Acute Neonatal Infections ‘Lock-In’ a Suboptimal CD8+ T Cell Repertoire with Impaired Recall Responses

<div><p>Microbial infection during various stages of human development produces widely different clinical outcomes, yet the links between age-related changes in the immune compartment and functional immunity remain unclear. The ability of the immune system to respond to specific antigens and mediate protection in early life is closely correlated with the level of diversification of lymphocyte antigen receptors. We have previously shown that the neonatal primary CD8+ T cell response to replication competent virus is significantly constricted compared to the adult response. In the present study, we have analyzed the subsequent formation of neonatal memory CD8+ T cells and their response to secondary infectious challenge. In particular, we asked whether the less diverse CD8+ T cell clonotypes that are elicited by neonatal vaccination with replication competent virus are ‘locked-in’ to the adult memory T cell, and thus may compromise the strength of adult immunity. Here we report that neonatal memory CD8+ T cells mediate poor recall responses compared to adults and are comprised of a repertoire of lower avidity T cells. During a later infectious challenge the neonatal memory CD8+ T cells compete poorly with the fully diverse repertoire of naïve adult CD8+ T cells and are outgrown by the adult primary response. This has important implications for the timing of vaccination in early life.</p></div

Comparison of the features of the gB-8p-specific CD8+ TCRβ repertoires for primary and secondary responses.

<p>The features of the gB-8p-specific TCRβ repertoires involved in the primary responses to VACV-gB in neonatal and adult mice and the secondary responses to HSV-1 in neonatal-vaccinated and adult-vaccinated mice. Shown are the percentage of gB-8p-specific CD8+ T cells per mouse that use the Vβ10 gene (A); the percentage of unique gB-8p-specific Vβ10+ TCRβ nucleotide (n.t.) clonotypes per mouse requiring no nucleotide additions (B); and the number of different TCRβ amino acid clonotypes (C), estimated for a standard sample size of 48 sequences per gB-8p-specific Vβ10+ TCRβ repertoire. The horizontal lines indicate the median values per age/infection group. A Mann-Whitney test was used for each of the pairwise comparisons between (i) primary and secondary responses in mice primarily challenged as adults, (ii) primary and secondary responses in mice primarily challenged as neonates, (iv) primary responses in adult and neonate mice, and (iv) secondary responses in adult-vaccinated and neonatal-vaccinated mice. Statistical significance was determined at p<0.0125 (*), using Bonferroni correction for multiple pairwise comparisons. The data shown for the neonatal and adult CD8+ T cell responses to primary infection were obtained in previous studies <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003572#ppat.1003572-Rudd1" target="_blank">[11]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003572#ppat.1003572-Rudd2" target="_blank">[12]</a> and shown here for comparison with the secondary CD8+ T cell responses.</p

Comparison of the features of the gB-8p-specific TCRβ repertoires between primary adult effector and secondary neonate memory CD8+ T cell populations responding to HSV-1 in congenic mice following the adoptive transfer of resting memory cells from neonatal mice previously infected with VACV-gB.

<p>For each recipient mouse the features of the paired primary adult effector and secondary neonate memory CD8+ T cell populations per mouse are shown. The percentage of gB-8p-specific CD8+ T cells that use the Vβ10 gene (A); the percentage of unique gB-8p-specific Vβ10+ TCRβ nucleotide (n.t.) clonotypes requiring no nucleotide additions (B); and the number of different TCRβ amino acid (a.a.) clonotypes (C), estimated for a standard sample size of 48 sequences per gB-8p-specific Vβ10+ TCRβ repertoire are shown. The horizontal lines indicate the median values per group. * p<0.05 (Wilcoxon test).</p

Summary of the gB-8p-specific Vβ10⁺ CD8⁺ TCRβ repertoire data.

a<p>These data were obtained in previous studies <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003572#ppat.1003572-Rudd1" target="_blank">[11]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003572#ppat.1003572-Rudd2" target="_blank">[12]</a> and are shown here for comparison with the other TCRβ repertoires.</p>b<p>Obtained at the peak of the CD8+ T cell response following primary VACV-gB challenge.</p>c<p>Obtained at the peak of the CD8+ T cell response following secondary HSV-1 challenge.</p>d<p>The resting memory CD8+ T cell population following VACV-gB infection of neonatal mice.</p>e<p>Obtained at the peak of the CD8+ T cell response to HSV-1 challenge in congenic mice following adoptive transfer of resting memory cells obtained from neonatal mice previously infected with VACV-gB.</p

Memory CD8+ T cells from neonatal mice exhibit impaired recall responses.

<p>Spleens were harvested from neonatal or adult vaccinated mice (day 42) and equal numbers of gB-8p-specific memory CD8+ T cells (∼2×10⁴ cells) were adoptively transferred into individual naïve congenic (CD45.1) recipient adult mice. Recipient mice were challenged the next day with HSV-1 (1×10⁶ pfu, i.p.). On day 6 post-infection, the spleens of recipient mice were harvested and the (A) absolute number of gB-8p+ CD8+ T cells (i.e. CD45.1 and CD45.2) and (B) the percentage of gB-8p+ CD8+ T cells that were donor-derived memory CD8+ T cells (i.e. CD45.2) were examined. All data is representative of at least 3 experiments, with n = 6–8 mice/group. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003572#s2" target="_blank">Results</a> depict mean ± SEM. ***, p<0.001.</p

TCR avidity of neonatal and adult memory gB-8p-specific CD8+ T cells.

<p>TCR:pMHC dissociation kinetics for splenic CD8+ T cells were also analyzed at 6–7 weeks after VACV-gB infection. Briefly, splenocytes were stained with Kb:gB-8p tetramer for 1 hour at 4°C, washed and incubated in the presence of anti-Kb antibody to prevent rebinding. Depicted here is the relative amount of CD8+ gB-8p+ T cells detected over time ± SEM. Assuming an exponential decay, there is a significant difference (p = 0.003, Mann-Whitney) between the decay rates for neonatal and adult gB-8p-specific CD8+ T cells. All data is representative of at least 2 separate experiments with n = 6–8 mice/group.</p

Magnitude of primary and secondary gB-8p-specific CD8+ T cell responses.

<p>Neonatal and adult B6 mice were vaccinated with VACV-gB and challenged 6 weeks later with HSV-1. The frequency (A) and total numbers (B) of gB-8p-specific CD8+ T cells were enumerated in the spleen by tetramer staining at the peak of VACV-gB infection (day 6 for adults, day 8 for neonates), at 6 weeks post-vaccination, and at the peak of HSV-1 challenge (day 6). Naive adult B6 mice were also infected with HSV-1 for comparison with the secondary CD8+ T cell responses. All data is representative of 3–4 separate experiments with n = 6–8 mice/group. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003572#s2" target="_blank">Results</a> depict mean ± SEM. The data shown for the neonatal and adult CD8+ T cell responses to primary VACV-gB infection were obtained in previous studies <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003572#ppat.1003572-Rudd1" target="_blank">[11]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003572#ppat.1003572-Rudd2" target="_blank">[12]</a>.</p

Memory CD8+ T cells from neonatal mice exhibit impaired protective immunity against <i>Listeria monocytogenes</i>.

<p>(A) Neonatal (CD45.2) and adult (CD45.1) mice were infected with ActA-/- LM-gB (1×10⁶, i.p.) and spleens were harvested from both age groups at 6 weeks post-infection. An equal number of gB-8p-specific memory CD8+ T cells (∼5×10³) were co-transferred into the same naïve congenic (Thy1.1) adult recipient and all mice were subsequently infected the next day with HSV-1 (1×10⁶ pfu, i.p.). On day 6 post-infection, spleens were harvested and the relative number of neonatal and adult gB-8p+ memory CD8+ T cells (i.e. CD45.1+ and CD45.2+) were expressed as a percentage of IFNg+ donor cells (Thy1.2). (B) In separate experiments, neonatal and adult mice were again infected with ActA-/- LM-gB (1×10⁶, i.p.) and allowed to transition in to the resting memory phase. At 6 weeks post-infection, neonatal (CD45.2) and adult (CD45.2) memory CD8+ T cells were transferred into separate congenic recipients (CD45.1) and challenged with wild-type LM-gB (5×10⁴, i.v.). On day 3 post-infection, livers were homogenized and the bacterial burden was determined. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003572#s2" target="_blank">Results</a> depict mean ± SEM. *, p<0.05.</p

Effect of age and early priming on 1⁰ and 2⁰ CD8⁺ T cell responses.

<p>(A) For the primary responses, naïve mice were infected i.n. with 1×10⁴ pfu of the HK (H3N2) influenza A virus either at a young (<3 months; mo) or extreme (22 mo) age. Analysis of CD8⁺ T cell responses was performed on d10 after the primary infection. (B) For the secondary responses of the early-primed mice, animals were primed at <2 mo i.p. with 1.5×10⁷ pfu the PR8 (H1N1) influenza A virus, then challenged 6 weeks (young) or >22 mo (aged) later i.n. with 1×10⁴ pfu of the HK virus. Analysis of CD8⁺ T cell responses was performed on d8 after the secondary infection. (C, D) Numbers of epitope-specific CD8⁺ T cells in the spleens recovered from young (filled symbols) or aged (>22 month, open symbols) B6 mice on d10 (1⁰, C) or d8 (2^o, D) following primary (1⁰) or secondary (primed young) (2⁰) i.n. infection with the HK (H3N2) influenza A virus. Memory mice had been injected i.p. with the PR8 (H1N1) influenza A virus at <2 mo and were challenged. Lymphocyte populations were stimulated with the NP₃₆₆ or PA₂₂₄ peptides in the presence of Brefeldin A for 5 hrs, then stained with the anti-CD8PerCPCy5.5 mAb, fixed/permeabilised and stained with anti-IFN-γ-FITC mAb. Cytokine (IFN-γ) production was calculated by subtracting background fluorescence for the no-peptide controls, and the numbers of IFN-γ⁺CD8⁺ D^bNP₃₆₆- and D^bPA₂₂₄-specifc CD8⁺ T cells were determined from the % cells staining and the total cell counts. Data represent individual mice (symbols) and the mean (line). Experiments were performed at least twice. * = p<0.05.</p

Immunodominance hierarchies in aged mice after 1⁰ infection or 2⁰ challenge of primed-early mice.

<p>The relative prevalence of the immunodominant D^bNP₃₆₆⁺CD8⁺ and D^bPA₂₂₄⁺CD8⁺ T cell population over the subdominant D^bPB1₇₀₃⁺CD8⁺ and K^bPB1-F2₆₂⁺CD8⁺ sets. <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002544#s2" target="_blank">Results</a> are shown for (A, B) 1⁰ and (C, D) 2⁰ HK infection in (A, C) young and (B, D) aged mice. The relative contributions of particular antigen-specific CD8⁺ T cells were analysed based on total cell responses (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002544#ppat-1002544-g001" target="_blank">Figure 1</a> for D^bNP₃₆₆⁺CD8⁺ and D^bPA₂₂₄⁺CD8⁺ and data not shown for D^bPB1₇₀₃⁺CD8⁺ and K^bPB1-F2₆₂⁺CD8⁺). Data represent the mean proportion of a particular peptide-specific CD8⁺ population. * = p<0.01 shows a difference between young and aged animals. Experimental outline as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002544#ppat-1002544-g001" target="_blank">Figure 1AB</a>.</p