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IntroductionTocilizumab, a humanized anti-interleukin-6 receptor (IL-6R) antibody, is recommended for the treatment of severe to critical coronavirus diseases 2019 (COVID-19). However, there were conflicting results on the efficacy of tocilizumab. Therefore, we hypothesized that the differences in tocilizumab efficacy may stem from the different immune responses of critical COVID-19 patients. In this study, we described two groups of immunologically distinct COVID-19 patients, based on their IL-6 response.MethodsWe prospectively enrolled critical COVID-19 patients, requiring oxygen support with a high flow nasal cannula or a mechanical ventilator, and analyzed their serial samples. An enzyme-linked immunosorbent assay and flow cytometry were used to evaluate the cytokine kinetics and cellular immune responses, respectively.ResultsA total of nine patients with critical COVID-19 were included. The high (n = 5) and low IL-6 (n = 4) groups were distinguished by their peak serum IL-6 levels, using 400 pg/mL as the cut-off value. Although the difference of flow cytometric data did not reach the level of statistical significance, the levels of pro-inflammatory cytokines and the frequencies of intermediate monocytes (CD14+CD16+), IFN-γ+ CD4+ or CD8+ T cells, and HLA-DR+PD-1+ CD4+ T cells were higher in the high IL-6 group than in the low IL-6 group.ConclusionThere were distinctive two groups of critical COVID-19 according to serum IL-6 levels having different degrees of cytokinemia and T-cell responses. Our results indicate that the use of immune modulators should be more tailored in patients with critical COVID-19.

Background Understanding the memory T-cell response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is crucial for assessing the longevity of protective immunity after SARS-CoV-2 infection or coronavirus disease 2019 (COVID-19) vaccination. However, the longitudinal memory T-cell response up to 8 months post–symptom onset (PSO) according to the severity of illness is unknown. Methods We analyzed peripheral blood mononuclear cells (PBMCs) from healthy volunteers or patients with COVID-19 who experienced asymptomatic, mild, or severe illness at 2, 5, and 8 months PSO. SARS-CoV-2 spike, nucleocapsid, and membrane protein-stimulated PBMCs were subjected to flow cytometry analysis. Results A total of 24 patients (7 asymptomatic, 9 with mild disease, and 8 with severe disease) and 6 healthy volunteers were analyzed. SARS-CoV-2–specific OX40+CD137+CD4+ T cells and CD69+CD137+CD8+ T cells persisted at 8 months PSO. Also, antigen-specific cytokine-producing or polyfunctional CD4+ T cells were maintained for up to 8 months PSO. Memory CD4+ T-cell responses tended to be greater in patients who had severe illness than in those with mild or asymptomatic disease. Conclusions Memory response to SARS-CoV-2, based on the frequency and functionality, persists for 8 months PSO. Further investigations involving its longevity and protective effect from reinfection are warranted.

Effector memory (EM) CD8+ T cells expressing lower levels of IL-7R α (IL-7Rαlow) from healthy individuals are partly compromised in vitro, but the identity of these cells has remained unclear. In this study, we demonstrate that human IL-7Rαlow EM CD8+ T cells are naturally occurring anergic cells in vivo and impaired in proliferation and IL-2 production but competent in IFN-γ and TNF-α production, a state that can be restored by IL-2 stimulation. IL-7Rαlow EM CD8+ T cells show decreased expression of GATA3 and c-MYC and are defective in metabolic reprogramming toward glycolysis, a process required for the proliferation of T cells. However, IL-7Rαlow EM CD8+ T cells can proliferate with TCR stimulation in the presence of IL-2 and IL-15, suggesting that these cells can be restored to normality or increased activity by inflammatory conditions and may serve as a reservoir for functional immunity.

SARS-CoV-2 infection is generally mild or asymptomatic in children but a biological basis for this outcome is unclear. Here we compare antibody and cellular immunity in children (aged 3–11 years) and adults. Antibody responses against spike protein were high in children and seroconversion boosted responses against seasonal Beta-coronaviruses through cross-recognition of the S2 domain. Neutralization of viral variants was comparable between children and adults. Spike-specific T cell responses were more than twice as high in children and were also detected in many seronegative children, indicating pre-existing cross-reactive responses to seasonal coronaviruses. Importantly, children retained antibody and cellular responses 6 months after infection, whereas relative waning occurred in adults. Spike-specific responses were also broadly stable beyond 12 months. Therefore, children generate robust, cross-reactive and sustained immune responses to SARS-CoV-2 with focused specificity for the spike protein. These findings provide insight into the relative clinical protection that occurs in most children and might help to guide the design of pediatric vaccination regimens., SARS-CoV-2 infection is milder in children, but direct comparison with adults is rare. Here the authors show that immune responses are higher in children, retained for 12 months or longer and can neutralize Alpha, Beta and Delta variants.

The Tec kinase IL-2–inducible T cell kinase (ITK) regulates the expression of TCR-induced genes. Itk−/− T cell responses are impaired but not absent. ITK inhibition prevented colitis disease progression and impaired T cell migration to the colon in mice. To examine the function of ITK in T cell migration to the intestine, we examined the number of gut T cells in Itk−/− mice and then evaluated their expression of gut-homing receptors. Combined with in vitro murine T cell stimulation and in vivo migration assay using congenic B6 mice, we demonstrated an essential role for ITK in T cell migration to the intestine in mice. Reconstitution of Itk−/− mouse CD8+ T cells with IFN regulatory factor 4 restored gut-homing properties, providing mechanistic insight into the function of ITK-mediated signaling in CD8+ T cell migration to the intestinal mucosa in mice.

At present, concerns that the recent global emergence of SARS-CoV-2 variants could compromise the current vaccines have been raised, highlighting the urgent demand for new vaccines capable of eliciting T cell-mediated immune responses, as well as B cell-mediated neutralizing antibody production. In this study, we developed a novel recombinant Mycobacterium paragordonae expressing the SARS-CoV-2 receptor-binding domain (RBD) (rMpg-RBD-7) that is capable of eliciting RBD-specific immune responses in vaccinated mice. The potential use of rMpg-RBD-7 as a vaccine for SARS-CoV-2 infections was evaluated in in vivo using mouse models of two different modules, one for single-dose vaccination and the other for two-dose vaccination. In a single-dose vaccination model, we found that rMpg-RBD-7 versus a heat-killed strain could exert an enhanced cell-mediated immune (CMI) response, as well as a humoral immune response capable of neutralizing the RBD and ACE2 interaction. In a two-dose vaccination model, rMpg-RBD-7 in a two-dose vaccination could also exert a stronger CMI and humoral immune response to neutralize SARS-CoV-2 infections in pseudoviral or live virus infection systems, compared to single dose vaccinations of rMpg-RBD or two-dose RBD protein immunization. In conclusion, our data showed that rMpg-RBD-7 can lead to an enhanced CMI response and humoral immune responses in mice vaccinated with both single- or two-dose vaccination, highlighting its feasibility as a novel vaccine candidate for SARS-CoV-2. To the best of our knowledge, this study is the first in which mycobacteria is used as a delivery system for a SARS-CoV-2 vaccine.

Interleukin-7 (IL-7), which is required for the development and survival of T cells in the thymus and periphery, plays a role in joint destruction. However, it remains unclear how IL-7 affects osteoclast formation. Thus, we investigated the mechanism by which IL-7 induced osteoclast formation through IL-7 receptor α (IL-7Rα) in osteoclast precursors. We cultured peripheral blood mononuclear cells or synovial fluid mononuclear cells with IL-7 in the presence or absence of an appropriate inhibitor to analyze osteoclast formation. We also constructed IL-7Rα-expressing RAW264.7 cells to uncover the mechanism(s) by which IL-7 induced osteoclast formation differed from that of receptor activator of nuclear factor κB ligand (RANKL). We found that IL-7 induced osteoclast formation of human monocytes from peripheral blood or synovial fluid in a RANKL-independent and a signal transducer and activator of transcription 5 (STAT5)-dependent manner. IL-7-induced osteoclasts had unique characteristics, such as small, multinucleated tartrate-resistant acid phosphatase positive cells and no alterations even when RANKL was added after IL-7 pretreatment. RAW264.7 cells, if overexpressing IL-7Rα, also were able to differentiate into osteoclasts by IL-7 through a STAT5 signaling pathway. Furthermore, IL-7-induced osteoclast formation was repressed by inhibitors of the IL-7R signaling molecules Janus kinase and STAT5. Our findings demonstrate that IL-7 is a truly osteoclastogenic factor, which may induce osteoclast formation via activation of STAT5, independent of RANKL. We also suggest the possibility that an IL-7R pathway blocker could alleviate joint damage by inhibiting osteoclast formation, especially in inflammatory conditions.

The voltage-gated potassium channel, Kv1.3, and the Ca2+-activated potassium channel, KCa3.1, regulate membrane potentials in T cells, thereby controlling T cell activation and cytokine production. However, little is known about the expression and function of potassium channels in human effector memory ( EM) CD8+ T cells that can be further divided into functionally distinct subsets based on the expression of the interleukin ( IL)-7 receptor alpha ( IL-7R alpha) chain. Herein, we investigated the functional expression and roles of Kv1.3 and KCa3.1 in EM CD8+ T cells that express high or low levels of the IL-7 receptor alpha chain ( IL-7R alpha(high) and IL-7R alpha(low), respectively). In contrast to the significant activity of Kv1.3 and KCa3.1 in IL-7Rahigh EM CD8+ T cells, IL-7Ralow EM CD8+ T cells showed lower expression of Kv1.3 and insignificant expression of KCa3.1. Kv1.3 was involved in the modulation of cell proliferation and IL-2 production, whereas KCa3.1 affected the motility of EM CD8+ T cells. The lower motility of IL-7Ralow EM CD8+ T cells was demonstrated using transendothelial migration and motility assays with intercellular adhesion molecule 1-and/or chemokine stromal cell-derived factor-1 alpha-coated surfaces. Consistent with the lower migration property, IL-7Ralow EM CD8+ T cells were found less frequently in human skin. Stimulating IL-7Ralow EM CD8+ T cells with IL-2 or IL-15 increased their motility and recovery of KCa3.1 activity. Our findings demonstrate that Kv1.3 and KCa3.1 are differentially involved in the functions of EM CD8+ T cells. The weak expression of potassium channels in IL-7Ralow EM CD8+ T cells can be revived by stimulation with IL-2 or IL-15, which restores the associated functions. This study suggests that IL-7Rahigh EM CD8+ T cells with functional potassium channels may serve as a reservoir for effector CD8+ T cells during peripheral inflammation.

The transcription factor Blimp-1 regulates the overall accumulation of virus-specific CD8+ T cells during acute viral infections. We found that increased proliferation and survival of Blimp-1-deficient CD8+ T cells resulted from sustained expression of CD25 and CD27 and persistent cytokine responsiveness. Silencing of these genes reduced the Blimp-1-deficient CD8+ T cell response. Genome-wide Chromatin immunoprecipitation (ChIP) sequencing analysis identified Il2ra and Cd27 genes as direct targets of Blimp-1. At the peak of the anti-viral response, but not earlier, Blimp-1 recruited the histone modifying enzymes G9a and HDAC2 to the Il2ra and Cd27 loci, thereby repressing expression of these genes. In the absence of Blimp-1, the Il2ra and Cd27 genes exhibited enhanced histone H3-acetylation and reduced histone H3K9-trimethylation. These data elucidate a central mechanism by which Blimp-1 acts as an epigenetic regulator, enhancing the numbers of short-lived effector cells while suppressing the development of memory precursor CD8+ T cells.

In T cells, the Tec kinases IL-2–inducible T cell kinase (ITK) and resting lymphocyte kinase (RLK) are activated by TCR stimulation and are required for optimal downstream signaling. Studies of CD4+ T cells from Itk−/− and Itk−/−Rlk−/− mice have indicated differential roles of ITK and RLK in Th1, Th2, and Th17 differentiation and cytokine production. However, these findings are confounded by the complex T cell developmental defects in these mice. In this study, we examine the consequences of ITK and RLK inhibition using a highly selective and potent small molecule covalent inhibitor PRN694. In vitro Th polarization experiments indicate that PRN694 is a potent inhibitor of Th1 and Th17 differentiation and cytokine production. Using a T cell adoptive transfer model of colitis, we find that in vivo administration of PRN694 markedly reduces disease progression, T cell infiltration into the intestinal lamina propria, and IFN-γ production by colitogenic CD4+ T cells. Consistent with these findings, Th1 and Th17 cells differentiated in the presence of PRN694 show reduced P-selectin binding and impaired migration to CXCL11 and CCL20, respectively. Taken together, these data indicate that ITK plus RLK inhibition may have therapeutic potential in Th1-mediated inflammatory diseases.

Notch receptors are processed by gamma-secretase acting in synergy with T cell receptor signaling to sustain peripheral T cell activation. Activated CD4+ T cells differentiate into T helper type 1 (TH1) or TH2 subsets. Molecular cues directing TH1 differentiation include expression of the TH1-specific transcription factor T-bet, encoded by Tbx21. However, the regulation of Tbx21 remains incompletely defined. Here we report that Notch1 can directly regulate Tbx21 through complexes formed on the Tbx21 promoter. In vitro, gamma-secretase inhibitors extinguished expression of Notch, interferon-gamma and Tbx21 in TH1-polarized CD4+ cells, whereas ectopic expression of activated Notch1 restored Tbx21 transcription. In vivo, administration of gamma-secretase inhibitors substantially impeded TH1-mediated disease progression in the mouse experimental autoimmune encephalomyelitis model of multiple sclerosis. Thus, using gamma-secretase inhibitors to modulate Notch signaling may prove beneficial in treating TH1-mediated autoimmunity.

T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10 and MALT1, which is required for TCR-mediated NF-kappaB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-kappaB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-kappaB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCtheta, a T cell-specific kinase important for CBM assembly and classical NF-kappaB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCtheta and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10 and MALT1 were lost. This failure in CBM assembly reduced IkappaBalpha phosphorylation and diminished NF-kappaB-DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-kappaB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-kappaB signaling.

IL-2-inducible T cell kinase (ITK) is the predominant Tec family kinase in T cells. ITK functions to regulate the magnitude of T cell receptor signaling. Deficiencies in ITK in humans leads to a fatal lymphoproilferative syndrome due to uncontrolled Epstein-Barr virus (EBV) infection. Yet, Itk−/− mice mount protective immune responses against several acute viral infections. To address this dichotomy, we have studied the role of ITK in anti-viral T cell responses to latent γ-herpesvirus infection using mouse γ-herpesvirus 68 (MHV68). Despite a delayed response at D7 post-infection, Itk−/− mice controlled MHV68 replication in the spleen at D14, and WT and Itk−/− mice showed no differences in their viral titers in the spleen during latency. However, MHV68-infected Itk−/− mice (n=11/24) spontaneously developed a severe gastrointestinal pathology after D100 post-infection, whereas WT mice (n=24/24) showed no disease symptoms. Following LPS-induced virus reactivation, Itk−/− mice also showed this pathology in the gut accompanied by a high viral titer in the intestine at various time points during viral latency, while WT mice remained healthy. Consistently, the number of gut-resident T cells were substantially less in Itk−/− mice than WT with or without infection. We also found that P-selectin binding on activated Itk−/− T cells or small molecule inhibitor of ITK (PRN694)-treated WT T cells is greatly impaired. Overall, these data suggest that ITK plays an important role in T cell migration to γ-herpesvirus-infected gut tissue following reactivation from latency.

The maturation of naive CD8+ T cells into effector CTLs is a critical feature of a functional adaptive immune system. Development of CTLs depends, in part, upon the expression of the transcriptional regulator eomesodermin (EOMES), which is thought to regulate expression of two key effector molecules, perforin and granzyme B. Although EOMES is important for effector CTL development, the precise mechanisms regulating CD8+ effector cell maturation remains poorly understood. In this study, we show that Notch1 regulates the expression of EOMES, perforin, and granzyme B through direct binding to the promoters of these crucial effector molecules. By abrogating Notch signaling, both biochemically as well as genetically, we conclude that Notch activity mediates CTL activity through direct regulation of EOMES, perforin, and granzyme B.

The transcription factor, ZBTB32, is not expressed in resting naïve CD8+ T cells, but is upregulated following TCR stimulation in the presence of cytokines IFNγ, IL-12 or IL-2. To investigate the role of ZBTB32 in the development of anti-viral CD8+ T cells, Zbtb32-/- mice were infected with LCMV-Armstrong or LCMV-Clone 13. Following infection with LCMV-Armstrong, Zbtb32-/- mice exhibited normal viral clearance, but generated increased numbers of virus-specific CD8+ T cells, relative to wild-type mice, resulting in an increased memory cell population. The Zbtb32-/- CD8+ memory T cell population showed rapid proliferation and enhanced protective memory potential to secondary challenge. In contrast, upon infection with LCMV-Clone 13, 70% of Zbtb32-/- mice succumbed to a fatal disease starting at day 9 post-infection, and exhibited severe immune pathology in the lung compared to WT mice. Examination of the surviving mice indicated reduced LCMV-clone 13 viral titers in Zbtb32-/- mice. Gene expression analysis showed up-regulation of Eomes, Cd27, Pvr and Cd7 in virus-specific Zbtb32-/- CD8+ T cells. ChIP assays confirmed that ZBTB32 bound to the regulatory regions of these genes and recruited HDAC1 and HDAC2 to promote repressive histone modifications. These data indicate that ZBTB32 acts an epigenetic regulator and negatively regulates T cell responses and memory generation during acute and chronic LCMV infection.

Repressor of GATA3 (ROG) is a member of the poxviruses and zinc finger family of transcriptional repressors, and is also known as ZBTB32. Several studies have suggested that ROG may regulate the activation and differentiation CD4+ and CD8+ T cells, and further, may also play a role in plasma cell differentiation. We have investigated the role of ROG in the development of anti-viral memory T cells. Upon acute infection with LCMV Armstrong, Rog-/- mouse accumulated higher proportions and numbers of virus-specific CD8+ T cells, resulting in an increased number of memory cells. Rog-/- mouse exhibited normal viral clearance, but as a consequence of the elevated primary response, the Rog-/- CD8+ T cell population contains enhanced protective memory potential. We also examined the response of Rog-/- mice to infection with a high dose of LCMV-clone 13, which causes a chronic infection leading to exhaustion of the CD8+ T cells. We found that approximately one-half of Rog-/- mice infected with high dose LCMV-clone 13 succumbed to this infection as early as day 9 post-infection and also exhibited severe immune pathology in the lung compared to WT mice. The higher frequency of IFNg-producing virus-specific CD8+ T cells in Rog-/- mice also resulted in enhanced viral control following LCMV-clone 13 infection. These data indicate that ROG plays a unique non-redundant role during acute and chronic LCMV infection, and is an important regulator of CD8+ effector and memory T cell generation.

CD8+ T cell development in the thymus generates a predominant population of conventional naïve cells, along with minor populations of ‘innate’ T cells that resemble memory cells. Recent studies analyzing a variety of knockout or knock-in mice have indicated that impairments in the TCR signaling pathway produce increased numbers of innate CD8+ T cells, characterized by their high expression of CD44, CD122, CXCR3, and the transcription factor Eomesodermin. One component of this altered development is a non-CD8+ T cell-intrinsic role for IL-4. To determine whether reduced TCR signaling within the CD8+ T cells might also contribute to this pathway, we investigated the role of the transcription factor IRF4. IRF4 is upregulated following TCR stimulation in wild-type T cells; however, this upregulation is impaired in Itk-/- T cells, which have reduced responses to TCR signaling. Further, analysis of IRF4-deficient CD8+ thymocytes showed normal development of thymic CD8+ T cells. Interestingly, IRF4-deficient peripheral CD8+ T cells acquired a memory phenotype and expressed the transcription factor Eomesodermin. We also show that activation of naïve IRF4-deficient CD8+ T cells leads to rapid and robust expression of Eomesodermin. Together, these data indicate that IRF4 upregulation following CD8+ T cell activation normally suppresses Eomesodermin expression, thereby regulating the differentiation pathway of CD8+ effector T cells.

During infections, the pathogen-induced cytokine milieu has been shown to determine T cell fate decisions between effector cells and memory precursor cells. However, how changes in cytokine receptor expression contribute to this process is poorly understood. Blimp-1 is known to be an important transcriptional repressor involved in the differentiation of short-lived CD8+ effector T cells following LCMV infection. Using a genome-wide ChIP-sequencing technique, we found that Blimp-1 directly controls cytokine receptor expression during the contraction phase of CD8+ T cells. During the expansion phase at day 5 post-infection (p.i.), CD8+ T cells are highly responsive to several cytokines, whereas at day 7 or 9 p.i. their responses are significantly decreased. In contrast, Blimp-1-/- CD8+ T cells maintain their cytokine responsiveness, and have increased cytokine receptor expression and increased formation of memory CD8+ T cells. Furthermore, Blimp-1 recruits histone modifying enzymes, HDAC2 and G9a to the genomic loci of cytokine receptors IL-2Rα and CD27. This study defines the mechanism by which Blimp-1 association with histone modifying enzymes controls cytokine receptor expression during the expansion and contraction phases of CD8+ T cells following LCMV infection, and thereby contributes to the generation of CD8+ effector and memory T cells.