New Flow Cytometric Methods for Monitoring STAT5 Signaling Reveal Responses to SARS-CoV-2 Antigen-Specific Stimulation in FOXP3+ Regulatory T Cells also in Patients with Advanced Chronic Lymphocytic Leukemia.

Increased frequency of CD4+CD25+ regulatory T-cells (Treg) has been associated with disease progression in chronic lymphocytic leukemia (CLL). Flow cytometric methods, which allow for the simultaneous analysis of their specific transcription factor Foxp3 and activated STAT proteins, together with proliferation can help to elucidate the signaling mechanisms driving Treg expansion and suppression of FOXP3- conventional CD4+T-cells (Tcon). Herein, we first report a novel approach in which STAT5 phosphorylation (pSTAT5) and proliferation (BrdU-FITC incorporation) could be analyzed specifically in FOXP3+ and FOXP3- responding cells after CD3/CD28 stimulation. The addition of magnetically purified CD4+CD25+ T-cells from healthy donors to cocultured autologous CD4+CD25- T-cells resulted in suppression of Tcon cell cycle progression accompanied by a decrease in pSTAT5. Next, a method using imaging flow cytometry is presented for the detection of cytokine-dependent pSTAT5 nuclear translocation in FOXP3-expressing cells. Finally, we discuss our experimental data obtained by combining Treg pSTAT5 analysis and antigen-specific stimulation with SARS-CoV-2 antigens. Applying these methods on samples from patients revealed Treg responses to antigen-specific stimulation and significantly higher basal pSTAT5 in CLL patients treated with immunochemotherapy. Thus, we speculate that through the use of this pharmacodynamic tool, the efficacy of immunosuppressive drugs and their possible off-target effects can be assessed.

[Changes of the T cell composition in the thymus during the COVID-19 pandemic]. / A csecsemomirigy T-sejtjeinek összetételében létrejövo változások a COVID­19-pandémia alatt.

INTRODUCTION: In our study, we aimed to investigate whether the COVID-19 infection itself or the vaccination against it affect the differentiation of T cells in the thymus, and whether the reduction in T cell counts observed in the blood of COVID-19-infected individuals is also observed at the tissue level in the thymus. METHOD: Data from a total of 55 thymectomy patients were processed to create three groups: 1) the pre-COVID-19 (PC) group included 22 patients, 12 women and 10 men, who underwent thymectomy between 2008 and 2013; 2) in the no-COVID-19 (NC) group (patients without verified infection or vaccination), 20 patients, 11 women and 9 men, underwent thymectomy in 2020-2021; 3) the vaccinated or infected COVID-19 (VIC) group included 13 patients, 4 women and 9 men, who underwent thymectomy also in 2020-2021. The pathological samples were immunohistochemically tested for CD4, CD8, CD25 and FOXP3 to verify the helper, cytotoxic and regulatory T cells. RESULTS: The VIC group had significantly lower values for CD4, compared to the PC and NC groups. The FOXP3 value was significantly lower in the VIC and NC groups compared to the PC group. No significant differences were found for CD8 and CD25 between the groups studied. DISCUSSION: The COVID-19 infection or vaccination affects the T cell composition of the thymus. Decreased expression of CD4 has been demonstrated in the VIC group, which confirms a decrease in the T cell counts that also occurs in the thymus. The low FOXP3 levels observed in the NC group during the COVID-19 era, compared to the PC group, may be indicative of a high rate of asymptomatic coronavirus infections and a worsening of immunetolerance. CONCLUSION: First in the world, we have verified that the helper T cell composition of the thymus in COVID-19 infection era is reduced, and in the asymptomatic patients the immune function is decreased as well. Orv Hetil. 2022; 163(52): 2062-2066.

New-onset type 1 diabetes and severe acute respiratory syndrome coronavirus 2 infection.

Type 1 diabetes (T1D) is a condition characterized by an absolute deficiency of insulin. Loss of insulin-producing pancreatic islet ß cells is one of the many causes of T1D. Viral infections have long been associated with new-onset T1D and the balance between virulence and host immunity determines whether the viral infection would lead to T1D. Herein, we detail the dynamic interaction of pancreatic ß cells with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the host immune system with respect to new-onset T1D. Importantly, ß cells express the crucial entry receptors and multiple studies confirmed that ß cells are infected by SARS-CoV-2. Innate immune system effectors, such as natural killer cells, can eliminate such infected ß cells. Although CD4+ CD25+ FoxP3+ regulatory T (TREG ) cells provide immune tolerance to prevent the destruction of the islet ß-cell population by autoantigen-specific CD8+ T cells, it can be speculated that SARS-CoV-2 infection may compromise self-tolerance by depleting TREG -cell numbers or diminishing TREG -cell functions by repressing Forkhead box P3 (FoxP3) expression. However, the expansion of ß cells by self-duplication, and regeneration from progenitor cells, could effectively replace lost ß cells. Appearance of islet autoantibodies following SARS-CoV-2 infection was reported in a few cases, which could imply a breakdown of immune tolerance in the pancreatic islets. However, many of the cases with newly diagnosed autoimmune response following SARS-CoV-2 infection also presented with significantly high HbA1c (glycated hemoglobin) levels that indicated progression of an already set diabetes, rather than new-onset T1D. Here we review the potential underlying mechanisms behind loss of functional ß-cell mass as a result of SARS-CoV-2 infection that can trigger new-onset T1D.

T-Cell Immune Responses and Immunological Factors Associated with Coronavirus Disease 2019 Progression as Predictors for the Severity of the Disease in Hospitalized Patients.

INTRODUCTION: The prevalence of coronavirus disease 2019 (COVID-19) has rapidly increased worldwide. More investigation is needed to progress toward understanding the exact role of immune responses in the pathology of the disease, leading to improved anticipation and treatment options. METHODS: In the present study, we examined the relative expression of T-bet, GATA3, RORγt, and FoxP3 transcription factors as well as laboratory indicators in 79 hospitalized patients along with 20 healthy subjects as a control group. In order to make an exact comparison between various degrees of severity of disease, patients were divided into critical (n = 12) and severe (n = 67) groups. To evaluate the expression of genes of interest by performing real-time PCR, blood samples were obtained from each participant. RESULTS: We found a significant increase in the expression of T-bet, GATA3, and RORγt and a reduction in the expression of FoxP3 in the critically ill patients compared to the severe and control groups. Also, we noticed that the GATA3 and RORγt expressions were elevated in the severe group in comparison with healthy subjects. Additionally, the GATA3 and RORγt expressions showed a positive correlation with elevation in CRP and hepatic enzyme concentration. Moreover, we observed that the GATA3 and RORγt expressions were the independent risk factors for the severity and outcome of COVID-19. DISCUSSION: The present study showed that the overexpression of T-bet, GATA3, and RORγt, as well as a decrease in the FoxP3 expression was associated with the severity and fatal outcome of COVID-19.

MicroRNA-155 and 194 alter expression of Th17 and T regulatory-related transcription factors in the patients with severe coronavirus disease 2019 (COVID-19).

INTRODUCTION: It has been demonstrated that the patients with severe acute respiratory syndrome coronavirus 2 (SARS­CoV­2) suffer from severe inflammation. Due to the ethnics, the immune responses may be different. Additionally, microRNAs may alter immune responses in the patients. The current study was aimed to evaluate the expression of T helper subsets-related transcription factors, some T helper 17 (Th17) products, and two microRNAs, including miR-155 and miR-194, in the Iranian hospitalized patients. METHODS: In this study, T-box expressed in T cells (T-bet), GATA binding protein 3, The retinoid orphan receptor gamma t (RORγt), forkhead box P3 (FOXP3), interleukin (IL)-17A, IL-8, and CC ligand 20 (CCL20) mRNA levels and, miR-155 and miR-194 levels were evaluated in 70 patients suffered from severe coronavirus disease 2019 (COVID-19) and 70 healthy subjects using Real-Time qPCR technique. RESULTS: The findings showed that RORγt, and FOXP3 mRNA levels were significantly increased, while IL-17A, IL-8, and CCL20 mRNA levels were significantly decreased in the hospitalized SARS-CoV-2 infected patients. Although the levels of miR-155 and miR-194 were not different between groups, miR-194 has negative and positive correlations with RORγt and IL-17A in the Iranian healthy controls. CONCLUSION: This study reports although RORγt was up-regulated, IL-17A, IL-8, and CCL20 mRNA levels were significantly decreased in the hospitalized SARS-CoV-2 infected patients. It may be concluded that up-regulation of FOXP3, via development of T regulatory lymphocytes suppresses Th17 functions and neutralizes Th17 activities. MiR-194 may play crucial roles in regulation of RORγt and IL-17A expression in healthy people, the phenomenon that is disrupted in the severe SARS-CoV-2 infected patients.

PD-1 and ICOS counter-regulate tissue resident regulatory T cell development and IL-10 production during flu.

Regulatory T cells that express the transcription factor Foxp3 (Treg cells) are a highly heterogenous population of immunoregulatory cells critical for maintaining immune homeostasis and preventing immunopathology during infections. Tissue resident Treg (TR-Treg) cells are maintained within nonlymphoid tissues and have been shown to suppress proinflammatory tissue resident T cell responses and promote tissue repair. Human populations are repetitively exposed to influenza infections and lung tissue resident effector T cell responses are associated with flu-induced long-term pulmonary sequelae. The kinetics of TR-Treg cell development and molecular features of TR-Treg cells during repeated and/or long-term flu infections are unclear. Utilizing a Foxp3RFP/IL-10GFP dual reporter mouse model along with intravascular fluorescent in vivo labeling, we characterized the TR-Treg cell responses to repetitive heterosubtypic influenza infections. We found lung tissue resident Treg cells accumulated and expressed high levels of co-inhibitory and co-stimulatory receptors post primary and secondary infections. Blockade of PD-1 or ICOS signaling reveals that PD-1 and ICOS signaling pathways counter-regulate TR-Treg cell expansion and IL-10 production, during secondary influenza infection. Furthermore, the virus-specific TR-Treg cell response displayed distinct kinetics, when compared to conventional CD4+ tissue resident memory T cells, during secondary flu infection. Our results provide insight into the tissue resident Foxp3+ regulatory T cell response during repetitive flu infections, which may be applicable to other respiratory infectious diseases such as tuberculosis and COVID.

Profound Treg perturbations correlate with COVID-19 severity.

The hallmark of severe COVID-19 is an uncontrolled inflammatory response, resulting from poorly understood immunological dysfunction. We hypothesized that perturbations in FoxP3+ T regulatory cells (Treg), key enforcers of immune homeostasis, contribute to COVID-19 pathology. Cytometric and transcriptomic profiling revealed a distinct Treg phenotype in severe COVID-19 patients, with an increase in Treg proportions and intracellular levels of the lineage-defining transcription factor FoxP3, correlating with poor outcomes. These Tregs showed a distinct transcriptional signature, with overexpression of several suppressive effectors, but also proinflammatory molecules like interleukin (IL)-32, and a striking similarity to tumor-infiltrating Tregs that suppress antitumor responses. Most marked during acute severe disease, these traits persisted somewhat in convalescent patients. A screen for candidate agents revealed that IL-6 and IL-18 may individually contribute different facets of these COVID-19-linked perturbations. These results suggest that Tregs may play nefarious roles in COVID-19, by suppressing antiviral T cell responses during the severe phase of the disease, and by a direct proinflammatory role.

Upregulation of FOXP3 is associated with severity of hypoxia and poor outcomes in COVID-19 patients.

The levels of messenger RNA (mRNA) transcription of FOXP3, IFN-γ, TNF, IL-6 and COX-2 from both COVID-19 infected and control subjects were evaluated using SYBRTM green real-time polymerase chain reaction (RT-PCR). Severe/critical cases showed significantly lower lymphocyte counts and higher neutrophil counts than the mild or moderate cases. There were significantly lower levels of mRNA expressions of IFN-γ, TNFα and FOXP3 in COVID-19 patients than in the control group. On the other hand, IL-6 and COX-2 expressions were significantly higher in patients suffering from severe disease. FOXP3 expressions were correlated with the severities of hypoxia and were excellent in predicting the disease severity. This was followed by the IL-6, COX-2 and TNFα expressions. FOXP3 expression was the only biomarker to show a significant correlation with patient mortality. It was concluded that SARS-CoV-2 infection is associated with the downregulation of FOXP3 and upregulations of IL-6 and COX-2.

Fast and Efficient Genome Editing of Human FOXP3+ Regulatory T Cells.

FOXP3+ regulatory T cells (Tregs) are central for maintaining peripheral tolerance and immune homeostasis. Because of their immunosuppressive characteristics, Tregs are a potential therapeutic target in various diseases such as autoimmunity, transplantation and infectious diseases like COVID-19. Numerous studies are currently exploring the potential of adoptive Treg therapy in different disease settings and novel genome editing techniques like CRISPR/Cas will likely widen possibilities to strengthen its efficacy. However, robust and expeditious protocols for genome editing of human Tregs are limited. Here, we describe a rapid and effective protocol for reaching high genome editing efficiencies in human Tregs without compromising cell integrity, suitable for potential therapeutic applications. By deletion of IL2RA encoding for IL-2 receptor α-chain (CD25) in Tregs, we demonstrated the applicability of the method for downstream functional assays and highlighted the importance for CD25 for in vitro suppressive function of human Tregs. Moreover, deletion of IL6RA (CD126) in human Tregs elicits cytokine unresponsiveness and thus may prevent IL-6-mediated instability of Tregs, making it an attractive target to potentially boost functionality in settings of adoptive Treg therapies to contain overreaching inflammation or autoimmunity. Thus, our rapid and efficient protocol for genome editing in human Tregs may advance possibilities for Treg-based cellular therapies.

SARS-CoV-2 infection induces the dedifferentiation of multiciliated cells and impairs mucociliary clearance.

Understanding how SARS-CoV-2 spreads within the respiratory tract is important to define the parameters controlling the severity of COVID-19. Here we examine the functional and structural consequences of SARS-CoV-2 infection in a reconstructed human bronchial epithelium model. SARS-CoV-2 replication causes a transient decrease in epithelial barrier function and disruption of tight junctions, though viral particle crossing remains limited. Rather, SARS-CoV-2 replication leads to a rapid loss of the ciliary layer, characterized at the ultrastructural level by axoneme loss and misorientation of remaining basal bodies. Downregulation of the master regulator of ciliogenesis Foxj1 occurs prior to extensive cilia loss, implicating this transcription factor in the dedifferentiation of ciliated cells. Motile cilia function is compromised by SARS-CoV-2 infection, as measured in a mucociliary clearance assay. Epithelial defense mechanisms, including basal cell mobilization and interferon-lambda induction, ramp up only after the initiation of cilia damage. Analysis of SARS-CoV-2 infection in Syrian hamsters further demonstrates the loss of motile cilia in vivo. This study identifies cilia damage as a pathogenic mechanism that could facilitate SARS-CoV-2 spread to the deeper lung parenchyma.

Exploring the therapeutic potential of forkhead box O for outfoxing COVID-19.

The COVID-19 pandemic has wreaked unprecedented societal havoc worldwide. The infected individuals may present mild to severe symptoms, with nearly 20% of the confirmed patients impaired with significant complications, including multi-organ failure. Acute respiratory distress imposed by SARS-CoV-2 largely results from an aggravated cytokine storm and deregulated immune response. The forkhead box O (FoxO) transcription factors are reported to play a significant role in maintaining normal cell physiology by regulating survival, apoptosis, oxidative stress, development and maturation of T and B lymphocytes, secretion of inflammatory cytokines, etc. We propose a potent anti-inflammatory approach based on activation of the FoxO as an attractive strategy against the novel coronavirus. This regime will be focused on restoring redox and inflammatory homeostasis along with repair of the damaged tissue, activation of lymphocyte effector and memory cells. Repurposing FoxO activators as a means to alleviate the inflammatory burst following SARS-CoV-2 infection can prove immensely valuable in the ongoing pandemic and provide a reliable groundwork for enriching our repertoire of antiviral modalities for any such complication in the future. Altogether, our review highlights the possible efficacy of FoxO activation as a novel arsenal for clinical management of COVID-19.

Nanocurcumin improves Treg cell responses in patients with mild and severe SARS-CoV2.

In Coronavirus disease 2019 (COVID-19), a decreased number of regulatory T (Treg) cells and their mediated factors lead to a hyperinflammatory state due to overactivation of the inflammatory cells and factors during the infection. In the current study, we evaluated the Nanocurcumin effects on the Treg cell population and corresponding factors in mild and severe COVID-19 patients. To investigate the Nanocurcumin effects, 80 COVID-19 patients (40 at the severe stage and 40 at the mild stage) were selected and classified into Nanocurcumin and placebo arms. In both the Nanocurcumin and placebo groups, the Treg cell frequency, the gene expression of Treg transcription factor forkhead box P3 (FoxP3), and cytokines (IL-10, IL-35, and TGF-ß), as well as the serum levels of cytokines were measured before and after treatment. In both mild and severe COVID-19 patients, Nanocurcumin could considerably upregulate the frequency of Treg cells, the expression levels of FoxP3, IL-10, IL-35, and TGF-ß, as well as the serum secretion levels of cytokines in the Nanocurcumin-treated group compared to the placebo group. The abovementioned factors were remarkably increased in the post-treatment with Nanocurcumin before pre-treatment conditions. By contrast, it has been observed no notable alteration in the placebo group. Our findings revealed the SinaCurcumin® effective function in a significant increase in the number of Treg cells and their mediated factors in the Nanocurcumin group than in the placebo group in both mild and severe patients. Hence, it would be an efficient therapeutic agent in rehabilitating COVID-19 infected patients.

T-cell dysregulation in COVID-19.

T-cells play key roles in immunity to COVID-19 as well as the development of severe disease. T-cell immunity to COVID-19 is mediated through differentiated CD4+ T-cells and cytotoxic CD8+ T-cells, although their differentiation is often atypical and ambiguous in COVID-19 and single cell dynamics of key genes need to be characterized. Notably, T-cells are dysregulated in severe COVID-19 patients, although their molecular features are still yet to be fully revealed. Importantly, it is not clear which T-cell activities are beneficial and protective and which ones can contribute to the development of severe COVID-19. In this article, we examine the latest evidence and discuss the key features of T-cell responses in COVID-19, showing how T-cells are dysregulated in severe COVID-19 patients. Particularly, we highlight the impairment of FOXP3 induction in CD4+ T-cells and how the impaired FOXP3 expression can lead to the differentiation of abnormally activated (hyperactivated) T-cells and the dysregulated T-cell responses in severe patients. Furthermore, we characterise the feature of hyperactivated T-cells, showing their potential contribution to T-cell dysregulation and immune-mediated tissue destruction (immunopathology) in COVID-19.

T-Cell Hyperactivation and Paralysis in Severe COVID-19 Infection Revealed by Single-Cell Analysis.

Severe COVID-19 patients show various immunological abnormalities including T-cell reduction and cytokine release syndrome, which can be fatal and is a major concern of the pandemic. However, it is poorly understood how T-cell dysregulation can contribute to the pathogenesis of severe COVID-19. Here we show single cell-level mechanisms for T-cell dysregulation in severe COVID-19, demonstrating new pathogenetic mechanisms of T-cell activation and differentiation underlying severe COVID-19. By in silico sorting CD4+ T-cells from a single cell RNA-seq dataset, we found that CD4+ T-cells were highly activated and showed unique differentiation pathways in the lung of severe COVID-19 patients. Notably, those T-cells in severe COVID-19 patients highly expressed immunoregulatory receptors and CD25, whilst repressing the expression of FOXP3. Furthermore, we show that CD25+ hyperactivated T-cells differentiate into multiple helper T-cell lineages, showing multifaceted effector T-cells with Th1 and Th2 characteristics. Lastly, we show that CD25-expressing hyperactivated T-cells produce the protease Furin, which facilitates the viral entry of SARS-CoV-2. Collectively, CD4+ T-cells from severe COVID-19 patients are hyperactivated and FOXP3-mediated negative feedback mechanisms are impaired in the lung, which may promote immunopathology. Therefore, our study proposes a new model of T-cell hyperactivation and paralysis that drives immunopathology in severe COVID-19.