Characterization of CD4+ T-cell expansion after cord blood transplantation and its role in anti-leukaemic effects

In the absence of serotherapy, cord blood transplantation (CBT) is followed by a rapid and unique CD4+ biased immune reconstitution derived from T cells within the graft. The mechanism for this enhanced CD4+ biased reconstitution which differs from that of other stem cell sources and correlates with rapid anti-viral and enhanced graft-versus-leukaemia responses is not known. Lymphopoiesis following stem cell transplantation may be derived from foetal or adult haematopoietic stem cells. We therefore sought to determine whether recapitulation of foetal lymphopoiesis mediates rapid expansion of cord blood CD4+ T cells into the lymphopenic environment created by transplant conditioning. We compared the foetal CD4+ T-cell transcriptome with the transcription profile of naïve CD4+ T cells from normal donor cord blood (n=9), normal donor peripheral blood (n=9), and reconstituting naïve CD4+ T cells following CBT (n=3) and bone marrow transplant (BMT) (n=3). Our findings confirm that cord blood CD4+ T cells and CD4+ T cells reconstituting after CBT retain the properties of foetal ontogenesis and can rapidly restore the CD4+ T-cell biased adaptive immunity through enhanced T-cell receptor (TCR) signalling. TCR sensitivity dictates the ability of T cells to respond to self-peptide and foreign antigens, and the emerging data suggests that unrelated CBT, particularly in the context of HLA-mismatching and a T-cell replete graft, may reduce leukaemic relapse. Therefore, I aimed to study the role of foetal-derived adaptive immune system following T-cell replete CBT in mediating graft-versus-tumour responses and dissect the underlying cellular mechanisms. To do this, the ability of HLA-mismatched cord blood and adult peripheral blood T cells to eliminate Epstein-Barr virus (EBV)-driven human B-cell lymphoma was compared, in a xenogeneic NOD/SCID/IL2rgnull mouse model. In our model, cord blood T cells mediated enhanced anti-tumour effects by rapid infiltration of the tumour with CCR7+ CD8+ T cells, and prompt induction of cytotoxic CD8+ and Th1 CD4+ T cells in the tumour microenvironment. Conversely, in the peripheral blood group, this anti-lymphoma effect was impaired because of delayed tumoural infiltration of peripheral blood T cells, and a relative bias toward suppressive Th2 and T regulatory cells. Our data suggest that, despite being naturally programmed toward tolerance, reconstituting T cells after unrelated T-cell replete CBT may provide superior Tc1-Th1 anti-tumour effects against high-risk haematological malignancies

Assessment of cross-talk between Notch and BCR-ABL activity in CD34+ primary CML cells.

<p><b>(a)</b><i>Hes1</i> gene expression in CD34+ cells isolated from GSI-responder CML patients. CD34+ cells were isolated from CML patients and cultured in the presence of 10 μM GSI for 72h. Live CD34+ cells were then sorted, and the gene expression of the Notch target gene <i>Hes1</i> was investigated by real-time PCR (*<i>p<0</i>.<i>01</i>). <b>(b)</b><i>Hes1</i> gene expression in CD34+ cells isolated from GSI-nonresponder CML patients. <b>(c)</b> Overexpression of P-crkl in CD34+ CML cells treated with GSI. CD34+ cells from five CML patients in chronic phase were cultured in the presence of 10 μM GSI. The change in BCR-ABL activity was assessed by the FACS-based P-crkl assay. <b>(d)</b> P-crkl expression was measured by mean fluorescence intensity (MFI) units in each condition. MFI of P-crkl in GSI-treated CD34+ cells were compared to a no-drug control in each sample, and the percentage of increase in P-crkl was calculated. Data shown here represent the mean of six CML samples (**<i>p<0</i>.<i>01</i>).</p

Cross-talk between Notch and BCR-ABL in the K562 and ALL-SIL cell line model.

<p><b>(a)</b> Assessment of IM efficacy in K562 cells using P-crkl assay. K562 cells were cultured in increasing concentrations of IM (10, 5, 1, 0.5, and 0.1 μM) for 48h. P-crkl expression in cells treated with IM is shown. Data shown is from one experiment representative of three separate experiments (n = 4). <b>(b)</b> Dose-dependent effect of IM on the expression of P-crkl in K562 cells. P-crkl expression of IM treated and untreated K562 cells represented as mean fluorescence intensity (MFI) uing FACS as described in <b>(a)</b> (n = 4). <b>(c)</b> Concentration-dependent effect of IM on P-crkl protein. K562 and Jurkat cells cultured in increasing concentrations of IM (10, 5, 1, 0.5, and 0.1 μM) for 48h. P-crkl protein levels were measured by western blotting. <b>(d)</b> Expression of <i>Hes1</i> in K562 cells, 48h posttreatment. Notch target gene <i>Hes1</i> was assessed after 48h treatment with 10 μM IM (**<i>p<0</i>.<i>01</i>). <b>(e)</b><i>Hes1</i> expression in K562 cells post valproic acid (VPA) treatment. K562 cells were treated with 4mM VPA for 72h and <i>Hes1</i> expression was measured by real-time PCR. Gene expression was normalised to the <i>GAPDH</i> (n = 3). Statistical significance was calculated using student t-test. (** = p <0.01). <b>(f)</b> Effect of VPA on BCR-ABL activity in K562 cells. K562 cells were treated with 4mM VPA for 72h and the activity of BCR-ABL was assessed by FACS analysis of P-crkl expression. (n = 3).</p

Expression of <i>Notch</i> receptors and its target genes in CD34+ cells isolated from BM of normal subjects and CML patients.

<p><b>(a)</b> Conventional PCR products are shown for four NBM (left panel) and four CML samples (right panel). <i>GAPDH</i> was used to assess the quality of cDNA. The lower left panel shows human genomic DNA (HGDNA) as a positive control for each set of oligonucleotides. (<b>b)</b> Real-time PCR analysis of <i>Hes1</i> expression on CD34+ cell subsets from NBM and CML patients. Results showed significant (* = <i>p<0</i>.<i>05</i>, ** = <i>p≤0</i>.<i>01</i>) upregulation of <i>Hes1</i> in all the CD34+ CML primary subset cells compared with NBM. <b>(c)</b> Summary of Notch1 expression profile in different cell lineages in CML and NBM. FACS analysis of Notch1 in different myeloid, lymphoid, and more primitive lineages in CML was done by co-staining mononuclear cells with both extracellular Notch1 (ECN1-EA1) antibody and a lineage-specific cell surface marker. Results shown here are representative of the total CD34+ cells in each sample. The mean of expression refers to the percentage of each cell population in the left column that was positive for EA1. The means of expression were measured from four different CML samples (n = 4).</p

Expression of Notch1 in the K562 cell line.

<p><b>(a)</b> Expression of <i>Notch1</i> in the K562 cell line model at transcriptional level. cDNA was prepared from K562 cells. The CEM cell line was used as a positive control for active Notch signalling. Transcript levels were measured by RT-PCR. RT-PCR products were resolved by agarose gel electrophoresis and visualised by Vistra Green (n = 3). <b>(b)</b> Analysis of Notch1 expression in K562 cells at protein level. Cells were stained with EA1 antibody to detect the extracellular domain of Notch1 (ECN1) and bTAN 20 antibody to detect intracellular domain of Notch1 (ICN1) using FACS. Appropriate isotype controls were used in each staining (n = 4). <b>(c)</b> Inhibition of Notch signalling by γ-seretase inhibitor (GSI) in K562 cells. The cDNA was prepared from cells treated with vehicle control (DMSO) and 10 μM GSI for 24h. Real-time PCR of the Notch target gene <i>Hes1</i> is shown (n = 5). ** = <i>p<0</i>.<i>01</i>. <b>(d)</b> The effect of Notch inhibition on BCR-ABL activity. K562 cells were cultured for 24h in the presence of GSI (10μM) and BCR-ABL activity was assessed by FACS based P-crkl assay (n = 4).</p

<i>Hes1</i> gene expression profile in CD34+ cells isolated from CML patients.

<p>The expression profiles of the Notch target gene <i>Hes1</i> was investigated by real-time PCR. Data is shown from CD34+ cells isolated from six CML patients (pt) in chronic phase and CD34+ control cells from three NBM samples. Relative gene expression was calculated using the DDCt method. ** = p<0.01; significant expression of <i>Hes1</i> in each CML sample was compared with <i>Hes1</i> expression in the NBM samples.</p