[Quantification and phenotypic analysis of hCMV specific CTL in peripheral blood from HLA-A2+ donors].
OBJECTIVE
To optimize tetramer staining condition using HLA-A*0201 tetramer (A2-NLV tetramer) loaded with NLV peptide (pp65(495-503)) derived from structural protein pp65 of human cytomegalovirus and to investigate its application in phenotyping of specific cytotoxic T lymphocytes (CTL).


METHODS
Peripheral blood from HLA-A2(+) donors was first stained with A2-NLV tetramer/PE under different conditions and then labeled with anti-CD3-FITC and anti-CD8-APC. The stained samples were analyzed with flow cytometry to find out the optimized staining condition. Meanwhile, the phenotype and activation antigen expression were determined.


RESULTS
Tetramer staining with whole blood was superior to peripheral blood mononuclear cells. The optimized condition for tetramer staining was incubating 100 muL of whole blood with 0.3 mug of A2-NLV tetramer for 1 h at 4 degrees Celsius. Under this condition the specific staining was strong while unspecific staining of CD8(-) T cells was quite weak. Phenotypic analysis under this condition showed that the ratio of CD28 positive A2-NLV tetramer specific CTL was lower than that of nonspecific CTL, whereas the ratio of CD57 positive specific CTL was higher than that of nonspecific CTL. CD25 molecules were only expressed on the activated specific CTL.


CONCLUSIONS
The optimized tetramer staining condition can increase the specificity of tetramer staining and decrease unspecific binding, therefore it is applicable for phenotyping and functional analysis of antigen-specific CTL.
Zha
Qing-bing
QB
Key Laboratory of Ministry of Education for Tissue Transplantation and Immunology, Jinan University, Guangzhou 510632, China.
He
Xian-hui
XH
Xu
Li-hui
LH
Chi
Xiao-yun
XY
Zeng
Yao-ying
YY
D000328:Adult; D018106:CD28 Antigens / Q000032:analysis; D018959:CD57 Antigens / Q000032:analysis; D003587:Cytomegalovirus / Q000737:chemistry*; D003586:Cytomegalovirus Infections / Q000276:immunology / Q000821:virology; D005434:Flow Cytometry; D015789:HLA-A2 Antigen / Q000097:blood*; D006801:Humans; D010641:Phenotype; D010750:Phosphoproteins / Q000032:analysis*; D011108:Polymers; D013602:T-Lymphocytes, Cytotoxic / Q000145:classification*; D014019:Tissue Donors; D014763:Viral Matrix Proteins / Q000032:analysis*; D014764:Viral Proteins / Q000032:analysis; D055815:Young Adult
D018106:CD28 Antigens; D018959:CD57 Antigens; D015789:HLA-A2 Antigen; D010750:Phosphoproteins; D011108:Polymers; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C065418:cytomegalovirus matrix protein 65kDa