Phenotypic and Functional Properties of Human Steady State CD14+ and CD1a+ Antigen Presenting Cells and Epidermal Langerhans Cells.

Cutaneous antigen presenting cells (APCs) are critical for the induction and regulation of skin immune responses. The human skin contains phenotypically and functionally distinct APCs subsets that are present at two separated locations. While CD1ahigh LCs form a dense network in the epidermis, the CD14+ and CD1a+ APCs reside in the dermal compartment. A better understanding of the biology of human skin APC subsets is necessary for the improvement of vaccine strategies that use the skin as administration route. In particular, progress in the characterization of uptake and activatory receptors will certainly improve APC-targeting strategies in vaccination. Here we performed a detailed analysis of the expression and function of glycan-binding and pattern-recognition receptors in skin APC subsets. The results demonstrate that under steady state conditions human CD1a+ dermal dendritic cells (DCs) were phenotypically most mature as measured by the expression of CD83 and CD86, whereas the CD14+ cells showed a higher expression of the CLRs DC-SIGN, mannose receptor and DCIR and had potent antigen uptake capacity. Furthermore, steady state LCs showed superior antigen cross-presentation as compared to the dermal APC subsets. Our results also demonstrate that the TLR3 ligand polyribosinic-polyribocytidylic acid (pI:C) was the most potent stimulator of cytokine production by both LCs and dDCs. These studies warrant further exploration of human CD1a+ dDCs and LCs as target cells for cancer vaccination to induce anti-tumor immune responses

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

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

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

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