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Supplementary Figure 1: Identification of pulmonary dendritic cell subsets by flow
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cytometry. (A) HDM-sensitized A/J mice were treated with AF405 HDM on day 14. 72
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hours later mice were sacrificed and their lungs were removed. Lungs cell suspensions
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were stained for flow cytometric analysis of DC population. (A) Gating strategy to
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identify pulmonary APC populations revealed 5 distinct CD11c+ cell populations.
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Alveolar Macrophages (AM - CD11cbright, CD11bneg, CD317neg), plasmacytoid DCs
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(pDCs
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CD11cbrightCD11bbrightGr1brightCD103neg),
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CD11cbrightCD11bbrightGr1dimCD103neg)
-
CD11cdimCD11bnegCD317+),
neutrophils
inflammatory
and
DCs
(PMNs
(Inf
myeloid
-
DCs
DCs
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(CD11cbrightCD11bbrightGr1negCD103neg). (B) Gated DC populations were subsequently
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examined for expression of PD-L2, and uptake of HDM. Representative plots from a
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single mouse shown.
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Supplementary Figure 2: In vivo PD-L2 blockade does not alter pulmonary mDC
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activity. A/J mice were treated with PBS, HDM, HDM + rat IgG2a control (Iso) or HDM
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+ anti PD-L2 (αPD-L2) as described in Materials and Methods. The frequency (A),
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allergen content (B), and activation status of pulmonary mDCs (C) was assessed by flow
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cytometry. n = 8 mice from 2 independent experiments. Mean + SEM shown.
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Supplementary Figure 3: In vivo PD-L2 blockade does not enhance IL-12 p40 levels
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in BAL. A/J mice were treated with PBS or HDM intratracheally on days 0 and 14, and
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250 µg of rat IgG2a control (Iso) or anti-PD-L2 (αPD-L2) intraperitoneally on days 0, 2,
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14 and 16, and mice were sacrificed on day 17. BAL was performed, and levels of IL-12
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p40 were assessed by ELISA. MEAN + SEM shown. n = 10 mice from 2 independent
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experiments.
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Supplementary Figure 4: In vivo PD-L2 or IL-12 blockade does not alter baseline
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tracheal pressure. A/J mice were treated with PBS or HDM intratracheally on days 0
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and 14. Mice were given 250 µg of rat IgG2a control (Iso) or anti-PD-L2 (αPD-L2)
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intraperitoneally on days 0, 2, 14 and 16 and/or 1 mg of rat IgG2a control (Iso) or anti-
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IL-12 (αIL-12) intraperitoneally on days -2 and 12. Mice were sacrificed on day 17 for
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assessment of baseline AHR by the APTI method. Tracheal pressure was monitored for 1
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minute prior to injection of intravenous acetylcholine. n = 10 mice from 2 independent
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experiments. Mean + SEM shown.
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Supplementary Figure 5: Changes in AHR observed in mice received PD-L2
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blocking and/or IL-12 blocking mAbs are not associated with changes in local
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changes in cytokine mRNA levels. A/J mice were treated with PBS or HDM
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intratracheally on days 0 and 14. Mice were given 250 µg of rat IgG2a control (Iso) or
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anti-PD-L2 (αPD-L2) intraperitoneally on days 0, 2, 14 and 16 and/or 1 mg of rat IgG2a
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control (Iso) or anti-IL-12 (αIL-12) intraperitoneally on days -2 and 12. Mice were
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sacrificed on day 17 and mRNA was isolated from snap-frozen lung samples for analysis
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of (A) IL-5 and (B) IL-13 expression by RT-PCR. n = 10 mice from 2 independent
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experiments. Mean + SEM shown.
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Supplementary Figure 6: IL-12 and IFNγ inhibits IL-13-driven gene expression, and
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IL-13 inhibits IFNγ, but not IL-12-driven gene expression. (A) BMDCs were cultured
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in the presence of medium, IL-12, IL-13, or IL-12 + IL-13 (all at 10 ng/ml) for 18 hours.
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mRNA was harvested from the cells and expression of IFNγ, a representative IL-12-
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induced gene, was examined by RT-PCR. (B) BMDCs were cultured in the presence of
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medium, IFNγ, IL-13, or IFNγ + IL-13 (all at 10 ng/ml) for 18 hours. mRNA was
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harvested from the cells and expression of IL-13-driven genes (Fizz1, Arg1, CD206) and
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IFNγ-driven genes (PD-L1) was examined by RT-PCR. (C) BMDCs were cultured in the
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presence of IL-13, IL-12 + IL-13 (all at 10 ng/ml), or IL-12 + IL-13 + αIFNγ (at 5 µg/ml)
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for 18 hours. mRNA was harvested from the cells and expression of IL-13-driven genes
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was examined by RT-PCR. *** and * indicate p < 0.001 and p < 0.5 respectively. n = 4
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replicates for each condition tested. 1 experiment of 2 performed shown.