Absence of an essential role for thymic stromal lymphopoietin receptor in murine B-cell development

Abstract

The murine cytokine thymic stromal lymphopoietin (TSLP) supports the development of B220+ IgM+ immature B cells and induces thymocyte proliferation in vitro. Human TSLP, by contrast, activates CD11c+ dendritic cells, but not B or T cells. Recent studies have demonstrated that the receptor for TSLP consists of a heterodimer of the interleukin 7 (IL-7) alpha chain and a novel protein that resembles the hematopoietic cytokine receptor common gamma chain. We examined signal transduction by the gamma-like chains using chimeric receptor proteins. The cytoplasmic domain of the human, but not of the murine, gamma-like chain, activates Jak2 and Stat5 and supports the proliferation of hematopoietic cell lines. In order to assess the role of the murine gamma-like chain in vivo, we generated gamma-like chain-deficient mice. Receptor-deficient mice are unresponsive to TSLP but exhibit no obvious phenotypic defects. In particular, hematopoietic cell development appeared normal. B-cell development, including the IgM+ compartment, was unaffected by loss of the TSLP pathway, as were T lymphopoiesis and lymphocyte proliferation in vitro. Cytokine receptors that utilize the common gamma chain signal through the lymphocyte-specific kinase Jak3. Mice deficient in Jak3 exhibit a SCID phenotype but harbor a residual B220+ splenic lymphocyte population. We demonstrate here that this residual lymphocyte population is lost in mice lacking both the gamma-like chain and Jak3.

FIG. 1.

Chimeric receptors. (A) The cytoplasmic domain of the human or murine γ-like chain was fused in frame to the extracellular and transmembrane portions of the murine EpoR (mEpoR) as indicated. The chimeric receptors contain 10 amino acids (aa) of the EpoR cytoplasmic domain. The box 1 and box 2 regions (black boxes) and the single cytoplasmic domain tyrosine (Y) are shown. The numbers are amino acid residue positions. TM, transmembrane domains. (B) Northern analysis of individual DA3 clones expressing either the EpoR-human γ-like chimeric receptor (EpoR/h γ-like) or the EpoR-murine γ-like chimeric receptor (EpoR/m γ-like). Expression levels of EpoR-human γ-like receptors were uniformly lower in all clones examined. rRNA loading controls are shown in the bottom blot. (C) Mass cultures of transfected DA3 or CTLL cells were cultured in medium without a cytokine (−) or in medium containing the indicated cytokine. The ability (+) or inability (−) to proliferate in long-term cultures is indicated.

FIG. 2.

Activation of Jak2 and Stat5 in transfected cell lines in response to Epo stimulation. DA3 and CTLL clones expressing the murine EpoR-human γ-like chimeric receptor (mEpoR/hγ-like) were used. Transfected cells were starved overnight and treated with the indicated stimuli or not treated (−). Lysates were prepared and subjected to immunoprecipitation (IP) with the anti-Jak2 antibody (αJak 2) and antibody against Stat5a and Stat5b (αSTAT 5A/B) shown. The activation of Jak2 (A) and Stat5 (B) was assessed by probing with antibodies to phosphotyrosine (αPTyr). Blots were stripped and reprobed with the indicated antibodies to assess protein levels.

FIG. 3.

γ-like chain targeting. (A) Structure of the γ-like chain targeting construct. The numbered black boxes represent exons 1 to 8. BamHI (B) sites are indicated, as is the location of the probe utilized in Southern analysis to distinguish wild-type (WT) and targeted alleles (black bar). The position and direction of the neomycin resistance cassette (Neo) and diphtheria toxin cassette (DTA) (30) are shown. (B) Southern blot of BamHI-digested genomic DNA from +/+ (wild type), +/− (heterozygote), and −/− (knockout [KO]) mice. Bands generated by the wild-type (WT), heterozygote (het), and targeted alleles are indicated. (C) Northern analysis of RNA isolated from +/+ (WT) or γ-like chain-deficient (KO) thymi. The blot was probed with a γ-like chain cDNA probe as indicated in Materials and Methods. The bottom blot shows the 28S and 18S RNAs as loading controls. (D) In vitro colony formation of bone marrow hematopoietic progenitors from wild-type (+/+) or γ-like chain-deficient (−/−) littermates. CFU-Mix, mixture of colonies.

FIG. 4.

Flow cytometric analysis of TSLP-R^+/+ (+/+) and TSLP-R^−/− (−/−) lymphocytes. Calculated percentages are displayed within each quadrant. Representative FACS analyses of at least six mice of each genotype are shown. (A) To assess B-lymphocyte development, bone marrow lymphocytes from wild-type and TSLP-R-deficient mice were stained with fluorochrome-conjugated antibodies as indicated. (B) Splenocytes from wild-type and TSLP-R-deficient mice were stained with anti-B220 and anti-CD3 antibodies (left). Alternatively, splenocytes were stained with anti-CD4 and anti-CD8 antibodies. The CD4/CD8 profile of splenic T cells is displayed (right). (C) Splenocytes from TSLP-R^+/+ (+/+) or TSLP-R^−/− (−/−) mice were stained with anti-IgD, anti-IgM, and anti-B220 antibodies. The IgM/IgD profile of splenocytes gated on B220⁺ cells is displayed. (D) Thymocytes from TSLP-R^+/+ or TSLP-R^−/− mice were stained with anti-CD4 and anti-CD8 antibodies. The CD4/CD8 profile of gated thymocytes is displayed.

FIG. 5.

Lymphocyte proliferation assays. (A) FACS-purified splenic B cells were cultured in the presence of the indicated stimuli (αIgM, anti-IgM; LPS, lipopolysaccharide) for 48 h. Cell proliferation was measured by adding 1 μCi of [³H]thymidine ([³H]Thy) to the cultures. The means ± standard deviations (error bars) for three samples are shown. The data are from one representative experiment of three experiments. (B) FACS-purified splenic T cells were cultured in the presence of the indicated stimuli for 48 h. The cells were stimulated with the indicated concentrations (in micrograms per milliliter) of anti-CD3 antibody or with phorbol myristate acetate and ionomycin (PMA + Ion). Cell proliferation was measured as in panel A.

FIG. 6.

Flow cytometric analysis of wild-type (WT), Jak3^−/−, and Jak3^−/− × TSLP-R^−/− lymphocytes. Calculated percentages are displayed within each quadrant. Representative FACS analyses of at least four mice of each genotype are shown. (A) To assess B-lymphocyte development, bone marrow lymphocytes from wild-type (WT), Jak3^−/−, and Jak3^−/− × TSLP-R^−/− mice were stained with fluorochrome-conjugated antibodies as indicated. (B) Splenocytes from wild-type (WT), Jak3^−/−, and Jak3^−/− × TSLP-R^−/− mice were stained with antibodies as indicated. Total numbers of splenocytes and percentages of gated lymphocytes for each sample are indicated below plots.