Virtual memory CD8+ T cell behavior is linked to peripheralization in the lymph node.

Introduction/Rationale: Virtual Memory (VM) CD8+ T cells constitute a distinct subset of antigen-inexperienced cells with memory-like functions. During infection, VM cells are the first to respond and differentiate into effector cells. However, whether this rapid activation is linked to distinct spatiotemporal niches within the lymph nodes (LNs) during infection remains unknown.

Methods: To address this, we used adoptive transfers, whole tissue clearing, and intravital three-photon microscopy (IVM-3P) to visualize VM cell dynamics within the draining lymph node (dLN) following footpad infection with Vaccinia virus (VV).

Results: Adoptive transfer experiments revealed that VM cells robustly produce IFNg within 24 hours post-infection (hpi) in the dLN. Tissue clearing of explanted whole LNs showed a pronounced peripheral clustering of VM cells as early as 9 hpi. IVM-3P imaging further showed VM cells migrating towards the LN capsule, near afferent lymphatics, where they formed clusters and exhibited a significantly reduced motility. These data suggest that VM cells respond rapidly to infection due to their enhanced ability to migrate out to antigen-bearing antigen-presenting cells (APCs) in the LN periphery. Interestingly, we also discovered that VM cells respond to infection akin to antigen-experienced memory CD8+ T cells, in terms of their ability to rapidly migrate to the outermost region of the LN and produce IFNg after infection.

Conclusion: Our results reveal that VM cells occupy distinct intranodal niches that enable rapid effector differentiation after infection. We are currently performing experiments to define the molecular cues guiding VM migration and activation. Overall, our findings challenge the traditional concept of a ‘homogeneous T cell zone’ within the LNs, instead highlighting spatial and temporal heterogeneity among antigen-inexperienced T cell subsets with distinct effector functions.