CD40 Ligand-Dependent T Cell Activation: Requirement of B7-CD28 Signaling Through CD40

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Science  27 Sep 1996:
Vol. 273, Issue 5283, pp. 1862-1864
DOI: 10.1126/science.273.5283.1862


  • Fig. 1.

    T helper functions. (A) Splenocytes (6 × 106) harvested from mice 10 days after infection were cultured with (stimulated) or without (unstimulated) antigen (that is, lacZ virus) in 24-well plates for 48 hours. C57BL/6 mice were left untreated or treated with a mixture of antibodies to B7.1 and B7.2. CD40L-deficient (CD40L−/−) mice were left untreated or treated with antibody to CD40 (anti-CD40). Culture supernatants were assayed for the production of IL-2, IL-4, IL-10, and IFN-γ (picograms per milliliter). (B) Splenocytes from the following mice—normal C57BL/6 (filled circles), CD40L−/− (open circles), CD40L−/− treated with anti-CD40 (filled triangles), and C57BL/6 (B6) mice treated with anti-B7.1 and anti-B7.2 (open squares) 10 days after administration of virus—were restimulated in vitro for 5 days and tested for specific lysis on mock-infected (left) or virus-infected (right) C57SV cells in a 6-hour 51Cr release assay.

  • Fig. 2.

    The formation of antiviral antibody. Serum samples (diluted 1:200) from normal (C57BL/6) mice, CD40L−/− mice either untreated or treated with anti-CD40, and C57BL/6 mice treated with anti-B7.1 and anti-B7.2 were harvested 24 days after infection and analyzed for the presence of adenovirus-specific IgM, IgG1, and IgG2a by isotype-specific ELISA (15). Absorbance was measured at a wavelength of 405 nm (A405) on a Bio-Rad model 450-microplate reader.

  • Fig. 3.

    Regulation of B7.2 expression. Single-cell suspensions of spleen were prepared from immunized (day 10) or naïve mice. Before staining, all cell preparations were spun through Ficoll-Hypaque. Cells were stained by incubation for 1 hour at 4°C with FITC-labeled anti-B7.2 (Pharmingen, San Diego, California) followed by washes. Cells were subsequently fixed in 1% formaldehyde in PBS and analyzed on a Coulter EPICS XL-MCL flow cytometer. A minimum of 10,000 cells were collected for each sample. The bar indicates the percent of B7.2-positive cells. Splenocytes were prepared from naïve mice (A) or immunized mice on day 10, including CD40L+/+ (B), CD40L−/− (C), and CD40L−/− mice treated with anti-CD40 (D). Cells were stained with FITC-labeled anti-B7.2 and analyzed for B7.2 expression by fluorescence-activated cell sorting.


  • Table 1.

    Morphometric analysis of liver sections for transgene expression and neutralizing antibody titer. Data were quantified with Leica Quantimet 500+ by analyzing a total of 15 lobes from three mice for the lacZ-expressing hepatocytes at days 3 and 24 and are represented as the mean ± SD. Neutralizing antibody titer (NAT) was determined by assessing the ability of sera to block transduction of 293 cells with the E1-deleted lacZ virus. The data are represented as the reciprocal dilution of serum samples harvested at day 24 with the end point defined as the dilution that inhibits transduction by 50%.

    Mice and treatmentlacZ-expressing hepatocytes (%)NAT
    Day 3Day 24
    C57BL/6 (B6)93.2 ± 1.60640
    CD40L−/−90.5 ± 3.882.4 ± 5.2<20
    CD40L−/− + anti-CD4092.9 ± 4.50320
    B6+anti-B7.1 and anti-B7.291.4 ± 3.582.3 ± 2.840