Previous studies reported associations of IFITM3 SNP rs12252 with severe influenza, but evidence of association and the mechanism of risk remains controversial. We prioritized SNPs in IFITM3 based on putative biological function and identified rs34481144 in the 5′ UTR. We found evidence of a novel association of rs34481144 with severe influenza in three influenza-infected cohorts characterized by different levels of influenza illness severity. We determined the role of rs34481144 as an expression quantitative trait loci (eQTL) for IFITM3, with the risk allele associated with lower mRNA expression. The risk allele was found to have decreased IRF3 binding and increased CTCF binding in promoter-binding assays, and risk allele carriage diminished transcriptional correlations among neighboring genes, indicative of CTCF boundary activity. Furthermore, the risk allele disrupts a CpG site that undergoes differential methylation in CD8 T-cell subsets. Carriers of the risk allele had reduced CD8 T-cells in their airways during natural influenza infection, consistent with IFITM3 promoting airway CD8 T-cell accumulation, indicating that a critical function for IFITM3 may be to promote immune cell persistence at mucosal sites. Our study identifies a new regulator of IFITM3 expression that associates with CD8 T-cell levels in the airways and a spectrum of clinical outcomes.

Influenza A, B and C viruses (IAV, IBV, ICV) circulate globally and infect humans, with IAV/IBV causing most severe disease. While CD8 + T-cells confer cross-protection against different IAV strains, CD8 + T-cell responses to IBV/ICV are understudied. We dissected the CD8 + T-cell cross-reactome against influenza viruses and provided the first evidence of CD8 + T-cell cross-reactivity across IAV, IBV and ICV. Using immunopeptidomics, we identified immunodominant CD8 + T-cell epitopes from IBV, protective in mice, and found prominent memory CD8 + T-cells towards both universal and influenza type-specific epitopes in blood and lungs of healthy humans, with lung-derived CD8 + T-cells displaying a tissue-resident phenotype. Importantly, effector CD38 + Ki67 + CD8 + T-cells against novel epitopes were readily detected in IAV-and IBV-infected pediatric and adult patients. Our study introduces a new paradigm, whereby CD8 + T-cells confer unprecedented cross-reactivity across all influenza viruses, a key finding for designing universal vaccines.

BackgroundThe bacterial communities of the nasopharynx play an important role in upper respiratory tract infections (URTIs). Our study represents the first survey of the nasopharynx during a known, controlled viral challenge. We aimed to gain a better understanding of the composition and dynamics of the nasopharyngeal microbiome during viral infection.MethodsRhinovirus illnesses were induced by self-inoculation using the finger to nose or eye natural transmission route in ten otherwise healthy young adults. Nasal lavage fluid samples (NLF) samples were collected at specific time points before, during, and following experimental rhinovirus inoculation. Bacterial DNA from each sample (N = 97 from 10 subjects) was subjected to 16S rRNA sequencing by amplifying the V1-V2 hypervariable region followed by sequencing using the 454-FLX platform.ResultsThis survey of the nasopharyngeal microbiota revealed a highly complex microbial ecosystem. Taxonomic composition varied widely between subjects and between time points of the same subject. We also observed significantly higher diversity in not infected individuals compared to infected individuals. Two genera – Neisseria and Propionibacterium – differed significantly between infected and not infected individuals. Certain phyla, including Firmicutes, Actinobacteria, and Proteobacteria, were detected in all samples.ConclusionsOur results reveal the complex and diverse nature of the nasopharyngeal microbiota in both healthy and viral-challenged adults. Although some phyla were common to all samples, differences in levels of diversity and selected phyla were detected between infected and uninfected participants. Deeper, species-level metagenomic sequencing in a larger sample is warranted.

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Compared to adults, infants suffer higher rates of hospitalization, severe clinical complications, and mortality due to influenza infection. We found that γδ T cells protected neonatal mice against mortality during influenza infection. γδ T cell deficiency did not alter viral clearance or interferon-γ production. Instead, neonatal influenza infection induced the accumulation of interleukin-17A (IL-17A)-producing γδ T cells, which was associated with IL-33 production by lung epithelial cells. Neonates lacking IL-17A-expressing γδ T cells or Il33 had higher mortality upon influenza infection. γδ T cells and IL-33 promoted lung infiltration of group 2 innate lymphoid cells and regulatory T cells, resulting in increased amphiregulin secretion and tissue repair. In influenza-infected children, IL-17A, IL-33, and amphiregulin expression were correlated, and increased IL-17A levels in nasal aspirates were associated with better clinical outcomes. Our results indicate that γδ T cells are required in influenza-infected neonates to initiate protective immunity and mediate lung homeostasis.

SARS-CoV-2 mRNA vaccines, including Pfizer/Biontech BNT162b2, were shown to be effective for COVID-19 prevention, eliciting both robust antibody responses in naive individuals and boosting pre-existing antibody levels in SARS-CoV-2-recovered individuals. However, the magnitude, repertoire, and phenotype of epitope-specific T cell responses to this vaccine, and the effect of vaccination on pre-existing T cell memory in SARS-CoV-2 convalescent patients, are still poorly understood. Thus, in this study we compared epitope-specific T cells elicited after natural SARS-CoV-2 infection, and vaccination of both naive and recovered individuals. We collected peripheral blood mononuclear cells before and after BNT162b2 vaccination and used pools of 18 DNA-barcoded MHC-class I multimers, combined with scRNAseq and scTCRseq, to characterize T cell responses to several immunodominant epitopes, including a spike-derived epitope cross-reactive to common cold coronaviruses. Comparing responses after infection or vaccination, we found that T cells responding to spike-derived epitopes show similar magnitudes of response, memory phenotypes, TCR repertoire diversity, and αβTCR sequence motifs, demonstrating the potency of this vaccination platform. Importantly, in COVID-19-recovered individuals receiving the vaccine, pre-existing spike-specific memory cells showed both clonal expansion and a phenotypic shift towards more differentiated CCR7-CD45RA+ effector cells. In-depth analysis of T cell receptor repertoires demonstrates that both vaccination and infection elicit largely identical repertoires as measured by dominant TCR motifs and receptor breadth, indicating that BNT162b2 vaccination largely recapitulates T cell generation by infection for all critical parameters. Thus, BNT162b2 vaccination elicits potent spike-specific T cell responses in naive individuals and also triggers the recall T cell response in previously infected individuals, further boosting spike-specific responses but altering their differentiation state. Overall, our study demonstrates the potential of mRNA vaccines to induce, maintain, and shape T cell memory through vaccination and revaccination.

Cancer arises from the accumulation of genetic alterations, which can lead to the production of mutant proteins not expressed by normal cells. These mutant proteins can be processed and presented on the cell surface by major histocompatibility complex molecules as neoepitopes, allowing CD8+ T cells to mount responses against them. For solid tumors, only an average 2% of neoepitopes predicted by algorithms have detectable endogenous antitumor T cell responses. This suggests that low mutation burden tumors, which include many pediatric tumors, are poorly immunogenic. Here, we report that pediatric patients with acute lymphoblastic leukemia (ALL) have tumor-associated neoepitope-specific CD8+ T cells, responding to 86% of tested neoantigens and recognizing 68% of the tested neoepitopes. These responses include a public neoantigen from the ETV6-RUNX1 fusion that is targeted in seven of nine tested patients. We characterized phenotypic and transcriptional profiles of CD8+ tumor-infiltrating lymphocytes (TILs) at the single-cell level and found a heterogeneous population that included highly functional effectors. Moreover, we observed immunodominance hierarchies among the CD8+ TILs restricted to one or two putative neoepitopes. Our results indicate that robust antitumor immune responses are induced in pediatric ALL despite their low mutation burdens and emphasize the importance of immunodominance in shaping cellular immune responses. Furthermore, these data suggest that pediatric cancers may be amenable to immunotherapies aimed at enhancing immune recognition of tumor-specific neoantigens.

A duplication variant within middle-ear-specific gene A2ML1 co-segregates with otitis media in an indigenous Filipino pedigree (LOD score=7.5 at reduced penetrance) and lies within a founder haplotype that is also shared by three otitis-prone European- and Hispanic-American children, but is absent in non-otitis-prone children and >62,000 next-generation sequences. Seven additional A2ML1 variants were identified in six otitis-prone children. Collectively our studies support a role for A2ML1 in the pathophysiology of otitis media.