Id3 and Id2 act as a dual safety mechanism in regulating the development and population size of innate-like γδ T cells

Abstract

The innate-like T cells expressing Vγ1.1 and Vδ6.3 represent a unique T cell lineage sharing features with both the γδ T and the invariant NKT cells. The population size of Vγ1.1(+)Vδ6.3(+) T cells is tightly controlled and usually contributes to a very small proportion of thymic output, but the underlying mechanism remains enigmatic. Deletion of Id3, an inhibitor of E protein transcription factors, can induce an expansion of the Vγ1.1(+)Vδ6.3(+) T cell population. This phenotype is much stronger on the C57BL/6 background than on the 129/sv background. Using quantitative trait linkage analysis, we identified Id2, a homolog of Id3, to be the major modifier of Id3 in limiting Vγ1.1(+)Vδ6.3(+) T cell expansion. The Vγ1.1(+)Vδ6.3(+) phenotype is attributed to an intrinsic weakness of Id2 transcription from Id2 C57BL/6 allele, leading to an overall reduced dosage of Id proteins. However, complete removal of both Id2 and Id3 genes in developing T cells suppressed the expansion of Vγ1.1(+)Vδ6.3(+) T cells because of decreased proliferation and increased cell death. We showed that conditional knockout of Id2 alone is sufficient to promote a moderate expansion of γδ T cells. These regulatory effects of Id2 and Id3 on Vγ1.1(+)Vδ6.3(+) T cells are mediated by titration of E protein activity, because removing one or more copies of E protein genes can restore Vγ1.1(+)Vδ6.3(+) T cell expansion in Id2 and Id3 double conditional knockout mice. Our data indicated that Id2 and Id3 collaboratively control survival and expansion of the γδ lineage through modulating a proper threshold of E proteins.

Figure 1

A single locus on mouse chromosome 12 strongly influences the development of Vδ6.3⁺ γδ T cells in Id3-deficient mice. (A) Deficiency of Id3 induces the accumulation of a large population of Vδ6.3⁺ γδ T cells in the thymus of mice with pure B6 background. However, this phenomenon persists in some but is absent in other mice with a B6/129 mix background, as two representative mice are showing here. (B) A breeding scheme was established to dissect potential modifying gene(s) in the B6 and 129 genetic backgrounds. Only F2 B6/129 hybrid Id3^−/− mice were used in the linkage analysis. (C) Percentages of Vδ6.3⁺ γδ T cells among total thymocytes were scored for individual mice of indicated genotype group. Each dot represents one mouse. Horizontal line indicates mean of the genotype group. (D) SNP analysis of the B6/129 hybrid Id3^−/− mice showed that one location on chromosome 12 is strongly correlated with the presence of more than 1% of Vδ6.3⁺ γδ T cells in the thymus. (E) Detailed SNP analysis of four mice with more than 1% of Vδ6.3⁺ γδ T cells in the thymus showed a linkage to B6 homozygocity within a 3 Mb region on chromosome 12. (F) A map of known features around the critical region on chromosome 12; arrows indicate the border of the region (25.16Mb-28.02Mb) as determined in E. Note that Id2 and Kidins220 are immediately outside of the border of this region.

Figure 2

Id2 is a major modifier of Vδ6.3⁺ γδ T cell development in Id3-deficient mice. (A) QPCR analysis showed that Id2 mRNA expression in Vδ6.3⁺ γδ T cells from Id3^−/− mice with B6 background on the chromosome 12 region encompassing Id2 is lower than those with B6/129 mix background. n=3 for independent sorting of each genotype group. (B) Restriction enzyme analysis of the SNP marker rs29191636 within the exon 3 of the Id2 gene. EcoRI digestion of Id2 cDNA made from Vδ6.3⁺ γδ T cells with B6/129 mix background generated abundant product specific to the 129 allele (marked by two arrows), indicating that the higher Id2 expression in these cells came from that allele. Data representative of 3 experiments. (C) Replacement of one copy of the Id2 B6 allele with the Id2^f allele of 129 origin is sufficient to reduce the population size of Vδ6.3⁺ γδ T cells. Id2^B indicates the wild type Id2 allele in B6 background. n≥3 for each group. (D) Analysis of Vδ6.3⁺ γδ T cells with various combinations of Id2^B and Id2^f alleles on Id3^f/f CD4Cre⁺ background. Data representative of 3 mice in each group. (E) Percentage and number of Vδ6.3⁺ γδ T cell in the thymus of genotype each group shown in D. N≥4 in each group. All error bars indicate SD.

Figure 3

Conditional knockout of both Id2 and Id3 impairs the proliferation and survival of Vδ6.3⁺ γδ T cells. (A) In the neonatal thymus, Vδ6.3⁺ γδ T cells from Id3^f/f CD4Cre⁺ mice are more highly proliferative than the Cre⁻ controls as shown by BrdU incorporation assay, regardless of their Id2 genotype. However, cells from the Id2^f/f Id3^f/f CD4Cre⁺ mice show a small but significant decrease in BrdU⁺ cell percentage compared to those from Id2^B/BId3^f/fCD4Cre⁺ mice. n=3 for each group. (B) Vδ6.3⁺ γδ T cells were sorted from the thymus of neonatal mice and cultured for 24 hours. Id2^f/f Id3^f/f CD4Cre⁺ cells showed increased cell death by 7AAD and Annexin V staining compared to Id2^f/B Id3^f/f CD4Cre⁺ cells. n=3 in each group. (C) QPCR analysis of a panel of cell death-related genes showed that Id2^f/f Id3^f/f CD4Cre⁺ Vδ6.3⁺ γδ T cells express more mRNA of pro-apoptotic genes Bim and Bax. n=3 in each group. *p<0.05. All error bars indicate SD.

Figure 4

Conditional knockout of Id2 alone results in expansion of γδ T cells. (A) Examination of Id2 expression in developing γδ T cells in the thymus with an Id2^GFP reporter showed that Id2 is up-regulated at the mature stage. DN2: Lin⁻CD25⁺CD44⁺. DN3: Lin⁻CD25⁺CD44⁻. Immature: TCRγδ⁺CD24⁺CD44^low. Mature: TCRγδ⁺CD24⁻CD44^high. (B) Id2 expression is higher in the Vγ1.1⁺Vδ6.3⁺ cells than in other γδ T cells. (C) In vitro culturing of sorted Id2^GFP negative γδ T cells from the thymus for 5 days with IL-7 and anti-TCR γδ stimulation resulted in more significant up-regulation of Id2 compared to culturing with IL-7 alone. For A-C, data representative of 3 mice in each group. (D) Id2^f/f CD4Cre⁺ mice have more γδ T cells in the spleen compared to Id2^f/f CD4Cre⁻ mice. Bar graphs show the percentage and number of γδ T cells in the spleen of mice in each group. Each dot represents one mouse. (E) Id2^f/f CD4Cre⁺ mice have a higher percentage of CD24⁻CD44^high mature γδ T cells in the thymus. Pre-gated on TCRγδ⁺ cells. (F) Id2^f/f CD4Cre⁺ mice have a higher percentage of Vγ1.1⁺Vδ6.3⁺ γδ T cells in the thymus. Pre-gated on TCRγδ⁺ cells. (G) The mature thymic γδ T cells from Id2^f/f CD4Cre⁺ mice show decreased cell death by 7AAD and Annexin V staining. (H) The mature thymic γδ T cells from Id2^f/f CD4Cre⁺ mice show similar proliferation rate to Id2^f/f CD4Cre⁻ mice in BrdU incorporation assay. Data representative of 3 mice in each group in E and F.

Figure 5

The effect of Id proteins on Vδ6.3⁺ γδ T cells is mediated by E proteins and is developmental stage-specific. (A) Although conditional knockout of both Id2 and Id3 limits the accumulation of Vδ6.3⁺ γδ T cells in the thymus, further deletion of HEB and/or E2A can restore the accumulation of those cells. N≥3 for each group. (B) Deletion of all alleles of HEB, E2A, Id2 and Id3 by CD4Cre can induce accumulation of Vδ6.3⁺ γδ T cells, but deletion by LckCre fails to induce a similar phenotype. N≥3 for each group. All error bars indicate SD.

Figure 6

A schematic diagram of Vγ1.1⁺Vδ6.3⁺ γδ T cell developmental control by Id2 and Id3. In the developing thymus, γδ T cells that express the Vγ1.1 and Vδ6.3 TCR segments receive strong TCR signaling, up-regulating Id2 and Id3 through Egr1/2 and PLZF. The Id proteins inhibit activity of E proteins, affecting the survival and proliferation of Vγ1.1⁺Vδ6.3⁺ γδ T cells. When Id3 is present, and Id2 is expressed from a more active allele, such as the one from the 129 genetic background (Id2^s, “strong”), E protein activity is very low and Vγ1.1⁺Vδ6.3⁺ γδ T cell population size is small. If Id3 is absent, and Id2 is expressed from a less active allele, such as the one from the B6 background (Id2^B, “B6”), E protein activity becomes higher and the Vγ1.1⁺Vδ6.3⁺ γδ T cells expand dramatically. However, if both Id2 and Id3 are completely absent, E protein activity becomes too high and again impairs the survival and proliferation of Vγ1.1⁺Vδ6.3⁺ γδ T cells, limiting its population size.