Cutting edge: Intrinsic programming of thymic γδT cells for specific peripheral tissue localization

Abstract

Various innate-like T cell subsets preferentially reside in specific epithelial tissues as the first line of defense. However, mechanisms regulating their tissue-specific development are poorly understood. Using the prototypical skin intraepithelial γδT cells (sIELs) as a model, we show in this study that a TCR-mediated selection plays an important role in promoting acquisition of a specific skin-homing property by fetal thymic sIEL precursors for their epidermal location, and the skin-homing potential is intrinsically programmed even before the selection. In addition, once localized in the skin, the sIEL precursors develop into sIELs without the requirement of further TCR-ligand interaction. These studies reveal that development of the tissue-specific lymphocytes is a hard-wired process that targets them to specific tissues for proper functions.

Figure 1

Impaired selection of Vγ3⁺ fetal thymocytes results in their acquisition of a different homing property. A. FACS analysis of CCR10(EGFP) and CD122 expression on E16 fetal thymic Vγ3⁺ vs. Vγ3⁻ T cells of CCR10^+/EGFP mice of Skint1^B6 or Skint1^FVB(Tac) background (N≥5 each). B. FACS analysis of Vγ3⁺ cells recovered from five-day in vitro cultured E15 fetal B6 thymic lobes reconstituted with fetal thymic CD122⁺CCR10⁻ or CD122⁻CCR10⁺ Vγ3⁺ cells of CCR10^+/EGFPSkint1^FVB(Tac) mice. N≥3 for each type.

Figure 2

A strong γδTCR signal promotes acquisition of the Vγ3-like skin-homing property by fetal thymic Tg Vγ2⁺ T cells. A. FACS analysis of CD122 and CCR10(EGFP) expression on E16-17 fetal KN6 or G8 Tg Vγ2⁺ thymocytes of different ligand-expressing backgrounds. At least two mice of each genotype were analyzed with the same results. B. FACS analysis of fetal vs. adult thymocytes for Tg Vγ2⁺ T cells in G8 or KN6 mice of B6 or Balb/c background. The histographs are gated on total thymocytes. N≥3. C. FACS analysis of fetal vs. adult G8 or KN6 Tg γδT cells of indicated backgrounds for the expression of different homing molecules. Histographs were gated on Vγ2⁺ cells. Gray areas were isotype controls.

Figure 3

Intrinsic properties of fetal vs. adult thymic KN6 γδT cells determine their different selection and differentiation processes. A. FACS of thymocytes of a five-day FTOC reconstituted with purified fetal thymic CD122⁻CCR10(EGFP)⁻ or adult thymic CD24^highCCR10(EGFP)⁻ Tg Vγ2⁺ cells of CCR10^+/EGFPβ2M^−/−KN6 mice. The gated regions in the top panels represent total thymocytes that are all of the Tg Vγ2⁺ donor origin, whose expression of CD122 and CCR10(EGFP) is shown in the bottom panels. N≥5. B. FACS analysis of thymocytes recovered from ten-day B6 FTOC reconstituted with purified E15 fetal or adult thymic αβTCR⁻γδTCR⁻ progenitor cells of CCR10^+/EGFPKN6 (B6) mice for the Tg Vγ2⁺ T cells. N≥ 5. C. FACS analysis of the Tg Vγ2⁺ T cells gated from the panel B for different homing molecules. N≥4. D–E. ChIP analyses of H3K4m2 and H3K9m3 modifications at the CCR10 locus in purified fetal EGFP(CCR10)⁻CD122⁻ and adult CD24^hiCD4⁻CD8⁻ Vγ2⁺ thymocytes of CCR10^+/EGFPβ2M^−/−KN6 mice. N ≥2. ND: non-detectable. F: fetal. A: adult.

Figure 4

Positively selected fetal thymic γδT cells develop into sIELs without the requirement of peripheral TCR/ligand interaction. A. FACS analysis of epidermal cell preparations for the Tg Vγ2⁺ sIELs in adult G8 and KN6 mice of different ligand backgrounds. N≥4 per genotype. B. Immunofluorescent microscopy of ear epidermal sheets of TCRδ^−/−β2M^−/−B6 or TCRδ^−/−B6 mice adoptively transferred with positively selected fetal thymic Tg γδT cells of G8 (B6) mice for the Tg Vγ2⁺ sIELs. C. High percentages of apoptotic Tg Vγ2⁺ sIELs in KN6 mice of B6 background. The percentages of apoptotic sIELs are of total sIELs based on in situ TUNEL analyses of ear epidermal sheets (N=4 each). D. FACS analysis of epidermal cell preparations for Vγ3⁺ sIELs in FVB(Tac) mice transferred with FVB(NCI) fetal thymic γδT cells (top panels). Increased percentages of the epidermal Vγ3⁺ cells expressing the “sIEL-specific” 17D1-epitope on gated γδTCR⁺ populations confirmed the reconstitution (bottom) (9). The data is representative of at least 3 experiments.