A Novel HLA-A*3303-Restricted Minor Histocompatibility
Antigen Encoded by an Unconventional Open Reading Frame
of Human TMSB4Y Gene1
Hiroki Torikai,2*‡Yoshiki Akatsuka,2,3* Mikinori Miyazaki,* Edus H. Warren III,§Taku Oba,¶
Kunio Tsujimura,* Kazuo Motoyoshi,‡Yasuo Morishima,†Yoshihisa Kodera,¶
Kiyotaka Kuzushima,* and Toshitada Takahashi*
Female-to-male hemopoietic stem cell transplantation (HSCT) elicits T cell responses against male-specific minor histocompati-
bility (H-Y) Ags encoded by the Y chromosome. All previously identified H-Y Ags are encoded by conventional open reading
frames, but we report in this study the identification of a novel H-Y Ag encoded in the 5?-untranslated region of the TMSB4Y gene.
An HLA-A*3303-restricted CD8?CTL clone was isolated from a male patient after an HSCT from his HLA-identical sister. Using
a panel of cell lines carrying Y chromosome terminal deletions, a narrow region controlling the susceptibility of these target
cells to CTL recognition was localized. Minigene transfection and epitope reconstitution assays identified an 11-mer peptide,
EVLLRPGLHFR, designated TMSB4Y/A33, whose first amino acid was located 405 bp upstream of the TMSB4Y initiation codon.
Analysis of the precursor frequency of CTL specific for recipient minor histocompatibility Ags in post-HSCT peripheral blood T
cells revealed that a significant fraction of the total donor CTL response in this patient was directed against the TMSB4Y epitope.
Tetramer analysis continued to detect TMSB4Y/A33-specific CD8?T cells at least up to 700 days post-HSCT. This finding
underscores the in vivo immunological relevance of minor histocompatibility Ags derived from unconventional open reading frame
products. The Journal of Immunology, 2004, 173: 7046–7054.
some-specific genes (1–3). Disparities in some minor H Ags in
allogeneic hemopoietic stem cell transplantation (HSCT) have
been shown to be associated with graft-vs-host disease (GVHD),
graft rejection, or graft-vs-leukemia/lymphoma (GVL) effect (4–
inor histocompatibility (minor H)4Ags are MHC-
bound peptides derived from cellular proteins and are
encoded by polymorphic genes, including Y chromo-
11). In the case of female to male HSCT, T cell clones specific for
Y chromosome-encoded (H-Y) Ags were generated from the pe-
ripheral blood of recipients during GVHD or graft rejection, and
their HLA class I or II epitopes have been identified, including
SMCY (12, 13), DFFRY (14, 15), UTY (16, 17), RPS4Y (18), and
DBY (19, 20). These five genes are among eight genes that have
been reported to lie in the nonrecombining region of the human Y
chromosome and have functional X homologues (21). Because all
eight genes are sufficiently polymorphic with their X chromosome
homologues to induce H-Y-specific T cell responses, it should be
possible that more H-Y epitopes can be encoded either by the five
genes that have proved to be immunogenic or by other Y chro-
mosome genes (i.e., ZFY, AMELY, and TMSB4Y) for which H-Y
epitopes have not yet been described.
In this study we report the identification of a novel human H-Y
Ag, recognized by an HLA-A*3303-resricted CTL clone isolated
from a male patient who developed chronic, but not acute, GVHD.
The identified H-Y Ag is an 11-mer peptide, EVLLRPGLHFR,
derived from TMSB4Y, a gene encoding thymosin ?-4, Y isoform
(22). Interestingly, the epitope identified in the TMSB4Y gene was
encoded by the polymorphic region located 405 bp upstream of the
initiation codon of the conventional open reading frame (ORF),
whereas all minor H Ags identified to date are encoded by con-
ventional ORF of the individual gene. There have been several
reports describing CTL epitopes encoded by unconventional
ORFs, such as untranslated regions (UTR) or alternative reading
frames, most of which have been identified in tumor cells (re-
viewed in Ref. 23). To our knowledge, this is the first demonstra-
tion of a minor H Ag encoded in a region other than conventional
coding region. Furthermore, we demonstrated, by CTL precursor
(CTLp) frequency analysis, that a significant fraction of the total
donor CTL responses in this patient was directed against the
TMSB4Y epitope, and that the precursor remained detectable up to
*Division of Immunology, Aichi Cancer Center Research Institute, and†Department
of Hematology and Chemotherapy, Aichi Cancer Center Hospital, Nagoya, Japan;
‡Third Department of Internal Medicine, National Defense Medical College, To-
korozawa, Japan;§Program in Immunology, Fred Hutchinson Cancer Research Cen-
ter, Seattle, WA 98109; and
Nagoya First Hospital, Nagoya, Japan
¶Department of Hematology, Japanese Red Cross
Received for publication August 4, 2004. Accepted for publication September
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1This work was supported in part by a Grant-in-Aid for Scientific Research (C) (to
Y.A.) and Scientific Research on Priority Areas (to T.T., K.T., and Y.A.), from the
Ministry of Education, Culture, Science, Sports, and Technology, Japan; Research on
Human Genome, Tissue Engineering Food Biotechnology (to Y.A.), and Second
Team Comprehensive 10-year Strategy for Cancer Control (to T.T.), from the Min-
istry of Health, Labor, and Welfare, Japan; a grant from Aichi Cancer Research
Foundation (to Y.A.); a Nagono Medical Research Grant (to K.K. and Y.A.); a grant
of Entrusted Research with DNA Bank, RIKEN BioResource Center (to Y.A.); and
a Lilly Clinical Investigator Award from the Damon Runyon Cancer Research Foun-
dation (to E.H.W.).
2H.T. and Y.A. contributed equally to this work.
3Address correspondence and reprint requests to Dr. Yoshiki Akatsuka, Division of
Immunology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku,
Nagoya 464-8681, Japan. E-mail address: email@example.com
4Abbreviations used in this paper: minor H Ag, minor histocompatibility Ag; CI,
confidence interval; CTLp, CTL precursor; DRiP, defective ribosomal product;
GVHD, graft-vs-host disease; GVL, graft vs leukemia/lymphoma; HSCT, hemopoi-
etic stem cell transplantation; H-Y Ag, Y chromosome-encoded Ag; LCL, B-lym-
phoblastoid cell line; ORF, open reading frame; UTR, untranslated region; STS, se-
The Journal of Immunology
Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00
700 days after HSCT. These findings underscore the in vivo im-
munological relevance of such a cryptic minor H Ag derived from
unconventional ORF products.
Materials and Methods
Cell cultures and Abs
The HLA-A*3303-restricted CD8?CTL clone, 1B6, was isolated by lim-
iting dilution from a cytotoxic T cell line generated from a PBMC sample
obtained on day 50 post-HSCT from a 54-year-old man (HLA-A*2402/
*3303, B*4403/*5401, Cw*0803/*1403) who had received his HLA-iden-
tical sister’s marrow for treatment of chronic myelocytic leukemia. He did
not develop acute GVHD, but did develop mild chronic GVHD of the skin
and liver. The CTL clone was expanded as previously described (24) and
frozen until use. B-lymphoblastoid cell lines (LCLs) were established from
the donor and recipient and from normal volunteers. All blood or tissue
samples were collected after obtaining written informed consent, and the
study was approved by the institutional review board of Aichi Cancer
The LCLs derived from individuals with Y chromosome deletions were
provided by Dr. D. C. Page (Howard Hughes Medical Institute, Whitehead
Institute, Massachusetts Institute of Technology, Cambridge, MA), and a
detailed analysis of these lines has been previously reported (25). LCLs
selected according to their deletion pattern and other cell lines including
Raji were retrovirally transduced with HLA-A*3303 cDNA as described
previously (26). LCLs were maintained in RPMI 1640 medium (Sigma-
Aldrich, St. Louis, MO) supplemented with 10% FCS (Immuno-Biological
Laboratory, Gunma, Japan), 2 mM L-glutamine, 1 mM sodium pyruvate,
and penicillin/streptomycin. Primary dermal fibroblast lines from skin and
oral mucosa, bone marrow stromal cell lines, and 293T cells were grown
in IMDM (Invitrogen Life Technologies, Carlsbad, CA) supplemented
with 10% FCS, 2 mM L-glutamine, and penicillin/streptomycin. mAbs,
W6/32 (anti-pan HLA class I), HDR-1 (anti-HLA-DR), and A11.1M (anti-
HLA-A24) were provided by Dr. K. Ito (Kurume University, Fukuoka,
Target cells were labeled with 0.1 mCi of51Cr for 2 h, and 1 ? 103target
cells/well were mixed with CTL at various E:T cell ratios in a standard 4-h
cytotoxicity assay using 96-well, round-bottom plates. All assays were per-
formed at least in duplicate. Cells were treated with IFN-? (100 U/ml;
Endogen, Woburn, MA) and TNF-? (10 ng/ml; Endogen) for 48 h where
indicated. The percent specific lysis was calculated as follows: ((experi-
mental cpm ? spontaneous cpm)/(maximum cpm ? spontaneous cpm)) ?
100. When necessary, allo-HLA-A24-specific CTL clones were used to
confirm the susceptibility of the target cells.
Mapping of Y chromosome deletion mutant LCLs
Oligonucleotide primer pairs specific for sequence-tagged sites (STSs) pre-
viously mapped to the Y chromosome (25) were used to PCR-amplify the
corresponding Y chromosomal target sequences from genomic DNA of
each LCL. Amplification of STSs was performed as reported previously
(16). Aliquots of each PCR were separated in 2% agarose or 5% acryl-
amide gels, and cell lines were scored as positive or negative for the pres-
ence of each STS. DNA extracted from LCLs derived from normal male
and female donors served as positive and negative controls, respectively.
Detection of expression of the candidate genes
An RT-PCR assay was used to examine the expression of the candidate
genes with cDNA synthesized from LCLs. PCR was performed in a total
volume of 20 ?l containing 1? PCR buffer, 1.5 mM MgCl2, 200 ?M of
each dNTP, 0.5 ?M of each gene-specific primer, and 1 U of AmpliTaq
DNA polymerase (Applied Biosystems, Foster City, CA) on a GeneAmp
PCR system 9700 (Applied Biosystems). The PCR products were separated
in 2% agarose gels and visualized with ethidium bromide staining.
PCR cloning of TMSB4Y gene
The conventional ORF sequence and full-length sequence of the TMSB4Y
(GenBank accession no. NM_004202) were amplified from cDNA pre-
pared from the recipient LCL and subcloned into a mammalian expression
plasmid. The primer sequences used were as follows (HindIII and NotI
sites are underlined, respectively): conventional ORF sense, 5?-TTA-
AGCTTCGCAGCCATGTCTGACAAACC-3?; conventional ORF anti-
sense, 5?-ATGCGGCCGCCATGCCTGTTTAAGATTCGC-3?; full-length
sense, 5?-TTAAGCTTTGGGAACAGACAGATCCTTTG-3?; and full-
length antisense, 5?-ATGCGGCCGCTAGATTTCACTGCCCTCCCA-3?.
PCR amplification was conducted in a total volume of 25 ?l of 1? buffer
containing 200 ?M of each dNTP, 1.0 mM MgSO4, 0.3 ?M of each
primer, and 1 U of KOD-Plus-DNA polymerase (TOYOBO, Osaka,
All products were digested with the restriction enzymes and ligated into
HindIII-NotI-cut pEAK10 vector (Edge Biosystems, Gaithersburg, MD).
The sequences of the cloned genes were verified by direct sequencing with
BigDye Terminator kit (version 3.0, Applied Biosystems) on an ABI
PRISM 3100 (Applied Biosystems).
Construction of truncated genes and minigenes for TMSB4Y
Expression plasmids encoding truncated forms of the TMSB4Y cDNA
were constructed by RT-PCR using antisense primers that produced 345,
552, 754, and 955 bp DNA fragments. All products were ligated into the
pEAK10 vector as described above. Minigene expression plasmids encod-
ing the minimal, N or C terminus-extended polypeptides of the epitope
predicted by BIMAS software (http://bimas.dcrt.nih.gov/molbio/hla_bind/)
(27) and SYFPEITHI software (http://syfpeithi.de) (28) were constructed
as previously described (29). The constructs all encoded a Kozak sequence
and initiator methionine (CCACC-ATG) and a stop codon (TAG). Pairs of
sense and antisense oligonucleotides were designed to form cohesive ends
for HindIII and NotI sites at the 5? and 3? ends after hybridization, respec-
tively, and all products were ligated into the pEAK10 vector and verified
Electroporation of LCL
The constructed vectors were introduced either into the donor LCL or into
293T cells. One million LCL were resuspended in 40 ?l of OPTI-MEM I
buffer (Invitrogen Life Technologies) and 4 ?g of each plasmid in a 2-mm
gap cuvette, and electroporated in an ECM 830 BTX Electro Square Po-
rator (BTX, San Diego, CA) at 350 V and a pulse length of 1 ms. Then,
cells were cultured in 4 ml of culture medium for 2 d, followed by selection
with puromycin (0.7 ?g/ml) for 3 d before use.
Transfection of 293T cells and cytokine release assays
293T cells were retrovirally transduced with HLA-A*3303 cDNA and se-
lected in the presence of 1 ?g/ml puromycin (referred to as 293T-A33).
Aliquots of the 293T-A33 cells were transiently cotransfected with
pEAK10 vectors encoding full-length TMSB4Y, a C-terminal deletion mu-
tant cDNA, or minigenes of TMSB4Y. 293T-A33 cells were plated the day
before transfection at 4 ? 104cells/100 ?l/well into 96-well, flat-bottom
microtiter plates and transfected with 6 ?l of RPMI 1640 containing 90 ng
of plasmid DNA and 0.27 ?l of FuGENE 6 (Roche, Indianapolis, IN).
After 24 h at 37°C, 100 ?l of a cell suspension containing 1 ? 104CTL
clone 1B6 in IMDM containing 20 U/ml IL-2 was added. Supernatants
from the cocultures were harvested after 24 h and assayed for the presence
of IFN-? by ELISA.
Epitope reconstitution assay
The candidate peptide epitope identified by the minigene experiments and
the homologous TMSB4X-encoded peptide were synthesized by standard
methods.51Cr-labeled donor LCL were incubated for 30 min in medium
containing 10-fold serial dilutions of the peptides and then used as target
cells in standard cytotoxicity assays.
Real-time PCR assay for TMSB4Y expression
cDNA from a panel of different human adult and fetal tissues were pur-
chased from BD Clontech (MTC panels human I and II; Palo Alto, CA) or
synthesized from total RNA of human lung (BD Clontech) or various cul-
tured cells. PCR amplification and real-time quantification analysis were
performed using the TaqMan assay according to the manufacturer’s in-
structions. The following sequences were used as primers and TaqMan
probe to detect the mRNA region encoding the epitope: 5?-GACTAGA
AAGCGGGCGCAG-3? (sense; nt 302–320), 5?-ACTTCCGCGTTCAA
GTGGTT-3? (antisense; nt 415–434), 5?-(FAM)-TCCCTTCTCGACACG
GAGTCTATGTGTAGT-(MGB)-3? (TMSB4Y probe; antisense; nt 366–
382). For the internal control, a primer and probe set for human GAPDH
(Applied Biosystems) was used. PCR was performed in a 1? TaqMan
Universal PCR master mix containing 10 pmol of each sense and antisense
primer and 2 pmol of probe in a total volume of 25 ?l in the ABI PRISM
7700HT Sequence Detector System (Applied Biosystems). The tempera-
ture profile was 50°C for 2 min, 95°C for 10 min, and then 95°C for 15 s
and 62°C for 1 min for 40 cycles. Samples were quantified using relative
standard curves for each amplification. All results are normalized with
7047 The Journal of Immunology
respect to the internal control and are expressed relative to the levels found
in a pool of male PBMC.
Limiting dilution-based CTLp frequency assay
The proportion of CTLp specific for the TMSB4Y peptide among the total
CTLp against the recipient minor H Ags was quantitated using a standard
limiting dilution assay. Purified CD8?T cells from the PBMC obtained at
days 50 and 146 post-HSCT were cultured at 2-fold serial dilutions with 33
Gy-irradiated 3 ? 104CD40-activated B (CD40-B) cells generated from
pre-HSCT recipient PBMC in 96-well, round-bottom plates in RPMI 1640
medium (Sigma-Aldrich) supplemented with 10% pooled human serum.
IL-2 (50 U/ml) was added on days 2 and 5 after each restimulation with
CD40-B cells. For each dilution, there were at least 12 replicates. After
three rounds of stimulation, a split-well analysis was performed for pep-
tide-specific cytotoxicity against51Cr-radiolabeled recipient PHA blasts or
donor PHA blasts pulsed with TMSB4Y peptide or unpulsed. The super-
natants were measured in a gamma counter after 4-h incubation. The wells
were considered to be positive for lytic activity if the total cpm released by
effector cells was ?2.5 ? SD above control wells (mean cpm released by
the target cells incubated with irradiated stimulator cells alone). The CTLp
frequency was calculated by L-Calc software (StemCell Technologies,
Tetramer construction and flow cytometric analysis
MHC-peptide tetramers were produced as described previously (30). In
brief, HLA-A*3303 H chain and ?2-microglobulin (cloned in pHN1?vec-
tor; provided by the late Dr. D. C. Wiley, Howard Hughes Medical Insti-
tute, Harvard University, Cambridge, MA) were produced in XA90. The C
terminus of the H chain was modified by the addition of a substrate se-
quence for the biotinylating enzyme BirA. Monomeric HLA/?2-micro-
globulin/peptide complexes were folded in vitro in the presence of the
peptide. The MHC complex was biotinylated and then converted into tet-
ramers with PE-labeled streptavidin. For staining, PBMC or T cell lines
were incubated with the tetramer at a concentration of 20 ?g/ml at room
temperature for 15 min, followed by FITC-conjugated anti-CD3 (BD
Biosciences, San Diego, CA) and Tricolor anti-CD8 mAb (Caltag Labo-
ratories, Burlingame, CA) on ice for 15 min. Cells were analyzed with a
FACSCalibur flow cytometer and CellQuest software (BD Biosciences).
A CD8?CTL clone shows cytotoxicity against an H-Y Ag
presented on HLA-A*3303?LCL
CD8?CTL clone 1B6 efficiently lysed recipient LCL and PHA
blasts, but not donor LCL (Fig. 1A). Addition of anti-pan HLA
class I mAb, but not anti-HLA-A24 or anti-HLA-DR mAbs, sig-
nificantly inhibited lysis of recipient LCL by 1B6. Transduction of
HLA-A*3303 cDNA into a male LCL conferred susceptibility to
1B6, indicating that the clone was restricted by HLA-A*3303 (Fig.
1B). 1B6 showed very weak cytotoxicity against dermal or oral
fibroblasts or against keratinocytes generated from HLA-A*3303-
positive male individuals, whereas these targets were lysed mod-
erately (i.e., 25–35%) by CTL specific for HLA-A24 alloantigen,
which is shared by these targets. Even when they were treated with
cytokines (IFN-? and TNF-?), 1B6 still demonstrated relatively
weak cytotoxicity, although HLA-A24-allospecific CTL induced
robust cytotoxicity (Fig. 1C). Finally, 1B6 showed lytic activity
only against male, but not female, LCLs transfected with HLA-
A*3303 cDNA, indicating that the clone was specific for a H-Y Ag
(data not shown).
The gene encoding the minor H Ag maps to deletion interval 5D
on the Y chromosome
Cytotoxicity assay-based mapping was conducted to determine the
location on the Y chromosome of the minor H gene encoding the
epitope for 1B6. First, various LCLs were typed for terminal de-
letions of the Y chromosomes using the technique of STS content
mapping (16, 21). Of these, a panel of LCLs with distinct terminal
deletions was selected for transfection with HLA-A*3303 cDNA
and assayed for susceptibility to 1B6.
Fig. 2A shows the 43-interval deletion map of the 7 LCLs and
their susceptibility to 1B6. LCL WHY10 and WHY12 that were
lysed by 1B6 share only deletion intervals 5C and 5D, indicating
that the region controlling the expression of this minor H Ag is
analyzed in standard51Cr release assays. A, 1B6 recognition of target cells
derived from recipient (Rt) LCL, PHA blasts, or donor (Do) LCL at the E:T
cell ratios indicated. B, Ab blocking of the cytolysis was performed with
anti-HLA mAbs (E:T cell ratio, 1:1). HLA-A*3303-negative male LCL
with or without HLA-A*3303 transduction were tested at an E:T cell ratio
of 10:1. C, Cytolytic activity of 1B6 or allo-HLA-A*2402-specific CTL
against cytokine-treated or untreated HLA-A*3303 and A*2402-positive
male B-LCL, dermal fibroblasts, oral fibroblasts, and keratinocytes was
tested at an E:T cell ratio of 10:1. The cytokine treatment used was incu-
bation of targets cells with 100 U/ml IFN-? and 10 ng/ml TNF-? for 48 h
before51Cr labeling. The lysis of cytokine-treated cells by 1B6 (f) or
allo-specific CTL (?) and of cytokine untreated cells by 1B6 (z) or allo-
specific CTL (u) is shown.
Specificity of the HLA-A*3303-restricted CTL clone, 1B6,
70485?UTR OF TMSB4Y GENE ENCODES HLA-A33-RESTRICTED MINOR Ag
located within these two deletion intervals. The region was further
narrowed down using the results from LCL WHY24, which was
found to lack deletion interval 5C, but was nevertheless lysed by
1B6. Collectively, these results indicate that the gene encoding the
minor H Ag maps to deletion interval 5D. Four genes, DFFRY,
DBY, UTY, and TMSB4Y, all of which have X homologues, are
encoded within deletion intervals 5C and 5D (21, 25). We exam-
ined mRNA expression of these four genes among the seven LCLs
by RT-PCR (Fig. 2B). As expected from the results of deletion
mapping, WHY6 and WHY11 were negative for the expression of
all four genes; WHY10, WHY9, WHY17, and WHY12 were all
positive. Because WHY24 was positive for the expression of
TMSB4Y and UTY and was lysed by 1B6, the minor H Ag was
encoded by either UTY or TMSB4Y. In addition, during the course
of specificity analysis, we found that HLA-A*3303-transduced
Raji cells were not killed by 1B6, although Raji cells are of male
origin. RT-PCR analysis showed that they were negative for ex-
pression of TMSB4Y as shown in Fig. 2B. Moreover, female LCLs
from the patient’s HSCT donor transfected with any of three iso-
forms of UTY cDNA were not lysed by 1B6 (data not shown).
These results indicated that TMSB4Y most likely encoded the mi-
nor H Ag.
The 5? untranslated region of the TMSB4Y gene encodes the
minor H Ag
To determine whether TMSB4Y indeed encodes the minor H
epitope recognized by 1B6, we first tested CTL recognition of the
female donor LCL transduced with the reported TMSB4Y ORF
comprising 43 aa. However, 1B6 did not lyse the transfectant (Fig.
3A), suggesting either that the epitope is encoded not by TMSB4Y
but by another gene located in deletion interval 5D, or that it is
encoded elsewhere in the ?1.7-kb TMSB4Y cDNA. Recently,
cryptic CTL epitopes encoded by alternative sources such as non-
coding regions and nonconventional ORF have been described
in both murine and human tumor cells (23). Thus, we cloned
NM_004202) and then transduced donor LCL with it. As shown in
Fig. 3B, female LCL expressing full-length TMSB4Y cDNA were
lysed efficiently. Because the alternative ORF that is able to en-
code the antigenic peptide was unknown, a series of 3? terminal
deletion mutants of the TMSB4Y cDNA were prepared and tested
for recognition by 1B6 by IFN-? ELISA. Although cells trans-
fected with TMSB4Y cDNA fragments extending from nt 1–552
were recognized when expressed in HLA-A*3303-transduced
293T cells, transfection of the fragment encoding nt 1–345 was not
(Fig. 3C). These results indicated that the epitope was encoded in
the 5?UTR between nt 346 and 552, which is at least 240 nt up-
stream of the reported ORF for the TMSB4Y protein (Fig. 4A).
Among three reading frames in this region, only one initiator
methionine (nt 362–364) was found in the same reading frame
encoding the TMSB4Y protein, followed by a polypeptide con-
sisting of 19 aa, EVLLRPGLHFRNSCPILTT. This 19-mer con-
tains a nonamer, LLRPGLHFR, which has the reported peptide-
binding motif for HLA-A*3303 (i.e., Ala, Ile, Leu, Phe, Tyr, or
Val at position 2, and Arg at C terminus) (31), with a predicted
dissociation score of 9.0 by BIMAS software (27). However, a
minigene construct encoding LLRPGLHFR failed to stimulate
1B6. Additional experiments using minigene constructs with
N or C extensions finally identified the minimal epitope as
EVLLRPGLHFR (Figs. 3D and 4A). Both Arg at the C terminus
and Glu at the N terminus were essential for recognition by 1B6,
indicating that Val and Arg are the likely N- and C-terminal an-
chors, respectively. The X homologue of TMSB4Y, TMSB4X
cDNA (GenBank accession no. NM_021109), encoding thymosin
?4, has much shorter 5?- and 3?UTR; thus, no corresponding re-
gion was found (Fig. 4B). However, a recently reported splice vari-
ant of TMSB4X, which includes 1076 bp of TMSB4X intron 1 (Gen-
Bank accession no. AK055976), has an initiator methionine and a
following 32 aa in its 5?UTR upstream TMSB4X conventional ORF,
and potentially encodes ETLFLPGLHFR, which differs from the1B6
terminal deletions of Y chromosome and their susceptibility to 1B6. Appropriate LCLs were selected based on their pattern of terminal deletions (16, 21,
25), transduced with HLA-A*3303 cDNA, and tested in standard51Cr release assays. The presence of the region encoding the minor H Ag in each LCL
line is determined by its susceptibility to 1B6 (indicated in the right column). Bidirectional arrows indicate the conserved region(s) from deletion and are
related to the 43-interval deletion map of the Y chromosome (21, 25). Vertical dotted bars indicate the region predicted to encode the minor H Ag. B, mRNA
expression of four genes encoded in the deletion intervals 5C and 5D in selected cell lines and their recognition by 1B6. Female donor LCL served as a
negative control. HLA-A*3303 transfected Raji cells, a Burkitt lymphoma cell line derived from a male patient, were also analyzed.
Mapping of the gene on the Y chromosome that encodes the minor H Ag recognized by 1B6. A, Genetic map of the LCLs carrying various
7049The Journal of Immunology
epitope, EVLLRPGLHFR, by three amino acids (underlined; Fig. 4,
B and C).
We next synthesized these two 11-mer peptides, EVLLRPGLHFR
and ETLFLPGLHFR, and tested the cytotoxicity of 1B6 against
donor LCL pulsed with serial dilutions of each peptide. The titra-
tion of peptide EVLLRPGLHFR recognized by 1B6 gave half-
maximal lysis at a concentration of 20 nM, whereas peptide
ETLFLPGLHFR failed to sensitize the donor LCL at any concentra-
tion tested (Fig. 5). Thus, EVLLRPGLHFR defines the HLA-
A*3303-restricted 1B6 epitope, and we designated it TMSB4Y/A33.
mRNA expression of the TMSB4Y gene is found in various
tissues and cell types
The X homologue of TMSB4Y, TMSB4X, has been shown to be
expressed in a broad range of tissue types in rodents, with very
high levels in spleen, thymus, and lung (32). To determine the
distribution of TMSB4Y expression in different tissues, quantitative
PCR analysis targeted to the 5?UTR of the mRNA was performed
using a large panel of test samples derived from different tissues.
PCR analysis demonstrated that the expression of TMSB4Y mRNA
assessed by its 5?UTR was indeed observed in a wide range of
normal tissues; from the highest expression in testis, prostate, pan-
creas, and hemopoietic cells to the lowest expression in dermal
fibroblasts and skeletal muscles (?50-fold less than that in hemo-
poietic cells; data not shown). Expression of the mRNA in a panel
of primary leukemic cells ranged from undetectable to levels sim-
ilar to those seen in normal hemopoietic cells (data not shown).
TMSB4Y/A33-specific CD8?T cells are detectable in recipient
A split-well assay was used to estimate the relative frequencies in
the post-HSCT PBMC of CTLp specific for the TMSB4Y/A33
minor H Ag and those specific for other minor H Ags expressed on
the recipient’s hemopoietic cells. As shown in Fig. 6A, the fre-
quencies of CTLp reactive with recipient PHA blasts and
TMSB4Y/A33 peptide-pulsed donor PHA blasts in peripheral
blood obtained on day 50 post-HSCT from which the 1B6 was
derived were 324 (95% confidence interval (CI), 213–493) and 96
(95% CI, 47–175) per 106peripheral blood CD8?cells, respec-
tively, indicating that nearly a quarter of the CTL responses to
recipient minor H Ags in this donor/recipient pair were indeed
directed at the TMSB4Y/A33 minor H Ag. On day 146, the fre-
quency of CTLp recognizing TMSB4Y peptide-pulsed donor
PHA-blasts was 316 (95% CI, 216–464), and that for CTLp rec-
ognizing recipient PHA blasts was 3215 (95% CI, 2150–4808) per
106peripheral blood CD8?cells, demonstrating that even at the
later time point the CTL responses against TMSB4Y/A33 contin-
ued to account for a significant fraction (10%) of the total donor
CTL responses against recipient minor H Ags in this donor/recip-
ient pair (Fig. 6B).
In additional experiments, an HLA/peptide tetramer was used to
confirm the presence of TMSB4Y/A33-specific CTL in unstimu-
lated post-HSCT PBMC (Fig. C, left column) as well as in T cell
lines prepared by stimulating these PBMC with the same stimu-
lators used in the CTLp assay (Fig. C, right column). The assays
clearly detected TMSB4Y/A33-specific CD8?T cells in PBMC
obtained on day 696 (0.35%), but for PBMCs obtained on day 50
and 146, the presence of TMSB4Y/A33-specific T cells was not
clear because of the low number of PBMC available. After in vitro
stimulation, tetramer-positive cells became detectable for the latter
two samples, although direct comparison with the CTLp results
was not possible due to the use of different culture conditions in the
In this study we have identified a gene, TMSB4Y, encoding a novel
HLA-A*3303-restricted, H-Y Ag by testing HLA-A*3303-trans-
fected cell lines carrying terminal deletions of the Y chromosome
in cytotoxicity assays. This approach has previously been used to
identify the HLA-B8-restricted H-Y Ag encoded by UTY (16).
TMSB4Y. A, Mammalian expression plasmid encoding the conventional
ORF (cORF) of TMSB4Y, as identified in the deletion mapping (Fig. 2),
was transfected into donor (Do) LCL, and recognition of transfected LCL
by 1B6 was determined in a standard51Cr release assay. Recipient (Rt)
LCL were used as a positive control. B, Plasmid encoding the full-length
TMSB4Y cDNA containing the 5?- and 3?UTR (TMSB4Y-full; GenBank
accession no. NM_004202) was transfected into donor LCL, and their sus-
ceptibility to 1B6 was tested as described above. C, Localization of the
region encoding the minor H Ag by 3? deletion mutants of the TMSB4Y
cDNA. HLA-A*3303-transduced 293 T cells were transfected with plas-
mids encoding various 3?-deleted TMSB4Y cDNAs terminating at 345,
552, 754, or 955 nt and cocultured with 1B6. Supernatants were harvested
and assayed for the presence of IFN-? by ELISA. The OD of each super-
natant is shown. D, Identification of the 1B6 epitope. The susceptibilities
of HLA-A*3303-transduced 293 T cells transfected with minigene con-
structs encoding nonamer peptide (LLRPGLHFR) predicted by BIMAS
software (27) and selected N- or C-terminally extended peptides were
tested by ELISA.
Localization of the minor H Ag epitope defined by 1B6 in
70505?UTR OF TMSB4Y GENE ENCODES HLA-A33-RESTRICTED MINOR Ag
With the current discovery, all four identified genes that are en-
coded in deletion intervals 5C and 5D of the nonrecombining re-
gion of the human Y chromosome have been shown to encode at
least one minor H Ag presented by class I or II HLA (14–17, 19,
20). Although the peptide sequence of the TMSB4Y/A33 minor H
Ag identified in this study was 11 residues in length, and half-
maximal lysis of peptide-pulsed female target cells was observed
at a relatively high peptide concentration (?20 nM), it is likely that
the 11-mer peptide is the minimal epitope, because it has a con-
sensus Arg at the C terminus and a Val at the auxiliary anchor
(position 2) (31). In addition, two computer algorithms predict that
cleavage after the C-terminal Arg would be correctly performed by
proteasomes (33, 34). Although all previously identified human
H-Y Ags have homologue peptide on the ORF of their X homol-
ogous gene, it is not yet clear whether TMSB4Y/A33 minor H Ag
has its homologue, because the longest cDNA clone (GenBank
accession no. AK055976) assigned to be one of the splice variants
of the TMSB4X gene containing the first intronic sequence was not
detected by RT-PCR, whereas the full-length cDNA encoding thy-
mosin ?4 was readily detectable (data not shown). Thus, it is con-
ceivable that the splice variant, AK055976, might be very rare or
derived from a precursor mRNA.
Recently, evidence has been accumulating that cryptic polypep-
tides derived from noncoding regions, such as UTRs or introns, or
encoded in alternative ORFs occasionally encode CTL epitopes for
tumor or viral Ags in humans or mice (reviewed in Ref. 23). Of
these, only one epitope is found in the 5?UTR of a cellular onco-
gene, c-akt, in the murine RL?1 leukemia system (35). This un-
usual epitope is generated by insertion of the murine leukemia
virus long terminal repeat into the exon of c-akt, resulting in tran-
scription initiated at the cap site of the long terminal repeat. To the
best of our knowledge, this is the first demonstration of a minor H
Ag encoded outside a conventional ORF of a nonmutated gene. Al-
though it is possible that the 19-residue ORF in the 5?UTR that en-
codes the epitope is an as yet unrecognized functional coding region,
a search of the protein database, including the Protein-Protein Blast
(http://www.ncbi.nlm.nih.gov/blast/), for amino acid sequence ho-
mology to this region did not identify any known functional domains.
accession no. NM_004202). A, The deduced amino acid sequence is shown in one-letter designation below the nucleotide sequence for conventional ORF
(the middle of the nucleotide sequence) and 5?UTR encoding the epitope recognized by 1B6. Asterisks indicate stop codons, and arrows indicate the region
initially mapped by TMSB4Y cDNAs with nested 3? deletions. The sequence corresponding to the identified peptide is boxed. B, Genomic organization
of TMSB4Y and its X homologue, TMSB4X, and the relationship with their mRNAs. E1, E2, and E3 indicate exon 1, exon 2, and exon 3, respectively. The
conventional ORF is indicated below the mRNA as cORF. The location of identified 1B6 epitope is shown below the 5?UTR of TMSB4Y cDNA. The
putative X homologue peptide in a reported splice variant of TMSB4X containing its intron 1 (GenBank accession no. AK055976) is indicated by
the arrowhead. C, Comparison of the deduced amino acid sequence in the 5?UTR encoding the 1B6 epitope in TMSB4Y and the putative X homologue
Location of the identified 1B6 epitope in the nucleotide and deduced amino acid sequences of the TMSB4Y cDNA (1702 bp; GenBank
7051 The Journal of Immunology
Moreover, when the whole TMSB4Y genomic region was ana-
lyzed using GENESCAN software (36) (http://genes.mit.edu/
GENSCAN.html), no ORF other than the reported ORF encoding the
34-mer polypeptide was predicted with a risk of ?1.6% of false neg-
ative. These results strongly suggest that theTMSB4Y/A33 minor H
Ag is not derived from a functional polypeptide, but, rather, that it is
a subsidiary translation product of the TMSB4Y transcript.
Defective ribosomal products (DRiPs) consist of prematurely
terminated polypeptides and misfolded polypeptides produced
from translation of genuine mRNAs in the proper reading frame or
are produced entropically due to the inevitable imperfections in-
herent to protein synthesis or folding (37). DRiPs, which account
for 30% of newly synthesized proteins, have been suggested to be
a major source of peptides presented on the cell surface by class I
MHC (38). TMSB4Y was expressed in normal cells as well as
transformed cells when assessed by quantitative PCR specific for
the region encoding the TMSB4Y/A33 epitope, and the full-length
mRNA was readily detected (data not shown). According to the
definition of DRiPs, which is defective products from genuine
mRNAs in the proper reading frame, TMSB4Y/A33 should be one
of epitopes derived from cryptic polypeptides rather than DRiPs.
In any case, the identification of a minor H Ag encoded outside the
conventional ORF has important implications for the identification
of other minor H Ag epitopes using genetic linkage analysis. Re-
cently, we identified two minor H Ags using a similar approach
(29), where we looked for peptides with potential HLA-binding
sequence motifs that spanned nonsynonymous single nucleotide
polymorphisms in the conventional ORF. However, the results of
the current study suggest that not only conventional ORFs but also
regions other than conventional ORFs should be taken into con-
sideration when attempting to identify the epitope within the re-
gion mapped by linkage analysis.
Although the function of TMSB4Y is not yet known, its X chro-
mosome homologue, TMSB4X, also known as thymosin ?4, en-
codes a protein that plays an important role in the organization of
the cytoskeleton, which binds to and sequesters actin monomers (G
actin), leading to inhibition of actin polymerization (22). As ex-
pected from its function, thymosin ?4 is highly expressed in met-
astatic melanoma cells together with fibronectin and RhoC, a
member of the Rho GTPase family (39). Because Rho-like
GTPases are suggested to be linked with HA-1 (40) and HA-3 (41)
proteins in cytoskeleton rearrangement and have myosin 1G en-
coding HA-2 minor H Ag (42) as one of the downstream effector
proteins (43), TMSB4Y/A33 derived from the Y homologue of
thymosin ?4 may also be classified as one of malignancy-associ-
ated minor H Ags according to the recent proposal by Spierings et
CTLp frequency assays revealed that the magnitude of the CTL
response to the TMSB4Y/A33 epitope was early after HSCT and
represented one-quarter of the measurable donor CTL responses to
activity. Donor LCL (female) were labeled with51Cr, then pulsed with
serial dilutions of either EVLLRPGLHFR or putative female X homologue
ETLFLPGLHFR (the mismatched amino acids are underlined) and used as
targets for CTL clone 1B6 in a standard51Cr release assay.
Evaluation of synthetic peptides for epitope reconstitution
analysis of post-HSCT PBMC. A and B, The proportion of CTL precursors
specific for the identified TMSB4Y peptide among total CTLp against the
recipient minor H Ags was quantitated using a standard limiting dilution
assay. CD8?T cells from the PBMC on day 50 (A) or day 146 (B) post-
HSCT were cultured at limiting dilution with irradiated CD40-B cells gen-
erated from pre-HSCT recipient PBMC. After three rounds of stimulation,
a split-well analysis was performed for peptide-specific cytotoxicity
against51Cr-radiolabeled recipient PHA blasts (E) or donor PHA blasts
pulsed with TMSB4Y peptide (Œ) or unpulsed (‚). The wells were con-
sidered to be positive for lytic activity if the total cpm released by effector
cells was ?2.5 ? SD above that in control wells (mean cpm released by
the target cells incubated with irradiated stimulator cells alone). The CTLp
frequency was calculated with L-Calc software. C, Thawed post-HSCT
(days 50, 146, and 696) PBMCs and T cell lines generated by stimulating
the PBMCs three times with irradiated CD40-B cells generated from pre-
HSCT recipient PBMC were stained with a PE-conjugated HLA-A33 tet-
ramer incorporating the TMSB4Y/A33 peptide. The percentage of tet-
ramer-positive cells of the total CD3?CD8?cells is shown.
TMSB4Y/A33-specific CTLp frequency assay and tetramer
70525?UTR OF TMSB4Y GENE ENCODES HLA-A33-RESTRICTED MINOR Ag
recipient minor H Ags in this donor/recipient pair. This illustrates
the extent to which such cryptic peptides may contribute to the
diversity and immunogenicity of the total class I MHC-associated
peptide pool in normal cells. In this regard, the relative immuno-
genicity of another minor H Ag, HB-1, which is derived from a
polypeptide whose translation is initiated at a CUG instead of a
conventional ATG codon exclusively in transformed B cells (44,
45), should also be of interest. Recently, Schwab et al. (46) have
shown that the insertion into a 3?UTR of a sequence encoding an
antigenic peptide elicits T cells specific for this peptide in vivo,
which recognize at least DCs, B cells, and fibroblasts from mice
carrying the transgene. In contrast, analysis of ?200 endogenously
derived HLA-B*1801-associated peptides from a human B cell
line revealed that all the peptides were encoded by conventional
ORFs from a wide variety of cellular genes (47), suggesting that
the frequency of cryptic peptides being presented on class I MHC
molecules is ?1/200. Identification of more minor H Ags may
answer the question of the significance of cryptic peptides over
A recent study has suggested that CTL responses against minor
H Ags encoded or regulated by genes on the Y chromosome con-
tribute to a selective GVL effect against myeloid and lymphoid
leukemias after female into male HSCT, even though recipients of
this combination experience increased GVHD (6). An HLA-B8-
restricted minor H Ag encoded by UTY has been shown to be a
potential target for immunotherapy against hematological malig-
nancies (16). It is noted in this regard that clone 1B6, used for
defining TMSB4Y, was isolated from a patient who did not de-
velop acute GVHD, and that its lytic activity against nonhemopoi-
etic cells, including dermal/oral fibroblasts and bone marrow stro-
mal fibroblasts, was significantly lower than that against LCL and
PHA blasts, suggesting that TMSB4Y/A33 is a potential target of
immunotherapy like UTY. However, the expression of the
TMSB4Y transcript was found to not be restricted to normal he-
mopoietic cells and leukemia/lymphoma cells. In addition, the in-
crease in A33/peptide tetramer-positive cells observed late after
HSCT during late GVHD may suggest that this minor H Ag could
be related to chronic GVHD rather than the GVL effect. Thus,
additional studies of the polypeptide expression level derived from
the TMSB4Y 5?UTR in various types of cells need to be conducted
to elucidate whether this cryptic product can serve as a target
We thank Dr. Stanley R. Riddell for critically reading the manuscript. We
also thank Yumi Nakao, Yasue Matsudaira, Keiko Nishida, and
Hiromi Tamaki for their expert technical assistance.
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