Avital L. Amir

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.

Allo-HLA reactive T cells inducing graft versus host disease are single peptide specific

T-cell alloreactivity directed against non-self-HLA molecules has been assumed to be less peptide specific than conventional T-cell reactivity. A large variation in degree of peptide specificity has previously been reported, including single peptide specificity, polyspecificity, and peptide degeneracy. Peptide polyspecificity was illustrated using synthetic peptide-loaded target cells, but in the absence of confirmation against endogenously processed peptides this may represent low-avidity T-cell reactivity. Peptide degeneracy was concluded based on recognition of Ag-processing defective cells. In addition, because most investigated alloreactive T cells were in vitro activated and expanded, the previously determined specificities may have not been representative for alloreactivity in vivo. To study the biologically relevant peptide specificity and avidity of alloreactivity, we investigated the degree of peptide specificity of 50 different allo-HLA-reactive T-cell clones which were activated and expanded in vivo during GVHD. All but one of the alloreactive T-cell clones, including those reactive against Ag-processing defective T2 cells, recognized a single peptide allo-HLA complex, unique for each clone. Down-regulation of the expression of the recognized Ags using silencing shRNAs confirmed single peptide specificity. Based on these results, we conclude that biologically relevant alloreactivity selected during in vivo immune response is peptide specific.

Multi-cistronic vector encoding optimized safety switch for adoptive therapy with T-cell receptor-modified T cells

T-cell receptor (TCR) gene transfer is an attractive strategy to equip T cells with defined antigen-specific TCRs using short-term in vitro procedures to target both hematological malignancies and solid tumors. TCR gene transfer poses different safety issues that might warrant the inclusion of a suicide gene. High affinity TCRs may result in on-target toxicity, and off-target reactivity directed against healthy tissue can be observed due to mixed TCR dimers. Inclusion of a suicide gene as a safety switch may abrogate these unwanted toxicities. Human CD20 has been proposed as a nonimmunogenic suicide gene targeted by widely used clinical-grade anti-CD20 antibodies that can additionally function as a selection marker. However, transduction of T cells with a multi-cistronic vector encoding both TCR and CD20 resulted in poor coexpression. In this study, we demonstrated that codon optimization of TCR and CD20 resulted in profound coexpression of both the preferentially expressed antigen in melanoma (PRAME)-TCR and CD20, allowing selective as well as efficient elimination of these engineered T cells in vitro. These results demonstrate the great potential of codon optimized CD20 to be broadly used in clinical trials as a safety switch.