A single-cell atlas of circulating immune cells over the first 2 months of age in extremely premature infants

Extremely premature infants (EPI), or those born prior to 30 weeks’ gestation, are highly susceptible to infection and inflammation. The impact of pre- and postnatal exposures on the immune response is poorly understood. We analyzed over 200, 000 circulating immune cells from 100-200L of whole blood collected from 25 donors including longitudinal samples from 10 EPI that were compared to healthy adults and to cord blood from term and preterm infants. We performed single-cell RNA-, T- and B-cell receptor sequencing, and phosphoprotein mass cytometry from matched samples and proteomics on dried blood spots from the same donors. Our data defined the trajectory of circulating T-, B-, myeloid and natural killer cells. Peripheral T cell development rapidly progressed over the first month of EPI’s life with an increase in the proportion of naïve CD4+, regulatory, and cycling T cells, accompanied by increased STAT5 signaling. Simultaneously, the transcription of IL2, which is essential for T cell activation and proliferation, increased in the lymphocytes. T cell repertoire diversity, clonality and gene usage increased rapidly over the first 60 days of life in EPI. CD38hi B cells were directly proportional to gestational age at birth. Interestingly, in utero exposure to inflammation (chorioamnionitis) altered the abundance, transcriptome and signaling of circulating T cells and monocytes specifically at 1 month of life in EPIs. Finally, we demonstrate the feasibility of a robust multi-omic longitudinal analysis in EPI from minute amounts of blood, developing a resource for studying the impact of early life exposures on immune development. Circulating immune cells were isolated from blood samples obtained within the first week, one month, and at two months of age from EPI and compared to cord blood from term and preterm infants and to samples from healthy adults. Single-cell cDNA with matched TCR and BCR VDJ libraries were prepared using the 10x Chromium Next GEM 5′ reagent kit (v2) according to the manufacturer’s instructions. Sequencing was performed on the NovaSeq 6000 sequencer (Illumina) to obtain paired end reads. PTCB is preterm cord blood samples TCB is term cord blood samples S1 is 1 week samples S2 and S3 are 1-month samples S5 is 2 months samples adult is adult samples