Generating AML-Specific Peripheral Blood Autologous Cytotoxic T-Lymphocytes (CTLs)
Authors: Mehta Rohtesh S. , Chen Xiaohua , Jeyaraj Antony , Szabolcs Paul 
Published: October 2019

Abstract
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)GeneΔΔ Ct(Post - Pre) (mean, SEM)P-valueFold change (mean, SEM)ΔΔ Ct(Post - Pre) (mean, SEM)P-valueFold change (mean, SEM)4-1BB-3.17 (0.76)0.02514 (7.7)1.98 (1.04)0.190.39 (0.22)HVEM-2.43 (0.61)0.0287.3 (3.7)0.14 (1.65)0.951.57 (1.28)LIGHT-3.62 (0.73)0.01617.3 (7.3)1.78 (1.84)0.441.1 (0.98)PRKC-α-2.03 (0.47)0.0234.6 (1.1)1.89 (0.36)0.0340.29 (0.08)PRKC-θ-3.36 (0.59)0.0113.7 (6.7)0.25 (0.59)0.710.99 (0.41)LAIR1-3.81 (0.42)0.00316.2 (5.6)-1.35 (2.20)0.6017.15 (16.5)PP2A-2.40 (0.57)0.0256.7 (2.6)0.49 (1.57)0.791.89 (1.52)2B4-1.53 (1.14)0.274.98 (1.82)-3.48 (0.11)0.0211.2 (0.9)LTA-α-1.18 (0.78)0.233.61 (2.11)2.69 (0.18)0.0430.16 (0.02)LTA-β-0.93 (0.63)0.242.49 (0.99)2.24 (0.47)0.0420.23 (0.08)
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
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.
Figure 2 Figure 2.
Figure 3 Figure 3.

Disclosures
No relevant conflicts of interest to declare.