Induction of Interleukin-9-Producing Mucosal Mast Cells Promotes Susceptibility to IgE-Mediated Experimental Food Allergy.

Experimental IgE-mediated food allergy depends on intestinal anaphylaxis driven by interleukin-9 (IL-9). However, the primary cellular source of IL-9 and the mechanisms underlying the susceptibility to food-induced intestinal anaphylaxis remain unclear. Herein, we have reported the identification of multifunctional IL-9-producing mucosal mast cells (MMC9s) that can secrete prodigious amounts of IL-9 and IL-13 in response to IL-33, and mast cell protease-1 (MCPt-1) in response to antigen and IgE complex crosslinking, respectively. Repeated intragastric antigen challenge induced MMC9 development that required T cells, IL-4, and STAT6 transcription factor, but not IL-9 signals. Mice ablated of MMC9 induction failed to develop intestinal mastocytosis, which resulted in decreased food allergy symptoms that could be restored by adoptively transferred MMC9s. Finally, atopic patients that developed food allergy displayed increased intestinal expression of Il9- and MC-specific transcripts. Thus, the induction of MMC9s is a pivotal step to acquire the susceptibility to IgE-mediated food allergy.

C5a receptor 1-/- mice are protected from the development of IgE-mediated experimental food allergy.

BACKGROUND: Food-induced anaphylaxis is a serious allergic reaction caused by Fcε-receptor activation on mast cells (MCs). The exact mechanisms breaking oral tolerance and the effector pathways driving food allergy remain elusive. As complement is activated in food-induced anaphylaxis, we aimed to assess the role of C5a in disease pathogenesis. METHODS: Oral antigen-induced food-induced anaphylaxis was induced in BALB/c wild-type (wt) and C5ar1-/- mice. Readouts included diarrhea development, changes in rectal temperature, hematocrit, antigen-specific serum IgE, MCPT-1, and intestinal MC numbers, as well as FcεR1-mediated MC functions including C5a receptor 1 (C5aR1) regulation. Further, histamine-mediated hypothermia and regulation of endothelial tight junctions were determined. RESULTS: Repeated oral OVA challenge resulted in diarrhea, hypothermia, increased hematocrit, high OVA-specific serum IgE, and MCPT-1 levels in wt mice. Male C5ar1-/- mice were completely whereas female C5ar1-/- mice were partially protected from anaphylaxis development. Serum MCPT-1 levels were reduced gender-independent, whereas IgE levels were reduced in male but not in female C5ar1-/- mice. Mechanistically, IgE-mediated degranulation and IL-6 production from C5ar1-/- BMMCs of both sexes were significantly reduced. Importantly, FcεR1 cross-linking strongly upregulated C5aR1 MC expression in vitro and in vivo. Finally, C5ar1-/- male mice were largely protected from histamine-induced hypovolemic shock, which was associated with protection from histamine-induced barrier dysfunction in vitro following C5aR targeting. CONCLUSIONS: Our findings identify C5aR1 activation as an important driver of IgE-mediated food allergy through regulation of allergen-specific IgE production, FcεR1-mediated MC degranulation, and histamine-driven effector functions preferentially in male mice.

Collaborative interactions between type 2 innate lymphoid cells and antigen-specific CD4+ Th2 cells exacerbate murine allergic airway diseases with prominent eosinophilia.

Type-2 innate lymphoid cells (ILC2s) and the acquired CD4(+) Th2 and Th17 cells contribute to the pathogenesis of experimental asthma; however, their roles in Ag-driven exacerbation of chronic murine allergic airway diseases remain elusive. In this study, we report that repeated intranasal rechallenges with only OVA Ag were sufficient to trigger airway hyperresponsiveness, prominent eosinophilic inflammation, and significantly increased serum OVA-specific IgG1 and IgE in rested mice that previously developed murine allergic airway diseases. The recall response to repeated OVA inoculation preferentially triggered a further increase of lung OVA-specific CD4(+) Th2 cells, whereas CD4(+) Th17 and ILC2 cell numbers remained constant. Furthermore, the acquired CD4(+) Th17 cells in Stat6(-/-)/IL-17-GFP mice, or innate ILC2s in CD4(+) T cell-ablated mice, failed to mount an allergic recall response to OVA Ag. After repeated OVA rechallenge or CD4(+) T cell ablation, the increase or loss of CD4(+) Th2 cells resulted in an enhanced or reduced IL-13 production by lung ILC2s in response to IL-25 and IL-33 stimulation, respectively. In return, ILC2s enhanced Ag-mediated proliferation of cocultured CD4(+) Th2 cells and their cytokine production, and promoted eosinophilic airway inflammation and goblet cell hyperplasia driven by adoptively transferred Ag-specific CD4(+) Th2 cells. Thus, these results suggest that an allergic recall response to recurring Ag exposures preferentially triggers an increase of Ag-specific CD4(+) Th2 cells, which facilitates the collaborative interactions between acquired CD4(+) Th2 cells and innate ILC2s to drive the exacerbation of a murine allergic airway diseases with an eosinophilic phenotype.

IL-25 and CD4(+) TH2 cells enhance type 2 innate lymphoid cell-derived IL-13 production, which promotes IgE-mediated experimental food allergy.

BACKGROUND: Food-mediated allergic reactions have emerged as a major health problem. The underlying mechanisms that promote uncontrolled type 2 immune responses to dietary allergens in the gastrointestinal tract remain elusive. OBJECTIVE: We investigated whether altering IL-25 signaling enhances or attenuates allergic responses to food allergens. METHODS: Mice of an IL-25 transgenic mouse line (iIL-25Tg mice), which constitutively overexpress intestinal IL-25, and Il17rb(-/-) mice, in which Il17rb gene expression is disrupted, were sensitized and gavage fed with ovalbumin (OVA). We assessed symptomatic characteristics of experimental food allergy, including incidence of diarrhea, incidence of hypothermia, intestinal TH2 immune response, and serum OVA-specific IgE and mast cell protease 1 production. RESULTS: Rapid induction of Il25 expression in the intestinal epithelium preceded onset of the anaphylactic response to ingested OVA antigen. iIL-25Tg mice were more prone and Il17rb(-/-) mice were more resistant to experimental food allergy. Resident intestinal type 2 innate lymphoid cells (ILC2s) were identified as the major producers of IL-5 and IL-13 in response to IL-25. Reconstituting irradiated wild-type mice with Rora(-/-) or Il17rb(-/-) bone marrow resulted in a deficiency or dysfunction of the ILC2 compartment, respectively, and resistance to experimental food allergy. Repeated intragastric antigen challenge induced a significant increase in numbers of CD4(+) TH2 cells, which enhance IL-25-stimulated IL-13 production by ILC2s ex vivo and in vivo. Finally, reconstituted IL-13-deficient ILC2s had reduced capability to promote allergic inflammation, resulting in increased resistance to experimental food allergy. CONCLUSION: IL-25 and CD4(+) TH2 cells induced by ingested antigens enhance ILC2-derived IL-13 production, thereby promoting IgE-mediated experimental food allergy.

Thermoneutrality Alters Gastrointestinal Antigen Passage Patterning and Predisposes to Oral Antigen Sensitization in Mice.

Food allergy is an emerging epidemic, and the underlying mechanisms are not well defined partly due to the lack of robust adjuvant free experimental models of dietary antigen sensitization. As housing mice at thermoneutrality (Tn) - the temperature of metabolic homeostasis (26-30°C) - has been shown to improve modeling various human diseases involved in inflammation, we tested the impact of Tn housing on an experimental model of food sensitization. Here we demonstrate that WT BALB/c mice housed under standard temperature (18-20°C, Ts) conditions translocated the luminal antigens in the small intestine (SI) across the epithelium via goblet cell antigen passages (GAPs). In contrast, food allergy sensitive Il4raF709 mice housed under standard temperature conditions translocated the luminal antigens in the SI across the epithelium via secretory antigen passages (SAPs). Activation of SI antigen passages and oral challenge of Il4raF709 mice with egg allergens at standard temperature predisposed Il4raF709 mice to develop an anaphylactic reaction. Housing Il4raF709 mice at Tn altered systemic type 2 cytokine, IL-4, and the landscape of SI antigen passage patterning (villus and crypt involvement). Activation of SI antigen passages and oral challenge of Il4raF709 mice with egg antigen under Tn conditions led to the robust induction of egg-specific IgE and development of food-induced mast cell activation and hypovolemic shock. Similarly, Tn housing of WT BALB/c mice altered the cellular patterning of SI antigen passage (GAPs to SAPs). Activation of SI antigen passages and the oral challenge of WT BALB/c mice with egg antigen led to systemic reactivity to egg and mast cell activation. Together these data demonstrate that Tn housing alters antigen passage cellular patterning and landscape, and concurrent oral exposure of egg antigens and SAP activation is sufficient to induce oral antigen sensitization.

Ssu72 is a T-cell receptor-responsive modifier that is indispensable for regulatory T cells.

The homeostatic balance between effector T cells and regulatory T cells (Tregs) is crucial for adaptive immunity; however, epigenetic programs that inhibit phosphorylation to regulate Treg development, peripheral expression, and suppressive activity are elusive. Here, we found that the Ssu72 phosphatase is activated by various T-cell receptor signaling pathways, including the T-cell receptor and IL-2R pathways, and localizes at the cell membrane. Deletion of Ssu72 in T cells disrupts CD4+ T-cell differentiation into Tregs in the periphery via the production of high levels of the effector cytokines IL-2 and IFNγ, which induce CD4+ T-cell activation and differentiation into effector cell lineages. We also found a close correlation between downregulation of Ssu72 and severe defects in mucosal tolerance in patients. Interestingly, Ssu72 forms a complex with PLCγ1, which is an essential effector molecule for T-cell receptor signaling as well as Treg development and function. Ssu72 deficiency impairs PLCγ1 downstream signaling and results in failure of Foxp3 induction. Thus, our studies show that the Ssu72-mediated cytokine response coordinates the differentiation and function of Treg cells in the periphery.

MG1141A as a Highly Potent Monoclonal Neutralizing Antibody Against SARS-CoV-2 Variants.

Since the coronavirus disease outbreak in 2019, several antibody therapeutics have been developed to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Antibody therapeutics are effective in neutralizing the virus and reducing hospitalization in patients with mild and moderate infections. These therapeutics target the spike protein of SARS-CoV-2; however, emerging mutations in this protein reduce their efficiency. In this study, we developed a universal SARS-CoV-2 neutralizing antibody. We generated a humanized monoclonal antibody, MG1141A, against the receptor-binding domain of the spike protein through traditional mouse immunization. We confirmed that MG1141A could effectively neutralize live viruses, with an EC50 of 92 pM, and that it exhibited effective Fc-mediated functions. Additionally, it retained its neutralizing activity against the alpha (UK), beta (South Africa), and gamma (Brazil) variants of SARS-CoV-2. Taken together, our study contributes to the development of a novel antibody therapeutic approach, which can effectively combat emerging SARS-CoV-2 mutations.

Vaccination with recombinant adenoviruses and dendritic cells expressing prostate-specific antigens is effective in eliciting CTL and suppresses tumor growth in the experimental prostate cancer.

BACKGROUND: Prostate cancer is currently the most commonly diagnosed cancer in men and the second leading cause of cancer-related death in men in the US. Immunological approaches may provide an alternative option for prevention and treatment of prostate cancer. METHODS: To develop vaccine against prostate cancer using mouse model, we constructed three recombinant adenoviruses expressing prostate-specific membrane antigen (rAd/PSMA), prostate stem cell antigen (rAd/PSCA) and six-transmembrane epithelial antigen of the prostate (rAd/STEAP), that were specifically up-regulated in the transgenic murine prostate cancer. RESULTS: Male C57BL/6 mice were immunized by intravenous injection of these recombinant adenoviruses and subsequently by subcutaneous injection of dendritic cells pulsed with TRAMP-C1 tumor lysate. After subcutaneous challenge with TRAMP-C1 cells, tumor growth was significantly delayed in the immunized mice compared to the control group. Surprisingly, significant numbers of STEAP-specific CD8 T cells were detected in the peripheral blood and the spleen of immune mice using MHC I tetramers, and injection of rAd/STEAP alone followed by pulsed DC was sufficient to inhibit tumor growth. Therapeutic vaccination also significantly delayed the growth of pre-established tumors. CONCLUSION: Our results suggest that STEAP is a good immunologic target antigen against prostate cancer and our vaccination regimen successfully elicits anti-tumor CTL responses and suppresses tumor growth. More studies will expedite the development of this vaccine toward clinical application.

T-bet expression is regulated by EGR1-mediated signaling in activated T cells.

T-bet is a Th1-specific transcription factor that is directly involved in three important pathways for Th1 cell differentiation, namely TCR signaling, and the IFN-gamma-STAT1 and IL-12-STAT4 pathways. A recent study also showed that T-bet plays a vital role in innate immunity. However, the molecular mechanism responsible for transcriptional activation of T-bet during T cell development is not yet known. Here, we characterize the essential human T-bet promoter elements and show that binding of EGR1 to this promoter induces T-bet transcription. Notably, overexpression of EGR1 transactivates and, synergistically in concert with TCR signaling, induces T-bet expression in activated T cells. In contrast, depletion of EGR1 significantly decreases T-bet induction. Finally, we report a positive correlation between EGR1 and T-bet expression during T helper cell differentiation. Collectively, these findings provide molecular insight into T-bet transcription and suggest that EGR1 is an upstream regulator of T-bet induction.

Single intranasal immunization with recombinant adenovirus-based vaccine induces protective immunity against respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine against RSV. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses. Here, a recombinant replication-deficient adenovirus-based vaccine, rAd/3xG, expressing the soluble core domain of G glycoprotein (amino acids 130 to 230) engineered by codon optimization and tandem repetition for higher-level expression, was constructed and evaluated for its potential as an RSV vaccine in a murine model. A single intranasal immunization with rAd/3xG provided potent protection against RSV challenge which lasted for more than 10 weeks. Strong mucosal immunoglobulin A responses were also induced by a single intranasal immunization but not by intramuscular or oral administration of rAd/3xG. Interestingly, neither gamma interferon- nor interleukin-4-producing CD4 T cells directed to I-E(d)-restricted epitope were detected in the lungs of rAd/3xG-immune mice upon challenge, whereas priming with vaccinia virus expressing RSV G (vvG) elicited strong Th1/Th2 mixed CD4 T-cell responses. Lung eosinophilia and vaccine-induced weight loss were significantly lower in the rAd/3xG-immune group than in the vvG-primed group. Together, our data demonstrate that a single intranasal administration of rAd/3xG elicits beneficial protective immunity and represents a promising vaccine regimen against RSV infection.

IL-9-producing cells in the development of IgE-mediated food allergy.

Food allergy is a harmful immune reaction driven by uncontrolled type 2 immune responses. Considerable evidence demonstrates the key roles of mast cells, IgE, and TH2 cytokines in mediating food allergy. However, this evidence provides limited insight into why only some, rather than all, food allergic individuals are prone to develop life-threatening anaphylaxis. Clinical observations suggest that patients sensitized to food through the skin early in life may later develop severe food allergies. Aberrant epidermal thymic stromal lymphopoietin and interleukin (IL) 33 production and genetic predisposition can initiate an allergic immune response mediated by dendritic cells and CD4+TH2 cells in inflamed skin. After allergic sensitization, intestinal IL-25 and food ingestion enhance concerted interactions between type 2 innate lymphoid cells (ILC2s) and CD4+TH2 cells, which perpetuate allergic reactions from the skin to the gut. IL-4 and cross-linking of antigen/IgE/FcεR complexes induce emigrated mast cell progenitors to develop into the multi-functional IL-9-producing mucosal mast cells, which produce prodigious amounts of IL-9 and mast cell mediators to drive intestinal mastocytosis in an autocrine loop. ILC2s and TH9 cells may also serve as alternative cellular sources of IL-9 to augment the amplification of intestinal mastocytosis, which is the key cellular checkpoint in developing systemic anaphylaxis. These findings provide a plausible view of how food allergy develops and progresses in a stepwise manner and that atopic signals, dietary allergen ingestion, and inflammatory cues are fundamental in promoting life-threatening anaphylaxis. This information will aid in improving diagnosis and developing more effective therapies for food allergy-triggered anaphylaxis.

Regulation of IgE-Mediated Food Allergy by IL-9 Producing Mucosal Mast Cells and Type 2 Innate Lymphoid Cells.

Due to the increasing prevalence and number of life-threatening cases, food allergy has emerged as a major health concern. The classic immune response seen during food allergy is allergen-specific IgE sensitization and hypersensitivity reactions to foods occur in the effector phase with often severe and deleterious outcomes. Recent research has advanced understanding of the immunological mechanisms occurring during the effector phase of allergic reactions to ingested food. Therefore, this review will not only cover the mucosal immune system of the gastrointestinal tract and the immunological mechanisms underlying IgE-mediated food allergy, but will also introduce cells recently identified to have a role in the hypersensitivity reaction to food allergens. These include IL-9 producing mucosal mast cells (MMC9s) and type 2 innate lymphoid cells (ILC2s). The involvement of these cell types in potentiating the type 2 immune response and developing the anaphylactic response to food allergens will be discussed. In addition, it has become apparent that there is a collaboration between these cells that contributes to an individual's susceptibility to IgE-mediated food allergy.

Cholera Toxin Promotes Th17 Cell Differentiation by Modulating Expression of Polarizing Cytokines and the Antigen-Presenting Potential of Dendritic Cells.

Cholera toxin (CT), an exotoxin produced by Vibrio cholera, acts as a mucosal adjuvant. In a previous study, we showed that CT skews differentiation of CD4 T cells to IL-17-producing Th17 cells. Here, we found that intranasal administration of CT induced migration of migratory dendritic cell (DC) populations, CD103+ DCs and CD11bhi DCs, to the lung draining mediastinal lymph nodes (medLN). Among those DC subsets, CD11bhi DCs that were relatively immature had a major role in Th17 cell differentiation after administration of CT. CT-treated BMDCs showed reduced expression of MHC class II and CD86, similar to CD11bhi DCs in medLN, and these BMDCs promoted Th17 cell differentiation more potently than other BMDCs expressing higher levels of MHC class II and CD86. By analyzing the expression of activation markers such as CD25 and CD69, proliferation and IL-2 production, we determined that CT-treated BMDCs showed diminished antigen-presenting potential to CD4+ T cells compared with normal BMDCs. We also found that CT-stimulated BMDCs promote activin A expression as well as IL-6 and IL-1ß, and activin A had a synergic role with TGF-ß1 in CT-mediated Th17 cell differentiation. Taken together, our results suggest that CT-stimulated DCs promote Th17 cell differentiation by not only modulating antigen-presenting potential but also inducing Th polarizing cytokines.

Dual role of respiratory syncytial virus glycoprotein fragment as a mucosal immunogen and chemotactic adjuvant.

Respiratory syncytial virus (RSV) is a major cause of severe lower respiratory tract disease in infancy and early childhood. Despite its importance as a pathogen, there is no licensed vaccine to prevent RSV infection. The G glycoprotein of RSV, a major attachment protein, is a potentially important target for protective antiviral immune responses and has been shown to exhibit chemotactic activity through CX3C mimicry. Here, we show that sublingual or intranasal immunization of a purified G protein fragment of amino acids from 131 to 230, designated Gcf, induces strong serum IgG and mucosal IgA responses. Interestingly, these antibody responses could be elicited by Gcf even in the absence of any adjuvant, indicating a novel self-adjuvanting property of our vaccine candidate. Gcf exhibited potent chemotactic activity in in vitro cell migration assay and cysteine residues are necessary for chemotactic activity and self-adjuvanticity of Gcf in vivo. Mucosal immunization with Gcf also provides protection against RSV challenge without any significant lung eosinophilia or vaccine-induced weight loss. Together, our data demonstrate that mucosal administration of Gcf vaccine elicits beneficial protective immunity and represents a promising vaccine regimen preventing RSV infection.

Evaluation of protective efficacy of respiratory syncytial virus vaccine against A and B subgroup human isolates in Korea.

Human respiratory syncytial virus (HRSV) is a significant cause of upper and lower respiratory tract illness mainly in infants and young children worldwide. HRSV is divided into two subgroups, HRSV-A and HRSV-B, based on sequence variation within the G gene. Despite its importance as a respiratory pathogen, there is currently no safe and effective vaccine for HRSV. In this study, we have detected and identified the HRSV by RT-PCR from nasopharyngeal aspirates of Korean pediatric patients. Interestingly, all HRSV-B isolates exhibited unique deletion of 6 nucleotides and duplication of 60 nucleotides in the G gene. We successfully amplified two isolates ('KR/A/09-8' belonging to HRSV-A and 'KR/B/10-12' to HRSV-B) on large-scale, and evaluated the cross-protective efficacy of our recombinant adenovirus-based HRSV vaccine candidate, rAd/3xG, by challenging the immunized mice with these isolates. The single intranasal immunization with rAd/3xG protected the mice completely from KR/A/09-8 infection and partially from KR/B/10-12 infection. Our study contributes to the understanding of the genetic characteristics and distribution of subgroups in the seasonal HRSV epidemics in Korea and, for the first time, to the evaluation of the cross-protective efficacy of RSV vaccine against HRSV-A and -B field-isolates.

CD43 Expression Regulated by IL-12 Signaling Is Associated with Survival of CD8 T Cells.

BACKGROUND: In addition to TCR and costimulatory signals, cytokine signals are required for the differentiation of activated CD8 T cells into memory T cells and their survival. Previously, we have shown that IL-12 priming during initial antigenic stimulation significantly enhanced the survival of activated CD8 T cells and increased the memory cell population. In the present study, we analyzed the mechanisms by which IL-12 priming contributes to activation and survival of CD8 T cells. METHODS: We observed dramatically decreased expression of CD43 in activated CD8 T cells by IL-12 priming. We purified CD43(lo) and CD43(hi) cells after IL-12 priming and analyzed the function and survival of each population both in vivo and in vitro. RESULTS: Compared to CD43(hi) effector cells, CD43(lo) effector CD8 T cells exhibited reduced cytolytic activity and lower granzyme B expression but showed increased survival. CD43(lo) effector CD8 T cells also showed increased in vivo expansion after adoptive transfer and antigen challenge. The enhanced survival of CD43(lo) CD8 T cells was also partly associated with CD62L expression. CONCLUSION: We suggest that CD43 expression regulated by IL-12 priming plays an important role in differentiation and survival of CD8 T cells.

Intranasal delivery of cholera toxin induces th17-dominated T-cell response to bystander antigens.

Cholera toxin (CT) is a potent vaccine adjuvant, which promotes mucosal immunity to protein antigen given by nasal route. It has been suggested that CT promotes T helper type 2 (Th2) response and suppresses Th1 response. We here report the induction of Th17-dominated responses in mice by intranasal delivery of CT. This dramatic Th17-driving effect of CT, which was dependent on the B subunit, was observed even in Th1 or Th2-favored conditions of respiratory virus infection. These dominating Th17 responses resulted in the significant neutrophil accumulation in the lungs of mice given CT. Both in vitro and in vivo treatment of CT induced strongly augmented IL-6 production, and Th17-driving ability of CT was completely abolished in IL-6 knockout mice, indicating a role of this cytokine in the Th17-dominated T-cell responses by CT. These data demonstrate a novel Th17-driving activity of CT, and help understand the mechanisms of CT adjuvanticity to demarcate T helper responses.

Phenotypic changes induced by IL-12 priming regulate effector and memory CD8 T cell differentiation.

In addition to TCR and co-stimulatory signals, inflammatory cytokines such as IL-12 provide important signals for differentiation and survival of activated CD8 T cells. In the present study, to investigate the mechanisms by which IL-12 priming contributes to activation and enhanced survival of CD8 T cells, we searched the differentially regulated genes and markers by IL-12 during antigenic stimulation. Here, we show that IL-12 priming results in the increased subpopulation of CD127(hi) cells, which differentiates into long-lived memory cells. We also found that IL-12 priming induces IL-10 expression from activated CD8 T cells, which is distinct from CD127 up-regulation. Direct IL-10 priming of CD8 T cells results in the significant increase of effector and memory CD8 T cell population after adoptive transfer, and this priming effect is closely associated with less susceptibility to apoptosis. Although IL-10 is known as a cytokine with anti-inflammatory and immunosuppressive properties, our results have shown that IL-10 has a direct and positive effect on the survival of CD8 T cells. Together, we suggest that IL-10-dependent and independent effects of IL-12 play important roles in regulating differentiation and survival of activated CD8 T cells into effector and memory cells.