Molecular Immunology

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.

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.

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.

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.

To understand the molecular and cellular mechanisms beyond B-cell depletion with the anti-CD20 monoclonal antibody ocrelizumab, we used comprehensive muti-modal flow cytometry and functional assays in a prospective longitudinal multiple sclerosis (MS) cohort. Ocrelizumab depleted the vast majority of B cells and showed selective effects on different B cells subsets. Analysis of residual/replenished B cells revealed relative enrichment of regulatory B cells like CD27+CD43+ B1 and CD24hiCD38hi transitional B cells, and reduction of CD27+ memory B cell subsets and CD19+IgD+CD27-naive B cells at early time points (1-3 month) and before subsequent infusions at 4-7 months, 11-14 months, and >18 months. CD20+ T cells and peripheral helper T-cells (Tph) were also reduced. RNA sequencing analysis showed B1 cells have significantly higher expression of LGALS1, KCNN4, ITGB1, and IL2RB. Compared to transitional B cells, B1 cells also displayed significantly higher expression of tissue homing molecules ITGAX (CD11c), S100A4, ITGB1, and CXCR3. IL10 signaling pathway is increased in these B cells. Ex vivo B cell functional assays indicated the residual/replenishing B cells were anergic following ocrelizumab, with increased IL10/TNF and IL10/IL6 ratios under BCR stimulation. Ocrelizumab treatment may create a self-reinforcing regulatory circuit: the reduction of Tph cells could alleviate suppression of regulatory B cells, which subsequently expand and further promote regulatory T cell networks via IL2RB, LGALS1, and an increased IL-10 signaling pathway.

BackgroundGene expression changes in response to developmental and environmental cues rely on cis-regulatory sequence elements (cREs). BRG1/BRM-Associated Factors (BAF) chromatin remodeling complexes maintain chromatin accessibility at many cREs, enabling binding by transcription factors (TFs). However, cREs exhibit a broad range of sensitivity to loss of BAF function, and the basis of this variability remains unknown. ResultsTo identify the characteristics of BAF-dependent cREs, we mapped chromatin accessibility changes following acute pharmacologic BAF inhibition in GM12878 lymphoblastoid cells. We integrated these results with over 100 TF and histone modification ChIP-seq datasets and used machine learning to identify features that predict chromatin accessibility changes. We found that Activator Protein 1 (AP-1) factors and lymphoid lineage-defining TFs including RUNX3 and PU.1 predicted BAF-dependence. Strikingly, we found that cREs bearing the chromatin signature of "primed" enhancers - enriched for H3K4me1 but lacking H3K27ac - were significantly more sensitive to BAF inhibition than typical active enhancers. As primed enhancers are known to facilitate transcriptional responses to stimuli, we tested the requirement of BAF activity in these responses. Acute BAF inhibition was sufficient to prevent both chromatin and transcriptional responses to interferon gamma and dexamethasone. cREs which normally gained accessibility in response to stimulation failed to do so with BAF inhibition, and these cREs were linked to genes with suppressed transcriptional induction. ConclusionsCollectively, our results demonstrate a requirement for continuous BAF activity to enable stimulus response and suggest that defective signal responsiveness may be a pathogenic mechanism in disease states caused by loss-of-function mutations in BAF subunits.

In mammals, most of the iron is found in the heme of red blood cells (RBCs), which must be recycled to support erythropoiesis in the bone marrow. Splenic red pulp macrophages (RPMs) play a crucial role in this process by phagocytosing senescent RBCs, metabolizing the heme and releasing iron back into the blood. Free cytoplasmic iron generates toxic reactive oxygen species, yet iron-specific adaptations of RPMs are not well documented. We previously reported that autophagy prevents ferroptosis in Langerhans cells, a cutaneous phagocyte subset. Thus, we hypothesized that autophagy may be important for the regulation of RPM metabolism and their maintenance of systemic iron homeostasis. To study this, we used Atg5flox/flox and Cd169cre mouse models to delete ATG5 in CD169+ macrophages, including RPMs. Atg5-deficient RPMs were decreased in number, and the remaining ones showed increased generation of toxic lipid peroxides. Spleens of Atg5{Delta}Cd169 mice were enlarged and contained more RBCs. Finally, autophagy impairment in RPMs exacerbated RBC loss in a model of phenylhydrazine-induced anemia. Our findings exemplify how dysregulation of macrophage metabolism alters their function and can disrupt tissue homeostasis upon challenge.

The Ube2J1 enzyme that mediates the ubiquitination and proteasomal degradation of misfolded proteins at the ER is phosphorylated at serine S184. Following anisomycin treatment of HEK293T cells, we observed an inverse relationship between phosphorylation and dephosphorylation at this site. This suggested a dynamic interchange between the two forms, and we show that S184 is a target for protein phosphatase 2A. The S184-phosphorylated protein is known to exhibit increased sensitivity to proteasomal degradation, and we found that mutation at K186R increased the ratio of S184-phosphorylated to S184-dephosphorylated protein. Although the K186R mutant retained some sensitivity to proteasomal inhibition, our results show that Ube2J1 steady state expression can be exercised at multiple levels, and can involve dynamic phosphorylation and dephosphorylation at S184.

Anemia is one of the most debilitating and frequent complications of inflammatory bowel diseases (IBD) and is often treated with iron supplementation, which has limited efficacy. Damaged intestinal barrier function is a hallmark of IBD and causes the translocation of endotoxins from gut bacteria into the bloodstream. In a previous study in mice, we reported that endotoxin suppresses erythropoiesis by reprogramming erythroblastic island macrophages (EBI M{varphi}). Here, we show that IBD patients and mice with acute colitis developed endotoxemia associated with anemia. Endotoxemia in IBD patients was negatively correlated with blood erythrocyte counts. In line with this, mice with acute colitis caused by drinking water containing dextrin sodium sulphate (DSS) had endotoxemia together with anemia characterized by reduced red blood cell counts, hemoglobin content and hematocrit., and reduced medullary erythropoiesis which was in part compensated by increased extramedullary erythropoiesis. As the endotoxin receptor TLR4 is expressed by CD169+ gut-resident macrophages and erythroid island macrophages in the bone marrow, we tested the hypothesis that TLR4 expressed by these CD169+ macrophages mediate both inflammatory colitis and anemia. Indeed, mice with conditional deletion of the Tlr4 gene specifically in CD169+ tissue-resident macrophages were protected from DSS-induced anemia and colitis. In addition, treatment of DSS mice with the TLR4 inhibitor C34 abated inflammation and anemia. These results suggest that endotoxins leaking from the inflamed gut may play a crucial role in IBD and associated anemia and that drugs targeting TLR4 may protect against IBD-associated anemia. Key pointsO_LIPatients with IBD and mice with acute colitis are anemic with increased endotoxemia and inflammation. C_LIO_LIEndotoxemia is inversely correlated with blood erythrocyte counts in IBD patients. C_LIO_LIConditional deletion of endotoxin receptor gene Tlr4 specifically in CD169+ tissue-resident macrophages or administration of synthetic TLR4 inhibitor significantly reduced colitis-induced anemia in mice. C_LI

BackgroundIn the course of testing mAb-based therapies to eradicate the persistent reservoir of HIV infection, we investigated the efficacy and mode of killing of HIV-infected cells by two categories of cytotoxic immunoconjugates (CICs) targeted by the same mAb, an immunotoxin (IT) and antibody-drug conjugate (ADC). MethodsWe performed metabolic and transcriptional analyses of treatment effects on the persistently-infected cell line H9/NL4-3. Cells were treated with CICs consisting of the anti-gp41 mAb 7B2 conjugated to either deglycosylated ricin A chain (dgA) or to the highly cytotoxic anthracycline derivative PNU-159682. At intervals up to 24 hr, intracellular metabolites were quantified by 1H nuclear magnetic resonance spectroscopy, and the transcriptome analyzed by RNA-Seq. ResultsSix hr post treatment, 7B2-dgA elicited both metabolic and transcriptional alterations, whereas 7B2-PNU treated cells did not differ from untreated cells. 7B2-dgA treated cells exhibited elevated intracellular levels of many amino acids, and activation of gene pathways for apoptosis, intracellular signaling, and immune activation. By 24 hr, both 7B2-dgA and 7B2-PNU treated cells differed markedly from untreated. Many of the changes observed following 7B2-PNU treatment at 24 hr were similar to those observed at 6hr following 7B2-dgA, likely indicating processes involved in cell death, but a number of alterations were unique to either IT or ADC treated cells. ConclusionsAn IT and ADC showed both similarities and differences in their cytotoxic effects. These results raise the question of whether the mode of cell killing could be a determinant of clinical efficacy. Although these studies were aimed at targeting the persistent reservoir of HIV infection, they have relevance for the design of CICs to treat cancer and other conditions. SUMMARYThe use of cytotoxic immunoconjugates, wherein an antibody is attached to a cellular poison, is effective in the treatment of cancer and other conditions. We seek to extend these results to treating HIV and other chronic viral infections. We analyzed the molecular mechanisms of cell killing when the same antibody was attached to different toxic structures. We report that each immunoconjugate induced both common and distinct patterns of killing. Such differences may have clinical relevance.

Unbiased classification of equine dendritic cells (DC) is necessary to address various research questions such as the role of DC subsets in immune-mediated diseases of horses. We applied single-cell RNA sequencing (scRNA-seq) on DC enriched from the blood of two horses. All main DC subsets were detected by key gene expression, including conventional DC type 1 (cDC1; XCR1) and type 2 (cDC2; FCER1A, CD1E) as well as plasmacytoid DC (pDC; TCF4). In addition, we detected a small cluster of hematopoietic progenitors, as well as transitional DC (tDC; FCER1A, TCF4) and putative DC type 3 (DC3; FLT3, CD163). Our data confirms the previously reported phenotype of equine pDC (Flt3+MHC-IIlowCADM1lowCD172aint), cDC1 (Flt3+MHC-IIhighCADM1highCD172alow-int) and cDC2 (Flt3+MHC-IIhighCADM1intCD172ahigh), while also highlighting considerable CD14 expression for cDC2. Two subclusters of equine cDC2 were found to be enriched in FCER1A or CX3CR1 transcripts (cDC2.1 and cDC2.2, respectively), with suggested enhanced extravasation and T-cell stimulatory capacities of the latter. Conservation of DC subsets across species (horse, pig, human, mouse) was illustrated by enrichment analyses with subset-specific gene signatures and by cross-species data integration with publicly available scRNA-seq datasets. Our atlas of equine blood DC is a valuable resource for comparative analyses, and it forms the foundation for understanding the involvement of distinct DC subsets in infections and immune-mediated pathologies.

MicroRNAs (miRNAs) play essential roles in the posttranscriptional regulation of gene expression in organisms. In the process of synthesizing mature miRNAs from miRNA precursors, the miRNA precursors are cleaved via Dicer at their loop structure, after which the miRNA precursors become mature and regulate transcription. However, the consequences of altering the loop sequence are not fully understood. The silkworm Bombyx mori is a lepidopteran insect with many genetic strains. We identified a mutant of the miRNA miR-3260 whose the part of the loop structure was lacking in a silkworm strain with translucent larval skin. Here, we aimed to analyze the role of wild-type miR-3260 and the influence of the mutation of the loop structure in B. mori. First, we identified the genomic region responsible for the translucent larval skin phenotype and determined that the mutated miR-3260 nucleotide sequences. Then, we predicted the binding partners of wild-type miR-3260 using the RNA hybrid tool and found two juvenile hormone (JH)-related genes as targets of wild-type miR-3260. Next, we assessed the relationships between miR-3260 and JH and found that miR-3260 was highly expressed in the Corpora allata and its expression responded to JH treatment. Meanwhile, miR-3260 mimic and inhibitor did not induce the typical phenotypes associated with JH in B. mori. Then, we compared the dicing products from wild-type and mutant miR-3260 precursors and observed that neither form underwent Dicer-mediated cleavage when the loop structure was altered. These results suggest that loop mutations in the miR-3260 precursor may not influence dicing activity, consistent with the lack of observable phenotypic effects.

Cancer remains a major therapeutic challenge despite substantial advances in diagnosis and treatment, including immune checkpoint blockade. Among emerging immunotherapeutic approaches, adoptive cell transfer (ACT) has attracted growing interest. Human peripheral V{gamma}9V{delta}2 T cells are promising candidates for ACT because they combine rapid and potent antitumor functions with major histocompatibility complex (MHC)-independent tumor recognition, enabling allogeneic use with limited risk of graft-versus-host disease. This raises the possibility of generating standardized V{gamma}9V{delta}2 T-cell banks from healthy donors for off-the-shelf immunotherapy. Here, we provide preclinical evidence supporting the suitability of allogeneic human V{gamma}9V{delta}2 T cells for ACT. We characterized peripheral blood V{gamma}9V{delta}2 T cells from healthy donors after successive antigen-specific and non-specific amplification steps, assessing their phenotype, effector functions, and metabolic state. Amplified cells maintained a strong pro-inflammatory Th1-like profile, preserved cytotoxic activity, and did not produce immunoregulatory cytokines. They also displayed high purity, a predominant effector memory phenotype, reduced expression of several inhibitory immune checkpoints, and sustained antitumor reactivity. Altogether, these findings support the development of allogeneic V{gamma}9V{delta}2 T-cell products as a scalable platform for next-generation cancer immunotherapies.

Dengue disease remains a significant global health threat, with current vaccines exhibiting variable efficacy and safety concerns. Virus-like particles (VLPs) offer a promising alternative by mimicking native virus structures without infectious genomes. We engineered a mammalian expression plasmid encoding Dengue-1 prM and E proteins, optimized for secretion using Japanese Encephalitis virus signal sequences, and transiently expressed it in HeLa cells. Purified VLPs exhibited spherical morphology ([~]39 nm diameter) consistent with native virions, as confirmed by transmission electron microscopy. Immunization of mice with these VLPs elicited robust Dengue-1 specific IgG antibody responses. Our study demonstrates production of immunogenic Dengue-1 VLPs in HeLa cells, highlighting their potential as a vaccine candidate and a tool for serodiagnosis. Further characterization of VLP epitopes and protective efficacy is warranted to advance vaccine development. ImportanceDengue remains a significant global health challenge, with serotype 1 being one of the dominant strains causing recurrent outbreaks in Nepal. Existing vaccines demonstrate limited efficacy and pose significant safety concerns, particularly in seronegative populations. To address these limitations, this study explores virus-like particles (VLPs) as a safer alternative vaccine platform. VLPs elicit robust immunogenicity by mimicking the structure of native virus while completely lacking genetic components. This study combines DENV1 structural proteins with optimized expression systems to enhance immunogenicity. This work is particularly significant as the first dengue vaccine research conducted in Nepal, directly addressing antigenic mismatches between existing commercial vaccines and locally circulating viral strains. Furthermore, the study provides scalable platform for developing region-specific dengue vaccines for other serotypes and flaviviruses.

Helminths are parasitic worms that secrete a variety of immune-regulating molecules to modulate the hosts inflammatory responses, enabling them to persist within the host over a long period of time, even decades. Their capacity to control host responses has prompted research into helminth-derived molecules as potential therapies for controlling excessive immune and inflammatory activity across a range of diseases. This systematic review with cross-study quantitative analysis aims to synthesize the published data on helminth-derived immunomodulatory peptides/polypeptides/proteins (HDIPs) with a focus on determining the extent of their disease-modifying and anti-inflammatory potential in in vivo animal models of inflammatory disease. In accordance with PRISMA 2020 guidelines, a predefined systematic search of the PubMed, Web of Science and Medline databases identified relevant studies published up to February 2026, and 65 articles were included after screening. We found that, although the HDIPs were assessed in multiple different disease models, most published studies assessed their potential in mouse models of colitis, asthma, arthritis and sepsis. Twenty species from which >65 isolated HDIPs were derived were tested in these models, with the trematode, Fasciola hepatica, and the nematode, Acanthocheilonema viteae, the most explored species. A common property of the HDIPs was the ability to significantly reduce disease severity across the in vivo animal models of inflammatory disease, underpinned by a decrease in pro-inflammatory cytokine levels and an increase in anti-inflammatory cytokine levels. Overall, this systematic review with cross-study quantitative analysis not only synthesizes the existing literature in this field but also highlights the disease-modifying and anti-inflammatory potential of HDIPs for a range of diseases in which immunoregulatory therapeutics may improve disease outcomes. It also encourages accelerated advancement of these helminth-derived molecules into first-in-human clinical trials.

Inflammation serves as a vital physiological process essential for preserving health and countering illness. Yet, persistent inflammation drives osteoclastogenesis and ongoing bone erosion in rheumatoid arthritis (RA), mainly via macrophage activation and overproduction of pro-inflammatory cytokines like TNF-, IL-1{beta}, and IL-6. Limitations of prolonged conventional treatments underscore the need for safer small-molecule inhibitors that address both inflammation and osteoclast formation. Chalcones, natural plant defense compounds, exhibit diverse pharmacological properties including anti-inflammatory, anticancer, antibacterial, antifungal, and antiparasitic actions, owing to their characteristic reactive , {beta}- unsaturated carbonyl moiety. This study assessed chalcone derivative 7a for its anti-inflammatory effects in vitro and in vivo, alongside its capacity to modulate osteoclast differentiation, offering the inaugural demonstration of its dual anti-inflammatory and anti-osteoclastogenic properties. In LPS-stimulated macrophages, 7a substantially curtailed nitric oxide production, curbed pro-inflammatory cytokines (TNF-, IL-1{beta}, IL-6), and concentration-dependently diminished iNOS and COX-2 expression while inhibiting reactive oxygen species levels. In vivo, oral 7a dosing potently alleviated carrageenan-evoked paw swelling and restored serum lactate dehydrogenase and C-reactive protein to normalcy. In LPS-exposed mice, it further lowered systemic cytokines and rectified dysregulated biomarkers such as LDH, ALP, ALT, AST, creatinine, and urea. Moreover, in RANKL-stimulated osteoclast cultures, 7a markedly suppressed osteoclastogenesis by downregulating pivotal markers like tartrate-resistant acid phosphatase (TRAP) and matrix metalloproteinase-9 (MMP-9). Derivative 7a also enhances antioxidant defense--superoxide dismutase and catalase--via blockade of NF-{kappa}B and MAPK pathways. Overall, chalcone derivative 7a displays robust anti-inflammatory and anti-osteoclastogenic activity, positioning it as a compelling candidate for RA therapy.

NLRP3 inflammasome is a cytosolic multi-protein complex that plays a crucial role in the immune system, responding to various exogenous and endogenous stimuli by triggering protective inflammatory responses. However, aberrant NLRP3 inflammasome activation is implicated in numerous inflammatory diseases. Therefore, the NLRP3 inflammasome is an important pharmacological target for the treatment of multiple diseases. In this context, we screened various US-FDA-approved drugs for NLRP3 inflammasome inhibition. We found that among various drugs, minoxidil hydrochloride (MXL) effectively inhibits NLRP3 inflammasome, evidenced by reduced secretion of IL-1{beta} and IL-18 in J774A.1 cells treated with MXL. The IC50 values of MXL for inhibition of IL-1{beta} and IL-18 were calculated to be 1.2 and 1.06 {micro}M, respectively. MXL was found to prevent ASC oligomerization, thereby inhibiting the NLRP3 inflammasome and leading to CASP1 cleavage. Further investigation revealed that MXL also utilizes AMPK-mediated autophagy to modulate NLRP3 inflammasome activity. Using siAMPK and bafilomycin A1, an end-stage autophagy inhibitor, we elucidated crosstalk between the NLRP3 inflammasome and autophagic pathways, which was modulated by MXL. Furthermore, we demonstrated the efficacy of MXL in two different mouse models of inflammation, involving the NLRP3 inflammasome. MXL at doses of 10 and 20 mg/kg effectively inhibited the activation of NLRP3 inflammasome by monosodium urate in the air pouch model and by ATP in the peritoneal inflammation model, as evidenced by reduced secretion of 1{beta} and IL-18 in the lavage. Our study identifies MXL as a potent NLRP3 inflammasome inhibitor, warranting further investigation as a potential therapeutic agent for inflammatory diseases.