Figure 1. Tet+ LDA reveals low-affinity CD4+ T cells in the naive repertoire.

(a) 6 × 10⁶ Nur77^gfp CD4+ T cells (6 × 10⁵ after a 10% park rate, Thy1.2+) were transferred into congenically distinct (Thy1.1+) mice and immunized with GP_61–81 peptide/CFA followed by tetramer enrichment and restimulations. (b) Flow cytometry of representative GP66-Tet+ enrichment of secondary lymphoid organs from day 7 immunized mice with a positive donor (Thy1.2+) clone identified. (c) After tetramer enrichment, the unbound cells were independently stimulated with 100 μg ml⁻¹ of specific (GP_61–81) or non-specific (Aasf_24–32, MOG_35–55 or NP_311–325) peptides and gated to identify donor CD44+ Tet− Thy1.2+ CD4+ T cells by flow cytometry. (d) Percent change over averaged background of Nur77^gfp+CD69+ CD4+ T cells after stimulation with individual antigens (n=7, two independent experiments, *=3 s.d. above background average, NS=no significance). (e) Bcl-6 expression measured by flow cytometry of CD4+ T cells from day 5 immunized mice within identified subsets. Frequency of cells expressing Bcl-6 within each subset identified (points represent individual mice, n=6–7, two experiments, one-way analysis of variance, NS=no significance, ***P<0.001,****P<0.0001).