Article Figures & Data
Figures
- FIGURE 1.
IL-12 allows optimal proliferation, and is required for CTL differentiation of purified naive CD8 T cells. A, CD44low (>99%) CD8+ (>95%, <0.3% CD4+) OT-1 T cells were stimulated with 5 μm-sized microspheres coated with 2C11 anti-CD3ε mAb (1 μg/ml/107 microspheres), in the absence of cytokines or with IL-2 (2.5 U/ml), IL-12 (2.0 U/ml), and/or a blocking anti-IL-2Rα mAb (20 μl of hybridoma supernatant), as indicated. Proliferation was determined on day 2 by measuring incorporation of [3H]TdR. B, Cells stimulated as in A were harvested on day 3, and a 4-h 51Cr release assay was done using OVA-transfected EL-4 cells (E.G.7 cell line) as targets. SDs of triplicate samples in the 51Cr release assay were less than 10% of the average values shown. The requirement for IL-12 to stimulate cytolytic activity under these conditions has been consistently observed in numerous experiments.
- FIGURE 2.
IL-12 enhances, but is not required for, expression of CD25 mRNA and protein, and IL-12Rβ2 mRNA. CD44low CD8+ OT-I T cells were stimulated with 2C11-coated microspheres, plus the indicated cytokines, along with blocking anti-IL-2Rα mAb, where indicated. Cells were harvested after 48 h and analyzed. A, Expression levels of CD25 and IL-12Rβ2 mRNA were determined by RT-PCR. B, Cells stimulated as indicated were stained with anti-CD25 mAb or isotype control mAb (dotted line) and analyzed by flow cytometry.
- FIGURE 3.
IL-12 increases and sustains CD25 expression, IL-12Rβ2 mRNA expression, and proliferation. CD44low CD8+ OT-1 T cells were stimulated with 2C11 (A, C, and E)- or Kb/OVA (B, D, and F)-coated microspheres, plus the indicated cytokines. CD25 surface expression (A and B), IL-12Rβ2 mRNA expression (C and D), and proliferation (E and F) were measured at the indicated times by flow cytometry, RT-PCR, and [3H]TdR incorporation, respectively. Mean fluorescence intensity (MFI) values were normalized to IgM isotype controls (A and B). mRNA abundance values were normalized to their respective β-actin controls (C and D).
- FIGURE 4.
The effects of IL-12 on CD25 expression and proliferation cannot be replaced by high levels of TCR engagement or IL-2. CD44low CD8+ OT-1 T cells were stimulated with IL-2 (2.5 U/ml), and microspheres were coated with varied amounts of 2C11 (A and C), or with 2C11-coated microspheres (1 μg/ml/107 microspheres) and varied amounts of IL-2 (B and D), in the presence or absence of IL-12 (2.0 U/ml). CD25 surface expression (A and B) and cell proliferation (C and D) were measured at day 2 by flow cytometry and [3H]TdR incorporation, respectively. Mean fluorescence intensity (MFI) values were normalized to IgM isotype controls (A and B).
- FIGURE 5.
The effects of IL-12 on CD25 expression and proliferation cannot be replaced by B7-dependent costimulation. CD44low CD8+ OT-1 T cells were stimulated with 2C11- or 2C11/B7-coated microspheres, plus the indicated cytokines. Surface CD25 expression (A and B) and cell proliferation (C) were measured at day 2 by flow cytometry and [3H]TdR incorporation, respectively. The dotted line in A and B is the IgG isotype control Ab.
- FIGURE 6.
IL-12 promotes clonal expansion and CD25 expression in vivo. CD44low CD25− CD8+2C transgenic T cells were adoptively transferred into congenic C57BL/6 mice, and the numbers and CD25 phenotype of the 2C cells were analyzed by flow cytometry 3 days after immunization. Recipient mice (two mice per group) were either not immunized (transfer only), or were immunized with SIYRYYGL peptide alone (peptide) or along with IL-12 (peptide + IL-12). One group of mice immunized with peptide and IL-12 also received CTLA-Ig by i.p. injection of 0.2 mg/mouse 6 h before immunization and again on days 0, 1, and 2 (total 0.8 mg/mouse). A, The number of 2C CD8 T cells in the lymph nodes was determined 3 days after immunization, as described in Materials and Methods. The results are expressed as the fold increase in number in comparison with the number in mice that had not been immunized, and the error bars indicate the range for duplicate mice. B, CD25 expression on 2C lymph node cells 3 days after immunization. Essentially the same results were obtained in an independent experiment.
- FIGURE 7.
IL-12 can enhance proliferation independently of effects on CD25 expression. CD44low CD8+ T cells from C57BL/6 wild-type or CD25−/− (ko) mice were stimulated with 2C11-coated microspheres, plus the indicated concentrations of IL-2, in the presence or absence of IL-12. Cell proliferation was measured at day 2 by [3H]TdR incorporation.
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