Retroviral-Mediated Expression of an MHC Class I-Restricted T Cell Receptor in the CD8 T Cell Compartment of Bone Marrow-Reconstituted Mice

Abstract

The introduction of cloned T cell receptor (TCR) genes into bone marrow cells could provide a way to increase the frequency of tumor- or pathogen-specific cytotoxic T lymphocyte (CTL) precursors. We demonstrate here the ability of a retroviral vector to direct expression of a Vα15/Vβ13 MHC class I-restricted TCR in lethally irradiated mice reconstituted with transduced bone marrow cells. We have detected retroviral-mediated TCR expression by flow cytometry 6–19 weeks after transplantation in C57L (Vβ13¯^/¯) and Rag1¯^/¯ bone marrow-reconstituted mice, and in C57BL/6 hosts reconstituted with transduced C57BL/6-Rag1¯^/¯bone marrow. Southern analysis confirmed the presence of integrated provirus and revealed that the frequency of transduction is greater than the frequency of cell surface TCR expression. Although TCR expression on Vβ13⁺ transduced cells is lower than endogenous TCR levels, it is largely confined to CD4⁺CD8⁺ (thymus) and CD8⁺ (thymus and spleen) T cells. In Rag1¯^/¯ mice, which display a developmental arrest of thymocytes at the immature CD4¯CD8¯ stage, retrovirus-mediated TCR expression selectively rescues CD4⁺CD8⁺ and CD8⁺ populations. These results indicate that the ectopically expressed TCR is functional during T cell development. Furthermore, we have observed Vβ13⁺ TCR expression by up to 13% of peripheral CD8⁺ T cells in C57L and C57BL/6 hosts. This represents a substantial increase relative to total Vβ13 frequency in normal C57BL/6 mice (3–5%), and an even greater increase over the estimated frequency of CTL precursors of a defined specificity (10¯⁵–10¯⁴). Our findings indicate that TCR gene transfer can be used to develop new approaches to immunotherapy, and provide the basis for further studies examining the contribution of retrovirus-mediated TCR expression to an antigen-specific CTL response.

Overview summary

Adoptive transfer of tumor-specific CTL contributes to tumor regression in a number of model systems. The transfer of genes encoding tumor-specific α/β TCR into hematopoietic stem cells could provide a valuable extension of this approach, particularly for malignancies treated with bone marrow transplantation and myeloablative doses of radiation or chemotherapy. We have previously constructed a retroviral vector that contains α- and β-chain genes encoding a mouse class 1 MHC-restricted TCR and demonstrated its ability to direct TCR expression in T hybridomas. We describe here the use of this vector to express the cloned TCR in T lymphoid cells of lethally irradiated mice reconstituted with transduced bone marrow cells. We also provide evidence that the ectopically expressed TCR is functional during T cell ontogeny, resulting in its selective expression by CD8⁺ T cells. The cassette design of our vector system allows insertion of PCR-generated V region cDNA fragments from any TCR, offering significant potential for the development of mouse models to evaluate TCR gene transfer into hematopoietic stem cells as a potential immunotherapeutic strategy.