Presentation on theme: "B. Activated CD56+ NK % CD56 + CD3 - cells% CD161 + CD3 - cells C. Activated CD161+ NK % CD56 + CD3 + cells D. Activated CD56+ NKT P=0.007 P=0.009 P=0.02."— Presentation transcript:
B. Activated CD56+ NK % CD56 + CD3 - cells% CD161 + CD3 - cells C. Activated CD161+ NK % CD56 + CD3 + cells D. Activated CD56+ NKT P=0.007 P=0.009 P=0.02 P=0.002 P=0.003 P=0.0001 P=0.0003 ND A Fold Expansion (Log) P=0.04 P=0.01 P=0.72
F. CD3+ Cells % total of CD3 + cells G. Tm cells % Tm cells P=0.8 P=0.02 P=0.006 P=0.02 P=0.003 P=0.001 % CD161 + CD3 + cells E. Activated CD161+ NKT P=0.004 P=0.05 ND Supplemental Figure 1. Determination of cryosensitivity of the reprogrammed cells. A) Fold expansion compared to day one of IL- 2 (n=13), B/I-Fresh (n=16) and Freeze-B/I (n=11) PBMCs. Cell counts were performed using trypan blue exclusion. Data represent Least Squares Means ± SEM. B)The percentage of activated CD56+ NK cells was determined in reprogrammed cells which included B/I-Fresh (n=6), B/I-Freeze (n=9), and Freeze-B/I (n=8). C) The percentage of activated CD161+ NK cells was determined in B/I-Freeze (n=5), and Freeze-B/I (n=3). D) The percentage of activated CD56+ NKT cells was determined in B/I-Fresh (n=6), B/I-Freeze (n=9), and Freeze-B/I (n=8). E) The percentage of activated CD161+ NKT cells was determined in B/I-Freeze (n=5), and Freeze-B/I (n=3). F) The percentage of total CD3 + cells on gated lymphocyte region was determined in B/I-Fresh (n=5), B/I-Freeze (n=7), and Freeze-B/I PBMCs (n=8). G) The percentage of Tm cells was determined in B/I-Fresh (n=5), B/I-Freeze (n=7), and Freeze-B/I PBMCs (n=8). Data represent Mean ± SEM. ND = not done.
P=0.2 A B Reprogrammed PBMC IL-2/7/15 PBMC P=0.2 % total of CD3 + cells CD3 % Tm cells CD62L CD44 Reprogrammed PBMC IL-2/7/15 PBMC P=0.07 IL-2/7/15 D. Activated CD161+ NK CD25 Reprogrammed P=0.12 ReprogrammedIL-2/7/15 C. Activated CD56+ NK CD25 % CD56+ CD25+ NK cells % CD161+ CD25+ NK cells Supplemental Figure 2. Determination of the role of B/I in cellular reprogramming. PBMCs (n=3) were split into two groups and then subjected to reprograming protocol in the presence (Reprogrammed PBMC) or absence (IL-2/7/15 PBMC) of B/I. The frequency of (A) total CD3 + cells, (B) Tm cells, (C) activated CD56+ NK cells, and (D) activated CD161+ NK cells was determined in Reprogrammed and IL2/7/15 PBMCs using flow cytometry. Data represent Mean ± SEM.
Baseline IL-2/7/15 PBMCReprogrammed PBMC V 9 J2-2 A B V 5-7 J2-3 V 11-3 J2-2 C Frequency J gene Supplemental Figure 3. Bryostatin 1 and Ionomycin selectively expand specific T cell clones. High-throughput sequencing of TcR Vβ was performed from one patients PBMCs at baseline, after culture with γ-c cytokines only (IL-2/7/15 PBMC), or after reprogramming with B/I and γ-c cytokines (Reprogrammed PBMC). Representative data for the effect of reprogramming on the expansion of dominant clone (A), skewed clonality (B), or no impact on specific clones (C).
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