The CTLp frequency reactive to the recipient alloantigen in the recipient after transplantation and the donor
| Samples . | Maximum CD8+ input* . | No. of growing wells† . | CTLp frequency−1 (95% confidence interval) . |
|---|---|---|---|
| Donor | 33 300 | 8 | 8.6 × 105 (1.49 × 106-5.0 × 105) |
| Day 100 | 35 500 | 0 | UD |
| Day 180 | 17 700 | 0 | UD |
| Day 300‡ | 86 000 | 0 | UD |
| Day 520§ | 95 000 | 7 | 4.3 × 105 (7.2 × 105-2.5 × 105) |
| Samples . | Maximum CD8+ input* . | No. of growing wells† . | CTLp frequency−1 (95% confidence interval) . |
|---|---|---|---|
| Donor | 33 300 | 8 | 8.6 × 105 (1.49 × 106-5.0 × 105) |
| Day 100 | 35 500 | 0 | UD |
| Day 180 | 17 700 | 0 | UD |
| Day 300‡ | 86 000 | 0 | UD |
| Day 520§ | 95 000 | 7 | 4.3 × 105 (7.2 × 105-2.5 × 105) |
Purified CD8+ T cells from the peripheral blood mononuclear cells obtained after transplantation from patient 2 and her donor were cultured at 2- or 3-fold serial dilutions with 33 Gy-irradiated 3 × 104 leukemic blasts cryopreserved at the time of initial diagnosis in 96-well, round-bottom plates in advanced RPMI 1640 medium supplemented with 4% pooled human serum, interleukin-6 ( IL-6), and IL-7 (10 ng/mL; both from R&D Systems). The IL-2 (50 U/mL) was added on day 7 with a half medium change. For each dilution, there were at least 12 replicates. On day 14 of culture, a split-well analysis was performed for recipient-specific cytotoxicity against 51Cr-radiolabeled recipient T-cell blasts, donor T-cell blasts, and leukemic blasts harvested at the time of initial diagnosis and at the time of relapse after DLI if indicated. The supernatants were measured in a γ counter after 4-hour incubation. The wells were considered to be positive for cytolytic activity if the total counts per minute released by effector cells was more than 3 SD above the control wells (mean counts per minute released by the target cells incubated with irradiated stimulator cells alone). The CTLp frequency was calculated using L-Calc software (StemCell Technologies). The CTLp frequencies reactive with recipient T-cell blasts in CD8+ T cells obtained around days 100, 180, and 300 (4 months before relapse) were undetectable, whereas the CTLp frequency obtained at day 520 (1 month after the third DLI or 2 weeks after remission confirmed by bone marrow aspirate) was close to the CTLp frequency in the donor CD8+ cells. Complete remission and more than 99% donor chimerism were confirmed on those days
CTLp indicates CTL precursor; and UD, undetermined because no growing wells are present.
Number of input CD8+ T cells seeded at the highest number per well.
Number of wells out of 12 wells that received the highest CD8+ cells and showed detectable growth.
Corresponds to 4 months before relapse.
Corresponds to 1 month after the third DLI or 2 weeks after complete remission was confirmed by bone marrow aspirate.