Accurate enumeration of pathogen-specific and virtual memory CD8 T cells after infection

Establishing the magnitude and kinetics of polyclonal Ag-specific CD8 T-cell responses, in addition to their functional fitness, is critical for evaluating a host's ability to respond to different kinds of infections and/or immunizations. To track CD8 T-cell responses during infection, a surrogate-activation-marker approach (CD8αloCD11ahi) is used to distinguish naïve and Ag-experienced effector/memory CD8 T cells in vivo. However, semidifferentiated virtual memory (Tvm) CD8 T cells have recently been identified in uninfected/unmanipulated mice that display a phenotype similar to Ag-experienced cells. Therefore, magnitude and breadth of CD8 T-cell responses may be overestimated when responses are profiled using only CD8α/CD11a markers. Thus, to precisely define and distinguish Tvm from pathogen-specific CD8 T cells during bacterial, parasitic, and viral infections, pathogen-specific sensor TCR-Tg cells were adoptively transferred prior to challenge. We demonstrate that Tvm CD8 T cells are found in CD8αloCD11ahi-defined Ag-experienced CD8 T cells but can be parsed out in infected host with their CD49d-CD44hiCD122hi expression pattern. However, this approach presents potential limitations as CD49d+ Ag-specific CD8 T cells can lose CD49d expression and adopt a Tvm-like phenotype depending on their Ag-stimulation history, age, and naïve CD8 T-cell precursor frequency before the infection. Importantly, Tvm cells contribute to the breadth of the CD8 T-cell response, and their contribution depends on type of infection, time after infection, and tissue examined. Thus, these data define limitations in our ability to resolve between pathogen/Ag-specific and Tvm CD8 T-cell responses during infection, a notion of direct relevance for experimental murine studies designed to follow CD8 T-cell responses in vivo.