Single-cell perforin and granzyme expression reveals the anatomical localization of effector CD8+ 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 CD8(+) 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 CD8(+) 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 CD8(+) cells from MLN, spleen, and lung parenchyma. Granzyme C expression was not detected. Individual activated or nucleoprotein peptide/class I tetramer-binding CD8(+) 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 CD8(+) 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.
Johnson
Barbara J
BJ
Cooperative Research Centre for Vaccine Technology and Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Queensland 4029, Australia.
Costelloe
Elaine O
EO
Fitzpatrick
David R
DR
Haanen
John B A G
JB
Schumacher
Ton N M
TN
Brown
Lorena E
LE
Kelso
Anne
A
D000818:Animals; D016827:CD8 Antigens / Q000096:biosynthesis; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D002860:Chromium Radioisotopes; D020552:Comet Assay; D005260:Female; D053804:Granzymes; D018960:Hyaluronan Receptors / Q000096:biosynthesis; D007371:Interferon-gamma / Q000096:biosynthesis; D008168:Lung / Q000276:immunology / Q000378:metabolism; D008198:Lymph Nodes / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000096:biosynthesis*; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D009975:Orthomyxoviridae / Q000378:metabolism*; D054353:Perforin; D010641:Phenotype; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000096:biosynthesis*; D013154:Spleen / Q000166:cytology; D013601:T-Lymphocytes / Q000276:immunology; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism
D016827:CD8 Antigens; D002860:Chromium Radioisotopes; D018960:Hyaluronan Receptors; D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D012333:RNA, Messenger; D054353:Perforin; D007371:Interferon-gamma; D053804:Granzymes; C509214:Gzmb protein, mouse; C509216:Gzmc protein, mouse; D012697:Serine Endopeptidases