Phenotypic Analysis of Antigen-Specific T Lymphocytes

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Science  04 Oct 1996:
Vol. 274, Issue 5284, pp. 94-96
DOI: 10.1126/science.274.5284.94


  • Fig. 1.

    Staining by MHC-peptide tetramers correlates with peptide-dependent cytotoxicity. Flow cytometric analysis (18) of CD8+ T cells (17, 20, 30) from (A) clone 20 stained with A2-Pol (solid line) and A2-Gag (dotted line) tetramers, (C) HIV-Gag-specific CTL line 868 stained with A2-Gag (solid line) and A2-Pol (dotted line) tetramers, and (E) an HLA-A2-restricted influenza matrix peptide CTL line (PG-001), stained with A2-MP tetramers and sorted into A2-MP+ and A2-MP populations, as indicated. Cytotoxicity assays with (B) clone 20 showed specific killing of autologous Epstein-Barr virus-transformed B cells pulsed with Pol peptide (closed squares) but not target cells without added peptide (closed circles). (D) The 868 Gag-specific CTL line killed cells pulsed with the Gag peptide (closed squares) but not target cells without added peptide (closed circles). (F) The sorted populations from (E) were assayed for killing of MP-pulsed target cells at an effector:target ratio of 1:1. At the same ratio, in cells not treated with peptide, no killing of target cells was seen.

  • Fig. 2.

    Correlation of antigen-specific staining in three of four patients with peptide-specific killing activity in CTL bulk cultures. Peripheral blood mononuclear cells from four healthy HIV-infected donors were separated as described (5). The CD4 counts for each patient at the time of analysis were as follows: patient 065, 410; patient 868, 330; patient 077, 270; and patient 606, 510. Two million peripheral blood cells from each patient were stained with anti-CD8a-CyChrome and phycoerythrin-labeled HLA-A2-Gag (solid line) or HLA-A2-Pol (dotted line) tetramers as indicated (A through D) (18). Software gates were set to display only CD8+ small lymphocytes. The percentages of CD8+ cells that were positive for either A2-Pol or A2-Gag within gates as displayed are included in each panel. The reproducibility of the A2-Pol+ and A2-Gag+ populations in patient 065 (A) was tested through analysis of five separate stains with each reagent; the standard deviations are reported. Bulk cultures were assayed for CTL activity (E through H) (19) on days 14 through 16 at an effector:target ratio of 50:1. Black bars, lysis of Gag-loaded targets; white bars, lysis of Pol-loaded targets.

  • Fig. 3.

    Analysis of surface phenotype of HLA-A2-Pol+ cells from patient 065. Peripheral blood cells were stained as described (18). Approximately 200,000 CD8+ small lymphocytes were analyzed for antigen specificity by using the A2-Pol tetramer and the expression of the following surface markers with fluorescein isothiocyanate-labeled antibodies as indicated: CD45RO (Dako, clone UCHL-1), CD38 (Serotec, MCA1019F), CD62L (Becton Dickinson, Leu-8, clone SK11), HLA-DR (Becton Dickinson, clone L243), and CD57 (Becton Dickinson, clone HNK-1). Contour plots were generated with CELLQuest software (Becton Dickinson) by using the 75% log density contour option to demonstrate the small populations that stain positive for HLA-A2-Pol.