A novel assay for assessment of HIV-specific cytotoxicity by multiparameter flow cytometry.
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).


CONCLUSIONS
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.
Godoy-Ramirez
Karina
K
Department of Immunology and Vaccinology, Swedish Institute for Infectious Disease Control, Sweden. karina.godoy@smi.ki.se
Mäkitalo
Barbro
B
Thorstensson
Rigmor
R
Sandström
Eric
E
Biberfeld
Gunnel
G
Gaines
Hans
H
D002478:Cells, Cultured; D002857:Chromium; D003602:Cytotoxicity, Immunologic; D005434:Flow Cytometry / Q000379:methods*; D005452:Fluoresceins; D015488:HIV Antigens / Q000276:immunology*; D015658:HIV Infections / Q000276:immunology*; D006679:HIV Seropositivity; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D007150:Immunohistochemistry; D011419:Propidium; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013388:Succinimides; D013602:T-Lymphocytes, Cytotoxic / Q000166:cytology / Q000276:immunology*
C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D005452:Fluoresceins; D015488:HIV Antigens; D013388:Succinimides; D002857:Chromium; D011419:Propidium