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. 2010 Nov;59(11):1633-44.
doi: 10.1007/s00262-010-0889-y. Epub 2010 Jul 3.

Differential gene expression in acute lymphoblastic leukemia cells surviving allogeneic transplant

Jessica C Shand  1 , Johan Jansson, Yu-Chiao Hsu, Andrew Campbell, Craig A Mullen
Affiliations

Differential gene expression in acute lymphoblastic leukemia cells surviving allogeneic transplant

Jessica C Shand et al. Cancer Immunol Immunother. 2010 Nov.

Abstract

The effectiveness of allogeneic graft-versus-leukemia (GVL) activity in control of acute lymphoblastic leukemia is generally regarded as poor. One possible factor is dynamic adaptation of the leukemia cell to the allogeneic environment. This work tested the hypothesis that the pattern of gene expression in acute lymphoblastic leukemia cells in an allogeneic environment would differ from that in a non-allogeneic environment. Expression microarray studies were performed in murine B lineage acute lymphoblastic leukemia cells recovered from mice that had undergone allogeneic MHC-matched but minor histocompatibility antigen mismatched transplants. A limited number of genes were found to be differentially expressed in ALL cells surviving in the allogeneic environment. Functional analysis demonstrated that genes related to immune processes, antigen presentation, ubiquitination and GTPase function were significantly enriched. Several genes with known immune activities potentially relevant to leukemia survival (Ly6a/Sca-1, TRAIL and H2-T23) were examined in independent validation experiments. Increased expression in vivo in allogeneic hosts was observed, and could be mimicked in vitro with soluble supernatants of mixed lymphocyte reactions or interferon-gamma. The changes in gene expression were reversible when the leukemia cells were removed from the allogeneic environment. These findings suggest that acute lymphoblastic leukemia cells respond to cytokines present after allogeneic transplantation and that these changes may reduce the effectiveness of GVL activity.

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Figures

Fig. 1
Fig. 1
Experimental design for recovery of leukemia cells from allogeneic or syngeneic transplant environments in vivo. C57BL/6 mice received total body irradiation and 5-fluorouracil chemotherapy as described in “Methods”. In the allogeneic group, they were then infused with allogeneic C3.SW donor bone marrow and spleen cells mixed with NSTY1 leukemia cells. The syngeneic control group received marrow and spleen cells from C57BL/6 mice mixed with leukemia. In each group there were 5 transplant recipients. 3 weeks later bone marrow containing leukemia was harvested from each transplant recipient and following overnight culture was infused into another set of transplant recipients. The re-isolated leukemias from each animal were kept separate. The process was repeated again. Flow cytometry was used to purify leukemia cells for RNA preparation. Leukemia 3 was leukemia that had passed through three transplant recipients and was used for the microarray experiments. In later independent experiments in which quantitative RT-PCR was used to validate upregulation of genes of interest flow sorting was used to purify Leukemia 1 which had passed through one generation of transplant recipients
Fig. 2
Fig. 2
Validation of differential expression of genes of interest by quantitative RT-PCR in freshly flow sorted leukemia cells and in cells placed in short term culture. 2 weeks after allogeneic or syngeneic transplant NSTY1 leukemia cells were purified by flow sorting. RNA was prepared immediately from 1 aliquot of the sorted cells, while the remaining aliquot was placed in culture for 96 h. After this short term culture RNA was prepared. Quantitative RT-PCR was performed for the genes Ly6a, H2-T23 and TNFSF10 using the fluorescence threshold detection method. mRNA copy numbers are expressed after normalization to the copy number of a GAPDH internal housekeeping gene. Because of limited cell numbers a single PCR reaction was performed for each gene and condition. The white bars represent gene expression in leukemia cells processed for RNA immediately at the time of cell sorting while the black bars represent gene expression following short term culture in normal tissue culture medium
Fig. 3
Fig. 3
Soluble factors from allogeneic mixed lymphocyte reactions induce similar changes in gene expression. Membrane inserts were placed in tissue culture wells that allowed free diffusion of soluble molecules but prevented passage of cells. NSTY1 leukemia cells (a), ASLN leukemia cells (b) or normal recipient B cells (c) were placed in the one chamber. In the other chamber were placed either a mixture of allogeneic donor C3.SW splenocytes and recipient C57BL/6 splenocytes (SW Allo), only syngeneic C57BL/6 splenocytes (B6 Syn), or only tissue culture medium (Control). After 72 h, leukemia cells and splenic B cells were removed from their chamber, RNA prepared and RT-PCR performed for the selected genes Ly6a, H2-T23 and TNFSF10 using the fluorescence threshold detection method. mRNA copy numbers are expressed after normalization to the copy number of a GAPDH internal housekeeping gene. PCR reactions were performed in duplicate and differences in mRNA expression between cells cultured in an allogeneic environment were compared to syngeneic controls using a paired Student’s t test. Significant P values are presented for the comparison of allogeneic to syngeneic wells. There was no significant difference (P > 0.05, values not shown) between Ly6a, H2-T23 or TNFSF10 expression in NSTY1, ASLN or splenic B cells cultured with control versus B6 syngeneic splenocytes. Data are presented from representative experiments. The number of independent experiments for each cell type is: (NSTY1, 3 experiments), (ASLN 1 experiment) and (nonmalignant B cells, 2 experiments)
Fig. 4
Fig. 4
Interferon gamma induces increased expression of Ly6a/Sca-1, H2-T23 and TRAIL in ALL cells. NSTY1 and ASLN acute lymphoblastic leukemia cells were incubated in interferon gamma 1 ng/ml for 48 h. At that time, cells were analyzed for expression of the genes by reverse transcriptase quantitative PCR (a, c and e) and for protein expression by flow cytometry or ELISA (b, d and f). RT-quantitative PCR was also performed for interferon-inducible GTPase 1 (Iigp1, g) and for macrophage activation 2 like, (Mpa2l, h). Data are presented from representative experiments. The number of independent experiments for each panel is: a, 6 experiments; b, 6 experiments; c, 2 experiments; d, 2 experiments; e, 3 experiments; f, 3 experiments; g, 3 experiments and h, 1 experiment
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