A critical role for Rictor in T lymphopoiesis

Abstract

Apart from a critical role for Notch and pre-TCR, the signaling pathway required for T lymphopoiesis is largely unknown. Given the potential link between Notch and mammalian target of rapamycin (mTOR) signaling in cancer cells, we used mice with conditional deletion of either Raptor or Rictor genes to determine potential contribution of the mTOR complex I and II in T lymphopoiesis. Our data demonstrated that targeted mutation of Rictor in the thymocytes drastically reduced the thymic cellularity, primarily by reducing proliferation of the immature thymocytes. Rictor deficiency caused a partial block of thymocyte development at the double-negative 3 stage. The effect of Rictor deficiency is selective for the T cell lineage, as the development of B cells, erythrocytes, and myeloid cells is largely unaffected. Analysis of bone marrow chimera generated from a mixture of wild-type and Rictor-deficient hematopoietic stem cells demonstrated that the function of Rictor is cell intrinsic. These data revealed a critical function of mTOR complex 2 in T lymphopoiesis.

Figure 1

Efficient deletion of Rictor in thymocytes. (A) Relative copy number of the floxed Rictor exon 11. BM cells and thymocytes were prepared from Ctrl mice and cKO mice on Day 20 after the final injection of pIpC. The deletion of exon11 in genomic DNA was assayed by real-time PCR, and the non-deleted exon 12 served as an internal control. N=5 for Ctrl mice; N=5 for cKO mice. (B) Deletion of Rictor exon11 in subsets of DN thymocytes. Genomic DNA isolated from sorted DN subsets was amplified by PCR. (C) Representative FACS profile for intracellular staining of pAkt 473 in CD3-DN thymocytes. (D) pAKT levels in DN thymocyte subsets as measured by phosphor-flow. Data shown are the relative mean fluorescence intensity (MFI) values from cKO mice compared with that from Ctrl mice. N=5 for Ctrl mice; N=3 for cKO mice. Similar data were obtained from another independent experiment.

Figure 2

Defective T-lymphopoiesis induced by targeted mutation of Rictor. (A) Significant reduction of thymic cellularity upon Rictor deletion. The weight (left) and absolute numbers (right) per thymus are shown. (B) Representative FACS profiles showing thymocyte subsets based on surface markers CD4, CD8, CD3, CD25 and CD44. (C) Percentages (left) and absolute numbers (right) of indicated thymocyte subpopulations in Ctrl and cKO mice. (D) Percentages (left) and absolute numbers (right) of DN1-DN4 subsets from Ctrl mice and cKO mice. N=8 for Ctrl mice, N=5 for cKO mice for data in (A), (C) and (D). These data have been reproduced in three independent experiments.

Figure 3

Rictor deletion reduced DN thymocytes proliferation. (A) Representative profiles of BrdU incorporation in DN thymocytes at 4 hours after BrdU pulse. (B) Percentages of BrdU⁺ cells among DN1-DN4 subsets. N=3 for Ctrl mice; N=3 for cKO mice. (C) Representative staining profiles for cell death in DN thymocytes. (D) Percentages of Annexin V⁺ DN thymocytes. N=5 for Ctrl mice; N=5 for cKO mice. Data shown have been reproduced in 2 independent experiments.

Figure 4

Rictor-deficiency reduces cell sizes of immature thymocytes. Panels A and C show representative histograms of forward scatters of area (FSC-A) of thymocytes. Thymocytes are divided into DN, DP and SP subsets (A) or DN1–4 subsets (C). Panels B and D show means and S.D, of relative cell sizes, involving 7 control and 5 cKO mice. The mean FSC-A of control mice is artificially defined as 1.0. These data have been reproduced in 5 independent experiments.

Figure 5

Abnormal T-lymphopoiesis induced by Rictor deletion was BM cell-intrinsic. (A) Diagram of experimental design. BM cells from either Ctrl or cKO donors were transplanted into lethally irradiated CD45.1 recipients (10⁶ cells/mouse). pIpC treatment commenced at 6 weeks after transplantation and lasted for two weeks. At 3 weeks after pIpC treatment, thymus was analyzed by flow cytometry. (B) Reduction of thymic cellularity in the recipients of Rictor cKO BM cells. The weight (left) and absolute numbers (right) of per thymus are shown. (C) Representative FACS profiles (upper panels) and summary data (lower panels) for T cell subsets of Rictor Ctrl and cKO recipients. (D) % and absolute number of DN subsets. N=4 for recipient mice of Ctrl donor; N=4 for recipient mice of cKO donor.

Figure 6

Cell-intrinsic requirement of Rictor for T-lymphopoiesis. A. Diagram of experimental design. BM cells were isolated from Ctrl or cKO mice at 20 days after pIpC treatment. They were mixed with equal number of recipient type BM cells and injected into lethally irradiated host (CD45.1⁺). B. Representative profiles of donor-type (CD45.2⁺) and recipient-type (CD45.1⁺) cells in the thymus of recipient mice at 12 weeks after BMT are shown on the left, while summary of percentages for donor-derived cells in recipient thymus are presented on the right. C. Donor-derived thymocyte subpopulations based on expression of CD4 and CD8 markers. Data shown are representative profiles. D. Percentages (left) and absolute numbers (right) of thymocyte subpopulations were shown. Data shown are representative of two independent similar bone marrow chimera studies.

Figure 7

A–C. Donor-derived DN subsets based on expression of CD25 and CD44 markers. The representative profiles (A), percentages (B) and absolute numbers (C) of DN subsets were shown. D. Proliferation rate of DN thymocytes, based on incorporation of BrdU at 4 hours after BrdU injection. N=3 for Ctrl recipients (Ctrl+CD45.1); N=4 for cKO recipients (cKO+CD45.1). Data shown are representative of two independent similar bone marrow chimera studies.

Figure 8

Targeted mutation of Rictor did not affect bone marrow cellularity (A) stem/progenitor cells (B) and development of B cells (C), erythrocytes and myeloid cells (D) in BM. LSK: Lin⁻Sca-1⁺c-Kit⁺; MP, Lin⁻Sca-1⁻c-Kit⁺, myeloid progenitor cells; MPP: Lin⁻Sca-1⁺c-Kit⁺ CD150⁻CD48⁺; multi-potent progenitors; LT-HSC: Lin⁻Sca-1⁺c-Kit⁺ CD150⁺CD48⁻, long-term hematopoietic stem cells; ST-HSC, Lin⁻Sca-1⁺c-Kit⁺ CD150⁺CD48⁺, short-term hematopoietic stem cells. The data have been reproduced in 3 independent experiments,N=7 for Ctrl, and N=6 for cKO.