Inhibitor of DNA binding 3 limits development of murine slam-associated adaptor protein-dependent "innate" gammadelta T cells

Abstract

Background: Id3 is a dominant antagonist of E protein transcription factor activity that is induced by signals emanating from the alphabeta and gammadelta T cell receptor (TCR). Mice lacking Id3 were previously shown to have subtle defects in positive and negative selection of TCRalphabeta+ T lymphocytes. More recently, Id3(-/-) mice on a C57BL/6 background were shown to have a dramatic expansion of gammadelta T cells.

Methodology/principal findings: Here we report that mice lacking Id3 have reduced thymocyte numbers but increased production of gammadelta T cells that express a Vgamma1.1+Vdelta6.3+ receptor with restricted junctional diversity. These Vgamma1.1+Vdelta6.3+ T cells have multiple characteristics associated with "innate" lymphocytes such as natural killer T (NKT) cells including an activated phenotype, expression of the transcription factor PLZF, and rapid production of IFNg and interleukin-4. Moreover, like other "innate" lymphocyte populations, development of Id3(-/-) Vgamma1.1+Vdelta6.3+ T cells requires the signaling adapter protein SAP.

Conclusions: Our data provide novel insight into the requirements for development of Vgamma1.1+Vdelta6.3+ T cells and indicate a role for Id3 in repressing the response of "innate" gammadelta T cells to SAP-mediated expansion or survival.

Figure 1. Altered γδ T cell development in Id3^−/− mice.

A) FACS analysis for TCRβ and TCRγδ on Id3 ^+/+ and Id3 ^−/− thymocytes. The frequency of TCRβ⁺ cells and TCRγδ⁺ cells is shown. B) Number of TCRγδ⁺ cells in the thymus of Id3^+/+ and Id3^−/− mice. Average +/− standard deviation was determined from >15 mice. p<0.0005. C) CD4 and CD8 expression on TCRγδ⁺ cells. D) Number of CD4, CD8, DN and DP TCRγδ⁺ cells in the thymus Id3^+/+ (grey) and Id3^−/− (black) mice. Average +/− standard deviation was determined from >15 mice. p<0.0005 in all Id3 ^+/+ to Id3 ^−/− comparisons. E) Analysis of Id3^−/− CD8α⁺ thymocytes for TCRβ and CD8β (left panel) or TCRγ (right panel) and CD8β. The TCRβ⁺ cells express CD8β whereas TCRγδ⁺ cells are CD8β⁻ (right panel) and presumably CD8αα. F) QPCR for Sox13a mRNA in sorted Id3^+/+ (grey) and Id3^−/− (black) TCRγ⁺ cells and DP thymocytes (standardized to Hprt). Bars are the average from 3 experiments +/− standard deviation.

Figure 2. Id3^−/− γδ T cells have an activated phenotype.

FACS analysis of Id3 ^+/+ DN thymocytes (A) or Id3 ^−/− DN, CD4 and CD8 thymocytes (B) for expression of TCRγδ and CD122, CD44, CD24 or NK1.1. The analysis shows that Id3 ^−/− γδ T cells have markers of activation independent of expression of CD4 or CD8. Data are representative of >10 experiments. C) FACS analysis showing intracellular staining for IFNγ (open histogram) in Id3^+/+ and Id3^−/− TCRγδ⁺ thymocytes 5 hours after stimulation with PMA and ionomycin. Shaded histogram shows isotype control. One of 3 experiments is shown.

Figure 3. γδ T cells with an activated phenotype are present in Id3^−/− neonates.

A) γδ T cell numbers in Id3^+/+ and Id3^−/− mice analyzed 7 days after birth. Average +/− standard deviation is derived from 6 mice. p<0.0005. B) TCRβ and TCRγδ expression on total thymocytes from 7 day old mice. C) Gated TCRγδ⁺ cells were analyzed for CD4 and CD8. D) Expression of CD122, CD44 or CD24 on TCRγδ⁺ thymocytes from 7 day old Id3^+/+ (grey) and Id3^−/− (black line) littermates. E) TCRγδ and NK1.1 expression total thymocytes from the same pair of mice as in D.

Figure 4. Id3^−/− γδ T cells are highly enriched for cells with a Vγ1.1/Vδ6.3 TCR.

A) FACS analysis of thymocytes for TCRγδ and Vγ1.1 (top panel). The frequency of TCRγδ⁺Vγ1.1⁺ cells is shown. TCRγδ⁺Vγ1.1⁺ cells were analyzed for expression of Vδ6.3 (bottom panel). The frequency of Vγ6.3⁺ cells in the TCRγδ⁺Vγ1.1⁺ population is shown. B) Frequency of Vγ1.1⁺Vδ6.3⁺ cells among TCRγδ⁺ cells in Id3 ^+/+ and Id3 ^−/− mice. Data were calculated by dividing the frequency of Vγ1.1⁺Vδ6.3⁺ cells by the frequency of TCRγδ⁺ cells (×100). Each dot represents the frequency from on mouse. The line represents the average value. C) Analysis of thymocytes for expression of TCRγδ and Vγ2, Vγ5 or Vγ3 in Id3^+/+ and Id3^−/− thymocytes. One of 4 representative experiments is shown.

Figure 5. Sequence analysis of Vγ1.1-Jγ4 and Vδ6-Jδ1 junctions.

Sequence of Vγ1.1 and Jγ4 (A) or Vδ6 and Jδ1 (B) junctions from TCRγδ⁺ T cells (upper, indicated on the right) or DN3 thymocytes (lower, indicated on the right). Data are cumulative from 2 independent experiments in which DNA was isolated 50,000 TCRγδ⁺ cells and 30,000 Lin⁻c-kit ⁻CD25⁺ (DN3) cells. In each experiment 3 independent PCR amplifications were performed on each population and cloned into pBSK for sequencing. A minimum of 30 sequences were analyzed for each population. Results from the two experiments were essentially identical. Only in-frame sequences are shown and the number of clones sharing the identical sequence is indicated on the right in parenthesis. The amino acid sequence is shown in bold below the DNA sequence. The Vδ6.3 primer also amplifies the Vδ6B gene segment and sequences derived from Vδ6B are indicated on the right. P/N represent potential P or N additions and the underlined sequences are potential P additions. Sequences derived from the Dδ1 and Dδ2 gene segments are also indicated.

Figure 6. Id3^−/− Vγ1.1⁺Vδ6.3⁺ T cells express PLZF and require SAP.

A) Intracellular FACS analysis for expression of PLZF in Id3 ^+/+ and Id3 ^−/− thymic Vγ1.1⁺Vδ6.3⁺ (black) and Vγ1.1⁻Vδ6.3⁻ (grey) γδ T cells. (B) Number of TCRγδ⁺ cells in the thymus of mice of the indicated genotype. Sh2d1a ^−/− mice fail to express the gene encoding SAP. Average +/− standard deviation derived from 6 mice. Id3 ^−/− mice have more TCRγδ⁺ cells than any of the other genotypes (p<0.05) (C) FACS analysis for TCRβ and TCRγ expression on total thymocytes from mice of the indicated genotype. The frequency of TCRγδ⁺ cells is shown. (D) Analysis of TCRγδ⁺ cells for expression of Vγ1.1 and Vδ6.3. The frequency of Vγ1.1⁺Vδ6.3⁺ cells among TCRγδ⁺ cells is shown.

Figure 7. γδ T cell development in Id3^−/− mice is E2A dependent.

A) Total number of TCRγδ⁺ cells in the thymus of Id3^+/+, E2A^−/−, Id3^−/− and Id3^−/−E47^−/− mice. Bars represent the average +/− standard deviation. p<0.001 for Id3 ^−/− compared to all other genotypes. B) Flow cytometric analysis for TCRβ and TCRγδ in mice with the indicated genotype. The frequency of TCRγδ⁺ cells is shown. (C) Analysis of TCRγδ⁺ cells for CD122 (upper plots) and NK1.1 (lower plots) expression. Genotypes are as indicated in (C) (for the open histogram) and are shown relative to Id3 ^+/+ TCRγδ⁺ cells (shaded histogram), which served as a negative control. Results are representative from at least 6 mice of each genotype.