Figure 4.
CD8+ T-cell subsets increase ALDH1A1 expression in mixed lymphocyte culture and after stimulation with low-dose IL-2. (A) T cells were isolated from healthy donor PBMCs and either taken directly for flow cytometric sorting as per supplemental Figure 1 or put in MLC as in Figure 1 and flow cytometrically sorted on day 3 or 7. Quantitative polymerase chain reaction (PCR) for ALDH1A1 or GAPDH was performed on RNA extracted from flow cytometrically-sorted CD8+ T-cell subsets, showing increased ALDH1A1 expression on day 3 that persisted but declined on day 7 of MLC. Each color shows a different healthy donor tested (n = 4). (B) ALDH functional activity was measured by Aldefluor positivity based on a DEAB negative control for each sample. There was overall increased Aldefluor positivity in CD8+ T-cell subsets on day 3 of MLC compared with freshly isolated healthy donor T cells (day 0); these differences were statistically significant on repeated-measure ANOVA for all but the CM, EM, and MAIT subsets. (C) A representative example of the positive shift in Aldefluor staining is shown for all CD8+ T cells on MLC day 3. The threshold of Aldefluor positivity is based on the sample-specific DEAB negative control. (D) Samples on day +3 after transplant from patients treated with myeloablative HLA-matched BM transplantation previously assessed for Aldefluor positivity within CD4+ T-cell subsets7 were assessed for Aldefluor positivity within CD4− T-cell subsets, also showing Aldefluor positivity within a subset of cells. (E) Human T cells were isolated and plated either in media alone; with 10 IU/mL IL-2, 10 ng/mL IL-7, or 10 ng/mL IL-15; in MLC; or with anti-CD3/CD28 stimulation beads ± IL-2, IL-7, or IL-15. T cells that were in MLC were immunomagnetically reisolated on day 3 to remove CD3− stimulator cells, after which ALDH1A1 and GAPDH expression were determined via quantitative PCR for all groups. The dose of IL-2 chosen (10 IU/mL) for the initial experiments resulted in a variable effect on ALDH1A1 expression, whereas bead stimulation decreased ALDH1A1 expression compared with cells cultured in media only. Relative expression is shown of ALDH1A1/GAPDH of the treatment divided by ALDH1A1/GAPDH of the vehicle-treated control group. (F) Therefore, a range of IL-2 doses was tested, showing increased ALDH1A1 expression at low doses and decreased ALDH1A1 expression at high doses. (G) A timecourse assessment showed that the peak increase in ALDH1A1 expression with low-dose IL-2 was at 48 hours in 3 of 4 healthy donors tested. Relative expression is shown of ALDH1A1/GAPDH of each time point divided by ALDH1A1/GAPDH of the pretreatment (time 0) sample. Combined results are shown from 2 independent experiments (n = 4) for (A,E-G) and from 3 independent experiments (n = 6) for (B). (D) n = 5.

CD8+ T-cell subsets increase ALDH1A1 expression in mixed lymphocyte culture and after stimulation with low-dose IL-2. (A) T cells were isolated from healthy donor PBMCs and either taken directly for flow cytometric sorting as per supplemental Figure 1 or put in MLC as in Figure 1 and flow cytometrically sorted on day 3 or 7. Quantitative polymerase chain reaction (PCR) for ALDH1A1 or GAPDH was performed on RNA extracted from flow cytometrically-sorted CD8+ T-cell subsets, showing increased ALDH1A1 expression on day 3 that persisted but declined on day 7 of MLC. Each color shows a different healthy donor tested (n = 4). (B) ALDH functional activity was measured by Aldefluor positivity based on a DEAB negative control for each sample. There was overall increased Aldefluor positivity in CD8+ T-cell subsets on day 3 of MLC compared with freshly isolated healthy donor T cells (day 0); these differences were statistically significant on repeated-measure ANOVA for all but the CM, EM, and MAIT subsets. (C) A representative example of the positive shift in Aldefluor staining is shown for all CD8+ T cells on MLC day 3. The threshold of Aldefluor positivity is based on the sample-specific DEAB negative control. (D) Samples on day +3 after transplant from patients treated with myeloablative HLA-matched BM transplantation previously assessed for Aldefluor positivity within CD4+ T-cell subsets7  were assessed for Aldefluor positivity within CD4− T-cell subsets, also showing Aldefluor positivity within a subset of cells. (E) Human T cells were isolated and plated either in media alone; with 10 IU/mL IL-2, 10 ng/mL IL-7, or 10 ng/mL IL-15; in MLC; or with anti-CD3/CD28 stimulation beads ± IL-2, IL-7, or IL-15. T cells that were in MLC were immunomagnetically reisolated on day 3 to remove CD3− stimulator cells, after which ALDH1A1 and GAPDH expression were determined via quantitative PCR for all groups. The dose of IL-2 chosen (10 IU/mL) for the initial experiments resulted in a variable effect on ALDH1A1 expression, whereas bead stimulation decreased ALDH1A1 expression compared with cells cultured in media only. Relative expression is shown of ALDH1A1/GAPDH of the treatment divided by ALDH1A1/GAPDH of the vehicle-treated control group. (F) Therefore, a range of IL-2 doses was tested, showing increased ALDH1A1 expression at low doses and decreased ALDH1A1 expression at high doses. (G) A timecourse assessment showed that the peak increase in ALDH1A1 expression with low-dose IL-2 was at 48 hours in 3 of 4 healthy donors tested. Relative expression is shown of ALDH1A1/GAPDH of each time point divided by ALDH1A1/GAPDH of the pretreatment (time 0) sample. Combined results are shown from 2 independent experiments (n = 4) for (A,E-G) and from 3 independent experiments (n = 6) for (B). (D) n = 5.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal