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. 2015 Nov 1;195(9):4273-81.
doi: 10.4049/jimmunol.1500939. Epub 2015 Sep 25.

PLZF Controls the Development of Fetal-Derived IL-17+Vγ6+ γδ T Cells

Ying Lu  1 , Xin Cao  1 , Xianyu Zhang  1 , Damian Kovalovsky  2
Affiliations

PLZF Controls the Development of Fetal-Derived IL-17+Vγ6+ γδ T Cells

Ying Lu et al. J Immunol. .

Abstract

Expression of promyelocytic leukemia zinc finger (PLZF) protein directs the effector differentiation of invariant NKT (iNKT) cells and IL-4(+) γδ NKT cells. In this study, we show that PLZF is also required for the development and function of IL-17(+) γδ T cells. We observed that PLZF is expressed in fetal-derived invariant Vγ5(+) and Vγ6(+) γδ T cells, which secrete IFN-γ and IL-17, respectively. PLZF deficiency specifically affected the effector differentiation of Vγ6(+) cells, leading to reduced numbers of mature CD27(-)CD44(+) phenotype capable of secreting IL-17. Although PLZF was not required for Vγ5(+) γδ T cells to develop, when these cells were reprogrammed into IL-17-secreting cells in Skint-1 mutant mice, they required PLZF for their effector maturation, similarly to Vγ6(+) γδ T cells. The impaired effector differentiation of PLZF-deficient Vγ6(+) γδ T cells was not due to increased apoptosis and it was related to reduced proliferation of immature CD27(+)CD44(-) Vγ6(+) γδ T cells, which was required for their differentiation into mature CD27(-)CD44(+) IL-17-secreting cells. Thus, the present study identifies that PLZF function is not restricted to NKT or IL-4(+) T cells, but it also controls the development of IL-17(+) γδ T cells.

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Figures

Figure 1
Figure 1. PLZF is expressed in fetal-derived Vγ5+ and Vγ6+ γδT-cells
a). PLZF intracellular staining of C57BL/6 fetal embryo day 16 (E16) and neonatal d1 thymus. Double negative (DN) thymocytes: ETP (CD4−CD8−Lin−CD44+ckithiCD25−); DN2a (CD4−CD8−Lin−CD44+ckithiCD25+); DN3 (CD4−CD8−Lin−CD44−CD25+); DN4 (CD4−CD8−Lin−CD44−CD25−) and γδT-cells (GL3+, 2C11+). b) RT-PCR of sorted T-cell subsets from adult, neonatal day 1 (d1) and fetal day 16 (E16) thymus. c) Gating strategy used to sort subpopulations of γδT-cells based on Vγ5 and 17D1 staining. d) RT-PCR from from sorted γδT-cell subsets indicated in (C). e) FACs analysis of Tdtomato label in the subpopulations indicated from different tissues. The numbers indicate the percentage of events within the gate from compound PLZF-CRE x Rosa-Tdtomato mice. (a and e) are representative of three independent experiments, (b, c, and d) are representative of two independent experiments. * Vγ6+ cells are identified as CD45.2+ GL3+2C11+Vγ5−17D1+ cells.
Figure 2
Figure 2. PLZF is required for the generation of Vγ6+ γδT-cells with a mature CD27−CD44+ phenotype and it does not affect the development of Vγ5+ γδT-cells
FACs analysis showing the proportion of Vγ5+ and Vγ6+ γδT-cells in fetal day 16 (E16) (a) and neonatal day 1 thymus (b). Gram plots showing thymic Vγ6+γδT-cell numbers (c), and Vγ6+γδT-cell numbers in Lung and uterus (d) comparing wild type (+/+) and PLZF-deficient (−/−) cells. e) Number of Vγ5+ γδT-cells in the Fetal (E16) thymus and in adult skin. f) FACs profile of Vγ6+ γδT-cells in the fetal (E16) and neonatal (d1) thymus. g) Gram plot indicating the cell numbers of subsets of Vγ6+ γδT-cells identified in panel (f). h) FACs analysis for intracellular IL-17 staining in Vγ6+γδT-cells. i) Gram plot indicating the number of IL-17+Vγ6+γδT-cells.j) Histogram analysis of Vγ6+γδT-cells in the neonatal thymus. The numbers within the dot-plots represent the proportion of events (a, b, f, h). Data correspond to two and three independent experiments. Each dot in the gram plots represents data from a fetus or neonate and horizontal lines represent the mean value. p values corresponding to the significance using T-test are shown. * Vγ6+ cells are identified as GL3+2C11+Vγ5−17D1+ cells.
Figure 3
Figure 3. PLZF is required for the effector maturation of Vγ6+ γδT-cells in vitro
a) Gating strategy indicating the subpopulations of Vγ6+ γδT-cells that were sorted and placed for 3 days in OP9-DL1 co-cultures in the presence of IL-7 to evaluate their ability to generate different subpopulations. FACs analysis showing the phenotypic profile of sorted cells post-sort and after co-culture. The numbers indicate the proportion of cells within the quadrants. b) FACs analysis for intracellular IL-17 staining after PMA plus Ionomycin stimulation of sorted CD27+CD44−Vγ6+γδT-cells from fetal (E16) thymus after sort (Ex vivo) or after a 3 day differentiation in OP9-DL1 co-cultures as in panel (a). The percentage of IL-17+ cells is indicated. c) FACs analysis of Anexin V positive cells after 3 day OP9-DL1 co-culture. The percentage of anexinV positive cells is indicated. d) Histogram showing the dilution of cell trace violet on sorted wild type (+/+) and PLZF-deficient (−/−) CD27+CD44− Vγ6+ γδT-cells after 3 day co-culture as in (a). The gray histogram represent non-proliferating T-cells. e) histograms showing the incorporation of BrdU in fetal E16 Vγ6+ γδT-cells 24 hours after a single BrdU injection to the mother. f) Histogram showing the dilution of cell trace violet on sorted wild type CD27+CD44− Vγ6+ γδT-cells after co-culture in the presence of the CDK4/6 inhibitor (PD0332991). g) FACs analysis showing the phenotypic profile of sorted cells after co-culture. h) FACs analysis showing intracellular IL-17 staining in sorted cells after co-culture. The numbers indicate the proportion of cells within the quadrants. i) RT-PCR analysis of sorted immature E16 Vγ6+CD27+CD44− γδT-cells for the indicated genes. Data correspond to two independent experiments. * Vγ6+ cells are identified as GL3+2C11+Vγ5−17D1+ cells.
Figure 4
Figure 4. Transgenic Bcl2 expression fails to rescue the effector maturation of PLZF-deficient Vγ6+γδT-cells
a) RT-PCR from wild type and Bcl2-TG neonatal Vγ6+γδT-cells. b) FACs analysis on γδT-cells from PLZF-wild type or PLZF-deficient neonatal thymus that are also transgenic for Bcl2 (Bcl2 TG). The numbers indicate the percentage of cells within the gate. c) Gram plot indicating the percentage of Vγ6+ γδT-cells represented in panel (b). d) FACs analysis for intracellular IL-17 staining after PMA plus Ionomycin stimulation of Vγ6+γδT-cells indicated in panel (b). Numbers represent the percentage of events within the gate. e) Gram plot of the percentage of IL-17+ Vγ6+γδT-cells as represented in panel (d). Data is representative of two independent experiments. Each dot in the gram plots represents data from a neonate and horizontal lines represent the mean value. p values corresponding to the significance using T-test are shown. * Vγ6+ cells are identified as GL3+2C11+Vγ5−17D1+ cells.
Figure 5
Figure 5. Impaired IL-17 effector maturation of PLZF-deficient SkinT-mutant Vγ5+ γδT-cells
a) FACs analysis of gated Vγ5+ γδT-cells from fetal (E16) and neonatal (d1) thymus in the indicated strain. The numbers indicate the percentage of cells within the gate. b) FACs analysis for intracellular IL-17 staining after PMA plus Ionomycin stimulation of Vγ5+ γδT-cells in Skint1-mutant (E16) thymus. c) Gram plot of the percentage of IL-17+ Vγ5+ γδT-cells as represented in panel (b). Data is representative of two independent experiments. Each dot in the gram plots represents data from a fetus and horizontal lines represent the mean value. p values corresponding to the significance using T-test are shown.
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