Cellular & Molecular Immunology

UNC93B1 is a crucial chaperone protein for the trafficking of TLRs and regulates antigen presentation in dendritic cells (DCs), which activates downstream immune responses. Here, we identified a novel homozygous gain-of-function (GOF) UNC93B1 variant in an early-onset lupus patient. The patient presented with elevated level of inflammation and auto-antibody, and organ damage. The Unc93b1R95L/R95L transgenic mice also exhibited with autoimmune and autoinflammatory phenotypes. The transcriptional analysis revealed increased inflammation and elevated activation of DCs in the patients PBMCs and bone marrow-derived DCs (BMDCs) from Unc93b1R95L/R95L mice. In addition to the selected TLR7/8 activation in previously reported UNC93B1 GOF variants, the single-cell transcriptome and flow cytometry of splenocytes from Unc93b1R95L/R95L mice demonstrated increased phagocytosis activity and T helper cell differentiation with altered ICAM and MHC signaling in DCs and T cells, respectively. These results suggest UNC93B1 GOF variant enhances antigen presentation from DCs to T cells in the pathogenesis of immune dysregulation. Our study expands the pathogenic variants spectrum of UNC93B1 and offers insight into the underlying mechanism of antigen presentation in immune dysregulation caused by UNC93B1 beyond its trafficking function of TLRs.

Neutralizing monoclonal antibodies (mAbs) are a key component of antiviral therapeutics against SARS-CoV-2; however, the contribution of Fc-mediated effector functions remains underexplored. Here, we compare the antiviral activities of the neutralizing and non-neutralizing mAbs CB6 and CR3022, respectively. The Fc regions of both plant-produced mAbs carried nonfucosylated, non-galactosylated complex glycans (pCB6 and pCR3022), and CR3022 was also produced with mammalian-typical galactosylated, fucosylated glycans (mCR3022). pCR3022 exhibited markedly enhanced antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cell-mediated virus inhibition (ADCVI) compared to mCR3022, indicating a significant impact of Fc glycosylation on antiviral activity despite the lack of neutralization. pCB6 exhibited potent neutralization while further enhancing virus clearance through synergistic Fc effector activity. Our findings suggest that Fc-mediated mechanisms, especially ADCC and ADCVI, can contribute substantially to viral control and may be particularly valuable against immune-evasive variants. These results advance our understanding of the functional roles that non-neutralizing antibodies can play in SARS-CoV-2 infection and highlight the potential of Fc glycoengineering to modulate the antiviral efficacy of both neutralizing and non-neutralizing mAbs.

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.

Human genetic studies have identified defects in multiple mechanisms that predispose the risk of developing inflammatory bowel diseases (IBD), which include alterations in adaptive and innate immune responses, epithelial integrity and regulation of the intestinal mucus layer. Despite the importance of intestinal barrier integrity in the pathogenesis of IBD, essentially all current therapies modulate the immune responses. In this study, we determined the high resolution cryo-EM structure of human NXPE1, a IBD associated protein. Based on the structural homology, we identified NXPE1 as an O-acetyltransferase. Since NXPE1 is a pseudo gene in mouse, we generated knockout mouse model that lacked two of the mouse NXPE1 homologs, Nxpe2 and Nxpe4. The O-acetylation of sialic acid on red blood cells was abolished in the double knockout mice, confirming the sialic acid O-acetyltransferase function of NXPE1 family members. These findings underscore the potential of NXPE1 as a novel therapeutic target of the intestinal barrier functions for the treatment of IBD.

Vaccination frequently elicits suboptimal immunogenicity in organ transplant recipients, particularly those on long-term immunosuppressive therapy, highlighting the need for improved understanding of immunosuppression mechanisms and optimized vaccination strategies. This study enrolled a cohort of 132 individuals and observed significantly lower antibody levels in kidney transplant recipients (KTRs) compared to non-transplant controls (non-KTRs). Antibody levels were inversely associated with both the dosage and duration of immunosuppressive therapy. Complementary small animal studies demonstrated that immunosuppressive treatment dosage-dependently and reversibly impaired antibody production, primarily by depleting immune cells, notably B cells. A single shot of adenoviral vector-based vaccines demonstrated enhanced immunogenicity relative to two shots of alum-adjuvanted protein vaccines, inducing potent neutralizing antibodies (NAbs) and a Th1-biased T-cell response even under continuous immunosuppression. The enhanced response was driven by reduced interference from pre-existing antibodies, sustained transgene expression, and the reprogramming of lipid metabolism to activate T and B cells. Our findings advocate for tailored vaccination strategies, positioning adenoviral vectors as a candidate modality for this vulnerable population.

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.

Aberrant activation of type I interferon (IFN-I) is closely related to the development of autoimmune diseases. The metabolic regulation of cytokine signaling is essential for immune homeostasis. In this study, we characterized Urolithin A(UA), a natural gut-derived metabolite, as an inhibitor of Janus kinase (JAK) signaling. UA was found to broadly dampen JAK phosphorylation and the downstream signaling induced by cytokines such as type I interferons (IFN-I), type II interferons (IFN-II), and interleukin-6 (IL-6). UA can directly bind to JAK1 JH1 domain and treatment with UA attenuated autoimmune pathogenesis in Trex1-KO mice, IMQ-induced SLE and psoriasis models. Our findings unveil that UA is an anti-inflammatory metabolite that promotes immune homeostasis and could be used to treat inflammatory and autoimmune diseases.

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.

Streptococcal pyrogenic exotoxin C (SpeC) is a prototypical superantigen produced by group A Streptococcus. It potently activates a broad subset of T lymphocytes via a bridging interaction involving TCR{beta}-SpeC-MHC-II. Our recent work demonstrated that SpeC induced profound release of IL-8 from human pharyngeal epithelial cells and this effect was reversible through a specific point mutation in SpeC. This study systematically investigated cellular signaling pathways using integrated transcriptomic profiling and Western blot analysis, with a focus on membrane-associated receptors and downstream intracellular signaling effectors. Our results demonstrate that this biological process is critically associated with the activation of Erk1/2, p38 MAPK and NF-{kappa}B signaling cascade. This study identifies a novel mechanism through which a bacterial superantigen target epithelial cells-the body primary physical barrier and first line of innate immune defense.

Human parainfluenza viruses (PIVs) are a leading cause of respiratory illness, particularly in vulnerable populations where infection can lead to severe disease. Despite their clinical impact, there are currently no licensed vaccines or effective antiviral treatments available. PIVs have two large surface proteins, the fusion and hemagglutinin-neuraminidase (HN) proteins, both of which are targets of neutralizing antibodies. In this study, we identified and characterized two human monoclonal antibodies (mAbs), 5217-2 and 5217-9, which bind recombinant PIV3 HN protein, bind PIV3-infected cells, and are neutralizing in vitro. We determined the binding epitopes of the PIV3 HN-specific mAbs via biolayer interferometry and found mAb 5217-9 targets a previously defined neutralizing epitope while mAb 5217-2 binds a unique epitope, enabling a more complete understanding of the antigenic landscape. To further understand the newly defined epitope, we determined a cryo-electron microscopy (cryo-EM) structure of mAb 5217-2, which revealed an epitope adjacent to the PIV3 HN protein active site. We also determined the structure of the previously discovered anti-PIV3 HN mAb PIV3HN-09, which was previously shown to be partially protective in vivo. In a hamster challenge model of PIV3, mAb 5217-2 was determined to significantly reduce lung viral titers, demonstrating its protective capacity. Furthermore, as the site 2-directed mAb PIV3HN-05 was previously shown to cross-neutralize PIV1, we evaluated its protective efficacy in an animal challenge model with PIV1, which demonstrated a reduction in lung viral titers. Overall, these findings provide new insights into the antigenic epitopes on the PIV3 HN protein to support structure-based vaccine design efforts and demonstrate new protective mAbs for both PIV3 and PIV1.

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.

T-cell senescence is a hallmark of immune dysfunction in persistent viral infections, characterized by DNA damage accumulation and telomere erosion. However, the mechanisms driving CD4 T-cell senescence in the context of chronic hepatitis B virus (HBV) infection remain poorly defined. In this study, we demonstrated that people with chronic HBV infection exhibited CD4 T-cell senescence, marked by elevated KLRG1, along with increased DNA damage and telomere shortening, compared to HS. Notably, activation of the MRN-ATM (MRE11/RAD50/NBS1-Ataxia Telangiectasia Mutated Protein) pathway was prominent in CD4 T cells from HBV patients. Importantly, suppression of MRN attenuated ATM phosphorylation and its downstream signaling molecules, and inhibition of ATM reduced the production of proinflammatory cytokines in CD4 T cells derived from both HBV patients and HS. These results suggest that in chronic HBV infection, the virus induced CD4 T-cell senescence, telomere erosion, and DNA damage, while concurrent activation of the MRN-ATM pathway may serve as a compensatory mechanism to preserve CD4 T-cell function. Elucidating this relationship between T-cell senescence and DNA damage repair helps to understanding the mechanisms underlying HBV persistence and providing potential therapeutic targets against chronic HBV infection.

The germinal center (GC) reaction requires tight regulation of B cell and T follicular helper (Tfh) cell interactions to ensure B cell expansion and antibody affinity maturation, while preventing oncogenesis. However, regulatory mechanisms fine-tuning B-T cell interactions within the GC to prevent aberrant activation and proliferation remain incompletely understood. Here, we identify Siglec-G, the mouse ortholog of human Siglec-10, as an immune checkpoint that restrains the GC by dampening B-T cell interactions. Selective and temporal ablation of Siglec-G on B cells after immunization triggers GC hyperplasia and enhanced plasma cell and antibody output. While Siglec-G is dispensable in B cell receptor (BCR)-mediated processes, it acts as an intrinsic inhibitory receptor of B-T cell interactions in the GC, ultimately limiting Myc and mTORC activation within positively selected GC B cells. Trans interactions of Siglec-G and its glycan ligands on Tfh likely contribute in fine-tuning the strength of bidirectional signaling following contact between GC B cells and Tfh cells. This interaction is further reinforced by glycan remodeling that occurs in the GC, resulting in concurrent decreased in glycan ligands on GC B cells and increased in glycan ligands on Tfh. This augmented binding of Siglec-G/10 on Tfh is mainly due to the upregulation of 2-6 linked sialic acid ligands. Moreover, APEX2-based proximity labeling revealed several candidate Siglec-G/10 binding partners on T cells, including BTLA, CD6, and Slamf6, which are known negative regulators of Tfh cell activation. Taken together, our findings identified that Siglec-G acts as a GC checkpoint receptor, restricting B cell proliferation by tuning T cell help following B-T cell interactions.

Chimeric Antigen Receptor (CAR) therapy for Acute Myeloid Leukemia (AML) is hindered by disease heterogeneity, antigen overlap with normal hematopoiesis, and the persistence of leukemic stem cells (LSCs). To overcome these barriers, we employed the IF-BETTER strategy to simultaneously target CD33 and the LSC-associated marker TIM-3 by pairing a second-generation CAR with a Cytokine-Costimulatory Receptor (CCR) in two dual CAR configurations (CD33.CAR/TIM-3.CCR and TIM-3.CAR/CD33.CCR). Both constructs displayed potent antigen-restricted cytotoxicity against AML cell lines and primary blasts, achieving leukemia clearance, while sparing normal immune and hematopoietic cells. Mechanistic studies revealed that the TIM-3.CAR single-chain fragment variable (scFv) recognizes a protein-proximal epitope whose interaction is selectively enhanced by AML-specific hyper-fucosylated and hyper-sialylated N-glycans. Fucosylation blockade reduced TIM-3.CAR avidity and cytotoxicity, confirming a glycosylation-modulated interaction. Integrating this glycosylation-tolerant TIM-3 scFv into a dual CAR framework enables selective targeting of AML cells, providing a rational strategy for safer and more effective AML-directed immunotherapy.

Mainland China experienced multiple waves of COVID-19 pandemic during 2020-2022, driven by emerging variants and changes in public health and social measures (PHSMs). We developed a hypergraph-based Susceptible-Vaccinated-Exposed-Infectious-Recovered-Susceptible (SVEIRS) model to reconstruct epidemic dynamics across 31 provinces, capturing transmission heterogeneity associated with clustered contacts. We assessed key characteristics of transmission at national and provincial levels during four outbreak periods: initial, localized pre-delta, Delta, and widespread Omicron, which accounted for 96.7% of all infections. We found significant diversity in transmission contributions across cluster sizes, with a small fraction of larger clusters responsible for a disproportionate share of infections. Counterfactual analyses showed that reducing cluster-size heterogeneity, while holding overall exposure constant, could have lowered national infections by 11.70-30.79%, with the largest effects during Omicron period. Ascertainment rates increased over time but remained spatially heterogeneous with a range: (14.40, 71.93)%. Population susceptibility declined following mass vaccination (to 42.49% in Aug 2021, nationally) and rebounded (to 89.89% in Nov 2022) due to waning immunity with variations across the provinces. Effective reproduction numbers displayed marked temporal and spatial variability, with higher estimates during Omicron. Overall, these results highlight critical role of group contact heterogeneity in shaping epidemic dynamics.

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.

Peptide-based cancer vaccines offer a promising strategy to target tumor-specific neoantigens, yet their clinical translation is restricted by poor metabolic stability, limited intracellular permeability, and stringent requirements for MHC-I binding and T cell receptor (TCR) recognition. Although peptidomimetic modifications have been widely explored to improve pharmacokinetics, their impact on antigen presentation and immune recognition remains poorly understood. Here, we systematically evaluate backbone N-methylation, peptoid substitution, and stereochemical inversion using the canonical MHC-I epitope from ovalalbumin (OVA), SIINFEKL. Through integrated assays measuring pMHC-I stability, T cell activation, cellular permeability, and serum stability, we demonstrate that tolerance to peptidomimetic modification is highly position-dependent. Specific N-methylated variants retained MHC binding and TCR engagement while exhibiting enhanced cytosolic accumulation, whereas peptoid and stereochemical substitutions were generally disruptive to TCR recognition and membrane permeability. Guided by these insights, we designed combinatorially modified peptides to probe the balance between immunogenicity and pharmacokinetic improvement, revealing that multiple modifications exert non-additive effects on immune recognition. Collectively, these findings establish design principles and provide a framework for balancing immune recognition with enhanced stability and permeability in peptidomimetic antigen design.