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Apoptosis and phagocytosis of apoptotic cells are crucial processes. At best the phagocytic machinery detects and swallows all apoptotic cells in a way that progression to secondary necrosis is avoided. Otherwise, inflammation and autoimmune diseases may occur. Most apoptotic cells are phagocytosed instantaneously in a silent fashion; however, some dying cells escape their clearance. If the cells are not cleared early, they lose membranes due to extensive shedding of membrane surrounded vesicles (blebbing) and shrink. It is unclear how apoptotic cells compensate their massive loss of plasma membrane. Here, we demonstrate that endoplasmic reticulum- (ER) resident proteins (calnexin, the KDEL receptor and a dysfunctional immunoglobulin heavy chain) were exposed at the surfaces of shrunken late apoptotic cells. Additionally, these cells showed an increased binding of lectins, which recognize sugar structures predominantly found as moieties of incompletely processed proteins in ER and Golgi. In addition the ER resident lipophilic ER-Tracker™ Blue-White DPX, and internal GM1 were observed to translocate to the cell surfaces during late apoptosis. We conclude that during blebbing of apoptotic cells the surface membrane loss is substituted by immature membranes from internal stores. This mechanism explains the simultaneous appearance of preformed recognition structures for several adaptor proteins known to be involved in clearance of dead cells.Cell Death and Differentiation (2007) 14, 733–742. doi:10.1038/sj.cdd.4402066; published online 15 December 2006 [ABSTRACT FROM AUTHOR]

The adaptor protein Swiprosin-1/Efhd2 that we identified recently in B cell lipid rafts exhibits its highest expression in immature B cells. Swiprosin-1/Efhd2 consists of a proline rich domain, two EF-hands and a C-terminal coiledcoil domain. Knock-down of Swiprosin-1/Efhd2 in an immature B cell line with a shRNA directed against the 3'-UTR (WEHI231shSw1) impaired spontaneous and BCR-elicited apoptosis. Due to co-clustering of a Swiprosin-1-EGFP fusion protein with the B-cell receptor we expected Swiprosin-1 to be a player in proximal BCR signaling. Indeed, downregulation of Swiprosin-1 attenuated total tyrosine phosphorylation and diminished BCR induced intracellular calcium flux. In contrast, WEHI231 cells ectopically expressing Swiprosin-1 showed increased, but shortened total tyrosine phosphorylation at early time points, as well as increased BCR induced calcium flux. Concomitantly, transient re-expression of Swiprosin-1 in WEHI231shSW1 cells restored BCR-induced calcium flux. These data suggested interactions of Swiprosin-1 with central elements of the BCR signaling cascade. In fact, GST-pulldown experiments showed that Swiprosin-1 can interact with tyrosine-phosphorylated proteins of ~145 and 70 kDa. With this assay we identified the protein tyrosine kinase Syk in its phosphorylated form as interaction partner of Swiprosin-1. The interaction of pSyk with Swiprosin-1 is mediated by the C-terminal part of Swiprosin-1 because a deletion mutant of Swiprosin-1 lacking the EF-hands and the coiled-coil domain of Swiprosin-1 did not show any interaction with pSyk. We next tested the kinase activity of Syk as a function of Swiprosin-1 levels. Interestingly, immune complex kinase assays showed that BCR-induced Syk activity was reduced in WEHI231shSw1 cells Hence, Swiprosin-1 could regulate proximal BCR signaling through maintenance of Syk activity. [ABSTRACT FROM AUTHOR]

Carma-1 is required for B cell receptor-/CD40- and T cell receptor-/CD28-induced B- and T-cell activation via JNK and NF-βB. In B cells, Carma-1 becomes phosphorylated by PKCβ, leading to its oligomerization. Subsequent Bcl10 binding induces IKKβ-activation and, thereby, canonical NF-κB signalling. Despite these findings it is still unknown how exactly Carma-1 is connected to the plasma membrane and to the IKK-complex. Therefore, we purified Carma-1 complexes from mouse CH12 B cells using anti-Carma-1 affinity columns. Mass spectrometric analyses of the column eluates demonstrated the presence of Carma-1 as well as three previously uncharacterized adaptor proteins in B cells, one of which was the Trk-fused gene (Tfg), an adaptor protein containing PB1 and coiledcoil domains. Whereas Tfg was originally identified as fusion partner of oncogenic Trk tyrosine kinase mutants, the normal cellular homologue of Tfg has so far not been described in B cells. However, Tfg has been shown in other systems to interact with IKKγ and to enhance TNF-induced NF-κB activation. Tfg and Carma-1 co-localized at the plasma membrane and perinuclear structures in B cells. We further corroborated the interactions of Tfg, IKKγ and Carma-1 by Blue Native gel electrophoresis, where Carma-1 and Tfg formed a 0.7-1 MDa complex. Ectopic expression of Tfg increased the molecular mass of IKKγ complexes, fused IKKγ, Bcl10 and Carma-1 complexes to a ~2 MDa complex, and increased basal and CD40-induced canonical activity of NF-κB and IKKβ. In contrast, shRNA-mediated silencing of Tfg decreased CD40-induced IKKβ activity. Very interestingly, in primary B cells, highest expression of Tfg was detected in marginal zone and B1 B cells, and Carma-1 and Tfg formed complexes in these B cells. Since Carma-1 is required for marginal zone B cell and B1 B cell development, we suggest that a functional interaction between Carma-1 and Tfg contributes to development and maintenance of these cells by means of canonical NF-κB signals. [ABSTRACT FROM AUTHOR]

A correction to the article "After shrinkage apoptotic cells expose internal membrane-derived epitopes on their plasma membranes," is presented.

The naturally occurring mutation E484D in the spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can render viral entry ACE2 independent and imdevimab resistant. Here, we investigated whether the cellular proteins ASGR1, DC-SIGN, and TMEM106B, which interact with the viral S protein, can contribute to these processes. Employing S protein-pseudotyped particles, we found that expression of ASGR1 or DC-SIGN jointly with TMEM106B allowed for robust entry of mutant E484D into otherwise non-susceptible cells, while this effect was not observed upon separate expression of the single proteins and upon infection with SARS-CoV-2 wild type (WT). Furthermore, expression of ASGR1 or DC-SIGN conferred ACE2 independence and imdevimab resistance to entry of mutant E484D but not WT, and entry under those conditions was dependent on endogenous TMEM106B. These results suggest that engagement of certain cellular lectins can direct SARS-CoV-2 mutant E484D to an ACE2-independent, TMEM106B-dependent entry pathway that is not inhibited by imdevimab.IMPORTANCEThe interaction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with the ACE2 receptor determines the viral cell tropism and is the key target of the neutralizing antibody response. Here, we show that SARS-CoV-2 with a single, naturally occurring mutation in the spike protein, E484D, can use the cellular lectins ASGR1 and DC-SIGN in conjunction with TMEM106B for ACE2-independent entry and evasion of therapeutic antibodies. These results suggest that engagement of cellular lectins might modulate target cell choice of SARS-CoV-2 and might allow evasion of certain neutralizing antibodies.

The potency of antibody neutralization in cell culture has been used as the key criterion for selection of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) for clinical development. As other aspects may also influence the degree of protection in vivo , we compared the efficacy of two neutralizing monoclonal antibodies (TRES6 and 4C12) targeting different epitopes of the receptor binding domain (RBD) of SARS-CoV-2 in a prophylactic setting in rhesus monkeys. All four animals treated with TRES6 had reduced viral loads in the upper respiratory tract 2 days after naso-oropharyngeal challenge with the Alpha SARS-CoV-2 variant. Starting 2 days after challenge, mutations conferring resistance to TRES6 were dominant in two of the rhesus monkeys, with both animals failing to maintain reduced viral loads. Consistent with its lower serum neutralization titer at the day of challenge, prophylaxis with 4C12 tended to suppress viral load at day 2 less efficiently than TRES6. However, a week after challenge, mean viral loads in the lower respiratory tract in 4C12-treated animals were lower than in the TRES6 group and no mutations conferring resistance to 4C12 could be detected in viral isolates from nasal or throat swabs. Thus, genetic barrier to resistance seems to be a critical parameter for the efficacy of prophylaxis with monoclonal antibodies against SARS-CoV-2. Furthermore, comparison of antibody concentrations in respiratory secretions to those in serum shows reduced distribution of the 4C12 antibody into respiratory secretions and a delay in the appearance of antibodies in bronchoalveolar lavage fluid compared to their appearance in secretions of the upper respiratory tract.IMPORTANCEMonoclonal antibodies are a powerful tool for the prophylaxis and treatment of acute viral infections. Hence, they were one of the first therapeutic agents licensed for the treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Oftentimes, the main criterion for the selection of antibodies for clinical development is their potency of neutralization in cell culture. By comparing two antibodies targeting the Spike protein of SARS-CoV-2, we now observed that the antibody that neutralized SARS-CoV-2 more efficiently in cell culture suppressed viral load in challenged rhesus monkeys to a lesser extent. Extraordinary rapid emergence of mutants of the challenge virus, which had lost their sensitivity to the antibody, was identified as the major reason for the reduced efficacy of the antibody in rhesus monkeys. Therefore, the viral genetic barrier to resistance to antibodies also affects their efficacy., Competing Interests: The authors declare no conflict of interest.

Signals initiated by the precursor B cell receptor (pre-BCR) are critical for B cell progenitors to mature into precursor B cells. The pre-BCR consists of a homodimer of microH chains, the covalently associated surrogate L (SL) chain composed of VpreB and lambda5, and the transmembrane signal molecules Ig(alpha) and Igbeta. One way to explain how maturation signals are initiated in late progenitor B cells is that the pre-BCR is transported to the cell surface and interacts from there with a ligand on stroma cells. To address this hypothesis, we first produced soluble Fab-like pre-BCR and BCR fragments, as well as SL chain, in baculovirus-infected insect cells. Flow cytometry revealed that, in contrast to Fab-like BCR fragments, the soluble pre-BCR binds to the surface of stroma and several other adherent cell lines, but not to B and T lymphoid suspension cells. The specific binding of the soluble pre-BCR to stroma cells is saturable, sensitive to trypsin digestion, and not dependent on bivalent cations. The binding of pre-BCR seems to be independent of the H chain of IgM (microH chain), because SL chain alone was able to interact with stroma cells. Finally, soluble pre-BCR specifically precipitated a 135-kDa protein from ST2 cells. These findings not only demonstrate for the first time the capacity of a pre-BCR to specifically bind to a structure on the surface of adherent cells, but also suggest that the pre-BCR interacts via its SL chain with a putative ligand on stroma cells.

The assembly of a pre-B cell receptor (pre-BCR) composed of an Ig mu heavy chain (mu H-chain), the surrogate light (SL) chain, and the Ig alpha/beta dimer is critical for late pro-B cells to advance to the pre-B cell stage. By using a transgenic mouse model, in which mu H-chain synthesis is solely driven by a tetracycline-controlled transactivator, we show that de novo synthesis of mu H-chain in transgenic pro-B cells not only induces differentiation but also proliferation. This positive effect of mu H-chain synthesis on proliferation requires the presence of SL chain and costimulatory signals provided by stromal cells or IL-7. We conclude that pre-BCR signaling induces clonal expansion of early pre-B cells.

The human lambda 5 (hu lambda 5) gene is the structural homologue of the murine lambda 5 (m lambda 5) gene and is transcriptionally active in pro-B and pre-B lymphocytes. The lambda 5 and VpreB polypeptides together with the Ig mu H chain and the signal-transducing subunits, Ig alpha and Ig beta, comprise the pre-B cell receptor. To further investigate the pro-B/pre-B-specific transcription regulation of hu lambda 5 in an in vivo model, we generated mouse lines that contain a 28-kb genomic fragment encompassing the entire hu lambda 5 gene. High levels of expression of the transgenic hu lambda 5 gene were detected in bone marrow pro-B and pre-B cells at the mRNA and protein levels, suggesting that the 28-kb transgene fragment contains all the transcriptional elements necessary for the stage-specific B progenitor expression of hu lambda 5. Flow cytometric and immunoprecipitation analyses of bone marrow cells and Abelson murine leukemia virus-transformed pre-B cell lines revealed the hu lambda 5 polypeptide on the cell surface and in association with mouse Ig mu and mouse VpreB. Finally, we found that the hu lambda 5 transgene is able to rescue the pre-B lymphocyte block when bred onto the m lambda 5-/- background. Therefore, we conclude that the hu lambda 5 polypeptide can biochemically and functionally substitute for m lambda 5 in vivo in pre-B lymphocyte differentiation and proliferation. These studies on the mouse and human pre-B cell receptor provide a model system to investigate some of the molecular requirements necessary for B cell development.

Ig gene rearrangements could generate V(H)-D-J(H) joining sequences that interfere with the correct folding of a mu-chain, and thus, its capability to pair with IgL chains. Surrogate light (SL) chain might be the ideal molecule to test the capacity of a mu-chain to pair with a L chain early in development, in that only pre-B cells that assemble a membrane mu-SL complex would be permitted to expand and further differentiate. We have previously identified two SL chain nonpairing V(H)81X-mu-chains with distinct V(H)-D-J(H) joining regions. Here, we show that one of these V(H)81X-mu-chains does not rescue B cell development in J(H) knock-out mice, because flow cytometric analysis of bone marrow cells from V(H)81X-mu transgenic J(H) knock-out mice revealed normal numbers of pro-B cells, but essentially no pre-B and surface IgM+ B cells. Immunoprecipitation analysis of transfected pre-B and hybridoma lines revealed that the same mu-chain fails to pair not only with SL chain but also with four distinct kappa L chains. These findings demonstrate that early pre-B cells are selected for maturation on the basis of the structure of a mu-chain, in particular its V(H)-D-J(H) joining or CDR3 sequence, and that one mechanism for this selection is the capacity of a mu-chain to assemble with SL chain. Therefore, we propose a new function of SL chain in early B cell development: SL chain is part of a quality control mechanism that tests a mu-chain for its ability to pair with conventional L chains.

Our studies examined the expression and DNA binding activity of myocyte enhancer factor 2 (MEF2A-D) transcription factors in lymphopoietic tissues, cell lines, and primary lymphocytes. Our analyses demonstrate that mef2C expression is restricted to B cells within the lymphocyte lineage. Using in situ hybridization, mef2C is detected in foci in fetal liver and postnatal thymic medulla, and both mef2B and mef2C are expressed in areas of the postnatal spleen and lymph node that also express kappa light chain (Ckappa), a B cell-specific marker. Reverse transcriptase-PCR (RT-PCR) analyses demonstrate that all mef2 family members are expressed in B cell lines, and all except mef2C are expressed in T cell lines. Immunoblot analyses of cell lines and primary thymic and splenic lymphocytes show that MEF2C and MEF2D proteins are expressed in B cells and that MEF2D is expressed in T cells; however, MEF2A protein is not detected in lymphocytes. Electrophoretic mobility shift assays (EMSA) demonstrate that B cell lines have MEF2C-containing, MEF2-specific DNA binding complexes whereas T cells do not. Our data is the first to describe mef2C expression in the lymphocyte lineage, and this finding suggests possible roles for MEF2C activity in B cell development and function.

CD30 engagement in human lymphoid cells induces pleiotropic cellular responses that affect cellular viability and proliferation, cytokine production, and nuclear factor kappaB (NF-kappaB) nuclear translocation. Studies examining the molecular basis for this pleiotropism thus far have relied on the use of antibodies and cells transfected with CD30L to trigger CD30, two methods of receptor induction that present important limitations: antibodies are not physiological receptor-triggering molecules and CD30L transfectants induce high background intracellular signaling in the cells under study. We have generated and expressed a functional soluble human CD30L molecule (sCD30L/CD8alpha) comprised of the extracellular domain of human CD30L fused to the extracellular domain of the human CD8alpha chain. Immunoprecipitation and Western blot analysis of sCD30L/CD8alpha revealed the existence of at least two forms of sCD30L/CD8alpha, which exhibited molecular sizes consistent with the existence of monomeric and trimeric forms of the molecule. Binding analyses performed using a soluble CD30 fusion protein (sCD30/gamma1) confirmed the ability of sCD30L/CD8alpha to bind to CD30. Functionally, immobilized sCD30L/CD8alpha-induced cell death in the CD30-expressing lines Karpas-299 and HDLM-2 and reduced proliferative levels in Karpas-299; these effects were inhibitable by the addition of sCD30/gamma1. These studies demonstrate the utility of sCD30L/CD8alpha in characterizing the normal function of CD30L and CD30 and indicate the natural ability of soluble forms of CD30L to trimerize.

Leflunomide is an immunosuppressive drug capable of inhibiting T and B cell responses in vivo. A number of studies demonstrate that leflunomide functions both as a pyrimidine synthesis inhibitor and as a tyrosine kinase inhibitor. We previously reported that leflunomide inhibits LPS-stimulated B cell proliferation, cell cycle progression, and IgM secretion. This inhibition can be reversed by the addition of exogenous uridine, suggesting that leflunomide functions as a pyrimidine synthesis inhibitor in B cells. We report here that while the addition of uridine restored proliferation and IgM secretion to leflunomide-treated LPS-stimulated B cells, as determined by metabolic labeling and immunoprecipitation, it did not completely restore secretion of IgG Ab. We hypothesized that leflunomide inhibits LPS-induced IgG secretion by inhibiting tyrosine kinase activity required for isotype switch. We tested this hypothesis in a well-defined model of isotype switch, LPS plus IL-4 induction of IgG1. Leflunomide inhibited IgG1 secretion in this model in a dose-dependent manner. The signal transduction pathway utilized by IL-4 to induce IgG1 involves tyrosine phosphorylation of the IL-4 receptor, JAK1, JAK3, and STAT6 proteins induced by IL-4 binding to the IL-4R. Leflunomide diminished the tyrosine phosphorylation of JAK3 and STAT6 in the absence or presence of uridine. In gel mobility shift studies, STAT6 binding to the STAT6 DNA binding site in the IgG1 promoter decreased in the presence of leflunomide or leflunomide plus uridine. Taken together, these data suggest that leflunomide acts as a tyrosine kinase inhibitor to block IgG1 production.

Levels of most nonsense mRNAs are normally reduced in prokaryotes and eukaryotes when compared with that of corresponding functional mRNAs. Genes encoding polypeptides that selectively reduce levels of nonsense mRNA have so far only been identified in simple eukaryotes. We have now cloned a human cDNA whose deduced amino acid sequence shows the highest degree of homology to that of UPF1, a bona fide Saccharomyces cerevisiae group I RNA helicase required for accelerated degradation of nonsense mRNA. Based on the total sequence of the shorter yeast UPF1 protein, the overall identity between the human protein and UPF1 is 51%. Besides NTPase and other RNA helicase consensus motifs, UPF1 and its human homolog also share similar putative zinc finger motifs that are absent in other group I RNA helicases. Northern blot analysis with the human cDNA probe revealed two transcripts in several human cell lines. Further, antibodies raised against a synthetic peptide of the human polypeptide detected a single 130 kDa polypeptide on Western blots from human and mouse cells. Finally, immunofluorescence and Western blot analyses revealed that the human and mouse polypeptides, like yeast UPF1, are expressed in the cytoplasm, but not in the nucleus. We have thus identified the first mammalian homolog of yeast UPF1, a protein that regulates levels of nonsense mRNA, and we tentatively name this protein human HUPF1 (for human homolog of UPF1).