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,
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Department of Microbiology and Carter Immunology Center, and
Department of Pathology, University of Virginia, Charlottesville, VA 22908;
Department of Chemistry, University of Virginia, Charlottesville, VA 22901;
Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, The Netherlands;
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Department of Microbiology and Immunology and Kimmel Cancer Institute, Thomas Jefferson University, Philadelphia, PA 19107; and
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Department of Pediatrics, Section on Medical Genetics, Wake Forest University School of Medicine, Winston-Salem, NC 27157
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We previously used a combination of peptide fractionation, T cell Ag reconstitution, and mass spectrometry to identify the first amino sequence of an autosomally encoded human mHag, known as HA-2 (7). This Ag is restricted by HLA-A*0201, present in 95% of the HLA-A*0201+ population, and expressed only on cells of hemopoietic origin (3, 5). The peptide sequence of HA-2 (YIGEVLVSV) did not yield an exact match in genetic databases available at the time, although it was highly homologous to a sequence present in several genes of the class I myosin family, one of the classes of nonfilament-forming myosins thought to play a role in intracellular transport and locomotion (8). The nearest human match, YIGSVLISV, was present in MYO1E and MYO1F (9). However, the ubiquitous tissue expression of these class I myosins was inconsistent with the possibility that either was an allelic homolog of HA-2. Failure to identify the gene encoding HA-2 has stood as an impediment to understanding the basis for its antigenicity and to the development of molecular methods to screen for its presence or absence.
In the present study, we relied on newly available genetic information from the Human Genome Project to identify a candidate genomic DNA sequence encoding the HA-2 epitope. Using this information, we have established that this candidate sequence does encode HA-2, and that it represents a new member of the class I human unconventional myosin family that is expressed only in cells of hemopoietic origin. We have also defined its nonantigenic allelic homolog and established the basis for its differential antigenicity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The origin and maintenance of the HA-2-specific CD8+ CTL clone 5H17 has been previously described (5). The HLA-A*0201+ EBV-transformed B lymphoblastoid cell lines (EBV-BLCL) Rp (HLA-A*0101, A*0201, B8, B27), JY (HLA-A*0201, B*0702 homozygous), Blk (HLA-A*0201, B44 homozygous), and Maja (HLA-A2, B35, C4) have all been described previously (7). C1R-A2 and JK-A2 are HLA-A*0201+ stable transfectants of C1R (10) and Jurkat (11), respectively. Other transformed cell lines used and their tissues of origin are described in the text and the figure legends. All transformed cell lines were cultured in RPMI 1640 containing 5% FBS, 4 mM HEPES, 0.125% SerXtend (Irvine Scientific, Santa Ana, CA), and 3 mM L-glutamine. To maintain the expression of the HLA-A*0201 gene in the C1R and Jurkat transfectants, this medium was supplemented with either 200 µg/ml Hygromycin (Mediatech, Herndon, VA) or 300 µg/ml G418, respectively.
Melanocytes, epidermal keratinocytes, HUVEC, and proximal tubular epithelial cells were all isolated and cultured as described elsewhere (3, 12, 13). PBMC were isolated by Ficoll-Isopaque density centrifugation of whole donor blood, washed twice with PBS, and used immediately.
PCR amplification and cloning of the HA-2 coding sequence
Poly(A)+ mRNA was prepared from 1 x
107 cells using the QuickPrep mRNA Purification
kit (Amersham Pharmacia Biotech, Piscataway, NJ). Alternatively, total
RNA was prepared from patient PBMC and normal tissue samples with the
RNAzol method (Cinaa/Biotecx Laboratories, Houston, TX). First-strand
cDNA was synthesized using an oligo(dT) primer as per the
manufacturer’s instructions (First Strand cDNA Synthesis kit; MBI
Fermentas, Hanover, MD). Amplifications were performed on cDNA using
0.5 µM the forward and reverse primers shown in Fig. 2
(Research
Genetics, Huntsville, AL) in 1.5 mM MgCl2, 0.2 mM
dNTPs, and 2.5 U Taq polymerase in 1x PCR buffer (all
obtained from Life Technologies, Rockville, MD). Cycle parameters were:
initial denaturation at 94°C for 2 min; 36 cycles of denaturation at
94°C for 1 min, annealing at 60.5°C for 1 min, extension at 72°C
for 1 min, and final extension at 72°C for 10 min. PCR products were
gel purified and isolated with the Wizard PCR-Preps DNA purification
system (Promega, Madison, WI). Purified fragments were cloned into the
pT-Adv plasmid using the AdvanTAge cloning kit (Clontech Laboratories,
Palo Alto, CA), and the inserts were sequenced at the University of
Virginia Biomolecular Core Facility using M13 forward and reverse
primers. At least seven individual clones were sequenced and analyzed
bidirectionally for each sample.
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For RFLP analysis, PCR fragments corresponding to
bases 1–206 in Fig. 2
were amplified from cDNA using the forward
primer 5'-AAGCTTTTCGAGAAGGGCCGCATCTA-3' and the reverse primer
5'-GAATTCGAGATGACGATGCAGGTGTC-3' under the conditions described
above. Samples were digested at 37°C for 12 h with either
Hsp92 II (Promega) or NlaIII (New England
Biolabs, Beverly, MA), and analyzed on a 4% agarose gel. The
MYO1GV derived sequence was not digested under
these conditions and ran as a fragment of 218 bp, whereas the
MYO1GM sequence gave fragments of 163 and 55 bp.
For tissue-specific expression, PCR fragments corresponding to bases
1–334 in Fig. 2 were amplified from cDNA using the forward primer
5'-AAGCTTTTCGAGAAGGGCCGCATCTA-3' and the reverse primer
5'-GAATTCCACAGGTGGACTTGAGCAGC-3'. Primers specific for
glucose-6-phosphate dehydrogenase (Stratagene, La Jolla, CA) were used
to amplify a 358-bp positive control fragment. The conditions
used were as above except that the MgCl2
concentration was adjusted to 1.0 mM, and the annealing temperatures
used were 66.2°C and 61.6°C for MYO1G and glucose-6-phosphate
dehydrogenase, respectively.
Fluorescence in situ hybridization
Three cloned PCR fragments representing bases 1–1246, 978–1397, and 1215–1900 were isolated from bacterial cultures with the Wizard Plus Minipreps DNA Purification System (Promega), and inserts were excised by digestion of the pT-Adv plasmid with BstXI. The products were separated by gel purification, and the excised inserts were isolated using the Wizard PCR Preps DNA Purification System (Promega). The fragments were labeled with biotin-14-dATP using the BioNick labeling system (Roche Molecular Biochemicals, Indianapolis, IN), and standard methodology was used for hybridization and detection of this probe (14).
Synthetic peptides
Peptides were synthesized on an AMS 422 multiple peptide synthesizer (Gilson, Middleton, WI) using solid-phase FMOC chemistry and Wang resins. Sequences of all synthetic peptide structures were confirmed by tandem mass spectrometry (MS/MS).
Cytotoxicity assays
51Cr release assays were conducted as previously described (7).
Class I MHC peptide affinity binding assays
The HLA-A*0201 peptide binding assay was conducted as previously described (15) using the iodinated indicator peptide FLPSDYFPSV.
Preparation of HLA-A*0201-associated peptides
HLA-A*0201 molecules were immunoaffinity purified from JY
(HLA-A*0201, B*0702 homozygous) or JK-A2 (HLA-A*0201) cells using the
mAb BB7.2 (16), and their associated peptides were
extracted as previously described (7, 17). Iodoacetamide
was omitted from the protease inhibitor mixture to avoid potential
amidocarboxy methylation of free cysteine residues. Peptides
were separated from class I heavy chains and
2-microglobulin by elution in 10% acetic acid
and passage through a 5-kDa-cutoff filter. Peptides were
fractionated using a HAISIL C18 column (2.1 x 40 mm, 5-µm
particles, 300-Å pore size; Higgins, Winter Park, FL) on an Applied
Biosystems 130A HPLC (Foster City, CA) using a gradient of acetonitrile
(HPLC grade; Mallinckrodt, Paris, KY) with trifluoroacetic acid (HPLC
grade; Applied Biosystems) as a modifying agent. Those fractions
expected to contain the synthetic peptide of interest (either
YIGEVLVSV or YIGSVLISV) were established by separating a mixture of
synthetic peptides including HA2V and
HA2M in a parallel HPLC fractionation
experiment.
Targeted MS/MS analysis of HLA-A*0201-associated peptides
Data-dependent MS/MS analysis was performed using an LCQ mass spectrometer (Thermo-Finnigan, San Jose, CA) equipped with sheathless nanoflow HPLC electrospray ionization as previously described (18). MS/MS data were acquired only on masses within 3-atomic-mass-unit (amu) windows centered on either 978.6 amu (the +1 mass of YIGEVLVSV) or 1010.6 amu (the +1 mass of YIGEVLVSM). Dynamic exclusion was not enabled for these experiments, such that multiple MS/MS spectra of the same mass were taken, increasing the quality of the data for masses of interest. The identity and amount of these peptides were established by comparison of fragmentation spectra with those of synthetic peptides.
Fourier transform mass spectrometry of HLA-A*0201-associated peptides
Mass spectrometric data were acquired on a home-built Fourier
transform ion cyclotron resonance mass spectrometer (FTMS)
(19) equipped with a nano-HPLC microelectrospray
ionization source. Nano-HPLC columns were 50 µm inside
diameter fused silica packed with 
8 cm of 5-µm diameter
reversed phase beads. An integrated microESI emitter tip
(1-µm diameter) was located a few millimeters from the column bed.
Samples were loaded onto the column and eluted into the FTMS
using a gradient of 0–60% B in 32 min and 60–100% B in the next 3
min, where solvent A is 0.1 M acetic acid (Sigma, St. Louis, MO) in
NANOpure water (Barnstead, Dubuque, IA), and solvent B is 0.1 M acetic
acid in 70% acetonitrile. Full scan mass spectra, over a
mass-to-charge (m/z) range 300 
m/z
2500, were acquired at a rate of 1 scan/s.
Streptolysin O peptide transport assay
In vitro assays of TAP-mediated peptide transport were performed
as previously described (20), with modifications.
T1 cells (1 x 106/sample) were
permeabilized on ice for 15 min with streptolysin O (15 U/ml; Murex,
Norcross, GA) and incubated for 5 min at 37°C with 100 ng of the
reporter peptide TVNKTERAY (21) (radiolabeled with
Na125I using the chloramine T method; Ref.
22), 10 µl of 100 mM ATP, and indicated dilutions of
competitor peptides. The reporter peptide contains an N-linked
glycosylation site (Asn-X-Thr/Ser), and will become glycosylated after
translocation by TAP into the endoplasmic reticulum (ER). Glycosylated
reporter peptide was isolated using Con A Sepharose (Pharmacia Biotech,
Uppsala, Sweden), eluted with 0.2 M methyl
-D-mannopyranoside (Sigma), and quantitated on a gamma
counter. Reporter peptide transport in TAP-negative T2 cells was
assessed as a negative control. Samples were tested in duplicate
except for T2 negative control and T1 cells with no inhibitor, tested
in triplicate.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The peptide sequence of HA-2 (YIGEVLVSV) was used to search the
high throughput genomic sequencing database, and a precise match
was identified in nucleotides 56,571–56,545 of GenBank accession no.
AC004847 (Fig. 1A).4
Additionally, the protein sequence encoded by nucleotides
56,598–56,388 of AC004847 was 44% identical and 67% homologous to
amino acids 42–111 of MYO1E, which had previously been identified as
containing a peptide that matched HA-2 at 7/9 residues (7, 9). We next searched AC004847 using 50- to 200-bp overlapping
stretches of the MYO1E cDNA sequence and low stringency parameters, and
regions of homology were further delimited based on putative splice
sites. This analysis led to the identification of 10 putative exons
each with amino acid sequence homology of greater than 40% to the
corresponding region of MYO1E (labeled 1–10 in Fig. 1B).
The apposition of these exons defined a putative partial cDNA
corresponding to amino acids 42–214, 260–285, 358–497, 513–533, and
575–647 of MYO1E (Fig. 1C). Therefore, we hypothesized that
AC004847 contained a previously unidentified unconventional myosin that
was the source of the HA-2 mHag.
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). Each primer combination generated a
product of the expected size (data not shown), which was then
sequenced. This analysis established 1900 bp of a cDNA sequence that
encompassed the 10 predicted exons, as well as four additional exons
(labeled I-IV in Fig. 1B). The amplified cDNA that was
sequenced reproducibly differed from the AC004847-derived sequence by
three nucleotides (one each in exons 8, 9, and 10) of 1900, none of
which occurred within the region encoding the HA-2 epitope. We
think it most likely that these three nucleotides represent
sequencing errors in AC004847.
In Fig. 2, the HA-2 epitope is encoded by nucleotides 25–51, which lie
entirely within a single exon, and represents amino acids 9–17 of the
corresponding coding sequence. The translated cDNA sequence also
includes three sequence elements that are conserved in all or most
myosins: the ATP-binding sequence GESGAGKT (23) at amino
acids 70–77; the consensus sequence EAFGNART (9) at amino
acids 114–121; and the actin-binding motif PFYVRCIKPNE
(23) at amino acids 564–574. It should also be noted that
the 43 nucleotides at the 3' end of the sequence in Fig. 2 may
represent one of two alternatively spliced transcripts. This sequence
is identical with that of a putative exon that matches to nucleotides
46,192–46,150 of AC004847. However, three human expressed sequence
tags (GenBank accession nos. AA310652, AW340433, and AA824566)
match our cDNA sequence for 60–250 nucleotides to the 5' side of this
sequence, but are then juxtaposed to AC004847 residues 45,807–45,598.
Nonetheless, the results of this analysis established convincingly that
a cDNA encoding an unconventional myosin and containing the HA-2
sequence was expressed in HA-2+ cells.
The gene encoding HA-2 encodes a novel human class I myosin located on chromosome 7
By comparison with the sequences of other human unconventional
myosins, the 617 amino acids encoded by the cDNA in Fig. 2 represent
89% of the head region of the protein. To determine the
relationship of the HA-2-encoding myosin to other known human
unconventional myosins, we constructed an unrooted phylogenetic tree,
once using that region spanned by the known HA-2 sequence and once
using the same spanned region, but with gaps produced in HA-2 by
insertions in other sequences removed. Both analyses gave similar
results, and the results of the latter alignment are shown in Fig. 3. This analysis demonstrates that this
new gene falls definitively within the MYO1 clade, and is most closely
related to MYO1C. In accordance with the nomenclature in this field, we
propose that this new gene be named MYO1G.
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. Ninety one
percent of 22 metaphase spreads showed hybridization to chromosome 7
band p12p13, with no evidence for additional loci on other chromosomes
(data not shown). These findings establish MYO1G as the first human
myosin, as well as the first mHag-encoding gene, to be localized to
this chromosome. MYO1G exists in two allelic forms
All of the RT-PCR-amplified molecular clones derived from the cell
lines Rp and C1R-A2 contained precisely the same sequence in the
interval encoding the HA-2 epitope. To determine the basis for
differential expression of this minor Ag, we RT-PCR amplified and
sequenced the region of MYO1G known to encode the HA-2 epitope from
several additional cell lines. Approximately 50% of the molecular
clones from the HA-2+ cell lines JY, Blk, and
Maja, and all of the clones derived from the
HA-2- cell line JK-A2, contained a single G-to-A
transition at nucleotide 49 of the sequence in Fig. 2. This was the
only variation observed in over 100 independent clones from 14 cell
lines of hemopoietic origin. This change alters the sequence of the
HA-2 epitope from YIGEVLVSV to
YIGEVLVSM. We designated the sequence
YIGEVLVSV as HA-2V and
YIGEVLVSM as HA-2M, and
the respective genes as MYO1GV and
MYO1GM.
To confirm the genotypes inferred from direct sequencing, we took
advantage of the fact that the restriction endonucleases Hsp92II and
NlaIII specifically recognize the sequence CATG, which is
present only in HA-2M. In keeping with this, the
RT-PCR product from C1R-A2 (HA-2V/V by
sequencing) representing nucleotides 1–206 of the sequence in Fig. 2, was not cut by either Hsp92II or NlaIII, whereas
that obtained from JK-A2 (HA-2M/M by sequencing)
was completely cut into two fragments of 163 and 55 bp by both enzymes
(Fig. 4, top). Digestion of
the RT-PCR products from Blk, JY, and Maja
(HA-2V/M by sequencing) resulted in a mixture of
both cleaved and uncleaved fragments. These results established
definitively that the HA-2+ cell lines Rp and
C1R-A2 are homozygous for HA-2V, whereas JY, Blk,
and Maja are heterozygous. The HA-2- cell line
JK-A2 is homozygous for HA-2M.
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, bottom). Four family members whose
lymphoid cells were recognized by 5H17 CTL were found to be
heterozygous or homozygous for HA-2V, whereas two
that were phenotypically HA-2- were homozygous
for HA-2M. Similar results were seen with a
second, unrelated family (data not shown). This genealogy also
confirmed the autosomal, Mendelian inheritance of the HA-2
gene, as both parents are heterozygous at this locus and produced
offspring in all three allelic combinations. Tissue-specific expression of the HA-2 gene
HA-2+ cells of hemopoietic origin are well
recognized by 5H17 CTL, but cells derived from other tissue sources are
not (3). Thus, we examined whether MYO1G expression was
restricted to hemopoietic cells. We used RT-PCR to amplify nucleotides
1–334 of the sequence in Fig. 2 (the primer pair used does not
discriminate between MYO1GV and
MYO1GM). Strong amplification of the
HA-2-specific fragment was consistently seen in lymphocytic lines of
both B cell (Rp, C1R-A2, JY, Blk, and 18LCL) and T cell (Jurkat) origin
(Fig. 5). Interestingly, however, MYO1G
was not expressed in K562 erythroleukemia cells, which represent
erythroblasts, and had not been assessed for HA-2 expression using CTL
because they are class I MHC negative. Conversely, no amplification of
the HA-2-specific fragment was observed in melanoma (18 Mel, DM93),
embryonal kidney (293K), melanocyte (MC292, MC293), proximal tubule
epithelial cells (PTEC29), HUVEC (HUVE59, HUVE62), and two samples of
epidermal keratinocytes (KC25, KC45). Very weak signals were seen in
lung carcinoma (VBT2), ovarian carcinoma (COV413), colorectal carcinoma
(SW480), osteosarcoma (Tk-), fibroblasts (GM0568), and two other
samples of epidermal keratinocytes (KC8, KC42). However, because
HA-2-specific CTL fail to recognize these nonhemopoietic cells
(3), this low level of gene expression does not result in
peptide presentation at the cell surface. Thus, high-level expression
of MYO1G is restricted to hemopoietic cells, consistent with the
expression of the HA-2 epitope.
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To more clearly define the basis for the
HA-2+ and HA-2-
phenotypes, we compared the recognition of HA-2V
and HA-2M by the HA-2-specific CTL clone 5H17.
Surprisingly, when these peptides were exogenously pulsed onto T2
(HA-2-, HLA-A*0201+)
target cells, we found that both were recognized at a similar peptide
concentration (Fig. 6). The difference in
recognition between the two allelic forms of the peptide ranged from 2-
to 10-fold in five independent experiments (data not shown). This
suggested that differences in the endogenous processing and
presentation of these two peptides, rather than T cell specificity,
accounted for the failure of cells that expressed
MYO1GM only to be recognized by 5H17
CTL.
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The cell surface density of peptide achieved under the short-term
pulsing conditions used to make CTL targets in the above experiments
minimize differences in peptide affinities for class I MHC molecules
that might be important during peptide binding in the ER
(24). Accordingly, we performed quantitative, cell-free
peptide binding assays to determine the affinity of the
HA-2V and HA-2M peptides
for HLA-A*0201. HA-2V half-maximally inhibited
the binding of an iodinated indicator peptide
(IC50) at a concentration of 4.4 nM, whereas
comparable inhibition by HA-2M required 15
times as much peptide (Table I). Thus,
substitution of Met for Val at position 9 of HA-2 reduces peptide
binding significantly, but this appeared unlikely to be large enough to
account for the difference in recognition of
HA-2+ and HA-2- cells.
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54 copies/cell (Fig. 7). Although JY also expresses mRNA
encoding HA-2M (Fig. 4), a similar analysis of
fragmentation spectra established that this peptide was not detected at
a level above 0.2 copies/cell (data not shown). The absence of the
HA-2M peptide was also established by analysis of
the JK-A2 extract using a FTMS, which is 10x more sensitive than
the LCQ. A single mass with an m/z of
1010.52+1 and an appropriate elution time for
HA-2M was detected in a single HPLC fraction of
this sample (Fig. 8). However, when
synthetic HA-2M was added to an aliquot of this
sample and a second analysis performed, the endogenous peptide eluted
almost a full half-minute before synthetic HA-2M.
No peptide of the appropriate mass was detected at this elution
position in the original sample. In these experiments, we also did not
detect appropriate masses at the elution position of HA-2 M with an
oxidized Met residue (25) (data not shown). Based on the
amount of peptidic material loaded and an instrument detection limit of
6 amol, the detection limit in these experiments is 0.04
copies/cell. We conclude that HA-2M peptide was
present at less than this level in the JK-A2 extract, despite the
expression of mRNA encoding HA-2M in this
cell.
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).
Although it is possible that the actual substrates for TAP transport
may correspond to precursors peptides rather than the mature 9 mer
epitope, these data are consistent with the hypothesis that factors
other than TAP transport determine the differential expression of the
HA-2V and HA-2M peptides in
association with HLA-A*0201.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The results of our work also provide an understanding of how HA-2 is
expressed as a mHag. By analyzing the sequence of MYO1G in cells from
different individuals, we identified only a single polymorphism that
encoded either a valine or a methionine at position 9 of the HA-2
peptide sequence. The inheritance of this polymorphism establishes two
alleles of MYO1G. Furthermore, there was complete concordance between
the presence MYO1GV and detection of HA-2 by CTL,
as well as the homozygous presence of MYO1GM and
the lack of detection of HA-2. Despite the differences in peptide
structure, both HA-2V and
HA-2M are recognized similarly by HA-2-specific
CTL when the synthetic peptides are exogenously pulsed onto
HLA-A*0201+ target cells. In addition, the
equilibrium binding of HA-2M to HLA-A*0201 is
only 12-fold lower than that of HA-2V. Based
on the detection of HA-2V at 54 copies/cell on
JY (HA-2V/M) and assuming that equilibrium
binding conditions exist in the ER, we would expect that
HA-2M should be detected at roughly 4–8
copies/cell and that such cells should be relatively well-recognized by
HA-2-specific CTL. However, we found no HA-2M
above a level of 0.04 copies/cell in the material derived from either
JY or JK-A2 (HA-2M/M).
One possible explanation for the failure of HA-2M to be presented is that it is transported into the ER relatively poorly by TAP. In fact, we have recently shown this to be the basis for differential expression of another human mHag, HA-8 (26). However, the V-to-M change is relatively conservative, and Met at P9 is not contraindicated for successful TAP transport (27, 28, 29). Furthermore, direct analysis of the ability of these two peptides to be transported by TAP was not consistent with this explanation. A second possibility is that MYO1GV and MYO1GM are processed differently by the proteasome, such that substitution of an M for a V results in either creation or loss of a cleavage site. Although the specific sites recognized by proteasomes are less well defined than are the binding motifs for TAP and HLA-A*0201, this amino acid change is rather minor, and it is not immediately evident that such a switch would have an effect on proteasomal cleavage (30). Finally, it should be noted that our analysis of polymorphism encompasses only the 24 bp immediately upstream of the HA-2 sequence. We have not defined the complete 5' end of MYO1G or the sequence of any relevant control sequences. Thus, it is possible that as yet unrecognized differences in MYO1GM influence proteolytic cleavage efficiency or proper translation of the gene. In addition, our RT-PCR conditions were not quantitative, such that there might be unrecognized differences in the levels of transcription of the two alleles. However, in this regard it should be noted that we were able to detect very low levels of transcription of MYO1G in a few cell lines, yet these were not recognized by HA-2-specific CTL. Further experimentation will be required to fully resolve this issue.
In keeping with previous work on HA-2 expression (5), we found significant transcription of MYO1G only in cells of hemopoietic origin. Such highly restricted tissue expression suggests great promise for immunotherapy. In addition to those derived posttransplant from the original patient, HA-2-specific T cells have now been generated ex vivo and shown to efficiently lyse EBV-BLCL and malignantly transformed leukemic blasts, while ignoring cells of nonhemopoietic origin (31). Thus, they represent excellent potential adoptive immunotherapy reagents in conjunction with BMT for hemologic malignancies. It is significant that we failed to detect expression of MYO1G in K562 erythroleukemia cells. The identity of the gene in the present work should enable a more thorough examination of the tissues in which it is expressed.
HA-2 is also expressed in nonmalignant leukocytes (PBL, thymocytes, monocytes, dendritic cells) and should thereby present a target on those cells that mediate autoimmune disease in patients with the proper haplotype. Although not yet common practice, studies in both mice and humans have suggested the potential of BMT as a curative treatment for various autoimmune diseases (32, 33, 34, 35, 36, 37). Therefore, it is feasible that HA-2-specific T cells could also be used to treat these diseases in the same way that they might be used to treat leukemia. Finally, the facts that the HA-2 epitope is so prevalent (95% in the HLA-A*0201+ population), and that disparity for this minor H Ag has not been shown to have a positive correlation with the incidence of GVHD, only reinforce the potential therapeutic value of T cells directed against this Ag.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Victor H. Engelhard, Carter Immunology Center, University of Virginia, P.O. Box 801386, Charlottesville, VA 22908. E-mail address: vhe{at}Virginia.edu
3 Abbreviations used in this paper: mHag, minor histocompatibility Ag; GVHD, graft-versus-host disease; BMT, bone marrow transplant; EBV-BLCL, EBV-transformed B lymphoblastoid cell line; MS/MS, tandem mass spectrometry; amu, atomic mass unit; m/z, mass-to-charge; ER, endoplasmic reticulum; FTMS, Fourier transform ion cyclotron resonance mass spectrometer.
4 The reverse numbering indicates that the HA-2 epitope is encoded on the complementary (reverse) strand of AC004847.
Received for publication April 17, 2001. Accepted for publication July 13, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 domain of class I influence the magnitude of the xenogeneic response. J. Exp. Med. 170:1091.This article has been cited by other articles:
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T. Kawase, Y. Akatsuka, H. Torikai, S. Morishima, A. Oka, A. Tsujimura, M. Miyazaki, K. Tsujimura, K. Miyamura, S. Ogawa, et al. Alternative splicing due to an intronic SNP in HMSD generates a novel minor histocompatibility antigen Blood, August 1, 2007; 110(3): 1055 - 1063. [Abstract] [Full Text] [PDF]
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E. H. Slager, M. W. Honders, E. D. van der Meijden, S. A. P. van Luxemburg-Heijs, F. M. Kloosterboer, M. G. D. Kester, I. Jedema, W. A. E. Marijt, M. R. Schaafsma, R. Willemze, et al. Identification of the angiogenic endothelial-cell growth factor-1/thymidine phosphorylase as a potential target for immunotherapy of cancer Blood, June 15, 2006; 107(12): 4954 - 4960. [Abstract] [Full Text] [PDF]
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 H. Torikai, Y. Akatsuka, M. Miyazaki, E. H. Warren III, T. Oba, K. Tsujimura, K. Motoyoshi, Y. Morishima, Y. Kodera, K. Kuzushima, et al. A Novel HLA-A*3303-Restricted Minor Histocompatibility Antigen Encoded by an Unconventional Open Reading Frame of Human TMSB4Y Gene J. Immunol., December 1, 2004; 173(11): 7046 - 7054. [Abstract] [Full Text] [PDF]
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S. S. Tykodi, E. H. Warren, J. A. Thompson, S. R. Riddell, R. W. Childs, B. E. Otterud, M. F. Leppert, R. Storb, and B. M. Sandmaier Allogeneic Hematopoietic Cell Transplantation for Metastatic Renal Cell Carcinoma after Nonmyeloablative Conditioning: Toxicity, Clinical Response, and Immunological Response to Minor Histocompatibility Antigens Clin. Cancer Res., December 1, 2004; 10(23): 7799 - 7811. [Abstract] [Full Text] [PDF]
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 A. W. Purcell and J. J. Gorman Immunoproteomics: Mass Spectrometry-based Methods to Study the Targets of the Immune Response Mol. Cell. Proteomics, March 1, 2004; 3(3): 193 - 208. [Abstract] [Full Text] [PDF]
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M. H. M. Heemskerk, M. Hoogeboom, R. A. de Paus, M. G. D. Kester, M. A. W. G. van der Hoorn, E. Goulmy, R. Willemze, and J. H. F. Falkenburg Redirection of antileukemic reactivity of peripheral T lymphocytes using gene transfer of minor histocompatibility antigen HA-2-specific T-cell receptor complexes expressing a conserved alpha joining region Blood, November 15, 2003; 102(10): 3530 - 3540. [Abstract] [Full Text] [PDF]
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E. Spierings, A. G. Brickner, J. A. Caldwell, S. Zegveld, N. Tatsis, E. Blokland, J. Pool, R. A. Pierce, S. Mollah, J. Shabanowitz, et al. The minor histocompatibility antigen HA-3 arises from differential proteasome-mediated cleavage of the lymphoid blast crisis (Lbc) oncoprotein Blood, July 15, 2003; 102(2): 621 - 629. [Abstract] [Full Text] [PDF]
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 Y. Akatsuka, T. Nishida, E. Kondo, M. Miyazaki, H. Taji, H. Iida, K. Tsujimura, M. Yazaki, T. Naoe, Y. Morishima, et al. Identification of a Polymorphic Gene, BCL2A1, Encoding Two Novel Hematopoietic Lineage-specific Minor Histocompatibility Antigens J. Exp. Med., June 2, 2003; 197(11): 1489 - 1500. [Abstract] [Full Text] [PDF]
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M. Murata, E. H. Warren, and S. R. Riddell A Human Minor Histocompatibility Antigen Resulting from Differential Expression due to a Gene Deletion J. Exp. Med., May 19, 2003; 197(10): 1279 - 1289. [Abstract] [Full Text] [PDF]
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H. Sahara and N. Shastri Second Class Minors: Molecular Identification of the Autosomal H46 Histocompatibility Locus as a Peptide Presented by Major Histocompatibility Complex Class II Molecules J. Exp. Med., February 3, 2003; 197(3): 375 - 385. [Abstract] [Full Text] [PDF]
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E. P. Hochberg, D. B. Miklos, D. Neuberg, D. A. Eichner, S. F. McLaughlin, A. Mattes-Ritz, E. P. Alyea, J. H. Antin, R. J. Soiffer, and J. Ritz A novel rapid single nucleotide polymorphism (SNP)-based method for assessment of hematopoietic chimerism after allogeneic stem cell transplantation Blood, January 1, 2003; 101(1): 363 - 369. [Abstract] [Full Text] [PDF]
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 T. Nebl, K. N. Pestonjamasp, J. D. Leszyk, J. L. Crowley, S. W. Oh, and E. J. Luna Proteomic Analysis of a Detergent-resistant Membrane Skeleton from Neutrophil Plasma Membranes J. Biol. Chem., November 1, 2002; 277(45): 43399 - 43409. [Abstract] [Full Text] [PDF]
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