Detection and Characterization of T Cells Specific for BDC2.5 T Cell-Stimulating Peptides

Abstract

Nonobese diabetic (NOD) mice expressing the BDC2.5 TCR transgene are useful for studying type 1 diabetes. Several peptides have been identified that are highly active in stimulating BDC2.5 T cells. Herein, we describe the use of I-Ag7 tetramers containing two such peptides, p79 and p17, to detect and characterize peptide-specific T cells. The tetramers could stain CD4+ T cells in the islets and spleens of BDC2.5 transgenic mice. The percentage of CD4+, tetramer+ T cells increased in older mice, and it was generally higher in the islets than in the spleens. Our results also showed that tetAg7/p79 could stain a small population of CD4+ T cells in both islets and spleens of NOD mice. The percentage of CD4+, tetramer+ T cells increased in cells that underwent further cell division after being activated by peptides. The avidity of TCRs on purified tetAg7/p79+ T cells for tetAg7/p79 was slightly lower than that of BDC2.5 T cells. Although tetAg7/p79+ T cells, like BDC2.5 T cells, secreted a large quantity of IFN-γ, they were biased toward being IL-10-producing cells. Additionally, <3% of these cells expressed TCR Vβ4. In vivo adoptive transfer experiments showed that NOD/scid recipient mice cotransferred with tetAg7/p79+ T cells and NOD spleen cells, like mice transferred with NOD spleen cells only, developed diabetes. Therefore, we have generated Ag-specific tetramers that could detect a heterogeneous population of T cells, and a very small number of NOD mouse T cells may represent BDC2.5-like cells.

Presence of Diabetes-Inhibiting, Glutamic Acid Decarboxylase-Specific, IL-10-Dependent, Regulatory T Cells in Naive Nonobese Diabetic Mice

“…In these previous experiments, we have used the same cell culture protocol that we have used to expand and isolate N221 ϩ cells described in this report. Additional functional studies showed that, unlike N221 ϩ cells, these p79-specific T cells did not inhibit diabetes development (30). In addition, in another report we demonstrated that T cells cultured under the same culture conditions displayed the same cytokine secretion profiles as freshly isolated T cells before culture in vitro (20).…”

“…The tetAg7/p221 tetramer did not stain CD4 ϩ T cells specific for an irrelevant peptide, p79, isolated using tetAg7/p79 (tetAg7/p79 ϩ cells; Fig. 1B) (30). Additionally, these N221 ϩ cells were not stained positively by another control tetramer, tetAg7/p206, specific for a control p206 peptide.…”

“…Together, these findings are consistent with the idea that p221-specific T cells are present in unimmunized naive NOD mice, but the percentage of these Ag-specific T cells was probably Ͻ0.1%, the lower detection limit of using the tetramers to detect these cells with FACS. Therefore, a combination of the use of tetramer together with CFSE labeling of Ag-activated T cells provides a useful method to detect Ag-specific T cells (30,37,38).…”

“…It is possible that the culture conditions used to expand these T cells could have provided signals for the acquisition of regulatory functions. However, as described in our previous report, the same experimental conditions were used to isolate from naive NOD mice the CD4 ϩ T cells specific for a BDC2.5 T cell-stimulating peptide, p79 (30). Using the tetAg7/p79 tetramer, we could detect p79-specific T cells in unimmunized naive NOD mice, and then could use this tetramer to isolate p79-specific T cells.…”

“…B, Isolated N221 ϩ T cells were stained with anti-CD4, tetAg7/p221, or control tetAg7/p206. Previously isolated tetAg7/p79 ϩ T cells (30), specific for a BDC2.5 cell-stimulating peptide p79, were used as a negative control. C, Phenotypic analyses of N221 ϩ T cells.…”