Diabetes Incidence Is Unaltered in Glutamate Decarboxylase 65-Specific TCR Retrogenic Nonobese Diabetic Mice: Generation by Retroviral-Mediated Stem Cell Gene Transfer

Abstract

TCR transgenic mice are valuable tools for dissecting the role of autoantigen-specific T cells in the pathogenesis of type 1 diabetes but are time-consuming to generate and backcross onto congenic strains. To circumvent these limitations, we developed a new approach to rapidly generate mice expressing TCR using retroviral-mediated stem cell gene transfer and a novel picornavirus-like 2A peptide to link the TCR α- and β-chains in a single retroviral vector. We refer to these as retrogenic (Rg) mice to avoid confusion with conventional transgenic mice. Our approach was validated by demonstrating that Rg nonobese diabetic (NOD)-scid mice expressing the diabetogenic TCRs, BDC2.5 and 4.1, generate clonotype-positive T cells and develop diabetes. We then expressed three TCR specific for either glutamate decarboxylase (GAD) 206–220 or GAD 524–538 or for hen egg lysozyme 11–25 as a control in NOD, NOD-scid, and B6.H2g7 mice. Although T cells from these TCR Rg mice responded to their respective Ag in vitro, the GAD-specific T cells exhibited a naive, resting phenotype in vivo. However, T cells from Rg mice challenged with Ag in vivo became activated and developed into memory cells. Neither of the GAD-reactive TCR accelerated or protected mice from diabetes, nor did activated T cells transfer or protect against diabetes in NOD-scid recipients, suggesting that GAD may not be a primary target for diabetogenic T cells. Generation of autoantigen-specific TCR Rg mice represents a powerful approach for the analysis of a wide variety of autoantigens.

Generation of T-cell receptor retrogenic mice

“…Therefore it is expected that transducing mouse bone marrow with TCR-2A retroviral producers will enable dissection of the role of different proteins in these processes. TCR Rg mice have also been used in the study of diabetes, clearly showing that this technique can be used to generate a large panel of different TCR specificities for the study of diseases involving T cells 10 . The speed of this procedure is a distinct advantage when the screening of different TCR combinations is required and when different TCRs need to be examined in the same system simultaneously.…”

“…Indeed, any studies that have previously been performed with conventional TCR Tg mice could conceivably be performed with Rg mice. We have used these mice to study positive and negative selection in the thymus, peripheral tolerance and effector function, and the diabetogenic potential of autoantigen-specific TCR in type-1 diabetes 10,11 . As such, it should be possible to create TCR Rg mice for any disease if the sequences of the TCR-a and TCR-b chains are known, including TCRs specific for viral or bacterial antigens.…”

Antigen-Specific CD8+ T Cells Respond to Chlamydia trachomatis in the Genital Mucosa

“…Total RNA was isolated from NR23.4 using Ultraspec RNA (Biotecx), treated with RNase-free DNase (Promega), and converted into cDNA using MMLV Reverse Transcriptase (Invitrogen Life Technologies). A TCR construct was produced by recombinant PCR and cloned into a 2A peptide-linked, GFP-labeled retroviral vector previously described (15,19,20). Expression of the construct was verified using TransIT-293 (Mirus) to transiently transfect 293T cells with each construct along with a multicistronic CD3 vector provided by D. Vignali (St. Jude Children's Research Hospital, Memphis, TN).…”

T cell receptors in an IL-10-secreting amino acid copolymer-specific regulatory T cell line that mediates bystander immunosuppression

“…Further in vivo experiments using these TCR to generate retrogenic mice (28,35,36) may provide an answer to this question. This approach may have therapeutic implications because the techniques used are adaptable to human studies.…”