Molecular Immunology

In chickens and humans, classical class I molecules of the major histocompatibility complex (MHC) can have a hierarchy of correlated properties, including cell surface expression and peptide repertoire. Chicken BF2 alleles that are less well-expressed on the cell surface and bind a very wide range of peptides are expressed by MHC haplotypes that confer protection from a variety of economically-important infectious diseases, while certain human HLA-B alleles that are well-expressed and bind a narrow range of peptides lead to slow progression from HIV infection to AIDS. Understanding the impact of these promiscuous generalists and fastidious specialists is of considerable interest. The promiscuous BF2 molecule from the chicken B21 haplotype, BF2*21:01, binds a wide range of peptides by remodelling the peptide-binding site, allowing co-variation of the anchor residues at peptide positions P2 and Pc-2, and binding of an anchor residue at Pc. By using in vitro refolding assays with peptides and peptide libraries, determining thermostability and crystal structures, and analysing a chicken B21 cell line by immunopeptidomics, we found that BF2*21:01 will accommodate many possible combinations at P2 and Pc-2, as well as several hydrophobic amino acids at Pc. However, marked preferences for particular peptide lengths, particular amino acids at the three anchor residues, combinations of amino acids at P2 and Pc-2, and amino acids at P3 and Pc-3 affecting stability lead to high frequencies of major peptides while still allowing the possibility of presenting a wide peptide repertoire. These simplifying principles may eventually allow predictions of pathogen peptides with stable binding for this iconic promiscuous class I molecule, as well as providing the data for more sophisticated peptide prediction methods.

Immunoglobulin M (IgM) is the most ancient and conserved antibody class in jawed vertebrates and is typically encoded by a single gene. In contrast, geckos and related lizards (infraorder Gekkota) possess multiple IgM genes within the immunoglobulin heavy chain locus. Here, we analyze 52 IgM constant-region sequences from 13 Gekkota species to clarify the evolutionary origin and functional consequences of this expansion. Phylogenetic reconstruction showed that IgM1 (the canonical form) is nearly monophyletic (86.7% clade purity), whereas internal-locus IgM2-6 variants display complex, lineage-specific duplication patterns. We identified 53 diagnostic amino acid positions distinguishing IgM1 from other variants, concentrated in CH1 (19 positions) and CH2 (25 positions). These differences are accompanied by a pronounced physicochemical shift in CH2: IgM1 carries a net positive charge (+2.01) while other IgMs are negatively charged (-2.13), a {Delta} of +4.14 charge units. Conservation analyses indicate stronger constraint on IgM1 in CH1/CH2, while internal-locus IgMs are more conserved in CH4, consistent with maintained polymerization function. Three-dimensional structural comparison of IgM1 and IgM4 supports functional divergence in assembly: IgM4 adopts an "open mouth" CH1-CH2 conformation with increased heavy-light chain contacts and a more electrostatically enriched interface, suggesting compensatory stabilization mechanisms. Together, these results support specialization of internal-locus IgMs through combined sequence and structural divergence.

T follicular helper (Tfh) cells are critical for germinal centers (GC), the specialised microenvironment where long-lived humoral immunity is generated in response to vaccination or infection. Within the GC, B cells engage with Tfh cells and elicit their help in the form of cytokines and cell-surface co-stimulator molecules. Tfh helper activity must be rapidly available in response to B cell engagement, yet the mechanisms controlling this helper activity remain poorly characterized. Post-transcriptional regulation of mRNA decay and translation offer one way to rapidly and temporally tune Tfh cell activity. ZFP36L1, a member of the ZFP family of RNA-binding proteins, is a candidate modulator of Tfh cell helper activity as it controls cytokine production and responses in other T cell lineages, modulating their differentiation and function. We sought to determine if ZFP36L1 is also important for Tfh cell biology. In this study, we show expression of ZFP36L1 by Tfh cells. We selectively delete ZFP36L1 from Tfh cells and analyze the effect this has on the GCs. Surprisingly, we find the GC response and affinity maturation is resilient to deletion of ZFP36L1 from Tfh cells.

Immunoglobulin Y (IgY) represents the major serum antibody in reptiles and birds, serving as the evolutionary precursor to mammalian IgG and IgE. While IgY diversification has been documented in several reptilian lineages, the structural basis underlying subclass divergence remains poorly understood. Here, we present a comprehensive phylogenetic and structural analysis of IgY sequences from 20 snake species, revealing two distinct evolutionary lineages (A and B) that arose through gene duplication. Structural modeling of the constant regions from Arizona elegans identified a fundamental difference in the light chain-heavy chain (CL-CH1) disulfide bond architecture between lineages. Lineage B utilizes CYS16 in the CH1 domain (alignment position 13) for the inter-chain disulfide bond with the light chain CYS98, whereas Lineage A employs CYS136 (alignment position 99), representing N-terminal versus C-terminal positioning within the CH1 domain. Analysis of 50 diagnostic amino acid positions between lineages revealed that changes are distributed across all constant domains (CH1-CH4), with 13 positions showing radical substitutions affecting charge or polarity. Sliding window dN/dS analysis demonstrated purifying selection ({omega} < 1) across both lineages, consistent with functional constraint following duplication. These findings provide structural evidence for subfunctionalization of snake IgY genes and suggest that alternative disulfide bond configurations may confer distinct biophysical or functional properties to each antibody subclass. This work advances our understanding of immunoglobulin evolution in reptiles and highlights the structural plasticity of antibody architecture.

It has been well established that females have a more active immune system. Females respond better to vaccines, are more resistant to somatic cell cancers, display better pathogen responses, present antigens better, and, conversely, are more prone to autoimmune diseases compared to male counterparts. Though these trends have been observed across normal and pathogenic states, the mechanisms underlying these sex differences have not been fully explained. Some hormonal effects on immune cell populations have been reported, but much less is known about effects contributed by genes on the sex chromosomes, for example those that are more highly expressed in females due to X inactivation escape, or Y-linked genes those unique to males. Here we use the Four Core Genotypes (FCG) mouse model and wildtype XY male mice to disentangle the effects of sex hormones, sex chromosome complement, and their interactions on baseline B and T cell populations in the periphery and T cells in the thymus. We test the effects of a previously described X-Y chromosomal translocation and those of the Sry transgene insertion on chromosome 3. We observe that mice harboring the Sry transgene show significant depletion of peripheral CD8+ T cell subpopulations. In the thymus, the XY XY,but not the XX males, show significant decrease to both CD8+ and CD4+ single positive T cells and an increase to CD4/CD8 double positive T cells. We also show that Y chromosome-bearing mice exhibit depletion in splenic marginal zone B cells. Our data suggests that the gonadal sex is the strongest contributor to this phenotype. Our studies define a critical framework for the use of this model and provide valuable data to assess the use of the FCGs model, especially for diseases involving the immune response.

AO_SCPLOWBSTRACTC_SCPLOWThe cellular transcription factor BCL11b (B-cell CLL/lymphoma 11b) interacts with numerous cellular and viral factors to modulate gene expression positively or negatively. Post-translational modifications of BCL11b, such as SUMOylation and phosphorylation, have been documented to switch its transcriptional activity from a repressor to an activator state. In the present study, we investigated the acetylation of BCL11b and we identified the histone acetyltransferase p300 as able to acetylate BCL11b. Subsequently, we observed that the mutation of the lysine K686 residue of BCL11b (BCL11b K686R) influenced its global acetylation. Furthermore, the BCL11b K686R mutation also modulated the transcriptional regulation of BCL11b, including its activity in regulating the p21 and IL-2 promoters. This effect on transcriptional regulation was due to the importance of the lysine K686 residue for BCL11b nuclear localization. Our results underscore the critical role of the lysine K686 residue in BCL11b for its interaction with p300 and its nuclear localization, suggesting a possible function of p300 in the nuclear transport of BCL11b. Collectively, our findings contribute to a better understanding of BCL11b-mediated gene expression and of the interactions of BCL11b with cellular partners.

GFI1 is a DNA-binding zinc finger transcription factor regulating the commitment of hematopoietic precursors to myeloid and lymphoid lineages. Here we report that GFI1 is expressed in {gamma}{delta} T cells and restricts the cellularity of ROR{gamma}t+V{gamma}6+ {gamma}{delta}T cells that produce high levels of IL-17A, while promoting the expansion of V{gamma}1+ and V{gamma}4+ {gamma}{delta}T cells. Absence of GFI1 results in a pronounced bias toward the production of {gamma}{delta}T17 cells, commencing post-birth. Additionally, we observe an expansion of ROR{gamma}t+/MAF+ cells within the thymic DN1e population of GFI1-deficient mice. The DN1e population, along with other DN subsets in GFI1 knock-out (KO) mice, exhibits a distinctive {gamma}{delta}T17 cell-specific transcriptomic profile. Specifically, DN1, DN3, and {gamma}{delta} T cells lacking GFI1 show upregulation of the B-ZIP transcription factor MAF, which regulates genes critical for {gamma}{delta}T cells, such as Il17a, Il22, and Blk that are all induced in Gfi1 deficient cells. The Maf gene is occupied by GFI1 in DN pre-T cells at cognate binding sites in its promoter region, suggesting that GFI1 acts as a direct repressor of Maf. We conclude that GFI1 functions as a novel regulator of V{gamma}6+ {gamma}{delta}T17 precursor cells restricting their peripheral expansion by acting upstream of a MAF dependent regulatory network. Highlights- GFI1 controls the expansion of {gamma}{delta} T cells in peripheral lymphoid organs and in barrier tissues. - GFI1 specifically restricts expansion of {gamma}{delta} T cells secreting IL-17 by acting upstream of a MAF dependent transcriptional regulatory network. - GFI1 plays a cell intrinsic role in controlling the generation of {gamma}{delta}T cells through the repression of Maf. - GFI1 controls a MAF+/ROR{gamma}t+ {gamma}{delta}T cell precursor cell population within the DN1e cell subset.

RATIONALEThe host-protective role of alpha-1-antitrypsin (AAT) against mycobacteria may be partly attributed to its binding to the cytoplasmic glucocorticoid receptor (GR) that results in gene regulation in macrophages that favors killing of ingested mycobacteria. The AAT-GR complex was found to be significantly responsible for limiting Mycobacterium avium complex (MAC) burden in macrophages; this host-protective function of AAT-GR is due, in part, to induction of COLONY STIMULATING FACTOR-2 (CSF-2) gene which encodes for granulocyte-monocyte colony stimulating factor (GM-CSF). METHODSTo better understand the role of AAT-GR binding during mycobacterial infection, we performed bulk RNA sequencing (RNA-seq) on four different groups of cells: (i) control THP-1 cells (THP-1control); (ii) THP-1control cells infected with Mycobacterium intracellulare (MAC); (iii) THP-1control cells incubated with MAC + AAT; and (iv) THP-1 cells knocked down for GR (THP-1GR-KD) incubated with MAC + AAT. RESULTSOur analyses revealed that MAC infection significantly upregulated 1,977 genes and significantly downregulated 2,303 genes in THP-1control cells. Additionally, AAT significantly upregulated 1,200 genes and downregulated 890 genes in MAC-infected THP-1control cells. Furthermore, the regulation of 1,624 genes that are regulated by AAT+GR in THP-1control cells was augmented in THP-1GR-KD cells, indicating that the regulation of these genes by AAT+MAC is inhibited by GR. Conversely, the regulation of 1,683 genes by AAT+MAC in THP-1control cells was attenuated in THP-1GR-KD cells, indicating that the regulation of these genes by AAT+MAC is enhanced by GR. MAC also induced both CSF2 (GM-CSF) and CSF1 (encodes for monocyte colony stimulating factor, M-CSF) expression. Whereas AAT inhibited MAC-induced M-CSF mRNA was dependent on GR, this inhibition of M-CSF protein was not dependent on GR. In contrast, AAT did not inhibit MAC-induced CSF2 (GM-CSF) expression. Since either MAC or AAT induced GM-CSF expression in macrophages, further investigation revealed that AAT-inhibition of cell-associated MAC burden was abrogated upon neutralization of endogenous GM-CSF. CONCLUSIONSThe ability of AAT to induce GM-CSF and to inhibit MAC-induced M-CSF may skew macrophages to a phenotype that is better endowed to control mycobacterial infection.

Adoptive cell therapy based on Natural Killer (NK) cells holds great promise for the treatment of cancer. For all approaches aiming at utilizing NK cells in immunotherapy, efficient ex vivo expansion technologies for the generation on of cytotoxic NK cells are a prerequisite for clinical translation. In this study, a novel multifunctional fusion protein consisting of a CD20-directed Fab-fragment, an agonistic anti-4-1BB single-chain Fragment variable (scFv), the Sushi domain of the interleukin (IL)-15 receptor and human IL-15 was generated. This molecule triggered strong NK cell expansion when bound to co-cultivated autologous B cells, due to trans-presentation of IL-15 and binding to 4-1BB/CD137. Expansion rates of up to 7,500-fold were achieved and the NK cells showed high cytotoxic capacity against a panel of tumor cell lines representing various tumor entities. Importantly, the activated NK cells did not show cytolytic activity against non-malignant B cells indicating that NK cells amplified by our novel approach were still physiologically regulated. The cytotoxic activity of the expanded NK cells was further enhanced by combination with therapeutic antibodies. Our molecule was additionally able to trigger efficient proliferation of NK cells from cord blood as well as multiple myeloma (MM) and acute myeloid leukemia (AML) patients. In conclusion, our novel platform technology provides ex vivo expansion of NK cells by using a single multifunctional fusion protein and may be well-suited for the development of NK cell-based immunotherapies. Key pointsA novel fusion protein that enables NK cell expansion from different sources including peripheral blood, bone marrow and cord blood

Atherosclerosis is a condition characterized by plaque growths in arteries, consisting of oxidized LDL (low-density lipoprotein) and localized cell cumulation. By the time of diagnosis for patients with atherosclerosis, the disease has often progressed into advanced stages. Statins are commonly prescribed; however, while these drugs can lower blood cholesterol levels, they cannot regress or stop the plaque growth. Currently, there are no treatments available to prevent the formation of new plaques. Such treatment options would require the identification of proteins that act during disease onset, initiating molecular mechanisms that promote plaque formation. Histone deacetylases (HDACs) and Ten Eleven Translocation (TET) demethylases are two important classes of epigenetic mediators. Some isoforms of these two classes of proteins have been found to transcriptionally regulate cellular inflammation, which may favor plaque formation. These transcriptional regulators seem to function early in the molecular mechanisms that are involved in disease progression. In the present work, we identified a clear role of these epigenetic proteins in foam cell formation. Foam cells have been implicated as part of the early steps which ultimately lead to atherosclerosis. Here we showed that in the presence of OxLDL (oxidized LDL), the protein isoform TET1 has a direct role in foam cell formation, while HDAC2 adopts a more indirect role. Using specific inhibitors of TET1 and HDAC2, we showed the inter-regulated molecular mechanisms between these proteins and how they regulate foam cell formation in vitro. In this study, we found that upon inhibition of TET1 in U937-derived macrophages, and subsequent foam cell formation via OxLDL treatment, a lower percentage of foam cells was observed. However, TET2 inhibition under the same treatment conditions had no effect on the inhibition of foam cell formation.

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.

Traumatic brain injury (TBI) is a significant risk factor for the development of Alzheimers disease (AD) and related dementia. In both TBI and AD, inflammation plays a pivotal role. Recent studies have found that TBI triggers activation of NLRP3 inflammasome in the brain whereas the NLRP3 inflammasome plays an important role in pathogenesis of AD. To evaluate the influence of TBI on the immune response and the importance of the NLRP3 inflammasome in mediating this process we have examined the immune profiles in the brain using a novel transgenic AD mouse line with the NLRP3 gene deleted. Briefly, a group of 3xTg and 3xTg/NLRP3-/- mice received a moderate TBI in the form of lateral fluid percusive injury FPI or sham surgery at the age of 4 months old, an age before the onset of AD. Injury-induced changes in immune profiling were assessed using flow cytometry, real-time quantitative PCR, or Western blot at the acute and subacute stages following TBI. We found that TBI altered immune profiles in 3xTg mice and NLRP3 gene knock out counteracts the injury effect with a significant sex-related difference. More specifically, at the acute stage after TBI in both male and female mice, TBI induced a significant infiltration of neutrophils, macrophages and {gamma}{delta} T-cells in the brains of 3xTg mice, however, NLRP3 knock down significantly blocked this injury effect in males and less so in females. We also found that NLRP3 gene knock down counteracted TBI-enhanced inflammatory cytokine IL1-{beta}, TNF-, and IL-17f expression. The abnormal immune profiling was not significant at 7 days post-injury, however, changes in cerebral vascular associated proteins including GFAP, AQP4, CD31, Occludin were observed in related to injury, NLRP3 and sex. In conclusion, our study has confirmed that TBI significantly alters immune profiles and vascular integrity in the context of predisposition of AD and the NLRP3 inflammasome is important in mediating these TBI-induced changes. Sex-related differences warrant further evaluation to define the role of NLRP3-related immune response and vascular pathology as a result to TBI and the predisposition to AD development.

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.

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.

The T Cell Receptor (TCR) is a highly variable component of the T cell immune response that recognizes unique epitopes presented on MHC molecules (pMHC). Random genetic recombination limits the ability for sequence homology to predict epitope specificity, which is more dependent on the strength of the TCR-pMHC binding interaction. Structures for understanding this interaction only exist for well characterized positive interactors, and there is no information available about the physical interaction of non-specific TCR-pMHCs. In this study, we explore the ability for structural prediction algorithms to generate interacting and non-interacting multimeric TCR-pMHC structures, then, examine features that can predict immune interaction. AlphaFold2 shows more consistent multimeric structure prediction compared to other deep learning structure generators or template based algorithms. Poor structure generation does not correlate with immune interaction, and non-interacting structures show similar structural properties to interacting structures. However, this results in less energetically stable conformations in non-interacting structures. Molecular dynamic simulation supports this finding and reveals a novel structural conformation that contributes mechanistically to proper immune synapse. We show that structural and physical features extracted from generated structures are more predictive of interaction than sequence based features. To support researchers in the prediction of TCR-epitope specificity we have made our structural prediction models available through an accessible notebook based webserver: https://github.com/RobbenLab/TCRSIP.

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.

It has been recognized a long time ago that the hedgehog (Hh) and Wnt signaling pathways have numerous similarities that suggest their common evolutionary origin. Although the Hh and Wnt proteins are unrelated they are similar in as much as they carry lipid modifications that are critical for their interaction with their receptors. In our earlier work we have shown that Wnt inhibitory factor 1 (WIF1), originally identified as a Wnt antagonist also binds to and inhibits the signaling activity of sonic hedgehog (Shh), raising the possibility that the lipid moieties of these unrelated morphogens play a dominant role in their interaction with WIF1. In the present work we have compared the interactions of human WIF1 protein with lipidated and non-lipidated forms of human sonic hedgehog (Shh) using Surface Plasmon Resonance spectroscopy and reporter assays monitoring the signaling activity of human Shh. Our studies have shown that human WIF1 protein has significantly higher affinity for lipidated than non-lipidated Shh, indicating that lipid modifications of Hhs are important for interactions with WIF1.

The IRE1-XBP1 axis is the most conserved of three major unfolded protein response (UPR) branches that are triggered by the endoplasmic reticulum (ER) stress. Although the transcription factor XBP1 is involved in the development and function of several hematopoietic lineages, the role of XBP1 in the activation of mast cells (MCs) that play key role in allergic response remains largely unknown. Because we have identified salicylaldehyde (SA), which inhibits IRE1 nuclease activity that is essential for production of XBP1, as an inhibitor of MC activation in our previous screening, we investigated the effects of additional IRE1 inhibitors, 3-methyl-6-bromo-salichylaldehyde (MBSA) and KIRA6, targeting nuclease domain and kinase domain, respectively, on MC activation. MBSA and KIRA6 suppressed IgE-dependent degranulation and cytokine release of bone marrow-derived MCs (BMMCs), whereas these inhibitors did not suppress the Ca2+ ionophore- or compound48/80-induced degranulation. Treatment with inhibitors against two other branches of UPR, the PERK and the ATF6 pathways, did not affect IgE-induced activation of BMMCs. Intraperitoneal administration of MBSA or KIRA6 significantly suppressed IgE-induced passive anaphylaxis in mice. Furthermore, to evaluate the effect of XBP1, siRNA-mediated knockdown was performed. It was confirmed that Xbp1 siRNA introduction reduced IgE-dependent degranulation of BMMCs in parallel with the knockdown level of Xbp1 mRNA. Taken together, the IRE1-XBP1 axis plays a significant role in IgE-dependent and MC-mediated allergic response, which is considered to be therapeutic target of allergic diseases.

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.

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.