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Blood, Vol. 109, Issue 10, 4320-4327, May 15, 2007
DC-HIL is a negative regulator of T lymphocyte activation
Blood Chung et al.
109: 4320
Supplemental materials for: Chung et al, Vol 109, Issue 10, 4320-4327
Files in this Data Supplement:
- Figure S1. Surface expression of DC-HIL by DCs (JPG, 73.8 KB)
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Bone marrow–derived DCs were harvested and treated or not treated with LPS (1 µg/mL) for 1 day. These DCs were then doubly stained with 1E4 rat anti–DC-HIL mAb (or control rat IgG) and anti-CD11c (or control hamster IgG). Immunostaining was assayed by FACS. Numbers in each quadrant represent percentages of the total cell population.
- Figure S2. Induction of DC-HIL expression by keratinocytes following IFN-γ stimulation (JPG, 73 KB)
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A keratinocyteline PAM212 was treated with IFN- plus PMA and cultured for indicated time periods. Cells were then analyzed by RT-PCR for mRNA expression of DC-HIL and  -actin (A) or by FACS for surface expression of DC-HIL (B).
- Figure S3. Dec2-Fc had no effect on T-cell activation (JPG, 46.4 KB)
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An irrelevant Fc fusion protein (Dec2-Fc, the extracellular domain of the C-type lectin receptor dectin-2 fused to the Fc portion of human IgG) does not bind to T cells. We examined effects of Dec2-Fc on T-cell activation. CD4+ T cells were treated with increasing doses of anti-CD3 Ab and a constant dose of DC-HIL-Fc or Dec2-Fc (None; cultures with anti-CD3 Ab alone). After culturing for 2 days, T-cell proliferative response was measured by 3H-thymidine incorporation. There was no significant difference in the proliferative responses of Dec2-Fc–treated and untreated T cells; by contrast, DC-HIL-Fc inhibited T-cell activation.
- Figure S4. Dec2-Fc has no effect on MLR (JPG, 38.4 KB)
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C57BL/6 spleen cells (5 × 104) were irradiated and mixed with CD4+ T cells (2 × 105) purified from BALB/c mouse spleens. Indicated doses of Dec2-Fc or hIgG were added to the MLR culture and incubated for 3 days prior to 3H-thymidine pulsing. Proliferative response of T cells was assayed by incorporation of 3H-thymidine. Neither an irrelevant Fc fusion protein nor hIgG enhanced MLR.
- Figure S5. Control hIgG binds very weakly to DCs (JPG, 33.7 KB)
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Mouse bone marrow–derived DCs (5 × 105 cells) were harvested and resuspended in 10% FCS-RPMI and then incubated with no protein (closed histograms) or with 20 µg/mL of hIgG or mouse IgG (mIgG; open histograms) and PE-goat F(ab′)2 anti-human (or mouse) IgG Ab. Binding was examined by FACS. hIgG showed very weak binding to DCs, while mIgG showed significant binding. These data indicate that Fc fusion proteins added to DC culture are unable to bind Fc receptors on DCs.
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