Single neonatal perturbations long-lastingly shape the transcriptome and TCR repertoire of the first wave of regulatory T cells

The first wave of Foxp3+ regulatory T cells (Tregs) generated in neonates has been reported to be indispensable for the life-long prevention of autoimmunity. Although it is widely accepted that neonates are highly susceptible to infections, the impact of neonatal infections on the first wave of Tregs is completely unknown. Here, we challenged newborn Treg fate-mapping mice (Foxp3eGFPCreERT2xRosa26-YFP) with the Toll-like receptor agonists LPS and poly I:C to mimic neonatal bacterial or viral infections, respectively, and subsequently administrated tamoxifen during the first eight days of life to selectively label the first wave of Tregs. Neonatally-tagged Tregs preferentially accumulated in non-lymphoid tissues when compared to lymphoid organs irrespective of the treatment. One week post perturbation, unexpectedly no differences in the frequency and phenotypes of neonatally-tagged Tregs were observed between challenged mice and untreated controls. However, upon aging, a decreased frequency of neonatally-tagged Tregs in both non-lymphoid tissues and lymphoid organs was detected in challenged mice when compared to untreated controls, and became significant twelve weeks post perturbation, with no signs of altered Foxp3 stability. Remarkably, this late decrease in the frequency of neonatally-tagged Tregs only occurred upon perturbations in newborns as challenging of eight-day-old mice did not result in long-lasting alterations of the first wave of Tregs. Combined single-cell T cell receptor (TCR)-seq and RNA-seq revealed that neonatal perturbations drastically diminished TCR diversity and long-lastingly altered the transcriptome of neonatally tagged Tregs, exemplified by lower expression of Tigit, Foxp3, and Il2ra. Together, our data demonstrate that a single, transient encounter with a pathogen in neonates can have long-lasting consequences for the first wave of Tregs, which might affect immunological tolerance, prevention of autoimmunity, and other non-canonical functions of tissue-resident Tregs. Newborn Foxp3EGFPCreERT2Rosa26YFP mice were intraperitoneally injected with LPS, poly I:C, or PBS, followed by three times intragastrical administrations of tamoxifen on days 2, 5, and 8 after birth. Single lymphocyte suspensions were prepared from the spleen, colon, and skin of twelve-week-old PBS-, LPS- or poly I:C neonatally treated mice, and were further stained with TotalSeq-C hashtag antibody (BioLegend) according to the manufacturer's protocol. The same type of organs was labeled by the same TotalSeq Hashtag antibodies, afterward spleen, colon, and skin samples from the same mouse were pooled together for sorting CD8-CD45R-CD11c-CD11b-NK1.1-CD4+YFP+ cells. Sorted cells were then uploaded onto a 10x Chromium Next GEM Chip G using reagents from the Chromium Single Cell 5' Library and Gel Bead Kit (10x Genomics) according to the manufacturer's protocol. Amplified cDNA was used for both 5' RNA-seq library generation and TCR V(D)J targeted enrichment using the Chromium Single-Cell V(D)J Enrichment Kit for Mouse T Cells (10x Genomics). 5' RNA-seq and TCR V(D)J libraries were prepared following the manufacturer's user guide (10x Genomics). Libraries were quantified by QubitTM 3.0 Fluorometer (ThermoFisher) and quality checked using 2100 Bioanalyzer with High Sensitivity DNA Kit (Agilent Technologies). scRNA-seq libraries were sequenced on an Illumina NextSeq500 sequencer to attain approximately 141,381 ± 63,000 reads per single cell and scTCR V(D)J libraries were sequenced on an Illumina NextSeq500 sequencer to obtain approximately 63,987 ± 6,131 reads per cell. The sequencing specifications for both single-cell RNA-seq and TCR V(D)J libraries were according to the manufacturer's specification (10x Genomics).