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Highlights • COVID-19 can deteriorate at the 2nd week of illness despite of decreasing viral loads • We analyzed PBMCs of severe or mild COVID-19 cases at the 1st and 3rd week of illness • Cellular responses were not different between two groups at the 1st week of illness • Higher T-cell proliferation, activation, and cytotoxicity was noted in severe cases at the 3rd week • Aberrant hyperactivation of cytotoxic T-cell may contribute to severe COVID-19 pneumonia, Objectives We hypothesized that immune response may contribute to progression of coronavirus disease-19 (COVID-19) at the second week of illness. Therefore, we compared cell-mediated immune (CMI) responses between severe and mild COVID-19 cases. Methods We examined peripheral blood mononuclear cells of laboratory-confirmed COVID-19 patients from their first and third weeks of illness. Severe pneumonia was defined as an oxygen saturation ≤ 93% at room air. Expressions of molecules related to T-cell activation and functions were analyzed by flow cytometry. Results The population dynamics of T cells at the first week were not different between the two groups. However, total numbers of CD4+ and CD8 + T cells tended to be lower in the severe group at the third week of illness. Expressions of Ki-67, PD-1, perforin, and granzyme B in CD4+ or CD8+ T cells were significantly higher in the severe group than in the mild group at the third week. In contrast to the mild group, the levels of their expression did not decrease in severe group. Conclusions Severe COVID-19 had higher degree of proliferation, activation, and cytotoxicity of T-cells at the late phase of illness without cytotoxic T-cell contraction, which might contribute to the development of severe COVID-19.

Highlights • MVP level were up-regulated in temozolomide-resistant glioblastoma cells and glioblastoma stem cells. • MVP decreased the sensitization to temozolomide of glioblastoma cells and glioblastoma stem cells. • Knockdown of MVP reduced temozolomide-resistance, sphere formation ability and invasive capacity. • Negative correlation between MVP expression and prognosis of glioblastoma patients, The resistance of highly aggressive glioblastoma multiforme (GBM) to chemotherapy is a major clinical problem resulting in a poor prognosis. GBM contains a rare population of self-renewing cancer stem cells (CSCs) that proliferate, spurring the growth of new tumors, and evade chemotherapy. In cancer, major vault protein (MVP) is thought to contribute to drug resistance. However, the role of MVP as CSCs marker remains unknown and whether MVP could sensitize GBM cells to Temozolomide (TMZ) also is unclear. We found that sensitivity to TMZ was suppressed by significantly increasing the MVP expression in GBM cells with TMZ resistance. Also, MVP was associated with the expression of other multidrug-resistant proteins in tumorsphere of TMZ-resistant GBM cell, and was highly co-expressed with CSC markers in tumorsphere culture. On the other hands, knockdown of MVP resulted in reduced sphere formation and invasive capacity. Moreover, high expression of MVP was associated with tumor malignancy and survival rate in glioblastoma patients. Our study describes that MVP is a potentially novel maker for glioblastoma stem cells and may be useful as a target for preventing TMZ resistance in GBM patients.

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.

Sox2 is a core transcription factor in embryonic stem cells (ESCs), and O-GlcNAcylation is a type of post-translational modification of nuclear-cytoplasmic proteins. Although both factors play important roles in the maintenance and differentiation of ESCs and the serine 248 (S248) and threonine 258 (T258) residues of Sox2 are modified by O-GlcNAcylation, the function of Sox2 O-GlcNAcylation is unclear. Here, we show that O-GlcNAcylation of Sox2 at T258 regulates mouse ESC self-renewal and early cell fate. ESCs in which wild-type Sox2 was replaced with the Sox2 T258A mutant exhibited reduced self-renewal, whereas ESCs with the Sox2 S248A point mutation did not. ESCs with the Sox2 T258A mutation heterologously introduced using the CRISPR/Cas9 system, designated E14-Sox2TA/WT, also exhibited reduced self-renewal. RNA sequencing analysis under self-renewal conditions showed that upregulated expression of early differentiation genes, rather than a downregulated expression of self-renewal genes, was responsible for the reduced self-renewal of E14-Sox2TA/WT cells. There was a significant decrease in ectodermal tissue and a marked increase in cartilage tissue in E14-Sox2TA/WT-derived teratomas compared with normal E14 ESC-derived teratomas. RNA sequencing of teratomas revealed that genes related to brain development had generally downregulated expression in the E14-Sox2TA/WT-derived teratomas. Our findings using the Sox2 T258A mutant suggest that Sox2 T258 O-GlcNAc has a positive effect on ESC self-renewal and plays an important role in the proper development of ectodermal lineage cells. Overall, our study directly links O-GlcNAcylation and early cell fate decisions., Stem cell development: hold the sugar Cells that can grow into any type of cell, called embryonic stem cells (ESCs), are signaled to stay in stem cell mode (maintain stemness) by addition of a single sugar molecule to Sox2, a regulatory protein coded for by a developmental gene. Sugar modification of Sox2 was known to be involved in maintaining stemness and sometimes implicated in cancer, but the mechanism was poorly understood. When Hyonchol Jang at the National Cancer Center in South Korea and co-workers prevented sugar modification of Sox2 by changing an amino acid at the sugar-binding site, ESCs showed reduced self-renewal. Rather than repressing genes related to stemness, the modification failed to repress developmental genes, permitting cells to grow into other cell types. These results illuminate both the role of Sox2 in cancer and the importance of sugar modification in stemness.

Precise mechanisms underlying interleukin-7 (IL-7)-mediated tumor invasion remain unclear. Thus, we investigated the role of IL-7 in tumor invasiveness using metastatic prostate cancer PC-3 cell line derivatives, and assessed the potential of IL-7 as a clinical target using a Janus kinase (JAK) inhibitor and an IL-7-blocking antibody. We found that IL-7 stimulated wound-healing migration and invasion of PC-3 cells, increased phosphorylation of signal transducer and activator of transcription 5, Akt, and extracellular signal-regulated kinase. On the other hand, a JAK inhibitor and an IL-7-blocking antibody decreased the invasiveness of PC-3 cells. IL-7 increased tumor sphere formation and expression of epithelial–mesenchymal transition (EMT) markers. Importantly, lentiviral delivery of IL-7Rα to PC-3 cells significantly increased bone metastasis in an experimental murine metastasis model compared to controls. The gene expression profile of human prostate cancer cells from The Cancer Genome Atlas revealed that EMT pathways are strongly associated with prostate cancers that highly express both IL-7 and IL-7Rα. Collectively, these data suggest that IL-7 and/or IL-7Rα are promising targets of inhibiting tumor metastasis.

Ethnopharmacological relevance Chamaecyparis obtusa (Siebold & Zucc.) Endl. (C. obtusa) has been used as folk medicine in East Asia and has been reported to alleviate inflammatory diseases. However, the detailed mechanisms for the anti-inflammatory effects of C. obtusa remain unclear. Aim of the study Although the anti-inflammatory mechanisms of natural products have been studied for decades, it is still important to identify the potential anti-inflammatory effects of natural sources. In this study, we investigated the anti-inflammatory effects and underlying mechanism of C. obtusa leaf extracts. Material &methods The cell viability was determined by MTT and crystal violet staining. NO production in the supernatant was measured using Griess reagent. The cell lysates were analyzed by immunoblotting and RT-qPCR. Secreted cytokines were analyzed using ELISA kit and cytokine array kit. mRNA expression from the GSE9632 database set. Z-scores were calculated for each gene and visualized by heat map. Results Among the extracts of C. obtusa obtained with different extraction methods, the 99% ethanol leaf extract (CO99EL) strongly inhibited lipopolysaccharide (LPS)-induced inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression and Janus kinase/signaling transducer and activator of transcription (JAK/STAT) phosphorylation in RAW264.7 cells. In addition, CO99EL strongly inhibited LPS-induced interleukin (IL)-1β, IL-6, IL-27, and C-C motif chemokine ligand (CCL)-1 production and directly inhibited LPS-induced JAK/STAT phosphorylation in RAW264.7 cells. Conclusions These findings demonstrate that CO99EL significantly prevents LPS-induced macrophage activation by inhibiting the JAK/STAT axis. Therefore, we suggest the use of C. obtusa extracts as therapeutic approach for inflammatory diseases.

Yes-associated protein 1 (YAP1) is a transcriptional regulator of the Hippo pathway, which regulates the development and progression of a number of types of cancer, including that of the colon. In the present study, the expression levels of Hippo pathway genes and their clinical significance were investigated in 458 patients with colon adenocarcinoma (COAD), the most frequently diagnosed neoplastic disease globally, using data obtained from The Cancer Genome Atlas database. Notably, mRNA expression of YAP1 was higher in COAD than in other types of gastrointestinal tract cancer. Expression of YAP1 mRNA was higher in COAD than in normal colon samples and was significantly higher in Tumor-Node-Metastasis (TNM) stages III-IV than in stages I-II. YAP1 protein levels, a protein primarily localized in the nucleus, was greater in TNM stages III-IV than in stages I-II. The level of pYAP1, which is inactive and localized in the cytoplasm, was significantly higher in TNM stages III-IV than in stages I-II. However, the YAP1/pYAP1 ratio, which is representative of activity, was higher in TNM stages III-IV than in stages I-II. High mRNA expression of YAP1, TAZ and TEAD4 was associated with a poor prognosis in patients with COAD. Bioinformatics analysis revealed that YAP1 was associated with DNA duplication, cell proliferation and development. Wnt signaling and transforming growth factor-β signaling were significantly higher in the high-YAP1 group, according to data from Gene Set Enrichment Analysis. Taken together, the results indicate that the subcellular distribution of YAP1 and high mRNA expression of YAP1, TAZ and TEAD4 may be associated with poorer overall survival rates in patients with COAD.

The antioxidant activity and chlorogenic acid and caffeic acid contents were investigated from different parts of Acanthopanax senticosus and A. koreanum. Antioxidant activity was assessed by various in vitro assays such as DPPH, ABTS, FRAP, reducing power assays and ORAC, and the chlorogenic acid and caffeic acid were validated by HPLC chromatography. Among the various extracts, the fruit extracts of A. senticosus and A. koreanum exhibited strongest antioxidant activities including ABTS, FRAP, reducing power and ORAC, however, strongest DPPH radical scavenging activity was observed from the leaf extract of A. senticosus. In addition, the antioxidant activities of various extracts were correlated with total phenolic and proanthocyanidin contents. The major phenolic contents from various parts of these plants observed that leaf extract of A. senticosus expressed higher levels of chlorogenic acid (14.86 mg/dry weigh g) and caffeic acid (3.09 mg/dry weigh g) than other parts. Therefore, these results suggest that the leaf of A. senticosus may be an excellent natural source for functional foods and pharmaceutical agents, and the validated method was useful for the quality control of A. senticosus.

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.

Scientific Reports 6: Article number: 39026; published online: 15 December 2016; updated: 31 January 2017. The original version of this Article contained an error in the spelling of Sin-Hyeog Im, which was incorrectly given as Sin-Hyeog Lm. This has now been corrected in the PDF and HTML versions ofthis Article.

Type I interferons (IFNs) play an important role in antiviral immunity as well as immunopathogenesis of diverse chronic viral infections. However, the precise mechanisms regulating the multifaceted effects of type I IFNs on the immune system and pathological inflammation still remain unclear. In order to assess the immunological dynamics associated with rapid viral clearance in chronic hepatitis C patients during the acute phase of type I IFN therapy, we analyzed multiple parameters of virological and immunological responses in a cohort of 59 Korean hepatitis C patients who received pegylated IFN-alpha and ribavirin (IFN/RBV). Most of the Korean patients had favorable alleles in the IFN-lambda loci for responsiveness to IFN/RBV (i.e., C/C in rs12979860, T/T in rs8099917, and TT/TT in rs368234815). Rapid virological response (RVR) was determined mainly by the hepatitis C virus genotype. Among the cytokines analyzed, higher plasma levels of IL-17A and FGF were observed in non-RVR patients infected with viral genotype 1 and IP-10 was consistently elevated in RVR group infected with genotype 2 during the early phase of antiviral therapy. In addition, these three cytokines were correlated each other, suggesting a functional linkage of the cytokines in antiviral responses during IFN/RBV therapy. A low baseline frequencies of regulatory T cells and.d T cells, but high level of group 2 innate lymphoid cells, in peripheral bloods were also significantly associated with the RVR group, implicating a potential role of the cellular immunity during the early phase of IFN/RBV therapy. Therefore, the immunological programs established by chronic hepatitis C and rapid disruption of the delicate balance by exogenous type I IFN might be associated with the subsequent virological outcomes in chronic hepatitis C patients.

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.

Age-associated immunological dysfunction (immunosenescence) is closely linked to perturbation of the gut microbiota. Here, we investigated whether syringaresinol (SYR), a polyphenolic lignan, modulates immune aging and the gut microbiota associated with this effect in middle-aged mice. Compared with age-matched control mice, SYR treatment delayed immunosenescence by enhancing the numbers of total CD3+ T cells and naïve T cells. SYR treatment induced the expression of Bim as well as activation of FOXO3 in Foxp3+ regulatory T cells (Tregs). Furthermore, SYR treatment significantly enhanced the Firmicutes/Bacteroidetes ratio compared with that in age-matched controls by increasing beneficial bacteria, Lactobacillus and Bifidobacterium, while reducing the opportunistic pathogenic genus, Akkermansia. In addition, SYR treatment reduced the serum level of lipopolysaccharide-binding protein, an inflammatory marker, and enhanced humoral immunity against influenza vaccination to the level of young control mice. Taken together, these findings suggest that SYR may rejuvenate the immune system through modulation of gut integrity and microbiota diversity as well as composition in middle-aged mice, which may delay the immunosenescence associated with aging.

The newly emerging Middle East respiratory syndrome coronavirus (MERS-CoV) causes a severe respiratory infection with a high mortality rate (~35%). MERS-CoV has been a global threat due to continuous outbreaks in the Arabian peninsula and international spread by infected travelers since 2012. From May to July 2015, a large outbreak initiated by an infected traveler from the Arabian peninsula swept South Korea and resulted in 186 confirmed cases with 38 deaths (case fatality rate, 20.4%). Here, we show the rapid emergence and spread of a mutant MERS-CoV with reduced affinity to the human CD26 receptor during the South Korean outbreak. We isolated 13 new viral genomes from 14 infected patients treated at a hospital and found that 12 of these genomes possess a point mutation in the receptor-binding domain (RBD) of viral spike (S) protein. Specifically, 11 of these genomes have an I529T mutation in RBD, and 1 has a D510G mutation. Strikingly, both mutations result in reduced affinity of RBD to human CD26 compared to wild-type RBD, as measured by surface plasmon resonance analysis and cellular binding assay. Additionally, pseudotyped virus bearing an I529T mutation in S protein showed reduced entry into host cells compared to virus with wild-type S protein. These unexpected findings suggest that MERS-CoV adaptation during human-to-human spread may be driven by host immunological pressure such as neutralizing antibodies, resulting in reduced affinity to host receptor, and thereby impairs viral fitness and virulence, rather than positive selection for a better affinity to CD26., IMPORTANCE Recently, a large outbreak initiated by an MERS-CoV-infected traveler from the Middle East swept South Korea and resulted in 186 confirmed cases with 38 deaths. This is the largest outbreak outside the Middle East, and it raised strong concerns about the possible emergence of MERS-CoV mutations. Here, we isolated 13 new viral genomes and found that 12 of them possess a point mutation in the receptor-binding domain of viral spike protein, resulting in reduced affinity to the human cognate receptor, CD26, compared to the wild-type virus. These unexpected findings suggest that MERS-CoV adaptation in humans may be driven by host immunological pressure.

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.

International audience; T follicular helper (TFH) and T helper 1 (Th1) cells generated after viral infections are critical for the control of infection and the development of immunological memory. However, the mechanisms that govern the differentiation and maintenance of these two distinct lineages during viral infection remain unclear. We found that viral-specific TFH and Th1 cells showed reciprocal expression of the transcriptions factors TCF1 and Blimp1 early after infection, even before the differential expression of the canonical TFH marker CXCR5. Furthermore, TCF1 was intrinsically required for the TFH cell response to viral infection; in the absence of TCF1, the TFH cell response was severely compromised, and the remaining TCF1-deficient TFH cells failed to maintain TFH-associated transcriptional and metabolic signatures, which were distinct from those in Th1 cells. Mechanistically, TCF1 functioned through forming negative feedback loops with IL-2 and Blimp1. Our findings demonstrate an essential role of TCF1 in TFH cell responses to viral infection.

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.

The expression of CD14, a monocyte receptor for the bacterial lipopolysaccharide (LPS), is upregulated during monocytic cell differentiation. Although a Sp1 site at −110 bp of the CD14 promoter was shown to be critical for activation of the promoter during the differentiation, how the Sp1 site is regulated has not been well understood. We have recently reported that expression of MEF2D protein increases during the differentiation of HL60 promyeloid cells to monocyte and that the upregulation of the protein is required for CD14 expression during the differentiation [Mol. Immunol. 36 (1999) 1209]. However, there is no obvious MEF2 binding site in the critical region of the CD14 promoter. In this study, which aimed to determine the regulatory role of MEF2D in monocytic cell differentiation, MEF2D was found to form a complex with Sp1 in U937 promyeloid cells. Transient transfection experiments showed that co-expression of MEF2D and Sp1 synergistically activated the CD14 promoter. The results support a model in which increased MEF2D protein during monocytic cell differentiation activates the CD14 promoter through interaction with Sp1.

Virus-specific CD8 + T cells in the lymphoid organs contract at the resolution of virus infections by apoptosis or by dissemination into peripheral tissues, and those residing in nonlymphoid organs, including the peritoneal cavity and fat pads, are more resistant to apoptosis than those in the spleen and lymph nodes. This stability of memory T cells in the nonlymphoid tissues may enhance protection to secondary challenges. Here, we show that lymphocytic choriomeningitis virus (LCMV)-specific CD8 + T cells in nonlymphoid tissues were enriched for memory precursors (expressing high levels of interleukin-7 receptor and low levels of killer cell lectin-like receptor G1 [IL-7R hi KLRG1 lo ]) and had higher expression of CD27, CXCR3, and T cell factor-1 (TCF-1), each a marker that is individually correlated with decreased apoptosis. CD8 + T cells in the peritoneal cavity of TCF-1-deficient mice had decreased survival, suggesting a role for TCF-1 in promoting survival in the nonlymphoid tissues. CXCR3 + CD8 + T cells resisted apoptosis and accumulated in the lymph nodes of mice treated with FTY720, which blocks the export of lymph node cells into peripheral tissue. The peritoneal exudate cells (PEC) expressed increased amounts of CXCR3 ligands, CXCL9 and CXCL10, which may normally recruit these nonapoptotic cells from the lymph nodes. In addition, adoptive transfer of splenic CD8 + T cells into PEC or spleen environments showed that the peritoneal environment promoted survival of CD8 + T cells. Thus, intrinsic stability of T cells which are present in the nonlymphoid tissues along with preferential migration of apoptosis-resistant CD8 + T cells into peripheral sites and the availability of tissue-specific factors that enhance memory cell survival may collectively account for the tissue-dependent apoptotic differences. IMPORTANCE Most infections are initiated at nonlymphoid tissue sites, and the presence of memory T cells in nonlymphoid tissues is critical for protective immunity in various viral infection models. Virus-specific CD8 + T cells in the nonlymphoid tissues are more resistant to apoptosis than those in lymphoid organs during the resolution and memory phase of the immune response to acute LCMV infection. Here, we investigated the mechanisms promoting stability of T cells in the nonlymphoid tissues. This increased resistance to apoptosis of virus-specific CD8 + T cells in nonlymphoid tissues was due to several factors. Nonlymphoid tissues were enriched in memory phenotype CD8 + T cells, which were intrinsically resistant to apoptosis irrespective of the tissue environment. Furthermore, apoptosis-resistant CD8 + T cells preferentially migrated into the nonlymphoid tissues, where the availability of tissue-specific factors may enhance memory cell survival. Our findings are relevant for the generation of long-lasting vaccines providing protection at peripheral infection sites.

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.

The regulatory role of MEF2 (myocyte enhancer binding factor 2) proteins in nonmuscle tissues has not been well characterized. We examined the expression of MEF2 family members, namely, MEF2A, -B, -C, and -D, in the differentiation of HL60 promyeloid cells and observed the remarkable increase in the expressions of MEF2A and MEF2D proteins during the differentiation process into monocytes. To examine the role of MEF2, we expressed a dominant-negative form of MEF2D, without its transactivation domain, in HL60 cells. When the HL60 cell line expressing the mutant MEF2D was induced to differentiate by VitD(3) treatment, cell surface expression of CD14 and the ability to reduce NBT, which are important characteristics of differentiated monocytes, were significantly decreased compared with control HL60 cells. These results show that MEF2D is required in the differentiation process along the monocyte/macrophage lineage

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.

Notch1 specifically upregulates expression of the cytokine interferon-gamma in peripheral T cells through activation of NF-kappaB. However, how Notch mediates NF-kappaB activation remains unclear. Here, we examined the temporal relationship between Notch signaling and NF-kappaB induction during T-cell activation. NF-kappaB activation occurs within minutes of T-cell receptor (TCR) engagement and this activation is sustained for at least 48 h following TCR signaling. We used gamma-secretase inhibitor (GSI) to prevent the cleavage and subsequent activation of Notch family members. We demonstrate that GSI blocked the later, sustained NF-kappaB activation, but did not affect the initial activation of NF-kappaB. Using biochemical approaches, as well as confocal microscopy, we show that the intracellular domain of Notch1 (N1IC) directly interacts with NF-kappaB and competes with IkappaBalpha, leading to retention of NF-kappaB in the nucleus. Additionally, we show that N1IC can directly regulate IFN-gamma expression through complexes formed on the IFN-gamma promoter. Taken together, these data suggest that there are two 'waves' of NF-kappaB activation: an initial, Notch-independent phase, and a later, sustained activation of NF-kappaB, which is Notch dependent.

Within a few minutes of T-cell activation, transcription of a set of genes including c-fos and c-jun is activated. For maximal induction of c-jun, at least two major signal pathways are required. One can be triggered by T-cell receptor engagement or phorbol esters and the other by anti-CD28 engagement. The c-jun promoter region between -117 and -50 contains binding sites for the transcription factors Spl, CTF, ATF/CREB, and MEF2. In this study, we sought to map the sequences in the c-jun promoter responsible for CD28-mediated induction in activated Jurkat T cell by point mutational analysis. We found that mutation of the c-jun MEF2 site strongly reduces CD28 induction of the promoter in Jurkat T cells and that MEF2D is the major binding molecule to the c-jun MEF2 site in Jurkat T cells. Mutation of the c-jun ATF site also partially reduced CD28 induction of the promoter. In addition, pretreatment with an endolysomotropic agent NH4Cl, an acidic sphingomyelinase inhibitor, completely inhibited the activation of the c-jun promoter by anti-CD28 antibody treatment, whereas pretreatment with wortmannin, a PI3-kinase inhibitor, did not affect the induction of the c-jun promoter. These results suggest that CD28 signaling leading to the c-jun promoter involves acidic sphingomyelinase, but not PI3-kinase, to activate factors binding to the MEF2 and ATF sites.