Molecular Immunology

Behcets disease (BD) is a systemic inflammatory disease. It is considered as an autoinflammatory disease triggered by innate immunity rather than adaptive immunity. Human leukocyte antigen-B51 (HLA-B51) is the strongest genetic factor associated with BD. This study investigated how HLA class 1 molecules interact with innate immune cells and induce cytokine secretion. For this purpose, 293T cells transfected with a plasmid encoding HLA-B51 were cultured with natural killer (NK) cells obtained from healthy human donors. Within 24 h, the concentrations of interleukin-4 (IL-4), IL-8, and interferon-{gamma} (IFN-{gamma}) in the medium increased, indicating that NK cells secreted cytokines without undergoing cellular expansion for cytolysis. NK cells stimulated by nonself HLA-B51 produced IFN-{gamma} levels comparable to those produced by NK cells stimulated by self HLA-B51. NK cells carrying HLA-B51 were accurately recognized by overexpressing HLA-B51 on 293T cells. Moreover, ample intracellular IFN-{gamma} levels were detected in NK cells after stimulation with phorbol 12-myristate-13-acetate (PMA) plus ionomycin. KLRK1 (CD314)-positive cells mainly primarily accounted for IFN-{gamma}-producing cells, whereas KLRK1-negative cells did not. In contrast, both NCR1 (CD335)-positive and -negative cells contributed to IFN-{gamma} production. We next investigated whether HLA-B51 on the surface of 293T cells stimulates KLRK1 as a ligand causing IFN-{gamma} secretion. In masking experiments using anti-KLRK1 antibodies, NK cells with high levels of cell surface KLRK1 decreased the production of IFN-{gamma}. Conversely, human NK cell line KHYG1 cells also produced IFN-{gamma} in culture with 293T cells, but did not increase IFN-{gamma} through HLA-B51 stimulation. The mRNA expression of the signal adaptor protein HCST (DAP10) in KHYG1 cells was lower than that in NK cells, whereas the relative expression of IL-2RA in KHYG1 cells was higher than that in NK cells. These findings suggest that HLA-B51 can interact with KLRK1 on the NK cells inducing IFN-{gamma} secretion, whereas IL-2 signals outweigh HLA-51 stimulation in KHYG1 cells.

Lymphocyte-extracellular matrix (ECM) interactions occur intermittently throughout the lymphocytes life cycle. Alterations in blood insulin levels following feeding modulates naive lymphocyte trafficking and adhesion to fibronectin via a pathway involving insulin-like growth factor-1 receptor (IGF-1R), phospholipase C gamma 1 (PLC-{gamma}1) and {beta}2 integrin activation. Lymphocytes exert traction forces, on the ECM during the process of extravasation. While these forces are essential for several homeostatic processes, the role of insulin in modulating lymphocyte-derived traction forces upon ECM adhesion is unknown. The aim of the current study was to investigate the effect of insulin on the traction generated by lymphocytes when adhered onto a fibronectin-coated substrate. Jurkat T-cells were placed on a fibronectin layer (50{micro}g/ml, 100{micro}m thickness) coated on polyacrylamide gels of stiffness 400Pa with red fluorescence beads as fiduciary markers. The cellular force generated by Jurkat T-cells was mapped using traction force microscopy. To elucidate the role of PLC-{gamma}1 in cellular force generation, the traction of Jurkat T-cells lacking PLC-{gamma}1, as well as those of a knockout cell where PLC-{gamma}1 was restored were quantified and compared with wild-type Jurkat T-cells. Lack of PLC-{gamma}1 attenuated adhesion when compared to wild-type Jurkat T-cells. Additionally, the traction force generated by each cell type decreased with increasing concentration of extracellular calcium. Treatment of adherent Jurkat T-cells with insulin increased traction in lower extracellular calcium condition while a dip was observed when a high extracellular calcium was present, in comparison to the untreated cells. However, the effect of insulin treatment was lost in the case of Jurkat T-cells lacking PLC-{gamma}1. Together these results indicate that insulin regulates traction force generated by adherent Jurkat T-cells via a process involving PLC-{gamma}1, in a calcium dependent manner.

Junctional adhesion molecule-like (JAML) is an adhesion molecule known to promote T cell activation and T cell-mediated tumor rejection. In the current study, we show that JAML expression is enriched on mouse intratumoral NK cells compared with splenic NK cells. JAML+ NK cells were associated with tissue residency and co-expressed the immune checkpoints PD-1 and LAG3. JAML expression could be induced on splenic NK cells by IL-2 and further enhanced by IL-21. JAML levels were inversely correlated with inhibitory signaling, as NK cells expressing self-recognizing Ly49 receptors had reduced JAML expression, suggesting regulation of JAML expression by MHC class I molecules. Interaction with the JAML ligand CXADR also reduced JAML surface expression, indicating that tumor-mediated membrane stripping may represent a mechanism of immunoediting. Although JAML RNA transcripts were detectable in human NK cells, JAML protein was found only intracellularly. Together, these findings identify the JAML-CXADR interaction as a potential regulatory pathway in NK cell-mediated killing of tumors.

T cell activation is associated with, and dependent upon, the upregulation of amino acid uptake from the extracellular environment. Uptake of the non-essential amino acid asparagine (Asn) is mediated via amino transporters such as Slc1a5 whilst Asn can be synthesized within cells that express asparagine synthetase (ASNS). Previous work demonstrated that initial activation of CD8+ T cells is perturbed in the absence of Asn, whereas effector cytotoxic T cells cells upregulate ASNS and lose their dependence on Asn uptake. By contrast, less is known of the role of Asn uptake and ASNS in CD4+ T cell responses. Here we demonstrate that CD4+ T cells are more reliant than CD8+ T cells on Asn uptake for initial activation, differentiation, metabolic reprogramming and regulation of autophagy. These phenotypes are associated with enhanced expression of ASNS in CD8+ as compared to CD4+ effector T cells.

Alternative pathways of antigen presentation are crucial in different immunological contexts such as autoimmunity and anti-microbial defense. Among these pathways, autophagy has a central role in delivering cytosolic substrates to the MHC class II compartment. However, its contribution to endogenous MHC class II presentation was only demonstrated for a few antigens. Here we focused our study on the contribution of autophagy to the peptidome of one major allele of the HLA-DR shared epitope, HLA DRB1*04:01 conferring the greatest risk factor for the development of rheumatoid arthritis (RA). We provide an extensive qualitative and quantitative mass spectrometry analysis of the autophagy related MHC class II peptide repertoire of the human DRB1*04:01 allele. A fraction of peptides representing 30% of the repertoire differ profoundly between autophagy sufficient and deficient cells. Our analysis demonstrates that autophagy contributes to MHC class II presentation of peptides from seven described RA autoantigens, the majority of them being related to the ER folding and stress response (calreticulin, calnexin, the 78 kDa glucose-regulated protein (GRP78)-also known as binding immunoglobulin protein (BiP) and several protein from the heat-shock-protein 70 family). Our results correlate with an increased activation of autophagy, in situ, in synovial biopsies and synovial fibroblast (SF) of RA patients. We could further show that SF upregulate MHC class II and present peptides from autophagy related auto-antigens to CD4 T cells from RA patients. Our finding identifies autophagy as a potential process that could contribute to the break of peripheral tolerance during RA.

The variable regions of antibody heavy chains (HCs) and light chains (LCs) are assembled by V(D)J recombination in progenitor B cells to generate an immense repertoire of primary B cell receptors (BCRs), the precursors of affinity-matured antibodies secreted in response to antigen stimulation. The complementarity determining region (CDR) 1, 2 and 3 of antibodies are the principal antigen contact sites, with CDR3 being highly diverse due to V(D)J junctional diversification by terminal deoxynucleotidyl transferase (TdT). The HC CDR3 (CDR H3) plays a prominent role in broadly neutralizing antibodies (bnAbs) against the human immunodeficiency virus-1 (HIV-1). BnAbs overcome the genetic heterogeneity of HIV-1 by recognizing conserved epitopes on the HIV-1 Envelope (Env) protein. Reaching these targets requires long CDR H3s that penetrate through the glycan shield or other structural hindrances on the Env protein. The shortage of human antibodies with such long CDR H3s poses a challenge for bnAb elicitation by vaccination. To aid immunogen design, we generated six mouse models for inducing bnAbs against particular HIV-1 Env epitopes. In each mouse model, we integrated the human HC VH, D, JH segments and LC VL, JL segments of a bnAb lineage into the mouse HC and LC loci, with each set engineered to undergo V(D)J recombination and to generate diverse human HC and LC variable regions. Combined action of V(D)J recombination and TdT- mediated junctional diversification in developing B cells generated a range of human variable region exons for a given bnAb lineage that contained highly diverse CDR3s in each mouse model. Moreover, these repertoires contained humanized antibodies that had bnAb-like long CDR H3s that could potentially serve as bnAb precursors. Therefore, these mouse models can be used to test whether immunogens can induce bnAbs from rare and diverse precursors in a complex antibody repertoire. Author summaryThe human immunodeficiency virus-1 (HIV-1) is the causative agent of acquired immunodeficiency syndrome (AIDS). An efficacious HIV-1 vaccine is needed to control the AIDS pandemic. However, in multiple clinical trials, vaccine candidates failed to confer protection against HIV-1 infection. The lack of efficacy is mainly due to the enormous heterogeneity of HIV-1 strains in human circulation. A breakthrough in the field has been the identification of broadly neutralizing antibodies (bnAbs) in a small fraction of HIV-1 infected patients. Because these antibodies recognize conserved targets on different HIV-1 strains, they can inhibit a wide spectrum of viruses. Eliciting HIV-1 bnAbs is a top priority for vaccine development. For this endeavor, a major difficulty is that most bnAbs have unusual properties. To induce bnAbs, vaccines must be highly selective for rare human antibodies that can develop into bnAbs. To facilitate this effort, we have generated a panel of mouse models that can produce potential precursors for major types of HIV-1 bnAbs. We engineered mouse models to produce diverse precursors in complex antibody repertoires, which mimic the challenging condition in human vaccination. These mouse models can be used to assess and optimize vaccine candidates at the preclinical stage.

Bordetella produce a wide array of virulence factors. These factors are involved in bacterial colonization and evasion of immune defenses. Our recent studies revealed that the bacteria produce an exoglycan, Bordetella oligosaccharide (BOS). B. petrii is the evolutionary early divergent species of the genus Bordetella. This study has focused on the investigation of two B. petrii type strains: clinical and environmental. We employed nuclear magnetic resonance (NMR) analyses to elucidate the structural differences between their lipopolysaccharides. Our findings revealed that the LPS of clinical B. petrii strain comprises a hexasaccharide unit, that was structurally identical to the BOS. This form of LPS is only a minor population in the bacterial outer membrane of the environmental strain. In addition to the cell-bound BOS, its secreted glycoform was also found in growth media of B. petrii. Anti-BOS neoglycoconjugate antibodies cross-reacted with B. petrii LPS. This suggest that the newly identified BOS associated with B. petrii PS would be a potential vaccine element against Bordetella.

NLRP3 is a cytosolic pattern recognition receptor that controls the formation of an inflammasome, a multimolecular complex that cleaves the pro-inflammatory cytokines interleukin-1{beta} and interleukin-18 into their bioactive forms. NLRP3 has been widely assumed to be conserved across vertebrates, suggesting it plays an indispensable role within the vertebrate innate immune system. Here, we used gene synteny, phylogeny, and structural analysis to examine the evolutionary origins of NLRP3 in greater detail. Our analysis revealed that modern NLRP3, defined by gene synteny and structural features, is unique to the eutherian lineage. Non-eutherian NLRP genes do not share synteny with the eutherian NLRP3 locus and lack conservation of key features including disc forming residues, cage interfaces, and membrane binding regions. NLRP3s characteristic regulatory architecture therefore appears to have evolved after eutherians split from marsupials 160 million years ago. These findings have important implications for understanding the beneficial roles of NLRP3 signalling and suggest that enhanced regulatory control of NLRP3 activity arose in response to distinct aspects of eutherian physiology.

Sarcoidosis is a heterogenous disease of unknown etiology characterized by non-caseating granulomas. Disease prevalence and presentation vary significantly by ancestry and ranges from acute, self-resolving disease to severe, chronic disease. Following previous reports suggesting B cells in the development and pathogenesis of sarcoidosis, we present here results of single-cell RNA sequencing, supporting B cell involvement in sarcoidosis through altered immediate early response, rewiring of MAPK signaling, and ancestry-specific preferential expansion of B cell receptors. Peripheral blood mononuclear cells were obtained from individuals of African or European Ancestry (AA and EA, respectively) including 48 healthy controls, 59 sarcoidosis patients, and 28 systemic lupus erythematosus (SLE) patients. SLE samples were used as a disease control. Differential expression analysis highlighted many differentially expressed genes (DEGs) with almost 5x more in the AA sarcoidosis versus AA control group compared to the EA sarcoidosis versus EA control group. B cells had the most DEGs of all cell types and expression patterns were similar between ancestries, however, sarcoidosis had an opposite transcription pattern than SLE, demonstrating an alternative immune response to acute activation than that seen in a prototypical autoinflammatory disease. This trend was maintained when examining specialized B cell subsets, with the most pronounced effect in the AA sarcoidosis versus AA control comparison. Our results strongly support further investigation of the role of humoral immune response in sarcoidosis and the potential to highlight patient groups likely to benefit from existing B cell therapies.

In germinal centers, activated B cells modify their antigen receptors through somatic hypermutation (SHM), followed by antigenic selection that favors expansion of high affinity B cells. The affinity maturation process is critical for development of broadly neutralizing antibodies (bnAbs) against the human immunodeficiency virus-1 (HIV-1). BnAbs have been isolated from some people living with HIV-1. Because these antibodies target conserved epitopes of the HIV-1 Envelope (Env) protein, they inhibit a broad spectrum of viruses. Eliciting bnAbs by vaccination is a top priority for HIV-1 prevention, but reproducing the lengthy maturation of bnAbs is a major challenge. The problem is typified by VRC01 class antibodies, which recognize the CD4 binding site of HIV-1 Env protein. To reach the CD4 binding site, antibodies need to navigate through adjacent glycans. Accommodating the glycans requires multiple SHMs in germinal center (GC) B cells, including infrequent events. For this reason, VRC01 vaccine development often stalls at this point. We have generated a mouse model aimed at providing a potential solution for navigating this vaccine design impediment. To this end, we made a mouse model that expresses a stalled VRC01 intermediate conditionally in GC B cells. This system has three advantages: 1) direct expression of the intermediate obviates prior immunization steps, thereby shortening the immunization scheme; 2) the conditional expression system bypasses tolerance control checkpoints that sometimes delete B cells expressing bnAbs; 3) the intermediate responds to immunization in GCs, the physiological site of affinity maturation. With this model, we established an immunization method to mature the VRC01 intermediate into heterologous neutralizing antibodies against viruses with a native glycan shield. Since high mutation load is common among bnAbs, the germinal center conditional expression system could provide a general tool for boost immunogen design to overcome roadblocks in the maturation pathway. Author summaryIn response to antigenic stimulation, cognate B cells become activated and form germinal centers in lymphoid tissues. Germinal center B cells modify their antigen receptors through somatic hypermutation (SHM) of immunoglobulin variable region gene exons, with antigen selecting for high affinity B cells by providing survival advantage. This mechanism accounts for antibody affinity maturaton over the gradual course of an immune response. Affinity maturation is critical for generating potent, neutralizing antibodies against diverse strains of the human immunodeficiency virus-1 (HIV-1). These broadly neutralizing antibodies (bnAbs) are heavily mutated, reflecting lengthy affinity maturation over years of chronic infection. Recapitulating the affinity maturation process is a major challenge for bnAb induction by vaccination. In immunization experiments, bnAb development often stalls at rate limiting steps that involve infrequent, but functionally important, mutational events. Overcoming such obstacles requires boost immunogens that can stimulate the stalled B cells to acquire the requisite mutations. To this end, we recapitulated the maturation arrest of a bnAb lineage by expressing a stalled antibody in mouse germinal center B cells. Using this mouse model, we developed boost immunization conditions that advanced the antibody maturation beyond a roadblock to attain neutralizing activities against heterogenous viruses.

B-cell leukemia/lymphoma 11B (BCL11B), despite its name, is a key regulator of T-cell development, specification, and T-cell malignancies. BCL11B contains a bipartite DNA binding domain composed of two C2H2 zinc finger arrays: low-affinity ZF2-3 and high affinity ZF4-6. These arrays function as homotypic modules that recognize similar six-nucleotide motifs, TG(O_SCPLOWNC_SCPLOW)CC(O_SCPLOWCC_SCPLOWO_SCPCAP/C_SCPCAPO_SCPLOWTC_SCPLOWO_SCPCAP/C_SCPCAPO_SCPLOWAC_SCPLOW), as seven of the eight DNA base-contacting residues are conserved between them. The most conserved interactions involve GG dinucleotides, contacted by arginine and lysine residues at key base-interacting positions in ZF3 and ZF5. The two ZF arrays are connected by a long [~]300-residue linker that provides flexibility in how the arrays engage DNA, allowing ZF2-3 and ZF4-6 binding to the same or opposite strands with variable orientation, spacing and positioning along the DNA. This extended linker is enriched in serine/threonine, acidic residues (aspartate/glutamate), and structural residues (glycine/proline), providing additional layers of transcriptional regulation possibly through post-translational modification, electrostatic modulation, and/or condensate formation. We also examined six missense mutations in base-interacting residues, that are associated with neurodevelopmental disorders. Substitutions replacing bulky, positively charged arginine or lysine with smaller or hydrophobic residues likely reduce DNA-binding affinity and/or specificity, whereas substitutions between asparagine and lysine may alter base recognition preferences.

Mechanosensing involves proteins detecting mechanical changes in the cytoskeleton or at cell adhesion sites. These interactions initiate signaling cascades that produce biochemical effects such as post-translational modifications or cytoskeletal rearrangements. Filamin is a ubiquitous mechanosensing protein that binds actin filaments and senses pulling forces within the cytoskeleton. Drosophila Filamin (Cheerio) is structurally similar to mammalian Filamin, with roles in egg chamber development, embryo cellularization, and integrity of muscle attachment sites and Z discs in Drosophila indirect flight muscles (IFMs). Here we report a potential novel binding partner of Drosophila Filamins: the death-associated protein kinase Drak that functions as a myosin light chain kinase. We found that Drak biochemically bound to an open mutant of Filamin that resembles the mechanically activated form partially bound to wild type Filamin and did not bind to closed mutant of Filamin. The interaction site was mapped to the intrinsically unfolded C-terminal region of Drak. To study the functional role of Drak-Filamin interaction, we studied two developmental events where Drak has been earlier shown to be expressed and where Filamin also functions: early embryonic cellularization and indirect flight muscle development at pupal stages. We found partial colocalization between Drak-GFP and Filamin-mCherry during the initiation of cellularization furrow, and at the time of myotube attachment site maturation in tendon cells. However, functionally we could not show direct correlation between Filamin and Drak. Our studies reveal interesting new expression patterns of Drak during Drosophila development and provide detailed information about Filamin localization during IFM development.

Immune memory responses are rapid and qualitatively distinct from primary responses. They typically develop in the presence of antigen-experienced memory T and B cells and pre-existing antibodies. Although the contribution of T and B cells to recall responses is well defined, the contribution of antibody "memory" and the mechanisms by which pre-existing antibodies modulate the development of germinal center and plasma cell responses is not precisely understood. Here we report on mechanisms that mediate antibody enhancement of germinal center (GC) and plasmablast (PB) compartments, and the parallel process by which they change the affinity threshold for B cell recruitment into immune responses. The data indicate that antibody-mediated enhancement of GC and PB responses is Fc gamma receptor (Fc{gamma}R) dependent and largely complement receptor 1 and 2 (CR1/2) independent. In contrast, the reduction in the affinity threshold for GC entry is independent of both Fc{gamma}Rs and CR1/2. SummaryCipolla et al. show that antibody can modulate immune responses via both Fc gamma receptor dependent and independent mechanisms. These mechanisms influence both the magnitude and composition of the germinal center response.

Triggering receptor expressed on myeloid cells 2 (TREM2) plays a crucial role in regulating various microglial functions, including phagocytosis, inflammation, chemotaxis, and proliferation. Recent studies have demonstrated that TREM2 cooperates with DAP12 to mediate intracellular signaling essential for these processes. Despite the importance of the TREM2-DAP12 complex in microglial physiology, the mechanisms controlling its expression and activity remain poorly understood. In this study, we report that the soluble ectodomain of TREM2 (sTREM2) regulates microglial phagocytic activity by attenuating the surface expression of DAP12. We found that stimulation of the microglial cell line BV2 with recombinant sTREM2 reduces the membrane expression of DAP12, but not that of TREM2. In addition, sTREM2 binds to full-length TREM2, leading to the uncoupling of TREM2 from DAP12. Furthermore, pre-treatment of BV2 cells with sTREM2 significantly inhibited amyloid-{beta} incorporation. These findings suggest that sTREM2 negatively regulates TREM2 signaling through the destabilization of the TREM2-DAP12 complex, and act as a novel bioactive molecule that modulates TREM2 signaling under physiological and pathological conditions.

Legionnaires disease (LD), a pneumonia caused by Legionella pneumophila intracellular bacterium, leads to intensive care unit (ICU) admission in 20-40% of cases. While these ICU-LD patients display severe lung injury or septic shock, their functional immune response remains poorly understood. The present study aimed, through a large immune gene expression assessment, to improve the understanding of immune cell functionality after whole blood LPS ex vivo stimulation in ICU-LD patients compared with non-ICU. Both ICU and non-ICU-LD displayed altered gene expression indicating that both patients immune cells are less able to respond to the LPS ex vivo stimulus than a healthy population. ICU-LD patients had 1.6-fold greater number of less-expressed genes (35/93 vs 22/93, p=0.039), and lower Log2(FC) of these genes (median [IQR]: -1.9 [-2.6;-1.5] vs -1.2 [-1.7;-0.9], p=0.0011) than non-ICU-LD. Seven genes were significantly less expressed by ICU-LD patients (IRF7, MX1, NFKBI2, NFKBIA, RELB, SRC, TIM3; p-value range: 0.029-0.0080). Top five gene ontology biological processes, subcellular localisations, and reactome pathways (STRING database) uniquely enriched in ICU-LD-patients and related less-expressed genes were cellular response to LPS (CCL2, NFKBIA, IRAK2, TIM3, SRC, NFKB1), regulation of IFN-{beta} production (IRF7, RIG1, OAS2, RELB), I-{kappa}B/NF-{kappa}B complex (NFKBIA, NFKB1, NFKB2), IFN regulatory factor complex (RIG1, IRF7), and TRAF6-mediated NF-{kappa}B activation pathway (NFKBIA, NFKB1, NFKB2, RIG1). Immune gene expression alterations in LD after LPS stimulation were found herein, with more pronounced alterations in ICU-LD patients. A reduced expression of key genes and pathways involved in controlling Legionella proliferation in ICU-LD patients may contribute to increased disease severity.

The Rho family of small GTPases plays a critical role in regulating actin cytoskeleton dynamics during endocytic processes in E. histolytica, including phagocytosis, pinocytosis, and trogocytosis. These proteins act as molecular switches, transitioning between inactive GDP-bound and active GTP-bound states, with guanine nucleotide exchange factors (GEFs) catalyzing this transition. Among the GEFs, EhFP10--a FYVE-domain-containing protein harbouring Dbl homology (DH) and pleckstrin homology (PH) domain was observed in phagocytosis along with seven functionally characterized Rho GTPases (EhRho1, EhRho2, EhRho4, EhRho5, EhRho6, EhRho8, and EhRho13). To study the specificity of FP10, a combination of GEF activity, binding affinity, and molecular dynamics simulations was used to characterize the interactions between EhFP10 and seven Rho GTPases systematically. The results revealed EhRho2 as the most specific and high-affinity interactor of EhFP10, with the highest nucleotide exchange rate and lowest dissociation constant (KD = 0.58 {micro}M). Structural modeling, sequence alignment, and interaction mapping further demonstrated that EhRho2 retains critical contact residues--such as Glu33, Arg4, and Leu69--that are variably absent in other isoforms, correlating with decreased GEF responsiveness. Molecular dynamics simulations and cross-correlation analyses supported the presence of a stable and coordinated interaction interface in the EhFP10-EhRho2 complex, distinguishing it from less active complexes. These findings indicate a highly selective GEF-GTPase module in E. histolytica, analogous to those in higher eukaryotes. The results uncover a potential regulatory mechanism specific to pathogenic amoebae and present EhFP10-EhRho2 as a novel therapeutic target for disrupting cytoskeleton-mediated processes crucial to virulence.

T cell development in the thymus is a tightly regulated process where epigenetic modifications, such as histone 3 lysine 27 acetylation (H3K27ac), play a crucial role in controlling the activation of genes. The epigenetic regulation of human mucosal-associated invariant T (MAIT) cell development is unknown; we mapped the regulatory chromatin landscape in the three developmental stages of thymic MAIT cells to identify the regulatory elements and enhancer activity involved in thymic maturation and analysed whether these chromatin dynamics are associated with the acquisition of effector programs in developing MAIT cells. Utilising cleavage under target and tagmentation (CUT&Tag), genome-wide H3K27ac profiles were generated and combined with transcriptome data from thymic MAIT cells, which revealed how developmental shifts in enhancer activity correspond to changes in gene expression. In total, 41,958 genomic regions with H3K27ac signal were identified in MAIT cells across the three development stages, of which 1,200 regions showed acetylation changes during differentiation from stage 1 to stage 3. At dynamic regions, the greatest differences were observed between stage 1 and stage 3, highlighting a progressive gain or loss of H3K27ac during MAIT cell development. Overall, MAIT cell maturation was associated with the gradual accumulation of H3K27ac at promoters and enhancers, which closely correlated with gene expression changes during development. Stage-specific enrichment of H3K27ac was observed at key transcription factor gene loci involved in MAIT cell development, including ZBTB16 (PLZF), EOMES, RUNX3, NFATC2, FOXO1, TGIF1, IRF1, and MAF genes. Epigenetic remodelling was also observed at cytokine and cytokine receptors (IL7R, IL18R1, IL23R, IFNG), chemokines and chemokine receptors (CCL4, CCL5, CCR5, CCR9, CXCR4, CXCR6), as well as several surface molecules with known immunological function. Our work reveals a previously uncharacterised epigenetic profile of human MAIT cells that regulates and inuences their development.

Muscle-specific kinase (MuSK) is a pivotal player in forming and maintaining healthy neuromuscular junctions (NMJ). In MuSK myasthenia gravis (MG), autoantibodies targeting MuSK disrupt its function, impairing neuromuscular transmission and causing fatigable skeletal muscle weakness. MuSK autoantibodies predominantly belong to the IgG4 subclass, which bind in a monovalent fashion due to Fab-arm exchange, although autoantibodies of other subclasses also exist. Polyclonal autoreactive IgG from patients may therefore harbor a variety of monovalent and bivalent MuSK antibodies with potentially distinct effects on MuSK signaling. To further unravel the pathomechanisms underlying MuSK MG, we have investigated how MuSK antibody-binding affects MuSK functioning with a diverse panel of (patient-derived) monoclonal MuSK antibodies. Our findings reveal that the valency of antibody-binding influences binding kinetics to MuSK, inhibition of agrin-induced MuSK activation, Dok7 binding to MuSK and NMJ gene expression. Monovalent binding to the frizzled domain of MuSK did not inhibit agrin-induced MuSK activation, while monovalent binding to the Ig-like domain 1 does. Moreover, the kinetics of Dok7 degradation induced by bivalent MuSK antibodies appear to depend on binding-epitope of MuSK. Surprisingly, none of the clones tested (both bivalent and monovalent) increased MuSK internalization. Taken together, the cumulative pathogenic effect of polyclonal MuSK antibodies in individual MuSK MG patients thus likely depends on autoantibody titer, affinity and the unique composition of MuSK autoantibodies varying in epitope and valency. This research enriches our understanding of the intricate interactions between antibodies and MuSK in MuSK MG and offers potential insights into novel therapeutic strategies using MuSK antibodies.

Although calmodulin is best known as an intracellular calcium sensor, it also possesses calcium-independent functions in unicellular organisms. This is exemplified by the budding yeast S. cerevisiae calmodulin, which binds its essential targets, the pericentrin-like protein Spc110 and type I and V myosins, without needing calcium. Whether such calcium-independent cellular functions are conserved in other yeasts and vertebrates nevertheless remains an open question. Here, we examined the calcium-independent functions of the fission yeast S. pombe calmodulin Cam1 by measuring its intracellular distribution. Using quantitative fluorescence microscopy, we assessed the intracellular localization of two cam1 mutants, where binding of Ca2+ had been compromised by mutations in their EF hands, compared to the wild type protein. Both Cam1-2V and -3V reduced their localization by 90% to the yeast microtubule-organizing center spindle pole bodies (SPB). In contrast, these two mutants did not affect the myosin-dependent localization to the equatorial division plane and to the cell tips. Replacing the endogenous cam1 with cam1-2V decreased the SPB localization of pericentrin Pcp1 by 69%, without changing the localization of either type V or I myosins. Over-expression of Pcp1 rescued the mitotic defects of cam1-2V cells at the restrictive temperature. Surprisingly, the cytokinesis of this cam1 mutant was largely normal. We concluded that fission yeast calmodulin Cam1 depends on Ca2+to be a component of SPBs, suggesting that calcium plays a critical role in the assembly of SPBs.

A proteomic analysis of Ixodes scapularis nymph saliva identified 252 proteins, including six tubular lipid-binding proteins (TULIPs). Comparing nymphs fed on mice that were uninfected or infected with Borrelia burgdorferi, twelve salivary proteins showed significant differences in the amounts detected, including XP_040079658.2, which we refer to as TULIP2. Considering the known immunity-related functions of some TULIPs, we expressed and purified TULIP2 from Escherichia coli and analyzed its interaction with B. burgdorferi lipids. The purification of TULIP2 from E. coli presented many obstacles, due to insolubility, which is consistent with previous reports from studies of other TULIP family members. The binding results showed specificity for B. burgdorferi lipids, with evidence for cholesteryl {beta}-galactoside as a major binding target. Molecular modeling of TULIP2 did not show any strong lipid binding sites. We used molecular dynamics simulation of TULIP2 to explore its conformational landscape by thermal unfolding. The earliest unfolding intermediate opened a hydrophobic pocket to which cholesteryl {beta}-galactoside was predicted to bind strongly. We propose that a specific lipid bilayer interaction with TULIP2 triggers the opening of the ligand-binding site.