Diverse immunological dysregulation, chronic inflammation, and impaired erythropoiesis in long COVID patients with chronic fatigue syndrome.

A substantial number of patients recovering from acute SARS-CoV-2 infection present serious lingering symptoms, often referred to as long COVID (LC). However, a subset of these patients exhibits the most debilitating symptoms characterized by ongoing myalgic encephalomyelitis or chronic fatigue syndrome (ME/CFS). We specifically identified and studied ME/CFS patients from two independent LC cohorts, at least 12 months post the onset of acute disease, and compared them to the recovered group (R). ME/CFS patients had relatively increased neutrophils and monocytes but reduced lymphocytes. Selective T cell exhaustion with reduced naïve but increased terminal effector T cells was observed in these patients. LC was associated with elevated levels of plasma pro-inflammatory cytokines, chemokines, Galectin-9 (Gal-9), and artemin (ARTN). A defined threshold of Gal-9 and ARTN concentrations had a strong association with LC. The expansion of immunosuppressive CD71+ erythroid cells (CECs) was noted. These cells may modulate the immune response and contribute to increased ARTN concentration, which correlated with pain and cognitive impairment. Serology revealed an elevation in a variety of autoantibodies in LC. Intriguingly, we found that the frequency of 2B4+CD160+ and TIM3+CD160+ CD8+ T cells completely separated LC patients from the R group. Our further analyses using a multiple regression model revealed that the elevated frequency/levels of CD4 terminal effector, ARTN, CEC, Gal-9, CD8 terminal effector, and MCP1 but lower frequency/levels of TGF-ß and MAIT cells can distinguish LC from the R group. Our findings provide a new paradigm in the pathogenesis of ME/CFS to identify strategies for its prevention and treatment.

The Role of Coinhibitory Receptors in B Cell Dysregulation in SARS-CoV-2-Infected Individuals with Severe Disease.

Severe SARS-CoV-2 infection is associated with significant immune dysregulation involving different immune cell subsets. In this study, when analyzing critically ill COVID-19 patients versus those with mild disease, we observed a significant reduction in total and memory B cell subsets but an increase in naive B cells. Moreover, B cells from COVID-19 patients displayed impaired effector functions, evidenced by diminished proliferative capacity, reduced cytokine, and Ab production. This functional impairment was accompanied by an increased apoptotic potential upon stimulation in B cells from severely ill COVID-19 patients. Our further studies revealed the expansion of B cells expressing coinhibitory molecules (PD-1, PD-L1, TIM-1, VISTA, CTLA-4, and Gal-9) in intensive care unit (ICU)-admitted patients but not in those with mild disease. The coinhibitory receptor expression was linked to altered IgA and IgG expression and increased the apoptotic capacity of B cells. Also, we found a reduced frequency of CD24hiCD38hi regulatory B cells with impaired IL-10 production. Our mechanistic studies revealed that the upregulation of PD-L1 was linked to elevated plasma IL-6 levels in COVID-19 patients. This implies a connection between the cytokine storm and altered B cell phenotype and function. Finally, our metabolomic analysis showed a significant reduction in tryptophan but elevation of kynurenine in ICU-admitted COVID-19 patients. We found that kynurenine promotes PD-L1 expression in B cells, correlating with increased IL-6R expression and STAT1/STAT3 activation. Our observations provide novel insights into the complex interplay of B cell dysregulation, implicating coinhibitory receptors, IL-6, and kynurenine in impaired B cell effector functions, potentially contributing to the pathogenesis of COVID-19.

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.

Transcriptomic profiling of peripheral blood cells in HPV-associated carcinoma patients receiving combined valproic acid and avelumab.

Human papillomavirus (HPV)-associated cancer continues to evade the immune system by promoting a suppressive tumor microenvironment. Therefore, immunotherapy appears to be a promising approach for targeting HPV-associated tumors. We hypothesized that valproic acid (VA) as an epigenetic agent combined with avelumab may enhance the antitumor immunity in HPV-associated solid tumors. We performed bulk RNA-sequencing (RNA-Seq) on total peripheral blood mononuclear cells (PBMCs) of seven nonresponders (NRs) and four responders (Rs). A total of 39 samples (e.g., pretreatment, post-VA, postavelumab, and endpoint) were analyzed. Also, we quantified plasma analytes and performed flow cytometry. We observed a differential pattern in immune response following treatment with VA and/or avelumab in NRs vs. Rs. A significant upregulation of transcripts associated with NETosis [the formation of neutrophil extracellular traps (NETs)] and neutrophil degranulation pathways was linked to the presence of a myeloid-derived suppressor cell signature in NRs. We noted the elevation of IL-8/IL-18 cytokines and a distinct transcriptome signature at the baseline and endpoint in NRs. By using the receiver operator characteristics, we identified a cutoff value for the plasma IL-8/IL-18 to discriminate NRs from Rs. We found differential therapeutic effects for VA and avelumab in NRs vs. Rs. Thus, our results imply that measuring the plasma IL-8/IL-18 and bulk RNA-Seq of PBMCs may serve as valuable biomarkers to predict immunotherapy outcomes.

CD71+ erythroid cells suppress T-cell effector functions and predict immunotherapy outcomes in patients with virus-associated solid tumors.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer. However, only a portion of patients respond to such treatments. Therefore, it remains a prevailing clinical need to identify factors associated with acquired resistance or lack of response to ICIs. We hypothesized that the immunosuppressive CD71+ erythroid cells (CECs) within the tumor and/or distant 'out-of-field' may impair antitumor response. METHODS: We studied 38 patients with cancer through a phase II clinical trial investigating the effects of oral valproate combined with avelumab (anti-programmed death-ligand 1 (PD-L1)) in virus-associated solid tumors (VASTs). We quantified the frequency/functionality of CECs in blood and biopsies of patients. Also, we established an animal model of melanoma (B16-F10) to investigate the possible effects of erythropoietin (EPO) treatment on anti-PD-L1 therapy. RESULTS: We found a substantial expansion of CECs in the blood of patients with VAST compared with healthy controls. We noted that the frequency of CECs in circulation was significantly higher at the baseline and throughout the study in non-responders versus responders to PD-L1 therapy. Moreover, we observed that CECs in a dose-dependent manner suppress effector functions of autologous T cells in vitro. The subpopulation of CD45+CECs appears to have a more robust immunosuppressive property compared with their CD45- counterparts. This was illustrated by a stronger expression of reactive oxygen species, PD-L1/PD-L2, and V-domain Ig suppressor of T-cell activation in this subpopulation. Lastly, we found a higher frequency of CECs in the blood circulation at the later cancer stage and their abundance was associated with anemia, and a poor response to immunotherapy. Finally, we report the expansion of CECs in the spleen and tumor microenvironment of mice with melanoma. We found that although CECs in tumor-bearing mice secret artemin, this was not the case for VAST-derived CECs in humans. Notably, our results imply that EPO, a frequently used drug for anemia treatment in patients with cancer, may promote the generation of CECs and subsequently abrogates the therapeutic effects of ICIs (eg, anti-PD-L1). CONCLUSIONS: Our results demonstrate that anemia by the expansion of CECs may enhance cancer progression. Notably, measuring the frequency of CECs may serve as a valuable biomarker to predict immunotherapy outcomes.

Depletion of polyfunctional CD26highCD8+ T cells repertoire in chronic lymphocytic leukemia.

BACKGROUND: CD8+ T cells play an essential role against tumors but the role of human CD8+CD26+ T cell subset against tumors, in particular, haematological cancers such as chronic lymphocytic leukemia (CLL) remains unknown. Although CD4+CD26high T cells are considered for adoptive cancer immunotherapy, the role of CD8+CD26+ T cells is ill-defined. Therefore, further studies are required to better determine the role of CD8+CD26+ T cells in solid tumors and haematological cancers. METHODS: We studied 55 CLL and 44 age-sex-matched healthy controls (HCs). The expression of CD26 on different T cell subsets (e.g. naïve, memory, effector, and etc.) was analyzed. Also, functional properties of CD8+CD26+ and CD8+CD26- T cells were evaluated. Finally, the plasma cytokine/chemokine and Galectin-9 (Gal-9) levels were examined. RESULTS: CD26 expression identifies three CD8+ T cell subsets with distinct immunological properties. While CD26negCD8+ T cells are mainly transitional, effector memory and effectors, CD26lowCD8+ T cells are mainly naïve, stem cell, and central memory but CD26high T cells are differentiated to transitional and effector memory. CD26+CD8+ T cells are significantly reduced in CLL patients versus HCs. CD26high cells are enriched with Mucosal Associated Invariant T (MAIT) cells co-expressing CD161TVα7.2 and IL-18Rα. Also, CD26high cells have a rich chemokine receptor profile (e.g. CCR5 and CCR6), profound cytokine (TNF-α, IFN-γ, and IL-2), and cytolytic molecules (Granzyme B, K, and perforin) expression upon stimulation. CD26high and CD26low T cells exhibit significantly lower frequencies of CD160, 2B4, TIGIT, ICOS, CD39, and PD-1 but higher levels of CD27, CD28, and CD73 versus CD26neg cells. To understand the mechanism linked to CD26high depletion, we found that malignant B cells by shedding Galectin-9 (Gal-9) contribute to the elevation of plasma Gal-9 in CLL patients. In turn, Gal-9 and the inflammatory milieu (IL-18, IL-12, and IL-15) in CLL patients contribute to increased apoptosis of CD26high T cells. CONCLUSIONS: Our results demonstrate that CD26+ T cells possess a natural polyfunctionality to traffic and exhibit effector functions and resist exhaustion. Therefore, they can be proposed for adoptive cancer immunotherapy. Finally, neutralizing and/or inhibiting Gal-9 may preserve CD26highCD8+ T cells in CLL.

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.

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.

Expanded antigen-experienced CD160+CD8+effector T cells exhibit impaired effector functions in chronic lymphocytic leukemia.

BACKGROUND: T cell exhaustion compromises antitumor immunity, and a sustained elevation of co-inhibitory receptors is a hallmark of T cell exhaustion in solid tumors. Similarly, upregulation of co-inhibitory receptors has been reported in T cells in hematological cancers such as chronic lymphocytic leukemia (CLL). However, the role of CD160, a glycosylphosphatidylinositol-anchored protein, as one of these co-inhibitory receptors has been contradictory in T cell function. Therefore, we decided to elucidate how CD160 expression and/or co-expression with other co-inhibitory receptors influence T cell effector functions in patients with CLL. METHODS: We studied 56 patients with CLL and 25 age-matched and sex-matched healthy controls in this study. The expression of different co-inhibitory receptors was analyzed in T cells obtained from the peripheral blood or the bone marrow. Also, we quantified the properties of extracellular vesicles (EVs) in the plasma of patients with CLL versus healthy controls. Finally, we measured 29 different cytokines, chemokines or other biomarkers in the plasma specimens of patients with CLL and healthy controls. RESULTS: We found that CD160 was the most upregulated co-inhibitory receptor in patients with CLL. Its expression was associated with an exhausted T cell phenotype. CD160+CD8+ T cells were highly antigen-experienced/effector T cells, while CD160+CD4+ T cells were more heterogeneous. In particular, we identified EVs as a source of CD160 in the plasma of patients with CLL that can be taken up by T cells. Moreover, we observed a dominantly proinflammatory cytokine profile in the plasma of patients with CLL. In particular, interleukin-16 (IL-16) was highly elevated and correlated with the advanced clinical stage (Rai). Furthermore, we observed that the incubation of T cells with IL-16 results in the upregulation of CD160. CONCLUSIONS: Our study provides a novel insight into the influence of CD160 expression/co-expression with other co-inhibitory receptors in T cell effector functions in patients with CLL. Besides, IL-16-mediated upregulation of CD160 expression in T cells highlights the importance of IL-16/CD160 as potential immunotherapy targets in patients with CLL. Therefore, our findings propose a significant role for CD160 in T cell exhaustion in patients with CLL.

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.

CD71+ Erythroid Suppressor Cells Promote Fetomaternal Tolerance through Arginase-2 and PDL-1.

Survival of the allogeneic pregnancy depends on the maintenance of immune tolerance to paternal alloantigens at the fetomaternal interface. Multiple localized mechanisms contribute to the fetal evasion from the mother's immune rejection as the fetus is exposed to a wide range of stimulatory substances such as maternal alloantigens, microbes and amniotic fluids. In this article, we demonstrate that CD71+ erythroid cells are expanded at the fetomaternal interface and in the periphery during pregnancy in both humans and mice. These cells exhibit immunosuppressive properties, and their abundance is associated with a Th2 skewed immune response, as their depletion results in a proinflammatory immune response at the fetomaternal interface. In addition to their function in suppressing proinflammatory responses in vitro, maternal CD71+ erythroid cells inhibit an aggressive allogeneic response directed against the fetus such as reduction in TNF-α and IFN-γ production through arginase-2 activity and PD-1/programmed death ligand-1 (PDL-1) interactions. Their depletion leads to the failure of gestation due to the immunological rejection of the fetus. Similarly, fetal liver CD71+ erythroid cells exhibit immunosuppressive activity. Therefore, immunosuppression mediated by CD71+ erythroid cells on both sides (mother/fetus) is crucial for fetomaternal tolerance. Thus, our results reveal a previously unappreciated role for CD71+ erythroid cells in pregnancy and indicate that these cells mediate homeostatic immunosuppressive/immunoregulatory responses during pregnancy.

Erythroid Suppressor Cells Compromise Neonatal Immune Response against Bordetella pertussis.

Newborns are highly susceptible to infection. The underlying mechanism of neonatal infection susceptibility has generally been associated with neonatal immune cell immaturity. In this study, we challenged this notion and built upon our recent discovery that neonates are physiologically enriched with erythroid TER119+CD71+ cells (Elahi et al. 2013. Nature 504: 158-162). We have used Bordetella pertussis, a common neonatal respiratory tract infection, as a proof of concept to investigate the role of these cells in newborns. We found that CD71+ cells have distinctive immune-suppressive properties and suppress innate immune responses against B. pertussis infection. CD71+ cell ablation unleashed innate immune response and restored resistance to B. pertussis infection. In contrast, adoptive transfer of neonatal CD71+ cells into adult recipients impaired their innate immune response to B. pertussis infection. Enhanced innate immune response to B. pertussis was characterized by increased production of protective cytokines IFN-γ, TNF-α, and IL-12, as well as recruitment of NK cells, CD11b+, and CD11c+ cells in the lung. Neonatal and human cord blood CD71+ cells express arginase II, and this enzymatic activity inhibits phagocytosis of B. pertussis in vitro. Thus, our study challenges the notion that neonatal infection susceptibility is due to immune cell-intrinsic defects and instead highlights active immune suppression mediated by abundant CD71+ cells in the newborn. Our findings provide additional support for the novel theme in neonatal immunology that immunosuppression is essential to dampen robust immune responses in the neonate. We anticipate that our results will spark renewed investigation in modulating the function of these cells and developing novel strategies for enhancing host defense to infections in newborns.