Detection of Autoreactive Myelin Proteolipid Protein 139–151-Specific T Cells by Using MHC II (IAs) Tetramers

Abstract

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.

An epitope from Acanthamoeba castellanii that cross-react with proteolipid protein 139-151-reactive T cells induces autoimmune encephalomyelitis in SJL mice

“…Derivation of MHC class II/IA s tetramers and flow cytometric analysis of antigen-specific T cells by tetramer staining PLP 139-151 and Theiler's murine encephalomyelitis virus (TMEV) 70-86 tetramers were generated as described (Reddy et al, 2003). To derive ACA 83-95 tetramers, IA s construct (gift from Dr. Vijay Kuchroo, Harvard Medical School, Boston, Massachusetts) was modified by excising PLP 139151 sequence using Kpn I and BamH I and replaced with the nt sequence encoding ACA 83-95 (5'-TATTTTCT-TCTTAAATGGCTTGGTCATCCTAATGTTTCT-3').…”

“…We sought to identify the disease-producing microbial mimics for PLP 139-151 from pathogens capable of inducing CNS disease and searched for homologous sequences between PLP 139-151 and pathogens in pro-fecting SF9 insect cells using a high-titer virus supernatant and the tetramers were derived as described previously (Reddy et al, 2003). To determine the frequency of Ag-specific T cells, LNC obtained from the mice immunized with PLP 139-151 or ACA 83-95 were restimulated with the corresponding peptides (20 µg/ml) for four to six days.…”

“…To determine the frequency of Ag-specific T cells, LNC obtained from the mice immunized with PLP 139-151 or ACA 83-95 were restimulated with the corresponding peptides (20 µg/ml) for four to six days. Viable lymphoblasts were harvested by Ficoll-Hypaque density gradient centrifugation and incubated with phycoerythrin (PE) conjugated IA s tetramers (30 µg/ml) at room temperature (RT) for 3 h followed by staining with anti-CD4 and 7-amino-actinomycin-D (7-AAD) (Eugene, Oregon) (Reddy et al, 2003(Reddy et al, , 2004. The frequency of tetramer + (tet + ) cells was then enumerated by four-color flow cytometric analysis (FACS caliber, BD Pharmingen, San Diego, California) and Flow Jo software (Tree star, Ashland, Oregon) in a live (7-AAD -) CD4 + population.…”

Tetramers reveal IL-17–secreting CD4+T cells that are specific for U1-70 in lupus and mixed connective tissue disease

“…Tetramer studies have provided insight into CD4 + T-cell molecular interactions, function, frequency, location, and response to therapy in other autoimmune diseases, including multiple sclerosis (32,33), type 1 diabetes (34, 35), rheumatoid arthritis (36), and celiac disease (37). Tetramers also have been used to detect IFNγ-secreting CD8 + T cells specific for myelin in neuropsychiatric SLE (38); however, the use of tetramers to identify and characterize self-reactive CD4 + T cells has not been reported in lupus.…”

Peptide 15-mers of defined sequence that substitute for random amino acid copolymers in amelioration of experimental autoimmune encephalomyelitis

“…Proliferative responses were measured as described in refs. 17 (17,29,30), peptide synthesis (13), isolation of empty HLA-DR2 (12,13), inhibition by peptide 15-mers of MBP 85-99 binding to HLA-DR2 (DRA͞DRB1*1501) (13,17), sorting of tg and endogenous (non-tg) T cells from humanized mice by flow cytometry (30), cytokine measurement by ELISA (30), and detection of PLP 139-151-reactive CD4 ϩ T cells by using I-A s ͞PLP 139-151 tetramers (31,32). (11,17).…”