A novel assay for assessment of HIV‐specific cytotoxicity by multiparameter flow cytometry

Abstract

BackgroundAssessment 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.MethodsTarget 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 (CFSEhiPI−) 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 51Cr‐release assay and IFN‐γ ELISpot were performed by standard procedures.ResultsA 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).ConclusionThis 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. © 2005 Wiley‐Liss, Inc.

Vγ9Vδ2 T Lymphocytes Efficiently Recognize and Kill Zoledronate-Sensitized, Imatinib-Sensitive, and Imatinib-Resistant Chronic Myelogenous Leukemia Cells

“…Cytotoxicity was measured by FACS analysis using CFSE and propidium iodide (PI; both from Molecular Probes) as described in Ref. 20. Briefly, a total of 50 ml of CFSE were added to 1 ml target cell suspension (5 3 10 5 cells/ml) in PBS to obtain the final concentration of 2.5 mM CFSE.…”

Impairment of Regulatory Capacity of CD4+CD25+ Regulatory T Cells Mediated by Dendritic Cell Polarization and Hyperthyroidism in Graves’ Disease

“…Apoptosis detection for T cells in an FCM assay was adapted from GodoyRamirez et al (33). First, freshly isolated DCs were prelabeled with 2.5 mM CFSE (Sigma-Aldrich) for 10 min, then freshly isolated CD4 + CD25 + T cells (1 3 10 5 cells/well) were cocultured with labeled autologous DCs (2 3 10 4 cells/well) in 96-well V-bottom plates containing 200 ml RPMI 1640 medium supplemented with 10% FBS, 100 U/ml penicillin, and 100 mg/ml streptomycin.…”

Immunological function of Langerhans cells in HIV infection

“…Recently, the detection of IFN-g by ELISPOT assay or intracellular cytokine staining using flow cytometry is replacing the traditional 51 Cr release cytolytic assay in vaccine trials using HIV [48,49]. Results between IFN-g production by virus-specific T cells and the cytotoxicity of virus-specific T cells determined by the 51 Cr-release assay were parallel upon virus infections, including cytomegalovirus, Epstein-Barr virus, influenza virus, and HIV [50][51][52][53]. IFN-g directly kill virus-infected cells, induces a wide range of proteins that are associated in regulating viral replication, and upregulates MHC class I on the cell surface, which increases antigenic recognition of intracellular pathogens by CTLs, thereby promoting anti-viral immunity [54][55][56].…”