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. 2006 Sep;26(17):6403-11.
doi: 10.1128/MCB.00755-06.

Generation and characterization of B7-H4/B7S1/B7x-deficient mice

Woong-Kyung Suh  1 , Seng Wang, Gordon S Duncan, Yoshiyuki Miyazaki, Elizabeth Cates, Tina Walker, Beata U Gajewska, Elissa Deenick, Wojciech Dawicki, Hitoshi Okada, Andrew Wakeham, Annick Itie, Tania H Watts, Pamela S Ohashi, Manel Jordana, Hiroki Yoshida, Tak W Mak
Affiliations

Generation and characterization of B7-H4/B7S1/B7x-deficient mice

Woong-Kyung Suh et al. Mol Cell Biol. 2006 Sep.

Abstract

Members of the B7 family of cosignaling molecules regulate T-cell proliferation and effector functions by engaging cognate receptors on T cells. In vitro and in vivo blockade experiments indicated that B7-H4 (also known as B7S1 or B7x) inhibits proliferation, cytokine production, and cytotoxicity of T cells. B7-H4 binds to an unknown receptor(s) that is expressed on activated T cells. However, whether B7-H4 plays nonredundant immune regulatory roles in vivo has not been tested. We generated B7-H4-deficient mice to investigate the roles of B7-H4 during various immune reactions. Consistent with its inhibitory function in vitro, B7-H4-deficient mice mounted mildly augmented T-helper 1 (Th1) responses and displayed slightly lowered parasite burdens upon Leishmania major infection compared to the wild-type mice. However, the lack of B7-H4 did not affect hypersensitive inflammatory responses in the airway or skin that are induced by either Th1 or Th2 cells. Likewise, B7-H4-deficient mice developed normal cytotoxic T-lymphocyte reactions against viral infection. Thus, B7-H4 plays a negative regulatory role in vivo but the impact of B7-H4 deficiency is minimal. These results suggest that B7-H4 is one of multiple negative cosignaling molecules that collectively provide a fine-tuning mechanism for T-cell-mediated immune responses.

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Figures

FIG. 1.
FIG. 1.
Generation of B7-H4 KO mice. (A) Strategy for B7-h4 gene targeting. We designed a targeting vector to replace all of B7-h4 exon (Ex) 3 (encoding the IgV domain) and one-third of exon 4 (encoding the IgC domain) with a neomycin resistance gene cassette. We prepared two homology arms by PCR as described in Materials and Methods. The location of the Southern blot probe is shown by an underline, and those of RT-PCR primers are shown by arrowheads. R, EcoRI; S, SalI; N, NotI; DT-A, diphtheria toxin A subunit gene cassette; neor, neomycin resistance gene cassette; Mut, mutant. (B) Southern blot analysis. Genomic DNAs from WT, heterozygous (Het), and KO mouse tails were digested with EcoRI and analyzed by Southern blot analysis with the probe depicted in panel A. (C) Lack of B7-H4 mRNA in cells derived from B7-H4 KO mice. First-strand cDNAs were prepared from the total RNA extracted from peritoneal macrophages and splenic B cells of WT, B7-H4 heterozygous (Het), and B7-H4 KO (KO) mice. B7-H4 cDNA was amplified by PCR along with glyceraldehyde-3-phosphate dehydrogenase cDNA as a positive control as described in Materials and Methods.
FIG. 2.
FIG. 2.
Normal T-cell proliferation in the absence of B7-H4. Single-cell suspensions of total LN cells were stimulated with soluble anti-CD3 (145-2C11; 0.1 or 1 μg/ml; BD Pharmingen) or concanavalin A (ConA; 1 or 2 μg/ml; Sigma) in U-bottom 96-well plates (1 × 105 cells/well) in triplicate. The cells were pulsed with [3H]thymidine (1 μCi/well) for the last 8 h of a 24- or 48-h incubation period. The data shown are the mean ± the standard deviation from two WT and two B7-H4 KO mice and are representative of four independent experiments.
FIG. 3.
FIG. 3.
Enhanced Th1 responses against L. major in B7-H4 KO mice. (A) Reduced footpad swelling in B7-H4 KO mice. BALB/c or B7-H4 KO mice in a BALB/c background (n = 6) were inoculated in the right hind footpad with L. major promastigotes, and footpad swelling was measured at the indicated time points. Two mice of each genotype died at day 21 and thus were excluded from the experiment. The data shown are the mean ± the standard deviation of footpad swelling and are representative of two independent experiments. *, P < 0.05; **, P < 0.01 (Student's t test). (B) Reduced parasite burden in B7-H4 KO mice. The number of L. major promastigotes in the footpad was assessed as described in Materials and Methods at day 14 postinfection. The data shown are the mean ± the standard deviation and are representative of two independent experiments with three mice per genotype. *, P < 0.05 by Student's t test. (C) Elevated IFN-γ production by T cells from B7-H4 KO mice. Popliteal LN cells were isolated at day 14 postinfection and then restimulated with or without antigen (Ag) for 3 days. The concentrations of IFN-γ and IL-4 in the culture supernatants were measured by ELISA. The data shown are means ± standard deviations and are representative of two independent experiments with three mice per genotype. **, P < 0.01 by Student's t test compared to data from the WT control at the same dose of antigen. (D) Increased T-bet expression in CD4+ T cells of B7-H4 KO mice. CD4 T cells were purified from popliteal LNs at day 14 postinfection, and the level of T-bet mRNA was analyzed by RT-PCR as described in Materials and Methods. The data shown are representative of three experiments with three mice per genotype.
FIG. 4.
FIG. 4.
Unaltered hypersensitivity reactions in B7-H4 KO mice. (A) Airway inflammation. B7-H4 KO and WT littermate mice in a BALB/c background (backcrossed for five generations) were repeatedly exposed to aerosolized ovalbumin under Th1- or Th2-polarizing conditions as described in Materials and Methods. Immune cells in BAL fluid were enumerated after differential staining. The data shown are means ± standard deviations from a combination of two independent experiments (n = 7 WT and 10 B7-H4 KO mice for Th1 and 7 WT and 9 B7-H4 KO mice for Th2). (B) Contact hypersensitivity reaction. WT and B7-H4 KO mice were sensitized by oxazolone on the abdominal skin and then challenged on the ear skin 5 days later. The data shown represent mean percentages ± standard deviations over the initial ear thickness obtained from seven mice of each genotype. (C) LCMV-mediated footpad swelling. WT and B7-H4 KO mice were injected with LCMV in the hind footpad, and the inflammatory reaction was monitored by measuring footpad thickness. The data shown represent the mean footpad thickness of four mice of each genotype ± the standard deviation.
FIG. 5.
FIG. 5.
Normal antiviral CTL responses in B7-H4 KO mice. (A, left side) WT or B7-H4 KO mice (n = 2) were infected with LCMV. Splenocytes were prepared at day 8 postinfection, and ex vivo cytotoxicity was measured as described in Materials and Methods. (A, right side) WT or B7-H4 KO mice (n = 2) were infected with LCMV. Thirty days later, splenocytes were prepared and restimulated for 5 days in vitro by the addition of antigenic peptide p33. Threefold serial dilutions of the restimulated splenocytes were used as effector cells for cytotoxicity assays as described in Materials and Methods. (B, left side) Expansion of NP-specific anti-influenza virus CD8 T cells in the spleen measured 7 days after primary or secondary influenza virus infection as described in Materials and Methods. The results shown represent the average percentages of tetramer+ T cells over the total CD8 T cells in the spleen from a combination of two independent experiments (n = 10 WT and 12 B7-H4 KO mice for primary infection and 7 WT and 10 B7-H4 KO mice for secondary infection). The error bars represent standard deviations. (B, right side) Cytotoxic activities in the spleens of WT or B7-H4 KO mice 7 days after a secondary influenza virus infection measured as described in Materials and Methods. The data shown represent average specific lysis ± the standard deviation (n = four WT and seven B7-H4 KO mice).
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