Single-cell atlas of fetal immunity across lung, spleen, and umbilical cord blood in nonhuman primates

The fetal immune system develops within a tightly regulated environment that balances immune tolerance with readiness for postnatal antigen exposure. However, limited fetal tissue access constrains understanding of immune ontogeny across compartments. Here we present a multi-tissue single-cell transcriptional atlas of the late-gestation (GD130-135) rhesus macaque fetal immune system, profiling leukocytes from lung, spleen, and umbilical cord blood (UCBMC) spanning myeloid, lymphoid, innate lymphoid, and hematopoietic stem cell lineages. Fetal lung is enriched in myeloid cells and ILC2s and shows a proinflammatory bias. Spleen is dominated by T and B cells, with B cells showing enhanced signatures of V(D)J recombination and isotype switching and CD4 T cells showing increased activation and Treg frequency. UCBMC is predominantly composed of T cells with a regulatory transcriptional landscape. Spectral flow cytometry and functional assays corroborate these tissue-specific differences revealing elevated secretion of inflammatory mediators in fetal lung at rest, while spleen and UCBMC show greater inducible responses following stimulation. Together, these data provide a comprehensive cross-tissue atlas of late-gestation primate fetal immunity, revealing tissue-specific immune specialization, functional capacity, and intercellular communication networks that shape immune readiness prior to birth. Investigations into fetal immune ontogeny across compartments are constrained by tissue inaccessibility during pregnancy. This rhesus macaque single-cell atlas profiles fetal lung, spleen, and cord blood, revealing tissue specific immune cell composition and activation at late-gestation validated by flow cytometry and functional assays.