Single-cell perforin and granzyme expression reveals the anatomical localization of effector CD[8.sup.+] T cells in influenza virus-infected mice

Influenza virus infection activates cytolytic T lymphocytes (CTL) that contribute to viral clearance by releasing perforin and granzymes from cytoplasmic granules. Virus-specific, perforin-dependent CD[8.sup.+] CTL were detected in freshly isolated cells from the mouse lung parenchyma but not from the mediastinal lymph nodes (MLN), where they are primed, or from the spleen during primary influenza virus infection. To determine whether this difference was due to the low frequency or incomplete maturation of effector CTL in MLN, we measured expression of perforin, granzymes A, B, and C, and IFN-[gamma] mRNAs in CD[8.sup.+] populations and single cells immediately after isolation from virus-infected mice. Quantitative PCR revealed significant expression of perforin, granzyme A, granzyme B, and IFN-[gamma] in activated CD[8.sup.+] cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramer-binding CD[8.sup.+] cells from the three tissues expressed diverse combinations of perforin, granzyme, and IFN-[gamma] mRNAs. Although cells from lung expressed granzymes A and B at higher frequency, each of the tissues contained cells that coexpressed perforin with granzymes A and/or B. The main difference between MLN and lung was the elevated frequency of activated CD[8.sup.+] T cells in the lung, rather than their perforin/granzyme expression profile. The data suggest that some CTL mature into perforin/granzyme-expressing effector cells in MLN but reach detectable frequencies only when they accumulate in the infected lung.