BackgroundWe designed an oral immunotherapy (OIT) clinical trial for cashew allergy to further our understanding of immunological responses with treatment, including changes in allergen-specific T cells. This information can further assist with the design of efficacious and safe treatments.MethodsParticipants were built up to and maintained on 1 g of cashew flour protein. Double-blind, placebo-controlled food challenges (DBPCFCs) were conducted before and after dosing completion (week 52) and 6 weeks after dosing discontinuation (week 58). Desensitization (DS) and sustained unresponsiveness (SU) were defined as tolerating DBPCFC to a cumulative dose of 2043 mg of the allergen at weeks 52 and 58, respectively. ClinicalTrials.gov, number NCT03504774.ResultsWe enrolled 40 cashew allergic participants. In the Intent-to-treat (ITT) population, both the DS and SU rate to cashew was 65% (26/40). Among cashew-reactive cells, CRTH2+ CD4+ T cells decreased at week 52 and week 58 compared to baseline. Additionally, we also saw reduced baseline expression of cytokines TARC, EGF and IP10 among participants that achieved SU at 4043mg compared to those who achieved SU at 2043mg.ConclusionCashew OIT have efficacy and safety outcomes similar to other published OIT studies. Reductions in pathogenic allergen-specific T cell populations may contribute to the immune mechanisms underlying tolerance achieved towards cashew post-treatment.Clinical trial registrationClinicalTrials.gov, identifier NCT03504774.

Despite the success of peanut oral immunotherapy (OIT), there is little insight into the safety, efficacy, and immune underpinnings of OIT to treat cashew or shrimp allergy. Cashew or shrimp allergic participants aged 7-55 years were enrolled in the phase 2 clinical trial - MOTIF (NCT03504774) and underwent cognate allergen-specific OIT to a maintenance dose of 1,000 mg until week 52. Oral food challenge (OFC) at weeks 52 and 58 evaluated desensitization and sustained unresponsiveness (SU) respectively to a cumulative allergen dose of 2043 mg. We examined immune changes in allergen-reactive CD4+ T cells and plasma at baseline, week 52, and week 58 using flow cytometry and Luminex. We enrolled 40 cashew and 12 shrimp allergic participants, of whom 25 cashew-treated (62.5%) and 7 shrimp-treated (58.3%) participants completed and passed week 58 OFC. Overall, cashew and shrimp OIT was safe with only mild (90%) or moderate (10%) adverse events, with gastrointestinal symptoms (31%) being the most common. OIT induced a significant increase in allergen-specific IgG4 at week 52 (q < 0.1). A trend of downregulation of Th2 and Th1 surrogate marker CRTH2 and CXCR3-expressing allergen-reactive CD4+ T cells was evident in cashew- and shrimp-treated participants respectively at week 52. Higher baseline plasma levels of TARC, EGF, and IP10 implied a lower likelihood of SU. Cashew and shrimp OIT could safely desensitize allergic study participants to respective allergen protein. Mechanistic analyses confirm readouts linked to successful desensitization and offer potential biomarkers for predicting SU that warrant further investigation.

Understanding antigen-specific T-cell responses, for example, following virus infections or allergen exposure, is of high relevance for the development of vaccines and therapeutics. We aimed on optimizing immunophenotyping of T cells after antigen stimulation by improving staining procedures for flow and mass cytometry. Our method can be used for primary cells of both mouse and human origin for the detection of low-frequency T-cell response using a dual-barcoding system for individual samples and conditions. First, live-cell barcoding was performed using anti-CD45 antibodies prior to an in vitro T-cell stimulation assay. Second, to discriminate between stimulation conditions and prevent cell loss, sample barcoding was combined with a commercial barcoding solution. This dual-barcoding approach is cell sparing and, therefore, particularly relevant for samples with low cell numbers. To further reduce cell loss and to increase debarcoding efficiency of multiplexed samples, we combined our dual-barcoding approach with a new centrifugation-free washing system by laminar flow (Curiox™). Finally, to demonstrate the benefits of our established protocol, we assayed virus-specific T-cell response in SARS-CoV-2–vaccinated and SARS-CoV-2–infected patients and compared with healthy non-exposed individuals by a high-parameter CyTOF analysis. We could reveal a heterogeneity of phenotypes among responding CD4, CD8, and gd-T cells following antigen-specific stimulations. Our protocol allows to assay antigen-specific responses of minute populations of T cells to virus-derived peptides, allergens, or other antigens from the same donor sample, in order to investigate qualitative and quantitative differences.

BACKGROUND:During the COVID-19 pandemic, novel nanoparticle-based mRNA vaccines were developed. A small number of individuals developed allergic reactions to these vaccines although the mechanisms remain undefined. METHODS:To understand COVID-19 vaccine-mediated allergic reactions, we enrolled 19 participants who developed allergic events within 2 h of vaccination and 13 controls, nonreactors. Using standard hemolysis assays, we demonstrated that sera from allergic participants induced stronger complement activation compared to nonallergic subjects following ex vivo vaccine exposure. RESULTS:Vaccine-mediated complement activation correlated with anti-polyethelyne glycol (PEG) IgG (but not IgM) levels while anti-PEG IgE was undetectable in all subjects. Depletion of total IgG suppressed complement activation in select individuals. To investigate the effects of vaccine excipients on basophil function, we employed a validated indirect basophil activation test that stratified the allergic populations into high and low responders. Complement C3a and C5a receptor blockade in this system suppressed basophil response, providing strong evidence for complement involvement in vaccine-mediated basophil activation. Single-cell multiome analysis revealed differential expression of genes encoding the cytokine response and Toll-like receptor (TLR) pathways within the monocyte compartment. Differential chromatin accessibility for IL-13 and IL-1B genes was found in allergic and nonallergic participants, suggesting that in vivo, epigenetic modulation of mononuclear phagocyte immunophenotypes determines their subsequent functional responsiveness, contributing to the overall physiologic manifestation of vaccine reactions. CONCLUSION:These findings provide insights into the mechanisms underlying allergic reactions to COVID-19 mRNA vaccines, which may be used for future vaccine strategies in individuals with prior history of allergies or reactions and reduce vaccine hesitancy.

BACKGROUND:Omalizumab (XOLAIR®)-assisted multi-food oral immunotherapy (mOIT) has been shown to safely, effectively, and rapidly desensitize patients with multiple food allergies. In our clinical trial (NCT02626611) on omalizumab-assisted mOIT, different desensitization outcomes (success or failure of desensitization) were observed following a period of either continued or discontinued mOIT. However, the association between the immunological changes induced by omalizumab-assisted mOIT and desensitization outcomes has not yet been fully elucidated. In this study, due to the key roles of regulatory T (Treg) cells and the type 2 helper T cell (Th2) pathway in immune tolerance to food allergens, we aimed to characterize their association with the desensitization outcomes of omalizumab-assisted mOIT. METHODS:Mass cytometry and multiplex cytokine assays were performed on blood samples obtained from participants with allergies to peanut, cashew, or milk in our phase 2 clinical study (NCT02626611). Comprehensive statistical and bioinformatic analyses were conducted on high-dimensional cytometry-based single-cell data and high-throughput multiplex cytokine data. RESULTS:Our results demonstrated that the frequency of HLA-DR+ Treg cells, and the production of Th2 cytokines (IL-4, IL-5, IL-13, and IL-9) as well as the immunoregulatory cytokine IL-10 by peripheral blood mononuclear cells (PBMCs) was significantly increased in cultures with allergen compared to cultures with media alone at baseline (Week 0). We also observed increased frequency of allergen responsive HLA-DR+ Treg cells and enhanced production of IL-10 by PBMCs in participants who achieved successful desensitization compared to those with failure of desensitization. However, the production of Th2 cytokines by PBMCs did not show significant differences between participants with different desensitization outcomes (success vs. failure of desensitization), despite omalizumab-assisted mOIT inducing a significant reduction in the production of Th2 cytokines. CONCLUSIONS:We demonstrated that the frequency of HLA-DR+ Treg cells and IL-10 cytokine production by PBMCs are associated with desensitization outcomes of omalizumab-assisted mOIT. These findings suggest potential immunological parameters that could be targeted to enhance desensitization success rates.

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To the Editor, Food allergy (FA) prevalence has expanded over recent decades and affects 220 million people worldwide.1 Although oral immunotherapy can help achieve desensitization,2 limited alternative treatment options exist outside of allergen avoidance. Clinical diagnosis of FA remains challenging today. Currently, the gold standard for FA diagnosis is the oral food challenge in which patients gradually ingest increasing amounts of their allergen in a supervised environment to test for an allergic response.3 Other diagnostic procedures (e.g., skin prick testing and Immunoglobulin E [IgE] blood testing) can be resourcedependent, timeconsuming, and distressing for patients. The medical literature has shown that FA status can be linked to increased levels of several antiinflammatory cytokines such as Interleukin13 (IL13). Recent studies have demonstrated that IL13 production is upregulated during IgEmediated allergic responses.4 This cytokine is also known to play a major role in the pathology of asthma, inducing goblet cell metaplasia, airway hyperresponsiveness, and airway remodeling.5 To this end, we aimed to identify stable biomarkers for FA that might improve diagnostic measures. We hypothesized that urinary secretions of antiinflammatory cytokines would be elevated in foodallergic participants compared to healthy control participants with no allergic disease. At the time of writing, no data on urinary cytokine output, including IL13, are available from foodallergic patients receiving oral immunotherapy. Twins with and without allergic disease were recruited for an observational study (NCT01613885) from the San Francisco Bay Area. Each twin pair shared similar lifestyles, diets, and environmental surroundings during the time of study participation, but these twins varied in allergy diagnosis and symptoms (Table S1). Informed consent was obtained from all 340 adult and pediatric participants who subsequently had their blood, nasal, buccal, saliva, skin, urine, and stool samples collected. In a subset analysis from our cohort, we ageand sexmatched 15 pairs of 30 foodallergic and healthy control participants (Figure S1) and analyzed their baseline urine samples on the Luminex EMD Millipore human 80plex immunoassay in duplicate according to the protocol (Table S2). We quantified urinary output for each cytokine in all 30 participants (Table S3). All foodallergic and healthy control participants were treatmentnaive and had healthy, functioning kidneys. Patients with diabetes were omitted to prevent confounding, as IL13 is linked to insulin resistance.6 Participants identified for the healthy control group had no atopic dermatitis, allergic rhinitis, FA, or asthma. Approximately, 56.7% of participants in the foodallergic group had asthma (Table 1). The median age of our entire cohort was 46.6 years (IQR 8.5– 56.4). Of the 15 pairs of foodallergic and healthy control participants, there were eight pairs of discordant twins, of which seven were dizygotic and one was monozygotic. To increase statistical power, additional individuals were included who were not twins. Nontwin pairs were ageand sexmatched individuals from our registry who were also from the San Francisco Bay Area. We investigated the association between FA status and urinary cytokine output using a generalized linear model and mixed effect models. We used mixed effect models to control for age and sex in our sensitivity analyses. No difference in urinary cytokine output was observed with respect to age or sex. Notably, our generalized linear model revealed that foodallergic individuals were more likely

Food allergies are a leading cause of anaphylaxis, and allergen-specific immune responses in both the innate and the adaptive immune system play key roles in its pathogenesis. We conducted a comprehensive phenotypic and functional investigation of immune cell responses from nonallergic (NA) and peanut allergic (PA) participants cultured with media alone or peanut protein and found, surprisingly, that NK cell activation was strongly associated with the immune response to allergen in PA participants. Peanut-responsive NK cells manifested a distinct expression pattern in PA participants compared with NA participants. Allergen-activated NK cells expressed both Th2 and immune regulatory cytokines, hinting at a potential functional role in mediating and regulating the Th2 allergic response. Depletion of CD3+ T cells attenuated the response of NK cells to peanut-allergen stimulation, suggesting that peanut-responsive NK cells are T cell dependent. We also showed that oral immune therapy was associated with decreased NK responses to peanut allergen stimulation in vitro. These results demonstrate that NK cells are associated with the food-allergic immune response, and the magnitude of this mobilized cell population suggests that they play a functional role in allergic immunity.

BACKGROUND:Conventional basophil activation tests (BATs) measure basophil activation by the increased expression of CD63. Previously, fluorophore-labeled avidin, a positively-charged molecule, was found to bind to activated basophils, which tend to expose negatively charged granule constituents during degranulation. This study further compares avidin versus CD63 as basophil activation biomarkers in classifying peanut allergy. METHODS:Seventy subjects with either a peanut allergy (N = 47), a food allergy other than peanut (N = 6), or no food allergy (N = 17) were evaluated. We conducted BATs in response to seven peanut extract (PE) concentrations (0.01-10,000 ng/mL) and four control conditions (no stimulant, anti-IgE, fMLP (N-formylmethionine-leucyl-phenylalanine), and anti-FcεRI). We measured avidin binding and CD63 expression on basophils with flow cytometry. We evaluated logistic regression and XGBoost models for peanut allergy classification and feature identification. RESULTS:Avidin binding was correlated with CD63 expression. Both markers discriminated between subjects with and without a peanut allergy. Although small by percentage, an avidin+ /CD63- cell subset was found in all allergic subjects tested, indicating that the combination of avidin and CD63 could allow a more comprehensive identification of activated basophils. Indeed, we obtained the best classification accuracy (97.8% sensitivity, 96.7% specificity) by combining avidin and CD63 across seven PE doses. Similar accuracy was obtained by combining PE dose of 10,000 ng/mL for avidin and PE doses of 10 and 100 ng/mL for CD63. CONCLUSIONS:Avidin and CD63 are reliable BAT activation markers associated with degranulation. Their combination enhances the identification of activated basophils and improves the classification accuracy of peanut allergy.

Participants successfully desensitized to peanut through the 2-year-long peanut OIT trial POISED were classified as sustained unresponsive (SU) vs. desensitized alone (DS) based upon oral peanut challenge outcome following a 3-month peanut avoidance period post-OIT. Mass cytometry-based phenotyping of PBMCs from these participants showed that a lower frequency of naïve CD8+ T cells positively associates with sustained unresponsiveness. We further probed CD8+ T cells from SU and DS participants to understand molecules and pathways influencing peanut tolerance. PBMCs from a subset of SU (n=8) and DS (n=7) participants were stimulated with peanut solution or with non-specific stimuli, namely anti-CD3/CD28 beads or PMA/Ionomycin. The cells were harvested, stained with a CD8+ and CD4+ subset-focused antibody panel, and acquired on a spectral flow cytometer. Data were analyzed by manual gating. Concomitant with the lower naïve CD8+ T cell frequency, SU participants showed a higher frequency of effector memory (EM) CD8+ T cells compared to DS post-OIT. In addition, post-OIT EM and terminally differentiated CD45RA+ EM (TEMRA) CD8+ T cells from SU participants expressed higher granzyme B and perforin than DS participants in response to both the non-specific stimuli tested. Notably, CD222 (mannose-6-phosphate receptor), a putative receptor for extracellular granzyme B, was exclusively expressed by ∼10% of peanut-reactive (pr) CD4+ T cells compared to other Th subsets and Treg cells. Our preliminary findings suggest a hypothesis that cytotoxic molecules such as granzyme B sourced from memory CD8+ T cells may target pathogenic pr CD4+ T cells, and thus support SU status.

Exposure to extreme conditions has been reported to catalyze the release and function of heat shock proteins (Hsps). However, little is known about the effects of temperature on the changes in immune cell type-specific expression of Hsps. PBMCs from healthy individuals were incubated at 26.5 oC, 37.0 oC or 40 oC with 5% CO2. After culture, expression of heat shock markers was examined on PBMCs using spectral flow cytometry. Hsp70 and Hsp90 were expressed in a wide range of immune cells, including T cells, B cells, natural killer (NK) cells, dendritic cells (DC) and monocytes when PBMCs were incubated at 26.5 oC, 37.0 oC or 40 oC. Among these cell types, monocytes had the highest expression of Hsp70 and Hsp90. Expression of Hsp70 in T cells, B cells, NK cells, DC and monocytes was significantly increased at 40oC compared to that at 37oC, whereas the expression of Hsp90 in T cells, B cells, NK cells and monocytes was significantly decreased at 40oC compared to that at 26.5oC. Our results also showed that Hsp70 expression in monocytes was positively correlated with NF-kB expression in monocytes (p=0.05), while Hsp90 expression was negatively correlated with NF-kB expression in monocytes (p=0.02) and B cells (p=0.0005). Expression of Hsp70 and Hsp90 in the immune cells was increased or decreased respectively upon temperature increase. Expression of Hsp70 and Hsp90 proteins were significantly correlated with NF-kB expression in monocytes and B cells, indicating cell-type specific expression of Hsp70 or Hsp90 may be associated with the regulation NF-kB.

The impact of exposure to air pollutants, such as fine particulate matter (PM), on the immune system and its consequences on pediatric asthma are not well understood. We investigated whether the ambient levels of fine PM with aerodynamic diameter ≤ 2.5 microns (PM ) are associated with alterations in circulating monocytes in children with or without asthma. Increased exposure to ambient PM was linked to specific monocyte subtypes, particularly in children with asthma. Mechanistically, we hypothesized that innate trained immunity is evoked by a primary exposure to fine PM and accounts for an enhanced inflammatory response after secondary stimulation in vitro . We determined that the trained immunity was induced in circulating monocytes by fine particulate pollutants, and it was characterized by upregulation of proinflammatory mediators, such as TNF, IL-6, and IL-8, upon stimulation with house dust mite or LPS. This phenotype was epigenetically controlled by enhanced H3K27ac marks in circulating monocytes. The specific alterations of monocytes after ambient pollution exposure suggest a possible prognostic immune signature for pediatric asthma, and pollution-induced trained immunity may provide a potential therapeutic target for asthmatic children living in areas with increased air pollution.

Peanut-reactive (pr) CD4+ T cells are pivotal for the onset of peanut allergy as well as for peanut oral immunotherapy (OIT)-induced desensitization (DS). However, the underlying immune mechanism of how pr CD4+ T cells impact the development of sustained unresponsiveness (SU) following a period of avoidance is largely unknown. To this end, we probed pr CD4+ T cells from the phase 2 peanut OIT trial- POISED participants, who achieved SU vs. those who did not (termed as SU and DS participants respectively based on no vs. with clinical reaction to peanut following 3 months of peanut avoidance post-OIT). Pre-OIT (baseline), post-OIT (week 104), and post-avoidance (week 117) PBMCs from 7 SU and 7 DS participants were stimulated with peanut solution for 18 hours. Single pr CD4+ T cells (CD4+CD137+ and/or CD4+CD154+) were captured using the BD Rhapsody single-cell multi-omic platform and evaluated through single-cell targeted mRNAseq, AbSeq, and TCRseq simultaneously. Th1/Th17 chemokine signaling-related (e.g. CCR6, CCL20, RORC) and cytotoxicity-related (e.g. PRF1, GZMB, GNLY, CST7) gene signatures in pr T effector memory cells positively associated with SU at baseline and week 117. On the other hand, higher Th2-related (e.g. IL4, IL3, IL5, IL9, IL13) gene signatures at baseline and week 117 suggested a lower likelihood of achieving SU. Also, higher expression of CD39 on pr Treg at week 117 was associated with SU. Our findings indicate that chemokine signaling-related, cytotoxicity-related, and Th2-related gene signatures and CD39 expression among pr CD4+ T cell subsets could influence the possibility of achieving SU.

IgE is a central molecule in type I hypersensitivity reactions. Currently, there is one approved anti-IgE therapeutic antibody, omalizumab. Next generation anti-IgE antibodies have primarily focused on improved affinity within the FceRI binding region. However, depending on the disease type, blocking IgE binding to both FceRI and FceRII may be important when treating atopy. We sought to create a therapeutic candidate which could neutralize soluble IgE and remove IgE bound to both FceRI and FceRII. A novel bivalent anti-IgE camelid derived single domain antibody, CX10759, was developed. The VHH binds distal to the known FceRI epitope overlapping the FceRII binding site. Binding and blocking of recombinant IgE was determined by surface plasmon resonance (SPR). Blocking and stripping of IgE from FceRI or FceRII was determined using RBL-2H3 cells expressing human FceRI or the RPMI-8866 cells which express FceRII. Basophil activation test (BAT) assays were conducted on human basophils from healthy donors. CX10759 binds soluble IgE with mid picomolar affinity. CX10759 blocked soluble IgE from associating with either FceRI or FceRII in SPR and cell-based assays. Additionally, CX10759 efficiently displaced prebound IgE from both FceRI or FceRII expressed on RBL-2H3 and RPMI8866 cells, respectively. Importantly, no basophil activation was observed with CX10759 at concentrations that mimic clinical exposures. CX10759 is a promising preclinical anti-IgE antibody that potently neutralizes soluble IgE and removes surface bound IgE from FceRI and FceRII. This molecule may have superior clinical utility by eliciting more complete IgE neutralization.

While food allergy oral immunotherapy (OIT) can provide safe and effective desensitization (DS), the immune mechanisms underlying development of sustained unresponsiveness (SU) following a period of avoidance are largely unknown. Here, we compare high dimensional phenotypes of innate and adaptive immune cell subsets of participants in a previously reported, phase 2 randomized, controlled, peanut OIT trial who achieved SU vs. DS (no vs. with allergic reactions upon food challenge after a withdrawal period; n = 21 vs. 30 respectively among total 120 intent-to-treat participants). Lower frequencies of naïve CD8 + T cells and terminally differentiated CD57 + CD8 + T cell subsets at baseline (pre-OIT) are associated with SU. Frequency of naïve CD8 + T cells shows a significant positive correlation with peanut-specific and Ara h 2-specific IgE levels at baseline. Higher frequencies of IL-4 + and IFNγ + CD4 + T cells post-OIT are negatively correlated with SU. Our findings provide evidence that an immune signature consisting of certain CD8 + T cell subset frequencies is potentially predictive of SU following OIT.

IgE-mediated food allergies in infants are a significant health concern, with peanut allergy being of particular interest due to its prevalence and severity. Among individuals who produce peanut-specific IgE some experience no adverse reaction on peanut consumption. This asymptomatic phenotype is known as sensitized tolerance. To elucidate the immune environment of peanut sensitized tolerant and clinically allergic one-year-olds, high-dimensional mass cytometry was conducted as part of the HealthNuts study. The resulting data includes peripheral blood mononuclear cells from 36 participants encompassing non-allergic, peanut sensitized with tolerance, and clinically peanut allergic infants. The raw mass cytometry data is described here and freely available for reuse through the Immunology Database and Analysis Portal (ImmPort). Additional allergy information and serum vitamin D levels of the participants were measured and are also included in the data upload. These high-dimensional mass cytometry data, when combined with clinical information, offer a broad immune profile of peanut allergic and sensitized tolerant infants.

Cross-contamination is a common cause of adverse food reactions that remains an ongoing challenge for the safety of patients with food allergies, especially those with concurrent diagnoses. It is concerning that oral immunotherapy (OIT) products are often manufactured in shared facilities and may risk cross-contamination. In this study, we explored potential cross-contamination in OIT products manufactured by the Stanford Sean N. Parker Center for Allergy and Asthma Research. Antigen-specific enzyme-linked immunosorbent assays (ELISAs) with no detectable cross-reactivity were used to quantify contaminant residues in: almond, cashew, cod, egg, hazelnut, milk, peanut, salmon, sesame, shrimp, soy, walnut, and wheat flour OIT samples. Gel electrophoresis was utilized to determine the presence of key allergenic proteins. Duplicate samples were tested for each contaminant-allergen pair. One-sided t-tests determined if contaminant residues exceeded 0 μg/mL. All tests were adjusted for multiple testing with the FDR method using Q-values to indicate adjusted p-values. ELISA results showed no significant contamination levels after adjustment for multiple testing (24.93±0.892 μg/mL, Q=0.14; 41.247±0.484 μg/mL, Q=0.11; 2.715±0.131 μg/mL, Q=0.14, respectively). In addition to the confirmation of expected key allergenic proteins in all samples, no other protein bands matching the molecular weights of cross-contaminants were detected. Contaminants are rare in food allergen flours manufactured for OIT. Their equivalency concentrations would remain below any medically significant threshold, i.e. the lowest median eliciting dose of 1 mg protein observed in 1,445 food challenges across 8 sites nationwide (p≤0.001). Our study highlights the importance of routine quality control and assurance using quantitative methods for cross-contamination.

Despite the success of the BNT162b2 mRNA vaccine, the immunological mechanisms that underlie its efficacy are poorly understood. Here we analyzed the innate and adaptive responses to BNT162b2 in mice, and show that immunization stimulated potent antibody and antigen-specific T cell responses, as well as strikingly enhanced innate responses after secondary immunization, which was concurrent with enhanced serum interferon (IFN)-γ levels 1 d following secondary immunization. Notably, we found that natural killer cells and CD8 + T cells in the draining lymph nodes are the major producers of this circulating IFN-γ. Analysis of knockout mice revealed that induction of antibody and T cell responses to BNT162b2 was not dependent on signaling via Toll-like receptors 2, 3, 4, 5 and 7 nor inflammasome activation, nor the necroptosis or pyroptosis cell death pathways. Rather, the CD8 + T cell response induced by BNT162b2 was dependent on type I interferon-dependent MDA5 signaling. These results provide insights into the molecular mechanisms by which the BNT162b2 vaccine stimulates immune responses.