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. 2012 Aug;97(8):1196-204.
doi: 10.3324/haematol.2011.049478. Epub 2012 Mar 14.

Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity

Marieke Griffioen  1 , M Willy Honders, Edith D van der Meijden, Simone A P van Luxemburg-Heijs, Ellie G A Lurvink, Michel G D Kester, Cornelis A M van Bergen, J H Frederik Falkenburg
Affiliations

Identification of 4 novel HLA-B*40:01 restricted minor histocompatibility antigens and their potential as targets for graft-versus-leukemia reactivity

Marieke Griffioen et al. Haematologica. 2012 Aug.

Abstract

Background: Patients with hematologic malignancies can be successfully treated with donor lymphocyte infusion after HLA-matched allogeneic hematopoietic stem cell transplantation. The effect of donor lymphocyte infusion is mediated by donor T cells recognizing minor histocompatibility antigens. T cells recognizing hematopoietic restricted minor histocompatibility antigens may induce selective graft-versus-leukemia reactivity, whereas broadly-expressed antigens may be targeted in graft-versus-host disease.

Design and methods: We analyzed in detail CD8(+) T-cell immunity in a patient with relapsed chronic myelogenous leukemia who responded to donor lymphocyte infusion with minimal graft-versus-host disease of the skin. CD8(+) T-cell clones specific for 4 HLA-B*40:01 restricted minor histocompatibility antigens were isolated which were identified by screening a plasmid cDNA library and whole genome association scanning. Detailed T-cell reactivity and monitoring experiments were performed to estimate the clinical and therapeutic relevance of the novel antigens.

Results: Three antigens were demonstrated to be expressed on primary leukemic cells of various origins as well as subtypes of non-malignant hematopoietic cells, whereas one antigen was selectively recognized on malignant hematopoietic cells with antigen presenting cell phenotype. Skin derived fibroblasts were only recognized after pre-treatment with IFN-γ by two T-cell clones.

Conclusions: Our data show evidence for different roles of the HLA-B*40:01 restricted minor histocompatibility antigens in the onset and execution of the anti-tumor response. All antigens may have contributed to a graft-versus-leukemia effect, and one minor histocompatibility antigen (LB-SWAP70-1Q) has specific therapeutic value based on its in vivo immunodominance and strong presentation on leukemic cells of various origins, but absence of expression on cytokine-treated fibroblasts.

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Figures

Figure 1.
Figure 1.
Isolation of CD8+ T-cell clones specific for HLA-B*40:01 restricted MiHA. (A) A number of selected CD8+ T-cell clones showed reactivity against patient, but not donor, EBV-B cells. Mean percentage of specific lysis in triplicate wells is shown at E:T ratios of 10:1 in 4 h 51Cr-release assays (upper) and of IFN-γ (ng/mL) in 50 μL culture supernatants of duplicate wells in ELISA (lower). Reactivity against patient and donor EBV-B cells by an allo-HLA-A*02:01 reactive T-cell clone is shown as control. (B) Selected CD8+ T-cell clones were specific for MiHA in HLA-B*40:01, as demonstrated by specific recognition of MiHA+ EBV-B cells after retroviral transfer of MP71-HLA-B*40:01-IRES-NGFR, but not mock MP71 vector, in IFN-γ ELISA. Specific production of IFN-γ (ng/mL) in 50 μL culture supernatants of duplicate wells in ELISA is shown.
Figure 2.
Figure 2.
Identification of MiHA by whole genome association scanning. (A) A panel of approx. 60 SNP-genotyped EBV-B cells expressing HLA-B*40:01 endogenously or after retroviral transfer of MP71-HLA-B*40:01-IRES-NGFR with more than 20% of marker gene positive cells were tested for recognition by T-cell clones ZRZ25, 3H1 and 12A2 in IFN-γ ELISA. EBV-B cells were divided into MiHA positive and negative groups based on a threshold of 5-fold the background production of IFN-γ (horizontal lines). Mean release of IFN-γ (ng/mL) in 50 μL culture supernatants of duplicate wells is shown. (B) Peptides comprising patient (filled symbols) type amino acids with predicted binding to HLA-B*40:01 as well as donor type peptide variants (gray symbols) were pulsed on donor EBV-B cells and tested for T-cell recognition in IFN-γ ELISA. LB-TRIP10-1EPC (GEPQDLCTL) contains 3 patient type amino acids and has been identified by cDNA library screening. LB-SON-1R (SETKQRTVL), LB-SWAP70-1Q (MEQLEQLEL) and LB-NUP133-1R (SEDLILCRL) contain single patient type amino acids encoded by exon SNPs identified by WGAs based on significant association with T-cell recognition. Donor type peptides SETKQCTVL and MEQLEELEL encoded by the genes for SON and SWAP70, respectively, were not recognized by the T-cell clones, whereas donor type peptide SEDLILCQL encoded by the NUP133 gene was similarly recognized as LB-NUP133-1R. Mean production of IFN-γ (ng/mL) in 50 μL culture supernatants of duplicate wells at various peptide concentrations (μg/mL) is shown. (C) NUP133 genes were isolated from patient and donor derived cDNA and cloned into expression vector pcDNA-3. Hela cells stably expressing HLA-B*40:01 were transiently transfected with patient and donor derived NUP133 genes encoding the R and Q at position 406, respectively, and incubated with T-cell clone 3H1. Mean production of IFN-γ (ng/mL) in 50 μL culture supernatants of duplicate wells is shown.
Figure 3.
Figure 3.
T-cell recognition of non-malignant hematopoietic and non-hematopoietic cells. CD8+ T-cell clones specific for LB-TRIP10-1EPC, LB-SON-1R, LB-SWAP70-1Q and LB-NUP133-1R were tested for recognition of non-malignant hematopoietic and non-hematopoietic cells in IFN-γ ELISA. (A) T-cell clones were tested against patient derived EBV-B cells and PHA-T blasts, as well as primary B cells, T cells and monocytes isolated from patient PBMC prior to alloSCT. (B) T-cell clones were tested against FB cultured from a skin biopsy obtained from the patient after alloSCT. FB were cultured with and without IFN-γ (100 IU/mL) for four days. Reactivity of T-cell clone 4D8 recognizing an unknown MiHA in HLA-B*08:01 is shown as control. Mean production of IFN-γ (ng/mL) in 50 μL culture supernatants of duplicate wells is shown.
Figure 4.
Figure 4.
T-cell recognition of leukemic cells of different origins. CD8+ T-cell clones specific for LB-TRIP10-1EPC, LB-SON-1R, LB-SWAP70-1Q and LB-NUP133-1R were tested for recognition of malignant hematopoietic cells in IFN-γ ELISA. (A) T-cell clones were tested against CD34+ CML cells directly after flowcytometric isolation from BM cells from 3 HLA-B*40:01 positive patients (gray bars) as well as after in vitro culture with growth factors to generate CML-APC (filled bars). (B) T-cell clones were tested against ALL cells from PB and BM samples from 5 HLA-B*40:01 positive patients directly after isolation by flowcytometry based on expression of CD19. (C) T-cell clones were tested against PB and BM samples from 10 HLA-B*40:01 positive patients with more than 40% CD33+ AML cells of different subtypes (M0–M6). Two HLA-B*40:01 negative AML-M4 and M5 samples were included as negative controls. The MiHA status of the samples is shown as +/+, +/− and −/−. Mean production of IFN-γ (ng/mL) in 50 μL culture supernatants of duplicate wells. Unpaired Student’s t-test showed a significant difference in T-cell recognition between MiHA positive (+/− or +/+) and MiHA negative (−/−) CML, ALL and AML samples for LB-SON-1R (P=5.6×10−5), LB-SWAP70-1Q (P=1.2×10−4), and LB-NUP133-1R (P=4.0×10−3). A significant difference for LB-TRIP10-1EPC (P=3.7×10−3) was demonstrated between MiHA positive (+/− and +/+) AML-M4/M5 and MiHA positive (+/+ and +/−) CML, ALL and AML samples of other subtypes.
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