Foxp1 is an essential transcriptional regulator for the generation of quiescent naive T cells during thymocyte development

Abstract

Proper thymocyte development is required to establish T-cell central tolerance and to generate naive T cells, both of which are essential for T-cell homeostasis and a functional immune system. Here we demonstrate that the loss of transcription factor Foxp1 results in the abnormal development of T cells. Instead of generating naive T cells, Foxp1-deficient single-positive thymocytes acquire an activated phenotype prematurely in the thymus and lead to the generation of peripheral CD4(+) T and CD8(+) T cells that exhibit an activated phenotype and increased apoptosis and readily produce cytokines upon T-cell receptor engagement. These results identify Foxp1 as an essential transcriptional regulator for thymocyte development and the generation of quiescent naive T cells.

Figure 1

Generation of the conditional Foxp1 allele. (A) Diagram of the targeting vector containing Foxp1 exons 11 to 14. (B) Southern blot analysis to identify embryonic cell lines carrying the targeted Foxp1 locus. XbaI-digested (left) and SpeI-digested (right) genomic DNA from embryonic stem cells, hybridized to the 2 probes in the short and long arms, respectively. (C) PCR analysis of heterozygous mice confirms germline transmission. Shown is a PCR result indicating the cointegration of the 3′ downstream loxP site (370 bp) and wild-type allele product (280 bp) using primers 1 and 2.

Figure 2

Peripheral Foxp1-deficient T cells have reduced cell numbers and an activated phenotype. (A) Total cell numbers of CD4⁺ T and CD8⁺ T cells in the lymph nodes and spleens of Foxp1^f/+Cd4^Cre and Foxp1^f/fCd4^Cre mice at different ages. Data represent average ± SD, weeks 4 to 5: n = 5, weeks 8-10: n = 7, and weeks 21-22: n = 5. *P < .05; **P < .01; ***P < .001. (B) Expression of CD44 or CD62L in splenic CD4⁺ T or CD8⁺ T cells from Foxp1^f/+Cd4^Cre and Foxp1^f/fCd4^Cre mice at different ages. Data are representative of at least 3 independent experiments. The histogram with solid gray represents Foxp1^f/+Cd4^Cre mice, and the histogram with dark line represents Foxp1^f/fCd4^Cre mice.

Figure 3

Peripheral Foxp1-deficient T cells respond like activated T cells and have increased apoptosis. (A) Total spleen cells from 4-week-old Foxp1^f/+Cd4^Cre and Foxp1^f/fCd4^Cre mice were stimulated with anti-CD3 plus anti-CD28 antibodies or PMA plus ionomycin for 4 hours and analyzed for IL-2 and IFN-γ production by intracellular staining. The data are gated on CD4⁺ T or CD8⁺ T cells, and are representative of various time points (weeks 4, 8, and 21) in at least 3 independent experiments. (B) Purified CD4⁺ T or CD8⁺ T cells from 4-week-old Foxp1^f/+Cd4^Cre and Foxp1^f/fCd4^Cre mice were labeled with CFSE and stimulated with 1 μg/mL anti-CD3 antibodies for 2 days. Cell proliferation was analyzed by measuring the CFSE profile. Data are representative of 2 independent experiments. (C) Thymic and lymph node cells from 8-week-old Foxp1^f/+Cd4^Cre and Foxp1^f/fCd4^Cre mice were analyzed for apoptosis by annexin V and PI staining. The bars represent the percentages of annexin V⁺ PI⁻ cells in gated CD4⁺ or CD8⁺ T cells. Data represent average ± SD of the data of 8-week-old mice, n = 4. *P < .05; **P < .01.

Figure 4

Foxp1-deficient Treg cells develop normally and are functional. (A) Staining of cell-surface CD4 and CD25 and intracellular Foxp3 in the thymuses, lymph nodes, and spleens of 10-week-old Foxp1^f/+Cd4^Cre and Foxp1^f/fCd4^Cre mice. The percentages of CD25⁺Foxp3⁺ cells in CD4⁺ T cells were shown. Data are representative of 2 independent experiments. (B) Total cell numbers of Treg cells in the thymuses, lymph nodes, and spleens of Foxp1^f/+Cd4^Cre and Foxp1^f/fCd4^Cre mice at different ages. Bars represent average ± SD, weeks 4-5: n = 5, weeks 8-10: n = 7, and weeks 21-22: n = 5. (C) In Treg cell suppression cocultures, CFSE-labeled control CD4⁺ T cells were incubated with various ratios of Treg cells from Foxp1^f/+Cd4^Cre or Foxp1^f/fCd4^Cre mice. The IFN-γ production by control CD4⁺ T cells was analyzed by intracellular staining. Data are representative of 2 independent experiments.

Figure 5

Foxp1-deficient CD4 SP and CD8 SP thymocytes have an activated phenotype. (A) Thymic CD4 and CD8 staining profile of 4-week-old Foxp1^f/+Cd4^Cre and Foxp1^f/fCd4^Cre mice. Data are representative of various time points (weeks 4, 5, 8, 10, 21, 22) in at least 4 independent experiments. (B) Total cell numbers of DP, CD4 SP, and CD8 SP thymocytes in Foxp1^f/+Cd4^Cre and Foxp1^f/fCd4^Cre mice at different ages. Data represent average ± SD, weeks 4-5: n = 7, weeks 8-10: n = 7, and weeks 21-22: n = 5. (C) Expression of TCR_β, CD3, CD5, CD44, CD62L, HSA, and Qa-2 in DP, CD4 SP, and CD8 SP thymocytes of 4- to 5-week-old Foxp1^f/+Cd4^Cre and Foxp1^f/fCd4^Cre mice. Data are representative of at least 2 independent experiments.

Figure 6

Foxp1-deficient T cells acquire the activated phenotype during the differentiation from DP stage to SP stage. (A) Diagram of intrathymic adoptive transfer. (B) CD44 expression of donor thymocytes 3 days after the intrathymic transfer by staining. Data are representative of various time points (days 3, 5, and 6) in at least 2 independent experiments. The histogram with solid gray represents Foxp1^f/+Cd4^Cre mice, the histogram with dark line represents Foxp1^f/fCd4^Cre mice. (C) Foxp1-deficient CD8 SP thymocytes readily produce IFN-γ ex vivo. FACS-sorted CD8 SP thymocytes from 6-week-old Foxp1^f/+Cd4^Cre and Foxp1^f/fCd4^Cre mice were stimulated with PMA and ionomycin for 4 hours and analyzed for IFN-γ production by intracellular staining. Data are representative of 2 independent experiments.

Figure 7

Foxp1-deficient T cells acquire an activated phenotype by a cell-autonomous mechanism. At 6 to 10 weeks after mixed bone marrow reconstitution and gating on congenic markers, the donor thymocytes and peripheral T cells in mixed chimeras were analyzed for (A) CD4 and CD8 profile; (B) expression of CD44 and CD62L; and (C) percentages of Foxp1^+/+Cd4^Cre and Foxp1^f/fCd4^Cre T cells recovered in the thymuses, spleens, and lymph nodes of the mixed chimeras. The thymic data are normalized to the ratios of Foxp1^+/+Cd4^Cre and Foxp1^f/fCd4^Cre B220⁺ cells in the bone marrow; the peripheral T-cell data are normalized to thymic T cells. Bars represent average ± SD. n = 10. **P < .01. (D) IL-2 and IFN-γ production in splenic CD4⁺ T and CD8⁺ T cells by intracellular staining after PMA plus ionomycin stimulation for 4 hours. (E) Cell size. All data are representative of 2 independent experiments.