FGL1 modulates macrophage function and enhances liver repair through LAG3-TNFR1 axis.

Influenza A viruses (FLUAV) utilize sialic acid to enter host cells via the envelope's hemagglutinin (HA), and its affinity for host-specific sialic acid linkage configuration is a major host range determinant. However, some FLUAV subtypes (H17, H18, H19) use the major histocompatibility complex class II (MHCII) as an entry receptor instead of sialic acid (SA), challenging our knowledge about FLUAV tropism and interspecies transmission potential. Here, we show that H3N2 viruses can use MHCII as an alternative entry receptor in a host-specific manner, and adaptation of human viruses to pigs increases affinity for the MHCII swine leukocyte antigen (SLA). By using two prototypic human-seasonal (hVIC/11) and swine-adapted (sOH/04) H3N2 viruses we found that expression of the human (HLA) but not the swine MHCII conferred replication of hVIC/11 in desialylated, non-susceptible cells. Further, expression of SLA in non-susceptible cells conferred susceptibility to infection by sOH/04. Introduction of point mutations near the hVIC/11 HA receptor-binding site (RBS) allowed the use of both human and swine MHCII. Our findings revealed that MHCII can serve as a sialic acid-independent entry receptor to H3N2 FLUAV in a host-specific manner, with potential implications for the viral pathogenesis and adaptation to a new species.

Respiratory diseases pose a severe threat to global health, with notable limitations in current diagnosis and treatment, such as insufficient sensitivity of diagnostic tools and a lack of effective targeted therapies. Due to their highly efficient information transmission capabilities and excellent safety profile, exosomes carrying non-coding RNA, particularly microRNA (miRNA), are increasingly attracting attention. Compared with free miRNAs, exosomes can protect miRNAs from nuclease degradation, prolong their circulation time in the body, thereby improving the stability and bioavailability of miRNAs. At the same time, they can also address the major bottleneck in the clinical application of miRNAs, including low in vivo delivery efficiency, poor stability, lack of targeting specificity, and off-target effects. Increasing evidence indicate that miRNAs play a significant role in respiratory diseases, including targeting multiple signaling pathways, regulating inflammation and oxidative stress, influencing tumor growth and apoptosis, and participating in tissue damage and repair, thus holding promising prospects for diagnosis and treatment in respiratory diseases. MSC-derived exosomes exhibit low tumorigenic risk because they originate from adult stem cells with limited differentiation ability, have low immunogenicity, and do not highly express major histocompatibility complex class II (MHC-II) molecules, making them suitable for allogeneic use. To enhance the therapeutic efficacy and specificity of exosomes in respiratory diseases, engineering modifications of MSC-exosomes (MSC-exos) are crucial. Current methods for engineering MSC-exos primarily include cargo loading and surface modification to improve therapeutic efficacy and targeting specificity. Through these engineering methods, more precise miRNA delivery can be achieved, reducing the side effects of traditional treatments and improving treatment efficacy. Although MSC-exos demonstrate significant potential in treating respiratory diseases, their clinical translation is hindered by critical hurdles, including individual differences in therapeutic efficacy, insufficient miRNA targeting specificity, challenges in large-scale production, and potential immunogenicity risks. To accelerate clinical application, future research should prioritize optimizing engineered targeting strategies (eg, precision surface modification), enhancing large-scale preparation efficiency of functional MSC-exos, and validating their long-term safety and efficacy in multi-center studies. At present, the good manufacturing practice (GMP) production process of MSC-exos has been established. Early clinical trials (Phase I/II) have shown its potential in respiratory diseases such as pulmonary fibrosis without serious adverse reactions. However, it has not yet been approved for clinical transformation and still faces challenges such as large-scale targeting and safety. Overall, MSC-exos carrying miRNAs show great promise in the treatment of respiratory diseases, but their true clinical application still requires more systematic research and validation.

Monocytes are key cells of the immune system; various cell subsets have distinct phenotypic characteristics and functions. We characterized canine monocyte subsets in different hematologic disorders by analyzing peripheral blood cells for their expression of CD14 and major histocompatibility complex class II (MHCII). Flow cytometry data of canine blood samples were evaluated retrospectively. We assigned 132 total cases to one of the following categories: healthy, inflammatory, T-zone lymphoma/leukemia (TZL), T-cell chronic lymphocytic leukemia (CLL), B-cell CLL, T-cell stage V lymphoma (SVL), B-cell SVL, acute lymphoblastic or undifferentiated leukemia (ALL/AUL), or acute myeloid leukemia (AML). We analyzed age, sex, breed, light scatter properties, WBC count, CD14pos cells, CD14posMHCIIpos cells, CD14posMHCIIneg cells, and the CD14posMHCIIpos/CD14posMHCIIneg ratio. All groups were compared with the healthy group. In healthy dogs, CD14posMHCIIpos cells were larger and had a more complex internal structure than CD14posMHCIIneg cells. Absolute numbers of CD14posMHCIIneg cells were significantly increased in all groups except the TZL and B-cell SVL groups. Absolute numbers of CD14posMHCIIpos cells were significantly increased in the T-cell CLL, B-cell CLL, and T-cell SVL groups, mildly increased in the inflammatory and TZL groups, and decreased in the ALL/AUL group. The CD14posMHCIIpos/CD14posMHCIIneg ratio was markedly decreased in both acute leukemia groups, being <1 in all cases and usually close to 0. We found significant changes in canine monocyte subsets in different hematologic conditions. To our knowledge, a decreased CD14posMHCIIpos/CD14posMHCIIneg ratio has not been identified previously as a hallmark of acute leukemia in dogs.

Aging and DNA damage increase the risk of chronic inflammation and autoimmunity, yet the molecular underpinnings remain unclear. In this study, we uncover a DNA damage-driven mechanism in macrophages that triggers immune autoreactivity. Here, using Er1Lyz2/- mice with a macrophage-specific DNA repair defect in ERCC1-XPF, we demonstrate that monocyte-derived macrophages accumulate DNA damage, activate the immune system, drive polyclonal T cell responses and generate antinuclear autoantibodies. Proteomic and immunopeptidomic analyses reveal a distinct major histocompatibility complex class II (MHC-II) antigen repertoire enriched in nuclear and ribosomal peptides, relying on autophagy for nuclear cargo delivery to MHC-II. Aged macrophages exhibit a similar lysosomal cargo profile, linking autophagy-driven nuclear antigen presentation to immune activation. Notably, inhibiting autophagy in Er1Lyz2/- mice suppresses autoimmune features, pinpointing autophagy-facilitated nuclear antigen processing as a central driver of age-related autoimmunity. These findings establish DNA damage-induced autophagy in macrophages as a pivotal mechanism linking aging to autoimmunity, unveiling potential therapeutic targets to mitigate age-related immune dysregulation.

PurposeThis study aimed to investigate whether 40-Hz light flicker could modulate the expression of major histocompatibility complex class II (MHC-II) and enhance the clearance of amyloid-β (Aβ) deposition.MethodsWe examined retinal MHC-II expression via RNA sequencing, immunofluorescence, and western blotting in mice 8 weeks, 9 months, and 18 to 20 months old. Retinal metabolic waste accumulation was induced by intravitreal and subretinal injections of Aβ oligomers. The impact of 40-Hz flicker on MHC-II expression, microglial activation, and retinal function was evaluated using immunofluorescence, western blotting, dot immunobinding assay, electroretinography, and optokinetic reflex (OKR) testing. Minocycline was used to inhibit microglial activity.ResultsThe 40-Hz light flicker upregulated MHC-II expression in the retinas of aged mice. MHC-II⁺ microglia accumulated along retinal veins and exhibited increased numbers and enlarged morphology in the subretinal space. Following intravitreal or subretinal Aβ injection, 40-Hz flicker enhanced microglial activation, further upregulated MHC-II expression, promoted Aβ clearance, and improved electroretinogram responses and OKR performance. These effects were abolished by minocycline treatment.ConclusionsWe observed that 40-Hz light flicker enhances retinal microglial clearance of Aβ oligomers by upregulating MHC-II expression. These findings support 40-Hz light flicker as a non-invasive therapeutic strategy for age-related retinal disorders by promoting metabolic waste clearance.

Single-cell RNA-sequencing has identified that Alzheimer's disease (AD) pathology in humans is associated with activation of disease-associated microglia (DAM). Microglial signatures of human AD have not been consistently identified in AD mouse models. Since the inflammatory response of rats is more like humans, we profiled microglial transcriptomes in aging TgF344-AD rats, which overexpress two human AD risk genes. Classic DAM gene activation (ApoE, Trem2, Gpnmb), and upregulation (MHC class-II) and downregulation (Ifngr1 and Fkbp5) of human AD microglial genes were identified in aging TgF344-AD rats. Thus, the TgF344-AD rat better recapitulates the microglial gene signature observed in human AD.

Isolation and characterization of mesenchymal stromal cells derived from equine ovarian follicular aspirates.

Class II major histocompatibility complexes (MHC-II) are a highly polymorphic and multigenic family of molecules that present exogenous peptides to CD4+ helper T cells, thereby activating the host adaptive immune system. In this study, we systematically analyzed the genomic distribution and tissue-specific expression of MHC-II genes in Carassius gibelio. The Cagi-DDA/DFA molecule was found to be highly expressed in the spleen and head kidney, moderately expressed in the intestine, gills, and trunk kidney, and expressed at low levels in the liver and brain. A polyclonal antibody was generated against the most prevalent Cagi-DDA/DFA allele in the population. Using immunopeptidomics, we identified viral peptides bound to Cagi-DDA/DFA molecules in the head kidney tissues of C. gibelio following Cyprinid herpesvirus 2 (CyHV-2) infection. A total of 276 antigen peptides were identified, originating from 39 viral proteins. Notably, viral proteins with high abundance and early expression profiles, such as ORF88, ORF121, and ORF141 proteins, were more likely to generate antigen peptides. The identified CyHV-2 peptide epitopes presented by C. gibelio MHC-II molecules provide candidate antigens required for anti-CyHV-2 vaccine development.IMPORTANCEVaccination represents a cornerstone in the prevention of infectious diseases, achieving substantial success in disease control. Upon immunization, protein-derived peptides are processed and presented by major histocompatibility complex class II (MHC-II) molecules, activating CD4+ T cells and triggering adaptive immune responses. Cyprinid herpesvirus 2 (CyHV-2), a pathogenic virus in crucian carp, poses a serious threat to global aquaculture. However, the absence of a comprehensive antigenic profile for CyHV-2 has hindered the development of effective vaccines. Here, we employed immunoaffinity purification coupled with mass spectrometry to systematically identify CyHV-2-derived peptides presented by MHC-II in Carassius gibelio. We identified 276 antigenic peptides originating from 39 viral proteins, which collectively delineate the antigenic landscape of CyHV-2 and provide a rational basis for the design of a vaccine against CyHV-2.

Enterococcus induces MHC-II expression by the intestinal epithelium during murine graft-versus-host disease.

Islet amyloid contributes to beta cell failure in type 2 diabetes through several mechanisms, one being the potent induction of local islet inflammation through activating inflammatory pathways in islet macrophages. As islet amyloid has recently been reported in pancreases of people with type 1 diabetes, and islet macrophages are thought to play a role in the pathogenesis of type 1 diabetes, we sought to understand the impact of islet amyloid on islet macrophages and beta cell autoimmunity. We performed an unbiased phenotypic investigation of islet macrophages in the early stage of islet amyloid formation using single-cell RNA-seq of resident islet macrophages in mice with and without the amyloidogenic form of human islet amyloid polypeptide (hIAPP). The role of islet amyloid in autoimmune diabetes and antigen presentation was assessed in hIAPP-expressing NOD mice and in antigen-presenting cells ex vivo. MHC class II (MHCII) antigen presentation genes were strongly downregulated in islet macrophages during islet amyloid formation. NOD mice expressing an hIAPP transgene had delayed diabetes relative to littermate controls (median onset 30.3 vs 19.5 weeks, p=0.016). Likewise, physiological expression of hIAPP by genetic knockin also delayed diabetes in NOD mice relative to littermate controls (median onset 28.2 vs 18.0 weeks, p=0.049), corresponding with decreased markers of antigen presentation and activation, as well as decreased immune cell infiltration in islets. Adoptive transfer studies showed that systemic autoimmune function remained intact and beta cells from hIAPP transgenic mice did not evade immune recognition by diabetogenic T cells, collectively indicating the protection from diabetes was mediated by decreased antigen presentation in the pancreas. Consistent with this, incubation of dendritic cells with islet amyloid polypeptide (IAPP) aggregates decreased MHCII surface expression and diminished antigen-specific T cell activation through a phagocytosis-dependent mechanism. Collectively, our data reveal a novel role for IAPP aggregates in decreasing MHCII antigen presentation and show that despite the well-established proinflammatory response of macrophages to IAPP aggregates, the uptake of IAPP aggregates during early amyloid formation also disrupts beta cell autoimmunity and delays diabetes in NOD mice.

Tumor-associated macrophages (TAMs) are abundant in the tumor microenvironment (TME) and often adopt an M2-like immunosuppressive phenotype that promotes tumor growth. Reprogramming TAMs toward an M1-like pro-inflammatory state is an attractive therapeutic strategy. Tumor Treating Fields (TTFields), an FDA-approved, electric-field-based therapy, has recently been suggested to modulate immune responses in addition to its established anti-mitotic activity. Here, we investigated the direct effects of TTFields on macrophage activation and function. Murine bone marrow-derived macrophages (BMDMs) were polarized toward a pro-inflammatory M1-like phenotype or an anti-inflammatory M2-like phenotype and exposed to TTFields. TTFields rapidly activated guanine nucleotide exchange factor-H1 (GEF-H1), and downstream nuclear factor kappa B (NF-κB) and activator protein-1 (AP-1, via c-Jun N-terminal kinase [JNK]) signaling. Functionally, TTFields reprogrammed M2-like macrophages by increasing major histocompatibility complex class II (MHC-II) and cluster of differentiation 80 (CD80); reducing arginase-1 (Arg1); and elevating secretion of chemokine (C-X-C motif) ligand 1 (CXCL1), interleukin-6 (IL-6), IL-1β, and IL-12 subunit p70 (IL-12p70). In interferon gamma (IFN-γ)-primed macrophages, TTFields provided a secondary signal, driving myeloid differentiation primary response 88 (MyD88)-dependent expression of inducible nitric oxide synthase (iNOS). In vivo, TTFields reduced tumor burden in an orthotopic murine lung cancer model and increased iNOS expression in both M1-like and a subset of M2-like TAMs. These findings demonstrate that TTFields directly reprogram macrophages toward a pro-inflammatory phenotype, suggesting a novel immunomodulatory mechanism that may enhance anti-tumor immunity in the TME.

Helper T cells (CD4 T cells) are lynchpins of adaptive immune responses. Each CD4 T cell expresses a single T-cell receptor, recognizing an epitope presented by major histocompatibility complex class II (MHC-II). Due to the enormous diversity of the T-cell repertoire, it is often desirable to study a population that responds to the same epitope. Here, we devised a novel method of selective immunization to produce a robust, monospecific helper T-cell response in mice using a modular mRNA vector. The vector encodes the target epitope attached via flexible linker to MHC-II. Immunization with this mRNA selectively elicits helper T-cell responses across a range of epitopes and MHC-II alleles. These CD4 T cells show robust, polyfunctional Th1 cytokine release when evaluated ex vivo. We tested the activity of these CD4 T cells in 2 disease models: Salmonella enterica and influenza virus. In both models, these monospecific CD4 T cells influenced the course of infection, demonstrating the utility of this experimental tool, which can produce a monospecific CD4 T-cell response.

B cell follicles in secondary lymphoid tissues are major sites of HIV expression in untreated people living with HIV (PLWH), but whether lymphoid B cells promote HIV expression in CD4+ T cells is unknown. Using a tonsil model and flow cytometry, germinal center B cells induced HIV expression in follicular helper CD4+ T cells (TFH) in a concentration-, contact-, and major histocompatibility complex class II (MHC-II)-dependent manner. Non-naïve tonsil B cells also induced HIV expression in nonTFH CD4+ T cells that was MHC-II restricted. In situ hybridization and immunofluorescent staining of lymph node sections from six PLWH on long-term antiretroviral therapy (ART) and six ART-naïve PLWH demonstrated most HIV RNA-expressing (vRNA+) cells were adjacent to at least one B cell. In both groups, vRNA+ cells per mm2 were elevated in B cell follicles, particularly after adjusting for CD3+CD4+ frequencies. TFH (PD-1+BCL6+) were a minority of vRNA+ cells in all PLWH. In contrast to ART-naïve PLWH, most vRNA+ cells in PLWH on ART resided outside follicles, and were preferentially adjacent to extrafollicular B cells. Thus, B cells upregulate HIV expression largely through cognate interactions in vitro and are linked to HIV expression in secondary lymphoid tissues.

Group 3 innate lymphoid cells: guardians of intestinal homeostasis.

The treatment of acute myeloid leukemia (AML) remains a challenge due to disease heterogeneity, which undermines efforts to develop targeted therapeutics, leaving conventional chemotherapy as the standard of care (SOC). Sialic acid binding Ig-like lectin 3 (CD33) is a myeloid cell surface glycoprotein that is highly expressed on AML blasts. However, while ~90% of AML cases express CD33, 50% of these patients harbor a single nucleotide polymorphism (SNP) that eliminates the antibody binding epitope for existing CD33-targeted antibody therapeutics. In this study, we developed an immunotherapy (M2T-CD33) that targets CD33 directly to MHC class II (MHCII) molecules on antigen presenting cells for enhanced presentation to the immune system. We found that M2T-CD33 induces a robust polyclonal anti-CD33 humoral response composed of the full immunoglobulin repertoire. M2T-CD33 induced an anti-AML response in a syngeneic mouse model that was dependent on CD4+ and CD8+ T cells. The immune response was elicited against both the full length and spliced version of CD33 and showed no evidence of toxicity at concentrations 40-fold higher than the efficacious dose. Finally, M2T-CD33 was enhanced by combinations with anti-PD-1 therapy. These experiments demonstrate the preclinical potential of M2T-CD33 in AML and emphasize the importance of MHCII for cancer immunotherapy.

Atherosclerosis is a chronic inflammatory disease of the arterial wall, where modified self-antigens cause sustained adaptive immune responses driven by clonal expansion of CD4+ effector T cells in situ. Although immune tolerance-inducing vaccination strategies show promise in animal models, their clinical translation is limited by the unclear role of adaptive immune responses in human atherosclerosis. RNA sequencing and immunohistochemistry were employed in human atherosclerotic plaques. Nuclear run-on, CRISPRi/a, RNA pull-down, ChIP-qPCR, and flow cytometry were utilized to investigate the lncRNA-dependent regulatory mechanism. Functional validation was performed in Apoe-/- mice through perivascular or intravenous siRNA delivery, as well as perivascular administration of macrophage-targeting lipid nanoparticles carrying dCas9-VP64 mRNA and the guide RNA. Major histocompatibility complex class II (MHCII)-dependent antigen presentation pathways were progressively activated in lesional macrophages as human atherosclerosis advanced. This activation was associated with increased CIITA expression, driven by enhanced transcription of its antisense-lncRNA (CIITA-AS1) and alternative splicing generating a non-coding transcript. Knockdown of these two lncRNAs reduced MHCII levels in human macrophages, thereby suppressing co-cultured T-cell activation. Mechanistically, CIITA-AS1 transcription promoted CIITA expression through STAT1 recruitment. Enforcing antisense-strand transcription of Ciita in lesional macrophages up-regulated Ciita and MHCII expression, exacerbating atherosclerosis in mice. Conversely, inhibition of macrophage antigen presentation via siRNA-mediated Ciita silencing attenuated atherosclerosis by reducing T-cell-mediated inflammation. This study reveals that CIITA-induced antigen presentation in macrophages activates local T-cell inflammatory response, promoting plaque progression. Targeting this pathway may improve vaccination efficacy by creating anti-inflammatory niches within plaques, offering a novel therapeutic strategy for atherosclerosis.

Pakistan currently holds the second-highest prevalence rate of Hepatitis C virus (HCV) globally. It makes it crucial to continuously monitor the circulating genotypes in the population, especially among the people who inject drugs (PWIDs), as they pose a significant risk of spreading new genotypes in the population. To address this issue, we identified the circulating HCV genotypes among PWIDs and non-PWIDs through Next Generation Sequencing (NGS). Additionally, a multi-epitope vaccine was designed through an immunoinformatic approach using NGS and Sanger sequencing results. The study indicated genotype 3a as the most prevalent genotype among the 61 HCV cases tested through NGS, followed by genotype 1a. The non-allergic and highly antigenic epitopes from both MHC Class-I and Class-II epitopes were retreived from non-structural proteins. Furthermore, B-cell epitopes were retrieved from the E2 protein. The selected epitopes showed 88.26% population coverage rate. Based on large conformational simulation analysis from NMSims, four best constructs suitable for vaccine design were further evaluated for their binding energies through all-atom molecular dynamics simulations and the MMGBSA. One of the constructs showed a low binding energy value with MHC, indicating its potential as a vaccine candidate. However, further experimental work is required to determine its efficacy and safety profile. This research emphasizes the promise of combining multiepitope vaccine design advanced computational methods to accelerate and improve vaccine development thereby filling a crucial gap in the fight against rising antibiotic resistance.

No currently licensed vaccine reliably prevents pulmonary tuberculosis (TB), a leading cause of infectious disease mortality. Developing effective new vaccines requires identifying which Mycobacterium tuberculosis (Mtb) proteins are presented on major histocompatibility complex class II (MHC-II) by infected human phagocytes (target cells) and defining their capacity for recognition by CD4+ T cells. Vaccine designs must elicit T cell responses recognizing the same peptide-MHC complexes presented by infected cells. Although many human CD4+ T cell Mtb epitopes have been described, presentation on MHC-II by infected cells in most cases has not been directly evaluated. Using mass spectrometry (MS), we demonstrated that Mtb type VII secretion system (T7SS) substrates are enriched in the MHC-II repertoire of Mtb-infected human monocyte-derived phagocytes and that many of these antigens are immunogenic in people with prior evidence of Mtb infection. We next used MS to guide TB messenger RNA (mRNA) vaccine design, increasing the presentation of target MHC-II epitopes by orders of magnitude by incorporating design features that mirror aspects of antigen presentation dynamics in infected phagocytes. Our results provide a strategy for TB vaccine design that is guided by bottom-up unbiased discovery. Our approach combines targeted evaluation of antigen presentation in human cells paired with rapid iterative testing of mRNA vaccine designs to optimize antigen presentation before animal studies or human clinical trials.

IFNγ-driven MHC class II expression by intestinal epithelial cells dictates local cytolytic Th1 cell differentiation and intestinal stem cell loss