Shared dependence on the DNA-binding factor TOX for the development of lymphoid tissue-inducer cell and NK cell lineages

Abstract

TOX is a DNA-binding factor required for development of CD4(+) T cells, natural killer T cells and regulatory T cells. Here we document that both natural killer (NK) cell development and lymphoid tissue organogenesis were also inhibited in the absence of TOX. We found that the development of lymphoid tissue-inducer cells, a rare subset of specialized cells that has an integral role in lymphoid tissue organogenesis, required TOX. Tox was upregulated considerably in immature NK cells in the bone marrow, consistent with the loss of mature NK cells in the absence of this nuclear protein. Thus, many cell lineages of the immune system share a TOX-dependent step for development.

Figure 1

Absence of lymph nodes in Tox^−/− mice. (a) Analysis of the gross anatomy of lymph nodes visualized by Chicago sky blue dye injection in Tox^+/− and Tox^−/− mice. Lymph nodes have never been observed in >100 Tox^−/− mice ranging from 4–12 weeks of age (analyzed in the absence of dye). (b,c) Quantification of Peyer’s patches by visual inspection was performed in adult control (wild-type or Tox^+/−) and Tox^−/− mice. Each symbol represents an individual mouse, and a Peyer’s patch from a representative mouse of each genotype is shown in the photograph; horizontal bars are the mean. *P = 1.4×10⁻⁸ (d) Hematoxylin and eosin staining of frozen sections of small intestine that included a Peyer’s patch from Tox^+/− and Tox^−/− mice. Magnification, 25X. (e) Immunofluorescence staining of Peyer’s patches in Tox^+/− and Tox^−/− mice. B cell and T cell areas are identified by B220 (green) and CD3 (red) staining. Magnification, 20X and 63X. Data (d,e) is representative of at least three animals of each genotype.

Figure 2

Defect in formation of lymph node structures in Tox^−/− mice. (a) Representative plots displaying donor Tox^+/− (n=5) and Tox^−/− (n=6) B (CD19⁺) and T (TCRβ⁺) cells in lymph nodes of reconstituted Tox^+/+ recipient mice. Frequencies refer to percentages of gated populations. The frequency of CD8⁺ T cells is also shown. (b) Analysis of Peyer’s patches as in (a) except that the frequency of CD4⁺ T cells is shown. (c) Presence of CD45.1⁺ donor wild-type cells in the spleen of chimeric Tox^−/− (n=2) or Tox^+/− (n=2) mice, four weeks post adoptive transfer.

Figure 3

TOX is required for the development of lymphoid tissue inducer (LTi) cells. (a) Representative analysis of LTi cells in neonatal (day 0) spleen. Frequencies refer to percentages of gated populations. (b,c) Quantification of the frequency (n=10 and 8 for Tox^+/− and Tox^−/−, respectively, *P = 3.2×10⁻⁵) and absolute cell numbers (n=3 and 4 for Tox^+/− and Tox^−/−, respectively, *P = 0.002) of LTi cells from neonatal spleen. (d) Analysis of fetal LTi cells (day E18) from small intestines. Frequencies refer to percentages of gated populations. (e) Quantification of the frequency of LTi cells from fetal (day E18) small intestines of Tox^+/− (n=6) and Tox^−/− (n=7) mice. *P = 0.012 (f,g,h) Quantitative RT-PCR gene expression analysis in Tox^+/+ LTi cells (CD45⁺CD3⁻B220⁻CD11c⁻ CD4⁺CD127⁺) purified from Rag1^−/− spleens compared to wild-type splenic B cells. Two independent experiments are shown. *<0.01 (i,j) Quantitative RT-PCR gene expression analysis in wild-type LTi cells, identified as in (f), purified from embryonic day E17 spleens and compared to wild-type adult splenic B cells. Two independent experiments using three to four pooled mice per experiment are shown.

Figure 4

Impaired development of NK cells in the absence of TOX. (a) Analysis of bone marrow NK cell populations. IL-2Rβ (CD122) or NK1.1 and DX5 expression is shown on Lin⁻ (CD3⁻CD4⁻CD8⁻CD19⁻Gr1⁻Ter119⁻) cells. Frequencies refer to percentages of gated populations. Data is representative of 6 mice for each genotype. (b) Absolute cell numbers of bone marrow NK cell subsets: NK progenitors (NKp), Lin⁻ CD122⁺NK1.1⁻ DX5⁻; immature NK (iNK), Lin⁻ CD122⁺NK1.1⁺DX5⁻; mature NK (mNK), Lin⁻ CD122⁺NK1.1⁺DX5⁺ subpopulations (*P = 0.012). Each symbol represents an individual mouse; horizontal lines indicate the mean. (c) Analysis of CD122 and DX5 expression on Lin⁻ splenic cells. Data is representative of five mice. (d) Absolute cell numbers of splenic NK cells. *P = 3.3×10⁻³ (e) Tox expression in isolated Tox^+/+ bone marrow NK cell subpopulations, Tox^+/+ splenic NK cells, and B cells. Two independent experiments are shown. (f) Expression of Id2 in bone marrow mNK cells. Two biological replicates are shown for Tox^−/− cells. (g) In vivo NK cell killing assay. Each symbol represents an individual mouse; horizontal lines indicate the mean. *P = 1.8×10⁻⁴.

Figure 5

Defect in NK cell development in the absence of TOX is cell-intrinsic. (a) Tox^+/− and Tox^−/− bone marrow LSK cells were purified and cultured in vitro to generate NK cells. Shown is staining of the LSK cell population at the initiation of culture and at day 12 (gated for Lin⁻ cells, except for NK lineage markers). Data is representative of four experiments. (b) Expression of CD122 on Lin⁻ Tox^+/− (red) and Tox^−/− (blue) enriched bone marrow progenitor cells cultured for six days and compared to Lin⁻ Tox^+/− cells at day 0 (black). Data is representative of four experiments. (c) Expansion of allelically marked 1:1 mixtures of Tox^+/+ and Tox^−/− LSK cells was assessed by cell counts and flow cytometry after six days of culture (n=2).

Figure 6

Expression of Tox but not Id2 can rescue the defect in NK cell development. (a) Retroviral gene transduction was used to express TOX, or the H-2K^b molecule as a control, in developing bone marrow progenitor Tox^+/− and Tox^−/− cells in NK-promoting cultures. Expression of NK1.1 versus Lin is shown on GFP⁺ gated cells. Data is representative of four experiments. (b) Retroviral gene transduction was used to express TOX, Id2, or the K^b molecule, in developing bone marrow progenitor cells in NK-promoting cultures. Expression of NK1.1 versus DX5 is shown on Lin⁻GFP⁺ gated cells. Frequencies of the gated populations are shown, and the frequency of the gated population within the total GFP⁺ subset is indicated in parentheses. Data is representative of four experiments. (c) Expression of CD19 versus GFP is shown on Tox^+/− and Tox^−/− cultured cells expressing H-2K^b or Id2. Data is representative of four experiments.