Introduction
Chronic myeloid leukemia (CML) is a malignant disease affecting a pluripotent hemapoietic precursor cell. CML is characterized by the Philadelphia chromosome, containing the t(9;22) translocation, resulting in the chimeric bcr-abl gene that encodes the P210 fusion protein. 1, 2 Depending on the exons involved in the breakpoint on chromosomes 9 and 22, variants of this fusion protein may occur of which b3a2 and b2a2 are the most frequent. 3, 4 Because these fusion proteins contain peptide sequencies that are novel to the immune system, they might generate a tumour-specific cytotoxic T cell response when presented by the HLA molecule of the patient. Based on binding experiments of P210-derived peptides in HLA class I and the generation of T cell responses against these CML-specific peptides in some HLA class I molecules [5] [6] [7] [8] [9] [10] we previously performed an association analysis and found a negative association between the class I molecules HLA-A3 and HLA-B8 and CML.
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More recently, T cell responses generated against P210-derived synthetic peptides in the HLA class II alleles HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4 and HLA-DR11 have also been reported. [12] [13] [14] [15] If the P210 protein is processed in the leukemic cell allowing presentation of parts of the protein in HLA class II molecules on the cell membrane, a T cell response against this fusion protein may occur in individuals expressing HLA class II molecules capable of binding these peptides.
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To assess a possibly protective effect of expression of class II molecules capable of binding bcr-abl fusion peptides for the development of CML, we performed a large multi-center study in which we compared the frequencies of HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4 and HLA-DR11 between patients with CML and healthy individuals.
Patients and methods

Patients and controls
HLA frequencies of patients with CML were obtained from two main sources: data of patients who underwent bone marrow transplantation were obtained from the database of the Chronic Leukemia Working Party of the EBMT and data of untransplanted patients from two centers also contributing to EBMT. Data from patients were restricted to countries which had contributed information for at least 15 patients. The HLA types of 1462 patients with CML were obtained. All HLA data derived from the EBMT were reviewed and confirmed by one of the authors (DN).
Normal individuals
HLA typing was available for 500 596 normal healthy individuals who had been tissue typed to assess the suitability as unrelated donors for allogeneic stem cell transplantation as present in the Bone Marrow Donors Worldwide Registries. [16] [17] [18] Patients and normal individuals were matched for their country of origin and ethnic origin.
Leukemia

Methods
Retrospective analysis was performed comparing frequencies of HLA class II molecules reported to bind P210-derived peptides and/or to which P210-specific CD4 T cell reponses have been generated between patients and controls: HLA-DR1, -DR2, -DR3, -DR4 or -DR11. [12] [13] [14] [15] The frequencies of these HLA molecules in patients and in healthy individuals were compared. The frequencies of other HLA class II molecules, without binding capacity for P210-derived peptides, were not compared because this would increase the number of comparisons and thereby the possibility of finding a difference just by chance. Since HLA-DR3 is known to have a linkage disequilibrium with HLA-B8 which was previously reported to be associated with a decreased incidence of CML we analyzed both the frequencies of individuals coexpressing HLA-DR3 and -B8 and the frequencies of HLA-DR3-positive/HLA-B8-negative individuals. To examine a possible synergistic effect of class I and class II protective types we also analyzed the combinations of HLA-A3 or HLA-B8 and HLA-DR4. Finally, we looked for a potential role for homozygosity for those alleles with a found negative association with CML in the above described analysis.
Statistical analysis
Differences, evaluated through odds ratios (OR) with 95% confidence intervals, were assessed by Woolf-Haldane analysis. 19, 20 The ORs of the different countries were combined using as study weights the inverse variance per country.
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Results
This multicenter study comprised 1462 patients with CML from eight different countries and of 500 596 healthy controls from the same countries (Table 1) .
Results
Two HLA-DR molecules gave a significantly lower OR for patients with CML: an OR of 0.86 (95% CI 0.75-0.98; P Ͻ 0.05) for HLA-DR3 and an OR of 0.80 (95% CI 0.71-0.89; P Ͻ 0.05) for HLA-DR4, resulting in protective effects of 14% and 20% for individuals expressing HLA-DR3 and HLA-DR4 (Figure 1) . The other HLA-DR molecules did not give a significantly lower risk on the acquirement of CML ( Table 2 ).
Results
The effect of the linkage disequilibrium of HLA-DR3 with HLA-B8 was analyzed ( Table 3) . Coexpression of HLA-DR3 and HLA-B8 gave a protective effect of 16% (OR 0.84; 95% CI 0.72-0.98) against the development of CML, whereas HLA- DR3-positive/HLA-B8-negative individuals did not have a diminished risk (OR 1.02; 95% CI 0.84-1.24) illustrating that the diminished incidence of HLA-DR3 is mediated by its linkage disequilibrium with HLA-B8. We also examined a possible synergistic effect of coexpression of protective class I and class II molecules on CML incidence. The effect of co-expression of HLA-A3 and HLA-DR4 gave an OR of 0.86 (95% CI 0.70-1.06), co-expression of HLA-B8 and HLA-DR4 an OR of 0.85 (95% CI 0.67-1.11), showing no evidence of a synergistic effect.
Results
Finally homozygosity for HLA-DR4 resulted in an OR of 0.18 (95% CI 0.12-0.27) ( Table 4) .
Discussion
The t(9,22) translocation characteristic for CML results from the fusion of the Bcr and Abl genes and leads to the production of the P210 protein.
Discussion
1,2 The junctional peptides of this protein are neoantigens. 3 If such tumor antigens are present in sufficient quantities after digestion of p210 by the proteasome presentation of tumor-specific peptides in HLA molecules could function as target antigens on the malignant cells for T lymphocytes. Although originally it was thought that endogenous intracellular proteins -like P210 -would only be presented by HLA class I molecules and exogenous proteins by HLA class II molecules, more recent evidence illustrates that also HLA class II molecules contain fragments of endogenous proteins. [22] [23] [24] In vitro studies have shown binding of, and subsequently induction of immune responses to synthetic bcr-abl-derived peptides presented in the context of the class I molecules HLA-A2, HLA-A3, HLA-A11 and HLA-B8. [5] [6] [7] [8] [9] [10] For class I we have previously shown that individuals expressing HLA-A3 and HLA-B8 have a diminished risk of the development of CML. 11 Based on the knowledge that also HLA class II can present endogenous proteins and the reported binding of and/or the generation of T cell responses to synthetic bcr-abl peptides in HLA-DR1, HLA-DR2, HLA-DR3, HLA-DR4 and HLA-DR11, [12] [13] [14] [15] we now compared these HLA-DR frequencies between patients and controls in a large multicenter study to determine if the presence of any of these class II antigens may protect an individual against the development of this disease. The results of this study showed a protective effect of HLA-DR4 against the acquisition of CML in vivo, corroborating the findings of in vitro studies. 14, 25 The protective effect of HLA-DR3 was shown to be mediated through the linkage disequilibrium with HLA-B8. The lack of protection by the other HLA-DR molecules may be explained by the inability of these MHC molecules to bind tumor-specific peptides or by the absence of immunogenicity of breakpointderived peptides in certain MHC molecules. The protective effect of HLA-DR4 in this study may even be underestimated, since the in vitro generated responses were directed against b3a2-derived peptides in the context of class II molecules. [12] [13] [14] [15] An even stronger protective effect of HLA-DR4 may be expected for this b3a2 subgroup while little or no effect might be observed in the b2a2 subgroup.
Discussion
Co-expression of HLA-DR4 with either HLA-A3 or HLA-B8 showed no synergistic effect. A possible explanation may be that when P210 is processed by the cell the presentation can be either in class I or class II or in both but the latter situation will not increase the likelihood of an immune response. When P210 is not sufficiently processed it cannot be presented in either HLA class I or class II and in this case no protection will occur. Leukemia Homozygosity for HLA-DR4 showed a stronger negative association with CML than heterozygosity making a causative role of HLA-DR4 more likely.
Discussion
In summary, the results of the presented study indicate that HLA-DR4 is associated with a diminished incidence of CML. This is in line with in vitro studies showing both a binding motif of b3a2 for HLA-DR4 24 and HLA-DR4 restricted T cell responses to synthetic b3a2-derived peptides.
Discussion
14 It remains unknown whether this is due to presentation of bcr/ablderived peptides in HLA-DR4; only revealing the presence of such peptides in the HLA of leukemic cells of patients with CML would provide the formal evidence.

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