![Figure 7. Cytotoxicity and granule exocytosis assays using stimulated PBMCs from the patient, his parents, an unrelated patient with CAEBV, and healthy donors. (A) PBMCs were stimulated with PHA and IL-2, cultured in IL-2 (effector cells), and flow cytometry showed lymphocyte phenotypes described in Figure 2B. The cells were incubated with [51Cr]-labeled L1210 target cells (Fas-deficient target cells) in the presence of anti-CD3 (UCHT-1; BD Pharmingen) and anti-Fas blocking antibody (ZB4; Beckman Coulter, Fullerton, CA) in a Fas-independent CTL killing assay. Cytotoxicity in this assay system is CD3-dependent, since T-cell receptor activation is required to trigger release of granules containing perforin and granzymes.20 The effector-to-target (E/T) ratio is indicated on the x axis and the percent specific lysis is indicated on the y axis. The percent lysis was calculated as follows: % lysis = (E–S)/(M–S) × 100, where E is the release from experimental samples, S is the spontaneous release, and M is the maximum release upon lysis with 2% NP-40. The gray band indicates the normal range of cytotoxicity seen in 4 healthy donors (data not shown). Cells from a healthy blood bank donor incubated in the absence of anti-CD3 antibody were assayed as a negative control. Flow cytometry showed the following lymphocyte phenotypes: donor 1: CD8 = 49%, CD56 = 42%; donor 2: CD8 = 55%, CD56 = 42%; patient: CD8 = 54%, CD56 = 25%; mother: CD8 = 50%, CD56 = 68%; father: CD8 = 91%, CD56 = 4%; unrelated patient with CAEBV: CD8 = 55%, CD56 = 40%. (B) PBMCs were stimulated with PHA and IL-2 followed by incubation in the presence (black bars) and absence (white bars) of plate-bound anti-CD3/anti-CD28 antibody. Secretion of β-hexosaminidase in the cell culture supernatant was measured to quantify the level of granule exocytosis.33 β-hexosaminidase release was expressed as a percentage of the enzyme in the supernatant divided by the total enzyme from cells lysed in 0.1% Triton X-100. Experiments in panels A and B were performed 3 times and a representative result is shown.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/103/4/10.1182_blood-2003-06-2171/6/zh80040456890007.jpeg?Expires=1724148544&Signature=ksl4~50U7EXcaJpblhoMTbaY1p3SBNID~HKWhUlyo8ibL9zFJB7Bi9i3GUDpW45YqslfiGPQJjmKr~DsaoJWcp9Usk9ckwc0tfGiO0GEG9C9q41a0qCVONeDGAvAkcebikPhsICRewOczpjcQKwbojZGpzFIs89PiDcQO8G6fQimuqh4d2y9xQ4LKLk3Cc6tgh2B9juj7tr1y2EyOKcnjAoLKZ8REcJimAgTq5NaTyAbx6E901ii65fGsNG5gk6p0U9WOlYLd0b4D~9rXLU2L8LnnH~JHNWIcLsk947p2zvPVNVQv9qIdrHrJiUb7J0R0hrhd8G34CXFAEDzcDc7Nw__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Cytotoxicity and granule exocytosis assays using stimulated PBMCs from the patient, his parents, an unrelated patient with CAEBV, and healthy donors. (A) PBMCs were stimulated with PHA and IL-2, cultured in IL-2 (effector cells), and flow cytometry showed lymphocyte phenotypes described in Figure 2B. The cells were incubated with [51Cr]-labeled L1210 target cells (Fas-deficient target cells) in the presence of anti-CD3 (UCHT-1; BD Pharmingen) and anti-Fas blocking antibody (ZB4; Beckman Coulter, Fullerton, CA) in a Fas-independent CTL killing assay. Cytotoxicity in this assay system is CD3-dependent, since T-cell receptor activation is required to trigger release of granules containing perforin and granzymes.20 The effector-to-target (E/T) ratio is indicated on the x axis and the percent specific lysis is indicated on the y axis. The percent lysis was calculated as follows: % lysis = (E–S)/(M–S) × 100, where E is the release from experimental samples, S is the spontaneous release, and M is the maximum release upon lysis with 2% NP-40. The gray band indicates the normal range of cytotoxicity seen in 4 healthy donors (data not shown). Cells from a healthy blood bank donor incubated in the absence of anti-CD3 antibody were assayed as a negative control. Flow cytometry showed the following lymphocyte phenotypes: donor 1: CD8 = 49%, CD56 = 42%; donor 2: CD8 = 55%, CD56 = 42%; patient: CD8 = 54%, CD56 = 25%; mother: CD8 = 50%, CD56 = 68%; father: CD8 = 91%, CD56 = 4%; unrelated patient with CAEBV: CD8 = 55%, CD56 = 40%. (B) PBMCs were stimulated with PHA and IL-2 followed by incubation in the presence (black bars) and absence (white bars) of plate-bound anti-CD3/anti-CD28 antibody. Secretion of β-hexosaminidase in the cell culture supernatant was measured to quantify the level of granule exocytosis.33 β-hexosaminidase release was expressed as a percentage of the enzyme in the supernatant divided by the total enzyme from cells lysed in 0.1% Triton X-100. Experiments in panels A and B were performed 3 times and a representative result is shown.
