Modulating cytokine microenvironment during T cell activation induces protective RSV-specific lung resident memory T cells in early life in mice

Abstract

Maternal immunisation against respiratory viruses provides protection in early life, but as antibodies wane, there can be a gap in coverage. This immunity gap might be filled by inducing pathogen-specific lung tissue-resident T cells (TRM). However, the neonatal mouse lung has a different inflammatory environment to the adult lung which affects T cell recruitment. We compared the factors affecting viral-specific TRM recruitment in the lungs of adult or neonatal mice. In contrast to adulthood, we demonstrated that RSV or influenza infection in neonatal mice recruited fewer TRM to the lungs. This was associated with reduced lung levels of CCL5 and CXCL10. Co-administration of CCL5 or CXCL10 at the time of primary T cell activation significantly increased RSV-specific TRM in the lung, protecting mice upon reinfection. These chemokine differences were reflected in responses to infection in human cord blood. Here we show a critical role for CCL5 and CXCL10 in the induction of lung TRM and a possible strategy for boosting responses.

Keywords: Immunopathogenesis; Vaccines.

Fig. 1. Differential lung response induced by RSV in neonates compared to adults.

Female 6–7 weeks old (adult) or mixed 7 days old (neonates) BALB/c mice were infected with RSV. Infection schedule made with BioRender (A). Percentage all CD8 Trm (B) and RSV specific (C). Airway cells were transferred from neonatal RSV-infected mice into naïve adult mice prior to the RSV challenge of the recipient mouse, weight at d7 (D). In a separate study, neonatal and adult mice were infected with RSV, on day 7, lungs were collected from the infected animals and RNAseq was carried out from RNA extracted. PCA of genes in different groups (E), pathways related to each PC (F) and loading genes driving PC2 and PC3 are shown in (G). Volcano plots of DEG from Adult (H) and Neonatal (I) mice. Bars in B and C represent mean ± SEM of n = 5 mice. *p < 0.05.

Fig. 2. Neonatal RSV infection induces a less pronounced cytokine response than adults.

Venn diagram showing the overlaps between neonates and adult sequencing data (A). KEGG Pathways upregulated in neonates and adults (B). Relative expression level of selected genes (C). Cytokines measured in the lung in adult or neonatal mice 24 h after RSV infection; *p < 0.05, **p < 0.01 compared between Ad RSV and NN RSV (D).

Fig. 3. Cytokines response to RSV stimulation in cells from mums, babies and non-pregnant women.

PBMC isolated from cord blood, maternal blood post-partum and non-pregnant mothers were stimulated with RSV for 24 h. Cytokines were measured in the supernatant by Luminex. PCA analysis of all data points (A). Heat map of cytokine responses (B). Individual cytokine responses (C). Number of donors, cord blood n = 21, maternal n = 13 and non-pregnant n = 9.

Fig. 4. Boosting primary RSV infection with chemokines enhanced tissue resident TRM production and confer protection.

Seven-day-old BALB/c mice were infected intranasally with RSV, and 20 µg of CCL5 or CXCL10 in 20 μl were given on days 7, 8 and 9 after infection. Mice were culled on day 21, and flow cytometry was performed on the lungs that were harvested from the animals. Lung cell count (A), CD8 (B), Trm epithelial CD8 T cells (C). In a separate study, neonatal mice were infected with RSV and subsequently received chemokines intranasally on d7, 8 and 9 of infection before re-challenge on day 21. Mice were followed for 4 days after infection, and weight loss (D), lung cell count (E), CD8 TRM% (F), Viral Load (G) and antibody (H) were recorded. The same set-up was repeated except CCL5 or CXCL10 was given only on day 7 post primary infection. Mice were followed for 4 days after infection, and weight loss (I), lung cell count (J), CD8 TRM% (K), Viral Load (L) and antibody (M) were recorded. *p < 0.05, **p < 0.01, ***p < 0.001; statistical analysis by one-way ANOVA except for panels D and I where 2 way ANOVA. N = 5 mice per study.

Fig. 5. Neonatal response to influenza virus is also blunted.

Female 6–7 weeks old (adult) or mixed 7 days old (neonates) BALB/c mice were infected with RSV. Percentage all CD8 (A), Trm (B) and RSV specific (C). Cytokines were measured in the lungs in adult or neonatal mice 24 h after influenza virus infection (D). PBMC isolated from cord blood, maternal blood post-partum and non-pregnant mothers were stimulated with RSV for 24 h. Cytokines were measured in the supernatant by Luminex (E).