Unleashing Type-2 Dendritic Cells to Drive Protective Antitumor CD4+ T Cell Immunity.

Differentiation of proinflammatory CD4+ conventional T cells (Tconv) is critical for productive antitumor responses yet their elicitation remains poorly understood. We comprehensively characterized myeloid cells in tumor draining lymph nodes (tdLN) of mice and identified two subsets of conventional type-2 dendritic cells (cDC2) that traffic from tumor to tdLN and present tumor-derived antigens to CD4+ Tconv, but then fail to support antitumor CD4+ Tconv differentiation. Regulatory T cell (Treg) depletion enhanced their capacity to elicit strong CD4+ Tconv responses and ensuing antitumor protection. Analogous cDC2 populations were identified in patients, and as in mice, their abundance relative to Treg predicts protective ICOS+ PD-1lo CD4+ Tconv phenotypes and survival. Further, in melanoma patients with low Treg abundance, intratumoral cDC2 density alone correlates with abundant CD4+ Tconv and with responsiveness to anti-PD-1 therapy. Together, this highlights a pathway that restrains cDC2 and whose reversal enhances CD4+ Tconv abundance and controls tumor growth.

Embryonic macrophages function during early life to determine invariant natural killer T cell levels at barrier surfaces.

It is increasingly recognized that immune development within mucosal tissues is under the control of environmental factors during early life. However, the cellular mechanisms that underlie such temporally and regionally restrictive governance of these processes are unclear. Here, we uncover an extrathymic pathway of immune development within the colon that is controlled by embryonic but not bone marrow-derived macrophages, which determines the ability of these organs to receive invariant natural killer T (iNKT) cells and allow them to establish local residency. Consequently, early-life perturbations of fetal-derived macrophages result in persistent decreases of mucosal iNKT cells and is associated with later-life susceptibility or resistance to iNKT cell-associated mucosal disorders. These studies uncover a host developmental program orchestrated by ontogenically distinct macrophages that is regulated by microbiota, and they reveal an important postnatal function of macrophages that emerge in fetal life.

Neutralizing anti-diphtheria toxin scFv produced by phage display.

BACKGROUND: Diphtheria can be prevented by vaccination, but some epidemics occur in several places, and diphtheria's threat is considerable. Administration of diphtheria antitoxin (DAT) produced from hyperimmunized animals is the most common treatment. Recombinant human antibody fragments such as single-chain variable fragments (scFv) produced by phage display library may introduce an interesting approach to overcome the limitations of the traditional antibody therapy. In the present study, B cells of immunized volunteers were used to construct a human single-chain fragment (HuscFv) library. MATERIALS AND METHODS: The library was constructed with the maximum combination of heavy and light chains. As an antigen, Diphtheria toxoid (DTd) was used in four-round phage bio-panning to select phage clones that display DTd bound HuscFv from the library. After panning, individual scFv clones were selected. Clones that were able to detect DTd in an initial screening assay were transferred to Escherichia coli HB2151 to express the scFvs and purification was followed by Ni metal ion affinity chromatography. Toxin neutralization test was performed on Vero cells. The reactivity of the soluble scFv with diphtheria toxin were done and affinity calculation based on Beatty method was calculated. RESULTS: The size of the constructed scFv library was calculated to be 1.3 × 106 members. Following four rounds of selection, 40 antibody clones were isolated which showed positive reactivity with DTd in an ELISA assay. Five clones were able to neutralize DTd in Vero cell assay. These neutralizing clones were used for soluble expression and purification of scFv fragments. Some of these soluble scFv fragments show neutralizing activity ranging from 0.6 to 1.2 µg against twofold cytotoxic dose of diphtheria toxin. The affinity constant of the selected scFv antibody was determined almost 107 M-1. CONCLUSION: This study describes the prosperous construction and isolation of scFv from the immune library, which specifically neutralizes diphtheria toxin. The HuscFv produced in this study can be a potential candidate to substitute the animal antibody for treating diphtheria and detecting toxins.

Production of IgY polyclonal antibody against diphtheria toxin and evaluation of its neutralization effect by Vero cell assay.

BACKGROUND: Diphtheria is a bacterial disease which is caused by Corynebacterium diphtheriae. The symptoms are due to the diphtheria toxin produced by the bacteria. Antibiotic therapy and the use of diphtheria antitoxin is a recommended strategy to control diphtheria. Although mammalian antibodies are used to treat patients, IgY antibody has advantages over mammalian ones, including cost-effectiveness and production through non-invasive means. Moreover, in contrast to mammalian antibodies, IgY does not bind to the rheumatoid factor and does not activate the complement system. The objective of this study was to evaluate the in vitro neutralizing effect of IgY against diphtheria toxin. RESULTS: Anti-DT IgY was produced by immunization of the laying white leghorn chickens. Indirect enzyme-linked immunosorbent assay revealed successful immunization of the animals, and the IgY was purified with a purity of 93% via polyethylene glycol precipitation method. The neutralizing activity of the purified IgY was evaluated by Vero cell viability assay. This assay confirmed that 1.95 µg (8.6 µg/ml of culture medium) of anti-DT IgY would neutralize 10 fold of cytotoxic dose 99% of DT, which was 0.3 ng (1.33 ng/ml of culture medium). CONCLUSION: This anti-DT IgY may be applicable for diphtheria treatment and quality controls in vaccine production.

Interleukin 1 beta and Matrix Metallopeptidase 3 Contribute to Development of Epidermal Growth Factor Receptor-Dependent Serrated Polyps in Mouse Cecum.

BACKGROUND & AIMS: Transgenic mice (HBUS) that express the epidermal growth factor receptor (EGFR) ligand HBEGF (heparin-binding epidermal growth factor-like growth factor) and a constitutively active G protein-coupled receptor (US28) in intestinal epithelial cells develop serrated polyps in the cecum. Development of serrated polyps depends on the composition of the gut microbiota and is associated with bacterial invasion of the lamina propria, accompanied by induction of inflammation and up-regulation of interleukin 1 beta (IL1B) and matrix metalloproteinase (MMP) 3 in the cecum. We investigated the mechanisms by which these changes contribute to development of serrated polyps. METHODS: We performed studies with C57BL/6 (control) and HBUS mice. To accelerate polyp development, we increased the exposure of the bacteria to the lamina propria by injecting HBUS mice with diphtheria toxin, which binds transgenic HBEGF expressed by the epithelial cells and causes apoptosis. Mice were given injections of IL1B-neutralizing antibody and the MMP inhibitor N-isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid. Intestinal tissues were collected from mice and analyzed by histology, reverse-transcription polymerase chain reaction, enzyme-linked immunosorbent assay, immunofluorescence, and flow cytometry. We examined fibroblast subsets in polyps using single-cell RNA sequencing. RESULTS: Administration of diphtheria toxin to HBUS mice accelerated development of serrated polyps (95% of treated mice developed polyps before 100 days of age, compared with 53% given vehicle). IL1B stimulated subsets of platelet-derived growth factor receptor alpha+ (PDGRFA+) fibroblasts isolated from cecum, resulting in increased expression of MMP3. Neutralizing antibodies against IL1B or administration of the MMP inhibitor reduced the number of serrated polyps that formed in the HBUS mice. Single-cell RNA sequencing analysis showed subsets of fibroblasts in serrated polyps that express genes that regulate matrix fibroblasts and inflammation. CONCLUSIONS: In studies of mice, we found that barrier breakdown and expression of inflammatory factors contribute to development of serrated polyps. Subsets of cecal PDGFRA+ fibroblasts are activated by release of IL1B from myeloid cells during the early stages of serrated polyp development. MMP3 produced by PDGFRA+ fibroblasts is important for serrated polyp development. Our findings confirm the functions of previously identified serrated polyp-associated molecules and indicate roles for immune and stromal cells in serrated polyp development.

Hydrogen-Deuterium Exchange Epitope Mapping Reveals Distinct Neutralizing Mechanisms for Two Monoclonal Antibodies against Diphtheria Toxin.

The diphtheria toxoid (DT) antigen is one of the major components in pediatric and booster combination vaccines and is known to raise a protective humoral immune response upon vaccination. However, a structurally resolved analysis of diphtheria toxin (DTx) epitopes with underlying molecular mechanisms of antibody neutralization has not yet been reported. Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and Biolayer Interferometry (BLI) assays, we have characterized two neutralizing anti-DTx monoclonal antibodies (mAbs), 2-25 and 2-18, by identifying the specific epitopes on the diphtheria toxin responsible for antibody binding. Our results show that both epitopes are conformational, and mechanistically distinct. Monoclonal antibody 2-25 binds selectively to the B-subunit (translocation and receptor domain) of DTx, blocking the heparin-binding EGF-like growth factor (HBEGF) binding site. In contrast, mAb 2-18 binds to the A-subunit (catalytic domain), partially covering the catalytic loop region that shuttles NAD during catalysis. The results are discussed in the context of antigen neutralization mechanisms and can ultimately help to reveal the underlying factors that contribute to Diptheria vaccine efficacy.

Regulatory T cells are required for normal and activin-promoted wound repair in mice.

Healing of skin wounds is orchestrated by various types of immune cells, but little is known about the role of FoxP3+ regulatory T cells (Tregs) in this process. Here, we determined if Tregs are important for wound healing in normal mice and if they contribute to the accelerated healing of mice overexpressing the growth and differentiation factor activin. Diphtheria toxin induced Treg depletion prior to injury caused impaired healing characterized by delayed reepithelialization, reduced wound contraction, and impaired vessel maturation. The accelerated wound repair of activin-transgenic mice was also abrogated. Mechanistically, we found a strong increase in IL-4 levels combined with overrepresentation of T-bet+ and GATA-3+ αß T cells in Treg-depleted 7-day wounds. In addition, numbers of IFN-γ- or IL-17A-producing CD4+ and CD4- T cells were elevated. These results demonstrate that Treg depletion in wounds facilitates the expansion of an αß T-cell population with features of Th1 and Th2 cells, and suggest that concomitant changes in the cytokine milieu disturb the healing process.

Seroepidemiology of diphtheria and pertussis in Chongqing, China: serology-based evidence of Bordetella pertussis infection.

OBJECTIVES: In order to gain further insight into the seroepidemiology of Bordetella pertussis infection and immunity against diphtheria in Chongqing, China, the concentrations of antibodies to pertussis toxin (PT) and diphtheria toxin (DT) were investigated in a healthy population. STUDY DESIGN: Cross-sectional study. METHODS: In total, 1080 healthy people were recruited into this study. Sera antibodies to DT and PT were measured quantitatively using commercial enzyme-linked immunosorbent assay kits. Age-specific incidence of infection with B. pertussis was estimated and compared with notified cases of pertussis. RESULTS: The mean concentration of anti-DT IgG was 0.71 IU/ml (95% confidence interval [CI] = 0.60-0.82), with a positive rate (>0.01 IU/ml) of 97.41% (1052/1080). The mean concentration of anti-PT IgG was 7.65 IU/ml (95% CI = 6.65-8.65), with a positive rate (>100 IU/ml) of 1.17% (11/944). The estimated pertussis infection rate was 7290/100,000, which was far higher than the reported incidence of 1.29/100,000 in 2015. The peaks of estimated incidence of infection were found in subjects aged 7-14 years (9971/100,000) and ≥20 years (13,898/100,000). CONCLUSIONS: B. pertussis infection occurs frequently in young infants and adolescents/adults; the latter are often responsible for the transmission of pertussis to young infants. The existing surveillance system may underestimate the true incidence of pertussis in older age groups in Chongqing, and the immunisation programme should be improved to provide protection against pertussis for adolescents and young adults.

T regulatory cells are critical for the maintenance, anamnestic expansion and protection elicited by vaccine-induced CD8 T cells.

T regulatory (Treg) cells are critical for preventing autoimmunity and suppressing immune responses during cancer and chronic infection. However, the role of Treg cells in the generation of vaccine-induced immune memory remains ill-defined. Using the mouse model of lymphocytic choriomeningitis virus (LCMV) infection, we demonstrate that transient absence of Treg cells during effector to memory CD8 T-cell transition results in a permanent impairment in the maintenance, function and recall capacity of CD8 T cells. Memory CD8 T cells in mice that were transiently depleted of Treg cells exhibited defective up-regulation of memory markers with a significant decrease in polyfunctionality. However, Treg-depleted mice showed no significant change in CD4 T-cell responses, and antibody levels relative to control. Altogether, this study evaluates the role of Treg cells in the formation of immune memory and demonstrates an important role for Treg cells in promoting memory CD8 T-cell differentiation and vaccine-induced immune protection against intracellular pathogens.

Altered lymph node composition in diphtheria toxin receptor-based mouse models to ablate dendritic cells.

Dendritic cells (DCs) are key regulators of innate and adaptive immunity. Our understanding of immune function has benefited greatly from mouse models allowing for selective ablation of DCs. Many such models rely on transgenic diphtheria toxin receptor (DTR) expression driven by DC-restricted promoters. This renders DCs sensitive to DT but is otherwise thought to have no effect on immune physiology. In this study, we report that, unexpectedly, mice in which DTR is expressed on conventional DCs display marked lymph node (LN) hypocellularity and reduced frequency of DCs in the same organs but not in spleen or nonlymphoid tissues. Intriguingly, in mixed bone marrow chimeras the phenotype conferred by DTR-expressing DCs is dominant over control bone marrow-derived cells, leading to small LNs and an overall paucity of DCs independently of the genetic ability to express DTR. The finding of alterations in LN composition and size independently of DT challenge suggests that caution must be exercised when interpreting results of experiments obtained with mouse models to inducibly deplete DCs. It further indicates that DTR, a member of the epidermal growth factor family, is biologically active in mice. Its use in cell ablation experiments needs to be considered in light of this activity.

Durability of Vaccine-Induced Immunity Against Tetanus and Diphtheria Toxins: A Cross-sectional Analysis.

BACKGROUND: Many adult immunization schedules recommend that tetanus and diphtheria vaccination be performed every 10 years. In light of current epidemiological trends of disease incidence and rates of vaccine-associated adverse events, the 10-year revaccination schedule has come into question. METHODS: We performed cross-sectional analysis of serum antibody titers in 546 adult subjects stratified by age or sex. All serological results were converted to international units after calibration with international serum standards. RESULTS: Approximately 97% of the population was seropositive to tetanus and diphtheria as defined by a protective serum antibody titer of ≥0.01 IU/mL. Mean antibody titers were 3.6 and 0.35 IU/mL against tetanus and diphtheria, respectively. Antibody responses to tetanus declined with an estimated half-life of 14 years (95% confidence interval, 11-17 years), whereas antibody responses to diphtheria were more long-lived and declined with an estimated half-life of 27 years (18-51 years). Mathematical models combining antibody magnitude and duration predict that 95% of the population will remain protected against tetanus and diphtheria for ≥30 years without requiring further booster vaccination. CONCLUSIONS: These studies demonstrate that durable levels of protective antitoxin immunity exist in the majority of vaccinated individuals. Together, this suggests that it may no longer be necessary to administer booster vaccinations every 10 years and that the current adult vaccination schedule for tetanus and diphtheria should be revisited.

Clearance of acute myeloid leukemia by haploidentical natural killer cells is improved using IL-2 diphtheria toxin fusion protein.

Haploidentical natural killer (NK) cell infusions can induce remissions in some patients with acute myeloid leukemia (AML) but regulatory T-cell (Treg) suppression may reduce efficacy. We treated 57 refractory AML patients with lymphodepleting cyclophosphamide and fludarabine followed by NK cell infusion and interleukin (IL)-2 administration. In 42 patients, donor NK cell expansion was detected in 10%, whereas in 15 patients receiving host Treg depletion with the IL-2-diphtheria fusion protein (IL2DT), the rate was 27%, with a median absolute count of 1000 NK cells/µL blood. IL2DT was associated with improved complete remission rates at day 28 (53% vs 21%; P = .02) and disease-free survival at 6 months (33% vs 5%; P < .01). In the IL2DT cohort, NK cell expansion correlated with higher postchemotherapy serum IL-15 levels (P = .002), effective peripheral blood Treg depletion (<5%) at day 7 (P < .01), and decreased IL-35 levels at day 14 (P = .02). In vitro assays demonstrated that Tregs cocultured with NK cells inhibit their proliferation by competition for IL-2 but not for IL-15. Together with our clinical observations, this supports the need to optimize the in vivo cytokine milieu where adoptively transferred NK cells compete with other lymphocytes to improve clinical efficacy in patients with refractory AML. This study is registered at clinicaltrials.gov, identifiers: NCT00274846 and NCT01106950.

Toxigenic Corynebacterium ulcerans isolated from a hunting dog and its diphtheria toxin antibody titer.

Toxigenic Corynebacterium ulcerans is a zoonotic pathogen that produces diphtheria toxin and causes a diphtheria-like illness in humans. The organism is known to infect and circulate among dogs, which can then transmit it to humans. Furthermore, previous studies have found that C. ulcerans is carried by wild animals, including game animals. In the present study, we tested hunting and companion dogs for the presence of toxigenic C. ulcerans and succeeded in isolating the bacterium from a hunting dog. Moreover, several hunting dogs had serum diphtheria antitoxin titers that were higher than the titers required for protection in humans, suggesting a history of exposure to toxigenic Corynebacterium strains. Notably, ribotyping, pulsed-field gel electrophoresis and tox gene sequencing demonstrated that the isolate from the hunting dog clustered with previously characterized C. ulcerans strains isolated from wild animals, as opposed to groups of isolates from humans and companion dogs. Interestingly, the wild animal cluster also contains an isolate from an outdoor breeding dog, which could have formed a bridge between isolates from wild animals and those from companion dogs. The results presented herein provide insight into the mechanism by which the zoonotic pathogen C. ulcerans circulates among wild animals, hunting and companion dogs, and humans.

Synthesis and immunological evaluation of N-acyl modified Tn analogues as anticancer vaccine candidates.

Tumor-associated carbohydrate antigens (TACAs), which are aberrantly expressed on the surface of tumor cells, are important targets for anticancer vaccine development. Herein, several N-acyl modified Tn analogues were synthesized and conjugated with carrier protein CRM197. The immunological results of these glycoconjugates indicated that 6-CRM197 elicited higher titers of antibodies which cross-reacted with native Tn antigen than the unmodified 2-CRM197 did. The IFN-γ-producing frequency of lymphocytes in mice treated with 6-CRM197 was obviously increased, compared to that of mice vaccinated with 2-CRM197 (p=0.016), which was typically associated with the Th1 response. Moreover, the elicited antisera against antigen 6-CRM197 reacted strongly with the Tn-positive tumor cells, implying the potential of this glycoconjugate as an anticancer vaccine.

Regulatory T cells limit induction of protective immunity and promote immune pathology following intestinal helminth infection.

Foxp3(+) regulatory T cells (Tregs) have a well-characterized role in limiting autoimmunity and dampening deleterious immune responses. However, a potential consequence of the immunosuppressive function of Tregs can be the limitation of protective immunity to infectious pathogens. Parasitic infections are a potent stimulus for the generation of Treg responses, which may be beneficial to both the parasite and the host by promoting persistence of infection and limiting immune-mediated pathology, respectively. In this study, we explore the functional role of Tregs post-low-dose infection with the intestinal helminth parasite Trichuris muris, which yields a chronic infection because of inefficient induction of Th2 responses. Early Treg depletion postinfection resulted in expedited worm clearance, and was associated with reduced Th1-mediated inflammation of the intestinal environment. Interestingly, this protective immunity was lost, and worm burden enhanced if Tregs were depleted later once the infection was established. Early and late Treg depletion resulted in enhanced Th2 and reduced Th1 cytokine and humoral responses. Blockade of the Th2 cytokine IL-4 resulted in a moderate increase in Th1 but had no effect on worm burden. Our findings suggest that Tregs preferentially limit Th2 cell expansion, which can impact infections where clear immune polarity has not been established. Thus, the impact of Treg depletion is context and time dependent, and can be beneficial to the host in situations where Th1 responses should be limited in favor of Th2 responses.

Development of a recombinant immunotoxin for the immunotherapy of autoreactive lymphocytes expressing MOG-specific BCRs.

OBJECTIVE: Myelin oligodendrocyte glycoprotein (MOG) is one of the major autoantigens in multiple sclerosis (MS), therefore selective depletion of autoreactive lymphocytes exposing MOG-specific B cell receptors (BCRs) would be beneficial in terms of MS treatment. RESULTS: Using E. coli we generated an efficient protocol for the purification of the recombinant immunotoxin DT-MOG composed of the extracellular Ig-like domain of MOG fused in frame with the catalytic and translocation subunits of diphtheria toxin (DT, Corynebacterium diphtheriae) under native conditions with a final yield of 1.5 mg per liter of culture medium. Recombinant DT-MOG was recognized in vitro by MOG-reactive antibodies and has catalytic activity comparable with wild-type DT. CONCLUSION: Enhanced pharmacokinetics (mean residence time in the bloodstream of 61 min) and minimized diminished nonspecific toxicity (LD50 = 1.76 mg/kg) of the DT-MOG makes it a potential candidate for the immunotherapy of MS.

Design of glycoconjugate vaccines against invasive African Salmonella enterica serovar Typhimurium.

Nontyphoidal salmonellae, particularly Salmonella enterica serovar Typhimurium, are a major cause of invasive disease in Africa, affecting mainly young children and HIV-infected individuals. Glycoconjugate vaccines provide a safe and reliable strategy against invasive polysaccharide-encapsulated pathogens, and lipopolysaccharide (LPS) is a target of protective immune responses. With the aim of designing an effective vaccine against S. Typhimurium, we have synthesized different glycoconjugates, by linking O-antigen and core sugars (OAg) of LPS to the nontoxic mutant of diphtheria toxin (CRM(197)). The OAg-CRM(197) conjugates varied in (i) OAg source, with three S. Typhimurium strains used for OAg extraction, producing OAg with differences in structural specificities, (ii) OAg chain length, and (iii) OAg/CRM(197) ratio. All glycoconjugates were compared for immunogenicity and ability to induce serum bactericidal activity in mice. In vivo enhancement of bacterial clearance was assessed for a selected S. Typhimurium glycoconjugate by challenge with live Salmonella. We found that the largest anti-OAg antibody responses were elicited by (i) vaccines synthesized from OAg with the highest glucosylation levels, (ii) OAg composed of mixed- or medium-molecular-weight populations, and (iii) a lower OAg/CRM(197) ratio. In addition, we found that bactericidal activity can be influenced by S. Typhimurium OAg strain, most likely as a result of differences in OAg O-acetylation and glucosylation. Finally, we confirmed that mice immunized with the selected OAg-conjugate were protected against S. Typhimurium colonization of the spleen and liver. In conclusion, our findings indicate that differences in the design of OAg-based glycoconjugate vaccines against invasive African S. Typhimurium can have profound effects on immunogenicity and therefore optimal vaccine design requires careful consideration.

Orchestration of CD4 T cell epitope preferences after multipeptide immunization.

A detailed understanding of the molecular and cellular mechanisms that underlie epitope preferences in T cell priming is important for vaccines designed to elicit a broad T cell response. Protein vaccinations generally elicit CD4 T cell responses that are skewed toward a small fraction of epitopes, a phenomenon known as immunodominance. This characteristic of T cell responses, which limits the diversity of CD4 T cell recognition, is generally attributed to intracellular Ag processing. However, we recently discovered that immunodominance hierarchies persist even after vaccination with synthetic peptides. In this study, we probed the regulatory mechanisms that cause diminished CD4 T cell responses to subdominant peptides after such multipeptide immunization in mice. We have found that the delivery of subdominant and dominant epitopes on separate dendritic cells rescues expansion of less favored CD4 T cells. Furthermore, through the use of genetic models and inhibitors, we have found that selective losses in CD4 T cell responses are mediated by an IFN-γ-induced pathway, involving IDO, and that regulatory T cell activities may also regulate preferences in CD4 T cell specificity. We propose that after multipeptide immunization, the expansion and differentiation of dominant T cells initiate complex regulatory events that determine the final peptide specificity of the elicited CD4 T cell response.

Development of a sensitive screening method for selecting monoclonal antibodies to be internalized by cells.

Antibody-drug conjugates (ADCs), drugs developed by conjugation of an anticancer agent to a monoclonal antibody (mAb), have lately attracted attention in cancer therapy because ADCs can directly bind cancer cells and kill them. Although mAbs for ADCs must be internalized by the target cells, few methods are available for screening mAbs for their ability to be internalized by cells. We have developed a recombinant protein, termed DT3C, which consists of diphtheria toxin (DT) lacking the receptor-binding domain but containing the C1, C2, and C3 domains of Streptococcus protein G (3C). When a mAb-DT3C conjugate, which functions in vitro like an ADC, reduces the viability of cancer cells, the mAb being tested must have been internalized by the target cells. DT3C can thus be a tool to identify efficiently and easily mAbs that can be internalized by cells, thereby enhancing the development of promising ADCs.

Eosinophil activities modulate the immune/inflammatory character of allergic respiratory responses in mice.

BACKGROUND: The importance and specific role(s) of eosinophils in modulating the immune/inflammatory phenotype of allergic pulmonary disease remain to be defined. Established animal models assessing the role(s) of eosinophils as contributors and/or causative agents of disease have relied on congenitally deficient mice where the developmental consequences of eosinophil depletion are unknown. METHODS: We developed a novel conditional eosinophil-deficient strain of mice (iPHIL) through a gene knock-in strategy inserting the human diphtheria toxin (DT) receptor (DTR) into the endogenous eosinophil peroxidase genomic locus. RESULTS: Expression of DTR rendered resistant mouse eosinophil progenitors sensitive to DT without affecting any other cell types. The presence of eosinophils was shown to be unnecessary during the sensitization phase of either ovalbumin (OVA) or house dust mite (HDM) acute asthma models. However, eosinophil ablation during airway challenge led to a predominantly neutrophilic phenotype (>15% neutrophils) accompanied by allergen-induced histopathologies and airway hyper-responsiveness in response to methacholine indistinguishable from eosinophilic wild-type mice. Moreover, the iPHIL neutrophilic airway phenotype was shown to be a steroid-resistant allergic respiratory variant that was reversible upon the restoration of peripheral eosinophils. CONCLUSIONS: Eosinophil contributions to allergic immune/inflammatory responses appear to be limited to the airway challenge and not to the sensitization phase of allergen provocation models. The reversible steroid-resistant character of the iPHIL neutrophilic airway variant suggests underappreciated mechanisms by which eosinophils shape the character of allergic respiratory responses.