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Familial hemophagocytic lymphohistiocytosis (FHL) is an inherited, often fatal immune deficiency characterized by severe systemic hyperinflammation. Although allogeneic bone marrow transplantation can be curative, more effective therapies are urgently needed. FHL is caused by inactivating mutations in proteins that regulate cellular immunity. Here, we used an adeno-associated virus–based CRISPR-Cas9 system with an inhibitor of nonhomologous end joining to repair such mutations in potentially long-lived T cells ex vivo. Repaired CD8 memory T cells efficiently cured lethal hyperinflammation in a mouse model of Epstein-Barr virus–triggered FHL2, a subtype caused by perforin-1 (Prf1) deficiency. Furthermore, repair of PRF1 and Munc13-4 (UNC13D)—whose deficiency causes the FHL subtype FHL3—in mutant memory T cells from two critically ill patients with FHL restored T cell cytotoxicity. These results provide a starting point for the treatment of genetic T cell immune dysregulation syndromes with repaired autologous T cells. Editor's summary: Familial hemophagocytic lymphohistiocytosis (FHL) is a spectrum of inherited, potentially fatal inflammatory diseases caused by loss-of-function mutations in the NK cell and T cell cytotoxic machinery. Although FHL can be treated with hematopoietic stem cell transplantation (HSCT), mortality is still high and more effective therapeutic approaches are needed. Li et al. developed an adeno-associated virus (AAV)–based CRISPR-Cas9 system combined with nonhomologous end joining (NHEJ) inhibition to repair T cells from a perforin (Prf1)–deficient Epstein-Barr virus (EBV)–triggered mouse model of FHL (FHL2). Repaired T cells could prevent or cure FHL2-like disease caused by EBV-driven B cell hyperproliferation. Moreover, this CRISPR-Cas9 system could be used to mend T cells from pediatric patients with FHL2 (PRF1 deficiency) and FHL3 (Munc13-4/UNC13D deficiency). This work demonstrates that gene repair of autologous T cells is a potentially promising treatment for human FHL. —Seth Thomas Scanlon [ABSTRACT FROM AUTHOR]

The CARMA1–Bcl10–MALT1 (CBM) signalosome is a crucial module of NF‐κB activation in B cell receptor (BCR) signaling. Biophysical studies have shown that the E3 ubiquitin ligase TRAF6 cooperatively modifies the CBM signalosome; however, the specific details regarding how TRAF6 is involved in BCR signal‐induced CBM formation remain unclear. In this study, we aimed to reveal the influences of TRAF6 on CBM formation and TAK1 and IKK activities using DT40 B cells which lack all the exons of TRAF6. In TRAF6‐null cells we found: (i) attenuation of TAK1 activity and abolishment of IKK activity and (ii) sustained binding of CARMA1 to Bcl10. To account for the molecular mechanism causing these dynamics, we performed a mathematical model analysis. The mathematical model analysis showed that the regulation of IKK activation by TRAF6 can reproduce TAK1 and IKK activities in TRAF6 null cells, and that the TRAF6 related signal‐dependent inhibitor suppresses CARMA1 binding to Bcl10 in wild‐type cells. These results suggest that TRAF6 contributes to the positive regulation of IKK activation via TAK1, alongside the negative signal‐dependent regulation of CARMA1 binding to Bcl10. [ABSTRACT FROM AUTHOR]

The immune evasion of SARS-CoV-2 Omicron variants caused by multiple amino acid replacements in the receptor-binding domain (RBD) of the spike protein wanes the effectiveness of antibodies elicited by current SARS-CoV-2 booster vaccination. The vaccines that target Omicron strains have been recently developed, however, there has been a concern yet to be addressed regarding the negative aspect of the immune response known as original antigenic sin. Here, we demonstrate that the breadth of neutralizing antibodies against SARS-CoV-2 variants is barely elicited by immunizing monovalent viral antigens via vaccination or natural infection in mice and human subjects. However, vaccination of Omicron BA.1 RBD to pre-immunized mice with the original RBD conferred sustained neutralizing activity to BA.1 and BA.2 not only original pseudoviruses. The acquisition of neutralizing antibody breadth was further confirmed in vaccinated-then-Omicron convalescent human sera in which neutralizing activity against BA.1 and BA.2 pseudoviruses was highly induced. Thus, our data suggest that Omicron-specific vaccines or the infection with Omicron viruses can boost potent neutralizing antibodies to the Omicron variants even in the host pre-vaccinated with the original antigen. [ABSTRACT FROM AUTHOR]

Simple Summary: The Epstein–Barr virus (EBV) is a γ-herpes virus that primarily infects human B cells, and more than 90% of adults have experienced infection. EBV+ B cells express several viral proteins, transmitting signals important for the transformation and tumorigenesis of the infected B cells. Immune surveillance by the host immune system is important to suppress such abnormal expansion of EBV-infected B cells. Here we found that both CD4+ T cells and CD8+ T cells show similar gene expression patterns relating to cytotoxicity towards EBV-infected B cells. EBV-specific cytotoxic CD4+ T cells markedly expressed T-bet, Granzyme B, and Perforin alongside killing activity, which could reflect mechanisms shared with cytotoxic CD8+ T cells. Our findings support the concept that, upon EBV and perhaps other viral infections, T cells of different subsets can be drawn into common pathways mediating immune surveillance through cytotoxicity. Most people infected by EBV acquire specific immunity, which then controls latent infection throughout their life. Immune surveillance of EBV-infected cells by cytotoxic CD4+ T cells has been recognized; however, the molecular mechanism of generating cytotoxic effector T cells of the CD4+ subset remains poorly understood. Here we compared phenotypic features and the transcriptome of EBV-specific effector-memory CD4+ T cells and CD8+ T cells in mice and found that both T cell types show cytotoxicity and, to our surprise, widely similar gene expression patterns relating to cytotoxicity. Similar to cytotoxic CD8+ T cells, EBV-specific cytotoxic CD4+ T cells from human peripheral blood expressed T-bet, Granzyme B, and Perforin and upregulated the degranulation marker, CD107a, immediately after restimulation. Furthermore, T-bet expression in cytotoxic CD4+ T cells was highly correlated with Granzyme B and Perforin expression at the protein level. Thus, differentiation of EBV-specific cytotoxic CD4+ T cells is possibly controlled by mechanisms shared by cytotoxic CD8+ T cells. T-bet-mediated transcriptional regulation may explain the similarity of cytotoxic effector differentiation between CD4+ T cells and CD8+ T cells, implicating that this differentiation pathway may be directed by environmental input rather than T cell subset. [ABSTRACT FROM AUTHOR]

Insulin receptor substrate (IRS)-1 and IRS-2 have dominant roles in the action of insulin (1), but other substrates of the insulin receptor kinase, such as Gabl, c-Cbl, SH2-B and APS, are also of physiological relevance (2-5). Although the protein downstream of tyrosine kinases-1 (Dok1) is known to function as a multisite adapter molecule in insulin signaling (6-8), its role in energy homeostasis has remained unclear. Here we show that Dok1 regulates adiposity. Expression of Dok1 in white adipose tissue was markedly increased in mice fed a high-fat diet, whereas adipocytes lacking this adapter were smaller and showed a reduced hypertrophic response to this dietary manipulation. Dok1-deficient mice were leaner and showed improved glucose tolerance and insulin sensitivity compared with wild-type mice. Embryonic fibroblasts from Dok1-deficient mice were impaired in adipogenic differentiation, and this defect was accompanied by an increased activity of the protein kinase ERK and a consequent increase in the phosphorylation of peroxisome proliferator-activated receptor (PPAR)-[gamma] on Ser112. Mutation of this negative regulatory site for the transactivation activity of PPAR-[gamma] blocked development of the lean phenotype caused by Dok1 ablation. These results indicate that Dok1 promotes adipocyte hypertrophy by counteracting the inhibitory effect of ERK on PPAR-[gamma] and may thus confer predisposition to diet-induced obesity., We and others have shown that tyrosine-phosphorylated Dok1 binds and recruits p120 Ras GTPase-activating protein (rasGAP) as well as the adapter protein Nck, thereby regulating mitogenic signaling mediated by Ras [...]

Isolation of both lymphocytes and myogenic cells from muscle tissue is required for elucidating the cellular and molecular mechanisms of muscle regeneration. Here, we aimed to establish an optimal method obtaining a high yield of lymphocytes during muscle regeneration. After the muscle injury, we observed higher infiltration of lymphocytic cells in the muscle on day 3 after injury. Then, we compared two different white blood cell isolation methods, the Percoll gradient and CD45-magnetic bead methods, to assess the percentage and number of T and B cells. Flow cytometry analysis showed that the CD45-magnetic bead method has a better efficiency in isolating CD4+, CD8+ T cells, and B cells from injured muscle tissues of wild-type and mdx mice than that by the Percoll gradient method. Moreover, we found that the CD45-negative fraction from wild-type and mdx mice includes myogenic cells. In conclusion, we report that the CD45-magnetic bead method is suitable to isolate T and B cells during muscle regeneration with higher purity and yield and can also isolate myogenic cells within the same sample. This method provides a technical basis for further studies on muscle regeneration, involving lymphocytes and muscle cells, with a wide range of clinical applications. [ABSTRACT FROM AUTHOR]

Although long noncoding RNAs (lncRNAs) are transcripts that do not encode proteins by definition, some lncRNAs actually contain small open reading frames that are translated. TINCR (terminal differentiation–induced ncRNA) has been recognized as a lncRNA that contributes to keratinocyte differentiation. However, we here show that TINCR encodes a ubiquitin-like protein that is well conserved among species and whose expression was confirmed by the generation of mice harboring a FLAG epitope tag sequence in the endogenous open reading frame as well as by targeted proteomics. Forced expression of this protein promoted cell cycle progression in normal human epidermal keratinocytes, and mice lacking this protein manifested a delay in skin wound healing associated with attenuated cell cycle progression in keratinocytes. We termed this protein TINCR-encoded ubiquitin-like protein (TUBL), and our results reveal a role for TINCR in the regulation of keratinocyte proliferation and skin regeneration that is dependent on TUBL. Author summary: Although, by definition, long noncoding RNAs (lncRNAs) are transcripts that do not encode proteins, recent studies have shown that some lncRNAs actually contain small open reading frames (ORFs) that are translated. Although TINCR (terminal differentiation–induced ncRNA) was originally identified as a lncRNA that contributes to keratinocyte differentiation, recent mass spectrometry–based analysis has suggested that TINCR is translated. Translation of the ORF within the TINCR lncRNA was validated by generating mice that harbor an in-frame insertion of the FLAG epitope tag sequence at the COOH-terminus of the ORF. The TINCR-encoded protein contains a ubiquitin-like (Ubl) domain and was designated TUBL (TINCR-encoded ubiquitin-like protein). TUBL accelerated cell cycle progression in keratinocytes, and TUBL-deficient mice manifested delayed wound healing after injury with a biopsy punch as a result of attenuated keratinocyte proliferation. TUBL plays a key role at the protein level in the maintenance of skin homeostasis after injury through promotion of keratinocyte proliferation. [ABSTRACT FROM AUTHOR]

The Epstein–Barr virus (EBV) is endemic in humans and can efficiently transform infected B cells under some circumstances. If an EBV carrier experiences immune suppression, EBV+ B cells can turn into lymphoblasts and exhibit growth expansion that may cause lymphoproliferative diseases which often develop into lymphoma. Our immune system conducts surveillance for EBV+ B cells in order to block spontaneous tumor formation. Here, we summarize the EBV products involved in tumorigenesis, EBV-associated lymphomas, and pathologically relevant mouse models. Preclinical mouse models for a range of EBV-associated diseases not only clear the path to new therapeutic approaches but also aid in our understanding of the nature of lymphomagenesis and immune surveillance. [ABSTRACT FROM AUTHOR]

Transforming growth factor-β-activated kinase ( TAK)1-binding proteins ( TAB) activate nuclear factor-κB by linking TAK1 to signaling molecules. We investigated the mechanisms underlying B-cell receptor ( BCR) signaling in TAB2- and TAB3-deficient and TAB3 domain deletion mutant DT40 B cell lines. Loss of TAB2 and TAB3 abolished BCR-induced inhibitor of κB kinase ( IKK) activation and TAK1 binding to caspase recruitment domain membrane-associated guanylate kinase protein ( CARMA)1. Deletion of TAB3, coupling of ubiquitin conjugation to ER degradation, coiled-coil, and zinc finger domains blocked IKK activation and association with CARMA1. Thus, TAB2 and TAB3 connect signaling molecules that activate IKK in BCR signaling. [ABSTRACT FROM AUTHOR]

Insulin receptor substrate (IRS)-1 and IRS-2 have dominant roles in the action of insulin, but other substrates of the insulin receptor kinase, such as Gab1, c-Cbl, SH2-B and APS, are also of physiological relevance. Although the protein downstream of tyrosine kinases-1 (Dok1) is known to function as a multisite adapter molecule in insulin signaling, its role in energy homeostasis has remained unclear. Here we show that Dok1 regulates adiposity. Expression of Dok1 in white adipose tissue was markedly increased in mice fed a high-fat diet, whereas adipocytes lacking this adapter were smaller and showed a reduced hypertrophic response to this dietary manipulation. Dok1-deficient mice were leaner and showed improved glucose tolerance and insulin sensitivity compared with wild-type mice. Embryonic fibroblasts from Dok1-deficient mice were impaired in adipogenic differentiation, and this defect was accompanied by an increased activity of the protein kinase ERK and a consequent increase in the phosphorylation of peroxisome proliferator–activated receptor (PPAR)-γ on Ser112. Mutation of this negative regulatory site for the transactivation activity of PPAR-γ blocked development of the lean phenotype caused by Dok1 ablation. These results indicate that Dok1 promotes adipocyte hypertrophy by counteracting the inhibitory effect of ERK on PPAR-γ and may thus confer predisposition to diet-induced obesity. [ABSTRACT FROM AUTHOR]

The B cell antigen receptor (BCR)-mediated activation of IκB kinase (IKK) and nuclear factor-κB require protein kinase C (PKC)β; however, the mechanism by which PKCβ regulates IKK is unclear. Here, we demonstrate that another protein kinase, TGFβ-activated kinase (TAK)1, is essential for IKK activation in response to BCR stimulation. TAK1 interacts with the phosphorylated CARMA1 (also known as caspase recruitment domain [CARD]11, Bimp3) and this interaction is mediated by PKCβ. IKK is also recruited to the CARMA1-Bcl10-mucosal-associated lymphoid tissue 1 adaptor complex in a PKCβ-dependent manner. Hence, our data suggest that phosphorylation of CARMA1, mediated by PKCβ, brings two key protein kinases, TAK1 and IKK, into dose proximity, thereby allowing TAK1 to phosphorylate IKK. [ABSTRACT FROM AUTHOR]

Summary: B cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach. [Copyright &y& Elsevier]

Abstract: We investigated the effects of a sublethal concentration of H2O2 on cancer cells by using sublines derived from human lung adenocarcinoma cell line A549 cells exposed to 200μM H2O2. These sublines (AST cells) showed an elongated morphology distinct from the rounded morphology of A549 cells. Notably, AST cells demonstrated telomere shortening despite displaying telomerase activity and expressing human telomerase reverse transcriptase (hTERT). This functional impairment of telomerase occurred due to perturbed subcellular localization of hTERT in AST cells. Endogenous as well as ectopically expressed hTERT was localized in the nuclei of A549 cells; however, in AST cells, the localization was mainly in the cytoplasm. Furthermore, these AST cells demonstrated decreased tumorigenic features both in vitro and in vivo. These results suggest that depletion of hTERT from nuclei not only endows cancer cells with a finite replicative life span accompanied by telomere shortening, but also decreases the tumorigenicity of cancer cells. [Copyright &y& Elsevier]

Senescent cells were recently shown to play a role in aging-related malfunctions and pathologies. This consensus has been facilitated by evidence from senolytic model mice capable of eliminating senescent cells in tissues using well-characterized senescent markers, such as p16INK4a (hereafter p16). However, since the incomplete or artificial gene expression regulatory regions of manipulated marker genes affect their cognate expression, it currently remains unclear whether these models accurately reflect physiological senescence. We herein describe a novel approach to eliminate p16 -expressing cells from mice at any given point in time, generating a new type of knock-in model, p16 hCD2 mice and a toxin-conjugated anti-human CD2 antibody (hCD2-SAP) as an inducer. p16 hCD2 mice possess an intact Cdkn2a locus that includes a p16 coding region and human CD2 (hCD2) expression unit. We confirmed cognate p16 -associated hCD2 expression in mouse embryonic fibroblasts (MEFs) and in several tissues, such as the spleen, liver, and skin. We detected chronological increases in the hCD2-positive population in T lymphocytes that occurred in a p16 -dependent manner, which reflected physiological aging. We then confirmed the high sensitivity of hCD2-SAP to hCD2 and validated its efficacy to remove p16 -positive cells, particularly in T lymphocytes. The multiple administration of hCD2-SAP for a prolonged p16 -positive cell deficiency partially restored aging-related phenotypes in T lymphocytes, such as the contraction of the CD4+ naïve population and expansion of senescence-associated T cells. Our novel approach of targeting p16-positive senescent cells will provide novel insights into the mechanisms underlying physiological aging in vivo. • A novel knock-in model p16 hCD2 mouse expressed the surface marker hCD2, which correlated with the induction of p16 INK4a . • A hCD2-targeting immunotoxin eliminated hCD2-positive MEFs derived from p16 hCD2 mice. • Age-associated p16 INK4a -positive T cells in p16 hCD2 mice were removed by hCD2-SAP in a hCD2 expression-dependent manner. • The administration of hCD2-SAP to p16 hCD2 mice partially recovered the age-related phenotypes of CD4+ T cells. [ABSTRACT FROM AUTHOR]

Summary Emerging evidence indicates that heterogeneity in ribosome composition can give rise to specialized functions. Until now, research mainly focused on differences in core ribosomal proteins and associated factors. The effect of posttranslational modifications has not been studied systematically. Analyzing ribosome heterogeneity is challenging because individual proteins can be part of different subcomplexes (40S, 60S, 80S, and polysomes). Here we develop polysome proteome profiling to obtain unbiased proteomic maps across ribosomal subcomplexes. Our method combines extensive fractionation by sucrose gradient centrifugation with quantitative mass spectrometry. The high resolution of the profiles allows us to assign proteins to specific subcomplexes. Phosphoproteomics on the fractions reveals that phosphorylation of serine 38 in RPL12/uL11, a known mitotic CDK1 substrate, is strongly depleted in polysomes. Follow-up experiments confirm that RPL12/uL11 phosphorylation regulates the translation of specific subsets of mRNAs during mitosis. Together, our results show that posttranslational modification of ribosomal proteins can regulate translation. Graphical Abstract Highlights • High-resolution polysome proteome profiling across ribosomal subcomplexes • Core ribosome of monosomes and polysomes does not differ significantly • Systematic identification of proteins associated with actively translating ribosomes • Phosphorylation of RPL12/uL11 regulates translation during mitosis Multiple regulatory layers shape gene expression. Imami et al. show that protein production in mitosis can be regulated by phosphorylation of the ribosomal protein RPL12/uL11. [ABSTRACT FROM AUTHOR]