Cytometry. Part A : the journal of the International Society for Analytical Cytology · 2005
A novel assay for assessment of HIV-specific cytotoxicity by multiparameter flow cytometry.
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Godoy-Ramirez Karina, Mäkitalo Barbro, Thorstensson Rigmor, Sandström Eric, Biberfeld Gunnel, Gaines Hans
Аннотация
BACKGROUND: Assessment of CD8(+) T-cell activity is of significant importance for the evaluation of cellular immune responses to viral infections, especially in HIV. We present a new assay for the assessment of HIV-specific cytotoxicity by multiparameter flow cytometry.
METHODS: Target cells, pulsed with peptide pools (Gag or Nef), were stained with 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), cultured with specific or nonspecific effector cells, and finally stained with propidium iodide (PI). Determination of cytolysis is based on the enumeration of viable target cells (CFSE(hi)PI(-)) in the test sample (target and specific effector cells) as compared with that of the viable target cells in the control sample (target and nonspecific effector cells). The (51)Cr-release assay and IFN-gamma ELISpot were performed by standard procedures.
RESULTS: A comparison with the Cr-release showed that the two assays were strongly correlated (r = 0.67; P < 0.001) but the sensitivity of the flow cytometric assay was significantly higher (P < 0.05), and the reproducibility good (CV, 7.7%). Good correlation was also found with the ELISpot assay (r = 0.66; P < 0.01).
CONCLUSION: This new assay provides both specific and sensitive results when employed for the detection of HIV-specific CTL and can be a valuable tool for the evaluation of cytolytic activity in vaccine trials or in HIV-infected subjects, especially if such responses are present at low levels.
METHODS: Target cells, pulsed with peptide pools (Gag or Nef), were stained with 5- (and -6)-carboxyfluorescein diacetate succinimidyl ester (CFSE), cultured with specific or nonspecific effector cells, and finally stained with propidium iodide (PI). Determination of cytolysis is based on the enumeration of viable target cells (CFSE(hi)PI(-)) in the test sample (target and specific effector cells) as compared with that of the viable target cells in the control sample (target and nonspecific effector cells). The (51)Cr-release assay and IFN-gamma ELISpot were performed by standard procedures.
RESULTS: A comparison with the Cr-release showed that the two assays were strongly correlated (r = 0.67; P < 0.001) but the sensitivity of the flow cytometric assay was significantly higher (P < 0.05), and the reproducibility good (CV, 7.7%). Good correlation was also found with the ELISpot assay (r = 0.66; P < 0.01).
CONCLUSION: This new assay provides both specific and sensitive results when employed for the detection of HIV-specific CTL and can be a valuable tool for the evaluation of cytolytic activity in vaccine trials or in HIV-infected subjects, especially if such responses are present at low levels.