Background: Ex-vivo expansion of CBT-cells using CD3/CD28 co-stimulatory beads, IL-2 + IL-7 and subsequent priming against leukemia cell lines using IL-15 generated specific CTLs. [1, 2]

Hypothesis: We hypothesized that (a) patient-derived AML-specific PB auto CTLs could be generated with immune-stimulatory culture condition (b) Resistant AML samples would possess gene expression profiles similar to MDSCs (myeloid-derived suppressor cells) (c) Frequency of Tregs (CD4+CD25brightFoxP3+) and T-cell co-signaling molecules gene expression will be different between effective and ineffective CTLs.

Methods: AML & auto T-cells were purified from cryopreserved PBMC of AML patients admitted with acute blast crisis (n=8). AML blasts were sustained in StemSpan™ Serum-Free media [STEMCELL Technologies] with MSC support + cytokine cocktail (IL-3, SCF, FLT3L, GMCSF, IL-4). T-cells were expanded in culture for 2 weeks as reported [1, 2] and 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 (lymphoid) and U937 (myeloid) cell lines, loaded with BATDA at an E:T ratio of 40:1, 20:1, 10:1 and 5:1 using DELFIA® EuTDA assay.[2] IFN-γ ELISPOT assay against same targets was also done.[2] RT-qPCR analysis was performed on AML & T-cells before and after priming, using Power SYBR® Green master mix (Thermo Fisher Scientific) and StepOne Plus system [Life Technologies]. Two-tailed student t-testcompared experimental groups.

Results

· T-cells expanded in all samples (n=8) with a median expansion of 155-fold (range 11-489), at EOW3.

· ELISPOT assay was positive in 4/8 samples. [Fig 1]

· CTL assay was difficult to standardize for primary AML blasts due to high degree of spontaneous apoptosis (>30% spontaneous release [SR]).

· 2/8 samples were deemed evaluable (SR<30%).

· Both samples showed AML-specific lysis. [Fig 2]

· Overall, AML-specific autologous CTL could be generated from 5 of 8 samples based on ELISPOT & CTL assays, regardless of original FAB immunophenotype, not shown.

· Tregs proportion declined significantly in effective CTLs post-priming as compared to pre-priming (56% to 24%, p-value 0.046, n=4). [Fig 3]

· T-cell gene expression profiling showed significant differences in effective vs ineffective CTLs. [Table 1]

· Resistant AML (n=3) had up-regulated downstream markers associated with MDSC generation compared to “non-resistant” AML (n=5). [Table 2]

Conclusions (a) AML-specific auto CTLs can be generated (b) Tregs decreased with priming in effective CTLs (c) differential T-cell gene expression profile exists between effective and ineffective CTLs (d) AML gene expression suggests MDSC-like profile in resistant samples.

Abstract 5806. TABLE 1:

T-CELL GENE EXPRESSION PROFILE (POST VS PRE-PRIMING)

 Effective CTLs (n=5) Ineffective CTLs (n=3) 
Gene ΔΔ Ct
(Post - Pre) (mean, SEM) 		P-value Fold change (mean, SEM) ΔΔ Ct
(Post - Pre) (mean, SEM) 		P-value Fold change (mean, SEM) 
4-1BB -3.17 (0.76) 0.025 14 (7.7) 1.98 (1.04) 0.19 0.39 (0.22) 
HVEM -2.43 (0.61) 0.028 7.3 (3.7) 0.14 (1.65) 0.95 1.57 (1.28) 
LIGHT -3.62 (0.73) 0.016 17.3 (7.3) 1.78 (1.84) 0.44 1.1 (0.98) 
PRKC-α -2.03 (0.47) 0.023 4.6 (1.1) 1.89 (0.36) 0.034 0.29 (0.08) 
PRKC-θ -3.36 (0.59) 0.01 13.7 (6.7) 0.25 (0.59) 0.71 0.99 (0.41) 
LAIR1 -3.81 (0.42) 0.003 16.2 (5.6) -1.35 (2.20) 0.60 17.15 (16.5) 
PP2A -2.40 (0.57) 0.025 6.7 (2.6) 0.49 (1.57) 0.79 1.89 (1.52) 
2B4 -1.53 (1.14) 0.27 4.98 (1.82) -3.48 (0.11) 0.02 11.2 (0.9) 
LTA-α -1.18 (0.78) 0.23 3.61 (2.11) 2.69 (0.18) 0.043 0.16 (0.02) 
LTA-β -0.93 (0.63) 0.24 2.49 (0.99) 2.24 (0.47) 0.042 0.23 (0.08) 
 Effective CTLs (n=5) Ineffective CTLs (n=3) 
Gene ΔΔ Ct
(Post - Pre) (mean, SEM) 		P-value Fold change (mean, SEM) ΔΔ Ct
(Post - Pre) (mean, SEM) 		P-value Fold change (mean, SEM) 
4-1BB -3.17 (0.76) 0.025 14 (7.7) 1.98 (1.04) 0.19 0.39 (0.22) 
HVEM -2.43 (0.61) 0.028 7.3 (3.7) 0.14 (1.65) 0.95 1.57 (1.28) 
LIGHT -3.62 (0.73) 0.016 17.3 (7.3) 1.78 (1.84) 0.44 1.1 (0.98) 
PRKC-α -2.03 (0.47) 0.023 4.6 (1.1) 1.89 (0.36) 0.034 0.29 (0.08) 
PRKC-θ -3.36 (0.59) 0.01 13.7 (6.7) 0.25 (0.59) 0.71 0.99 (0.41) 
LAIR1 -3.81 (0.42) 0.003 16.2 (5.6) -1.35 (2.20) 0.60 17.15 (16.5) 
PP2A -2.40 (0.57) 0.025 6.7 (2.6) 0.49 (1.57) 0.79 1.89 (1.52) 
2B4 -1.53 (1.14) 0.27 4.98 (1.82) -3.48 (0.11) 0.02 11.2 (0.9) 
LTA-α -1.18 (0.78) 0.23 3.61 (2.11) 2.69 (0.18) 0.043 0.16 (0.02) 
LTA-β -0.93 (0.63) 0.24 2.49 (0.99) 2.24 (0.47) 0.042 0.23 (0.08) 
View Large

TABLE 2:

GENE EXPRESSION PROFILE RESISTANT VS NON-RESISTANT AML

Gene ΔΔ Ct
(mean, SEM) 		95% CI P-value Relative fold change 
JAK1 -4.63 (1.98) -9.48 0.21 0.0579 24.83 
JAK2 -5.38 (0.94) -7.67 -3.08 0.0012 41.52 
JAK3 -5.90 (2.17) -12.81 1.01 0.0726 59.77 
S100A8 -7.16 (2.66) -14.01 -0.32 0.0432 143.27 
S100A9 -8.31 (2.75) -15.04 -1.59 0.0233 318.37 
c-myc -2.78 (0.59) -4.24 -1.33 0.0034 6.89 
Gene ΔΔ Ct
(mean, SEM) 		95% CI P-value Relative fold change 
JAK1 -4.63 (1.98) -9.48 0.21 0.0579 24.83 
JAK2 -5.38 (0.94) -7.67 -3.08 0.0012 41.52 
JAK3 -5.90 (2.17) -12.81 1.01 0.0726 59.77 
S100A8 -7.16 (2.66) -14.01 -0.32 0.0432 143.27 
S100A9 -8.31 (2.75) -15.04 -1.59 0.0233 318.37 
c-myc -2.78 (0.59) -4.24 -1.33 0.0034 6.89 
View Large

Refs:

1.Davis et al. Cancer Research 2010;70(13):5249

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 TCRγδ Cells. ASH 2012 Annual Meeting

Figure 1
Figure 1.
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Figure 2
Figure 2.
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Figure 3
Figure 3.
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Disclosures

No relevant conflicts of interest to declare.

Topics:
aldesleukin, antigens, cd25, apoptosis, blast cells, blast phase, cancer research, cd28 antigens, cell lines, cognitive-behavioral therapy, conflict of interest

Author notes

*

Asterisk with author names denotes non-ASH members.

© 2014 by The American Society of Hematology
2014
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