
Figure 1 Generation and evaluation of J15 TCR transgenic mice. (A) Diagram depicting cassette vectors pTα and pTβ. Each vector has V-region (Vα and Vβ, respectively) promoter and the complete constant-region (Cα and Cβ, respectively) gene sequences and were subcloned with Vα13D-1 and Jα34-02 for the α chain (using XmaI and NotI), as well as Vβ13-1-02, Dβ2-01, and Jβ2-7-01 for the β chain (using XhoI and SacII). (B) J15 TCR transgenic mice were screened by staining the peripheral blood lymphocytes with anti-V β8.3 mAb, anti-CD8 mAb, and H60-tetramer, followed by flow-cytometric analysis. Vβ8.3 by CD8 and H60-tetramer by CD8 profiles of peripheral-blood lymphocytes from J15 and WT mice. Numbers in plots indicate the frequency of Vβ8.3 expression or H60-tetramer binding of CD8 T cells in the peripheral blood from J15 and WT mice.

Figure 2 Positive selection of J15 TCR transgenic T cells specific for H60. (A) CD4 by CD8 profiles (left) of thymocytes and numbers (right) of each thymic population from J15, OT-I, and WT mice on the B6 background. Numbers at the top of plots indicate total number of thymocytes. (B) Surface expression of Vβ8.3 and Vβ5.2, as well as (C) the binding of the H60- and OVA-tetramer of DN, DP, and CD8 SP thymocytes from J15, OT-I, and WT mice. (D) CD4 by CD8 profiles (left) of splenocytes and numbers (right) of CD4 and CD8 T cells in the spleen from J15, OT-I, and WT mice. (E) Surface expression of Vβ8.3 and Vβ5.2, as well as the binding of the H60- and OVA-tetramer of CD8 T cells in the spleen from J15, OT-I, and WT mice. Numbers in plots indicate the percentage of cells in each quadrant. ***p<0.001; **p<0.01; *p<0.05. Data are representative of three independent experiments and depict the mean±SD.

Figure 3 Negative selection of J15 TCR transgenic T cells specific for H60. (A) CD4 by CD8 profiles (left) of thymocytes and numbers (right) of each thymic population from J15-H60 Tg, J15, OT-I-OVA Tg, and OT-I mice on the B6 background. Numbers at the top of the plots indicate total number of thymocytes. (B) Surface expression of Vβ8.3 and Vβ5.2, as well as (C) the binding of the H60- and OVA-tetramer of DN, DP, and CD8 SP thymocytes from J15-H60 Tg, J15, OT-I-OVA Tg, and OT-I mice. (D) CD4 by CD8 profiles (left) of splenocytes and numbers (right) of CD4 and CD8 T cells in the spleen from J15-H60 Tg, J15, OT-I-OVA Tg, and OT-I mice. (E) Surface expression of Vβ8.3 and Vβ5.2, as well as the binding of the H60- and OVA-tetramer of CD8 T cells in the spleen from J15-H60 Tg, J15, OT-I-OVA Tg, and OT-I mice. Numbers in plots indicate the percentage of cells in each quadrant. ***p<0.001; **p<0.01. Data are representative of three independent experiments and depict the mean±SD.

Figure 4 Lack of negative selection of thymocytes expressing J15 TCR in the presence of H60H. (A) Cytotoxicity assays (left) tested affinities of several different single-amino-acid variants of the H60 epitope by changing the amino acid asparagine (N) residue at P4 of the H60 epitope to different amino acids. The amino acid sequences of the normal H60 and H60H epitope (right), LTFNYRNL and LTFHYRNL, respectively. (B) CD4 by CD8 profiles (left) of thymocytes and numbers (right) of each thymic population from J15-H60H Tg, J15, and H60HTg mice on the B6 background. Numbers at the top of plots indicate the total number of thymocytes. (C) H60-tetramer binding of DN, DP, and CD8 SP thymocytes from J15-H60H Tg, J15, and H60HTg mice. (D) CD4 by CD8 profiles (left) of splenocytes and numbers (right) of CD4 and CD8 T cells in the spleen from J15-H60H Tg, J15, and H60HTg mice. (E) Surface expression of Vβ8.3 and the binding of the H60-tetramer of CD8 T cells in the spleen from J15-H60H Tg, J15, and H60HTg mice. Numbers in plots indicate the percentage of cells in each quadrant. **p< 0.01; *p<0.05; ns, non-significant. Data are representative of two independent experiments and depict the mean±SD.