mHAgs play a pivotal role in inducing T cell responses mediating GVL reactivity after HLA-identical allogeneic SCT (4
). Molecular definition of mHAg peptides is crucial for the development of immunotherapeutic protocols to either selectively boost the GVL reaction or to inhibit GVHD. In this study, we have identified a novel hematopoietic cell–restricted mHAg, designated LRH-1, which is presented by HLA-B*0702 and encoded by the P2X5
purinergic receptor gene. CTLs specific for LRH-1 (RP1 clone) were isolated from a CML patient that was successfully treated with high-dose DLI for relapse in accelerated phase after allogeneic SCT. Here, we show a direct association between in vivo expansion of LRH-1–specific CTLs and the disappearance of Bcr-Abl–positive CML tumor cells following DLI. These findings indicate a causative role for LRH-1 in GVL reactivity. Although the patient achieved a complete cytogenetic remission after high-dose DLI, low levels of Bcr-Abl transcripts remained detectable (Figure ). Therefore, it remains to be investigated whether targeting of LRH-1 in patients with CML might be able to eradicate the most undifferentiated leukemic stem cells. GVHD was not increased by the emergence of LRH-1–specific CTLs despite interruption of CsA treatment. However, it is difficult to conclude that LRH-1–specific CTLs had no role in GVHD, since the patient required readministration of CsA for the treatment of chronic skin GVHD.
The level of expression of mHAgs may determine whether it is involved in GVL and/or GVHD (5
). Previous studies by RNA dot blot analysis showed that P2X5 is selectively expressed in brain and lymphoid tissues (28
). Here, we have used sensitive real-time quantitative PCR to determine relative expression levels of the P2X5 splice variant 1 encoding mHAg LRH-1. Our results confirm that P2X5 is highly expressed in various cell types of lymphoid origin and lymphoid tissues. Interestingly, we observed that the P2X5
gene is significantly expressed in leukemic CD34+
subpopulations from most CML as well as AML patients. We were unable, however, to demonstrate in vitro CTL RP1 recognition of CD34+
cells of the CML patient from whom the LRH-1–specific CTL was isolated (data not shown). This may be explained by the relatively low P2X5 expression level (0.61) in the leukemic CD34+
progenitor subset of this CML patient, which represented approximately 50% of the total CD34+
population (data not shown). But, P2X5 expression in vivo could be higher, resulting in increased sensitivity to CTL recognition. The efficiency of killing of leukemic CD34+
progenitor cell subsets by LRH-1–specific CTLs is currently under investigation using sensitive in vitro progenitor cell cytotoxicity assays and could be studied in vivo in the NOD/SCID leukemia model (14
Importantly, P2X5 is not expressed in prominent GVHD tissues such as skin, liver, colon, and small intestine. Furthermore, mature cells of myeloid origin and various nonhematopoietic cell types were negative for P2X5. In nonlymphoid tissues, we detected only high P2X5 expression levels in fetal brain, whereas in adult tissues, we observed low expression in brain, cerebellum, and skeletal muscle, which could not be explained by the presence of hematopoietic cells (Table ). This low level of P2X5 expression could be caused by a small subset of cells present in these tissues (30
). But based on the fact that both brain and skeletal muscle are tissues that are not very likely to be infiltrated with lymphocytes without significant inflammation (32
), it seemed unlikely that LRH-1–specific CTLs damage these cells in vivo. This is supported by our finding that emergence of LRH-1–specific CTLs was not accompanied by significant toxicity to these organs.
Most previously described human mHAgs result from disparities in protein sequences between donor and recipient due to nonsynonymous SNPs in their encoding genes. The resulting aa substitutions may affect either peptide processing by the proteasome (10
), transporter associated with antigen processing translocation of the peptide into the ER (23
), binding of the peptide to MHC (8
), or recognition of the MHC-peptide complex by mHAg-specific T cells (20
). In the case of an mHAg encoded by the UGT2B17
gene, disparity resulted from differential protein expression due to a homozygous gene deletion in the donor, although the genetic basis for this gene deletion remains to be elucidated (25
). Here, we demonstrate for the first time to our knowledge that mHAgs can arise from frameshift polymorphisms that are disparate between recipients and their HLA-identical transplant donors.
P2X5 is a member of the P2X purinergic receptor family consisting of at least 7 proteins, P2X1 to P2X7, which are capable of forming ATP-gated cation channels by homo- or heteromultimerization (34
). Transcription of the human P2X5
gene results in expression of 2 major isoforms as a result of differential splicing (28
). Splice variant 1 encodes a long isoform consisting of 422 aas, whereas translation of splice variant 2 results in a shorter isoform lacking exon 3 (397 aas). Our results indicate that a donor-specific, homozygous frameshift polymorphism in exon 3 of splice variant 1 leads to the formation of a truncated aberrant version of the long P2X5 isoform. Hence, donor-derived T lymphocytes are not tolerant of the wild-type exon 3 of the recipient. The high genotype frequency (i.e., 46%; unpublished observations) of healthy individuals that are homozygous for the cytosine deletion in exon 3 indicates that deficiency for the long P2X5 isoform is common without any significant phenotypic characteristics. The short P2X5 isoform as well as redundancy among P2X family members may compensate for the deficiency of the long P2X5 isoform.
Hematopoietic cell–restricted mHAgs are suitable targets for the application of immunotherapy to prevent or to treat recurrences of hematopoietic malignancies after allogeneic SCT. Based on its hematopoietic-specific expression, the P2X5-encoded mHAg LRH-1 could be an attractive candidate. P2X5 is highly expressed in a broad range of lymphoid malignancies including T and B cell acute lymphoblastic leukemia, chronic lymphocytic leukemia, various types of B cell lymphoma, and multiple myeloma (unpublished observations). Furthermore, CD34+CD38– and CD34+CD38+ myeloid leukemic progenitor cells were found to have enriched levels of P2X5 transcripts compared with mature nonleukemic monocytes (P < 0.05; ANOVA; Figure A), suggesting that LRH-1–specific CTLs may preferentially target immature myeloid leukemia cells. Therefore, vaccination with the LRH-1 peptide would be an attractive approach for induction of GVL reactivity. Recently we developed a genotyping assay for the LRH-1 mHAg, and of the 45 HLA-B7–positive SCT donors and recipients analyzed so far, 6 patients (~13%) positive for LRH-1 mHAg received a transplant from an LRH-1–negative donor (unpublished observations). These patients would be eligible for LRH-1 peptide–based vaccination. Studies are underway to determine whether LRH-1–specific CD8+ T cells can be detected or induced ex vivo in these LRH-1 disparate transplant recipients.
In conclusion, we describe a novel hematopoietic cell–restricted mHAg, designated LRH-1, that can function as a GVL-specific antigen for hematological malignancies. The significance of mHAg LRH-1 for cellular immunotherapy of relapsed leukemia is demonstrated by the direct correlation between expansion of LRH-1–specific CTLs and disappearance of CML, and the expression of its encoding gene in leukemic CD34+ progenitor cells. In addition, we describe a novel mechanism responsible for antigenicity of mHAg LRH-1. Our data demonstrate for the first time to our knowledge that differential protein expression as a consequence of a homozygous frameshift polymorphism is the basis for mHAg disparity. This novel mechanism may apply to other mHAgs involved in alloresponses after HLA-identical allogeneic SCT.