Human minor histocompatibility antigens (mHags) play an important role in the induction of
cytotoxic T lymphocyte (CTL) reactivity against leukemia after human histocompatibility
leukocyte antigen (HLA)-identical allogeneic bone marrow transplantation (BMT). As most
mHags are not leukemia specific but are also expressed by normal tissues, antileukemia reactivity is often associated with life-threatening graft-versus-host disease (GVHD). Here, we describe a novel mHag, HB-1, that elicits donor-derived CTL reactivity in a B cell acute lymphoblastic leukemia (B-ALL) patient treated by HLA-matched BMT. We identified the gene
encoding the antigenic peptide recognized by HB-1-specific CTLs. Interestingly, expression of
the HB-1 gene was only observed in B-ALL cells and Epstein-Barr virus-transformed B cells.
The HB-1 gene-encoded peptide EEKRGSLHVW is recognized by the CTL in association
with HLA-B44. Further analysis reveals that a polymorphism in the HB-1 gene generates a single amino acid exchange from His to Tyr at position 8 within this peptide. This amino acid
substitution is critical for recognition by HB-1-specific CTLs. The restricted expression of the
polymorphic HB-1 Ag by B-ALL cells and the ability to generate HB-1-specific CTLs in vitro
using peptide-loaded dendritic cells offer novel opportunities to specifically target the immune system against B-ALL without the risk of evoking GVHD.
Key words:
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Introduction |
Transplantation with HLA-identical sibling bone marrow is successfully used to treat patients with leukemia. The therapeutic effect can be partly attributed to the
elimination of residual host leukemic cells by donor-
derived T cells, termed the graft-versus-leukemia reactivity
(1). However, this donor-derived T cell reactivity generally
causes GVHD, a life-threatening complication in allogeneic bone marrow transplantation (BMT)1 (1). The ability
of donor-derived CTLs to recognize host minor histocompatibility Ags (mHags) as foreign peptides plays an important role in both GVHD and graft-versus-leukemia reactivity (2). mHags are HLA-associated peptides generated
from polymorphic regions of proteins present in the target
cells (5). Interestingly, as well as mHags with ubiquitous
tissue distribution, mHags with expression restricted to hematopoietic cells or leukemic cells have been characterized
(2, 8).
Although allogeneic BMT may cure many patients with
leukemia, relapse of the tumor occurs in a significant number of patients, indicating that in these patients not all tumor cells are sufficiently eliminated or suppressed. Therefore, there is a continuous search for additional and more
specific immunotherapeutic strategies in the treatment of
leukemia in order to eliminate leukemic cells without inducing GVHD. We have focused our attention on CTLs
that selectively recognize leukemic but not normal cells.
Recently, we isolated from a BMT recipient an HLA-B44-
restricted CTL clone recognizing a novel mHag named
HB-1, which shows specificity for B cell acute lymphoblastic leukemia (B-ALL). Using this CTL, we have identified
both the nucleotide sequence encoding the HB-1 Ag and
the polymorphic CTL epitope. Furthermore, we show that the expression of the HB-1 gene is restricted to B-ALL and
EBV-transformed B cells.
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Materials and Methods |
Cell Culture.
All cell lines were cultured in IMDM (GIBCO
BRL) supplemented with 10% FCS. CTL clone MP1 was isolated
from PBLs of leukemia patient MP after HLA-identical BMT and
grown in IMDM supplemented with 10% pooled human serum,
irradiated EBV transformed-lymphoblastoid cell line (EBV-LCL)
of the patient pre-BMT (106/ml), irradiated allogeneic PBMCs
(106/ml), 100 U/ml IL-2 (Glaxo), and 0.4 µg/ml PHA.
cDNA Library Construction.
The cDNA library was constructed
using the Superscript Plasmid System (GIBCO BRL). Total
RNA was isolated from EBV-LCL MP and poly(A)+ mRNA
was prepared by oligo-dT binding (Qiagen). mRNA was converted into cDNA using an oligo-dT primer that contains a NotI
site at its 5' end. The cDNA was ligated to SalI adaptors, digested
with NotI, and ligated into SalI and NotI sites of the expression
vector pSV-Sport1. Escherichia coli DH10B were electroporated
with the recombinant plasmids and clones were selected with
ampicillin. This library was divided into 1,500 pools of ~100
cDNA clones. Each pool was amplified for 4 h and plasmid DNA
was extracted with Bio-Rad miniprep kits (Bio-Rad).
Isolation of the HLA-B*4403 Gene.
Total RNA from EBV-LCL MP was extracted using the Trizol method (GIBCO BRL).
Reverse transcription was performed on 2 µg of total RNA using an oligo-dT primer and Moloney's murine leukemia virus
(Mo-MuLV) reverse transcriptase (GIBCO BRL). HLA cDNA
was amplified by PCR using 50 pmol HLA-5UTR primer
(5'-GACTCAGA(AT)TCTCCCCAGACG-3'), 50 pmol HLA-3UTR primer (5'-CTCAGTCCCTCACAAGGCA-3'), 0.5 mM
dNTPs, and 2.5 U Taq polymerase as previously described (12).
After separation of the PCR product on a 1% agarose gel, the
1.2-kb DNA fragment was isolated and cloned into pCR3 vector
by using the TA cloning kit (Invitrogen). This cloned HLA-B*4403 gene was sequenced by the dideoxynucleotide chain termination method.
Transfection of COS-1 Cells and CTL Stimulation Assay.
Transfections were performed by the lipofection method. In brief, 1.5 × 104 COS-1 cells were plated in a flat-bottomed 96-well plate and incubated for 18 h at 37°C. 200 ng of plasmid pCR3 containing the HLA-B*4403 gene and ~400 ng of plasmid pSV-Sport1 containing a pool of the cDNA library were mixed with 1 µl of lipofectamine (GIBCO BRL) in 100 µl IMDM. Transfection mixtures were incubated for 30 min and 50 µl was added in duplicate
wells to the COS-1 cells. After 4 h of incubation, 50 µl of
IMDM/20% FCS was added. Transfectants were tested for their
ability to stimulate the production of IFN-
by the CTLs. In
brief, 3,000 CTLs were added to the wells containing transfected
cells or 3 × 104 stimulator cells in 200 µl IMDM/10% FCS and
25 U/ml IL-2. After 18 h of incubation at 37°C, 100 µl of supernatant was collected and the IFN-
concentration was determined
by ELISA (Endogen).
Production of Truncated and Mutated cDNA Constructs.
Deletion
and mutation constructs were produced by PCR using specific
primers, and amplified products were subsequently cloned into
vector pCR3 using the unidirectional TA cloning method (Invitrogen).
HB-1 Polymorphism.
Total RNA from cells was extracted using
the Trizol method (GIBCO BRL). Reverse transcription was
performed on 2 µg of total RNA using an oligo-dT primer and
Mo-MuLV reverse transcriptase (GIBCO BRL). HB-1 cDNA
was amplified by PCR using 50 pmol HB1-F5 primer (5'-GAGCCTTCTGACCTCACATC-3'), 50 pmol HB1-GSP3 primer
(5'-TTGTCCCTGCTCATCCACACC-3'), 0.5 mM dNTPs, and
2.5 U Taq polymerase (GIBCO BRL). The PCR was performed
for 33 cycles (1 min at 94°C, 1 min 60°C, and 1 min 72°C). PCR
products were digested with NlaIII to discriminate between HB-1H
and HB-1Y alleles.
Peptides and Cr-release Assay.
Peptides were synthesized with
a free COOH terminus by Fmoc peptide chemistry using a multiple synthesizer (ABIMED). Peptides (>90% pure as indicated by
analytical HPLC) were dissolved in DMSO and stored at
20°C.
Cr-release assays were performed as previously described (13). In
peptide recognition assays, target cells were preincubated with
various concentrations of peptide for 1 h at 37°C in a volume of
100 µl before the addition of effector cells. After 4 h of incubation at 37°C, 100 µl supernatant was collected and radioactivity
was measured by a gamma counter.
TaqmanTM PCR Assay for HB-1 Expression.
Total RNA from
cells was extracted using the Trizol method (GIBCO BRL). Reverse transcription was performed on 2 µg of total RNA using an
oligo-dT primer and Mo-MuLV reverse transcriptase (GIBCO
BRL). Of the total cDNA volume of 20 µl, 1 µl was used for
each PCR reaction. PCR amplification and real time quantitation analysis were performed using the TaqmanTM assay (14, 15). The following sequences were used as primers and TaqmanTM
probes: 5'-GAGCCTTCTGACCTCACATC-3' (HB1-F5, sense,
nucleotide [nt] 58-77), 5'-TTGTCCCTGCTCATCCACACC-3'
(HB1- GSP3, antisense, nt 271-291), 5'-(FAM)-TCCCTTCTCGACACGGAGTCTATGTGTAGT-(TAMRA)-3' (HB1-probe, antisense, nt 188-217), 5'-GGCAATGCGGCTGCAA-3' (Pbgd-F),
5'-GGGTACCCACGCGAATCAC-3' (Pbgd-R), and 5'-(JOE)-
CTCATCTTTGGGCTGTT T TCT T CCGCC-(TAMRA)-3' (Pbgd-probe). The reaction mixture contains 1.25 U AmpliTaq
Gold (PE-Applied Biosystems), 250 µM dNTPs, 15 pmol sense,
and 15 pmol antisense primer in a total reaction volume of 50 µl.
The enzyme was activated by heating for 10 min at 95°C. A two-step PCR procedure of 60 s at 60°C and 15 s at 95°C was applied
for 40 cycles. 6 mM MgCl2 and 100 nM probe for the HB-1
PCR, and 5 mM MgCl2 and 100 nM probe for the Pbgd PCR,
was used. The PCR and TaqmanTM analysis were performed in the
ABI/PRISM 7700 Sequence Detector System (PE-Applied Biosystems). The system generates a real time amplification plot based
upon the normalized fluorescence signal. Subsequently the threshold cycle (CT), i.e., the fractional cycle number at which the number of amplified target reaches a fixed threshold, is determined.
The CT is proportional to the initial number of target copies in the
sample (14, 15). We used the expression of the porphobilinogen
deaminase (Pbgd ) gene to normalize the HB-1 expression. Pbgd was
used as an endogenous reference to correct for differences in the
amount of total RNA added to the reaction. The Pbgd primers
only allows amplification of cDNA derived from the Pbgd housekeeping gene (16, 17). HB-1 mRNA expression was quantified by
determining calibration functions for HB-1 and Pbgd expression of
a reference cell line. Therefore, 2 µg of RNA of the B-ALL cell
line KM3 was reverse transcribed into cDNA and serially diluted
into water. This cDNA serial dilution was prepared once, stored at
20°C, and used in all tests performed in this study. The linear calibration functions between the CT and the logarithm of the initial
starting quantity (N) were CT =
3.44log(N) + 25.8 and CT =
3.44log(N) + 21.9 for HB-1 and Pbgd, respectively. HB-1 and
Pbgd mRNA expression in all test samples were quantified using
these calibration functions. At the same level of Pbgd expression the level of HB-1 expression of test samples was determined as a
percentage of the HB-1 expression in cell line KM3.
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Results |
Identification of a cDNA Coding for the HB-1 Antigenic Peptide.
We generated a cDNA library from an EBV-LCL
that expresses the HB-1 Ag since it was efficiently lysed by
CTL MP1. Using expression cloning, we isolated from this
library a cDNA that upon transfection along with the
HLA-B44 cDNA into COS-1 cells stimulated CTL MP1 to release IFN-
(Fig. 1). The IFN-
release was as high as
that induced by EBV-LCL MP, from which the cDNA library was generated, and even higher than that induced by
EBV-LCL as well as B-ALL cells of the HLA-B44, HB-1-positive patient VR. Untransformed CD40-stimulated B
cells of patient VR could not stimulate CTL MP1 to release IFN-
. Transfection of either of the aforementioned cDNAs alone failed to induce the production of IFN-
by
CTL MP1, indicating that the isolated cDNA clone encodes the HB-1 Ag.

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Fig. 1.
Identification of the cDNA encoding the antigenic peptide
recognized by CTL clone MP1. Production of IFN- is shown upon
stimulation with the following stimulator cells: B-ALL cells, EBV-transformed B cells, and B cells stimulated with CD40 plus 100 U/ml TNF-
for 2 d of the HLA-B44, HB-1-positive patients MP and VR, and COS-1
cells cotransfected with the HB-1 cDNA plus HLA-B44 cDNA, and
COS-1 cells transfected either with the HLA-B44 cDNA alone or with
HB-1 cDNA alone. Release of IFN- was measured by ELISA.
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The cDNA encoding the HB-1 Ag consists of 397 nucleotides with no significant homology to sequences presently recorded in data banks. To localize the region encoding the HB-1 epitope, we transfected COS-1 cells with
truncated HB-1 cDNA constructs in combination with the
HLA-B44 cDNA and tested their capacity of inducing
IFN-
release by the CTLs (Fig. 2). The smallest truncated construct that still encoded both the translation initiation
codon and the peptide coding region contains the nucleotide sequence 100 to 165 (Fig. 2). The three possible
translational reading frames within this sequence did not
code for a peptide according to the described HLA-B44
binding motif, a Glu at position 2 and a Phe or Tyr at position 9 or 10 (18, 19). We next synthesized all 9-, 10-, and
11-mer peptides with a Glu residue at position 2 encoded
by the translational reading frames within nucleotide sequence 100 to 165, and tested their ability to induce lysis by CTL MP1 upon loading on HB-1-negative target cells.
The 10-mer EEKRGSLHVW was specifically recognized
by CTL MP1, but not by HLA-B44-restricted, EBNA3C-specific CTLs (Fig. 3, A and B). CTL MP1 did not recognize the 9-mer peptide EEKRGSLHV (data not shown).

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Fig. 2.
Location of the nucleotide sequence coding for the translation initiation codon and the antigenic peptide recognized by CTL MP1.
HB-1 cDNA deletion constructs were cloned into an expression vector
and cotransfected with the HLA-B44 cDNA into COS-1 cells. Transfected cells and CTL MP1 were incubated for 18 h and the release of
IFN- was measured by ELISA.
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Fig. 3.
Identification of the
HB-1 antigenic peptide. (A) Cytolytic activity by CTL clone
MP1 against HLA-B44-positive
target cells incubated with 5 µM
of HB-1 peptide EEKRGSLHVW. Controls included the
HLA-B44-positive target cells
incubated either without peptide
or with the EBNA3C 281-290
peptide EENLLDFVRF. (B)
Cytolytic activity by the
EBNA3C-specific CTL against
HLA-B44-positive target cells
incubated with 5 µM of
EBNA3C peptide EENLLDFVRF. Controls included the
HLA-B44-positive target cells
incubated either without peptide
or with the HB-1 peptide EEKRGSLHVW.
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The open reading frame (ORF) encoding the EEKRGSLHVW CTL epitope contains a CTG translation initiation codon resulting in a putative 41-amino acid protein
(Fig. 4). Substitution of this CTG into AAG resulted in a
complete loss of the ability to stimulate IFN-
release by
CTL MP1 upon transfection into COS-1 cells (data not
shown). Together, these data led us to conclude that within the HB-1 sequence the CTG at position 108-110 initiates
the translation of a 41-amino acid protein from which the
EEKRGSLHVW CTL epitope is generated.

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Fig. 4.
Sequence of HB-1 cDNA and of the 41 amino acid-
encoded protein starting from the CTG start codon (underlined) at nucleotide positions 108-110. The sequence corresponding to the HLA-B44-
restricted HB-1 peptide is boxed. These sequence data are available from
EMBL/GenBank/DDBJ under accession number AF103884.
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The HB-1 Gene Is Only Expressed by Transformed B Cells.
Interestingly, we observed that CTL MP1 exhibits specific
cytotoxicity towards leukemia- and EBV-transformed B
cells, but not against untransformed B cells, T cells, monocytes, and fibroblasts (8). Therefore, we studied the level of
HB-1 gene expression in a large panel of tumor and nonmalignant cells using real time quantitative reverse transcriptase PCR. All B-ALL samples expressed the HB-1
gene at a significant level exceeding 10% of that found in
the reference B-ALL cell line KM3 (Fig. 5 A). In addition, 2 out of 14 B cell lymphomas and 2 out of 5 acute undifferentiated leukemias showed significant HB-1 expression.
In contrast, all T-ALLs, multiple myelomas, acute myeloid
leukemias, and nonhematological solid tumors lacked HB-1
expression. Since the number of HB-1 transcripts in B-ALL
cells is even lower than that of the low copy gene Pbgd we
concluded that HB-1 is a rare mRNA species. This notion
was confirmed by the observation that we could not detect
HB-1 expression by Northern blot analysis, whereas
-actin mRNA was readily detected (data not shown).

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Fig. 5.
Expression of the HB-1 gene in tumor cells and nonmalignant
cells. (A) Measurement of the HB-1 expression obtained by real time quantitative reverse transcriptase PCR in tumor cells and cell lines. The following cell lines were used: B-ALL (KM3, BV173); B cell lymphoma (Daudi,
Raji, Ramos, SU-DHL6); multiple myeloma (RPMI1758); T-ALL (Jurkat,
CEM, HSB-2); and acute myeloid leukemia (Lama, Kasumi, K562, HL60,
KG-1). The expression levels were determined by a calibration function
generated from RNA of the HB-1-positive B-ALL cell line KM3 and expressed relative to the HB-1 level measured in these cells. The Pbgd gene
was used as standard to correct for RNA quantity and quality. The detection limit is indicated with a solid line. Samples showed significant HB-1
gene expression if they exceeded 10% of that found in the B-ALL cell line
KM3. This arbitrary threshold is indicated with a dashed line. (B) Measurement of the HB-1 expression obtained by real time quantitative reverse
transcriptase PCR in freshly isolated cells or primary cell cultures.
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Analysis of the expression of the HB-1 gene in a panel of
nonmalignant cells revealed significant levels in 90% of the
EBV-transformed B cell lines, whereas no significant HB-1
transcription was observed in all other nonmalignant cell
types (Fig. 5 B). Some B cell and PHA-stimulated T cell
samples express very low levels of HB-1 (<10% of that
found in the reference B-ALL cell line KM3; Fig. 5 B).
PHA-stimulated T cell blasts with an HB-1 expression levels of 8 and 2.5% of that found in the KM3 cell line were
not recognized by CTL MP1 (Fig. 6). Loading of these
PHA-stimulated T cell blasts with the EEKRGSLHVW
peptide results in lysis that is as high as the killing of the
HB-1-positive EBV-LCL. Analysis of HB-1 expression in
normal tissues revealed only low (<10%) transcription
levels in testis samples (Table I). Together, these results
demonstrate that substantial HB-1 mRNA expression is
observed only in B-ALL cells and in EBV-transformed B
cells and that very low mRNA expression of HB-1 does
not result in lysis by CTL MP1.

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Fig. 6.
Cytolytic activity by CTL clone MP1 against EBV-LCL and
PHA-stimulated T cell blasts of HLA-B44, HB-1-positive individuals.
PHA-stimulated T cell blasts were preincubated either without peptide or
with the HB-1 peptide EEKRGSLHVW. The level of HB-1 gene expression in EBV-LCL VH and MT was 140 and 122% of that found in
the B-ALL cell line KM3, respectively, and in PHA-stimulated T cell
blasts VH and MT was 8 and 2.5%, respectively. Both individuals express
homozygous the HB-1H allele and the HLA-B44 subtype of VH is
B*4403 and of MT B*4402. The E/T cell ratio was 3:1.
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A Polymorphism in the HB-1 Gene Determines CTL Recognition.
CTL MP1 lyse EBV-transformed B cells derived
from the patient MP, whereas EBV-transformed B cells
derived from the HLA-identical sibling donor BP are not
recognized (8). Since the HB-1 gene was significantly
expressed by both cell lines, these results suggested the
presence of a polymorphism in this sequence. Analysis of
the HB-1 sequence of the donor revealed that it differs
from that of the patient at only one nucleotide (position
153: C to T), leading to an amino acid change from H to Y
in the HB-1 peptide (Fig. 7 A). The corresponding alleles
were named HB-1H and HB-1Y, respectively.

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Fig. 7.
The mHag HB-1 is encoded by the HB-1H allele of the HB-1
gene. (A) Sequence of the peptide coding region of the HB-1 gene of patient MP and donor BP. The nucleotide and amino acid polymorphism
are underlined. (B) Correlation between expression of HB-1 alleles and
cytolytic activity by CTL clone MP1 against EBV-transformed B cell
lines of relatives of patient MP. Filled circles (females) or squares (males)
indicate strong lysis by CTL MP1. Open symbols indicate no lysis. Expression of HB-1 alleles was determined by reverse transcriptase PCR
amplification and digestion of PCR products with restriction enzyme
NlaIII.
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Studies of the HB-1 gene polymorphism in relatives of
patient MP resulted in a clear correlation between recognition by CTL MP1 of EBV-LCL of HLA-B44-positive
family members and the expression of the HB-1H allele
(Fig. 7 B). To verify that only expression of the HB-1H
allele leads to recognition by CTL MP1, we transfected
HB-1H or HB-1Y cDNA along with HLA-B44 cDNA into
COS-1 cells. Cells transfected with the HB-1H cDNA
stimulated IFN-
release by the CTL, whereas cells transfected with the HB-1Y cDNA did not (Fig. 8 A). In addition, EBV-transformed B cells incubated with the peptide
encoded by the HB-1Y allele were not lysed by CTL MP1
(Fig. 8 B). This probably is not the result of defective binding of the HB-1Y peptide to HLA-B44 molecules, as both
peptides appeared to bind with equal affinity to these HLA
molecules (data not shown). These results demonstrate that
at least two allelic forms of the HB-1 gene exist, and that
only the HB-1H allele encodes the peptide that is recognized by CTL MP1.

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Fig. 8.
The single amino acid exchange within the HB-1 antigenic
peptide is critical for recognition by CTL clone MP1. (A) Production of
IFN- is shown upon stimulation with EBV-transformed B cells of patient MP and donor BP, and COS-1 cells cotransfected with the HB-1H
or HB-1Y cDNA and HLA-B44 cDNA. (B) Cytolytic activity by CTL
clone MP1 against HLA-B44-positive target cells incubated with the
HB-1H or HB-1Y peptide. The E/T cell ratio was 10:1.
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Discussion |
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, 7, 21). 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, 23). 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
2 domain (18, 31, 32).
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, 19).
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 and HB-1Y 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.
Address correspondence to Harry Dolstra, Department of Hematology, University Hospital Nijmegen,
Geert Grooteplein 8, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: 31-24-361-9449; Fax:
31-24-354-2080; E-mail: h.dolstra{at}chl.azn.nl
We thank Rick Brouwer for cloning the B*4403 gene, Aukje Zimmerman for help in screening the cDNA
library, and Wendy Unger for generation of the EBNA3C peptide-specific CTL clone. We thank Louis van
de Locht and Ellen Linders for technical assistance with the ABI/PRISM 7700 Sequence Detector System.
We thank Han Zendman (Department of Pathology, University Hospital Nijmegen, The Netherlands) for
providing RNA samples from primary cell cultures.
This work was supported by grants from the Dutch Cancer Society (KUN 97-1508), the Ank van Vlissingen Foundation, and the Maurits and Anna de Kock Foundation.
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