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Blood, 1 May 2007, Vol. 109, No. 9, pp. 4089-4096. Prepublished online as a Blood First Edition Paper on January 18, 2007; DOI 10.1182/blood-2006-08-043935.
TRANSPLANTATION Multiple myeloma–reactive T cells recognize an activation-induced minor histocompatibility antigen encoded by the ATP-dependent interferon-responsive (ADIR) gene1 Laboratory of Experimental Hematology, Department of Hematology, Leiden University Medical Center, The Netherlands; 2 Department of Immunohematology and Blood Transfusion, Centre for Medical Systems Biology, Leiden University Medical Center, The Netherlands; 3 Medisch Spectrum Twente, Enschede, The Netherlands
Minor histocompatibility antigens (mHags) play an important role in both graft-versus-tumor effects and graft-versus-host disease (GVHD) after allogeneic stem cell transplantation. We applied biochemical techniques and mass spectrometry to identify the peptide recognized by a dominant tumor-reactive donor T-cell reactivity isolated from a patient with relapsed multiple myeloma who underwent transplantation and entered complete remission after donor lymphocyte infusion. A frequently occurring single nucleotide polymorphism in the human ATP-dependent interferon-responsive (ADIR) gene was found to encode the epitope we designated LB-ADIR-1F. Although gene expression could be found in cells from hematopoietic as well as nonhematopoietic tissues, the patient suffered from only mild acute GVHD despite high percentages of circulating LB-ADIR-1F–specific T cells. Differential recognition of nonhematopoietic cell types and resting hematopoietic cells as compared with activated B cells, T cells, and tumor cells was demonstrated, illustrating variable LB-ADIR-1F expression depending on the cellular activation state. In conclusion, the novel mHag LB-ADIR-1F may be a suitable target for cellular immunotherapy when applied under controlled circumstances.
Allogeneic stem cell transplantation (SCT) is a curative treatment in patients with hematologic cancers. In addition to the antitumor effects of chemotherapy, antibody treatment and/or irradiation administered as the conditioning regimen prior to transplantation, an allogeneic graft-versus-tumor (GVT) immunoreactivity significantly contributes to the curative potential of this therapy.1–5 This GVT reactivity following HLA-matched SCT has been demonstrated to be mediated primarily by T cells from the donor. All reactive T cells from donor origin may not only mediate the beneficial GVT effect but are also responsible for the development of graft-versus-host disease (GVHD), which is the major detrimental complication after allogeneic SCT.6,7 T-cell depletion of the stem cell graft removes both GVHD and GVT effect.8–10 The antitumor reactivity can be reintroduced in case of persistent or relapsed hematologic malignancies after transplantation by donor lymphocyte infusion (DLI).11–15 Whereas profound antitumor effects are frequently associated with GVHD in patients responding to DLI, postponed administration of DLI has been associated with a decreased risk of severe GVHD, and clinical observations indicate that more subtle antitumor reactivities can also be observed in the absence of GVHD.16–18 The main targets of both GVHD and GVT reactivity after HLA-matched allogeneic SCT are minor histocompatibility antigens (mHags).19–21 mHags are peptides differentially expressed by donor and recipient that can be recognized in the context of self-HLA molecules. mHags may arise from differential processing of peptides due to polymorphisms in the gene encoding the protein or by direct polymorphisms in the peptide sequence that is presented in the HLA molecules.22,23 The clinical manifestation of immune responses against mHags is likely to be determined by the specific tissue expression of the proteins encoding these antigens. Whereas mHags constitutively expressed in many tissues are likely to be targets for combined alloreactive GVHD and GVT responses, T-cell responses directed against antigens that are restricted to the hematopoietic cell lineages including the malignant T cells of hematopoietic origin are likely to mediate a GVT reactivity without severe GVHD.21,24–31 However, antigens with variable expression in tissues may also be targets for relatively specific GVT responses because these proteins may not be expressed or recognized on normal tissues under steady state conditions.19,32,33 By specific stimuli like inflammatory reactions they may be induced in target tissues of GVHD, leading to a detrimental local immune response.7,34,35 Characterization of mHags from patients responding to cellular immunotherapeutic interventions following allogeneic SCT in the presence or absence of GVHD will lead to a better understanding of the pathogenesis of GVHD and GVT and may lead to the development of specific antitumor T-cell therapy.
Recently, we studied in detail several patients treated for relapsed hematologic malignancies after allogeneic SCT with DLI. During the clinical GVT response, we isolated tumor-reactive T cells based on their ability to produce IFN- In the present study we identified the mHags recognized by this CTL clone to be encoded by the ATP-dependent interferon-responsive (ADIR) gene.37 This gene was found to be highly expressed in the MM cells, in other hematopoietic tumors, as well as nonhematopoietic tumor cell lines. Recognition of normal nonmalignant cells appears to be minor under steady state conditions, but activation of the target cell populations resulted in enhanced recognition by the CTL clone. We hypothesize that T-cell responses against mHags encoded by the ADIR gene may lead to a strong GVT reactivity, potentially coincident with GVHD depending on the activation state of the target tissues.
CTL generation and culture
The HLA-A2–restricted mHag-specific CTL clone RDR2 was previously isolated using the IFN- Target-cell populations
After informed consent was obtained in accordance with the Declaration of Helsinki, with approval from Leiden University Medical Center's Institutional Review Board, BM and PB samples were collected from patients and donors, and MNCs were isolated by Ficoll-Isopaque separation. Recognition of target cells was measured in cytotoxicity assays, and stimulation of responder cells was measured using INF- Cytotoxicity assays CTL-induced specific cytotoxicity of defined cell types in a heterogeneous target cell population was determined in CFSE-based cellular cytotoxicity assays as described before.40 Briefly, target cells were labeled with 2.5 µM CFSE (Molecular Probes, Leiden, The Netherlands) and incubated with target cells at a 1:1 ratio. After 4 and 24 hours, specific cell populations were counterstained with PE- or APC-labeled CD138, CD3, CD4, CD8, CD14, or CD19 mAb (Becton Dickinson, Erembodegem-Aalst, Belgium), and propidium iodide was added to exclude dead cells. To allow quantification of the surviving cell numbers in each sample, 104 Flow-count Fluorospheres (Coulter, Miami, FL) were added immediately before flow cytometric analysis. Cytotoxicity of CTL clones in standard 51Cr release assays was performed as described previously.41 HPLC-purified natural peptides or diluted synthetic peptides were tested for reactivity by loading 51Cr-labeled T2 cells for 1 hour at 37°C and 5% CO2 prior to addition of CTLs.
IFN-
Quantification of CTL stimulation was performed by IFN- Peptide isolation, purification, and characterization Purification of peptides from 8 x 1010 EBV-LCLs was performed as described before.24,42 Briefly, frozen cell pellets were lysed and affinity purified. Peptides were separated from proteins by size-exclusion centrifugation, and peptide concentrates were injected on a Smart System (Amersham Biosciences, Freiburg, Germany) and subjected to reverse-phase high-performance liquid chromatography (RP-HPLC) on a 10 cm x 2.1 mm C2/C18 3 µm particle column at 0.2 mL/min. A gradient from 20% to 50% organic phase containing 0.1% TFA was run while 0.1 mL fractions were collected in siliconized vials and stored at –80°C. Isopropanol or acetonitrile was used as organic phase. Reactive fractions were injected on a 15 cm x 75 um C18-Pepmap nano column (LC Packings/Dionex, Breda, The Netherlands), which was directly coupled to a QTOF1 mass spectrometer (Micromass Waters, Manchester, United Kingdom). The flow was delivered by a conventional JASCO binary gradient system (JASCO, Maarssen, The Netherlands), which was reduced to approximately 300 nL/min by an in-house–constructed flow splitter. Injections were performed with a FAMOS (LC Packings/Dionex). On-line HPLC tandem mass spectrometry analysis on the HCTplus (Bruker Daltonics, Bremen, Germany) was performed in a similar fashion. In this case the flow was delivered by an Ultimate (LC Packings/Dionex). Sequence analysis of the ADIR gene Trizol reagent (Invitrogen) was added to patient and donor cell pellets, RNA was isolated and purified, and 4 µg was reverse transcribed into cDNA for 1 hour at 37°C using Moloney murine leukemia virus (M-MLV) reverse transcriptase (Invitrogen) in accordance with the manufacturer's instructions. Polymerase chain reactions (PCRs) of nucleotides 1 to 327 of the ADIR gene were performed in 50 µL GeneAmpII PCR buffer containing 1.5 mM MgCl2, 250 µM dNTPs, 800 nM forward primer (nucleotides 1 to 18), 800 nM reverse primer (nucleotides 309 to 327), 2% DMSO, and 1.5 units of AmpliTaq DNA polymerase. Amplification on an Applied Biosystems GeneAmp PCR system 2400 was achieved in 35 cycles of 15 seconds at 95°C and 30 seconds at 58°C. Sequence reactions were performed on 1 µL of purified PCR product using the Big Dye Terminator v3.1 sequencing kit (Applied Biosystems, Foster City, CA) and 1 µM reverse primer in 25 cycles of 10 seconds at 96°C, 5 seconds at 58°C, and 4 minutes at 60°C. After DNA purification, sequencing was performed using an ABI310 sequencer. Transfection of constructs containing the LB-ADIR-1F epitope
Constructs from donor and patient cDNA containing the ADIR gene were generated using 3 different forward primers and 1 reverse primer and ligated in pCR3.1 expression vector (Invitrogen) as described previously.31 A mixture of 100 ng plasmid and 0.8 µL Lipofectamine (Invitrogen) was incubated for 30 minutes at room temperature and used to transiently transfect 2 x 104 Hela/A2 cells. After 5 hours of incubation at 37°C, 50 µL IMDM/10% FBS was added. After 24 hours, 104 RDR2 cells were added and again after 24 hours 50 µL of supernatant was harvested and tested for IFN- Ex vivo detection of LB-ADIR-1F–specific T cells and TCRBV analysis Recombinant biotinylated HLA-A*0201 monomers were folded with both ADIR peptide variants. Streptavidin-APC tetramers were produced as described previously.43 Tetramer-positive events in thawed patient samples were sorted by fluorescence-activated cell sorting (FACS) to single cells per well, expanded, and tested for cytotoxicity as described previously.21 T-cell receptor V-B expression of cytotoxic clones and tetramer-positive T cells in patient material was determined by staining with FITC-conjugated mAb to TCRBV7 (Beckman Coulter, Mijdrecht, The Netherlands). Sequences of the TCRBV were determined as described previously,36 and TCR chains were named in accordance with the nomenclature described by Arden et al.44 Quantitative real-time-PCR to analyze gene expression Quantitative real-time PCR analysis was performed as described previously.45 To normalize for variations in the procedures for RNA isolation and cDNA synthesis, 1% mouse spleen cells were added to each cell sample. Quantitative real-time PCR was performed on an ABI/PRISM 7700 Sequence Detector System (Applied Biosystems) using qPCR-Core Kit (Eurogentec, Seraing, Belgium). Human ADIR and PBGD results were normalized using the murine GAPDH expression. For each sample, relative gene expression was calculated using the results obtained from a serial diluted EBV-LCL. EBV-LCL expression was set to 100. Primers for human ADIR were designed spanning exons 1 to 3: forward 5'-GACGACTGTGACGAGGACGA-3', reverse 5'-CAAATGCTGGCCATGCAG-3', and probe 5'-(TET)-CTGGGCTGGCGCCTTCCTCTGT-(TAMRA)-3'. Primers and probe for human PBGD were as follows: forward 5'-GCAATGCGGCTGCAA-3', reverse 5'-GGGTACCCACGCGAATG-3', and probe 5'-(TET)-CTCATCTTTGGGCTGTTTTCTTCCGCC-(TAMRA)-3'. Primers and probe for murine GAPDH were as follows: forward 5'-GGGCTCATGACCACAGTCCA-3', reverse 5'-ATACTTGGCAGGTTTCTCCAGG-3', and probe 5'-(TET)-TCCTACCCCCAATGTGTCCGTCGT-(TAMRA)-3'.
Isolation of the HLA-A2–restricted CD8+ CTL clone RDR2 recognizing a frequently expressed mHags
We previously described the isolation of various CTL clones from a female patient who was successfully treated with DLI after relapsed MM.36 CTL clones were generated by direct cloning of IFN-
Because RDR2 was isolated from an MM patient, we investigated the sensitivity of her MM cells to lysis by the T-cell clone. The CFSE-based cytotoxicity assay was performed to allow quantitative measurement of lysis of MM cells that were present in relatively low frequencies within the heterogeneous bone marrow samples.40 Figure 1A shows dot plots of a quantitative CFSE-based cytotoxicity assay performed on thawed bone marrow cells from an mHag-positive patient containing 10% MM cells. Lysis was measured after incubation with a control CMV-pp65-A2–specific CTL clone or with CTL RDR2. CD138 was used as a marker for the malignant MM cells. A strong decrease of the number of CD138+ cells was observed. In a sample containing 1% MM cells that was taken from the patient from which CTL RDR2 was isolated, lysis was measured of bone marrow cells using CD138 as a marker for the malignant MM cells and CD3 as a marker for nonmalignant patient-derived T cells. Figure 1B illustrates that MM cells from the patient were strongly lysed whereas lysis of normal unstimulated T cells was low. Activated T cells (PHA blasts) and EBV-LCLs from the patient were strongly recognized. Donor-derived PHA blasts and EBV-LCLs were not recognized (data not shown). To further study susceptibility of normal hematopoietic cells to lysis by RDR2, PBMC subpopulations from HLA-A2–positive mHag-positive donors were tested for recognition by RDR2 and an allo-A2 clone. Whereas similar recognition of PHA blasts and EBV-LCLs by the allo-A2 clone and RDR2 was observed, RDR2-mediated lysis of normal B cells and T cells was lower as compared with allo-A2–mediated lysis (Figure 1C). In addition, PBMCs were separated by magnetic bead cell sorting into CD4+ T cells, CD8+ T cells, monocytes, and B cells and were used to stimulate RDR2 and allo-A2 CTL in IFN-
In conclusion, RDR2 recognized an HLA-A2–restricted epitope causing strong lysis of MM cells and activated T cells and B cells. In contrast, reactivity with normal nonactivated hematopoietic cells was relatively low as measured both by direct cytotoxicity and by IFN-  secretion. Purification and mass spectrometric identification of the peptide To identify the epitope that was recognized by CTL clone RDR2, EBV-LCL cells expressing the antigen were lysed, and peptide-HLA complexes were affinity purified using HLA-A2–specific BB7.2 mAb. After elution of peptides, size centrifugation was performed to separate peptides from HLA monomers and ß2-microglobulin. After freeze drying, the peptide mixture was subjected to RP HPLC using isopropanol as organic solvent and fractions were collected. 51Cr-labeled T2 cells were loaded with a small sample of each fraction. RDR2 was added, and a single positive fraction could be detected. This fraction was subsequently subjected to RP HPLC with acetonitrile as organic solvent and fractionated. Fractions were tested for reactivity, and again a positive fraction was found. To determine the most abundant masses present in this fraction, part of the fraction was injected on a nano–liquid chromatography system directly coupled to a Q-TOF1 mass spectrometer. Abundantly present masses were fragmented by collision-activated dissociation on an HCTplus mass spectrometer. Analysis of obtained fragmentation patterns led to the sequence of several candidate peptides that were subsequently synthesized. RDR2 recognized an [M+2H]++ candidate peptide with mass/charge of 528.8 and sequence SVAPALALFPA at levels as low as 10 pM (data not shown). Furthermore, the fragmentation patterns of the synthetic peptide and the natural eluted peptide were identical (data not shown). Identification of a polymorphic gene responsible for RDR2 recognition
A basic local alignment sequence tool (BLAST) search of sequence SVAPALALFPA against a 6-frame translation of the European Molecular Biology Laboratory (EMBL) nucleotide database revealed 100% identity to amino acids 13 to 23 from an alternative open reading frame (ORF) of the ADIR gene, also known as TOR3A37 (National Center for Biotechnology Information [NCBI] mRNA Reference Sequence [RefSeq] NM_022371
[GenBank]
). A known single nucleotide polymorphism (SNP) in ADIR at nucleotide 78 from C>T (NCBI dbSNP cluster ID rs2296377) results in an amino acid change in an alternative transcript from serine (S) to phenylalanine (F) at position 21, corresponding to position 9 of the eluted peptide (Figure 2A). Both peptides were synthesized and loaded on T2 cells. Patient-type peptide SVAPALAL-F-PA but not donor-type peptide SVAPALAL-S-PA was recognized by RDR2 (Figure 2B). Sequence analysis of the ADIR PCR product flanking the SNP revealed that the donor was CC homozygous and the patient CT heterozygous. To demonstrate that patient-type T but not donor-type C of this gene was responsible for recognition by RDR2, constructs were generated from both patient and donor cDNA. Because RDR2 recognized a peptide arising from an alterative ORF controlled by a start codon 5' upstream from the normal start codon, 3 different forward primers were composed. The first primer was chosen at the normal start codon, thus lacking the alternative start codon. The second primer was chosen at the start of the transcript, thus providing both start codons. The third primer was chosen at the alternative start codon and also contained the normal start codon. The constructs were transiently transfected into Hela/A2 cells. Only patient-derived constructs induced IFN-
Tetramer staining and FACS of LB-ADIR-1F–specific CTLs Both peptide LB-ADIR-1F and peptide SVAPALAL-S-PA were able to bind to recombinant HLA-A*0201 molecules, and tetrameric complexes were produced. RDR2 specifically bound the LB-ADIR-1F tetramer whereas SVAPALAL-S-PA tetramers and irrelevant HA-1 tetramers were negative (data not shown). LB-ADIR-1F tetramers were used to analyze a series of peripheral blood samples that were taken from the patient before and after DLI. Serum paraprotein levels were analyzed as a marker for disease activity. Whereas LB-ADIR-1F–specific T cells were not detectable prior to DLI, at 7 weeks after DLI high numbers of LB-ADIR-1F–specific CD8+ T cells could be detected (Figure 3A). The appearance of LB-ADIR-1F–specific T cells coincided with development of acute GVHD grade II and resulted in complete remission. GVHD was treated successfully with a short course of 1 mg prednisone per kilogram of body weight and cyclosporin. Tetramer-positive T cells were clonally isolated by FACS and expanded. All tetramer-positive CTL clones were able to lyse both patient EBV-LCLs and LB-ADIR-1F–pulsed donor EBV-LCLs (data not shown). TCR characterization of RDR2 showed usage of V-beta BV7S1, J-region BJ1S4, and identical CDR3 regions in 43 of 44 growing clones analyzed. One clone, however, expressed TCRBV6S4 (Figure 3B). Analysis of the patient sample at 7 weeks after DLI revealed that a low percentage of LB-ADIR-1F–positive T cells did not stain with mAb directed against TCR-BV7 (Figure 3C). Functional comparison of the original RDR2, newly isolated identical TCR-BV7S1 clones, and TCR-BV6S4–expressing clones was performed. Whereas cytotoxicity of the original RDR2 and newly isolated BV7S1 clones was similar (data not shown), the BV6S4 clone showed diminished recognition of peptide-loaded T2 cells (Figure 3D). When HLA-A2–positive and LB-ADIR-1F–expressing EBV-LCL cells were used as target cells, again the BV6S4 clone displayed lower cytotoxicity (Figures 3E). Similar results were obtained using HLA-A2–positive and LB-ADIR-1F–expressing PHA blasts (data not shown). To further investigate differential recognition, reactivity of both TCR BV7S1 and TCR BV6S4 was tested on EBV-LCL–expressing HLA-A*0205 and HLA-A*0220 subtypes. Lysis of LB-ADIR-1F–positive HLA-A*0205 EBV-LCLs was absent whereas identical strong lysis between both TCR types was observed of HLA-A*0220 EBV-LCLs. Pulsing of HLA-A*0205 EBV-LCLs with synthetic peptide could only partially restore the recognition to identical levels by both TCR types (data not shown).
Modulation of recognition by IFN-  and ADIR gene expression
Because previous studies on the ADIR gene indicated that IFN-
Recognition of normal nonhematopoietic cells by LB-ADIR-1F–specific CTLs
Recognition of nonhematopoietic normal tissues was studied using BECs and MSCs expressing the LB-ADIR-1F SNP variant in both direct cytotoxicity assays and stimulation assays. Whereas direct cytotoxicity against patient EBV-LCLs was strong, lysis of MSCs (Figure 5A) and BECs (Figure 5B) was low. LB-ADIR-1F peptide pulsing of MSCs and BECs did not significantly increase lysis, suggesting overall low susceptibility of these target cells to direct cytotoxicity. When MSCs (Figure 5C) and BECs (Figure 5D) were used as stimulator cells in IFN-
Recognition of malignant hematopoietic cells and solid tumor cell lines To investigate susceptibility to lysis by LB-ADIR-1F–specific CTLs, a panel of HLA-A2–positive malignant hematopoietic cells was subjected to sequence analysis on the LB-ADIR-1F polymorphism and lysis by CTL RDR2 and allo-A2 CTL. Prominent recognition was found of MM cells. Additionally, leukemic cells expressing the LB-ADIR-1F polymorphism were susceptible to lysis by RDR2 (Figure 6A). Furthermore, when a panel of LB-ADIR-1F–positive HLA-A2–expressing solid tumor lines was tested for recognition by RDR2, lysis at levels comparable to lysis by the allo-A2 CTL was observed (Figure 6B).
In this study, we identified the ATP-dependent interferon-responsive gene to encode an activation-induced minor histocompatibility antigen that was recognized by MM-reactive T cells. A dominant HLA-A2–restricted CD8+ CTL clone designated RDR2 was isolated from a patient with relapsed MM after allogeneic SCT responding to DLI, resulting in a long-lasting complete remission. Biochemical analysis of peptides eluted from HLA molecules revealed that this CTL clone recognized the peptide SVAPALALFPA, which was encoded by an alternative ORF of the human ADIR gene. This mHag, designated LB-ADIR-1F, differed only by an S>F substitution from the donor-type allelic counterpart. A population study with 76 HLA-A2 individuals revealed a 100% correlation between SNP occurrence and recognition of target cells, confirming that the CTL clone was directed against this ADIR-derived epitope, which had a population frequency of 57%. The function of the ADIR gene is unknown. When T-cell recognition of target cells and expression levels of ADIR mRNA were analyzed in various cell types, we demonstrated a relatively low expression of the gene in resting cells corresponding with low recognition. When proliferation was induced in PB T cells and B cells, strong gene up-regulation was observed, resulting in high recognition by the LB-ADIR-1F–specific T cells. Although concurrent expression of normally and alternatively transcribed ADIR proteins cannot be directly determined in the cell, the strong correlation between the ADIR SNP and susceptibility to lysis illustrates the absence of specific expression regulation of the alternatively translated protein.
Because the patient was treated with IFN- Not only MM cells but also other hematologic malignancies and nonhematopoietic solid tumor cell lines were recognized by LB-ADIR-1F–specific T cells. MSCs and BECs, which may represent possible target tissues of GVHD, displayed low recognition by the ADIR-specific T cells. Our results illustrate broad antitumor reactivity of LB-ADIR-1F–specific T cells asshown by the lysis of malignant hematopoietic cells as well as solid tumor cell lines combined with limited recognition of nonactivated tissues. Kinetic studies of PB samples isolated from the patient before and after DLI using tetramers recognizing various mHags differentially expressed in donor and recipient illustrated a strong correlation between the clinical response and the occurrence of not only a high frequency of LB-ADIR-1F–specific T cells but also of HA-1 and LB-ECGF-1H–specific T cells as studied previously.21,28,31,36 The LB-ADIR-1F–specific T cells were the most dominant, comprising almost 3% of the CD8+ T cells. Approximately 1% LB-ECGF-1H–specific T cells and 0.9% HA1-specific T cells were found with similar kinetics. These in vivo circulating mHag-specific T cells represent 80% of activated PB CD8+ T cells as identified by coexpression of HLA-DR. Furthermore, because HA-1, ECGF, and ADIR proteins are highly expressed in MM cells and their relevant polymorphism can be recognized by T cells, we hypothesize that the 3 combined strong reactivities are likely to have caused the clinical course of the major response to DLI resulting in a complete remission lasting now for more than 6 years. In conclusion, we characterized a novel mHag encoded by a frequently occurring SNP in the ADIR gene that is recognized by tumor-reactive CTLs. We hypothesize that the balance between GVT responses and GVHD is not only determined by the specificity of T cells but also by the activation state of GVHD target tissues.33–35,46 Up-regulated tissue susceptibility to mHag-specific T cells may be caused by the cytokine storm shortly after stem celltransplantation due to conditioning regimens resulting in tissue damage and the presence of high numbers of recipient-originated antigen-presenting cells. Homeostatic proliferation of these T cells during the lymphopenic phase may further amplify the magnitude of such responses. This state may result in severe GVHD if these T cells are administered as part of the stem cell graft. Immunotherapy of tumor cells by postponed adoptive transfer of LB-ADIR-1F–specific T cells following initially T-cell–depleted transplantation may result in a strong GVT response with limited risk of GVHD.
Contribution: C.A.M.v.B., M.G.D.K., P.A.v.V., R.W., and J.H.F.F. designed the study; C.A.M.v.B., M.G.D.K., I.J., M.H.M.H., S.A.P.v.L.H., F.M.K., A.H.d.R., P.A.v.V., and J.H.F.F. performed research and analyzed data; W.A.F.M. and M.R.S. provided and analyzed the patient's materials; and C.A.M.v.B., R.W. and J.H.F.F. wrote the manuscript. Conflict-of-interest disclosure: The authors declare no competing financial interests. P.A.v.V. and J.H.F.F. contributed equally to this paper. Correspondence: Cornelis A. M. van Bergen, Laboratory of Experimental Hematology, Department of Hematology, Leiden University Medical Center, PO Box 9600 2300 RC Leiden, The Netherlands; e-mail: c.a.m.van_bergen{at}lumc.nl.
This work was supported by the Centre for Medical Systems Biology, a center of excellence approved by the Netherlands Genomic Initiative/Netherlands Organisation for Scientific Research (A.H.d.R. and P.A.v.V.), and AlloStem (EU grant 503319). The authors thank E. G. A. Lurvink and A. B. Kruisselbrink for their expert technical assistance with EBV-LCL cell culture and BEC cell culture and R. van der Linden for expert technical assistance with flow cytometric isolation.
Submitted August 29, 2006; accepted December 20, 2006.
Prepublished online as Blood First Edition Paper, January 18, 2007
DOI: 10.1182/blood-2006-08-043935
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Abstract
Introduction
) and control allo-A2 CTL (
) was tested in CFSE-based cytotoxicity assays and by IFN-
) (A). LB-ADIR-1F tetramer-positive cells present at week 7 after DLI were single-well sorted and expanded. TCRBV sequence analysis was performed on 44 reactive clones revealing TCRBV7S1 in 43 clones and TCRBV6S4 in 1 clone (B). Reanalysis of the patient sample was performed using counterstaining with TCRBV7 confirming a low percentage of TCRBV7-negative cells in the LB-ADIR-1F–positive population (C). Reactivity of TCRBV7S1- (
) expressing clones was determined using 51Cr release assays on peptide-pulsed T2 cells (D) and EBV-LCL cells (E), demonstrating that TCRBV6S4-expressing T cells displayed lower cytotoxicity.
