Immunology

Hematopoietic stem cells and progenitors of chronic myeloid leukemia express leukemia-associated antigens: implications for the graft-versus-leukemia effect and peptide vaccine-based immunotherapy

Abstract

The cure of chronic myeloid leukemia (CML) patients following allogeneic stem cell transplantation (SCT) is attributed to graft-versus-leukemia (GVL) effects targeting alloantigens and/or leukemia-associated antigens (LAA) on leukemia cells. To assess the potential of LAA-peptide vaccines in eliminating leukemia in CML patients, we measured WT1, PR3, ELA2 and PRAME expression in CD34+ progenitor subpopulations in CML patients and compared them with minor histocompatibility antigens (mHAgs) HA1 and SMCY. All CD34+ subpopulations expressed similar levels of mHAgs irrespective of disease phase, suggesting that in the SCT setting, mHAgs are the best target for GVL. Furthermore, WT1 was consistently overexpressed in advanced phase (AdP) CML in all CD34+ subpopulations, and mature progenitors of chronic phase (CP) CML compared to healthy individuals. PRAME overexpression was limited to more mature AdP-CML progenitors only. Conversely, only CP-CML progenitors had PR3 overexpression, suggesting that PR1-peptide vaccines are only appropriate in CP-CML. Surface expression of WT1 protein in the most primitive hematopoietic stem cells in AdP-CML suggest that they could be targets for WT1 peptide-based vaccines, which in combination with PRAME, could additionally improve targeting differentiated progeny, and benefit patients responding suboptimally to tyrosine kinase inhibitors, or enhance GVL effects in SCT patients.

Introduction

The graft-versus-leukemia (GVL) effect mediated by donor lymphocytes may be the principal mechanism of cure after allogeneic stem cell transplantation (SCT) for chronic myeloid leukemia (CML).1, 2 Furthermore, T-cell responses to leukemia-associated antigens (LAA) are detectable after SCT and have been induced in non-transplanted patients using leukemia vaccines.3 Recently, because of their low toxicity and impressive efficacy, tyrosine kinase inhibitors such as imatinib are replacing SCT as first-line therapy for CML. However, although over 85% of imatinib-treated patients with chronic phase (CP) CML achieve a complete cytogenetic remission, a proportion of patients have persisting molecular disease as assessed by RQ-PCR (real-time quantitative polymerase chain reaction) for BCR-ABL transcripts.4, 5 Ph-positive CD34+ progenitor cells have been identified in such patients in complete cytogenetic remission.6

We hypothesized that the GVL effect is mainly orchestrated by T cells targeting alloantigens and LAA, and that similar mechanisms could be exploited to eliminate the leukemic progenitors that evade eradication by tyrosine kinase inhibitors. We, therefore, studied primitive CD34+ leukemic progenitors for expression of minor histocompatibility antigens (mHAgs) as well as LAA to ascertain if LAA-peptide vaccines could have the potential to target these residual cells. Furthermore, as the expression of CD7 on CML CD34+ cells has been associated with a poor outcome and clonal evolution,7 we also investigated the effect of this surface marker on the expression of LAA in primitive progenitors.

We chose to study the WT1, PRAME, PR3 and ELA2 genes, which code for WT1 (Wilms' tumor-1), PRAME (preferentially expressed antigen of melanoma), proteinase 3 and neutrophil elastase proteins, respectively. Immunogenic peptides have been isolated from each of these proteins,8, 9, 10, 11, 12, 13, 14 and are currently being investigated as peptide vaccines in clinical trials.15, 16, 17 We found that the gene expression pattern of these LAAs is associated with CML disease phase, and differ depending on the maturation of the progenitors, in contrast to mHAgs which are ubiquitously expressed. Of the studied LAAs, WT1 and PRAME expression were consistently upregulated in advanced phase (AdP) CML, whereas PR3 and ELA2 were only overexpressed in CP. Our results should assist in optimizing patient-specific immunotherapy as an adjunct to tyrosine kinase inhibitors or SCT, and suggest the need for combined peptide vaccine strategies to maximize therapeutic efficacy.

Materials and methods

Patients and samples

Nucleated cells from 8 healthy individuals who were donors for SCT (granulocyte colony-stimulating factor mobilized peripheral blood) and 21 CML patients (8 CP, 13 AdP (7 accelerated phase and 6 blast crisis)) were collected by leukapheresis. The majority of patients were referred for SCT and non-responders to prior therapy (Table 1). Informed consent for the use of these cells for research was obtained according to the requirements of the Institutional Review Board of the National Heart, Lung and Blood Institute. Mononuclear cells were isolated by density gradient centrifugation (Organon Teknika, Durham, NC, USA) and cryopreserved in RPMI1640 supplemented with 20% fetal calf serum and 10% dimethyl sulfoxide. After thawing, CD34+ cells were selected by binding to immunomagnetic beads (MiniMACS, Miltenyi Biotech, Auburn, CA, USA) according to the manufacturer's instructions.

Table 1 Patient characteristics

Flow cytometry

Fluorescence-activated cell sorting (FACS) was performed with the FACS Vantage sorter utilizing the FACSDiva software (BD Biosciences, San Jose, CA, USA). Monoclonal antibodies for cell surface markers were fluorescein isothiocyanate (FITC)-conjugated CD38 (Beckman Coulter, Fullerton, CA, USA); phycoerythrin (PE)-conjugated lineage markers;7 PE-Cy5-conjugated CD7 and IL3Rα, allophycocyanin (APC)-conjugated CD90, APC-Cy7-conjugated CD34 and CD33 (all from BD Biosciences), ECD (PE-Texas Red)-conjugated CD45RA and CD34 (Beckman-Coulter) and APC-conjugated CD7 (Caltag, Burlingame, CA, USA).

CD38+ and CD38− cells were gated off the total CD34+ population. CD34+CD38-Lin−CD90+ hematopoietic stem cells (HSC),18 CD34+CD38− Lin− CD7+ and CD34+CD38+ Lin−CD7+ primitive progenitor populations were collected. In addition, CD34+CD38+Lin− cells were divided into common myeloid progenitor (CMP) CD34+CD38+Lin− IL3Rα+CD45RA-, granulocyte-macrophage progenitor (GMP) CD34+CD38+Lin− IL3Rα+CD45RA+, CD33+ and CD33− populations. CD7+ and CD7− subpopulations from these subsets of CD34+CD38+Lin− cells were collected (Supplementary Figure 1).

Analysis of WT1 protein was performed on the LSRII flowcytometer (BD Biosciences) staining 1 × 106 CD34+ cells using rabbit polyclonal WT1 (C19): sc-192 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and FITC-conjugated goat-anti-rabbit (GAR) secondary antibody (Sigma-Aldrich, St Louis, MO, USA). Monoclonal antibodies for cell surface markers were PE-conjugated lineage markers, PE-Cy7- conjugated CD38, PE-Cy5-conjugated IL3Rα, APC-conjugated CD90 and CD45RA, and APC-Cy7-conjugated CD34 (BD Biosciences). WT1 surface protein expression was calculated as a relative fluorescence intensity from the ratio of the mean fluorescence intensity (MFI) of gated WT1-stained cells against negative control (GAR-FITC) using the formula ((MFI specific staining–MFI control)/MFI control).

The proportion of BCR-ABL-positive cells in CML samples was investigated by FISH (fluorescence in situ hybridization) using the BCR-ABL LSI dual-color dual-fusion translocation probes (Vysis, Downers Grove, IL, USA).

Real-time quantitative polymerase chain reaction

Total RNA was extracted from sorted CD34+ subpopulations using the RNeasy kit (Qiagen, Valencia, CA, USA), and cDNA synthesized using the Advantage RT-for-PCR kit (Clontech, Mountain View, CA, USA). TaqMan Assays-on-demand probe-and-primer reagents (Applied Biosystems, Foster City, CA, USA) for HA1, Hs00299628_m1, SMCY, Hs00190491_m1, PRAME, Hs00196132_m1, ELA2, Hs00357734_m1 and PR3, Hs00160521_m1 were utilized according to the manufacturer's instructions. Primers and probes for BCR-ABL,19 WT120 and ABL7 as the endogenous cDNA quantity control for all samples have been described previously. All RQ-PCR reactions were performed in triplicate on 10 μl volume using standard conditions on the ABI PRISM 7900 sequence detection system (Applied Biosystems).

Statistical methods

The correlation of gene expression in CD34+ subpopulations was calculated with Spearman's ρ. The Mann–Whitney U-test was used to compare two discrete groups. All quoted P-values are from two-sided tests with values <0.05 considered significant. Data analysis was performed using GraphPad Prism 4.0 (GraphPad Software, San Diego, CA, USA).

Results

CD34+ subpopulations

A median of 155 × 106 mononuclear cells (range 27.5–2000 × 106) were utilized for selection of CD34+ cells from each patient per session, with a median of 10 × 106 CD34+ cells (range 1–33 × 106) sorted. All samples with less than 70 × 106 mononuclear cells available had greater than 10% CD34+ cells, and were mainly from AdP-CML patients. Using the same gating strategy, in general, similar cell numbers were obtained from sorting individual CD34+ subpopulations in healthy donors and CML patients irrespective of disease phase. However, significantly greater numbers of HSCs, and mature progenitors were obtained from healthy individuals than CML patients, with the exception of the GMP CD7+ subpopulation in AdP-CML, where there was an expansion of this population compared to normal (Supplementary Figure 1). As the subpopulations available for study were very small, usually comprising less than 5% of the total CD34+ cell pool, gene expression between healthy and CML groups were compared on a background of acceptably similar numbers of cells from these groups. In particular, as aberrantly increased gene expression for LAAs in CML has been described in comparison to healthy individuals, any ‘undetectable’ levels in healthy individuals in small CD34+ subpopulations was not due to the disparity of cell number between CML and healthy individuals. Purity of sorted cell populations was >96% in larger sorted populations where purity could be assessed.

BCR-ABL transcripts were detected in all sorted subpopulations of CD34+ progenitors from CML patients (data not shown). Furthermore, FISH for six representative samples (four CP and two AdP) with adequate biological material revealed greater than 80% BCR-ABL positivity in all primitive CML CD34+ progenitor subpopulations.

Gene expression of minor histocompatibility antigens

Both mHAgs studied, HA1 and SMCY (male patients only), were ubiquitously expressed in all CD34+ subpopulations from the most primitive HSC to the most mature progenitors in CML patients. Individual patients appeared to have a relatively narrow range of expression of these mHAgs irrespective of CD34+ progenitor maturation stage (Figure 1). There was no significant difference in mHAg expression between patients with CP-CML or AdP-CML in any CD34+ subpopulation.

Figure 1
figure1

Expression of genes encoding minor histocompatibility antigens in CD34+ subpopulations of CML patients. Pattern of HA1 (upper panel) and SMCY (male patients only, lower panel) expression in CD34+ subpopulations of individual CML patients. AP, accelerated phase; AdP, advanced phase; BC, blast crisis; CP, chronic phase.

Gene expression of leukemia-associated antigens

The expression of PR3 and ELA2 in all CD34+ subpopulations studied was highly correlated (Spearman's correlation coefficient 0.93, P<0.0001). Expression of both PR3 and ELA2 was reduced in AdP-CML compared to CP-CML generally in CD34+ subpopulations, but this difference was more marked in the more mature progenitors (Figure 2). PR3 was significantly overexpressed in HSCs of CP-CML in comparison to healthy individuals. Although there was a trend for PR3 overexpression in most mature CD34+ subpopulations of CP-CML in comparison to healthy individuals, this did not reach statistical significance in some, most probably due to the small numbers of samples in the respective groups compared. Conversely, in AdP-CML, PR3 expression was indistinguishable from that of healthy individuals in all studied CD34+ subpopulations.

Figure 2
figure2figure2

Expression of genes encoding leukemia-associated antigens in CD34+ subpopulations of healthy individuals and CML patients. CD34+ progenitor maturity increases from left to right. Values of genes represent the RQ-PCR expression as a ratio of the gene of interest to the ABL control gene. Clear circles=healthy individuals, n=8; black circles=chronic phase, n=8; red diamonds=advanced phase (accelerated phase and blast crisis), n=13. Square brackets=Mann–Whitney U-test. *P<0.05, **P<0.01, ***P<0.001. WT1, PRAME and PR3 expression in (a) hematopoietic stem cells (HSC) and primitive progenitors; (b) common myeloid progenitors (CMP) and granulocyte-macrophage progenitors (GMP); (c) CD33+ and CD33- subpopulations of CD34+CD38+Lin− cells. (d) Upper panel: WT1 protein surface expression on hematopoietic stem cells (HSC) in a representative CML patient (UPN 452). HSCs are gated off the CD34+CD38-Lin− population. Lower panel: correlation between WT1 gene (as a ratio to the ABL control gene) and WT1 surface protein expression (as the ratio of the mean fluorescence intensity of gated WT1-stained cells against negative control as detailed in Materials and methods) in two representative patients (UPN 314, accelerated phase, black symbols; UPN 57, blast crisis, red symbols).

In contrast to PR3 and ELA2, WT1 was overexpressed in AdP-CML as compared to healthy individuals in all CD34+ subpopulations from the most primitive HSC to the most mature progenitors studied. No WT1 differential expression was found between CP-CML and healthy donors of HSCs, but in more mature progenitors, WT1 was overexpressed in CP-CML (Figures 2b and c). Furthermore, surface expression of WT1 protein was demonstrated on HSC, CMP and GMP from AdP-CML and correlated with WT1 gene expression (Figure 2d).

In comparison to healthy individuals, PRAME was significantly overexpressed mainly in AdP-CML, and in more mature progenitors (Figure 2). In contrast to the expression of PR3, ELA2 or WT1, there was no generalized consistent pattern of PRAME expression—some patients only expressed PRAME in very early progenitors, others only in more mature progenitors, and a minority expressed PRAME in all CD34+ subpopulations studied.

CD7 expression in either primitive CD34+CD38-Lin− progenitors or in more mature CMP and GMP populations did not adversely influence the expression levels of any of the LAAs studied.

Discussion

The immunological eradication of CML by the GVL effect following SCT and potentially by vaccines requires the expression of target antigens on primitive leukemic progenitors for effective T-cell control of the leukemia. Alloreactive T-cell responses against mHAgs on leukemia cells are considered the main effectors of GVL.2 However, WT1 and proteinase 3 expression by CML hematopoietic progenitors have been implicated in GVL effects, and peptide vaccines derived from WT1 and proteinase 3 have also been used with some success, either alone or in combination with SCT, to control CML.15, 16, 21 Recently, peptides derived from the PRAME protein, which is aberrantly overexpressed in acute leukemias and AdP-CML,22 have also been shown to be immunogenic.13

The focus of our vaccine research involves the development of vaccines applicable either alone or in conjunction with SCT, as in current clinical practice, immunotherapeutic approaches using vaccines would be an adjunct in patients who have minimal residual disease following primary treatment with tyrosine kinase inhibitors or SCT. We studied mainly patients who were referred to our institution for SCT, who were non-responders to prior therapy, including tyrosine kinase inhibitors. We determined which progenitors in the leukemic hierarchy express LAAs and mHAgs. In view of the very small populations of early progenitors within the bulk CD34+ pool available physiologically, formal cytotoxicity assays using these cells were not feasible. However, abundant data exist demonstrating the efficacy of mHAg- or LAA-specific cytotoxic T lymphocytes in lysing bulk populations of CD34+ leukemia cells.9, 23, 24 Our purpose here was to delineate the spectrum of expression of mHAgs and the available LAAs which have been sufficiently well-characterized, within the CD34+ primitive stem cell and committed progenitor pool to help optimize the strategy for further clinical studies using LAAs as peptide vaccines for immunotherapeutic eradication of minimal residual disease in CML. We, therefore, studied the expression of LAAs encoded by WT1, PRAME as well as ELA2 and PR3 (which encode elastase and proteinase 3 proteins respectively, both of which contain the PR1 nonapeptide sequence (VLQELNVTV) capable of eliciting cytotoxic T lymphocyte responses)12, 25 in defined CD34+ subpopulations. We found that mHAgs are ubiquitously expressed in all CD34+ subpopulations, irrespective of CML disease phase. This suggests that in the SCT setting, mHAgs are likely the best target for total leukemia eradication, as their expression is undiminished in the most primitive stem cells (HSCs). It is likely that the greater spectrum of mHAgs recognized to the level of the most primitive leukemic stem or progenitor cell accounts for improved elimination of leukemia following SCT, as seen in reports of reduced relapse rates in male recipients of female stem cell grafts, where additional Y-chromosome associated mHAgs are recognized.26, 27, 28 In the well-characterized mHAgs studied, HA1 and SMCY, the degree of mHAg expression tended to be patient-specific. Some patients have greater than one-log higher expression of mHAgs than others in CD34+ subpopulations, which is independent of CML disease phase. We speculate that patients who have a physiologically lower mHAg expression in CD34+ subpopulations may be at greater risk of relapse, but this requires greater numbers of samples to clarify.

In contrast to the expression of mHAgs, in general, the expression of the LAAs studied is heterogeneous and varies depending on CML disease phase and CD34+ progenitor maturity. The expression of ELA2 and PR3 is highly correlated in all CD34+ subpopulations, as has been reported for bulk CD34+ populations.29 The expression of these genes in AdP-CML is not significantly different from that of healthy individuals. Aberrant overexpression of ELA2 and PR3 in CD34+ subpopulations is chiefly found in CP-CML, suggesting that PR1 peptide vaccines would only be appropriate in CP-CML. Furthermore, as PR3 is overexpressed in HSCs of CP-CML compared to healthy individuals, PR1 vaccination may be useful for minimal disease eradication of CP-CML patients in complete cytogenetic remission following treatment with tyrosine kinase inhibitors. Interestingly, we recently found that higher expression of PR3 and ELA2 in total CD34+ progenitors confers a better outcome post-SCT for AdP-CML and may improve PR1 peptide-driven GVL effects.29 GMPs that are expanded in CML18 may aberrantly express elastase and proteinase 3 and present a significant target for GVL effects.

The expression of both WT1 and PRAME is higher in AdP-CML than CP-CML, as has been reported previously.22 In particular, WT1 is significantly overexpressed in all CD34+ subpopulations in AdP-CML as compared to healthy individuals. This range of overexpression encompassing the most primitive HSC to the most mature progenitor is the closest to the ubiquitous expression of mHAgs. Conversely, in the same sample population, PRAME overexpression was mainly found in more mature progenitors, and most prominent in AdP-CML as well. These findings support the use of WT1 and PRAME peptide vaccines in AdP-CML whether in the post-SCT setting to enhance GVL without exacerbating graft-versus-host disease, or in patients not eligible for SCT as an adjunct to therapy such as tyrosine kinase inhibitors. Combining peptide vaccines for increased efficacy may be a valuable therapeutic strategy. We recently reported the safety of combining WT1 and PR1 vaccines in myeloid malignancies.17 Such combination vaccine strategies should also appropriately include PRAME peptide.

The overexpression of CD7 on total CD34+ progenitors has been shown in several studies to be associated with adverse prognosis in CML.7, 30, 31 We found that CD7 expression did not affect the expression of mHAgs, nor did it adversely affect expression of any of the LAAs studied. Thus, the previously reported poor prognostic impact of aberrant CD7 expression on CD34+ CML cells may not be relevant in the immunotherapeutic setting, such as in the context of SCT, as has been shown in a recent retrospective study.32

Our results highlight significant differences in the representation of LAAs in the CD34+ progenitor cell hierarchy of CML and is summarized in Figure 3. Our observations suggest that leukemia eradication could more likely be achieved by T cells directed against a combination of immunogenic targets, which are expressed by the most primitive HSCs as well as more mature committed progenitors. Previous studies have demonstrated that both WT1-specific T cells9 and PR1-specific T cells33 can lyse a proportion of total CD34+ cells from CML patients, with concomitant sparing of total CD34+ cells from healthy individuals. However, primitive HSCs, which are only a very small proportion of the CD34+ progenitor cell pool, are an essential target as these cells not only contribute to minimal residual disease, but they also have the capability to maintain and propagate leukemia. Our findings also suggest that not all LAAs are similarly expressed between CP-CML and AdP-CML. Thus, peptide vaccines should be optimized according to disease phase for maximal efficacy, and combination peptide vaccines with effects on both early and late progenitors, might work synergistically to control and eradicate CML as an adjunct to tyrosine kinase inhibitor treatment or SCT, and these studies are now being planned.

Figure 3
figure3

Spectrum of graft-versus-leukemia target antigen expression according to disease phase and CD34+ progenitor maturation in chronic myeloid leukemia (CML). Maturation progression of CD34+ leukemic progenitors from hematopoietic stem cells (HSC) to common myeloid progenitors (CMP) to granulocyte-macrophage progenitors (GMP) in chronic phase (CP) and advanced phase (AdP) CML and expression of target antigens depicted with red triangles (minor histocompatibility antigens (mHAg)), green triangles (proteinase 3 (PR3)), blue triangles (WT1) and yellow triangles (PRAME).

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Acknowledgements

We thank Ms Loretta Pfannes for technical advice on the assessment of WT1 surface protein by flowcytometry. This research was supported by the Intramural Research Program of the National Heart, Lung and Blood Institute of the NIH.

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Correspondence to A S M Yong.

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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Yong, A., Keyvanfar, K., Eniafe, R. et al. Hematopoietic stem cells and progenitors of chronic myeloid leukemia express leukemia-associated antigens: implications for the graft-versus-leukemia effect and peptide vaccine-based immunotherapy. Leukemia 22, 1721–1727 (2008). https://doi.org/10.1038/leu.2008.161

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Keywords

  • chronic myeloid leukemia
  • CD34+ progenitor cells
  • leukemia-associated antigens
  • graft-versus-leukemia effect

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