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Blood, 1 December 2005, Vol. 106, No. 12, pp. 3955-3957. Prepublished online as a Blood First Edition Paper on August 11, 2005; DOI 10.1182/blood-2004-09-3749.
NEOPLASIA Up-regulated expression in nonhematopoietic tissues of the BCL2A1-derived minor histocompatibility antigens in response to inflammatory cytokines: relevance for allogeneic immunotherapy of leukemiaFrom the Department of Hematology, Leiden University Medical Center, Leiden, The Netherlands.
T cells directed against hematopoietic-restricted minor histocompatibility antigens (mHags) may mediate graft-versus-leukemia (GVL) reactivity without graft-versus-host disease (GVHD). Recently, the HLA-A24–restricted mHag ACC-1 and the HLA-B44–restricted mHag ACC-2 encoded by separate polymorphisms within the BCL2A1 gene were characterized. Hematopoietic-restricted expression was suggested for these mHags. We demonstrate BCL2-related protein A1 (BCL2A1) mRNA expression in mesenchymal stromal cells (MSCs) that was up-regulated by the inflammatory cytokines tumor necrosis factor  (TNF-) and/or interferon  (IFN-). Analysis of cytotoxicity and IFN- production illustrated that ACC-2–specific T cells did not recognize untreated MSCs or IFN-–treated MSCs but showed specific recognition and killing of MSCs treated with TNF- plus IFN-. We hypothesize that under steady-state circumstances BCL2A1-specific T cells may exhibit relative specificity for hematopoietic tissue, but reactivity against nonhematopoietic cells may occur when inflammatory infiltrates are present. Thus, the role of BCL2A1-specific T cells in differential induction of GVL reactivity and GVHD may depend on the presence of inflammatory responses that may occur during GVHD.
Allogeneic stem cell transplantation (SCT) and donor lymphocyte infusion (DLI) are effective treatments for patients with hematologic malignancies,1-4 but can be complicated by graft-versus-host-disease (GVHD).5,6 Following HLA-identical SCT both the graft-versus-leukemia (GVL) effect and GVHD can be mediated by donor-derived T cells recognizing polymorphic minor histocompatibility antigens (mHags) on recipient cells. Immunotherapy directed against mHags with hematopoietic tissue-restricted expression may eradicate the leukemic cells without inducing GVHD.7
Recently, the HLA-A24–restricted mHag ACC-1 and the HLA-B44–restricted mHag ACC-2, encoded by separate polymorphisms within the BCL2A1 gene, were identified, and hematopoietic-restricted expression was suggested for these mHags.8 However, in a retrospective analysis, GVHD relapse and disease-free survival were not statistically different between patients receiving ACC-1–compatible or –incompatible transplants.9 Previous studies have indicated expression of the human BCL2A1 gene not only in hematopoietic cells, but also in nonhematopoietic tissues, including lung, small intestine, and testis, and expression could be induced in endothelial cells by the inflammatory cytokines tumor necrosis factor Induction of BCL2A1 expression in nonhematopoietic tissues by inflammatory cytokines may have important consequences for the role of T cells recognizing ACC-1 and ACC-2 in differential induction of GVL and GVHD. Local inflammatory cytokine production can be triggered by T cells recognizing host antigen-presenting cells (APCs) in nonhematopoietic tissues.13-15 In this study, we characterized the recognition pattern of mHag ACC-2–specific T cells isolated from a patient during a combined GVHD and GVL response after DLI. We show that ACC-2–specific T cells recognized not only leukemic cells but also nonhematopoietic cells under inflammatory conditions and, therefore, could play a role in the etiology of GVHD.
Isolation of T-cell clones and cell culture Cytotoxic T-lymphocyte (CTL) clones were isolated from an HLA-A2 A3 B7 B44+ male patient with relapsed chronic myeloid leukemia (CML) during a clinical response to DLI from his HLA-identical female donor.16
Epstein-Barr virus (EBV)–transformed B-cell lines (EBV-LCLs) and phytohemagglutinin (PHA)–activated T cells (PHA blasts) were generated as described.17 Mesenchymal stromal cells (MSCs) were derived from bone marrow of healthy donors or CML patients as previously described.18 To induce gene expression in response to inflammatory cytokines, MSCs were cultured for 48 hours with 10 ng/mL TNF-
Genotyping of BCL2A1 polymorphisms and quantitative real-time RT-PCR RNA isolation and cDNA synthesis were performed as described.19 A 311-bp amplication product containing all known BCL2A1 polymorphisms was generated using forward primer 5'-ttggatatatttacaggctggctca-3' and reverse primer 5'-gggcaatttgctgtcgtagaag-3'. Polymerase chain reaction (PCR) products were sequenced as described.20 Quantitative real-time reverse transcriptase (RT)–PCR for BCL2A1 was performed as described21 using primers 5'-gtcccgtagacactgccagaacact-3' and 5'-ccattttcccagcctccgt-3' and probe 5'-TET-ttctacgacagcaaattgccccgg-TAMRA-3'.
Analysis of cytotoxicity and IFN-
To determine cytotoxicity, 51Cr-release assays were performed.22 As effector cells, CTL clone 65 and the HLA-A2–specific control CTL clone MBM13 were used. To analyze cytotoxicity against ACC-2 peptide KEFEDDIINW or ACC-2 control peptide KEFEDGIINW, donor-derived EBV-LCLs were loaded with 1 µg/mL peptide during 51Cr-labeling. IFN-
Recently, we isolated mHag-specific CTL clones from a male patient with relapsed CML during the clinical response to DLI from his HLA-identical female donor.16,23 The DLI induced a complete remission but was complicated by GVHD resulting in organ damage. Not only CTL clones specific for the mHags HA-1 and HY-B7 were isolated. CTL clone 65 appeared to be specific for ACC-2 because it lysed donor-derived EBV-LCLs labeled with ACC-2 peptide KEFEDDIINW (Figure 1A). The ACC-2–specific T cells lysed patient-derived EBV-LCLs and CML cells and leukemic cells from unrelated HLA-B44+ mHag ACC-2+ individuals but not HLA-B44+ mHag ACC-2- leukemic cells (Figure 1). These data are in line with the reactivity reported by Akatsuka et al.8
To investigate potential cytolytic activity against nonhematopoietic cells, first B-cell lymphoma 2 (BCL2)–related protein A1 (BCL2A1) mRNA expression levels in nonhematopoietic and hematopoietic cells were measured by quantitative real-time RT-PCR. A screen of commercially available tissue samples indicated BCL2A1 expression in normal and leukemic hematopoietic cells, lung, and heart (data not shown). We analyzed in more detail hematopoietic cells, ASM cells, and MSCs. High BCL2A1 mRNA expression was found in patient-derived EBV-LCLs and PHA blasts. In primary ASM cells expression was 15 to 40 times lower but treatment with TNF-
To determine whether this up-regulation of BCL2A1 mRNA expression in MSCs was sufficient for recognition of these nonhematopoietic cells, we measured lysis of untreated or inflammatory cytokine-treated MSCs and IFN- In this report, we demonstrate that in nonhematopoietic tissue under steady-state circumstances expression of BCL2A1 is relatively low but can be up-regulated during inflammation. Although we could not determine whether the ACC-2–specific T cells were involved in the GVHD in the patient studied, which may also have been due to the presence of several other mHag-specific T cells including male-specific T cells, we hypothesize that the concomitant disease state of a patient poses a risk factor for GVHD and may therefore determine whether BCL2A1–specific T cells exclusively exhibit a GVL effect or also contribute to GVHD. Especially the presence of inflammatory cells such as monocytes or dendritic cells in nonhematopoietic tissues may pose a risk factor for inducing or enhancing GVHD. Recognition of the inflammatory cells by donor-derived T cells may provide the necessary cytokines leading to up-regulated expression of the BCL2A1-derived mHags in damaged tissue.13-15 The subsequent recognition by ACC-1– or ACC-2–specific T cells may then enhance GVHD. Accordingly, the patient from whom we isolated the ACC-2–specific CTLs suffered from skin and pulmonary GVHD. A recent report24 on patients treated with mHag-specific T-cell clones may also be indicative of inflammatory cytokine-induced expression in nonhematopoietic tissue of mHags that under steady-state circumstances are preferentially expressed in hematopoietic tissue. In one of the patients treated for relapsed leukemia with donor T cells recognizing a mHag preferentially expressed in hematopoietic cells, pulmonary toxicity developed, which was attributed to recognition of the mHag in the lung microenvironment.24
Our study does not necessarily exclude the use of BCL2A1-specific T cells in adoptive immunotherapy to eliminate leukemic cells. In the absence of inflammatory cells in nonhematopoietic tissues, ACC-1 or ACC-2 CTLs may act as hematopoietic-specific T cells eradicating leukemia. However, when patients suffer from signs of GVHD the production of inflammatory cytokines may be triggered, resulting in the up-regulated expression of BCL2A1 in target tissues of GVHD.
Submitted September 28, 2004; accepted June 19, 2005.
Prepublished online as Blood First Edition Paper, August 11, 2005; DOI 10.1182/blood-2004-09-3749.
F.M.K. designed the research, performed research, analyzed data, and wrote the paper; S.A.P.v.L.-H., R.A.v.S., and H.M.E.v.E. performed research and analyzed data; R.W. designed research and wrote the paper; and J.H.F.F. designed research, analyzed data, and wrote the paper.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Freke M. Kloosterboer, Department of Hematology, Leiden University Medical Center, C2-R, PO Box 9600, 2300 RC Leiden, The Netherlands; e-mail: f.m.kloosterboer{at}lumc.nl.
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Abstract
Introduction
(IL-1
