Rapid identification of MHC class I-restricted antigens relevant to autoimmune diabetes using retrogenic T cells

J Immunol Methods. 2008 Jun 1;335(1-2):106-15. doi: 10.1016/j.jim.2008.03.007. Epub 2008 Apr 8.


The method described herein provides a novel strategy for the rapid identification of CD8(+) T cell epitopes relevant to type 1 diabetes in the context of the nonobese diabetic (NOD) mouse model of disease. Obtaining the large number of antigen-sensitive monospecific T cells required for conventional antigen discovery methods has historically been problematic due to (1) difficulties in culturing autoreactive CD8(+) T cells from NOD mice and (2) the large time and resource investments required for the generation of transgenic NOD mice. We circumvented these problems by exploiting the rapid generation time of retrogenic (Rg) mice, relative to transgenic mice, as a novel source of sensitive monospecific CD8(+) T cells, using the diabetogenic AI4 T cell receptor on NOD.SCID and NOD.Rag1(-/-) backgrounds as a model. Rg AI4 T cells are diabetogenic in vivo, demonstrating for the first time that Rg mice are a means for assessing the pathogenic potential of CD8(+) T cell receptor specificities. In order to obtain a sufficient number of Rg CD8(+) T cells for antigen screens, we optimized a method for their in vitro culture that resulted in a approximately 500 fold expansion. We demonstrate the high sensitivity and specificity of expanded Rg AI4 T cells in the contexts of (1) specific peptide challenge, (2) islet cytotoxicity, and (3) their ability to resolve previously defined mimotope candidates from a positional scanning peptide library. Our method is the first to combine the speed of Rg technology with an optimized in vitro Rg T cell expansion protocol to enable the rapid discovery of T cell antigens.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CD8-Positive T-Lymphocytes / immunology*
  • Cell Culture Techniques
  • Cell Proliferation
  • Cells, Cultured
  • Cytotoxicity, Immunologic
  • Diabetes Mellitus, Type 1 / immunology*
  • Diabetes Mellitus, Type 1 / pathology
  • Disease Models, Animal
  • Flow Cytometry
  • Hematopoietic Stem Cell Transplantation
  • Histocompatibility Antigens Class I / analysis*
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Humans
  • Immunologic Techniques*
  • Islets of Langerhans / immunology*
  • Islets of Langerhans / pathology
  • Mice
  • Mice, Inbred NOD
  • Mice, Knockout
  • Mice, SCID
  • Peptide Library
  • Receptors, Antigen, T-Cell, alpha-beta / genetics
  • Receptors, Antigen, T-Cell, alpha-beta / metabolism
  • T-Lymphocytes, Cytotoxic / immunology
  • Time Factors
  • Transfection


  • Histocompatibility Antigens Class I
  • Homeodomain Proteins
  • Peptide Library
  • Receptors, Antigen, T-Cell, alpha-beta
  • RAG-1 protein