Figure 4. Spontaneous lymphocyte activation in Tln1^fl/flCd4^Cre mice is Treg cell-dependent.

(A–E) Lethally irradiated RAG1-deficient mice were reconstituted with bone marrow cells from a CD45.2 wild-type donor in combination with cells from a CD45.1 wild-type donor (“WT:WT”, n=4), wild-type (CD45.2) bone marrow cells in combination with Foxp3-deficient (CD45.1) bone marrow cells (“WT:Foxp3null”, n=3), bone marrow cells from Tln1^fl/flCd4^Cre mice (CD45.2) alone (“Cd4^Cre”, n=5), bone marrow cells from Tln1^fl/flCd4^Cre mice (CD45.2) in combination with wild-type (CD45.1) bone marrow cells (“Cd4^Cre:WT”, n=14), and bone marrow cells from Tln1^fl/flCd4^Cre mice (CD45.2) in combination with Foxp3-deficient deficient (CD45.1) bone marrow cells (“Cd4^Cre:Foxp3-null”, n=5). Mice were analyzed by flow cytometry 8–10 weeks after reconstitution. (A) Activation status of splenic CD4⁺ T cells based on CD62L and CD44 expression; displayed cells were gated on CD4⁺CD45.1⁺ events in WT:WT, WT:Foxp3null, Cd4 ^Cre:WT and Cd4 ^Cre:Foxp3null bone marrow chimeric mice and on CD4⁺CD45.2⁺ events in Cd4^cre bone marrow chimeras. (B) Percentage of splenic CD4⁺ cells expressing Foxp3⁺; displayed cells were gated on CD4⁺CD45.2⁺ cells to identity talin-deficient Treg cells in Cd4^cre and Cd4 ^Cre:WT bone marrow chimeras and on CD4⁺CD45.1⁺ cells in WT:WT and Cd4 ^Cre:WT chimeras to identity wild-type Treg cells. (C) Absolute numbers of talin-deficient splenic CD4⁺CD45.2⁺ Foxp3⁺ Treg cells in Cd4^cre and Cd4^cre:WT bone marrow chimeras and wild-type CD4⁺CD45.1⁺ Foxp3⁺ cells in WT:WT and Cd4 ^cre:WT chimeras. (D) Foxp3 expression on a per cell basis (MFI) from talin-deficient (CD45.2⁺) or wild-type (CD45.1⁺) CD4⁺Foxp3⁺ splenic Treg cells from WT:WT, Cd4^cre and Cd4 ^Cre:WT bone marrow chimeras. (E) Expression of suppressive molecules IL-2Rα, CD39, CD73, GITR and CTLA4 by splenic talin-deficient (CD45.2⁺) or wild-type (CD45.1⁺) CD4⁺Foxp3⁺ splenic Treg cells from WT:WT, Cd4^cre and Cd4 ^cre:WT bone marrow chimeras. (F) Experimental approach for the adoptive transfer of CD4⁺CD45.2⁺ T cells isolated from Tln1^fl/flCd4^CreFoxp3^GFP mice into CD45.1⁺ wild-type recipients. Expression (G) and MFI (H) of Foxp3 in talin-deficient CD4⁺CD45.2⁺ Treg cells isolated from the spleens of recipient mice. (I) Expression of IL-2Rα, CD39, CD73, GITR and CTLA4 by CD4⁺CD45.2⁺GFP⁺ Treg cells isolated from the spleen of recipient mice. Data shown are mean ± SEM and are representative of at least 2 independent experiments. *, P < 0.05; **, P <0.01; ***, P<0.001.