Transgenic TCR expression: comparison of single chain with full-length receptor constructs for T-cell function

Genetic modification of T lymphocytes with T-cell receptor (TCR) genes provides a novel tool for adoptive immunotherapy. However, the efficiency of full-length TCR (flTCR)-transduced T cells could be limited by factors such as incorrect pairing between exogenous and endogenous TCR chains and downregulation of the CD3 complex. To overcome these hurdles, one promising strategy is to use three-domain single-chain TCRs (3D-scTCR), in which TCR Vα and Vβ chains are joined by a linker with signal transduction domains fused at the carboxyl termini as signal transducers and amplifiers. Our results showed that surface expression of scTCRs on T cells after retroviral transduction was affected by the origin of the transmembrane (TM) region and placement of signaling domains. scTCR-modified T cells were functional as shown by cytokine (IL-2 and IFN-γ) release in response to antigen stimulation and cytolytic activity against specific target cells. CD8 and CD28, but not the complete CD3 complex, could enhance the scTCR-induced T cell activation. Compared with flTCR-modified T cells and native CTLs, scTCR-modified T cells require higher thresholds of antigen stimulation (∼10−8 M peptide) to be functional. Despite the low efficiency of scTCRs, our data provide insight into further improvements in generating efficient scTCRs for in vivo applications.

[1]  J. Trapani,et al.  Rejection of Syngeneic Colon Carcinoma by CTLs Expressing Single-Chain Antibody Receptors Codelivering CD28 Costimulation , 2003, The Journal of Immunology.

[2]  D. Margulies,et al.  A three-domain T cell receptor is biologically active and specifically stains cell surface MHC/peptide complexes. , 1997, Journal of immunology.

[3]  J. Bonifacino,et al.  Transmembrane helical interactions and the assembly of the T cell receptor complex. , 1990, Science.

[4]  M. Weijtens,et al.  Functional balance between T cell chimeric receptor density and tumor associated antigen density: CTL mediated cytolysis and lymphokine production , 2000, Gene Therapy.

[5]  M. Nishimura,et al.  Transfer of TCR Genes into Mature T Cells Is Accompanied by the Maintenance of Parental T Cell Avidity , 2003, The Journal of Immunology.

[6]  David J. Cole,et al.  CD8-Independent Tumor Cell Recognition Is a Property of the T Cell Receptor and Not the T Cell 1 , 2003, The Journal of Immunology.

[7]  D. Hafler,et al.  Functional three-domain single-chain T-cell receptors. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[8]  K D Wittrup,et al.  High affinity T cell receptors from yeast display libraries block T cell activation by superantigens. , 2001, Journal of molecular biology.

[9]  J. Trapani,et al.  Redirecting mouse CTL against colon carcinoma: superior signaling efficacy of single-chain variable domain chimeras containing TCR-zeta vs Fc epsilon RI-gamma. , 2001, Journal of immunology.

[10]  Dirk Homann,et al.  Detection of Low-Avidity CD4+ T Cells Using Recombinant Artificial APC: Following the Antiovalbumin Immune Response1 , 2003, The Journal of Immunology.

[11]  T. Tsang,et al.  Efficient transduction of murine primary T cells requires a combination of high viral titer, preferred tropism, and proper timing of transduction. , 2003, Journal of hematotherapy & stem cell research.

[12]  T. Schumacher,et al.  Immunotherapy through TCR gene transfer , 2001, Nature Immunology.

[13]  A. Miller,et al.  Production of high-titer helper virus-free retroviral vectors by cocultivation of packaging cells with different host ranges , 1991, Journal of virology.

[14]  D. Kranz,et al.  Specificity and binding properties of a single-chain T cell receptor. , 1996, Journal of molecular biology.

[15]  T. Kitamura,et al.  Functional reconstitution of class II MHC-restricted T cell immunity mediated by retroviral transfer of the alpha beta TCR complex. , 2000, Journal of immunology.

[16]  K D Wittrup,et al.  In vitro evolution of a T cell receptor with high affinity for peptide/MHC. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[17]  K. Liao,et al.  Design of transgenes for efficient expression of active chimeric proteins on mammalian cells. , 2001, Biotechnology and bioengineering.

[18]  R. Flavell,et al.  Integrated src kinase and costimulatory activity enhances signal transduction through single-chain chimeric receptors in T lymphocytes. , 2001, Blood.

[19]  P. Goodfellow,et al.  Surface expression of the T cell receptor complex requires charged residues within the α chain transmembrane region , 1989, European journal of immunology.

[20]  R. Siliciano,et al.  A soluble, single-chain T-cell receptor fragment endowed with antigen-combining properties. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[21]  L. Bolliger,et al.  Identification and functional characterization of the zeta-chain dimerization motif for TCR surface expression. , 1999, Journal of immunology.

[22]  Y. Yarden,et al.  Targeting of T lymphocytes to Neu/HER2-expressing cells using chimeric single chain Fv receptors. , 1993, Journal of immunology.

[23]  T. Kitamura,et al.  Functional Reconstitution of Class II MHC-Restricted T Cell Immunity Mediated by Retroviral Transfer of the αβ TCR Complex1 , 2000, The Journal of Immunology.

[24]  S. Rosenberg,et al.  Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity. , 1999, Journal of immunology.

[25]  J. Marchalonis,et al.  Generation of diverse single-chain proteins using a universal (Gly4-Ser)3 encoding oligonucleotide. , 1995, BioTechniques.

[26]  G. Nolan,et al.  Retroviral transduction of a T cell receptor specific for an Epstein-Barr virus-encoded peptide. , 2001, Clinical immunology.

[27]  B. Malissen,et al.  The T cell receptor/CD3 complex is composed of at least two autonomous transduction modules , 1992, Cell.

[28]  R. Zamoyska,et al.  The CD8 coreceptor revisited: one chain good, two chains better. , 1994, Immunity.

[29]  Hans J. Stauss,et al.  Circumventing tolerance to a human MDM2-derived tumor antigen by TCR gene transfer , 2001, Nature Immunology.

[30]  M. Bevan,et al.  Introduction of soluble protein into the class I pathway of antigen processing and presentation , 1988, Cell.

[31]  J W Gratama,et al.  Grafting primary human T lymphocytes with cancer-specific chimeric single chain and two chain TCR , 2000, Gene Therapy.

[32]  A. Lawson,et al.  Chimeric receptors providing both primary and costimulatory signaling in T cells from a single gene product. , 1998, Journal of immunology.

[33]  T. Whiteside.  Signaling defects in T lymphocytes of patients with malignancy , 1999, Cancer Immunology, Immunotherapy.

[34]  L. Pease,et al.  A retroviral vector that directs simultaneous expression of alpha and beta T cell receptor genes. , 1998, Human gene therapy.

[35]  Simon J Davis,et al.  Molecular interactions mediating T cell antigen recognition. , 2003, Annual review of immunology.

[36]  David M Kranz,et al.  Quantitative analysis of the contribution of TCR/pepMHC affinity and CD8 to T cell activation. , 2003, Immunity.

[37]  B. Groner,et al.  Cytotoxic T lymphocytes with a grafted recognition specificity for ERBB2-expressing tumor cells. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[38]  Mark J. Smyth,et al.  Redirecting Mouse CTL Against Colon Carcinoma: Superior Signaling Efficacy of Single-Chain Variable Domain Chimeras Containing TCR-ζ vs FcεRI-γ1 , 2001, The Journal of Immunology.

[39]  K. D. Hardman,et al.  Characterization of a single-chain T-cell receptor expressed in Escherichia coli. , 1992, Proceedings of the National Academy of Sciences of the United States of America.