ImmunoHorizons最新文献

Tuberculosis (TB) is a serious public health concern in many regions of the world and the only approved vaccine to prevent TB is the live-attenuated BCG vaccine. Despite being widely used, the BCG vaccine fails to prevent pulmonary TB in adults. The BCG vaccine is administered during the neonatal period when levels of the immunosuppressive cytokine interleukin (IL)-27 are elevated, and previous studies have demonstrated that the source of IL-27 can impact downstream immune responses. We therefore sought to characterize the specific subpopulations of myeloid cells that produce IL-27 following BCG vaccination. To investigate this, we administered the BCG vaccine to neonatal IL-27p28eGFP mice that report IL-27 production. Our studies demonstrated that BCG vaccination steadily increased IL-27 production throughout the weeks post-vaccination. We also showed that a predominantly CD11b+ F4/80+ population of IL-27 producers increased MHC class II expression following BCG vaccination in both the spleen and the lung. However, producers of IL-27 in these tissues differ, with a population of CD11c+ MHC II+ cells emerging in the spleen and a subset of Ly6G/C+ MHC II+ emerging in the lung. 10x scMultiome analysis further validated the increase in MHC class II expression and demonstrated improved antigen presentation functionality following vaccination. The sequencing analysis also revealed subpopulations of IL-27 producers with immunosuppressive functions such as a population of macrophages with increased Mrc1 expression post-vaccination. Our findings suggest that IL-27 producers are a heterogenous population of myeloid cells that impact the development of protective immune responses induced by the BCG vaccine.

Whether epigenetic factor UTX, a histone H3 lysine 27 (H3K27) demethylase, is critical for type 2 immunity, including allergic sensitization and antigen-driven anaphylaxis, is unclear. We used UTXfl/fl x Lck-Cre mice with UTX-deficient T cells (UTX-TCD) to determine whether T cell-specific UTX expression regulates antigen-specific IgE production after airway sensitization to peanut and anaphylaxis following intraperitoneal (i.p.) peanut challenge. UTX-TCD mice sensitized via the airway with peanut and lipopolysaccharide (LPS), a bacterial component and environmental adjuvant found in house dust, made 2-fold less peanut-IgE and 3.5-fold less peanut-IgG1 than comparably sensitized UTXfl/fl mice, despite higher total IgE and total IgG1 serum antibody levels pre-sensitization. Peanut-induced anaphylaxis was blunted in UTX-TCD mice, with maximum drop in core body temperature after i.p. peanut challenge two-fold lower than in UTXfl/fl mice. Compared to UTXfl/fl controls, UTX-TCD mice had reduced frequencies of CD4+ T-follicular helper (Tfh) cells and germinal center B cells, but higher frequencies of IL-4+ T-helper (Th)2, Tfh2, and IL-13+ Tfh13 cells in airway-draining mediastinal lymph nodes. UTX-TCD mice also skewed toward type 2 antibody and T-helper immune responses independent of allergic sensitization, with fewer IL-10-producing splenic Treg and T-follicular regulatory (Tfr) cells. Our results suggest that UTX expression in T cells impact the production of antigen-specific antibody responses required for allergic sensitization and antigen-specific allergic reactions, suggesting a role for H3K27 histone demethylase UTX in regulating type 2 immunity.

C3 glomerulopathy (C3G), a rare kidney disease caused by dysregulation of alternative pathway complement activation, is characterized by glomerular C3 deposition, proteinuria, crescentic glomerulonephritis, and renal failure. The anti-C5 monoclonal antibody (mAb) drug eculizumab has shown therapeutic effects in some but not all patients with C3G, and no approved therapy is currently available. Here, we developed and used a triple transgenic mouse model of fast progressing lethal C3G (FHm/mP-/-hFDKI/KI) to compare the therapeutic efficacy of a bifunctional anti-C5 mAb fused to a functional factor H (FH) fragment (short consensus repeat 1-5 [SCR1-5]) and the anti-C5 mAb itself. The new C3G mouse model is derived by humanizing factor D (hFDKI/KI) in a previously described FHm/mP-/- mouse that developed lethal C3G. We tested the effectiveness of these 2 complement inhibitors in triple transgenic mice with established C3G and glomerular disease. No FHm/mP-/-hFDKI/KI mice treated with vehicle survived the 30-d study period. All FHm/mP-/-hFDKI/KI mice treated with the C5 mAb-FH SCR1-5 fusion protein and 50% of mice treated with the anti-C5 mAb survived the 30-d treatment period. Moreover, mice treated with the C5 mAb-FH SCR1-5 fusion protein, but not those treated with the anti-C5 mAb, showed restored plasma alternative pathway complement control. The C5 mAb-FH SCR1-5 fusion protein reversed glomerular disease to a greater degree than the anti-C5 mAb. These data suggest that simultaneously inhibiting the terminal and proximal complement pathways, by anti-C5 mAb and FH SCR1-5, respectively, can reverse established C3G and is more efficacious than inhibiting the terminal pathway alone. A similar approach may be effective in treating human C3G.

Antibody (Ab) crosslinking of HLA class II (HLA II) molecules on the surface of endothelial cells (ECs) triggers proliferative and prosurvival intracellular signaling, which are implicated in promoting chronic Ab-mediated rejection (cAMR). Despite the importance of cAMR in transplant medicine, the mechanisms involved remain incompletely understood. Here, we examined the regulation of yes-associated protein (YAP) nuclear cytoplasmic localization and phosphorylation in human ECs challenged with Abs that bind HLA II, which are strongly associated with cAMR. To examine changes in YAP localization in response to Ab-mediated engagement of HLA II, we used an adenoviral vector to express the class II transactivator or treatment with interferon γ. In unstimulated ECs expressing HLA II, YAP localized mainly in the cytoplasm. Stimulation with HLA II Ab (0.1-1 µg/mL) induced marked translocation of YAP to the nucleus. HLA II signaling triggered by high concentrations of HLA II Ab (1 µg/mL) also induced prominent YAP localization in cytoplasmic punctate structures that were disassembled by exposure to 1,6-hexanediol, suggesting that these structures are biomolecular condensates. Using multiple treatments, including stimulation with serum, thrombin or HLA I Ab and conditions (eg ECs plated at different densities) indicate that formation of YAP cytoplasmic puncta can be dissociated from YAP nuclear localization and phosphorylation at Ser127, a site in YAP targeted by the Hippo kinases LATS1/2. The results revealed that HLA II signaling regulates YAP subcellular distributions in ECs and demonstrate, for the first time, that HLA II Ab selectively stimulates YAP concentration in punctate structures.

Enzyme-linked immunosorbent spot analysis is frequently used to investigate immune responsiveness during clinical trials. However, ELISpot classically utilizes peripheral blood mononuclear cell isolates from whole blood, requiring relatively high blood draw volumes and removing both granulocytes and bound drug. Here, we describe a novel protocol whereby CD45 cells are magnetically isolated from human whole blood and co-incubated with serum isolated from the same subject. Infliximab is a well characterized anti-tumor necrosis factor α (TNF-α) antibody in clinical use since the late 1990s. We demonstrated that TNF-α inhibition by infliximab in spiked whole blood is lost on peripheral blood mononuclear cell isolation but remains in serum, and that combining serum from infliximab spiked whole blood with magnetically isolated CD45 immune cells inhibited PMA/ionomycin-stimulated TNF-α secretion. This novel protocol has important implications for enzyme-linked immunosorbent spot analysis in clinical trials in which blood volume is limited, and keeping drug responses intact provides critical information.

The global dissemination of SARS-CoV-2 led to a worldwide pandemic in March 2020. Even after the official downgrading of the COVID-19 pandemic, infection with SARS-CoV-2 variants continues. The rapid development and deployment of SARS-CoV-2 vaccines helped to mitigate the pandemic to a great extent. However, the current vaccines are suboptimal; they elicit incomplete and short-lived protection and are ineffective against evolving virus variants. Updating the spike antigen according to the prevailing variant and repeated boosters is not the long-term solution. We have designed a lipopeptide-based, mucosal, pan-coronavirus vaccine candidate, derived from highly conserved and/or functional regions of the SARS-CoV-2 spike, nucleocapsid, and membrane proteins. Our studies demonstrate that the designed lipopeptides (LPMix) induced both cellular and humoral (mucosal and systemic) immune responses upon intranasal immunization in mice. Furthermore, the antibodies bound to the wild-type and mutated S proteins of SARS-CoV-2 variants of concern, including Alpha, Beta, Delta and Omicron, and also led to efficient neutralization in a surrogate viral neutralization assay. Our sequence alignment and 3-dimensional molecular modeling studies demonstrated that spike-derived epitopes, P1 and P2, are sequentially and/or structurally conserved among the SARS-CoV-2 variants. The addition of a novel mucosal adjuvant, heat-killed Caulobacter crescentus, to the lipopeptide vaccine significantly bolstered mucosal antibody responses. Finally, the lipopeptide-based intranasal vaccine demonstrated significant improvement in lung pathologies in a hamster model of SARS-CoV-2 infection. These studies are fundamentally important and open new avenues in the investigation of an innovative, broadly protective intranasal vaccine platform for SARS-CoV-2 and its variants.

Adjuvants play a central role in enhancing the immunogenicity of otherwise poorly immunogenic vaccine antigens. Combining adjuvants has the potential to enhance vaccine immunogenicity compared with single adjuvants, although the cellular and molecular mechanisms of combination adjuvants are not well understood. Using the influenza virus hemagglutinin H5 antigen, we define the immunological landscape of combining CpG and MPLA (TLR-9 and TLR-4 agonists, respectively) with a squalene nanoemulsion (AddaVax) using immunologic and transcriptomic profiling. Mice immunized and boosted with recombinant H5 in AddaVax, CpG+MPLA, or AddaVax plus CpG+MPLA (IVAX-1) produced comparable levels of neutralizing antibodies and were equally well protected against the H5N1 challenge. However, after challenge with H5N1 virus, H5/IVAX-1-immunized mice had 100- to 300-fold lower virus lung titers than mice receiving H5 in AddaVax or CpG+MPLA separately. Consistent with enhanced viral clearance, unsupervised expression analysis of draining lymph node cells revealed the combination adjuvant IVAX-1 significantly downregulated immune homeostasis genes, and induced higher numbers of antibody-producing plasmablasts than either AddaVax or CpG+MPLA. IVAX-1 was also more effective after single-dose administration than either AddaVax or CpG+MPLA. These data reveal a novel molecular framework for understanding the mechanisms of combination adjuvants, such as IVAX-1, and highlight their potential for the development of more effective vaccines against respiratory viruses.

Atopic dermatitis (AD) is characterized by dysregulated T cell immunity and skin microbiome dysbiosis with predominance of Staphylococcus aureus, which is associated with exacerbating AD skin inflammation. Specific glycosylation patterns of S. aureus cell wall structures amplify skin inflammation through interaction with Langerhans cells (LCs). Nevertheless, the role of LCs in AD remains poorly characterized. Here, we performed single cell RNA sequencing of primary epidermal LCs and dermal T cells, isolated from skin biopsies of AD patients and healthy control subjects, alongside specific glycoanalysis of S. aureus strains isolated from the AD lesions. Our findings revealed 4 LC subpopulations ie, 2 steady-state clusters [LC1 and LC1H] and 2 proinflammatory/matured subsets [LC2 and migratory LCs]. The latter 2 subsets were enriched in AD skin. AD LCs showed enhanced expression of C-type lectin receptors, the high-affinity IgE receptor, and activation of prostaglandin and leukotriene biosynthesis pathways, upregulated transcriptional signatures related to T cell activation pathways, and increased expression of CCL17 compared with healthy LCs. Correspondingly, T helper 2 and T regulatory cell populations were increased in AD lesions. Complementary, we performed bulk RNA sequencing of primary LCs stimulated with the S. aureus strains isolated from the AD lesions, which showed upregulation of T helper 2-related pathways. Our study provides proof-of-concept for a role of LCs in connecting the S. aureus-T cell axis in the AD inflammatory cycle.

Respiratory syncytial virus (RSV) is a major contributor to morbidity and mortality in infants. We developed an in vitro model of human respiratory infection to study cellular immune responses to RSV in infants, children, and adults. The model includes human lung epithelial A549 cells or human fetal lung fibroblasts infected with a clinical strain of RSV at a multiplicity of infection of 0.3, cocultured with human cord blood mononuclear cells (CBMCs) or peripheral blood mononuclear cells (PBMCs). Mononuclear cells were collected at multiple ages ranging from birth to adulthood. After 20 h of incubation, flow cytometry was used to measure CBMC/PBMC responses to RSV. A549s were more permissive to RSV and when infected produced more CCL5, CCL11, and CXCL9; less CSF-3, CXCL10, interleukin (IL)-1α, IL-1RA, and IL-6; and similar CCL2, CCL3, CCL4, CCL7, CXCL1, CXCL11, IL-1β, IL-7, IL-8, and tumor necrosis factor α compared with fibroblasts; A594s were used for subsequent experiments. CBMCs/PBMCs upregulated multiple markers of activation, maturation, and degranulation upon exposure to RSV-infected A549s. Interferon γ expression in natural killer, CD4, and CD8 cells and CD107a expression in natural killer cells showed a gradual increase from infancy to adulthood. IL-12 expression in dendritic cells and monocytes was highest in adult PBMCs. Our in vitro model of human RSV infection recapitulated the expected bias away from T helper 1 and effector responses to RSV infection in infancy and revealed changes in innate and adaptive RSV-specific cellular immune responses over time.