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* 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, Tokorozawa, Japan;
Program in Immunology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109; and
¶ Department of Hematology, Japanese Red Cross Nagoya First Hospital, Nagoya, Japan
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Introduction |
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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 conventional 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 (reviewed in Ref.23). To our knowledge, this is the first demonstration 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 700 days after HSCT. These findings underscore the in vivo immunological relevance of such a cryptic minor H Ag derived from unconventional ORF products.
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Materials and Methods |
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The HLA-A*3303-restricted CD8+ CTL clone, 1B6, was isolated by limiting 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-identical 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 Center.
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, Japan).
Cytotoxicity assays
Target cells were labeled with 0.1 mCi of 51Cr for 2 h, and 1 x 103 target 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 performed 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: ((experimental cpm – spontaneous cpm)/(maximum cpm – spontaneous cpm)) x 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) previously 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% acrylamide gels, and cell lines were scored as positive or negative for the presence 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 µl containing 1x PCR buffer, 1.5 mM MgCl2, 200 µM of each dNTP, 0.5 µM 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 prepared 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'-TTAAGCTTCGCAGCCATGTCTGACAAACC-3'; conventional ORF antisense, 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 µl of 1x buffer containing 200 µM of each dNTP, 1.0 mM MgSO4, 0.3 µM of each primer, and 1 U of KOD-Plus-DNA polymerase (TOYOBO, Osaka, Japan).
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 encoding 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, respectively, and all products were ligated into the pEAK10 vector and verified by sequencing.
Electroporation of LCL
The constructed vectors were introduced either into the donor LCL or into 293T cells. One million LCL were resuspended in 40 µl of OPTI-MEM I buffer (Invitrogen Life Technologies) and 4 µg of each plasmid in a 2-mm gap cuvette, and electroporated in an ECM 830 BTX Electro Square Porator (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 µg/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 selected in the presence of 1 µg/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 mutant cDNA, or minigenes of TMSB4Y. 293T-A33 cells were plated the day before transfection at 4 x 104 cells/100 µl/well into 96-well, flat-bottom microtiter plates and transfected with 6 µl of RPMI 1640 containing 90 ng of plasmid DNA and 0.27 µl of FuGENE 6 (Roche, Indianapolis, IN). After 24 h at 37°C, 100 µl of a cell suspension containing 1 x 104 CTL 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 purchased from BD Clontech (MTC panels human I and II; Palo Alto, CA) or synthesized from total RNA of human lung (BD Clontech) or various cultured cells. PCR amplification and real-time quantification analysis were performed using the TaqMan assay according to the manufacturer’s instructions. The following sequences were used as primers and TaqMan probe to detect the mRNA region encoding the epitope: 5'-GACTAGAAAGCGGGCGCAG-3' (sense; nt 302–320), 5'-ACTTCCGCGTTCAAGTGGTT-3' (antisense; nt 415–434), 5'-(FAM)-TCCCTTCTCGACACGGAGTCTATGTGTAGT-(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 1x 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 µl in the ABI PRISM 7700HT Sequence Detector System (Applied Biosystems). The temperature 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 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 x 104 CD40-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 peptide-specific cytotoxicity against 51Cr-radiolabeled recipient PHA blasts or donor PHA blasts pulsed with TMSB4Y peptide or unpulsed. The supernatants 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 x 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, Vancouver, Canada).
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+ vector; provided by the late Dr. D. C. Wiley, Howard Hughes Medical Institute, Harvard University, Cambridge, MA) were produced in XA90. The C terminus of the H chain was modified by the addition of a substrate sequence for the biotinylating enzyme BirA. Monomeric HLA/2-microglobulin/peptide complexes were folded in vitro in the presence of the peptide. The MHC complex was biotinylated and then converted into tetramers with PE-labeled streptavidin. For staining, PBMC or T cell lines were incubated with the tetramer at a concentration of 20 µg/ml at room temperature for 15 min, followed by FITC-conjugated anti-CD3 (BD Biosciences, San Diego, CA) and Tricolor anti-CD8 mAb (Caltag Laboratories, Burlingame, CA) on ice for 15 min. Cells were analyzed with a FACSCalibur flow cytometer and CellQuest software (BD Biosciences).
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Results |
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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, significantly 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 moderately (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).
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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 deletions 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 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 examined 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 expression of TMSB4Y as shown in Fig. 2B. Moreover, female LCLs from the patient’s HSCT donor transfected with any of three isoforms of UTY cDNA were not lysed by 1B6 (data not shown). These results indicated that TMSB4Y most likely encoded the minor H Ag.
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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 noncoding regions and nonconventional ORF have been described in both murine and human tumor cells (23). Thus, we cloned the full-length TMSB4Y cDNA (GenBank accession no. 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 encode 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 transfected 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 upstream of the reported ORF for the TMSB4Y protein (Fig. 4A).
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4, has much shorter 5'- and 3'UTR; thus, no corresponding region was found (Fig. 4B). However, a recently reported splice variant of TMSB4X, which includes 1076 bp of TMSB4X intron 1 (GenBank 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 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 titration 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 concentration tested (Fig. 5). Thus, EVLLRPGLHFR defines the HLA-A*3303-restricted 1B6 epitope, and we designated it TMSB4Y/A33.
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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, pancreas, and hemopoietic cells to the lowest expression in dermal fibroblasts and skeletal muscles (50-fold less than that in hemopoietic cells; data not shown). Expression of the mRNA in a panel of primary leukemic cells ranged from undetectable to levels similar to those seen in normal hemopoietic cells (data not shown).
TMSB4Y/A33-specific CD8+ T cells are detectable in recipient post-transplant PBMC
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 frequencies 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 106 peripheral blood CD8+ cells, respectively, 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 frequency of CTLp recognizing TMSB4Y peptide-pulsed donor PHA-blasts was 316 (95% CI, 216–464), and that for CTLp recognizing recipient PHA blasts was 3215 (95% CI, 2150–4808) per 106 peripheral blood CD8+ cells, demonstrating that even at the later time point the CTL responses against TMSB4Y/A33 continued to account for a significant fraction (10%) of the total donor CTL responses against recipient minor H Ags in this donor/recipient pair (Fig. 6B).
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20 nM), it is likely that the 11-mer peptide is the minimal epitope, because it has a consensus 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 homologous 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 thymosin 4 was readily detectable (data not shown). Thus, it is conceivable that the splice variant, AK055976, might be very rare or derived from a precursor mRNA.
Recently, evidence has been accumulating that cryptic polypeptides 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 oncogene, c-akt, in the murine RL
1 leukemia system (35). This unusual epitope is generated by insertion of the murine leukemia virus long terminal repeat into the exon of c-akt, resulting in transcription 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. Although it is possible that the 19-residue ORF in the 5'UTR that encodes 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 homology to this region did not identify any known functional domains. Moreover, when the whole TMSB4Y genomic region was analyzed 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 negative. 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 inherent 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. Recently, 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 consideration when attempting to identify the epitope within the region mapped by linkage analysis.
Although the function of TMSB4Y is not yet known, its X chromosome homologue, TMSB4X, also known as thymosin 4, encodes 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 expected from its function, thymosin 4 is highly expressed in metastatic 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 encoding 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-associated minor H Ags according to the recent proposal by Spierings et al. (43).
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 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 immunogenicity 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 conventional peptides.
A recent study has suggested that CTL responses against minor H Ags encoded or regulated by genes on the Y chromosome contribute 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 malignancies (16). It is noted in this regard that clone 1B6, used for defining TMSB4Y, was isolated from a patient who did not develop acute GVHD, and that its lytic activity against nonhemopoietic cells, including dermal/oral fibroblasts and bone marrow stromal 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 hemopoietic cells and leukemia/lymphoma cells. In addition, the increase 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 for GVL.
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Acknowledgments |
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Footnotes |
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1 This 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 Ministry 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 Foundation (to E.H.W.). 
2 H.T. and Y.A. contributed equally to this work.
3 Address 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: yakatsuk{at}aichi-cc.jp
4 Abbreviations 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, hemopoietic stem cell transplantation; H-Y Ag, Y chromosome-encoded Ag; LCL, B-lymphoblastoid cell line; ORF, open reading frame; UTR, untranslated region; STS, sequence-tagged site.
Received for publication August 4, 2004. Accepted for publication September 3, 2004.
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4 gene: structure, promoter identification, and chromosome localization. Genomics 32:388.[Medline]
4 gene: preliminary characterization and expression in tissues, thymic cells, and lymphocytes. J. Immunol. 143:2740.[Abstract]
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) or allo-specific CTL (
) and of cytokine untreated cells by 1B6 (
) or allo-specific CTL (
) is shown.
) or donor PHA blasts pulsed with TMSB4Y peptide (
) or unpulsed (
). The wells were considered to be positive for lytic activity if the total cpm released by effector cells was >2.5 x 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 tetramer incorporating the TMSB4Y/A33 peptide. The percentage of tetramer-positive cells of the total CD3+CD8+ cells is shown.