Separation of Notch1 promoted lineage commitment and expansion/transformation in developing T cells

Abstract

Notch1 signaling is required for T cell development. We have previously demonstrated that expression of a dominant active Notch1 (ICN1) transgene in hematopoietic stem cells (HSCs) leads to thymic-independent development of CD4(+)CD8(+) double-positive (DP) T cells in the bone marrow (BM). To understand the function of Notch1 in early stages of T cell development, we assessed the ability of ICN1 to induce extrathymic T lineage commitment in BM progenitors from mice that varied in their capacity to form a functional pre-T cell receptor (TCR). Whereas mice repopulated with ICN1 transduced HSCs from either recombinase deficient (Rag-2(-/)-) or Src homology 2 domain--containing leukocyte protein of 76 kD (SLP-76)(-/)- mice failed to develop DP BM cells, recipients of ICN1-transduced Rag-2(-/)- progenitors contained two novel BM cell populations indicative of pre-DP T cell development. These novel BM populations are characterized by their expression of CD3 epsilon and pre-T alpha mRNA and the surface proteins CD44 and CD25. In contrast, complementation of Rag-2(-/)- mice with a TCR beta transgene restored ICN1-induced DP development in the BM within 3 wk after BM transfer (BMT). At later time points, this population selectively and consistently gave rise to T cell leukemia. These findings demonstrate that Notch signaling directs T lineage commitment from multipotent progenitor cells; however, both expansion and leukemic transformation of this population are dependent on T cell-specific signals associated with development of DP thymocytes.

Figure 1

Notch1-induced extrathymic T cells resemble normal DP thymocytes. (A) Flow cytometric analysis for CD4 and CD8 expression on normal thymocytes versus BM cells from a recipient of ICN1-transduced wild-type progenitor cells at 21 d after BMT. Also shown is TCRβ expression (B), forward scatter (C), and DNA content (D). The TCRβ expression (B) and forward scatter (C) results are based on DP GFP⁺ cells as shown in panel A. Because GFP is lost after cell permeabilization, the analysis in D used ungated BM cells; however, 95% of the cells in the BM at the time of analysis were DP GFP⁺. Data are representative of three recipients.

Figure 2

Notch1 induction of extrathymic DP T cells requires pre-TCR assembly and signaling. Progenitor cells from wild-type, Rag-2^−/−, Rag-2^−/− × TCRβ, and SLP-76 mice were transduced with MigR1 control or MigR1-ICN1 viruses and then transferred to lethally irradiated C57BL/6 recipients. BM cells were stained 20–30 d after transfer with the indicated antibodies and analyzed by flow cytometry. GFP gates were set as described in Materials and Methods. The CD4⁺ and CD8⁺ SP thymocytes present in all GFP⁻ populations are recipient derived, as shown by prior congenic analysis (data not shown). Data are representative of 3–12 recipients for each donor genotype.

Figure 4

Notch1 induction of T cell leukemia requires a functional TCRβ chain. Progenitor cells from wild-type, Rag-2^−/−, and Rag-2^−/− × TCRβ (DO11.10) donors were transduced with MigR1 or MigR1-ICN1 before transfer to irradiated adoptive hosts. (A) Leukemia onset in mice reconstituted with retrovirally transduced BM cells. Leukemia onset is defined as the time after BMT when CD4⁺CD8⁺ DP GFP⁺ T cells appear in the peripheral circulation, and/or the WBC exceeds 50 × 10⁶ cells/ml. Mice typically survived for ∼1–2 mo after the onset of leukemia, during which time the fraction and number of DP GFP⁺ cells continued to increase (not shown). None of the mice reconstituted with the Rag-2^−/− receiving Mig ICN or any of the mice receiving the empty MigR1 virus developed circulating DP GFP⁺ cells or any evidence of neoplasia. Each dot on the Mig ICN Rag-2^−/− × TCRβ (DO11.10) curve represents the analysis of two mice. Although this figure shows leukemia-free survival to 100 d, these mice remained leukemia-free for the duration of this study (see Table ) (B). Western blot analysis of Notch1 expression in BM cells of recipients of ICN1-transduced progenitors from Rag-2^−/− and Rag-2^−/− × TCRβ (DO11.10) donors.

Figure 4

Notch1 induction of T cell leukemia requires a functional TCRβ chain. Progenitor cells from wild-type, Rag-2^−/−, and Rag-2^−/− × TCRβ (DO11.10) donors were transduced with MigR1 or MigR1-ICN1 before transfer to irradiated adoptive hosts. (A) Leukemia onset in mice reconstituted with retrovirally transduced BM cells. Leukemia onset is defined as the time after BMT when CD4⁺CD8⁺ DP GFP⁺ T cells appear in the peripheral circulation, and/or the WBC exceeds 50 × 10⁶ cells/ml. Mice typically survived for ∼1–2 mo after the onset of leukemia, during which time the fraction and number of DP GFP⁺ cells continued to increase (not shown). None of the mice reconstituted with the Rag-2^−/− receiving Mig ICN or any of the mice receiving the empty MigR1 virus developed circulating DP GFP⁺ cells or any evidence of neoplasia. Each dot on the Mig ICN Rag-2^−/− × TCRβ (DO11.10) curve represents the analysis of two mice. Although this figure shows leukemia-free survival to 100 d, these mice remained leukemia-free for the duration of this study (see Table ) (B). Western blot analysis of Notch1 expression in BM cells of recipients of ICN1-transduced progenitors from Rag-2^−/− and Rag-2^−/− × TCRβ (DO11.10) donors.

Figure 3

Notch1 signaling induces extrathymic T cell commitment. Progenitor cells from Rag-2^−/− donors were transduced with MigR1 or MigR1-ICN1 then analyzed 30 d after transfer. (A) Flow cytometric analysis for CD44 and CD25 expression on BM cells from recipients. Data are representative of six recipients of ICN1-transduced Rag-2^−/− progenitors. (B) RT-PCR analysis for CD3ε and pre-Tα mRNA was performed as described in Materials and Methods on sorted populations using the indicated gates. In the analysis of pre-Tα expression cDNA from the SCID-derived early T cell line SCID.ADH (reference 18) was included as a positive control (lane marked “+”), and from Hardy Fraction D (B220⁺CD43⁻IgM⁻ pre-B cells) as a negative control (lane marked “Fr. D”). Thy III contains cDNA from purified CD25⁺CD44⁻CD3⁻ thymocytes.