The PANE1 gene encodes a novel human minor histocompatibility antigen that is selectively expressed in B-lymphoid cells and B-CLL

Abstract

Minor histocompatibility antigens (mHAg's) are peptides encoded by polymorphic genes that are presented by major histocompatibility complex (MHC) molecules and recognized by T cells in recipients of allogeneic hematopoietic cell transplants. Here we report that an alternative transcript of the proliferation-associated nuclear element 1 (PANE1) gene encodes a novel human leukocyte antigen (HLA)-A(*)0301-restricted mHAg that is selectively expressed in B-lymphoid cells. The antigenic peptide is entirely encoded within a unique exon not present in other PANE1 transcripts. Sequencing of PANE1 alleles in mHAg-positive and mHAg-negative cells demonstrates that differential T-cell recognition is due to a single nucleotide polymorphism within the variant exon that replaces an arginine codon with a translation termination codon. The PANE1 transcript that encodes the mHAg is expressed at high levels in resting CD19(+) B cells and B-lineage chronic lymphocytic leukemia (B-CLL) cells, and at significantly lower levels in activated B cells. Activation of B-CLL cells through CD40 ligand (CD40L) stimulation decreases expression of the mHAg-encoding PANE1 transcript and reciprocally increases expression of PANE1 transcripts lacking the mHAg-encoding exon. These studies suggest distinct roles for different PANE1 isoforms in resting compared with activated CD19(+) cells, and identify PANE1 as a potential therapeutic target in B-CLL.

Figure 1.

CTL Clone CTL-7A7 recognizes a HLA-A^*0301-restricted mHAg that is selectively expressed in B-lymphoid cells. (A) The cytolytic activity of CTL-7A7 CTLs against EBV-LCLs, PHA-stimulated T-cell blasts, unfractionated peripheral blood mononuclear cells (PBMCs), and dermal fibroblasts derived from the transplant recipient (Recip) or donor was evaluated in a 4-hour ⁵¹Cr release assay at the indicated effector-target ratios. (B) Cytolytic activity at E/T 10:1 of CTL-7A7 CTLs against a panel of EBV-LCLs derived from unrelated individuals, each of whom shared 1 class I MHC allele with the recipient-donor pair from whom the CTL clone CTL-7A7 was derived. The specific class I MHC allele shared with the recipient/donor by each individual is indicated.

Figure 2.

Reconstitution of the CTL-7A7 mHAg epitope with HPLC-fractionated peptides extracted from HLA-A3 molecules. HLA-A3-associated peptides were purified from 5 × 10¹⁰ mHAg⁺ EBV-LCLs and fractionated by RP-HPLC as described in “Materials and methods.” Aliquots of each fraction corresponding to 4 × 10⁹ cell equivalents in panels A and B and 6 × 10⁹ cell equivalents in panels C and D were preincubated with ⁵¹Cr-labeled donor EBV-LCLs and tested for their ability to sensitize targets to lysis by CTL-7A7 CTLs. An E/T ratio of 10:1 was used. (A) First-dimension separation of extracted peptides was achieved using TFA as the ion-pairing agent. (B) Fractions 61 and 62 from panel A were pooled and rechromatographed using HFBA as the ion-pairing agent. (C) Fractions 70 and 71 from panel B were pooled and rechromatographed on a microcapillary column using TFA as the ion-pairing agent. (D) Determination of candidate peptides via mass spectrometry correlated with ⁵¹Cr release assay. Fractions 52 and 53 from panel C were pooled and rechromatographed using nanoflow effluent splitter technology. Ion abundances of candidate masses within the MS scan window 806-856 were plotted and correlated to the percent specific ⁵¹Cr release in that same region. Background lysis of donor EBV-LCLs by CTL-7A7 CTLs in the absence of peptide was 10% in panel A, 3% in panel B, 16% in panel C, and 10% in panel D. Lysis of recipient EBV-LCLs (positive control) was 54% in panel A, 67% in panel B, 86% in panel C, and 66% in panel D.

Figure 3.

Identification of the antigenic peptide recognized by the CTL clone CTL-7A7. (A) CAD mass spectrum of candidate peptide (M+2H)²⁺ ion with monoisotopic m/z of 591.865 as eluted from HLA-A^*0301-homozygous mHAg⁺ EBV-LCLs. (B) CAD mass spectrum of synthetic peptide RVWDLPGVLK. Mass spectra were recorded on a Finnigan LCQ ion trap MS operating with a 3.0 atomic mass unit isolation window and 35% collision energy. The b and y ions are labeled above and below the amino acid sequence, respectively. Ions observed in the spectrum are underlined. (C) CTL-7A7 mHAg epitope reconstitution with synthetic RVWDLPGVLK peptide. ⁵¹Cr-labeled donor EBV-LCLs or T2A3 target cells were pulsed for thirty minutes at 37°C with the indicated concentrations of synthetic peptides RVWDLPGVLK or SLPRGTSTPK, then tested for recognition by CTL-7A7 CTLs in a 4-hour ⁵¹Cr-release assay at an E/T ratio of 10:1. The control peptide SLPRGTSTPK corresponds to the HLA-A^*0301-restricted epitope recognized by CTL clone DRN-7 (E.H.W., manuscript in preparation).

Figure 4.

Genomic structure and gene/protein alignment of human PANE1 transcripts. (A) PANE1 transcripts k and c share a common last exon. The first exon of transcript k is unique from all other PANE1 transcripts, and the sequence encoding the CTL-7A7 mHAg RVWDLPGVLK is contained entirely within this first exon, as indicated by the vertical arrow. (B) PANE1 transcript k nucleotide sequence, and sequence of its predicted 58-amino acid isoform. The polymorphic nucleotide generating the CTL-7A7 mHAg is indicated by a vertical arrow. These sequence data are available from Entrez PubMed (http://www.ncbi.nlm.nih.gov/entrez) under accession number BQ056829. Italicized nucleotides represent additional 5′ and 3′ sequence determined by RACE PCR. (C) Comparison of the predicted protein sequences of PANE1 isoforms k and c. Asterisks indicate amino acid identity; bold type indicates CTL-7A7 epitope, with polymorphic residue indicated by an arrow.

Figure 5.

PANE1 transcript k is selectively expressed in resting CD19⁺ cells and B-CLL. (A) Relative expression levels of the CTL-7A7 mHAg-encoding PANE1 transcript k in different human tissues. Expression was determined by real-time quantitative RT-PCR as described in “Materials and methods.” The GAPDH gene was used as a standard to correct for RNA quantity and quality. (B-C) Relative expression levels of PANE1 transcripts c () and k (▪) in resting and activated fractions of human peripheral blood leukocytes (B), and in primary ALL and B-CLL samples (C).

Figure 6.

The CTL-7A7 mHAg encoded by PANE1 transcript k is expressed in normal CD19⁺ cells and CD19⁺ B-CLL. Expression of the CTL-7A7 antigen in purified CD19⁺ cells from the peripheral blood of 2 HLA-A^*0301-positive healthy donors and in B-CLL cells from 2 patients as determined in a standard 4-hour ⁵¹Cr release assay at the indicated effector-target (E/T) ratios.

Figure 7.

CD40L stimulation of B-CLL cells differentially regulates expression of PANE1 transcripts c and k. Effect of CD40L stimulation on relative expression levels of PANE1 transcripts k and c in 6 primary B-CLL samples. Data are representative of those from 2 independent experiments. CD40L stimulation was performed as described in “Materials and methods.”