Respiratory viral infections contribute substantially to global infant losses and disproportionately affect preterm neonates. Using our previously established neonatal murine model of influenza infection, we demonstrate that three-day old mice are exceptionally sensitive to influenza virus infection and exhibit high mortality and viral load. Intranasal pre- and post-treatment of neonatal mice with Lactobacillus rhamnosus GG (LGG), an immune modulator in respiratory viral infection of adult mice and human preterm neonates, considerably improves neonatal mice survival after influenza virus infection. We determine that both live and heat-killed intranasal LGG are equally efficacious in protection of neonates. Early in influenza infection, neonatal transcriptional responses in the lung are delayed compared to adults. These responses increase by 24 hours post-infection, demonstrating a delay in the kinetics of the neonatal anti-viral response. LGG pretreatment improves immune gene transcriptional responses during early infection and specifically upregulates type I IFN pathways. This is critical for protection, as neonatal mice intranasally pre-treated with IFNβ before influenza virus infection are also protected. Using transgenic mice, we demonstrate that the protective effect of LGG is mediated through a MyD88-dependent mechanism, specifically via TLR4. LGG can improve both early control of virus and transcriptional responsiveness and could serve as a simple and safe intervention to protect neonates.
Currently, there is little consensus regarding the most appropriate animal model to study acute infection and the virus–specific CD8+ T cell (CTL) responses in neonates. TCRβ high-throughput sequencing in naïve CTL of differently aged neonatal mice was performed, which demonstrated differential Vβ family gene usage. Using an acute influenza infection model, we examined the TCR repertoire of the CTL response in neonatal and adult mice infected with influenza type A virus. Three-day old mice mounted a greatly reduced primary NP(366–374)-specific CTL response when compared to 7-day old and adult mice, while secondary CTL responses were normal. Analysis of NP(366–374)-specific CTL TCR repertoire revealed different Vβ gene usage and greatly reduced public clonotypes in 3-day old neonates. This could underlie the impaired CTL response in these neonates. To directly test this, we examined whether controlling the TCR would restore neonatal CTL responses. We performed adoptive transfers of both non-transgenic and TCR-transgenic OVA(257–264)-specific (OT-I) CD8+ T cells into influenza-infected hosts, which revealed that naïve neonatal and adult OT-I cells expand equally well in neonatal and adult hosts. In contrast, non-transgenic neonatal CD8+ T cells when transferred into adults failed to expand. We further demonstrate that differences in TCR avidity may contribute to decreased expansion of the endogenous neonatal CTL. These studies highlight the rapid evolution of the neonatal TCR repertoire during the first week of life and show that impaired neonatal CTL immunity results from an immature TCR repertoire, rather than intrinsic signaling defects or a suppressive environment.
Respiratory support improvements have aided survival of premature neonates, but infection susceptibility remains a predominant problem. We previously reported that neonatal mice have a rapidly evolving T-cell receptor (TCR) repertoire that impairs CD8+ T cell immunity. To understand the impact of prematurity on the human CD8+ TCR repertoire, we performed next-generation sequencing of the complementarity-determining region 3 (CDR3) from the rearranged TCR variable beta (Vβ) in sorted, naïve CD8+ T cells from extremely preterm neonates (23–27 weeks gestation), term neonates (37–41 weeks gestation), children (16–56 months), and adults (25–50 years old). Strikingly, preterm neonates had an increased frequency of public clonotypes shared between unrelated individuals. Public clonotypes identified in preterm infants were encoded by germline gene sequences, and some of these clonotypes persisted into adulthood. The preterm neonatal naïve CD8+ TCR repertoire exhibited convergent recombination, characterized by different nucleotide sequences encoding the same amino acid CDR3 sequence. As determined by Pielou’s evenness and iChao1 metrics, extremely preterm neonates have less clonality, and a much lower bound for the number of unique TCR within an individual preterm neonate, which indicates a less rich and diverse repertoire, as compared to term neonates, children, and adults. This suggests that T cell selection in the preterm neonate may be less stringent or different. Our analysis is the first to compare the TCR repertoire of naïve CD8+ T cells between viable preterm neonates and term neonates. We find preterm neonates have a repertoire immaturity which potentially contributes to their increased infection susceptibility. A developmentally regulated, evenly distributed repertoire in preterm neonates may lead to the inclusion of public TCR CDR3β sequences that overlap between unrelated individuals in the preterm repertoire.
Background CD31, expressed by the majority of the neonatal T cell pool, is involved in modulation of T-cell Receptor signalling by increasing the threshold for T cell activation. Therefore, CD31 could modulate neonatal tolerance and adaptive immune responses. Methods Lymphocytes were harvested from murine neonates at different ages, human late preterm and term cord blood, and adult peripheral blood. Human samples were activated over a five-day period to simulate acute inflammation. Mice were infected with influenza; lungs and spleens were harvested at days 6 and 9 post-infection and analyzed by flow cytometry. Results CD31 expressing neonatal murine CD4+ and CD8a+ T cells increase over the first week of life. Upon in vitro stimulation, human infants’ CD4+ and CD8a+ T cells shed CD31 faster in comparison to adults. In the context of acute infection, mice infected at 3-days old have an increased number of naive and activated CD31+ T lymphocytes at the site of infection at day 6 and 9 post-infection, as compared to 7-days old; however, the opposite is true in the periphery. Conclusion Differences in trafficking of CD31+ Cytotoxic T Lymphocytes (CTLs) during acute influenza infection could modulate tolerance and contribute to a dampened adaptive immune response in neonates.
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