Marleen M. van Loenen

Allo-HLA reactivity of virus-specific memory T-cells is common

Graft-versus-host disease and graft rejection are major complications of allogeneic HLA-mismatched stem cell transplantation or organ transplantation that are caused by alloreactive T cells. Because a range of acute viral infections have been linked to initiating these complications, we hypothesized that the cross-reactive potential of virus-specific memory T cells to allogeneic (allo) HLA molecules may be able to mediate these complications. To analyze the allo-HLA reactivity, T cells specific for Epstein-Barr virus, cytomegalovirus, varicella zoster virus, and influenza virus were tested against a panel of HLA-typed target cells, and target cells transduced with single HLA molecules. Eighty percent of T-cell lines and 45% of virus-specific T-cell clones were shown to cross-react against allo-HLA molecules. The cross-reactivity of the CD8 and CD4 T-cell clones was directed primarily against HLA class I and II, respectively. However, a restricted number of CD8 T cells exhibited cross-reactivity to HLA class II. T-cell receptor (TCR) gene transfer confirmed that allo-HLA reactivity and virus specificity were mediated via the same TCR. These results demonstrate that a substantial proportion of virus-specific T cells exert allo-HLA reactivity, which may have important clinical implications in transplantation settings as well as adoptive transfer of third-party virus-specific T cells.

Mixed T cell receptor dimers harbor potentially harmful neoreactivity

Adoptive transfer of T cell receptor (TCR)-transduced T cells may be an attractive strategy to target both hematological malignancies and solid tumors. By introducing a TCR, large numbers of T cells with defined antigen (Ag) specificity can be obtained. However, by introduction of a TCR, mixed TCR dimers can be formed. Besides the decrease in TCR expression of the introduced and endogenous TCR, these mixed TCR dimers could harbor potentially harmful specificities. In this study, we demonstrate that introduction of TCRs resulted in formation of neoreactive mixed TCR dimers, composed of the introduced TCR chains pairing with either the endogenous TCR α or β chain. Neoreactivities observed were HLA class I or class II restricted. Most neoreactive mixed TCR dimers were allo-HLA reactive; however, neoreactive mixed TCR dimers with autoreactive activity were also observed. We demonstrate that inclusion of an extra disulfide bond between the constant domains of the introduced TCR markedly reduced neoreactivity, whereas enhanced effectiveness of the introduced TCR was observed. In conclusion, TCR transfer results in the formation of neoreactive mixed TCR dimers with the potential to generate off-target effects, underlining the importance of searching for techniques to facilitate preferential pairing.

αβ T-Cell Receptor Engineered γδ T Cells Mediate Effective Antileukemic Reactivity

Retroviral transfer of T-cell receptors (TCR) to peripheral blood–derived T cells generates large numbers of T cells with the same antigen specificity, potentially useful for adoptive immunotherapy. One drawback of this procedure is the formation of mixed TCR dimers with unknown specificities due to pairing of endogenous and introduced TCR chains. We investigated whether γδ T cells can be an alternative effector population for TCR gene transfer because the γδTCR is not able to form dimers with the αβTCR. Peripheral blood–derived γδ T cells were transduced with human leukocyte antigen (HLA) class I– or HLA class II–restricted minor histocompatibility antigen (mHag) or virus-specific TCRs. Because most γδ T cells do not express CD4 and CD8, we subsequently transferred these coreceptors. The TCR-transduced γδ T cells exerted high levels of antigen-specific cytotoxicity and produced IFN-γ and IL-4, particularly in the presence of the relevant coreceptor. γδ T cells transferred with a TCR specific for the hematopoiesis-specific mHag HA-2 in combination with CD8 displayed high antileukemic reactivity against HA-2–expressing leukemic cells. These data show that transfer of αβTCRs to γδ T cells generated potent effector cells for immunotherapy of leukemia, without the expression of potentially hazardous mixed TCR dimers. (Cancer Res 2006; 66(6): 3331-7)

PRAME specific allo-HLA restricted T-cells with potent antitumor reactivity useful for therapeutic T cell receptor gene transfer

Purpose: In human leukocyte antigen (HLA)–matched stem cell transplantation (SCT), it has been shown that beneficial immune response mediating graft-versus-tumor (GVT) responses can be separated from graft-versus-host disease (GVHD) immune responses. In this study, we investigated whether it would be possible to dissect the beneficial immune response of allo-HLA–reactive T cells with potent antitumor reactivity from GVHD-inducing T cells present in the detrimental immune response after HLA-mismatched SCT. Experimental Design: The presence of specific tumor-reactive T cells in the allo-HLA repertoire was analyzed at the time of severe GVHD after HLA-mismatched SCT, using tetramers composed of different tumor-associated antigens (TAA). Results: High-avidity allo-HLA-restricted T cells specific for the TAA preferentially expressed antigen on melanomas (PRAME) were identified that exerted highly single-peptide–specific reactivity. The T cells recognized multiple different tumor cell lines and leukemic cells, whereas no reactivity against a large panel of nonmalignant cells was observed. These T cells, however, also exerted low reactivity against mature dendritic cells (DC) and kidney epithelial cells, which was shown to be because of low PRAME expression. Conclusions: On the basis of potential beneficial specificity and high reactivity, the T-cell receptors of these PRAME-specific T cells may be effective tools for adoptive T-cell therapy. Clinical studies have to determine the significance of the reactivity observed against mature DCs and kidney epithelial cells. Clin Cancer Res; 17(17); 5615–25. ©2011 AACR.

Increased deposition of von Willebrand factor in the rat heart after local ionizing irradiation

Background and Purpose:Von Willebrand factor (vWf), a glycoprotein involved in blood coagulation, is synthesized by endothelial cells. Increased amounts of vWf in blood plasma or tissue samples are indicative of damaged endothelium. In the present study, mRNA expression and localization of vWf were determined in irradiated rat heart tissue.Material and Methods:Sprague-Dawley rats received local heart irradiation with a single dose of 0, 15, or 20 Gy. Hearts were dissected at different time points (up to 16 months) after irradiation. In a second experiment, rats were injected with the radioprotector amifostine (160 mg/kg, i. p.) 15–20 min before irradiation and sacrificed after 6 months. Immunohistochemistry was performed using a polyclonal anti-vWf antibody. Serial sections were subjected to a general rat endothelial cell immunostaining (RECA-1) or a collagen staining (picrosirius red). mRNA expression was determined by using PCR.Results:In control tissue, all endothelial cells lining the lumen of the endocardium and coronary arteries, but not capillary endothelial cells, were stained for vWf. 1 month after irradiation with both 15 and 20 Gy, myocardial capillaries became immunoreactive. From 3 months onward, staining was observed also within the extracellular matrix (ECM) of fibrotic areas. At mRNA level, no changes in vWf could be observed at all time points after irradiation, suggesting that vWf deposition was not due to increased biosynthesis of the protein. In sections of amifostine-treated rat hearts, vWf staining was increased to a lesser extent.Conclusion:These dose- and time-dependent increases in deposition of vWf indicate the presence of damaged endothelium in the irradiated rat heart. These increases in vWf accumulation precede development of fibrosis in the subendocardial layer and myocardium of the left ventricles, right ventricles, and atria.Hintergrund und Ziel:Das Glykoprotein Von-Willebrand-Faktor (VWF) spielt eine wichtige Rolle bei der Blutgerinnung und wird durch Endothelzellen synthetisiert. Erhöhte VWF-Konzentrationen im Blutplasma oder in Gewebeproben weisen auf geschädigtes Endothel hin. In der hier vorgestellten Studie werden die mRNA-Expression und VWF-Lokalisation im Gewebe bestrahlter Rattenherzen bestimmt.Material und Methodik:Sprague-Dawley-Ratten erhielten eine lokale Bestrahlung des Herzens mit einer Dosis von 0, 15 oder 20 Gy. Die Herzen wurden zu definierten Zeitpunkten von bis zu 16 Monaten nach der Bestrahlung seziert. In einem zweiten Experiment wurden Ratten seziert, denen 15–20 min vor der Bestrahlung der Radioprotektor Amifostin (160 mg/kg, i. p.) injiziert worden war. Immunhistochemische Tests wurden unter Verwendung des polyklonalen Anti-VWF-Antikörpers durchgeführt. Serielle Schnitte wurden einer allgemeinen Ratten-Endothelzellen-Immunfärbung (RECA-1) oder Kollagenfärbung (Picrosirius-Rot) unterzogen. Die mRNA-Expression wurde mittels PCR bestimmt.Ergebnisse:Im Kontrollgewebe wurden alle Endothelzellen des Endokards und der Kranzarterien, nicht jedoch die Endothelzellen der Kapillargefäße auf VWF gefärbt. 1 Monat nach Bestrahlung mit 15 und 20 Gy wurden die Kapillaren des Myokards immunreaktiv. Nach Zeiträumen von ≥ 3 Monaten ließ sich auch eine Färbung der extrazellulären Matrix (ECM) innerhalb fibrotischer Bereiche beobachten. Bezüglich des mRNA-Levels konnten unabhängig vom Zeitpunkt der Untersuchung nach der Bestrahlung keine Veränderungen für den VWF festgestellt werden. Dies lässt vermuten, dass die VWF-Deposition nicht auf eine erhöhte Biosynthese des Proteins zurückzuführen ist. In den Schnitten der mit Amifostin behandelten Rattenherzen war die Zunahme der VWF-Färbung weniger ausgeprägt.Schlussfolgerung:Die von der Strahlendosis und Zeit abhängige Zunahme der VWF-Einlagerung deutet auf das Vorliegen von geschädigtem Endothel in den bestrahlten Rattenherzen hin. Die Zunahme der VWF-Akkumulation geht der Fibrosierung in der Subendokardschicht und im Myokard der linken und rechten Ventrikel sowie im Atrium voraus.

Functional Human Antigen-Specific T Cells Produced In Vitro Using Retroviral T Cell Receptor Transfer into Hematopoietic Progenitors

In vitro production of human T cells with known Ag specificity is of major clinical interest for immunotherapy against tumors and infections. We have performed TCRαβ gene transfer into human hemopoietic progenitors from postnatal thymus or umbilical cord blood, and subsequently cultured these precursors on OP9 stromal cells expressing the Notch human ligand Delta-like1. We report here that fully mature, functional T cells with controlled Ag specificity are obtained from such cultures. Using vectors encoding TCRαβ-chains directed against melanoma (MART-1), viral (CMV), and minor histocompatibility (HA-2) Ags, we show that the obtained Ag-specific T cells exert cytolytic activity against their cognate Ag and expand in vitro upon specific TCR stimulation. Therapeutic applications may arise from these results because they provide a way to produce large numbers of autologous mature Ag-specific T cells in vitro from undifferentiated hemopoietic progenitors.

A Good Manufacturing Practice procedure to engineer donor virus-specific T-cells into potent anti-leukemic effector cells

A sequential, two-step procedure in which T-cell-depleted allogeneic stem cell transplantation is followed by treatment with donor lymphocyte infusion at 6 months can significantly reduce the risk and severity of graft-versus-host disease, with postponed induction of the beneficial graft-versus-leukemia effect. However, patients with high-risk leukemia have a substantial risk of relapse early after transplantation, at a time when administration of donor lymphocytes has a high likelihood of resulting in graft-versus-host disease, disturbing a favorable balance between the graft-versus-leukemia effect and graft-versus-host disease. New therapeutic modalities are, therefore, required to allow early administration of T cells capable of exerting a graft-versus-leukemia effect without causing graft-versus-host disease. Here we describe the isolation of virus-specific T cells using Streptamer-based isolation technology and subsequent transfer of the minor histocompatibility antigen HA-1-specific T-cell receptor using retroviral vectors. Isolation of virus-specific T cells and subsequent transduction with HA-1-T-cell receptor resulted in rapid in vitro generation of highly pure, dual-specific T cells with potent anti-leukemic reactivity. Due to the short production procedure of only 10–14 days and the defined specificity of the T cells, administration of virus-specific T cells transduced with the HA-1-T-cell receptor as early as 8 weeks after allogeneic stem cell transplantation is feasible. (This clinical trial is registered at www.clinicaltrialsregister.eu as EudraCT number 2010-024625-20).

Optimization of the HA-1-specific T-cell receptor for gene therapy of hematologic malignancies

To broaden the applicability of adoptive T-cell therapy for the treatment of hematologic malignancies, we aim to start a clinical trial using HA-1-TCR transferred virus-specific T cells. TCRs directed against the minor histocompatibility antigen (MiHA) HA-1 are good candidates for TCR gene transfer to treat hematologic malignancies because of the hematopoiesis-restricted expression and favorable frequency of HA-1. For optimal anti-leukemic reactivity, high cell-surface expression of the introduced TCR is important. Previously, however, we have demonstrated that gene transferred HA-1-TCRs are poorly expressed at the cell-surface. In this study several strategies were explored to improve expression of transferred HA-1-TCRs.

Kinetic Preservation of Dual Specificity of Coprogrammed Minor Histocompatibility Antigen-Reactive Virus-Specific T Cells

Adoptive transfer of antigen-specific T cells is an attractive strategy for the treatment of hematologic malignancies. It has been shown that T cells recognizing minor histocompatibility antigens (mHag) selectively expressed on hematopoietic cells mediate antileukemic reactivity after allogeneic stem cell transplantation. However, large numbers of T cells with defined specificity are difficult to attain. An attractive strategy to obtain large numbers of leukemia-reactive T cells is retroviral transfer of mHag-specific T-cell receptors (TCR). TCR transfer into T cells specific for persistent viruses may enable these T cells to proliferate both after encountering with viral antigens as well as mHags, increasing the possibility of in vivo survival. We analyzed whether the dual specificity of the TCR-transferred T cells after repetitive stimulation via either the introduced antileukemic HA-2-TCR or the endogenous cytomegalovirus (CMV) specific CMV-TCR was preserved. We show that after repetitive stimulation, T cells skew to a population predominantly expressing the triggered TCR. However, HA-2-TCR-transferred CMV-specific T cells with high antileukemic HA-2-TCR expression but low CMV-TCR expression were able to persist and proliferate after repetitive stimulation with pp65. Moreover, HA-2-TCR-transferred CMV-specific T cells remained dual specific after repetitive stimulation and TCR expression could be reverted after additional stimulation via the previously nonstimulated TCR, restoring high-avidity interactions. These data imply persistence of TCR-transferred virus-specific T cells with both antileukemic and antivirus reactivity in vivo.

Extracellular domains of CD8α and CD8ß subunits are sufficient for HLA class I restricted helper functions of TCR-engineered CD4(+) T cells.

By gene transfer of HLA-class I restricted T-cell receptors (TCRs) (HLA-I-TCR) into CD8+ as well as CD4+ T-cells, both effector T-cells as well as helper T-cells can be generated. Since most HLA-I-TCRs function best in the presence of the CD8 co-receptor, the CD8αß molecule has to be co-transferred into the CD4+ T-cells to engineer optimal helper T-cells. In this study, we set out to determine the minimal part of CD8αβ needed for optimal co-receptor function in HLA-I-TCR transduced CD4+ T-cells. For this purpose, we transduced human peripheral blood derived CD4+ T-cells with several HLA-class I restricted TCRs either with or without co-transfer of different CD8 subunits. We demonstrate that the co-transduced CD8αβ co-receptor in HLA-I-TCR transduced CD4+ T-cells behaves as an adhesion molecule, since for optimal antigen-specific HLA class I restricted CD4+ T-cell reactivity the extracellular domains of the CD8α and ß subunits are sufficient.