A novel method for isolation of human lung T cells from lung resection tissue reveals increased expression of GAPDH and CXCR6.
Lung T lymphocytes are important in pulmonary immunity and inflammation. It has been difficult to study these cells due to contamination with other cell types, mainly alveolar macrophages. We have developed a novel method for isolating lung T cells from lung resection tissue, using a combination of approaches. Firstly the lung tissue was finely chopped and filtered through a nylon mesh. Lymphocytic cells were enriched by Percoll density centrifugation and the T cells purified using human CD3 microbeads, resulting in 90.5%+/-1.9% (n=11) pure lymphocytes. The T cell yield from the crude cell preparation was 10.8+/-2.1% and viability, calculated using propidium iodide (PI) staining and trypan blue, was typically over 95%. The purification process did not affect expression of CD69 or CD103, nor was there a difference in the proportion of CD4 and CD8 cells between the starting population and the purified cells. Microarray analysis and real time RT-PCR revealed upregulation of GAPDH and CXCR6 of the lung T cells as compared to blood-derived T cells. This technique highly enriches lung T cells to allow detailed investigation of the biology of these cells.
Day
C E
CE
Institute for Lung Health, Department of Infection, Immunity and Inflammation, University of Leicester, Glenfield Hospital, Leicester, United Kingdom. caroline2day@gmail.com
Zhang
S D
SD
Riley
J
J
Gant
T
T
Wardlaw
A J
AJ
Guillen
C
C
D017252:CD3 Complex; D002469:Cell Separation / Q000379:methods*; D005434:Flow Cytometry; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating) / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000166:cytology* / Q000276:immunology; D008863:Microspheres; D020411:Oligonucleotide Array Sequence Analysis; D010641:Phenotype; D011205:Povidone; D000075744:Receptors, CXCR6; D019707:Receptors, Chemokine / Q000378:metabolism*; D011991:Receptors, Virus / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012822:Silicon Dioxide; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism
D017252:CD3 Complex; C106330:CXCR6 protein, human; D000075744:Receptors, CXCR6; D019707:Receptors, Chemokine; D011991:Receptors, Virus; C016039:Percoll; D012822:Silicon Dioxide; D024581:Glyceraldehyde-3-Phosphate Dehydrogenase (Phosphorylating); D011205:Povidone