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Background: Ex-vivo expansion of CBT-cells using CD3/CD28 co-stimulatory beads, IL-2 + IL-7 & subsequent priming against leukemia cell lines using IL-15 generated specific CTLs. [1, 2] Using similar immune-stimulatory culture conditions, we generated auto PB-derived CTLs from AML patients in 5 of 8 samples. [Abstract #5836] The aim of present study was to find source of disparity between “resistant” (n=3) & non-resistant AML (n=5).
Hypothesis: We hypothesized (a) in contrast to AML cells that are susceptible to cytotoxicity by auto CTLs (n=5), the “resistant” AML cells (n=3), would possess features similar to myeloid-derived suppressor cells (MDSCs).
Methods: AML blasts were purified from PBMCs of AML patients admitted with blast crisis (n=8). AML-specific auto CTLs were generated using immune-stimulatory culture conditions as described. [Abstract #5836] RNA purification & RT-qPCR from blasts was done using Trizol reagent (Life Technologies) & Power SYBR® Green master mix (Applied Biosystems) with StepOne Plus system (Life Technologies) as suggested by manufacturers. Genes with defined role in MDSC generation [2] were selected for analysis. Primers were designed using NCBI primer blast. [3] GAPDH was used as house-keeping gene. Student t-test was used to compare the groups.
Results: Resistant blasts had significant up-regulation of JAK-2, S100A8, S100A9 and c-myc compared to the susceptible blasts. [Table 1] Although JAK-1 and JAK-3 were up-regulated notably, statistical significance was not met.
Discussion: JAK-2 signaling pathway is critical for MDSC generation and survival, which induces c-myc expression, while S100A8 & S100A9 potentiate the suppressive effects of MDSC. [Figure 1] [2] Many solid tumors assume MDSC-like phenotype, [2] but this is rarely reported in hematological malignancies. [3, 4] Our findings mandate further validation. If replicated, therapeutic roles of JAK and S100 pathway inhibitors could be explored.
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Figure 1: Signalling pathways involved in the expansion of mDSCs [2] (reproduced with written permission from the Nature Publishing Group) |
Refs:
1. Jeyaraj A et al. IL-15 Induced Polyclonal CTL Generated From Expanded CBT Cells Against Leukemia Cell Lines Constitutes IFN-γ Producing Cells and TCRγd Cells. ASH 2012 Annual Meeting
2. Gabrilovich DI et al. Nat Rev Immunol. (2009) Myeloid-derived suppressor cells as regulators of the immune system.
3. Primer BLAST http://www.ncbi.nlm.nih.gov/tools/primer-blast/
4. Alex AA et al. Myeloid Derived Suppressor Cells in Acute Leukemia and Its Association with Conventional Cytogenetic and Molecular Risk Factors. ASH 2010 Annual Meeting; 1446.
5. Miner S et al. Myeloid Leukemias Directly Suppress T Cell Proliferation Through STAT3 and Arginase Pathways. Blood Nov 15, 2013; 122 (21)