Article Figures & Data
Figures
- FIGURE 1.
Expression of cell-surface markers by human DC cultures. A, CD34+ cells were seeded at 1 × 104 cells/ml and cultured for 8 days in the described growth medium (serum-free X-VIVO plus cytokines). Cells were then harvested, and the expression of HLA-DR, CLA, CD13, CD1a, CD14, and CD66b was determined by flow cytometry. Thin lines represent control Abs. B, Undisturbed control cells were stained at days 10 and 12 (day 10, thin line; day 12, thick line). Alternatively (right two panels), day 10 cultures were harvested at day 10 and recultured for 2 days at the same density in fresh growth medium (X-VIVO plus cytokines) or RPMI 1640 plus 5% FCS. Cells were stained for CD1a on day 10 (thin line) and day 12 (thick line). Dotted lines represent isotype controls. CD1a expression, consistent with the LC phenotype, was not lost upon reculture.
- FIGURE 2.
DCs within cell clusters exhibit a LC phenotype. Proliferating cultures (day 8) contain abundant multicellular aggregates of HLA-DR+ cells that express the LC markers CD1a and Lag as shown by immunofluorescence microscopy (B and D), while single cells outside of the clusters generally expressed only HLA-DR (A and C). Note how one cell in the center of the cluster shown in A and B, most likely a proliferating precursor, is negative for both differentiation markers. E, Day 10 cells were harvested and subjected to sedimentation on 7.5% BSA columns resulting in an enrichment of CD1a+ cells from 55 to 89%. Cells were then replated for 2 days in the same medium. The majority (≈90%) of cells after cluster purification and reculture are also Lag+. F, Electron microscopy analysis of LC in culture reveal the presence of abundant Birbeck granules (indicated by arrowheads). G, Only BGs (and not other structures such as multilamellar lysosomes, indicated by asterisks) were heavily decorated by anti-Lag Abs. However, the multilamellar structures did label with Abs to MHC class II molecules (not shown).
- FIGURE 3.
Maturation profile of human LC-type DCs. Purified clusters were replated at 5 × 105 cell/well and cultured for 2 days. Replating was performed very carefully in order not to break the clusters (A) or after cluster disaggregation by repeated pipetting (B). Thin lines are cells stained immediately after cluster purification; thick lines are cells stained after 2 days in culture. Breakage of clusters induced spontaneous up-regulation of specific maturation markers. C, Unbroken clusters can be induced to mature by addition of 250 ng/ml LPS (thin line) or 12.5 ng/ml TNF-α (thick line) to the regular growth medium (dotted line). D, Clusters were cocultured with resting (thin line) or activated (thick line) platelets in RPMI 1640/5% FCS or simply replated in regular growth media (dotted line).
- FIGURE 4.
Different proinflammatory agents induce morphologically distinct maturational states of human LCs. Purified clusters were cultured for 2 days in fresh growth media alone (top row) or in presence of 12.5 ng/ml TNF-α (second row), 250 ng/ml LPS (third row), or activated platelets (bottom row). Cells were then fixed and stained for confocal immunofluorescence microscopy. In the merged images, HLA-DR staining is shown in green and Lamp-1 (CD107a) staining in red.
- FIGURE 5.
LC maturation induces enhanced CD4 and CD8 T cell alloreaction. Representative data from three experiments is shown. A, Differentially treated DCs were harvested and cocultured with freshly isolated CD8+ T cells (see Results and Materials and Methods for details). Proliferation at day 2, 4, and 6 was measured by [3H]thymidine incorporation. B, Proliferation of freshly isolated CD8 and CD4 T cells were measured in response to fixed DCs. C, IL-2 production was also measured for CD4+ T cells. D, Proliferation at day 6 of CD4+ T cells (300,000 cells/well) was measured in response to the indicated amount of stimulating DC. E, The ability of TNF-α and LPS to induce differential T cell responses did not reflect differences in the quantitative expression levels of surface markers. Surface staining for MHC class I, MHC class II, CD58, and CD83 are shown for control cells (dotted line), TNF-α-treated cells (thick line), and LPS-treated cells (thin line).
- FIGURE 6.
A, Differential expression of TLR genes following maturation by LPS and TNF-α. Cluster-purified LCs were replated in X-VIVO (control, C) or X-VIVO containing LPS (L) or TNF-α (T) for 48 h, and expression of TLR1–5 was determined by RT-PCR as described in Materials and Methods. Amplification of actin mRNA was included as an internal control. B, LPS-matured, but not TNF-matured, LCs produce IL-12. Cells were matured (as above) with either 12.5 ng/ml of TNF-α or 10 μg/ml of LPS. Supernatants were assayed for IL-12 content by ELISA. Representative data from two experiments is shown.
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