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. 2003 May 19;197(10):1279-89.
doi: 10.1084/jem.20030044. Epub 2003 May 12.

"V体育官网" A human minor histocompatibility antigen resulting from differential expression due to a gene deletion

Makoto Murata  1 , Edus H Warren, Stanley R Riddell
Affiliations

A human minor histocompatibility antigen resulting from differential expression due to a gene deletion

Makoto Murata et al. J Exp Med. .

Abstract

Minor histocompatibility antigens (minor H antigens) are targets of graft-versus-host disease and graft-versus-leukemia responses after allogeneic human leukocyte antigen identical hematopoietic stem cell transplantation. Only a few human minor H antigens have been molecularly characterized and in all cases, amino acid differences between homologous donor and recipient proteins due to nucleotide polymorphisms in the respective genes were responsible for immunogenicity. Here, we have used cDNA expression cloning to identify a novel human minor H antigen encoded by UGT2B17, an autosomal gene in the multigene UDP-glycosyltransferase 2 family that is selectively expressed in liver, intestine, and antigen-presenting cells. In contrast to previously defined human minor H antigens, UGT2B17 is immunogenic because of differential expression of the protein in donor and recipient cells as a consequence of a homozygous gene deletion in the donor. Deletion of individual members of large gene families is a common form of genetic variation in the population and our results provide the first evidence that differential protein expression as a consequence of gene deletion is a mechanism for generating minor H antigens in humans VSports手机版. .

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Figures

Figure 1.
Figure 1.
Identification of a cDNA encoding the minor H antigen recognized by PL8 CTL. (A) Pools of a cDNA library containing ∼50 cDNA clones were transiently transfected into COS cells in individual wells of a 96-well plate together with a plasmid encoding HLA-A*2902. COS transfectants were cocultured with PL8 CTL and IFN-γ production was measured in supernatant collected after 24 h. Two pools (indicated by arrows) stimulated IFN-γ production by PL8 CTL. (B) Isolation of a cDNA that stimulates IFN-γ production by PL8 CTL. The two pools of cDNA were subcloned in E. coli, and individual plasmids were transfected into COS cells alone or with HLA-A*2902. Two clones, designated 2H9 and 4A2 stimulated IFN-γ production by PL8 CTL only when transfected with HLA-A*2902. Data is shown as the mean of triplicate determinations.
Figure 2.
Figure 2.
Identification of the antigenic epitope encoded by UGT2B17. (A) Location of the epitope-containing region by transfection of truncated UGT2B17 constructs. Alignment of 4A2 cDNA with the UGT2B17 cDNA (NM_001077), the nucleotide numbering of the constructs used for transfection corresponds to that provided for the UGT2B17 GenBank sequence. Constructs I, II, and III contained the indicated UGT2B17 sequences and were transfected with HLA-A*2902 into COS cells. IFN-γ production by PL8 CTL was measured after coculture with COS transfectants and is indicated by (+) or (−) in the right-hand column. (B) UGT2B17 493–564 encodes a decamer peptide with anchor residues for HLA-A*2902. The amino acid sequence encoded by UGT2B17 493–564 is shown and a putative epitope for PL8 CTL is boxed. (C) CTL recognition of donor B-LCL cultured with synthetic peptides corresponding to UGT2B17 sequences. The concentration of peptide that elicited half-maximal lysis was ∼0.7 pM for AELLNIPFLY, ∼6 pM for ELLNIPFLY, ∼50 pM for LAELLNIPFLY, and ∼80 nM for AELLNIPFL. Specific lysis is shown as the mean of triplicate cultures at an E:T ratio of 5:1. (D) Transfection of minigene constructs define a requirement for tyrosine at the COOH terminus of the naturally processed UGT2B17 epitope. Donor B-LCL were transfected by electroporation with either UGT2B17 493–564 that encodes the 24 amino acid polypeptide described in B or UGT2B17 493–561 that encodes 23 amino acids with the COOH-terminal tyrosine deleted. Transfected B-LCL were selected for 3 d with puromycin (0.6 μg/ml). The lysis of UGT2B17 493–564-transfected donor B-LCL (solid circles), UGT2B17 493–561-transfected donor B-LCL (triangles), recipient B-LCL (squares), and untransfected donor B-LCL (open circles) is shown as the mean of triplicate cultures at various E:T ratios.
Figure 3.
Figure 3.
Donor cells pulsed with synthetic peptides corresponding to homologous sequences of other UGT2B family members are not recognized by PL8 CTL. Donor B-LCL were pulsed with various concentrations of the following synthetic peptides: AELLNIPFLY (encoded by UGT2B17), AELFNIPFLY (UGT2B15), AALLNIRFVY (UGT2B11), AELFNIPFVY (UGT2B10 and UGT2B7), AELLKIPFVY (UGT2B4), or AALLNIPFVY (UGT2B28), and tested as targets for PL8 CTL in a cytotoxicity assay. Specific lysis is shown as the mean of triplicate cultures at an E:T ratio of 5:1.
Figure 4.
Figure 4.
UGT2B17 mRNA is transcribed in minor H antigen positive but not minor H antigen negative cells. (A) Northern blot analysis of total RNA from recipient and donor B-LCL, and from HLA-A29+ B-LCLs from 4 unrelated donors (designated 1–4). The blot was hybridized with a 32P-labeled UGT2B17 probe for 16 h at 68°C. Detection of GAPDH mRNA was performed as a control. (B) Cytotoxicity assay for B-LCL by PL8 CTL. The B-LCL from unrelated donors (–4) are the same lines used for analysis of gene expression in Fig. 3 A. Specific lysis is shown as the mean of triplicate cultures at an E:T ratio of 5:1. (C) Transfection of UGT2B17 cDNA into donor B-LCL restores recognition by PL8 CTL. Donor B-LCL were transfected by electroporation with the full-length construct of UGT2B17, selected for 3 d with puromycin (0.6 μg/ml), and assayed as targets for PL8 CTL. The lysis of UGT2B17-transfected donor B-LCL (triangles), untransfected donor B-LCL (circles), and recipient B-LCL (squares) is shown at various E:T ratios.
Figure 5.
Figure 5.
The UGT2B17 gene is deleted in donor cells and HLA-A*2902 positive cells from unrelated individuals that are not recognized by PL8 CTL. (A) UGT2B17 consists of six exons encoded over 27 Kb of DNA on chromosome 4. Primer pairs used for PCR to detect exon 1 and exon 6 sequences were selected such that at least one primer of each pair contained nucleotides that were mismatched with all other known UGT family members. The primer pair for the region immediately 5′ to the UGT2B17 start site was selected to amplify nt −370 to −30. (B) SSP-PCR for exon 1 and exon 6 sequences of UGT2B17 and PCR for 5′ sequences upstream of UGT2B17 on genomic DNA prepared from B-LCL. PCR products for exon 1, exon 6 and the 5′ region upstream of UGT2B17 were detected in the recipient B-LCL and unrelated HLA-A29+ B-LCL that were recognized by PL8 CTL. These PCR products were sequenced and found to be identical to UGT2B17 (unpublished data). No PCR products were detected in donor B-LCL or unrelated HLA-A29+ B-LCL that were not lysed by PL8 CTL. The B-LCL from unrelated donors (indicated as 1–4) are the same lines used for analysis of gene expression and cytotoxicity in Fig. 4, A and B. PCR for GAPDH was performed as a control.
Figure 6.
Figure 6.
Tissue expression of UGT2B17. (A) Expression of UGT2B17 in tissues by RT-PCR. First-strand cDNA prepared from a series of different human tissues pooled from multiple donors was analyzed by SSP-PCR for UGT2B17 using the exon 1(a) primer pair. The highest level of expression of UGT2B17 was observed in cDNA from liver and colon. Detectable bands were also obtained from lung, skeletal muscle, pancreas, spleen, thymus, prostate, testis, ovary, and small intestine. No band was obtained from heart, brain, placenta, and kidney. PCR for GAPDH was performed as a control. (B) Expression of UGT2B17 in subsets of PBMC. First-strand cDNA was isolated from PBMCs or from subsets of PBMCs and analyzed by SSP-PCR using the exon 1(a) primer pair. A PCR product of the correct size was obtained from PBMCs, immature and mature DCs, resting and activated CD19+ B cells and B-LCL. No bands were obtained from resting or activated CD4+ and CD8+ T cells, or from CD14+ cells (monocytes). PCR for GAPDH was performed on all samples as a control. (C) APCs stimulate IFN-γ production by PL8 CTL. PL8 CTL (5 × 103) were cocultured with B-LCL, mature DCs, and activated B cells (0.625 × 103) in triplicate wells of a 96-well plate for 24 h at 37°C. IFN-γ production was measured in the supernatant by ELISA. The mean and SD of triplicate measurements are shown.
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