presentation_1_Toll-Like Receptor 4 Triggering Promotes Cytosolic Routing of DC-SIGN-Targeted Antigens for Presentation on MHC Class I.PDF

<p>DC-SIGN is an antigen uptake receptor expressed on dendritic cells (DCs) with specificity for glycans present on a broad variety of pathogens and is capable of directing its cargo to MHC-I and MHC-II pathways for the induction of CD8⁺ and CD4⁺ T cell responses, respectively. Therefore, DC-SIGN is a very promising target for the delivery of antigen for anti-cancer vaccination. Although the endocytic route leading to MHC-II presentation is characterized to a large extent, the mechanisms controlling DC-SIGN targeted cross-presentation of exogenous peptides on MHC-I, are not completely resolved yet. In this paper, we used imaging flow cytometry and antigen-specific CD8⁺ T cells to investigate the intracellular fate of DC-SIGN and its cargo in human DCs. Our data demonstrates that immature DCs and toll-like receptor 4 (TLR4) stimulated DCs had similar internalization capacity and were both able to cross-present antigen targeted via DC-SIGN. Interestingly, simultaneous triggering of TLR4 and DC-SIGN on DCs resulted in the translocation of cargo to the cytosol, leading to proteasome-dependent processing and increased CD8⁺ T cell activation. Understanding the dynamics of DC-SIGN-mediated uptake and processing is essential for the design of optimal DC-SIGN-targeting vaccination strategies aimed at enhancing CD8⁺ T cell responses.</p

Gene expression analysis of human skin APC populations.

<p>A. LCs and dermal APCs were allowed to migrate from human skin for 3 days where after the samples were purified using CD1a and HLA-DR MACS beads, respectively. Cell purity was verified using flow cytometry. Data is shown for one representative LC and dDC sample. N = 3. B. Microarray gene array analyses on emigrated LCs and dermal APCs samples. Data are shown for 3 independent LCs and dermal APC samples.</p

Human skin APC subsets do not phenotypically mature <i>in vitro</i> upon inflammatory conditions.

<p>A. Percentages of CD14⁺ dermal cells, CD1a⁺ dDCs and LCs (gated on HLR-DR⁺ cells) directly after enzymatic isolation or after 24 h of culture of epidermal and dermal suspensions in medium (IMDM), GM-CSF and IL-4 (GM/4), pI:C or LPS. Mean values ± SEM; n = 3. B. Surface expression of molecules associated with DC maturation, CD86, CD83 and CD70 and molecules association with T cell activation, MHC class I and II measured directly after enzymatic isolation of the skin APC subsets or after 24 h of culture of epidermal or dermal suspensions in the presence of indicated reagents. Mean values ± SEM; n = 3. *p<0.05 and ***p<0.001, as measured by the one-way ANOVA followed by the Bonferroni multiple comparison test.</p

The CD1a⁺ dDC subset is the phenotypically most mature human skin APC subset under steady conditions.

<p>A. Subset distribution in percentages of CD1a⁺ and CD14⁺ dermal cells (upper panel) or CD1a⁺ LCs (lower panel) after gating on isolated HLA-DR⁺ cells. Dot plots of a representative experiment are shown. N = 8. B. Surface expression of MHC class I and II, CD86 and CD83 was measured by flow cytometry on isolated HLA-DR⁺CD14⁺ cells, HLA-DR⁺CD1a⁺ dDCs and HLA-DR⁺CD1a^high LCs. Grey histograms depict matching isotype controls. Histograms of a representative experiment are shown. N = 3. C. Relative mRNA levels of CD83 and CD86 compared to the housekeeping gene GAPDH are shown present in FACS-sorted, steady state DN, CD14⁺ or CD1a⁺ dermal cells and LCs. Due to low cell numbers of each subset after sorting, the subsets contain combined cells of at least 4 skin donors. Mean values ± SEM; n = 3. ***p<0.001, as measured by the one-way ANOVA followed by the Bonferroni multiple comparison test.</p

Dermal APCs and LCs secrete cytokines after culture in the presence of GM/4 or the TLR ligands pI:C and LPS.

<p>Enzymatically isolated LCs and dermal APCs were cultured for 24 h in medium or medium containing GM/4, LPS or pI:C. Secretion of cytokines was measured by ELISA. Mean values ± SEM; n = 3, each experiment measured in triplicate. *p<0.05, **p<0.01 and ***p<0.001 as measured by the one-way ANOVA followed by the Bonferroni multiple comparison test.</p

Antigen internalization and CD8⁺ T cell stimulatory capacities of steady state isolated human skin APC subsets.

<p>A. Internalization of fluorescently labeled OVA by the enzymatically isolated skin APC subsets after 2 h as measured by flow cytometry. A representative experiment is shown. Mean values ± SEM; n = 3. ***p<0.001 as measured by the two-way ANOVA followed by the Bonferroni multiple comparison test. B. Antigen-pulsed, MACS-isolated human skin APC subsets were co-cultured with HLA-A2⁺ GP100-specific CD8⁺ T cells. After 24 h, IFN-y levels were analyzed in the supernatants as measure for T cell activation using ELISA. A representative experiment is shown. Mean values ± SEM; n = 3, each experiment performed in triplicate. *p<0.05 as measured by the one-way ANOVA followed by the Bonferroni multiple comparison test. C. MACS-isolated human skin DC subsets were pulsed with the 9 aa long GP100 peptide for 2 h, washed, and co-cultured with the HLA-A2⁺ GP100-specific CD8⁺ T cell clone. IFN-y production by the T cells was measured using ELISA. Mean values ± SEM; n = 3, each measured in triplicate. *p<0.05 and ***p<0.001 as measured by the two-way ANOVA followed by the Bonferroni multiple comparison test.</p

Expression of CLRs and TLRs by highly purified, FACS-sorted human skin APC subsets.

<p>mRNA expression of various CLRs (A) or TLRs (B) was examined on FACS-sorted human skin APC subsets using RT-PCR analyses. Due to low cell numbers of each subset after sorting, the distinct subsets contain combined cells of at least 4 skin donors. Mean values ± SEM; n = 4. Highly significant differences between the skin APC subsets were observed for all CLRs (p<0.001, as measured by the one-way ANOVA followed by the Bonferroni multiple comparison test).</p