TCR-inducible PLZF transcription factor required for innate phenotype of a subset of gammadelta T cells with restricted TCR diversity

Abstract

Some gammadelta and alphabeta T lymphocytes exhibit an "innate" phenotype associated with rapid cytokine responses. The PLZF transcription factor is essential for the innate phenotype of NKT cells. This report shows that PLZF is likewise responsible for the innate, NKT-like phenotype of Vgamma1+Vdelta6.3/Vdelta6.4+ cells. TCR cross-linking induced PLZF expression in all polyclonal immature gammadelta thymocytes, suggesting that agonist selection might be required for PLZF induction. Transgenic expression of Vgamma1Vdelta6.4 TCR was sufficient to support the development of large numbers of PLZF+ T cells, further supporting the importance of the TCR for PLZF induction. Interestingly, expression of this TCR transgene led to the development of spontaneous dermatitis.

Fig. 1.

PLZF is expressed by a subset of γδ T cells. (A) Cells isolated from thymi, spleens, and livers of wt or PLZF-deficient mice were stained for surface expression of B220, TCRγδ, TCRβ, and for intracellular PLZF and analyzed by FACS. TCRγδ versus TCRβ staining on B220− population is shown (Left and Center). PLZF expression in TCRγδ+ population is shown (Right). (B) Wt thymocytes were stained for surface expression of TCRγδ and individual Vγ, and intracellular expression of PLZF was analyzed in Vγ+ and Vγ− populations as indicated. (C) Cells were stained as in A with addition of anti-Vγ1 and anti-Vδ6.3. Plots in the first two columns are gated on TCRγδ+ population, additional gates applied in the other plots as indicated. Representative FACS plots from 1 of 3 independent experiments are shown.

Fig. 2.

PLZF-deficient Vγ1+Vδ6.3+ cells show impaired cytokine responses. TCRγδ+ thymocytes (Left) or splenocytes (Right) from wt or PLZF-deficient mice were sorted and stimulated with PMA/ionomycin as described in Material and Methods. Surface expression of Vγ1 and Vδ6.3 and intracellular expression of IFNγ and IL-4 were analyzed. Gates applied as indicated. Representative FACS plots from 1 of 3 independent experiments are shown.

Fig. 3.

T cells from Vγ1Vδ6.4 TCR transgenic mice express PLZF. (A) CD4/CD8a expression profiles of thymocytes from Vγ1Vδ6.4 TCR transgenic mice with (Right) or without (Left) LTH-1 transgene. (B) Intracellular expression of PLZF in thymocytes of these mice. Gates applied as indicated. Representative FACS plots from 1 of 8 independent experiments are shown (A and B). (C) Thymocytes from LTH-1/TCR double-transgenic mouse were stimulated with PMA/ionomycin (Right) or left unstimulated (Left) and stained for intracellular expression of PLZF, IL-4, and IFNγ; gated on PLZF+ cells. Representative FACS plots from 1 of 2 independent experiments are shown (C).

Fig. 4.

PLZF can be induced in γδ thymocytes by strong TCR signal. (A) Wt and PLZF−/− TCRγδ thymocytes (Left and Center) of indicated phenotype or splenocytes (Right) were sorted and cocultured with irradiated OP9-DL1 cells in plates precoated with 0.1 μg/mL anti-TCRγδ or left uncoated. PLZF expression was analyzed on day 5 of culture. Representative FACS plots from 1 of 4 independent experiments are shown. (B) CD25+ TCRγδ+ thymocytes were sorted and cultured with irradiated OP9-DL1 cells in plates precoated with 0.01 μg/mL anti-Vγ1 (Top), 0.1 μg/mL anti-Vγ4 (Middle), or 0.01 μg/mL anti-Vγ7 (Bottom). PLZF expression was analyzed on day 5 of culture. (C) CD25+ TCRγδ+ thymocytes were sorted and cultured as in A and frequency of Vγ1, Vγ4, and Vγ7 positive cells among total TCRγδ+ cells was analyzed by FACS. (D) Ex vivo CD5 expression on Vγ1+Vδ6.3+ and Vγ1−Vδ6.3− γδ thymocytes of individual mouse (Left) or average of median fluorescent intensity for 3 individual mice (±SD). *, indicates statistical significance (P < 0.05) with a paired t-test. (E) Sorted TCRγδ+ wt thymocytes were stimulated with PMA/ionomycin and analyzed for expression of PLZF and IL-17.

Fig. 5.

Vγ1Vδ6.4 TCR transgenic mice develop spontaneous dermatitis. (A) Tails of 4-month-old LTH-1/TCR double-transgenic (Right) or TCR single-transgenic (Left) littermates are shown. (B–E) HE staining of tails from single-transgenic (B and D) or double-transgenic (C and E) mice are shown. Arrows and brace indicate infiltrate, P indicate a pustule.