Improved outcome following allogeneic stem cell transplantation in chronic myeloid leukemia is associated with higher expression of BMI-1 and immune responses to BMI-1 protein

Patients and healthy controls

All consecutive CML patients who received T-cell depleted SCT from their HLA-identical sibling between September 1993 and May 2006 in the Hematology Branch, National Heart, Lung and Blood Institute, with available pre-SCT biological material were studied. The pre-SCT disease status of either CP or advanced phase (AdP, accelerated and blast phase) was determined using the International Bone Marrow Transplant Registry criteria(19). All patients and donors gave written informed consent before enrolling in myeloablative (n=84) or non-myeloablative (n=2) Institutional Review Board (IRB)-approved transplantation protocols, details of which have been reported previously(20–22). Current leukemia free survival (LFS) was defined as the survival without evidence of leukemia at the time of the most recent assessment(23). Diagnosis and staging of acute and chronic GVHD was graded according to standard criteria(24, 25). Immune responses were studied in cells from HLA-A*0201⁺ patients pre-SCT (n=19) and healthy sibling donors (n=25). Furthermore, 8 patients with sufficient biological material were studied post-SCT, and 8 HLA-A*0201⁺ healthy blood donors were assessed for short-term expansion of leukemia-associated antigen (LAA)-specific CTL.

Sample preparation

Patient cells from leukapheresis products, except in nine patients where bone marrow cells were used, were collected within two months prior to SCT. Donor leukaphereses were collected prior to stem cell mobilization. Post-SCT patient cells were obtained from venous sampling at designated timepoints during clinic follow-up. Mononuclear cells (MNCs) from either leukapheresis product, bone marrow harvest or venous whole blood were isolated using Ficoll-Hypaque density gradient centrifugation (Organon Teknika, Durham, NC) and cryopreserved in RPMI1640 supplemented with 20% fetal calf serum and 10% dimethyl sulfoxide. For gene expression studies, CD34⁺ cells from thawed MNCs were selected by binding to immunomagnetic beads (MiniMACS, Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions as previously reported(20). Total RNA was extracted from CD34⁺ and CD34-negative cells using the RNeasy kit (Qiagen, Valencia, CA) and reverse transcription performed with the Advantage RT-for-PCR kit (Clontech, Mountain View, CA). In HLA-A*0201⁺ patients and donors, MNCs were thawed, washed and resuspended in complete media (CM) (RPMI1640 supplemented with 10% pooled human AB serum (Sigma-Aldrich, St Louis, MO)). High resolution HLA Class I typing was performed by sequence-specific polymerase chain reaction (PCR) using genomic DNA (HLA Laboratory, Department of Transfusion Medicine, NIH, Bethesda, MD). The presence of immunoglobulin-G (IgG) and IgM cytomegalovirus (CMV) antibodies in the samples was analyzed by passive latex agglutination (CMVSCAN kit; BD Becton Dickinson Microbiology System, Cockeysville, MD) according to the manufacturer’s instructions.

Peptide synthesis

Peptides were manufactured by Biosynthesis (Lewisville, TX) to a minimum purity of 95%. The identity of each peptide was confirmed by mass spectrometry. Two peptides each derived from BMI-1 (BMI-1 _74–82; TLQDIVYKL [BMI-T] and BMI-1 _271–279; CLPSPSTPV [BMI-C]) and EZH2 (EZH2 _666–674; YMCSFLFNL [EZH-Y] and EZH2 _734–742; SQADALKYV [EZH-S])(17, 18) were tested. Comparative studies were performed with peptides from two well-characterized LAAs - Wilms’ tumor 1 (WT1 _126–134; RMFPNAPYL) and proteinase 3 (PR1 _169–177; VLQELNVTV)(26). In patients and donors who had CMV antibodies, the CMVpp65 -derived peptide NLVPMVATV was used as positive control.

Peptide-HLA Class I dextramer complexes and immunophenotyping

Freshly thawed MNCs from HLA-A*0201⁺ patients and donors were stained with peptide/HLA Class I dextramer complexes for 30 min according to the manufacturer (Immunodex, Dako, Glostrup, Denmark)’s instructions using allophycocyanin (APC)- conjugated BMI-C/HLA-A*0201 or EZH-Y/HLA-A*0201, phycoerythrin (PE)-conjugated BMI-T/HLA-A*0201 or EZH-S/HLA-A*0201, with positive control dextramer complexes of CMVpp65-derived peptides NLVPMVATV/HLA-A*0201-APC and VLEETSVML/HLA-A*0201-PE and negative controls human immunodeficiency virus (HIV) envelope-derived peptides SLYNTVATL/HLA-A*0201-APC and ILKEPVHGV/HLA-A*0201-PE. Between 1 – 3 × 10⁶ MNCs were stained in 100μL 5% FCS/phosphate buffered saline (PBS) at room temperature. Subsequently, cells were stained with a titrated panel of directly conjugated monoclonal antibodies to CD3, CD4, CD8, CD27, CD45RO (all from BD Biosciences, San Jose, CA) using fluorescein isothiocyanate (FITC), peridinin chlorophyll protein (PerCP), PE-Cy7, APC-H7 and Pacific Blue as fluorochromes. An amine-reactive dead cell stain detected in the ultra-violet channel (BIVID, Molecular Probes, Invitrogen, Carlsbad, CA) was used to gate out non-viable cells. A minimum of 0.5 × 10⁶ cells were acquired using the LSRII flowcytometer on the FACSDiva software (BD Biosciences, San Jose, CA). All individuals studied were serologically negative for HIV antibodies, and a 2-fold or greater response in comparison to the negative control (HIV dextramers) was defined as a positive immune response.

Detection of functional antigen-specific CD8⁺ T cells

MNCs from HLA-A*0201⁺ patients and donors were rested overnight at 37°C in CM after thawing. Costimulatory antibodies anti-CD28 and 49d, (1μg/mL, Fastimmune, BD Biosciences), monensin (Golgistop, BD Biosciences), brefeldin (10μg/mL, Sigma-Aldrich, St Louis, MO) and CD107a-FITC (BD Biosciences) were added. The cells were loaded with 0.1 and 10.0μM of test peptides BMI-T, BMI-C, EZH-Y and EZH-S. Negative control consisted of unstimulated cells, and two positive controls, Staphylococcus enterotoxin B (SEB) (1μg/mL, Toxin Technology, Sarasota, FL) and the CMVpp65 -derived peptide NLVPMVATV, were included for each assay. Cells were incubated for 5 hours at 37°C, and then washed and stained with CD3-FITC, CD8-PerCP and BIVID, fixed and permeabilised and subsequently stained for intra-cellular cytokines with monoclonal antibodies against interferon (IFN)-γ, tumor- necrosis factor (TNF)-α, interleukin (IL)-2, IL-4, IL-10 conjugated with APC or PE (all from BD Biosciences).

Real-time quantitative reverse-transcription polymerase chain reaction (RQ-PCR)

TaqMan^™ Assays-on-demand^™ probe-and-primer reagents (Applied Biosystems, Foster City, CA) for BMI-1, Hs00180411_m1, EZH2, Hs01016789_m1, and CDKN2A, Hs00233365_m1 were used according to the manufacturer’s instructions. WT1 and BCR-ABL expression were measured as previously described for assessment of minimal residual disease post-SCT(26). ABL expression was used as the endogenous cDNA quantity control as previously described(27). All reactions were performed in triplicate on 10 μL volume using standard conditions with 40 cycles of amplification using the ABI PRISM 7900 sequence detection system.

Short-term expansion of BMI-1 and EZH2-specific CD8⁺ T cells in vitro and IFN-γELISPOT assay

The frequency of antigen-specific CTLs in freshly thawed MNCs and in MNCs expanded for seven days in-vitro was assessed using a modification of the ELISPOT assay as comprehensively described previously (28, 29). Cells were plated in 96-well round bottom microtiter plates (100,000 MNCs or 25,000 CD8⁺ T-cells/well) and loaded with test peptides (0.1, 1.0 and 10μM), or medium alone as negative control. Test peptides BMI-T, BMI-C, EZH-Y, EZH-S, PR1, WT1 and control peptides gp100_{209–217(210M)} and CAP1-6D (irrelevant HLA-A*0201-binding peptide- negative controls), and CMVpp65 -derived peptide NLVPMVATV (positive control for CMV-responsive patients/donors) were added directly to each well. Cells were harvested on day 7 for detection of IFN-γ by ELISPOT analysis, and weekly subsequently to assess longer-term expansion following weekly restimulation with the relevant peptide. A positive ELISPOT response was defined as fulfilling 3 criteria: (a) at least 20 spots per 100,000 MNCs or 25,000 CD8⁺ T-cells, (b) at least 2-fold the number of spots in background negative control wells, and (c) significant by t-test (p<0.05), in the presence of viable negative and positive controls.

Statistical methods

Patient groups were compared using the χ² test for categorical data or Mann-Whitney U test for continuous data. Survival curves were calculated using the Kaplan-Meier method and patient groups were compared using the log-rank test. Patients were divided into groups delineated by the median cutoffs of RQ-PCR expression values for EZH2, BMI-1 and CDKN2A. RQ-PCR results from CD34⁺ and CD34-negative populations in patient samples were compared using Spearman’s ρ. P-values were from two-sided tests with values <0.05 considered significant. Data analysis was performed using SPSS 14 for Windows software (SPSS, Chicago, IL) and GraphPad Prism 4.0 (GraphPad Software, San Diego, CA).

Transplant outcome

The characteristics of the patients included in this study, conditioning regimens, GVHD prophylaxis and parameters of transplant outcome have been reported in detail recently (20). In brief, this cohort of CML patients received T-cell depleted grafts (containing 0.2 – 2 × 10⁵/kg T cells) from their HLA-identical sibling donors. The median follow-up time for this cohort after SCT was 62.6 months (range 0.5 – 159.2 months). Patients were transplanted at a median of 12.8 months from diagnosis (range, 1.6–147 months). According to IRB-approved protocols(30), in the absence of GVHD or unless molecular remission was documented, 1 – 5 × 10⁷/kg T-cells were added back on day 30–60 (n=67) or day 100 (n=14). Only 12 patients had T-cells added at both timepoints. On multivariate analysis, the factors associated with improved leukemia free survival (LFS) and overall survival (OS) in this cohort of CML patients were disease phase, CD34⁺ cell dose of the graft and lymphocyte count at Day 30 post-SCT(21). In particular, there was no difference in LFS, OS or relapse rate post-SCT between older and newer transplant protocols, despite modest improvements in transplant-related mortality with the era of transplantation. In this cohort, we had previously reported that a higher expression of PR3 or ELA2 in pre-SCT leukemic CD34⁺ cells of AdP-CML patients was associated with a better outcome post-transplantation. However, PR3 and ELA2 expression were not prognostic in CP-CML patients(20).

BMI-1, EZH2 and CDKN2A expression as predictors of SCT outcome in CML patients

Biological material for isolation of CD34⁺ cells was available in 82 of 86 CML patients pre-SCT, of which 51 were in CP and 31 AdP-CML (6 blast phase, 25 accelerated phase) at the time of SCT. The expression of EZH2, BMI-1, and its target for repression, CDKN2A (which encodes for p16INK4A) was highly correlated in CD34⁺ and CD34-negative cells (p<0.005, Supplementary Figure 1). In a univariate analysis of all CML patients in this cohort, a lower CDKN2A expression in CD34-negative cells was associated with greater incidence of chronic GVHD (cGVHD) (25/32 patients below median value with cGVHD [limited = 20, extensive = 5] vs. 16/34 above median with cGVHD [limited = 13; extensive = 3], p= 0.009). However, there was no corresponding association of BMI-1 or EZH2 expression with either acute GVHD or cGVHD in the entire cohort. In a logistic regression model, death-related-to-disease (DRD) was lower in patients with low CDKN2A in either CD34⁺ or CD34-negative populations (p=0.0001 for both). Correspondingly, a lower CDKN2A in both the CD34⁺ population (p= 0.013 and p=0.005, respectively) and CD34-negative population (p=0.03 and p=0.048, respectively) was associated with superior LFS and OS in the entire cohort of CML patients. In subgroup analysis of CP-CML patients, a higher expression of BMI-1, and accordingly, lower CDKN2A was associated with improved LFS (p=0.019 and p=0.011, respectively) and OS (p=0.005 and p=0.006, respectively). Transplant-related mortality (TRM) was also lower in CP-CML patients with high BMI-1 (p=0.0058) (Figure 1). There were no significant associations of EZH2 expression with disease outcome.

Ex vivo detection of CD8⁺ T-cell responses directed against HLA-A*0201-restricted BMI-1 and EZH2 epitopes

All HLA-A*0201⁺ patients and donors with available biological material were studied for immune responses to BMI-1- and EZH2-derived peptides. BMI-1-specific CTL responses were detected in 8 of 19 (42%) HLA-A*0201⁺ CML patients before SCT, and 5 of 25 (20%) healthy sibling donors. Of the two BMI-1 derived peptides, nine CTL responses were to BMI-T and eight to BMI-C. Four individuals had CTL responses to both BMI-1 peptides. On the other hand, native EZH2-specific CTL responses were detected in 6 of 19 (32%) CML patients and 3 of 25 (12%) healthy donors. There were eight EZH-Y-CTL responses and four EZH-S-CTL responses, with three individuals having CTL responses to both EZH2 peptides (Table 1). Polyfunctionality of CTLs was studied in 14 patients and 4 donors where sufficient biological material was available. The majority of detected PcG-specific CTLs were interferon-γ producing only upon exposure to the relevant peptide. However, in the minority, TNF-α and CD107a responses were also detected (Figure 2). IL-2, IL-4 or IL-10 responses were not detected in samples with concordant interferon-γ, TNF-α or CD107a responses to specific peptides.

Immune responses to PcG-specific peptides were studied in eight patients with available biological material from the first 120 days post-SCT. Four patients received SCT from donors with PcG-CTL responses (UPN 25, UPN 262, UPN 273 and UPN 365), three had donors without PcG-CTL responses (UPN 469, UPN 483 and UPN 283) and one patient (UPN 199) did not have available donor material for study. PcG-specific CTLs were detected post-SCT in 4/4 patients whose donors had detectable PcG-CTLs, and in none of the patients whose donors did not have PcG-CTLs. Donor myeloid and T cell chimerism was achieved in the time period studied, suggesting that post- SCT PcG-CTL responses were donor-derived. These PcG-CTLs were of memory phenotype and could be cultured short-term in ELISPOT assays (Figure 3). However, in three patients (UPN19, UPN 262, UPN273) with available biological material, PcG-CTL responses were no longer detectable in the later period post-SCT (at 13, 12 and 10 months post-SCT respectively) in the absence, or reduction of residual leukemia by PCR in these patients. Concordance of the ELISPOT assay with the detection of PcG-specific CTLs using intracytoplasmic cytokine or dextramer staining was found in 75% of individual responses (9 patients, 7 healthy donors) (Supplementary Table 1). In 50% (5/10) of individuals where direct ex-vivo immune responses to PcG-peptides were detected, PcG-specific CTLs could also be expanded in short-term 7-day cultures. In two of three patients (UPN 273, UPN 365 and UPN 319) with adequate biological material, expansion of PcG-specific CTLs were detected beyond two weeks culture. CML patients whose donors had immune responses to BMI-1 peptides had improved leukemia-free survival post-SCT compared with those whose donors had no BMI-1 immune response (80% vs 60%); however, as this was a small cohort, this difference was not statistically significant.

CTL responses to two concentrations of peptide were studied to assess the avidity of responses. A high avidity CTL response was defined as that provoked by a low concentration of peptide (0.1μM), whereas a low avidity response required the higher concentration of 10 μM peptide as previously reported(28, 31). More high avidity responses were found upon stimulation with BMI-1 peptides compared to EZH2-peptides. BMI-T peptides generated the greatest frequency of high avidity CTL responses (Figure 4). There was no appreciable association of PcG expression on CML cells, or CML disease phase with the frequency of higher or lower avidity CTL responses. As PcG protein expression is important in the maintenance of self-renewal in leukemia stem cells, a relationship between the expression of BMI-1 and EZH2 in CD34⁺ CML cells and PcG-specific CTL responses was sought. There was a significantly higher expression of BMI-1 in CD34⁺ cells of patients with detectable BMI-1-CTL responses (p=0.02). On the other hand, there was no significant association between EZH2 expression and EZH2-CTL responses (Supplementary Figure 2).

Comparison of immune responses to BMI-1 and EZH2 with established leukemia-associated antigens

In an additional 8 healthy HLA-A*0201⁺ blood donors, the frequency of immune responses to BMI-1 and EZH2 was compared to that obtained with well-characterized LAAs WT1 and PR1 peptides using ELISPOT assay for short-term peptide-specific CTL expansion weekly for up to six weeks. Under the same conditions, the short-term expansion of PcG-specific CTLs was not inferior to that obtained by cells stimulated with WT1 or PR1. Five of 8 (63%) healthy blood donors mounted immune responses to BMI-1 peptides, and 3 to EZH2 peptides. WT1- and PR1-specific CTL expansions were found in 2 and 3 donors respectively (Figure 5).