Renate de Boer

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.

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.

The molecular bases of δ/αβ T cell–mediated antigen recognition

Godfrey, Rossjohn, and colleagues define a population of T cells in healthy humans that express T cell receptors (TCRs) comprised of δ variable gene segments fused to α joining and constant domains and paired with a variety of TCR-β chains. Functional and structural analyses reveal how components of αβ and γδ TCR gene loci combine to create T cells with unique patterns of antigen recognition.

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.

Mixed functional characteristics correlating with TCR-ligand koff -rate of MHC-tetramer reactive T cells within the naive T-cell repertoire.

The low frequency of antigen‐specific naïve T cells has challenged numerous laboratories to develop various techniques to study the naïve T‐cell repertoire. Here, we combine the generation of naïve repertoire‐derived antigen‐specific T‐cell lines based on MHC‐tetramer staining and magnetic‐bead enrichment with in‐depth functional assessment of the isolated T cells. Cytomegalovirus (CMV) specific T‐cell lines were generated from seronegative individuals. Generated T‐cell lines consisted of a variety of immunodominant CMV‐epitope‐specific oligoclonal T‐cell populations restricted to various HLA‐molecules (HLA‐A1, A2, B7, B8, and B40), and the functional and structural avidity of the CMV‐specific T cells was studied. Although all CMV‐specific T cells were isolated based on their reactivity toward a specific peptide‐MHC complex, we observed a large variation in the functional avidity of the MHC‐tetramer positive T‐cell populations, which correlated with the structural avidity measured by the recently developed Streptamer koff‐rate assay. Our data demonstrate that MHC‐tetramer staining is not always predictive for specific T‐cell reactivity, and challenge the sole use of MHC‐tetramers as an indication of the peripheral T‐cell repertoire, independent of the analysis of functional activity or structural avidity parameters.

Identification of Varicella-Zoster Virus-Specific CD8 T Cells in Patients after T-Cell-Depleted Allogeneic Stem Cell Transplantation

ABSTRACT To study the role of CD8 T cells in the control of varicella-zoster virus (VZV) reactivation, we developed multimeric major histocompatibility complexes to identify VZV-specific CD8 T cells. Potential HLA-A2 binding peptides from the putative immediate-early 62 protein (IE62) of VZV were tested for binding, and peptides with sufficient binding capacity were used to generate pentamers. Patients with VZV reactivation following stem cell transplantation were screened with these pentamers, leading to the identification of the first validated class I-restricted epitope of VZV. In 42% of HLA-A2 patients following VZV reactivation, these IE62-ALW-A2 T cells could be detected ex 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.

Cytomegalovirus-Induced Expression of CD244 after Liver Transplantation Is Associated with CD8+ T Cell Hyporesponsiveness to Alloantigen

The chronic presence of viral Ags can induce T cell exhaustion, which is characterized by upregulation of coinhibitory receptors and loss of T cell function. We studied whether a similar phenomenon occurs after liver transplantation (LTx), when there is continuous exposure to alloantigen. Expression of coinhibitory receptors on circulating CD4+ and CD8+ T cells was analyzed longitudinally in 19 patients until 6 mo after LTx and cross-sectionally in 38 patients late (1–12 y) after LTx. Expression of the coinhibitory receptors CD160 and CD244 on circulating CD8+ T cells was already higher 6 mo after LTx compared with pre-LTx, and the elevated expression was sustained late after LTx, with CD244 showing the more prominent increase. The strongest upregulation of CD244 on circulating CD8+ T cells was observed in patients who experienced CMV infection after LTx. CMV infection also was associated with reduced CD8+ T cell proliferation and cytotoxic degranulation in response to alloantigen late after LTx. Purified CD244+CD8+ T cells from LTx patients showed lower proliferative responses to alloantigen, as well as to polyclonal stimulation, than did their CD244− counterparts. In addition, the CD244+CD8+ T cell population contained the majority of CMV peptide–loaded MHC class I tetramer-binding cells. In conclusion, CMV infection after LTx, rather than persistence of alloantigen, induces the accumulation of dysfunctional CD244+CD8+ T cells in the circulation that persist long-term, resulting in reduced frequencies of circulating alloreactive CD8+ T cells. These results suggest that CMV infection restrains CD8+ T cell alloresponses after LTx.