T1/ST2-deficient mice demonstrate the importance of T1/ST2 in developing primary T helper cell type 2 responses

Abstract

We have generated mice with a deficiency in T1/ST2 expression to clarify the roles of T1/ST2 in T helper cell type 2 (Th2) responses. Using immunological challenges normally characterized by a Th2-like response, we have compared the responses of T1/ST2-deficient mice with those generated by wild-type mice. Using a primary pulmonary granuloma model, induced with Schistosoma mansoni eggs, we demonstrate that granuloma formation, characterized by eosinophil infiltration, is abrogated in T1/ST2-deficient mice. Furthermore, we clearly demonstrate that in the absence of T1/ST2 expression, the levels of Th2 cytokine production are severely impaired after immunization. Thus, in a secondary pulmonary granuloma model, draining lymph node cells from the T1/ST2-deficient animals produced significantly reduced levels of IL-4 and IL-5, despite developing granulomas of a magnitude similar to those of wild-type mice and comparable antigen-specific immunoglobulin isotype production. These data clearly demonstrate that T1/ST2 expression plays a role in the development of Th2-like cytokine responses and indicate that effector functions are inhibited in its absence.

Figure 1

Inactivation of the T1/ST2 gene by homologous recombination. (A) The structure of the T1/ST2 locus, the targeting vector, and the predicted homologous recombination event are shown. Targeted disruption results in the deletion of the majority of exons 4 and 5. Neo^R, neomycin resistance cassette; TK, thymidine kinase cassette; B, BamHI; X, XbaI; N, NotI. Intron–exon boundaries are inferred from the rat gene sequence 16. (B) Southern blot of F₂ tail genomic DNA. The indicated probe detects a 5.7-kb BamHI fragment in the wild-type T1/ST2 gene and a 2.7-kb fragment as a result of the correct homologous recombination event. (C) Analysis of T1/ST2 expression by mast cells. Mast cells were cultured from bone marrow in the presence of IL-3 and stem cell factor. Biotinylated anti-T1/ST2 (DJ8) was used to stain for T1/ST2 expression.

Figure 2

Analysis of naive T helper cell differentiation. Cytokine production after in vitro Th cell differentiation. Mesenteric lymph node cells or splenocytes from three wild-type or three IL-13^−/− mice were stimulated under Th1 or Th2 culture conditions for 5 d, washed, and restimulated on anti-CD3 antibody–coated plates for 24 h. Supernatants were analyzed by ELISA. Data are representative of three repeat experiments.

Figure 3

Total serum Ig isotype production from naive wild-type (□) and T1/ST2^−/− (•) mice. Represented data points indicate individual animals.

Figure 4

Analysis of primary pulmonary inflammatory response. (A) Determination of granuloma volumes in immunized mice. Cohorts of four to five mice were injected intravenously with 5,000 schistosome eggs to induce synchronous pulmonary granuloma. Mice were killed 11 d later. Lung sections were stained with hematoxylin and eosin, and at least 100 individual granulomas were measured per group. Data are presented as means less egg volumes plus SD. (B) Morphological analysis of granuloma formation in wild-type (WT) and T1/ST2^−/− (KO) mice. Lung sections were stained with hematoxylin and eosin. Magnification, 63. Data are representative of two repeat experiments.

Figure 5

Analysis of presensitized pulmonary inflammatory response. Cohorts of four to five mice were sensitized by intraperitoneal injection of 5,000 S. mansoni eggs. After 11 d, these mice were injected intravenously with 5,000 eggs to induce synchronous pulmonary granuloma. Mice were killed 11 d later. (A) Cytokine responses to pulmonary challenge. Cytokine responses from activated lymph node cells. Draining mediastinal lymph node cells were stimulated with soluble egg antigen or anti-CD3 antibody, and supernatants were assayed for cytokines by ELISA. Data are presented as means plus SD. Open bars, wild-types; filled bars, ST2^−/−. (B) Determination of granuloma volumes in sensitized mice. Lung sections were stained with hematoxylin and eosin, and at least 100 individual granulomas were measured per group. Data are presented as means less egg volumes plus SD and are representative of two repeat experiments. WT, wild-type; KO, knockout.

Figure 6

Antigen-specific Ig response to immunization. Cohorts of four to five animals were immunized intraperitoneally with schistosome eggs, followed by secondary administration of eggs intravenously. □, wild-type; ▪, T1/ST2^−/−. Serum samples were assayed 11 d after the secondary antigen challenge by ELISA for Ig isotypes. Representative data from two repeat experiments are shown.