A human minor histocompatibility antigen specific for B cell acute lymphoblastic leukemia

Abstract

Human minor histocompatibility antigens (mHags) play an important role in the induction of cytotoxic T lymphocyte (CTL) reactivity against leukemia after human histocompatibility leukocyte antigen (HLA)-identical allogeneic bone marrow transplantation (BMT). As most mHags are not leukemia specific but are also expressed by normal tissues, antileukemia reactivity is often associated with life-threatening graft-versus-host disease (GVHD). Here, we describe a novel mHag, HB-1, that elicits donor-derived CTL reactivity in a B cell acute lymphoblastic leukemia (B-ALL) patient treated by HLA-matched BMT. We identified the gene encoding the antigenic peptide recognized by HB-1-specific CTLs. Interestingly, expression of the HB-1 gene was only observed in B-ALL cells and Epstein-Barr virus-transformed B cells. The HB-1 gene-encoded peptide EEKRGSLHVW is recognized by the CTL in association with HLA-B44. Further analysis reveals that a polymorphism in the HB-1 gene generates a single amino acid exchange from His to Tyr at position 8 within this peptide. This amino acid substitution is critical for recognition by HB-1-specific CTLs. The restricted expression of the polymorphic HB-1 Ag by B-ALL cells and the ability to generate HB-1-specific CTLs in vitro using peptide-loaded dendritic cells offer novel opportunities to specifically target the immune system against B-ALL without the risk of evoking GVHD.

Figure 1

Identification of the cDNA encoding the antigenic peptide recognized by CTL clone MP1. Production of IFN-γ is shown upon stimulation with the following stimulator cells: B-ALL cells, EBV-transformed B cells, and B cells stimulated with CD40 plus 100 U/ml TNF-α for 2 d of the HLA-B44, HB-1–positive patients MP and VR, and COS-1 cells cotransfected with the HB-1 cDNA plus HLA-B44 cDNA, and COS-1 cells transfected either with the HLA-B44 cDNA alone or with HB-1 cDNA alone. Release of IFN-γ was measured by ELISA.

Figure 2

Location of the nucleotide sequence coding for the translation initiation codon and the antigenic peptide recognized by CTL MP1. HB-1 cDNA deletion constructs were cloned into an expression vector and cotransfected with the HLA-B44 cDNA into COS-1 cells. Transfected cells and CTL MP1 were incubated for 18 h and the release of IFN-γ was measured by ELISA.

Figure 3

Identification of the HB-1 antigenic peptide. (A) Cytolytic activity by CTL clone MP1 against HLA-B44–positive target cells incubated with 5 μM of HB-1 peptide EEKRGSLHVW. Controls included the HLA-B44–positive target cells incubated either without peptide or with the EBNA3C 281–290 peptide EENLLDFVRF. (B) Cytolytic activity by the EBNA3C-specific CTL against HLA-B44–positive target cells incubated with 5 μM of EBNA3C peptide EENLLDFVRF. Controls included the HLA-B44–positive target cells incubated either without peptide or with the HB-1 peptide EEKRGSLHVW.

Figure 3

Identification of the HB-1 antigenic peptide. (A) Cytolytic activity by CTL clone MP1 against HLA-B44–positive target cells incubated with 5 μM of HB-1 peptide EEKRGSLHVW. Controls included the HLA-B44–positive target cells incubated either without peptide or with the EBNA3C 281–290 peptide EENLLDFVRF. (B) Cytolytic activity by the EBNA3C-specific CTL against HLA-B44–positive target cells incubated with 5 μM of EBNA3C peptide EENLLDFVRF. Controls included the HLA-B44–positive target cells incubated either without peptide or with the HB-1 peptide EEKRGSLHVW.

Figure 4

Sequence of HB-1 cDNA and of the 41 amino acid– encoded protein starting from the CTG start codon (underlined) at nucleotide positions 108–110. The sequence corresponding to the HLA-B44– restricted HB-1 peptide is boxed. These sequence data are available from EMBL/GenBank/DDBJ under accession number AF103884.

Figure 5

Expression of the HB-1 gene in tumor cells and nonmalignant cells. (A) Measurement of the HB-1 expression obtained by real time quantitative reverse transcriptase PCR in tumor cells and cell lines. The following cell lines were used: B-ALL (KM3, BV173); B cell lymphoma (Daudi, Raji, Ramos, SU-DHL6); multiple myeloma (RPMI1758); T-ALL (Jurkat, CEM, HSB-2); and acute myeloid leukemia (Lama, Kasumi, K562, HL60, KG-1). The expression levels were determined by a calibration function generated from RNA of the HB-1–positive B-ALL cell line KM3 and expressed relative to the HB-1 level measured in these cells. The Pbgd gene was used as standard to correct for RNA quantity and quality. The detection limit is indicated with a solid line. Samples showed significant HB-1 gene expression if they exceeded 10% of that found in the B-ALL cell line KM3. This arbitrary threshold is indicated with a dashed line. (B) Measurement of the HB-1 expression obtained by real time quantitative reverse transcriptase PCR in freshly isolated cells or primary cell cultures.

Figure 6

Cytolytic activity by CTL clone MP1 against EBV-LCL and PHA-stimulated T cell blasts of HLA-B44, HB-1–positive individuals. PHA-stimulated T cell blasts were preincubated either without peptide or with the HB-1 peptide EEKRGSLHVW. The level of HB-1 gene expression in EBV-LCL VH and MT was 140 and 122% of that found in the B-ALL cell line KM3, respectively, and in PHA-stimulated T cell blasts VH and MT was 8 and 2.5%, respectively. Both individuals express homozygous the HB-1^H allele and the HLA-B44 subtype of VH is B*4403 and of MT B*4402. The E/T cell ratio was 3:1.

Figure 7

The mHag HB-1 is encoded by the HB-1^H allele of the HB-1 gene. (A) Sequence of the peptide coding region of the HB-1 gene of patient MP and donor BP. The nucleotide and amino acid polymorphism are underlined. (B) Correlation between expression of HB-1 alleles and cytolytic activity by CTL clone MP1 against EBV-transformed B cell lines of relatives of patient MP. Filled circles (females) or squares (males) indicate strong lysis by CTL MP1. Open symbols indicate no lysis. Expression of HB-1 alleles was determined by reverse transcriptase PCR amplification and digestion of PCR products with restriction enzyme NlaIII.

Figure 8

The single amino acid exchange within the HB-1 antigenic peptide is critical for recognition by CTL clone MP1. (A) Production of IFN-γ is shown upon stimulation with EBV-transformed B cells of patient MP and donor BP, and COS-1 cells cotransfected with the HB-1^H or HB-1^Y cDNA and HLA-B44 cDNA. (B) Cytolytic activity by CTL clone MP1 against HLA-B44–positive target cells incubated with the HB-1^H or HB-1^Y peptide. The E/T cell ratio was 10:1.