Cellular & Molecular Immunology

Lymph nodes (LN) are key secondary lymphoid organs (SLO) for a coordinated immune response. They have been extensively characterized by numerous investigative techniques chiefly as single cell suspensions because they are composed of vagile yet crowded hematolymphoid elements, unfriendly to spatial tissue organization-saving techniques. We comprehensively classify in situ all cells of 19 human LN free of pathology with a 78-marker antibody panel, an hyperplexed cyclic staining method, MILAN, and an analytical bioinformatic pipeline, BRAQUE. A total of 77 cell types were classified, encompassing T, B, innate immune and stromal cells. CD4 and CD8 T-cells were classified into 27 unique subsets by leveraging the expression profiles of TCF7, the presence of co-inhibitory receptors and the spatial distribution. CD5 and TCF7 expression defined novel B-cell types. CD27+ mature B-cells occupied previously unrecognized nodal spaces non-overlapping with the cortex and the plasma-cell rich medullary cords. Type 2 conventional dendritic cells were located in nodular paracortical aggregates. Statistically controlled pairwise neighborhood analysis showed sparse cell-cell interactions, known and new neighbors, established and novel LN landscape niches. A high-dimensional proteomic interrogation of the normal human LN provides spatial allocation of known cell types, novel interactions and the landscape organization.

Autoimmunity is emerging as a new etiology for early-onset gastric cancer (GC). However, it remains unclear what molecular pathways drive the initiation and progression of autoimmune tumorigenesis. Given that Major Histocompatibility Complex Class II (MHCII) is the strongest genetic risk factor for many autoimmune diseases, we hypothesized that MHCII-mediated autoantigen presentation drives tumorigenic differentiation of epithelial cells. Here we show that epithelial MHCII, rather than MHCII from immune cells, plays an essential role in the initiation of autoimmunity-driven tumorigenic differentiation of gastric epithelial cells, which was characterized by increased expression of cancer-associated markers with immune-evasive and stem-like features that potentiate premalignant progression. In addition, we show that early gastric premalignancy is reversible upon the removal of epithelial MHCII. This study reveals that epithelial MHCII antigen presentation is essential in the early stages of autoimmune-driven gastric tumorigenesis and highlight epithelial MHCII as a potential biomarker or therapeutic target in early interventions of autoimmunity-driven cancer development.

The establishment of mixed hematopoietic chimerism is a promising way to induce immune tolerance for islet replacement therapy and to treat the underlying autoimmunity in Type 1 diabetes (T1D). Mixed chimerism not only promotes effective thymic negative selection of autoreactive cells but also restores regulatory T cell (Treg) function and peripheral tolerance. In the current study, we determined that a novel class of donor-derived CD8+CD44+CD122+ Tregs (d-CD8+CD122+ Tregs) plays a crucial role in controlling autoimmunity in non-obese diabetic (NOD) mice with induced mixed chimerism. Using adoptive T cell transfer experiments, we showed that d-CD8+CD122+ Tregs abrogate autoimmunity by selectively depleting the exogenously injected diabetogenic T cells in Recombination-Activating Gene deficient NOD mice. These d-CD8+CD122+ Tregs from NOD chimeras show upregulation of Helios, Programmed cell death protein 1, perforin, granzyme-B, CD39, Folate receptor 4, and downregulation of proinflammatory markers like Scart1 and Scart2. Using in vitro assays, we show that d-CD8+CD122+ Tregs respond specifically to a Complementarity-Determining Region-3 peptide sequence derived from T cell receptors of islet antigen-specific autoreactive T cells. Thus, mixed chimerism might be a method to revitalize CD8+CD122+ Tregs which are decreased in number and functionality in NOD mice. Similarly, we found that individuals with T1D have a deficiency in CD8+CD122+ Tregs, suggesting a potential loss of regulatory function accompanies disease onset. Revitalizing CD8+CD122+ Tregs may offer a new therapeutic strategy of restoring immune tolerance in autoimmune diabetes. One sentence summary Inducing mixed donor chimerism in NOD mice generates donor-derived CD8+CD122+ Tregs that suppress autoimmunity and restore immune tolerance by selectively eliminating autoreactive T cells.

African swine fever (ASF) is a highly pathogenic disease caused by the African swine fever virus (ASFV) infection, which can affect pigs of all ages and breeds, posing significant threat to the global pig farming industry. The ASFV p30 protein is an early-expressed viral structural protein; however, its function is not fully understood. In this study, the interaction of viral p30 with host TRIM21 was identified. The ectopic TRIM21 inhibited ASFV replication, while knockdown or knockout of TRIM21 promoted ASFV replication. Further, p30 was found to interact with RIG-I-like receptor (RLR) signaling adaptor MAVS, and during ASFV infection, p30-TRIM21-MAVS interacted with each other. Mechanistically, TRIM21 activated the K27 polyubiquitination of MAVS to induce IRF3 mediated type I interferon (IFN) production, whereas p30 counteracted TRIM21 activated MAVS K27 polyubiquitination to evade RLR signaling mediated antiviral IFN induction. In summary, our study revealed a novel function of ASFV p30, and provided new insights into the immune evasion of ASFV.

Maladaptive repair of acute kidney injury (AKI) may lead to the development of chronic kidney disease (CKD) characterized by renal fibrosis. Macrophages play roles in AKI-to-CKD progression; however, the interplay between inflammation and fibrosis after AKI remains controversial and the precise role of the distinct macrophage subsets remains elusive. In the present study we identified a unique population of Trem2hi macrophages derived from the bone marrow as a mediator bridging inflammation resolution and fibrosis establishment after kidney injury. Trem2 deficient mice exhibited mitigated renal fibrosis after ischemia-reperfusion injury (IRI) while the renal injury and inflammation persisted. Mechanistically, Trem2 promoted renal inflammation resolution by facilitating macrophage efferocytosis to remove apoptotic tubule cells and reshaping the macrophage cytokine production profile. Loss of Trem2 expression led to excessive cholesterol accumulation in macrophages via Lxr-Abca1/Abcg1 axis and thus sustained pro-inflammatory cytokines production. Moreover, Trem2hi macrophages orchestrated the pro-fibrotic tubular epithelial cells and the activation of myofibroblasts through SPP1 to promote the establishment of renal fibrotic niche. Based on our findings, Trem2hi macrophages may serve as a potential therapeutic target for AKI-to-CKD in combination with anti-inflammatory remedies.

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.

Viral infections are one of the main environmental factors triggering type 1 diabetes (T1D). Pancreatic alpha cells are more resistant than beta cells to diabetogenic viruses, partially explaining their survival in T1D. Similarly, bats have enhanced viral resistance, suggesting putative convergent evolution in antiviral mechanisms. Herein, we compared global gene expression in bat fibroblasts under basal conditions or exposed to double-stranded RNA to human alpha and beta cells and found that alpha cells exhibit greater similarity than beta cells to the antiviral responses of bat cells, as well as stronger intrinsic resistance to viral infection. Interferon-stimulated gene 15 (ISG15), a key regulator of antiviral responses in humans and bats, has higher expression in alpha compared to beta cells in five single-cell RNASeq datasets from human islet cells and in human induced pluripotent stem cell (hiPSC)-derived alpha-like cells. ISG15 knockdown in human insulin-producing EndoC-{beta}H1 cells and human islets increases apoptosis under basal conditions and after IFN exposure, exacerbates IFN responses and increases cell death and viral production after infection with the diabetogenic virus coxsackievirus B1, while its overexpression protects EndoC-{beta}H1 cells from the virus. Collectively, the present results demonstrate that alpha cells but not beta cells have similarities with the virus resistance gene program present in bats and identify ISG15 as an important factor for islet cells to cope with viral and diabetogenic stresses.

The cGAS-STING pathway has been widely recognized as a critical DNA-sensing pathway that plays a broad-spectrum antiviral role. Livestock, especially pigs, represents one of the most important meat sources. In this study, we identified a key lysine 61 (K61) of porcine STING (pSTING) that plays an essential role in its degradation and antiviral signaling in a species-specific manner, with K61 as the major lysine of pSTING for K48-linked ubiquitination. After virus infection, pSTING recruits the E3 ligase, RNF5, which specifically assembles a K48-linked ubiquitin chain at K61, thereby mediating pSTING proteasomal degradation and reducing its antiviral activity. Meanwhile, the deubiquitylation of K61 is mediated mainly by deubiquitinase USP20, which enhances the stability and antiviral activity of pSTING. Together, given the relatively few lysine numbers in livestock STINGs and species-specific K61 regulation of pSTING stability and antiviral function, the K61 and its specific regulatory enzymes of pSTING could serve as potential targets for breeding of antiviral pigs and design of antiviral drugs, respectively.

Follicular helper T (Tfh) cells orchestrate germinal centre-derived humoral immunity by providing essential help to B cells. Despite their key role in humoral immunity, the metabolic processes that guide Tfh differentiation and functions in human remain poorly understood. In this study, we used a human ex vivo Tfh differentiation model to investigate how key metabolic pathways influence Tfh cell differentiation and helper function. Human naive CD4 T cells were differentiated into Tfh cells, and glycolysis was selectively inhibited during early differentiation to assess its effects on cell fate, function, and transcriptomic landscape. Unexpectedly, early glycolytic inhibition enhanced Tfh cell differentiation but significantly impaired their function, including reduced IL-21 secretion and diminished B cell help. Transcriptomic analyses further revealed downregulation of fatty acid metabolic pathways when glycolysis was inhibited. To better understand this link, we disrupted fatty acid synthesis and oxidation and observed a marked decline in helper functions, including IL-21 and IFN-{gamma} production. Interestingly, acetate supplementation partially restored IFN-{gamma} secretion in glycolysis inhibited conditions, but not IL-21, suggesting that some functional requirements cannot be compensated by alternative metabolic sources. These findings identify glycolysis during early differentiation as a key regulator of human Tfh cells fate and reveal a glycolysis-dependent fatty acid metabolic axis that selectively controls Tfh function. This metabolic checkpoint provides mechanistic insight into tuning Tfh cells and Tfh-driven humoral immunity in vaccination and autoimmunity.

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.

West Nile virus (WNV) is a mosquito-borne pathogen of global concern that can cause fatal neuroinvasive disease. No specific prophylaxis or treatment exists for WNV or related orthoflavivirus infections, and the determinants of human disease severity remain poorly understood. Here, we report that neutralizing autoantibodies against type I interferons do not impair antiviral antibody development. Among the fully human monoclonal antibodies with potent neutralizing activity against WNV that were discovered, W010 targets a unique epitope within the envelope protein domain III (EDIII) and confers both pre- and post-exposure protection in a murine WNV model, even when interferon signaling is impaired. A second protective antibody, W014, exhibits broad cross-neutralization of other pathogenic orthoflavivirus members, including Japanese encephalitis virus, Murray Valley encephalitis virus, Saint Louis encephalitis virus, and Usutu virus. These findings identify key neutralizing epitopes on WNV EDIII and provide candidates for the development of antibody-based interventions against encephalitic orthoflavivirus infections.

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.

Interleukin-6 (IL-6) is a central regulator of vertebrate immunity, yet its existence in jawless vertebrates has remained obscure because of extreme sequence divergence. The extant jawless vertebrates (lampreys and hagfish), which deploy variable lymphocyte receptors (VLRs) instead of immunoglobulins and T cell receptors, provide a unique window into the earliest interface between cytokines and adaptive immunity. Here, we identify IL-6-like genes in jawless vertebrates by combining computational structural comparisons, phylogenetic reconstruction, and conserved local and long-range synteny analyses. The encoded proteins adopt the canonical four-helix bundle characteristic of immune-related IL-6 family cytokines and align topologically with mammalian IL-6. In sea lamprey, three IL-6 paralogs show distinct leukocyte and tissue expression patterns and are differentially induced by pathogen-associated molecular patterns and skin injury, indicating early functional diversification of IL-6-mediated inflammatory responses. Stimulation of myeloid peritoneal leukocytes with recombinant lamprey IL-6 induces STAT5 phosphorylation and rapid upregulation of SOCS1/3 genes, consistent with an IL-6/STAT5/SOCS regulatory axis. These findings extend the repertoire of immune-related four-helix bundle cytokines to jawless vertebrates and indicate that IL-6-dependent inflammatory programs were already in place before the divergence of VLR-based and Ig/TCR-based adaptive immune systems in vertebrates.

GPR65 has been shown to be a critical regulator of Th17 cell pathogenicity. Loss of GPR65 in mice results in a decrease in Th17 cells and reduced susceptibility to a mouse model of multiple sclerosis. The CREB/CRTC2 pathway has emerged as an important regulator of immune function. We have previously shown that the CREB/CRTC2 pathway modulates autoimmune disease by promoting differentiation of Th17 cells. In this study we performed RNA-seq to identify Th17 genes regulated by the CREB/CRTC2 pathway. Our RNA-seq analysis led us to uncover the first mechanism of regulation of the orphan receptor GPR65 by the CREB/CRTC2 pathway. We show that GPR65 is a target of the CREB/CRTC2 pathway through expression studies and chromatin immunoprecipitation. In addition, we show that targeting GPR65 with small molecules alters the expression of IL-17A. Understanding the regulation of GPR65 will be crucial in developing small molecules to treat patients with Th17 cell-mediated disorders.

Tolerogenic dendritic cells (TolDCs) are essential for immune tolerance and offer promise for treating autoimmune diseases. Despite the clinical evidence of their therapeutic potential, the key molecular pathways guiding their differentiation and tolerogenic phenotype remain elusive due to complex interactions identified in functional assays. Here we investigated the molecular profiles and regulatory programs underlying the functional status of tolerogenic dendritic cell populations in response to known tolerizing agents. We identified CD86 as a consistent and robust marker downregulated in tolerogenic state. Using CD86 blocking antibodies or CRISPR-mediated gene inactivation we demonstrated that CD86 is functionally required for TolDC-mediated suppression of T cell proliferation and cytokine secretion, establishing CD86 as both a consensus phenotypic and functional screening marker and a mechanistic regulator of tolerance. Leveraging CD86 as a scalable readout, we performed a pooled genome-wide CRISPR-Cas9 knockout screen to identify regulators of TolDC function. This approach uncovered UBE2L6 as a novel modulator that promotes the tolerogenic phenotype and restricts TolDC-mediated T cell activation. Mechanistically, UBE2L6 deficiency leads to coordinated upregulation of ISG15 and USP18, indicating a possible ISGylation-dependent pathway regulating CD86 expression and tolerogenic function. Together, this study identifies pathways that can be targeted to promote immune tolerance in immune-mediated inflammatory diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/713621v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@4c3636org.highwire.dtl.DTLVardef@17b27e5org.highwire.dtl.DTLVardef@783d95org.highwire.dtl.DTLVardef@1337ce_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

ABSTRACTSHeterozygous gain-of-function (GOF) mutations in signal transducer and activator of transcription 1 (STAT1) cause an inborn error of immunity characterized by immune dysregulation, recurrent infections and various autoimmune manifestations. However, the precise pathogenic mechanism by which STAT1 GOF contributes to autoimmunity remains elusive. In our cohort, STAT1-GOF patients exhibit biased circulating follicular helper T (cTfh) populations with CXCR3+ Tfh1-like features. Using a Stat1 GOF mouse model that spontaneously developed autoimmunity, we found that overactivated STAT1 promotes Tfh differentiation and disrupted T cell-dependent humoral responses with skewed immunoglobulin class switching towards IgG2. Furthermore, STAT1 GOF directly targets to Tfh and Th1 cell signature genes and thereby drives the development of Tfh1 cells with excessive IFN-{gamma} production, which implicated in autoantibody production and the development of autoimmunity. Notably, IFN-{gamma} neutralization significantly alleviated autoimmune cellular responses and autoantibody levels in mutant mice, highlighting IFN-{gamma} blockade as a promising targeted therapy for the STAT1-GOF patients with autoimmunity. Our findings suggest that proper regulation of STAT1 activity within a reasonable magnitude is crucial for ensuring optimal host-protective humoral immunity. One-sentence summaryOveractivated STAT1 promotes Tfh1 differentiation to drive autoimmunity.

Allergic responses to the major cat allergen Fel d 1 are primarily driven by immunoglobulin E (IgE) cross-linking on effector cells. While conventional allergen-specific immunotherapies often rely on whole-allergen extracts with inherent risks of systemic reactions, rationally engineered variants offer a safer path to desensitisation. We present a structure-guided strategy to deimmunise Fel d 1 by selectively disrupting crucial IgE-binding interfaces. Using a computational pipeline integrating structural mapping and immunoinformatics, we designed 30 single-point mutations targeting immunodominant regions. Ex vivo functional evaluation using sera from cat-allergic individuals demonstrated that specific variants, notably K29G in Chain 1, exhibited significantly diminished IgE reactivity and profoundly suppressed basophil activation. Furthermore, CRISPR/Cas9-mediated introduction of the K29G mutation into feline fibroblasts confirmed that the substitution preserves fundamental cellular proliferation. This work demonstrates that targeted computational design can not only yield superior candidates for safer immunotherapeutics but also establish a viable foundation for generating genetically edited, hypoallergenic cats.

The emergence of new SARS-CoV-2 variants and breakthrough infections underscores the need for next-generation vaccines capable of protecting from natural infection and/or preventing virus transmission to others. Intranasal vaccination offers a promising approach by eliciting local immune responses in the nasal mucosa, the primary site of infection and reservoir for transmissible virus. We evaluated two live-attenuated, respiratory syncytial virus vectored vaccines in which the RSV F and G surface glycoproteins were replaced with a chimeric SARS-CoV-2 Spike protein from either the ancestral USA/WA-1/2020 strain (MV-014-212) or the Delta variant (MV-014-212-delta). A single intranasal dose of either vaccine elicited systemic and mucosal immunity in K18-hACE2 mice, including serum neutralizing antibodies, Spike-specific memory B cells and plasmablasts, and Spike-specific CD8 lung-resident memory T cells. Although MV-014-212-delta vaccination provided the best protection against Delta variant virus challenge, both vaccines decreased viral loads in nasal discharge, lung and brain, and reduced weight loss and mortality. In naturally acquired infection studies, vaccinated hamsters exposed to infected cagemates exhibited minimal weight loss, limited viral replication within the nasal mucosa, and attenuated lung pathology. Therefore, intranasal RSV-vectored vaccines can elicit broad protective respiratory immunity, suggesting that this platform could be leveraged for other respiratory pathogens.