Heterogeneity in the Naïve CD8⁺ T Pool Shapes the Infection Response

Introduction/Rationale: Naïve CD8⁺ T cells are classically viewed as a uniform population that expands upon infection to protect the host against infection and cancer. However, growing evidence demonstrates that naïve CD8⁺ T cell heterogeneity influences the infection response. Recent single-cell studies revealed transcriptional heterogeneity among naïve CD8⁺ T cells, but whether this diversity reflects functionally distinct subsets possessing characteristic responses to infection remains unclear. We hypothesized that the naïve CD8⁺ T cell pool is composed of distinct subsets with underlying molecular programs that shape response kinetics and fate decisions during infection.

Methods: We combined single-cell multi-omic profiling (gene expression, surface protein, TCR), conventional and spectral flow cytometry, and in vivo adoptive transfer and infection models to identify, isolate, and functionally interrogate previously uncharacterized naïve CD8⁺ T cell subsets.

Results: scRNA-seq analysis identified multiple naïve CD8⁺ T cell subsets enriched for signatures of quiescence, effector priming, memory potential, interferon responsiveness, NF-κB signaling, regulatory function, and metabolic remodeling. Using subset-defining surface markers, we isolated these populations and assessed their in vivo responses to infection. Individual subsets showed differences in expansion, effector differentiation and memory formation, demonstrating that pre-existing regulatory states influence the infection response. Notably, further multi-omic profiling revealed that the relative abundance of naïve subsets varies with genetic background and age.

Conclusion: Our findings redefine the naïve CD8⁺ T cell pool as a heterogeneous compartment composed of functionally distinct subsets with intrinsic fate biases. Thus, variation in naïve pool composition represents a key mechanism that shapes CD8⁺ T cell immunity and may contribute to age- and genotype-dependent differences in susceptibility to infection and cancer.