers (e.g. IL-1α, IL-6, TNF-α, Flt-1, and sCD14) but the reduction in ATP in LC patients. Our results demonstrate that LC patients exhibit persistent metabolomic abnormalities 12 months after the acute COVID-19 disease. Of note, such metabolomic alterations can be observed in the R group 12 months after the acute disease. Hence, the metabolomic recovery period for infected individuals with SARS-CoV-2 might be long-lasting. In particular, we found a significant reduction in sarcosine and serine concentrations in LC patients, which was inversely correlated with depression, anxiety, and cognitive dysfunction scores. Conclusion: Our study findings provide a comprehensive metabolomic knowledge base and other soluble biomarkers for a better understanding of the pathophysiology of LC and suggests sarcosine and serine supplementations might have potential therapeutic implications in LC patients. Finally, our study reveals that LC disproportionally affects females more than males, as evidenced by nearly 70% of our LC patients being female.

Immunological responses in SARS-CoV-2 and HIV co-infection versus SARS-CoV-2 mono-infection: case report of the interplay between SARS-CoV-2 and HIV.

BACKGROUND: There is an urgent need to understand the interplay between SARS-CoV-2 and HIV to inform risk-mitigation approaches for HIV-infected individuals. OBJECTIVES: We conclude that people living with HIV (PLWH) who are antiretroviral therapy (ART) naïve could be at a greater risk of morbidity or mortality once co-infected with SARS-CoV-2. METHODS: Here, we performed extensive immune phenotyping using flow cytometry. Moreover, to compare the range of values observed in the co-infected case, we have included a larger number of mono-infected cases with SARS-CoV-2. We also quantified soluble co-inhibitory/co-stimulatory molecules in the plasma of our patients. RESULTS: We noted a robust immune activation characterized by the expansion of CD8+ T cells expressing co-inhibitory/stimulatory molecules (e.g. PD-1, TIM-3, 2B4, TIGIT, CD39, and ICOS) and activation markers (CD38, CD71, and HLA-DR) in the co-infected case. We further found that neutrophilia was more pronounced at the expense of lymphopenia in the co-infected case. In particular, naïve and central memory CD8+ T cells were scarce as a result of switching to effector and effector memory in the co-infected case. CD8+ T cell effector functions such as cytokine production (e.g. TNF-α and IFN-γ) and cytolytic molecules expression (granzyme B and perforin) following anti-CD3/CD28 or the Spike peptide pool stimulation were more prominent in the co-infected case versus the mono-infected case. We also observed that SARS-CoV-2 alters T cell exhaustion commonly observed in PLWH. CONCLUSION: These findings imply that inadequate immune reconstitution and/or lack of access to ART could dysregulate immune response against SARS-CoV-2 infection, which can result in poor clinical outcomes in PLWH. Our study has implications for prioritizing PLWH in the vaccination program/access to ART in resource-constrained settings.

Analysis of SARS-CoV-2 isolates, namely the Wuhan strain, Delta variant, and Omicron variant, identifies differential immune profiles.

There is an urgent need to better understand the impact of different severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants on immune response and disease dynamics to facilitate better intervention strategies. Here, we show that SARS-CoV-2 variants differentially affect host immune responses. The magnitude and quantity of cytokines and chemokines were comparable in those infected with the Wuhan strain and the Delta variant. However, individuals infected with the Omicron variant had significantly lower levels of these mediators. We also found an elevation of plasma galectins (Gal-3, Gal-8, and Gal-9) in infected individuals, in particular, in those with the original strain. Soluble galectins exert a proinflammatory role in COVID-19 pathogenesis. This was illustrated by their correlation with the plasma levels of sCD14, sCD163, enhanced TNF-α/IL-6 secretion, and increased SARS-CoV-2 infectivity in vitro. Moreover, we observed enhanced CD4+ and CD8+ T cell activation in Wuhan strain-infected individuals. Surprisingly, there was a more pronounced T cell activation in those infected with the Omicron in comparison to the Delta variant. In line with T cell activation status, we observed a more pronounced expansion of T cells expressing different co-inhibitory receptors in patients infected with the Wuhan strain, followed by the Omicron and Delta variants. Individuals infected with the Wuhan strain or the Omicron variant had a similar pattern of plasma soluble immune checkpoints. Our results imply that a milder innate immune response might be beneficial and protective in those infected with the Omicron variant. Our results provide a novel insight into the differential impact of SARS-CoV-2 variants on host immunity. IMPORTANCE There is a need to better understand how different SARS-CoV-2 variants influence the immune system and disease dynamics to facilitate the development of better vaccines and therapies. We compared immune responses in 140 SARS-CoV-2-infected individuals with the Wuhan strain, the Delta variant, or the Omicron variant. All these patients were admitted to the intensive care unit and were SARS-CoV-2 vaccination naïve. We found that SARS-CoV-2 variants differentially affect the host immune response. This was done by measuring soluble biomarkers in their plasma and examining different immune cells. Overall, we found that the magnitude of cytokine storm in individuals infected with the Wuhan strain or the Delta variant was greater than in those infected with the Omicron variant. In light of enhanced cytokine release syndrome in individuals infected with the Wuhan strain or the Delta variant, we believe that a milder innate immune response might be beneficial and protective in those infected with the Omicron variant.

SARS-CoV-2 Mpro Protease Variants of Concern Display Altered Viral Substrate and Cell Host Target Galectin-8 Processing but Retain Sensitivity toward Antivirals.

The main protease of SARS-CoV-2 (Mpro) is the most promising drug target against coronaviruses due to its essential role in virus replication. With newly emerging variants there is a concern that mutations in Mpro may alter the structural and functional properties of protease and subsequently the potency of existing and potential antivirals. We explored the effect of 31 mutations belonging to 5 variants of concern (VOCs) on catalytic parameters and substrate specificity, which revealed changes in substrate binding and the rate of cleavage of a viral peptide. Crystal structures of 11 Mpro mutants provided structural insight into their altered functionality. Additionally, we show Mpro mutations influence proteolysis of an immunomodulatory host protein Galectin-8 (Gal-8) and a subsequent significant decrease in cytokine secretion, providing evidence for alterations in the escape of host-antiviral mechanisms. Accordingly, mutations associated with the Gamma VOC and highly virulent Delta VOC resulted in a significant increase in Gal-8 cleavage. Importantly, IC50s of nirmatrelvir (Pfizer) and our irreversible inhibitor AVI-8053 demonstrated no changes in potency for both drugs for all mutants, suggesting Mpro will remain a high-priority antiviral drug candidate as SARS-CoV-2 evolves.

Differential effects of age, sex and dexamethasone therapy on ACE2/TMPRSS2 expression and susceptibility to SARS-CoV-2 infection.

ACE2 and TMPRSS2 are crucial for SARS-CoV-2 entry into the cell. Although ACE2 facilitates viral entry, its loss leads to promoting the devastating clinical symptoms of COVID-19 disease. Thus, enhanced ACE2/TMPRSS2 expression is likely to increase predisposition of target cells to SARS-CoV-2 infection. However, little evidence existed about the biological kinetics of these two enzymes and whether dexamethasone treatment modulates their expression. Here, we show that the expression of ACE2 at the protein and mRNA levels was significantly higher in the lung and heart tissues of neonatal compared to adult mice. However, the expression of TMPRSS2 was developmentally regulated. Our results may introduce a novel concept for the reduced susceptibility of the young to SARS-CoV-2 infection. Moreover, ACE2 expression but not TMPRSS2 was upregulated in adult female lungs compared to their male counterparts. Interestingly, the ACE2 and TMPRSS2 expressions were upregulated by dexamethasone treatment in the lung and heart tissues in both neonatal and adult mice. Furthermore, our findings provide a novel mechanism for the observed differential therapeutic effects of dexamethasone in COVID-19 patients. As such, dexamethasone exhibits different therapeutic effects depending on the disease stage. This was supported by increased ACE2/TMPRSS2 expression and subsequently enhanced infection of normal human bronchial epithelial cells (NHBE) and Vero E6 cells with SARS-CoV-2 once pre-treated with dexamethasone. Therefore, our results suggest that individuals who take dexamethasone for other clinical conditions may become more prone to SARS-CoV-2 infection.

Differential Impact of SARS-CoV-2 Isolates, Namely, the Wuhan Strain, Delta, and Omicron Variants on Erythropoiesis.

SARS-CoV-2 variants exhibit different viral transmissibility and disease severity. However, their impact on erythropoiesis has not been investigated. Here, we show SARS-CoV-2 variants differentially affect erythropoiesis. This is illustrated by the abundance of CD71+ erythroid cells (CECs) in the blood circulation of COVID-19 patients infected with the original Wuhan strain followed by the Delta and Omicron variants. We observed the CD45+CECs are the dominant subpopulation of CECs expressing the receptor, ACE2, and coreceptor, TMPRSS2, and thus, can be targeted by SARS-CoV-2. Also, we found CECs exhibit immunosuppressive properties, specifically CD45+CECs are the dominant immunosuppressive cells and via reactive oxygen species (ROS) and arginase I expression can impair CD8+ T cell functions. In agreement, we observed CECs suppress CD8+ T cell effector (e.g., Granzyme B expression and degranulation capacity [CD107]), which was partially but significantly reversed with l-arginine supplementation. In light of the enriched frequency of CECs, in particular, CD45+CECs in patients infected with the original (Wuhan) strain, we believe this strain has a more prominent impact on hematopoiesis compared with the Delta and Omicron variants. Therefore, our study provides an important insight into the differential impact of SARS-CoV-2 variants on erythropoiesis in COVID-19 patients. IMPORTANCE Silent hypoxia has been the hallmark of SARS-CoV-2 infection. Red blood cells (RBCs) work as gas cargo delivering oxygen to different tissues. However, their immature counterparts reside in the bone marrow and normally absent in the blood circulation. We show SARS-CoV-2 infection is associated with the emergence of immature RBCs so called CD71+ erythroid cells (CECs) in the blood. In particular, we found these cells were more prevalent in the blood of those infected with the SARS-CoV-2 original strain (Wuhan) followed by the Delta and Omicron variants. This suggests SARS-CoV-2 directly or indirectly impacts RBC production. In agreement, we observed immature RBCs express the receptor (ACE2) and coreceptor (TMPRSS2) for SARS-CoV-2. CECs suppress T cells functions (e.g., Granzyme B and degranulation capacity) in vitro. Therefore, our study provides a novel insight into the differential impact of SARS-CoV-2 variants on erythropoiesis and subsequently the hypoxia commonly observed in COVID-19 patients.

Elevated ATP via enhanced miRNA-30b, 30c, and 30e downregulates the expression of CD73 in CD8+ T cells of HIV-infected individuals.

CD8+ T cells play a crucial role against chronic viral infections, however, their effector functions are influenced by the expression of co-stimulatory/inhibitory receptors. For example, CD73 works with CD39 to convert highly inflammatory ATP to adenosine. However, its expression on T cells in the context of viral infections has not been well defined. Here, we analyzed the expression of CD73 on human T cells in a cohort of 102 HIV-infected individuals including those on antiretroviral therapy (ART), ART-naïve, and long-term non-progressors who were not on ART. We found that the frequency of CD73+ T cells was markedly lower among T cell subsets (e.g. naïve, effector or memory) in the peripheral blood of all HIV-infected individuals. Notably, CD73 was decreased at the cell surface, intracellular and gene levels. Functionally, CD8+CD73+ T cells exhibited decreased cytokine expression (TNF-α, IFN-γ and IL-2) upon global or antigen-specific stimulation and impaired expression of cytolytic molecules at the gene and protein levels. In contrast, CD8+CD73+ T cells expressed elevated levels of homing receptors such as CCR7, α4ß7 integrin, which suggests a migratory advantage for these cells as observed in vitro. We also observed significant migration of CD73+CD8+ T cells into the cerebrospinal fluids of multiple sclerosis (MS) patients at the time of disease relapse. Moreover, we found that elevated levels of ATP in the plasma of HIV-infected individuals upregulates the expression of miRNA30b-e in T cells in vitro. In turn, inhibition of miRNAs (30b, 30c and 30e) resulted in significant upregulation of CD73 mRNA in CD8+ T cells. Therefore, we provide a novel mechanism for the downregulation of CD73 via ATP-induced upregulation of miRNA30b, 30c and 30e in HIV infection. Finally, these observations imply that ATP-mediated downregulation of CD73 mainly occurs via its receptor, P2X1/P2RX1. Our results may in part explain why HIV-infected individuals have reduced risk of developing MS considering the role of CD73 for efficient T cell entry into the central nervous system.

Sex Matters: Physiological Abundance of Immuno-Regulatory CD71+ Erythroid Cells Impair Immunity in Females.

Mature erythrocytes are the major metabolic regulators by transporting oxygen throughout the body. However, their precursors and progenitors defined as CD71+ Erythroid Cells (CECs) exhibit a wide range of immunomodulatory properties. Here, we uncover pronounced sexual dimorphism in CECs. We found female but not male mice, both BALB/c and C57BL/6, and human females were enriched with CECs. CECs, mainly their progenitors defined as CD45+CECs expressed higher levels of reactive oxygen species (ROS), PDL-1, VISTA, Arginase II and Arginase I compared to their CD45- counterparts. Consequently, CECs by the depletion of L-arginine suppress T cell activation and proliferation. Expansion of CECs in anemic mice and also post-menstrual cycle in women can result in L-arginine depletion in different microenvironments in vivo (e.g. spleen) resulting in T cell suppression. As proof of concept, we found that anemic female mice and mice adoptively transferred with CECs from anemic mice became more susceptible to Bordetella pertussis infection. These observations highlight the role of sex and anemia-mediated immune suppression in females. Notably, enriched CD45+CECs may explain their higher immunosuppressive properties in female BALB/c mice. Finally, we observed significantly more splenic central macrophages in female mice, which can explain greater extramedullary erythropoiesis and subsequently abundance of CECs in the periphery. Thus, sex-specific differences frequency in the frequency of CECs might be imprinted by differential erythropoiesis niches and hormone-dependent manner.

Neutrophils promote T-cell activation through the regulated release of CD44-bound Galectin-9 from the cell surface during HIV infection.

The interaction of neutrophils with T cells has been the subject of debate and controversies. Previous studies have suggested that neutrophils may suppress or activate T cells. Despite these studies, the interaction between neutrophils and T cells has remained a largely unexplored field. Here, based on our RNA sequencing (RNA-seq) analysis, we found that neutrophils have differential transcriptional and functional profiling depending on the CD4 T-cell count of the HIV-infected individual. In particular, we identified that neutrophils in healthy individuals express surface Galectin-9 (Gal-9), which is down-regulated upon activation, and is consistently down-regulated in HIV-infected individuals. However, down-regulation of Gal-9 was associated with CD4 T-cell count of patients. Unstimulated neutrophils express high levels of surface Gal-9 that is bound to CD44, and, upon stimulation, neutrophils depalmitoylate CD44 and induce its movement out of the lipid raft. This process causes the release of Gal-9 from the surface of neutrophils. In addition, we found that neutrophil-derived exogenous Gal-9 binds to cell surface CD44 on T cells, which promotes LCK activation and subsequently enhances T-cell activation. Furthermore, this process was regulated by glycolysis and can be inhibited by interleukin (IL)-10. Together, our data reveal a novel mechanism of Gal-9 shedding from the surface of neutrophils. This could explain elevated plasma Gal-9 levels in HIV-infected individuals as an underlying mechanism of the well-characterized chronic immune activation in HIV infection. This study provides a novel role for the Gal-9 shedding from neutrophils. We anticipate that our results will spark renewed investigation into the role of neutrophils in T-cell activation in other acute and chronic conditions, as well as improved strategies for modulating Gal-9 shedding.

Plasma Extracellular Vesicles Enhance HIV-1 Infection of Activated CD4+ T Cells and Promote the Activation of Latently Infected J-Lat10.6 Cells via miR-139-5p Transfer.

HIV latency is a challenge to the success of antiretroviral therapy (ART). Hence patients may benefit from interventions that efficiently reactivate the latent virus to be eliminated by ARTs. Here we show that plasma extracellular vesicles (pEVs) can enhance HIV infection of activated CD4+ T cells and reactivate the virus in latently infected J-Lat 10.6 cells. Evaluation of the extravesicular miRNA cargo by a PCR array revealed that pEVs from HIV patients express miR-139-5p. Furthermore, we found that increased levels of miR-139-5p in J-Lat 10.6 cells incubated with pEVs corresponded with reduced expression of the transcription factor, FOXO1. pEV treatment also corresponded with increased miR-139-5p expression in stimulated PD1+ Jurkat cells, but with concomitant upregulation of FOXO1, Fos, Jun, PD-1 and PD-L1. However, J-Lat 10.6 cells incubated with miR-139-5p inhibitor-transfected pEVs from HIV ART-naïve and on-ART patients expressed reduced levels of miR-139-5p than cells treated with pEVs from healthy controls (HC). Collectively, our results indicate that pEV miR-139-5p belongs to a network of miRNAs that can promote cell activation, including latent HIV-infected cells by regulating the expression of FOXO1 and the PD1/PD-L1 promoters, Fos and Jun.

The Quality of SARS-CoV-2-Specific T Cell Functions Differs in Patients with Mild/Moderate versus Severe Disease, and T Cells Expressing Coinhibitory Receptors Are Highly Activated.

Understanding the function of SARS-CoV-2 Ag-specific T cells is crucial for the monitoring of antiviral immunity and vaccine design. Currently, both impaired and robust T cell immunity is described in COVID-19 patients. In this study, we explored and compared the effector functions of SARS-CoV-2-reactive T cells expressing coinhibitory receptors and examine the immunogenicity of SARS-CoV-2 S, M, and N peptide pools in regard to specific effector T cell responses, Th1/Th2/Th17, in COVID-19 patients. Analyzing a cohort of 108 COVID-19 patients with mild, moderate, and severe disease, we observed that coinhibitory receptors (e.g., PD-1, CTLA-4, TIM-3, VISTA, CD39, CD160, 2B4, TIGIT, Gal-9, and NKG2A) were upregulated on both CD4+ and CD8+ T cells. Importantly, the expression of coinhibitory receptors on T cells recognizing SARS-CoV-2 peptide pools (M/N/S) was associated with increased frequencies of cytokine-producing T cells. Thus, our data refute the concept of pathological T cell exhaustion in COVID-19 patients. Despite interindividual variations in the T cell response to viral peptide pools, a Th2 phenotype was associated with asymptomatic and milder disease, whereas a robust Th17 was associated with severe disease, which may potentiate the hyperinflammatory response in patients admitted to the Intensive Care Unit. Our data demonstrate that T cells may either play a protective or detrimental role in COVID-19 patients. This finding could have important implications for immune correlates of protection, diagnostic, and prophylaxis with respect to COVID-19 management.

Differential transcriptional and functional properties of regulatory T cells in HIV-infected individuals on antiretroviral therapy and long-term non-progressors.

OBJECTIVES: Regulatory T cells (Tregs) are widely recognised as a subset of CD4+CD25+FOXP3+ T cells that have a key role in maintaining immune homeostasis. The impact of HIV-1 infection on immunological properties and effector functions of Tregs has remained the topic of debate and controversy. In the present study, we investigated transcriptional profile and functional properties of Tregs in HIV-1-infected individuals either receiving antiretroviral therapy (ART, n = 50) or long-term non-progressors (LTNPs, n = 24) compared to healthy controls (HCs, n = 38). METHODS: RNA sequencing (RNAseq), flow cytometry-based immunophenotyping and functional assays were performed to study Tregs in different HIV cohorts. RESULTS: Our RNAseq analysis revealed that Tregs exhibit different transcriptional profiles in HIV-infected individuals. While Tregs from patients on ART upregulate pathways associated with a more suppressive (activated) phenotype, Tregs in LTNPs exhibit upregulation of pathways associated with impaired suppressive properties. These observations may explain a higher propensity for autoimmune diseases in LTNPs. Also, we found substantial upregulation of HLA-F mRNA and HLA-F protein in Tregs from HIV-infected subjects compared to healthy individuals. These observations highlight a potential role for this non-classical HLA in Tregs in the context of HIV infection, which should be investigated further in other chronic viral infections and cancer. CONCLUSION: Our study has provided a novel insight into Tregs at the transcriptional and functional levels in different HIV-infected groups.

Galectin-9, a Player in Cytokine Release Syndrome and a Surrogate Diagnostic Biomarker in SARS-CoV-2 Infection.

The outbreak of SARS-CoV-2 infection has enormously impacted our lives. Clinical evidence has implicated the emergence of cytokine release syndrome as the prominent cause of mortality in COVID-19 patients. In this study, we observed massive elevation of plasma Galectin-9 (Gal-9) in COVID-19 patients compared to healthy controls (HCs). By using the receiver operating characteristic (ROC) curve, we found that a baseline of 2,042 pg/ml plasma Gal-9 can differentiate SARS-CoV-2-infected from noninfected individuals with high specificity/sensitivity (95%). Analysis of 30 cytokines and chemokines detected a positive correlation of the plasma Gal-9 with C-reactive protein (CRP) and proinflammatory cytokines/chemokines such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IP-10, MIP-1α, and MCP-1 but an inverse correlation with transforming growth factor ß (TGF-ß) in COVID-19 patients. In agreement, we found enhanced production of IL-6 and TNF-α by monocytes and NK cells of COVID-19 patients once treated with the recombinant human Gal-9 in vitro Also, we observed that although the cell-membrane expression of Gal-9 on monocytes does not change in COVID-19 patients, those with higher Gal-9 expression exhibit an activated phenotype. Furthermore, we noted significant downregulation of surface Gal-9 in neutrophils from COVID-19 patients compared to HCs. Our further investigations indicated that immune activation following SARS-CoV-2 infection results in Gal-9 shedding from neutrophils. The strong correlation of Gal-9 with proinflammatory mediators suggests that inhibition of Gal-9 may severe as a therapeutic approach in COVID-19 infection. Besides, the plasma Gal-9 measurement may be used as a surrogate diagnostic biomarker in COVID-19 patients.IMPORTANCE The outbreak of SARS-CoV-2 infection has enormously impacted our lives. Clinical evidence has implicated the emergence of cytokine release syndrome as the prominent cause of mortality in COVID-19 patients. We observed substantial elevation of the plasma Galectin-9 (Gal-9) in COVID-19 patients compared to healthy controls. Gal-9 is an abundant protein in many immune and nonimmune cells. We found that Gal-9 detection assay can differentiate SARS-CoV-2-infected from noninfected individuals with a specificity/sensitivity of 95%. Importantly, we found a positive correlation of the plasma Gal-9 with a wide range of proinflammatory biomarkers in COVID-19 patients. In agreement, we found enhanced expression and production of such proinflammatory molecules by immune cells of COVID-19 patients once treated with Gal-9 in vitro Our results propose Gal-9 as an important contributing factor in cytokine release syndrome; therefore, Gal-9 inhibition may serve as a beneficial therapeutic approach by suppressing the hyperimmune activation in COVID-19 patients.

Erythroid precursors and progenitors suppress adaptive immunity and get invaded by SARS-CoV-2.

SARS-CoV-2 infection is associated with lower blood oxygen levels, even in patients without hypoxia requiring hospitalization. This discordance illustrates the need for a more unifying explanation as to whether SARS-CoV-2 directly or indirectly affects erythropoiesis. Here, we show significantly enriched CD71+ erythroid precursors/progenitors in the blood circulation of COVID-19 patients. We found that these cells have distinctive immunosuppressive properties. In agreement, we observed a strong negative correlation between the frequency of these cells with T and B cell proportions in COVID-19 patients. The expansion of these CD71+ erythroid precursors/progenitors was negatively correlated with the hemoglobin levels. A subpopulation of abundant erythroid cells, CD45+ CD71+ cells, co-express ACE2, TMPRSS2, CD147, and CD26, and these can be infected with SARS-CoV-2. In turn, pre-treatment of erythroid cells with dexamethasone significantly diminished ACE2/TMPRSS2 expression and subsequently reduced their infectivity with SARS-CoV-2. This provides a novel insight into the impact of SARS-CoV-2 on erythropoiesis and hypoxia seen in COVID-19 patients.

Selective Upregulation of CTLA-4 on CD8+ T Cells Restricted by HLA-B*35Px Renders them to an Exhausted Phenotype in HIV-1 infection.

HLA-B*35Px is associated with HIV-1 disease rapid progression to AIDS. However, the mechanism(s) underlying this deleterious effect of this HLA allele on HIV-1 infection outcome has not fully understood. CD8+ T cells play a crucial role to control the viral replication but impaired CD8+ T cells represent a major hallmark of HIV-1 infection. Here, we examined the effector functions of CD8+ T cells restricted by HLA-B*35Px (HLA-B*35:03 and HLA-B*35:02), HLA-B*27/B57 and non-HLA-B*27/B57 (e.g. HLA-A*01, A*02, A*03, A*11, A*24, A*26, B*40, B*08, B*38, B*44). CD8+ T cells restricted by HLA-B*35Px exhibited an impaired phenotype compared with those restricted by HLA-B*27/B57 and even non-HLA-B*27/B57. CD8+ T cells restricted by non-HLA-B*27/B57 when encountered their cognate epitopes upregulated TIM-3 and thus became suppressed by regulatory T cells (Tregs) via TIM-3: Galectin-9 (Gal-9). Strikingly, CD8+ T cells restricted by HLA-B*35Px expressed fewer TIM-3 and therefore did not get suppressed by Tregs, which was similar to CD8+ T cells restricted by HLA-B*27/B57. Instead, CD8+ T cells restricted by HLA-B*35Px upon recognition of their cognate epitopes upregulated CTLA-4. The transcriptional and impaired phenotype (e.g. poor effector functions) of HIV-specific CD8+ T cells restricted by HLA-B*35 was related to persistent CTLA-4, elevated Eomes and blimp-1 but poor T-bet expression. As such, anti-CTLA-4 antibody, Ipilimumab, reversed the impaired proliferative capacity of antigen-specific CD8+ T cells restricted by HLA-B*35Px but not others. This study supports the concept that CD8+ T resistance to Tregs-mediated suppression is related to allele restriction rather than the epitope specificity. Our results aid to explain a novel mechanism for the inability of HIV-specific CD8+ T cells restricted by HLA-B*35Px to control viral replication.

Galectin-9 and VISTA Expression Define Terminally Exhausted T Cells in HIV-1 Infection.

We report significant upregulation of Galectin-9 (Gal-9) and VISTA on both CD4+ and CD8+ T cells in HIV-infected human patients. Gal-9 and VISTA expression was associated with impaired T cells effector functions. Although Gal-9 was coexpressed with other coinhibitory receptors such as TIGIT, CD160, CD39, and VISTA, it was simultaneously coexpressed with PD-1. Coexpression of Gal-9 with PD-1 was associated with a more terminally exhausted T cell phenotype in HIV-1 patients. This was marked by higher expression of EOMES, blimp1, and Glut1 in Gal-9+ versus Gal-9- T cells, which is consistent with an exhausted T cell phenotype. Gal-9+ T cells exhibited the phenotype characteristics of effector T cells (CD45RA+, CD45RO-/lo, CD62L-, CD27lo) with higher T-bet expression. A positive correlation between the plasma viral load with the plasma Gal-9 levels in treatment-naive HIV patients and an inverse correlation between CD4 count with the frequency of CD4+Gal-9+ T cells were observed. Increased percentages of Gal-9+ T cells was evident in HIV-treated patients. Enhanced expression of Gal-9 on T cells following PMA stimulation via protein kinase C suggests persistent TCR stimulation as a potential contributing factor in Gal-9 upregulation in HIV patients. This was supported by the constant degranulation of Gal-9+ T cells. Moreover, CD44 clustering by Gal-9 may influence cytoskeleton rearrangement and coclustering of CD3, which likely impact initiation of signal transduction via TCR. Our preliminary data also confirm upregulation of Gal-9 on T cells in hepatitis B virus and HPV infections. These results demonstrate a novel role for Gal-9 and VISTA in HIV pathogenesis.

CD71+ Erythroid Cells Exacerbate HIV-1 Susceptibility, Mediate trans-Infection, and Harbor Infective Viral Particles.

CD71+ erythroid cells (CECs) have a wide range of immunomodulatory properties. Here, we show that CECs are expanded in the peripheral blood of HIV patients, with a positive correlation between their frequency and the plasma viral load. CECs from HIV patients and human cord blood/placenta exacerbate HIV-1 infection/replication when cocultured with CD4+ T cells, and that preexposure of CD4+ T cells to CECs enhances their permissibility to HIV infection. However, mature red blood cells (RBCs) do not enhance HIV replication when cocultured with CD4+ T cells. We also found CECs express substantial levels of the NOX2 gene and via a mitochondrial reactive oxygen species (ROS)-dependent mechanism possibly upregulate NF-κB in CD4+ T cells once cocultured, which affects the cell cycle machinery to facilitate HIV-1 replication. The complement receptor-1 (CD35) and the Duffy antigen receptor for chemokines (DARC) as potential HIV target molecules are expressed significantly higher on CECs compared to mature red blood cells. Blocking CD35 or DARC substantially abolishes HIV-1 transmission by RBCs to uninfected CD4+ T cells but not by CECs. In contrast, we observed CECs bind to HIV-1 via CD235a and subsequently transfer the virus to uninfected CD4+ T cells, which can be partially blocked by the anti-CD235a antibody. More importantly, we found that CECs from HIV-infected individuals in the presence of antiretroviral therapy harbor infective viral particles, which mediate HIV-1 trans-infection of CD4+ T cells. Therefore, our findings provide a novel insight into the role of CECs in HIV pathogenesis as potential contributing cells in viral persistence and transmission.IMPORTANCE Immature red blood cells (erythroid precursors or CD71+ erythroid cells) have a wide range of immunomodulatory properties. In this study, we found that these erythroid precursors are abundant in the human cord blood/placental tissues, in the blood of HIV-infected and anemic individuals. We observed that these cells exacerbate HIV-1 replication/infection in target cells and even make HIV target cells more permissible to HIV infection. In addition, we found that HIV gets a free ride by binding on the surface of these cells and thus can travel to different parts of the body. In agreement, we noticed a positive correlation between the plasma viral load and the frequency of these cells in HIV patients. More importantly, we observed that infective HIV particles reside inside these erythroid precursors but not mature red blood cells. Therefore, these cells by harboring HIV can play an important role in HIV pathogenesis.

Galectin-9 Expression Defines a Subpopulation of NK Cells with Impaired Cytotoxic Effector Molecules but Enhanced IFN-γ Production, Dichotomous to TIGIT, in HIV-1 Infection.

NK cell functions are tightly regulated by the balance between the inhibitory and stimulatory surface receptors. We investigated the surface expression of galectin-9 (Gal-9) and its function in NK cells from HIV-infected individuals on antiretroviral therapy, long-term nonprogressors, and progressors compared with healthy controls. We also measured the expression of TIGIT and TIM-3 on different NK cell subpopulations and compared their functionality to Gal-9 + NK cells. Our data demonstrated significant upregulation of Gal-9 on NK cells in HIV-infected groups versus healthy controls. Gal-9 expression was associated with impaired expression of cytotoxic effector molecules granzyme B, perforin, and granulysin. In contrast, Gal-9 expression significantly enhanced IFN-γ expression in NK cells of HIV-1-infected individuals. We also found an expansion of TIGIT + NK cells in HIV-infected individuals; however, dichotomous to Gal-9 + NK cells, TIGIT + NK cells expressed significantly higher amounts of cytotoxic molecules but lower IFN-γ. Moreover, lower expression of cytotoxic effector molecules in Gal-9+ NK cells was associated with higher CD107a expression, which suggests indiscriminate degranulation. Importantly, a positive correlation between the plasma viral load and Gal-9+ NK cells was observed in progressors. Finally, we found that a cytokine mixture (IL-12, IL-15, and IL-18) can improve effector functions of Gal-9+ NK cells in HIV-infected individuals, although, such an effect was observed for Gal-9- NK cells, as well. Overall, our data highlight the important role of Gal-9 in dysfunctional NK cells and, more importantly, a dichotomy for the role of Gal-9 versus TIGIT and suggest a potential new avenue for the development of therapeutic approaches.

18F-Sodium Fluoride Imaging of Coronary Atherosclerosis in Ambulatory Patients With Diabetes Mellitus.

Objective- Although patients with diabetes mellitus (DM) are considered at high risk of cardiovascular events, there is growing evidence that this notion is incorrect. Atherosclerosis imaging may identify patients at risk. Approach and Results- We performed coronary atherosclerosis with 18F-sodium fluoride (NaF) positron emission tomography/computed tomography and gated chest computed tomography for coronary artery calcium in 88 consecutive ambulatory patients with DM on a stable medical regimen. NaF has been shown to localize avidly in culprit lesions of patients with acute coronary syndromes and may identify unstable plaques. NaF activity was measured as target (coronary arteries)-to-background (left ventricular pool) ratio (TBR). High TBR was defined as ≥1.5. The mean age of the cohort was 54±14 years, 55% had type 2 DM, 65% were men, the median HgbA1c (hemoglobin A1c) and LDL (low-density lipoprotein) cholesterol were 7.5% (interquartile range, 7.1-8.5) and 1.9 mmol/L (interquartile range, 1.5-2.6), respectively. Mean coronary artery calcium score was 374±773, and median TBR was 1.2. Coronary artery TBR ≥1.5 was detected in 13 (15%) patients. In univariable analyses, male sex ( P=0.0002), estimated glomerular filtration rate ( P=0.02), and total coronary artery calcium score ( P=0.04) were associated with TBR. In multivariable analyses, TBR >median was associated with male sex ( P=0.0001) and statin use ( P=0.042). Conclusions- In ambulatory patients with DM asymptomatic for cardiovascular disease, the prevalence of potentially vulnerable plaques detected with NaF was low, but in the absence of follow-up data at this stage, we cannot assess the import of this information. Future research will establish whether NaF imaging helps risk stratify patients with DM. Clinical Trial Registration- URL: http://www.clinicaltrials.gov . Unique identifier: NCT03530176.

CD71+VISTA+ erythroid cells promote the development and function of regulatory T cells through TGF-ß.

Cell-surface transferrin receptor (CD71+) erythroid cells are abundant in newborns with immunomodulatory properties. Here, we show that neonatal CD71+ erythroid cells express significant levels of V-domain Immunoglobulin (Ig) Suppressor of T Cell Activation (VISTA) and, via constitutive production of transforming growth factor (TGF)- ß, play a pivotal role in promotion of naïve CD4+ T cells into regulatory T cells (Tregs). Interestingly, we discovered that CD71+VISTA+ erythroid cells produce significantly higher levels of TGF-ß compared to CD71+VISTA- erythroid cells and CD71+ erythroid cells from the VISTA knock-out (KO) mice. As a result, CD71+VISTA+ erythroid cells-compared to CD71+VISTA- and CD71+ erythroid cells from the VISTA KO mice-significantly exceed promotion of naïve CD4+ T cells into induced Tregs (iTreg) via TGF-ß in vitro. However, depletion of CD71+ erythroid cells had no significant effects on the frequency of Tregs in vivo. Surprisingly, we observed that the remaining and/or newly generated CD71+ erythroid cells following anti-CD71 antibody administration exhibit a different gene expression profile, evidenced by the up-regulation of VISTA, TGF-ß1, TGF-ß2, and program death ligand-1 (PDL-1), which may account as a compensatory mechanism for the maintenance of Treg population. We also observed that iTreg development by CD71+ erythroid cells is mediated through the inhibition of key signaling molecules phosphorylated protein kinase B (phospho-Akt) and phosphorylated mechanistic target of rapamycin (phospho-mTOR). Finally, we found that elimination of Tregs using forkhead box P3 (FOXP3)-diptheria toxin receptor (DTR) mice resulted in a significant expansion in the frequency of CD71+ erythroid cells in vivo. Collectively, these studies provide a novel, to our knowledge, insight into the cross-talk between CD71+ erythroid cells and Tregs in newborns. Our results highlight the biological role of CD71+ erythroid cells in the neonatal period and possibly beyond.