Molecular cloning and biological characterization of a novel murine lymphoid growth factor

Abstract

Using a bioassay consisting of the proliferation of a murine B cell line, a cDNA of a gene whose product supports the growth of that cell line was isolated from a thymic stromal cell line. This factor, termed thymic stromal lymphopoietin (TSLP), is a protein of 140 amino acids. The gene encoding TSLP was mapped to murine chromosome 18. Purified recombinant TSLP supported the growth of pre-B cell colonies in vitro, but had no myelopoietic activity. TSLP had comitogenic activity for fetal thymocytes, but was not as potent as interleukin 7 in lobe submersion cultures. Injection of TSLP into neonatal mice induced the expansion of B220(+)BP-1(+) pre-B cells.

Figure 1

The cDNA and predicted amino acid sequence of murine TSLP. (A) The sequence given is a composite of three clones (see Materials and Methods). The signal peptide is in italics; amino acid numbering begins with the NH₂ terminus of the mature protein (Tyr). Potential sites of N-linked carbohydrate addition are underlined. Cysteines are double underlined. Although there is no stretch of polyA at the 3′ end of our clones, it is likely that the AATAAA sequence at nucleotides 1111–1116 represents a polyadenylation signal, since the clone containing it was isolated from an oligo-dT–primed cDNA library. TSLP sequence data are available from EMBL/GenBank/DDBJ under accession no. AF232937. (B) Schematic representation of cysteine residues and potential glycosylation sites.

Figure 1

The cDNA and predicted amino acid sequence of murine TSLP. (A) The sequence given is a composite of three clones (see Materials and Methods). The signal peptide is in italics; amino acid numbering begins with the NH₂ terminus of the mature protein (Tyr). Potential sites of N-linked carbohydrate addition are underlined. Cysteines are double underlined. Although there is no stretch of polyA at the 3′ end of our clones, it is likely that the AATAAA sequence at nucleotides 1111–1116 represents a polyadenylation signal, since the clone containing it was isolated from an oligo-dT–primed cDNA library. TSLP sequence data are available from EMBL/GenBank/DDBJ under accession no. AF232937. (B) Schematic representation of cysteine residues and potential glycosylation sites.

Figure 2

Analysis of TSLP mRNA expression in various tissues. (A) Northern analysis of indicated tissue or cell line polyA⁺ mRNA with an antisense TSLP probe. (B) RT-PCR of TSLP mRNA expression in various tissues: (a and b) TSLP RT-PCR reaction products from indicated samples of cDNA amplified for 35 or 40 cycles, respectively; (c) normalization of cDNA sample amount using HPRT PCR primers and an HPRT competitor; (d) PCR of mock-transcribed samples. BM, bone marrow; L. node, lymph node; Skel., skeletal.

Figure 3

Tslp maps in the proximal region of mouse chromosome 18. Tslp was placed on mouse chromosome 18 by interspecific backcross analysis. The segregation patterns of Tslp and flanking genes in 148 backcross animals that were typed for all loci shown at the top of the figure. For individual pairs of loci, >148 animals were typed. Each column represents the chromosome identified in the backcross progeny that was inherited from the (C57BL/6J × M. spretus)F₁ parent. The black boxes represent the presence of a C57BL/6J allele and the white boxes represent the presence of the M. spretus allele. The number of offspring inheriting each type of chromosome is listed at the bottom of each column. A partial chromosome 18 linkage map showing the location of Tslp in relation to linked genes is shown at the bottom of the figure. The number of recombinant animals is presented over the total number of animals typed to the left of the chromosome map between each pair of loci. Recombination frequencies expressed as genetic distance in cM (±1 SE) The positions of loci in human chromosomes, where known, are shown to the right. References for the human map positions of loci cited in this study can be obtained from Genome Data Base (www.gdb.org), a computerized database of human linkage information maintained by The William H. Welch Medical Library of The Johns Hopkins University (Baltimore, MD).

Figure 4

Cell surface phenotype of bone marrow colonies grown in TSLP. Whole bone marrow cells were cultured for 7 d in methylcellulose plus TSLP (20 ng/ml). Colonies were plucked from the semisolid media, pooled, and disrupted. Cells were stained with the appropriate reagents as indicated and analyzed via flow cytometry.

Figure 5

Analysis of bone marrow pre-B cells from mice treated with TSLP or IL-7. Neonatal C57BL/6 mice were injected with MSA, TSLP, or IL-7 for 10 d as described above (see Materials and Methods). 1 d after cessation of treatment, bone marrow was harvested, stained, and analyzed. (A) BP-1 and B220 expression on sIgM⁻sIgD⁻ bone marrow cells. (B) CD43 (S7) and HSA (M1/69) expression of IgM⁻B220⁺ bone marrow cells.