The microRNA biogenesis machinery modulates lineage commitment during αβ T cell development

Abstract

Differentiation of CD4(+) helper and CD8(+) cytotoxic αβ T cells from CD4(+)CD8(+) thymocytes involves upregulation of lineage-specifying transcription factors and transcriptional silencing of CD8 or CD4 coreceptors, respectively, in MHC class II or I (MHCII or I)-restricted thymocytes. In this study, we demonstrate that inactivation of the Dicer RNA endonuclease in murine thymocytes impairs initiation of Cd4 and Cd8 silencing, leading to development of positively selected MHCI- and MHCII-restricted mature CD4(+)CD8(+) thymocytes. Expression of the antiapoptotic BCL2 protein or inactivation of the p53 proapoptotic protein rescues these thymocytes from apoptosis, increasing their frequency and permitting accumulation of CD4(+)CD8(+) αβ T cells in the periphery. Dicer-deficient MHCI-restricted αβ T cells fail to normally silence Cd4 and display impaired induction of the CD8 lineage-specifying transcription factor Runx3, whereas Dicer-deficient MHCII-restricted αβ T cells show impaired Cd8 silencing and impaired induction of the CD4 lineage-specifying transcription factor Thpok. Finally, we show that the Drosha RNA endonuclease, which functions upstream of Dicer in microRNA biogenesis, also regulates Cd4 and Cd8 silencing. Our data demonstrate a previously dismissed function for the microRNA biogenesis machinery in regulating expression of lineage-specifying transcription factors and silencing of Cd4 and Cd8 during αβ T cell differentiation.

Figure 1. Generation of aberrant peripheral CD4⁺CD8⁺ cells in Dicer deficient mice expressing a BCL2 transgene or lacking Trp53

A, Representative CD4 and CD8 staining on CD24^loTCRβ⁺ splenocytes of WTBCL2LD, or LBD mice. B, Average percentages of CD4⁺CD8⁺, and CD4⁺CD8⁺ cells amongst CD24^loTCRβ⁺ splenocytes of WTBCL2LD, or LBD mice. C, Total numbers of CD4⁺CD8⁺CD24^loTCRβ⁺ splenocytes in mice of the indicated genotypes. D and E, Frequency of CD4⁺CD8⁺ cells among TCRβ⁺ cells in lymph nodes (D) or blood (E) of LBD or control mice. F, Representative CD4 and CD8 staining on CD24^loTCRβ⁺ splenocytes of WT or LckCre:EμBCL2 mice. G, Representative CD4 and CD8 staining on CD24^loTCRβ⁺ splenocytes of WT or LPD mice. H, Average frequency of CD4⁺CD8⁺ cells amongst CD24^loTCRβ⁺ splenocytes of WT or LPD mice. B, C, D, E, and H, The total numbers of mice analyzed are indicated. B, C, D, E, F, and H, each experiment was performed at least 3 independent times.

Figure 2. Impaired initiation of co-receptor silencing in mature thymocytes of Dicer deficient mice expressing a BCL2 transgene or lacking Trp53

A, Representative CD4 and CD8 staining on CD24^loTCRβ^hi mature thymocytes of WTBCL2LD, or LBD mice. B, Average percentages of CD4⁺CD8⁺and CD4⁺CD8⁺ cells amongst CD24^loTCRβ^hi mature thymocytes of WTBCL2LD, or LBD mice. C, Total numbers of CD4⁺CD8⁺CD24^loTCRβ^hi mature thymocytes in mice of the indicated genotypes. D, Representative CD4 and CD8 staining on CD24^loTCRβ^hi mature thymocytes of WT or LPD mice. E, Average frequency of CD4⁺CD8⁺ cells amongst CD24^loTCRβ⁺ mature thymocytes of WT or LPD mice. B, C, and E, the numbers of mice analyzed are indicated; each experiment was performed at least 3 independent times.

Figure 3. Normal initiation of Cd4 and Cd8 silencing in thymocytes and T cells with Dicer deletion initiating in DP thymocytes

A and C, Representative CD4 and CD8 staining on mature thymocytes (A) or CD24^loTCRβ⁺ splenocytes (C) of WT or CBD mice. B and D, Average percentages of CD4⁺CD8⁺and CD4⁺CD8⁺ cells amongst mature thymocytes (B) or CD24^loTCRβ⁺ splenocytes (D) of WTBCL2CD, and CBD mice. B and D, the numbers of mice analyzed are indicated. Each experiment was performed at least 3 independent times.

Figure 4. Dicer is required for appropriate initiation of Cd4 silencing in MHCI-restricted cells and Cd8 silencing in MHCII-restricted cells

A-D, Representative CD4 and CD8 staining on mature thymocytes (A) or CD24^loTCRβ⁺ splenocytes (C) of MHCI^−/− mice reconstituted with BCL2 or LBD bone marrow. Average frequencies of CD4⁺CD8⁺ cells amongst mature thymocytes (B) or CD24^loTCRβ⁺ splenocytes (D) of MHCI^−/− mice reconstituted with BCL2 or LBD bone marrow. E-H, Representative CD4 and CD8 staining on mature thymocytes (E) or CD24^loTCRβ⁺ splenocytes (G) of MHCII^−/− mice reconstituted with BCL2 or LBD bone marrow. Average frequencies of CD4⁺CD8⁺ cells among mature thymocytes (F) or CD24^loTCRβ⁺ splenocytes (H) of MHCII^−/− mice reconstituted with BCL2 or LBD bone marrow. B, D, F and H, The numbers of mice analyzed are shown. The experiment was performed twice with at least 4 recipient mice per group; a representative experiment is shown.

Figure 5. Dicer regulates Cd4 and Cd8 silencing and expression of Runx3 and Zbtb7b in positively-selected αβ T cells

A, Representative CD4 and CD8 staining on CD24^loTCRβ^hi mature thymocytes of R1 OT-I and LBD R1 OT-I mice. B, Average percentages of CD4⁺CD8⁺, and CD4⁺CD8⁺ cells amongst CD24^loTCRβ^hi mature thymocytes of R1 OT-I and LBD R1 OT-I mice. C, Representative CD4 and CD8 staining on CD24^loTCRβ⁺ splenocytes of BD R1 OT-I and LBD R1 OT-I mice. D, Average percentages of CD4⁺CD8⁺, and CD4⁺CD8⁺ cells amongst CD24^loTCRβ⁺ splenocytes of R1 OT-I and LBD R1 OT-I mice. E, Representative CD4 and CD8 staining on CD24^loTCRβ^hi mature thymocytes of R1 OT-II and LBD R1 OT-II mice. F, Average percentages of CD4⁺CD8⁺, and CD4⁺CD8⁺ cells amongst CD24^loTCRβ^hi mature thymocytes of R1 OT-II and LBD R1 OT-II mice. G, Representative CD4 and CD8 staining on CD24^loTCRβ⁺ splenocytes of BD R1 OT-II and LBD R1 OT-II mice. H, Average percentages of CD4⁺CD8⁺, and CD4⁺CD8⁺ cells amongst CD24^loTCRβ⁺ splenocytes of R1 OT-II and LBD R1 OT-II mice. I, qRT-PCR for primary (un-spliced) Cd4 transcripts in sorted splenic populations from WTR1 OT-I, or LBD R1 OT-I mice. J, Runx3 Western blot in sorted splenic CD4⁺ or CD8⁺ cells from WT mice and CD8⁺ or CD4⁺CD8⁺ DP) cells from LBD R1 OT-I. Three independent replicates were performed; a representative blot is shown. K, Zbtb7b qRT-PCR in sorted populations from R1 OT-II or LBD R1 OT-II mice. B, D, F, H, I, and K, The numbers of mice analyzed are indicated. At least 3 independent experiments were performed in each case.

Figure 6. Drosha is required for appropriate initiation of Cd4 and Cd8 silencing after positive selection

Representative CD4 and CD8 staining on CD24^loTCRβ^hi mature thymocytes (A) or TCRβ⁺ splenocytes (C) of WT or LBDr mice. Average percentages of CD4⁺CD8⁺, and CD4⁺CD8⁺ cells among mature thymocytes (B) or TCRβ⁺ splenocytes (D) of WT and LBDr mice. B and D, The numbers of mice analyzed are indicated; each experiment was performed at least 3 independent times.