Single-Cell Atlas of Fetal Immune Development 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 access to fetal tissues has constrained our understanding of immune ontogeny across distinct anatomical compartments. Here, we present a high-resolution, multi-tissue single-cell transcriptional atlas of the late-gestation (GD130–135) rhesus macaque ( Macaca mulatta ) fetal immune system, profiling leukocytes from lung, spleen, and umbilical cord blood mononuclear cell (UCBMC) compartments spanning myeloid, lymphoid, innate lymphoid, and hematopoietic stem cell (HSPC) lineages. The fetal lung was enriched in myeloid populations and ILC2 cells while fetal spleen was comprised primarily of T- and B-cells and UCBMC were dominated by T-cells. Despite reduced overall intercellular communication in lung compared to spleen and UCBMC, lung immune networks showed proinflammatory bias, suggesting preparation for postnatal environmental exposure. Splenic B cells showed strong transcriptional signatures associated with V(D)J recombination and isotype switching, while CD4 T cells displayed increased activation, and increased Tregs, consistent with the spleen's role as a secondary lymphoid organ which integrates antigen monitoring with immune tolerance to prevent overactivation. UCBMC showed a predominantly regulatory immune landscape. Together, this atlas provides a foundational resource defining tissue-specific immune specialization and intercellular communication in the late-gestation primate fetus.