T cell responses to minor H antigens have been implicated in both GVHD and GVL responses after allogeneic HLA-identical HSCT, and a more precise understanding of the genetics and tissue expression of these determinants could potentially lead to improvements in transplant outcome. However, the polymorphisms in the genome that encode minor H antigens and the basis for antigenicity have been defined for only a few human determinants. In all cases described so far, human minor H antigens have been derived from genes that are expressed in both donor and recipient cells but have polymorphism in their coding sequences. The resulting changes in amino acid sequence may alter the processing of recipient and donor peptides (17
), the ability of the peptide to bind MHC (5
), or the ability of T cells to recognize the MHC/peptide complex (6
). Here, we have identified a new human minor H antigen encoded by the UGT2B17
gene and demonstrated that differential expression in donor and recipient cells as a consequence of a homozygous gene deletion in the donor is responsible for immunogenicity. This is the first example in humans of differential expression of a protein as the basis for generating a minor H antigen. The murine minor H antigens, H60 and H28, that result in rejection of BALB/B skin grafts by C57Bl/6 recipients, are also immunogenic as a result of differential transcription, although the genetic basis for absent expression of these genes in some strains of mice remains to be elucidated (4
The optimal peptide of UGT2B17 for recognition by PL8 CTL was AELLNIPFLY, which contains preferred amino acid residues at both of the anchor positions for binding to HLA-A*2902, and sensitized donor B-LCL for lysis at pM concentrations. Peptides with one amino acid deleted or added to the NH2 terminus of AELLNIPFLY, respectively, or with one amino acid deleted from the COOH terminus also sensitized donor B-LCL for recognition by PL8 CTL, although a much higher concentration of peptide was required. Donor B-LCL that were transfected with UGT2B17493–564 encoding AELLNIPFLY but not UGT2B17493–561 encoding AELLNIPFL, were recognized by PL8 CTL suggesting tyrosine is the anchor residue at the COOH terminus of the naturally processed UGT2B17 epitope presented by HLA-A*2902. The elution of peptides from HLA-A29 molecules of recipient cells that reconstitute recognition by PL8 CTL will be required to conclusively define the NH2 terminus of the naturally processed peptide, since the synthetic peptides LAELLNIPFLY and ELLNIPFLY were both able to sensitize target cells for recognition by PL8 CTL, albeit at 1–2 log10 higher concentration than AELLNIPFLY.
The UDP glycosyl transferases are comprised of two subfamilies, UGT1 and UGT2 (36
), and serve a major role in the conjugation and subsequent elimination of endogenous compounds including steroid hormones and bilirubin, and potentially toxic exogenous compounds (37
). A single gene located on chromosome 2 encodes several UGT1 isoforms that arise by differential splicing of the encoded mRNA. Mutations of UGT1 family members have been described and are responsible for the Crigler-Najjar and Gilbert syndromes (38
). Individual genes located on chromosome 4 encode the UGT2 family members, but clinical syndromes resulting from mutation or deletion of UGT2 members have not been described. The lack of UGT2B17 in the adult donor in this study was not clinically significant since this individual had a normal physical exam, complete blood count, and serum chemistry at the time of evaluation for donation of hematopoietic stem cells. UGT2B17 has been shown to catalyze the conjugation of the 17β-hydroxy position of dihydrotestosterone (DHT), testosterone, and androstane-3α17β-diol (3α-Diol), and glucuronidates androsterone (33
). Other UGT family members, such as UGT2B7 and UGT2B15 also conjugate DHT, testosterone, and 3α-Diol, and UGT2B7 can glucuronidate androsterone (25
). Thus, redundancy of other UGT2B family members for these substrates may compensate for the deficiency of UGT2B17
in normal individuals and be responsible for the absence of a clinical phenotype associated with homozygous UGT2B17
Despite the redundancy in substrate specificity, there is significant variation in the nucleotide and amino acid sequences of UGT2B17
compared with other UGT2B family members. The region of UGT2B17
that encodes the epitope recognized by PL8 CTL contains one or more unique amino acids compared with all other family members and synthetic peptides corresponding to the sequences of other family members failed to sensitize donor cells for recognition by PL8 CTL. We have analyzed other regions of UGT2B17 that also differ in amino acid sequence with all other family members using computer algorithms that identify peptides predicted to bind to class I HLA molecules based on preferred anchor and secondary anchor residues (45
). Several peptides were identified that are predicted to bind to common HLA alleles such as HLA-A2 and -B44. Thus, studies in donor/recipient pairs that express these HLA molecules and are discordant for UGT2B17
gene expression are warranted to determine if UGT2B17
encodes additional minor H antigens.
Establishing a causative role for individual human minor H antigens in GVHD after allogeneic HSCT has proven difficult for several reasons. Until recently, few minor H antigens were molecularly characterized and reagents and methods to detect T cells of selected antigen specificities at tissue sites of GVHD were not available. Additionally, other factors such as the intensity of the conditioning regimen, polymorphism in genes that encode cytokines or cytokine receptors, the intensity of posttransplant immunosuppression, and the relative immunodominance of individual minor H antigens may influence the development of clinical GVHD even when disparity for defined minor H antigens is known to exist (46
). Studies in an situ model of human skin GVHD have demonstrated that T cells specific for minor H antigens expressed by both epithelial cells and APC induced the histologic features of GVHD whereas T cells specific for minor H antigens that are selectively expressed in APC did not cause GVHD (47
). The requirement that the target antigen be expressed in APC for the induction of GVHD by CD8+
CTL is consistent with prior results obtained in vivo in murine models (34
). The patient from whom the PL8 CTL clone was isolated had acute GVHD involving the liver and gastrointestinal tract and we found UGT2B17 was highly and preferentially expressed in both of these target organs. An in situ model is not available to address the potential for PL8 CTL to induce GVHD in the liver or intestine, but based on the known function of UGT2B17, it seemed unlikely this gene would fulfill the requirement that it be expressed in APCs. Surprisingly, our data showed that both dendritic cells and activated B cells express sufficient levels of UGT2B17 to stimulate PL8 CTL in vitro. While these results are consistent with the principles suggested by murine studies and in situ models of human GVHD involving the skin, they do not definitively establish UGT2B17 as a target of GVHD. However, the development of the SSP-PCR assay used to demonstrate the homozygous deletion of UGT2B17 in the donor in this study will permit prospective genotyping of HSCT donors and recipients and analysis of the occurrence of GVHD in UGT2B17 positive recipients who undergo HSCT from donors that lack this gene.
The demonstration in this study that differential expression of proteins in donor and recipient cells provides a mechanism for generating minor H antigens in humans has potentially broad implications for understanding T cell responses that mediate GVHD after allogeneic HSCT. Deficiencies of members of other enzyme families have been described in humans. These include deletion or lack of expression of the cytochrome P450 (CYP) family genes CYP2A6
, and CYP2C19
, which metabolize foreign chemicals as well as endogenous steroids (48
), and deletion of the glutathione S-transferase (GST) T1
genes, which belong to the GST
gene family that detoxify mutagenic hydrophobic and electrophilic compounds (52
). Deletions of GSTT1 and GSTM1 are especially common, occurring in 38 and 50% of individuals, respectively, and these proteins share only 55% amino acid identity. Both GSTT1 and GSTM1 are expressed in the gastrointestinal tract and liver and could potentially be targets of GVHD involving these organs after HSCT from a donor with a deficiency of one of these enzymes into a recipient who expresses the protein (54
). Thus, the novel mechanism responsible for antigenicity of UGT2B17 may apply to other minor H antigens involved in allogeneic reactions after HSCT.