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Background: Ex-vivo expansion of CBT-cells with CD3/28 co-stimulatory beads, IL-2 & IL-7 & subsequent priming against leukemia cell lines using IL-15 created specific CTLs. [1, 2]
Hypothesis: We hypothesized (a) patient-derived AML-specific PB auto CTLs could be generated (b) Tregs proportion (CD4+CD25brightFoxP3+) & T-cell co-signaling markers' gene expression will be different between effective and ineffective CTLs.
Methods: AML & auto T-cells were purified from PBMC of AML patients admitted with acute blast crisis (n=8). AML blasts were sustained in Serum-Free media (STEMCELL Tech) with MSC support & cytokines (IL-3, SCF, FLT3L, GMCSF, IL-4). T-cells were expanded in culture for 2 weeks as reported [1, 2] & subsequently primed with γ-irradiated auto AML weekly X 3 with IL15 & CD28ab [BD Biosciences]. At the end of week 3 (EOW3), cytotoxicity was assessed against AML and irrelevant targets - IM9 & U937 cell lines, at an E:T ratio of 40:1, 20:1, 10:1 & 5:1 using DELFIAŽ EuTDA assay.[2] IFN-γ ELISPOT assay against same targets was done.[2] RT-qPCR analysis was done on T-cells before & after priming, with Power SYBRŽ Green master mix (Thermo Fisher) & StepOne Plus system (Life Tech). Student t-test compared the groups.
Results
1. T-cells expanded in all samples (n=8) by median of 155-fold (range 11-489) at EOW3.
2. ELISPOT assay was positive in 4/8 samples. [Fig 1]
3. CTL assay was difficult to standardize for AML blasts due to high degree of spontaneous apoptosis (>30% spontaneous release [SR]).
i. 2/8 samples were evaluable (SR<30%).
ii. Both samples showed AML-specific lysis. [Fig 2]
4. Overall, AML-specific auto CTLs could be generated in 5 of 8 samples based on ELISPOT & CTL assays.
5. Tregs declined notably in effective CTLs post-priming compared to pre-priming (56% to 24%, p-value 0.046, n=4). [Fig 3]
6. T-cell co-signaling molecules' gene expression differed in effective vs ineffective CTLs. [Table 1]
Conclusions (a) AML-specific auto CTLs can be generated (b) Tregs decreased after priming in effective CTLs (c) Gene expression of T-cell co-stimulatory markers differ notably in effective & ineffective CTLs
TABLE 1: T-CELL GENE EXPRESSION (POST VS PRE-PRIMING)
| ||||
| Effective CTLs (n=5) | Ineffective CTLs (n=3)
| ||
Gene | Fold-change (mean, SEM) | P-value | Fold-change (mean, SEM) | P-value |
4-1BB | 14 (7.7) | 0.025 | 0.39 (0.22) | 0.19 |
HVEM | 7.3 (3.7) | 0.028 | 1.57 (1.28) | 0.95 |
LIGHT | 17.3 (7.3) | 0.016 | 1.1 (0.98) | 0.44 |
PRKC-α | 4.6 (1.1) | 0.023 | 0.29 (0.08) | 0.034 |
PRKC-θ | 13.7 (6.7) | 0.01 | 0.99 (0.41) | 0.71 |
LAIR1 | 16.2 (5.6) | 0.003 | 17.15 (16.5) | 0.60 |
PP2A | 6.7 (2.6) | 0.025 | 1.89 (1.52) | 0.79 |
2B4 | 4.98 (1.82) | 0.27 | 11.2 (0.9) | 0.02 |
LTA-α | 3.61 (2.11) | 0.23 | 0.16 (0.02) | 0.043 |
LTA-β | 2.49 (0.99) | 0.24 | 0.23 (0.08) | 0.042 |
Ref
1.Davis CC et al. Cancer Res. (2010)
2.Jeyaraj A, Chen X, Szabolcs P. IL-15 Induced Polyclonal CTL Generated From Expanded CBT Cells Against Leukemia Cell Lines Constitutes IFN-γ Producing Cells and TCRγd Cells. ASH 2012 Meeting
