Targeted disruption of the interferon-gamma receptor 2 gene results in severe immune defects in mice

Abstract

To study the role of the interferon- (IFN) gammaR2 chain in IFN-gamma signaling and immune function, IFN-gammaR2-deficient mice have been generated and characterized. Cells derived from IFN-gammaR2 -/- mice are unable to activate either JAK/STAT signaling proteins or gene transcription in response to IFN-gamma. The lack of IFN-gamma responsiveness alters IFN-gamma-induced Ig class switching by B cells from these mice. In vitro cultures of T cells demonstrate that the T cells from the IFN-gammaR2 -/- mice have a defect in Th1 cell differentiation. The IFN-gammaR2 (-/-) mice also produce lower amounts of IFN-gamma in response to antigenic challenge. In addition, IFN-gammaR2 -/- mice are defective in contact hypersensitivity and are highly susceptible to infection by Listeria monocytogenes. These results demonstrate that the IFN-gammaR2 is essential for IFN-gamma-mediated immune responses in vivo.

Figure 1

Gene-targeted mutagenesis of IFNGR2 gene. (A) Schematic drawing of the IFNGR2 gene genomic locus. The abbreviation for restriction enzymes are X: XbaI, E: EcoRI, B: BamHI, S: SmaI. TK is the cassette of herpes thymidine kinase driven by PGK promoter. Probe A was generated by PCR with primers flanking exon 3 of the IFNGR2 gene. Probe B is the PCR-amplified fragment which represents exon 1 of the IFNGR1 gene. (B) The detection of the targeted IFNGR2 gene genomic locus by Southern blot analysis. Genomic DNA generated from mouse tails were digested with BamHI. The Southern blot was hybridized to probe A described above.

Figure 2

The IFN-γR2 chain disruption results in defective JAK-STAT activation by IFN-γ. (A) IFN-γR2 is required for activation of Jak1 and Jak2 by IFN-γ. Embryonic fibroblasts from both wild-type mice (lanes 1 and 2) and IFN-γR2 −/− mice (lanes 3 and 4) were cultured with IFN-γ (lanes 2 and 4) for 10 min. The extracts from these cells were immunoprecipitated with anti-Jak1 or anti-Jak2 antisera and blotted with either the 4G10 antibody and anti-Jak1 or anti-Jak2 antisera. (B) IFN-γR2 is required for Stat1 activation by IFN-γ. Splenocytes from wild-type mice (lanes 1 and 2) and IFN-γR2 −/− mice (lanes 3 and 4) were either cultured without (lanes 1 and 3) or with IFN-γ (lanes 2 and 4) for 15 min. (C) Total splenocytes were isolated from mice and cultured with IFN-γ for 2 hr. The total RNA was analyzed by Northern blotting with specific IRF-1, GBP-1, and glyceraldehyde-3-phosphate dehydrogenase probes.

Figure 3

Defect in class switching in B cells isolated from IFN-γR2 −/− mice. IgM⁺ B cells were isolated from total splenocytes and then cultured for 6 days in either LPS, or LPS plus IL-4, or LPS plus IFN-γ, or LPS plus IL-4 plus IFN-γ. The culture supernatants were then collected and assayed for IgG2a (A), IgE (B), and IgG1 (C) production by ELISA.

Figure 4

Altered Th1 responses in IFN-γR2 −/− mice. +/+, wild-type mice. −/−, IFN-γR2-deficient mice. (A) IFN-γ production of in vitro differentiated CD4 T cells. Naive (Mel14⁺) CD4⁺ T cells were cultured for 7 days in anti-CD3 antibody-coated wells with IL-2 (bar set 1), or IL-2, IL-4, and anti-IFN-γ antibody (bar set 2); or IL-2, IFN-γ, and anti-IL-4 antibody (bar set 3); or IL-12 and anti-IL-4 antibody (bar set 4). The results represent the average of three independent experiments. (B) IL-4 production of the in vitro differentiated CD4⁺ T cell cultures described above in A. The results represent the average of three independent experiments. (C and D) Antigen-specific T cell cytokine production in local LNCs isolated from KLH-immunized mice. Both wild-type mice (bars 1 and 2) and IFN-γR2 −/− mice (bars 3 and 4) were immunized with KLH in complete Freund’s adjuvant. The LNCs were isolated on day 10 and cultured with (bars 2 and 4) or without KLH (at 100 μg/ml) for 2 days. The supernatants were then assayed for IFN-γ production (C). The duplicated wells of cells were pulsed with ³H on day 2 for 8 hr and proliferation was assayed on day 3 (D). The results represent the average of four independent experiments.

Figure 5

Impaired cellular immunity of IFN-γR2 −/− mice. (A) Defective contact hypersensitivity in IFN-γR2 −/− mice. Five 6-wk-old female wild-type mice and five 6-wk-old IFN-γR2 −/− mice were used in this assay. The assay was done as described in Materials and Methods. Contact hypersensitivity is expressed in the change of ear thickness (T). T = (ear thickness 24 hr after elicitation) − (baseline ear thickness). The t test was used in comparison of T in IFN-γR2 +/+ and IFN-γR2 −/− mice. (B) IFN-γR2 −/− mice were susceptible to L. monocytogenes infection. Six-week-old female mice from each genetic background were used. For each genotype, five mice were injected i.p. with 1 × 10⁴ live L. monocytogenes. Percentage of survival represents the percentage of live mice each day.