Over the past few years, a large number of CTL-defined tumor Ags and their encoding genes have been identified in melanoma (20
). However, little is known about CTL-defined Ags encoded by genes with expression restricted to leukemia. Using biochemical methods to isolate and sequence antigenic peptides presented by MHC class I molecules, the first CTL epitopes on leukemic cells have recently been identified (6
). Expression of the identified polymorphic Ags HA-1 and HA-2 is restricted to hematopoietic cells but they are not leukemia specific (2
). Although these mHags have this limited tissue distribution, mismatching for HA-1 is associated with the occurrence of severe GVHD (22
). To the best of our knowledge, the polymorphic HB-1
gene product described here is the first identified leukemia-associated mHag that is only significantly expressed by leukemia- and EBV-transformed B cells. In some normal cells, few HB-1 transcripts are present, but expression is too low for recognition by HB-1–specific CTLs, as was also observed for a number of other tumor-associated Ags (20
). Low levels of transcription of the melanocyte-associated Ag gp100 have been found in nearly all normal tissues and tumor cell lines of nonmelanocytic origin, but no gp100 protein could be detected by either Western blot or cytotoxicity assays (23
). Similarly, the Ag encoded by the N-acetylglucosaminyl-transferase V (GnTV)
gene was not recognized by specific CTLs when transcription levels did not exceed 8% of that of the reference melanoma cell line (24
). Finally, MAGE-1–specific CTLs are unable to recognize tumor cell lines expressing low levels (<10%) of the MAGE-1
gene when compared with melanoma cells that were efficiently lysed (25
). These reported data and the observation that PHA-stimulated T cells expressing low levels of the HB-1
gene (2.5 and 8% of that of the reference B-ALL cell line) are not lysed by CTL MP1 indicate that only significant HB-1 transcription leads to CTL recognition.
The HB-1 antigenic peptide is encoded by a sequence which starts with a CUG codon resulting in the translation of a short protein of 41 amino acids. Such an unusual initiation of translation at a CUG has been reported previously (26
). Whether this product is the only protein encoded by the HB-1
gene or whether there are more proteins encoded by alternative ORFs is currently unknown. Although most eukaryotic mRNAs have a single ORF of which translation is usually initiated by an AUG codon, several human genes are bicistronic, encoding two proteins (27
). For instance, the gp75
gene in melanoma has two overlapping ORFs resulting in two completely different proteins: (i) gp75 as recognized by IgG antibodies in the serum from melanoma patients, and (ii) a short protein of 24 amino acids from which an antigenic peptide recognized by CTLs is generated (27
). Whether the generation of the short 41-amino acid protein from the HB-1
gene is the result of translation of an alternative ORF awaits further characterization of the gene.
At present we can not exclude the possibility that the HB-1
gene–encoded protein is a B cell differentiation Ag that is overexpressed in B-ALL cells and lost in mature B cells. Alternatively, the malignant transformation of progenitor B cells itself may induce HB-1 expression. This latter possibility is supported by the finding that the HB-1
gene also shows significant expression in B cells that are transformed in vitro with EBV. These EBV-transformed B cell lines express all EBV gene–encoded proteins, whereas EBV-positive Burkitt's lymphoma cell lines express only the EBNA1 protein (30
). Since HB-1 is significantly expressed by EBV-transformed B cell lines and not by Burkitt's lymphoma cell lines, it is tempting to speculate that expression of EBV gene–encoded proteins other than EBNA1 may induce HB-1 mRNA transcription in mature B cells. Studies dealing with expression levels during B cell differentiation and the role of EBV transformation in inducing HB-1 expression are currently under investigation.
The antigenic peptide EEKRGSLHVW encoded by the HB-1
gene is recognized in association with HLA-B44, a common HLA-B allele expressed by 23% of the Caucasian population. Five subtypes of HLA-B44 have been identified, but the most frequently expressed subtypes are HLA-B*4402 and -B*4403. These two subtypes differ only by a single amino acid substitution from Asp (*4402) to Leu (*4403) in position 156 of the α2 domain (18
). Both HLA-B*4402 and -B*4403 are able to present the HB-1 peptide to CTL clone MP1. The consensus peptide binding motif for HLA-B44 shows a predominance for Glu at position 2, and Tyr or Phe at position 9 or 10 (18
). The HB-1 peptide contains a Glu at position 2, but in contrast to the consensus motif it has a Trp at position 10, indicating that all amino acids with aromatic side chains facilitate binding to HLA-B44. A polymorphism in the HB-1
gene resulted in a single amino acid exchange from His to Tyr at position 8 of the HB-1 peptide. The anchor residues of the peptide are not involved in this substitution, and both the HB-1H
peptide bind with similar affinity to HLA-B44 molecules. Since the HB-1Y
peptide is not recognized by CTL clone MP1, the polymorphism appears to influence a TCR contact residue.
The molecular identification and characterization of the leukemia-associated mHag HB-1 allows the opportunity to treat leukemia patients with immunotherapy specifically targeted to the tumor without the risk of inducing GVHD. For this, patients must be typed for the presence of the HB-1H allele and their tumor cells must significantly express the HB-1 gene. The mHag HB-1 might turn out to be an excellent target against which specific CTLs can easily be generated from allogeneic donors and adoptively transferred into BMT recipients with relapsed B-ALL. We have already succeeded in generating HB-1 specific CTLs in vitro from peripheral blood of healthy HLA-B44–positive individuals by stimulating CD8-positive T cells with peptide-loaded autologous dendritic cells (our unpublished results). These induced CTLs displayed lysis of both peptide-loaded HB-1–negative target cells, and autologous HLA-B44–positive EBV-transformed B cells endogenously expressing the HB-1 Ag. The presence of HB-1–specific CTLs in the T cell repertoire of HB-1–positive individuals and the restricted expression of the HB-1 gene by B-ALL cells may also allow the use of HB-1–encoded Ags for vaccination protocols to induce specific immunity in B-ALL patients.