Unaltered negative selection and Treg development of self-reactive thymocytes in TCR transgenic Fyn-deficient mice

Abstract

The tyrosine kinase Fyn has been implicated as playing an important role in the generation of both stimulatory and inhibitory signaling events induced by TCR engagement. To assess the role of Fyn for antigen-driven negative selection and Treg development, which are both dependent on the strength and nature of TCR signaling, we generated mice that co-express the transgenes for OVA and the OT-II TCR, which recognizes a peptide from OVA. In mice expressing both transgenes, negative selection, Treg development in the thymus, and the number of Treg in the periphery were each unaffected by ablation of Fyn. Moreover, fyn(-/-) Treg were functional, as assessed in vitro. We further tested the role of Fyn for the adaptor function of c-Cbl, using mice containing a point mutation in c-Cbl that abolishes its E3 ubiquitin ligase function but maintains its adaptor function. The functional and signaling properties of this mutant c-Cbl were unaltered in fyn(-/-) thymocytes. Combined, these data indicate that Fyn was not required for the induction of central tolerance by negative selection, the adaptor protein role of c-Cbl, or the normal development and function of Treg.

Figure 1. Fyn-deficiency does not alter CD4⁺ T cell development in RIP-Ova OT-II TCR transgenic mice

Total number of thymocytes (A) and the number of Vα2⁺CD4^-SP thymocytes (B) for OT-II WT, Ova OT-II WT, OT-II fyn^-/-, Ova OT-II fyn^-/- mice. Error bars show mean ± SD for each genotype. (C, D) Cell surface expression of Vα2 on CD4 SP thymocytes from (C) OT-II WT and RIP-Ova OT-II WT, and (D) OT-II fyn^-/- mice and RIP-Ova fyn^-/- mice. Also shown is the expression of Vα2 on CD4 SP thymocytes from OT-II fyn^-/- mice (C) or from OT-II WT mice (D) from samples that were all stained and analyzed in the same experiment. (E, F) Expression of CD5 on Vα2⁺ CD4 SP thymocytes from OT-II WT and RIP-Ova OT-II WT mice (E), and from OT-II fyn^-/- and RIP-Ova OT-II fyn^-/- mice (F). Expression of Foxp3 in Vα2⁺CD4 SP thymocytes from OT-II WT mice (G) and OT-II fyn^-/- mice (H), and from RIP-Ova OT-II WT mice (G) and RIP-Ova OT-II fyn^-/- mice (H) are also shown. Four to eight mice were assessed from each genotype in three separate experiments.

Figure 2. Fyn-deficiency does not alter the functional and phenotypic effects observed in thymocytes from c-Cbl RING finger mutant mice

C-Cbl^+/- and c-Cbl^A/- littermates expressing (A-C) or lacking (D-F) Fyn were assessed for cell surface expression of TCR, CD5, and CD69 on CD4⁺CD8⁺ thymocytes by flow cytometry. (G) Western blot analysis for phospho-S473 Akt and tyrosine phosphorylated c-Cbl from total cell lysates from thymocyes stimulated with anti-CD3 or anti-CD3 and anti-CD4, as indicated. Total Akt, c-Cbl and Fyn protein levels were also assessed by western blot as controls.

Figure 3. Fyn-deficiency does not alter the frequency of antigen-specific Treg in the periphery in RIP-Ova OT-II mice, or the suppressive function of non-transgenic Treg

The effect of cognate antigen expression on total spleen cell number (A) and the number of splenic Vα2⁺CD4⁺ T cells (B) from OT-II WT, RIP-Ova OT-II WT, OT-II fyn^-/-, and RIP-Ova OT-II fyn^-/- mice. (C) Frequency of Vα2⁺CD4⁺ splenic T cells that expressed Foxp3 from OT-II WT, OT-II fyn^-/-, RIP-Ova OT-II WT, and RIP-Ova OT-II fyn^-/- mice. (D) Frequency of CD4⁺ splenic T cells that express Foxp3 in C57BL/6 mice and G15 fyn^-/- mice. Error bars show mean ± SD from 2-3 independent experiments. (E, F) Anti-CD3 induced ³H-TdR incorporation of naïve CD4⁺ T cells from C57BL/6 mice (open bar, E) or from G15 fyn^-/- mice (open bar, F). Also shown is ³H-TdR incorporation of cultures containing both naïve and Treg at various ratios, as indicated. In these cultures, the number of naïve WT (E) or fyn^-/- (F) CD4⁺ T cells was held constant at 5×10⁴ cells/well, and between 0.5-5×10⁴ cells/well of Treg from C57BL/6 (stippled bars) or from G15 fyn^-/- mice (filled bars) were added. ³H-TdR was added for the last 8hrs of a 72hr culture. Data show mean ± SEM from two independent experiments.