Figure 7. IL-4 producing CD1d-tetramer^neg PLZF⁺ αβ T cells develop in the absence of Itk.

(A) Thymocytes from WT or itk^-/- mice were stained with CD1d-tetramer and antibodies to CD4, CD8, TCRδ, TCRβ HSA, Eomesodermin, and PLZF.

Dot-plots (left) show PLZF versus Eomes staining on CD4SP TCRδ^neg TCRβ^high HSA^low CD1d-tetramer^neg thymocytes.

Graphs (right) show frequencies and absolute numbers of CD4SP TCRδ^neg TCRβ^high CD1d-tetramer^neg HSA^low PLZF⁺ thymocytes.

n = 20-23 mice per group from 8 independent experiments. Statistical analysis was performed using a Mann-Whitney student t test. ****p < 0.0001

(B) Itk KO mice were crossed to IL-4 reporter (4get) mice as previously described (31). Thymocytes from WT and Itk KO expressing the IL-4 reporter (positive) or without the transgene (negative) were harvested, processed, and stained with CD1d tetramer and with antibodies against CD4, CD8, TCRδ, TCRβ, HSA, and CD44. Flow cytometry plots are gated on CD4 SP TCRδ^neg TCRβ^high CD1d tetramer^neg HSA^low thymocytes.

Results are representative of four independent experiments involving 2-3 mice per group per experiment.

(C-D) Thymocytes from WT or Itk KO mice were harvested, processed, and stimulated with PMA and ionomycin (right) or remained unstimulated (left) in the presence of brefeldin A and monensin for 5-6 hours at 37°C before staining with CD1d tetramer and with antibodies against CD4, CD8, Eomesodermin, PLZF, IFNγ, and IL-4.

(C) Gated on CD4SP CD1d-tetramer^neg Eomesodermin⁺ thymocytes.

(D) Gated on CD4 SP CD1d-tetramer^neg PLZF⁺ thymocytes.

Results are representative of two independent experiments involving 5-7 mice per group.