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

Extremely premature infants (EPIs) who are born before 30 weeks of gestation are susceptible to infection; however, the trajectory of their peripheral immunity is poorly understood. Here, we undertook longitudinal analyses of immune cells from 250 μl of whole blood at 1 week, 1 month, and 2 months from 10 EPIs and compared these with samples from healthy adults and with preterm and full-term cord blood samples. Single-cell suspensions from individual samples were split to perform single-cell RNA sequencing, T and B cell receptor sequencing, and phosphoprotein mass cytometry. The trajectories of circulating T, B, myeloid, and natural killer cells in EPIs over the first 2 months of life were distinct from those of full-term infants. In EPIs, peripheral T cell development rapidly progressed over the first month of life, with an increase in the proportion of naïve CD4 + , regulatory, and cycling T cells, accompanied by greater STAT5 (signal transducer and activator of transcription 5) signaling. EPI memory CD4 + T cells showed a T helper 1 (T H 1) predominance compared with T H 2 skewing of central memory–like T cells in full-term infants, and B cells from 2-month-old EPIs exhibited increased signatures of activation and differentiation. Both B and T cells from 2-month-old EPIs displayed increased interferon signatures compared with cells from full-term infants. In conclusion, we demonstrated the feasibility of longitudinal multiomic analyses in EPIs using minute amounts of blood and developed a resource describing peripheral immune development in EPIs that suggested ongoing activation in early life.