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. 2013 Sep 12;8(9):e74019.
doi: 10.1371/journal.pone.0074019. eCollection 2013.

Differential requirement for CCR4 in the maintenance but not establishment of the invariant Vγ5(+) dendritic epidermal T-cell pool

Kyoko Nakamura  1 , Andrea J White, Sonia M Parnell, Peter J Lane, Eric J Jenkinson, William E Jenkinson, Graham Anderson
Affiliations

Differential requirement for CCR4 in the maintenance but not establishment of the invariant Vγ5(+) dendritic epidermal T-cell pool

Kyoko Nakamura et al. PLoS One. .

Abstract

Thymocytes expressing the invariant Vγ5 γδT-cell receptor represent progenitors of dendritic epidermal T-cells (DETC) that play an important immune surveillance role in the skin. In contrast to the bulk of αβT-cell development, Vγ5(+) DETC progenitor development occurs exclusively in fetal thymus. Whilst αβT-cell development is known to require chemokine receptor mediated migration through distinct thymus regions, culminating in medullary entry and thymic egress, the importance and control of intrathymic migration for DETC progenitors is unclear. We recently revealed a link between Vγ5(+) DETC progenitor development and medullary thymic epithelial cells expressing Aire, a known regulator of thymic chemokine expression, demonstrating that normal Vγ5(+) DETC progenitor development requires regulated intramedullary positioning. Here we investigate the role of chemokines and their receptors during intrathymic Vγ5(+) DETC progenitor development and establishment of the DETC pool in the skin. We report that thymic medullary accumulation of Vγ5(+) DETC progenitors is a G-protein coupled receptor dependent process. However, this process occurs independently of Aire's influences on intrathymic chemokines, and in the absence of CCR4 and CCR7 expression by DETC progenitors. In contrast, analysis of epidermal γδT-cells at neonatal and adult stages in CCR4(-/-) mice reveals that reduced numbers of DETC in adult epidermis are not a consequence of diminished intrathymic embryonic development, nor deficiencies in initial epidermal seeding in the neonate. Collectively, our data reveal differences in the chemokine receptor requirements for intrathymic migration of αβ and invariant γδT-cells, and highlight a differential role for CCR4 in the maintenance, but not initial seeding, of DETC in the epidermis.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Inhibition of G-Coupled Receptor Signaling Prevents Accumulation of Vγ5+ DETC Progenitors in the Thymic Medulla.
(A) Experimental design. Freshly isolated E14 C57BL6 thymus lobes were treated with or without 250 ng/ml Pertussis toxin (PTX) for 30 minutes, cultured as FTOC for 48 hr, then harvested for flow cytometry or confocal analysis. (B) Vγ5+ thymocyte numbers from PTX treated or non-treated FTOC. Control; n = 8, PTX; n = 9. (C and D) Confocal analysis of Vγ5+ thymocyte distribution in control and PTX treated FTOC. Error bars represents SEM, and asterisks signify a significant difference, where p<0.0001. Scale bars in D on upper panel represent 200 µm and on lower panel represent 50 µm.
Figure 2
Figure 2. Aire is not Required for Vγ5+ DETC Progenitor Clustering with mTEC.
(A) Representative confocal analysis of E18 WT and Aire−/− thymus sections. Vγ5+ cells are shown in green, CD8β+ cells are shown in red and EpCAM+ medullary areas are shown in blue. M denotes medulla, C denotes cortex. Scale bars in A represent 100 µm. (B) Quantification of Vγ5+ cells per mm2 thymic medulla. WT; n = 6, Aire−/−; n = 6. Error bars represents SEM. (C) Individual thymus lobes of E18 WT and Aire−/− embryos were teased apart and stained for Vγ5, CD3, CD24 and CD45RB expression. Numbers shown on FACS plot are the mean percentages +/− SD. WT; n = 9, Aire−/−; n = 4.
Figure 3
Figure 3. Intrathymic Medullary Accumulation of Vγ5+ DETC Progenitors occurs Independently of CCR4 and CCR7.
(A) Expression of CCR4 and CCR7 on total Vγ5+ thymocytes and CD45RBhigh/CD45RBlow Vγ5+ thymocyte subsets from E18 thymus. Black histograms show the levels of antibody staining using E18 thymocytes from the indicated chemokine receptor knockout mice as a control. Red histograms show the expression level of each chemokine receptor in WT E18 thymocytes. Numbers represent the mean fluorescent intensity of CCR4/7 KO then WT cells. (B-D) Representative confocal images of E18 WT (B), CCR4−/− (C), CCR7−/− (D) thymus. M denotes medulla, C denotes cortex. Scale bars in B-D represent 50 µm. (E) Confocal quantification of Vγ5+ thymocytes per in mm2 medullary area in WT (n = 6), CCR4−/− (n = 6) and CCR7−/− (n = 6) E18 thymus. Error bars represent SEM.
Figure 4
Figure 4. Absence of CCR4 or CCR7 causes an Increase in Mature Vγ5+ T Cells in E18 Fetal Thymus.
(A) Total number of thymocytes from both thymus lobes of individual E18 mouse embryos of the indicated strain. (B) Total cell number of Vγ5+ thymocytes from E18 thymus. (C) Shows the ratio of CD45RBhigh and CD45RBlow subsets within total CD3+ Vγ5+ thymocytes. A minimum of 10 mice of each strain were analyzed. Error bars represent SEM, with asterisks signifying a significant difference, where p<0.001.
Figure 5
Figure 5. Vγ5+ DETC are Selectively Reduced in the Epidermis of Adult, but not Neonatal, CCR4−/− Mice.
(A) Day 0 newborn epidermal sheets were digested and stained for Vγ5TCR and CD3 expression. The graph shows the percentage of T-cells within DAPI- cells of the indicated strains. WT; n = 12, CCR4−/−; n = 8, CCR7−/−; n = 9. (B) Percentages of Vγ5+ DETC within CD3+ T-cells in d0 newborn epidermis. (C) Adult ear epidermal sheets were digested with Collagenase D and stained for Vγ5TCR and CD3 expression. The graph shows the percentage of CD3+ T-cells within DAPI- cells. WT; n = 19, CCR4−/−; n = 14, CCR7−/−; n = 12. (D) Percentage of Vγ5+ DETC within CD3+ T-cells in adult epidermis. (E) Percentage of Annexin V+ cells in adult ear, after gating on Vγ5+ DETC. WT n = 4, CCR4−/− n = 3. (F) shows the percentage of T-cells in the dermis of adult ear skin in WT and CCR4−/− mice, while (G) shows the proportion of Vγ5+ cells within dermal T cells. WT; n = 4, CCR4−/−; n = 4. Asterisks signify a significant difference, where ** p<0.001, * p<0.01.
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