ZBTB1 is a determinant of lymphoid development

Abstract

In this study, we describe a chemically induced mouse mutation that caused a complete and cell-intrinsic T cell deficiency. Development of other lymphoid lineages was also partially impaired and was severely compromised under competitive conditions. Positional cloning, retroviral transduction, and a somatic reversion event revealed that the causative mutation lay within Zbtb1 (zinc finger and BTB domain containing 1), a gene conserved throughout vertebrate evolution. Our data establish ZBTB1 as a critical determinant of T cell development and lymphopoiesis in general, most likely by acting as a transcriptional regulator.

Figure 1.

T cell aplasia in scanT mice. (A) Initial generations of the scanT pedigree. Black symbols, T-deficient phenotype; gray symbols, wild-type phenotype; open symbols, not tested. (B) Percentages of B (CD19⁺) and T (CD3ε⁺) cells in the peripheral blood of 8-wk-old mice as measured by flow cytometry. (C–F) Relative size (C), histological appearance (H&E; D), and cellular composition (E and F) of the thymus of wild-type and mutant siblings at 8 wk of age. Lineage markers in E and F were CD11b, CD3ε, B220, Ter119, Ly6G, NK1.1, and CD8α. Subsets in F were gated as follows: ETP (Lin⁻CD44⁺CD25⁻CD117⁺), DN2 (Lin⁻CD44⁺CD25⁺), DN3 (Lin⁻CD44⁻CD25⁺), DN4 (Lin⁻CD44⁻CD25⁻), DP (CD4⁺CD8α⁺), CD4SP (CD4⁺CD8α⁻), and CD8SP (CD4⁻CD8α⁺). Data are representative of one (A and D), two to three (E and F), or more than three (B and C) independent experiments. Error bars represent standard error, and symbols in F represent individual mice. Numbers in parentheses in E represent mean thymic cellularity and standard error. Bars, 100 µm.

Figure 2.

Identification of a missense mutation in ZBTB1. (A and B) Chromosomal mapping (A) and fine mapping (B) of the scanT phenotype. LOD, logarithm of odds score. (C) DNA sequence chromatograms of the mutated nucleotide in Zbtb1 (thymine to cytosine), resulting in the substitution of arginine for cysteine at codon 74. (D) Predicted ZBTB1 protein domain structure. (E) Conservation of ZBTB1 sequence across multiple vertebrates, with the amino acid corresponding to ZBTB1^C74 highlighted in red. ZBTB1 counterparts were not found in nonvertebrate proteomes. (F) Schematic of the MIG-Zbtb1 and control retroviral constructs. MIG, MSCV-IRES-GFP; LTR, long terminal repeat. (G) Bone marrow cells from scanT mice or heterozygous controls (CD45.2⁺) were transduced with a retroviral construct expressing Zbtb1 and transferred into irradiated CD45.1⁺ recipients (∼2 × 10⁶ cells/mouse). Transduced CD3ε⁺ cells were identified in blood 4 mo after transfer by coexpression of GFP. (H) Phenotypic reversion associated with somatic mutation of Zbtb1. DNA was prepared from tail clippings and flow-sorted CD19⁺ and CD3ε⁺ lymph node cells and sequenced at the locus mutated in the scanT pedigree. Contour plots were obtained from blood. Data in G are representative of three independent experiments. (C and H) Yellow highlighting indicates the position of the nucleotide mutated in the scanT pedigree..

Figure 3.

scanT mice have generalized deficiencies of lymphoid but not myeloid cells. (A) Total numbers of major lymphoid and myeloid cell subsets in the spleen of 8-wk-old sex-matched littermates, gated as follows: T (CD3ε⁺), B (CD19⁺), NK (NK1.1⁺CD3ε⁻), plasmacytoid DCs (pDC; Lin⁻PDCA1⁺SiglecH⁺), CD8α⁻ DCs (Lin⁻CD11c⁺CD11b⁺CD8α⁻), CD8α⁺ DCs (Lin⁻CD11c⁺CD11b⁻CD8α⁺), macrophages (Lin⁻CD11b⁺F4/80⁺SSC^lo), neutrophils (Lin⁻CD11b⁺F4/80⁻Ly6G^hi), and eosinophils (Lin⁻CD11b⁺F4/80⁺SSC^hi). Lineage markers included CD19, TCR-β, NK1.1, and the viability dye 7-AAD. (B–D) Frequencies (B and C) and numbers (D) of major B cell subsets in bone marrow and spleen. Subsets in bone marrow were gated as follows: A (CD11b⁻CD3e⁻Ter119⁻Ly6G⁻NK1.1⁻IgM⁻B220⁺CD19⁻CD24⁻BP-1⁻), B (B220⁺IgM⁻CD43⁺CD24⁺BP-1⁻), C (B220⁺IgM⁻CD43⁺CD24⁺BP-1⁺), D (B220⁺CD43⁻IgM⁻IgD⁻), E (B220⁺CD43⁻IgM⁺IgD⁻), and F (B220⁺CD43⁻IgM⁺IgD⁺). Subsets in spleen were gated as follows: T1 (B220⁺CD93⁺CD23⁻), T2 (B220⁺CD93⁺CD23⁺), Fo (follicular; B220⁺CD93⁻CD23⁺CD21/35^int), and MZ (marginal zone; B220⁺CD93⁻CD23⁻CD21/35^hi). (E) NP (4-hydroxy-3-nitrophenylacetyl)-specific antibodies in the serum of mice immunized with NP-Ficoll or NP-CGG at 7 or 14 d (NP-Ficoll) or 14 d (NP-CGG) after immunization, presented as absorbance at 450 nm (A₄₅₀). (A–E) Data are representative of one (E) or two (A–D) independent experiments with four or more mice per group. Error bars represent standard error, and symbols represent individual mice.

Figure 4.

A cell-intrinsic T cell deficiency and competitive failure of lymphoid reconstitution. (A and B) Lethally irradiated Rag1 mutant mice (CD45.2⁺) were reconstituted with unmixed scanT or wild-type bone marrow (CD45.2⁺; A) or a mixture of scanT or wild-type marrow (CD45.2⁺) with wild-type bone marrow (CD45.1⁺; B). Chimerism was measured 8 wk after transplant. Panels in B have been gated on the indicated cell subset. (C) Lethally irradiated wild-type recipients (CD45.1⁺) were transplanted with an equal mixture of wild-type (CD45.1⁺) and heterozygous or homozygous mutant (CD45.2⁺) bone marrow. Wild-type donor chimerism (CD45.1⁺) was measured 8 wk later in the spleen, bone marrow, and thymus. Subsets were gated as in Fig. 3, with the addition of NK progenitors (NKP; 7-AAD⁻CD19⁻TCR-β⁻CD122⁺NK1.1⁻). (A–C) Data are representative of one (A) or three (B and C) independent experiments with at least three mice per group. Error bars represent standard error.

Figure 5.

Normal development and function of hematopoietic progenitors. (A and B) Frequencies (A) and numbers (B) or hematopoietic progenitors in heterozygous and homozygous Zbtb1 mutant bone marrow. LT-HSC (long-term HSC; CD135⁻CD34⁻ or CD150⁺CD48⁻), ST-HSC (short-term HSC; CD135⁻CD34⁺ or CD150⁺CD48⁺), MPP (multipotent progenitors; CD135⁺CD34⁺ or CD150⁻CD48⁺), CMP (common myeloid progenitors; CD16/32⁻CD34⁺), GMP (granulocyte/monocyte progenitors; CD16/32⁺CD34⁺), MEP (megakaryocyte/erythroid progenitors; CD16/32⁻CD34⁻), and CLP (CD117^intSca-1^int) were gated as indicated. (C) Chimerism of a subset of hematopoietic progenitors outlined in A, 8 wk after reconstitution of lethally irradiated CD45.1⁺ recipients with an equal mixture of CD45.1⁺ and Zbtb1^+/scanT (CD45.2⁺) or CD45.1⁺ and Zbtb1^scanT/scanT (CD45.2⁺) bone marrow cells. (D) Incorporation of the thymidine analogue EdU 4 h after injection. (E) Spleen colony forming units (CFU-S) counted 8 or 12 d after transfer of 10⁵ bone marrow cells (or media alone) into lethally irradiated recipients. (F) Engraftment of 2 × 10⁶ CD45.1⁺ bone marrow cells in unconditioned recipients 8 wk after injection. Lineage markers in A–D were CD11b, CD3ε, B220, Ter119, Ly6G, NK1.1, and CD8α. (A–F) Data are representative of one (D and F), two (A–C), and three (E) independent experiments with at least three mice per group. Error bars represent standard error, and symbols in D and E represent individual mice.