Detection of autoreactive T cells using MHC II tetramers is difficult because of the low affinity of their TCR. We have generated a class II tetramer using the IAs class II molecule combined with an autoantigenic peptide from myelin proteolipid protein (PLP; PLP139-151) and used it to analyze myelin PLP139-151-reactive T cells. Using monomers and multimerized complexes labeled with PE, we confirmed the specificity of the reagent by bioassay and flow cytometry. The IAs tetramers stimulated and stained the PLP139-151-specific 5B6 TCR transgenic T cells and a polyclonal cell line specific for PLP139-151, but not a control T cell line specific for PLP178-191. We used this reagent to optimize conditions to detect low affinity autoreactive T cells. We found that high pH (∼8.0) and neuraminidase treatment enhances the staining capacity of PLP139-151 tetramer without compromising specificity. Furthermore, we found that induction of calcium fluxing by tetramers in T cells may be used as a sensitive measure to detect autoreactive T cells with a low affinity. Taken together, the data show that the tetrameric reagent binds and stimulates PLP139-151-reactive T cells with specificity. This tetrameric reagent will be useful in studying the evolution of PLP139-151-specific repertoire in naive mice and its expansion during the autoimmune disease experimental autoimmune encephalomyelitis.
ASJC Scopus subject areas
- Immunology and Allergy