Matched skin and sentinel lymph node samples of melanoma patients reveal exclusive migration of mature dendritic cells

Abstract

Mature and immature myeloid dendritic cells (DCs) are thought to differentially modulate T-cell responses in secondary lymphoid tissues. Although mature DCs are believed to induce T-cell activation under proinflammatory conditions, immature DCs are believed to maintain a state of T-cell tolerance under steady state conditions. However, little is known about the actual activation state of human DCs under these different conditions. Here, we compare the frequency and activation state of human DCs between matched skin and sentinel lymph node (SLN) samples, after intradermal administration of either granulocyte/macrophage colony-stimulating factor (GM-CSF) or saline, at the excision site of stage I primary melanoma. Although DCs remained immature (CD1a+CD83-) and mostly situated in the epidermis of the saline-injected skin (fully consistent with a quiescent steady state), mature (CD1a+CD83+) DC frequencies significantly increased in the GM-CSF-injected skin and correlated with the number of mature DCs in the SLN, indicative of increased DC migration. Interestingly, irrespective of GM-CSF or saline administration, all CD1a+ myeloid DCs in the SLN were phenotypically mature (ie, CD83+). These data are indicative of migration of small numbers of phenotypically mature DCs to lymph nodes under steady state conditions.

Figure 1

DC phenotype and localization in primary tumor re-excision skin samples after intradermal administration of saline or GM-CSF. Immunohistochemical analysis for the indicated DC markers from two representative melanoma patients (stage I) after intradermal injections of either saline or GM-CSF. Original magnifications, ×400.

Figure 2

Cutaneous DC, macrophage, and T-cell frequencies in relation to saline or GM-CSF administration. Numbers of DCs and T cells in the epidermis (A), superficial (B), and deep dermis (C), counted in paraffin-embedded slides after immunohistochemical analysis of the indicated markers. All tissue samples were obtained through re-excision of the scar of the primary melanoma excision. Five patients were given GM-CSF around the scar tissue and six patients were given saline. Results are shown as mean number per 10 HPFs (HPF at ×400 magnification), except for CD3: mean per five HPFs. *Significant in a Mann-Whitney U-test at P < 0.05.

Figure 3

Correlation between DC numbers in the epidermis or superficial dermis and in the SLN. The numbers of DCs positive for CD1a, CD83, and S100 in the SLN (expressed as number per 600 T cells: quantified on cytospins from SLN cell suspensions), correlated to the number of DCs positive for CD1a (A), CD83 (B), and S100 (C) in the epidermis and CD1a (D), CD83 (E), and S100 (F) in the superficial dermis (all shown as mean numbers per 10 HPFs, HPF at ×400 magnification). ⋄Patients treated with saline; ▴patients treated with GM-CSF. Correlations were calculated using Spearman’s ρ test: r and P values are displayed.

Figure 4

Myeloid DCs in skin-draining SLNs have a CD83⁺ mature phenotype, irrespective of intradermal GM-CSF or saline administration. A: Correlation between the number of CD1a⁺ DCs and CD83⁺ DCs in the SLN (expressed as number per 600 T cells) of patients treated with saline (⋄) and patients treated with GM-CSF (▴) reveals a linear relationship (Spearman’s ρ r = 0.836, P = 0.001). B: Flow cytometric analysis of CD83 expression on myeloid DCs in SLN single cell suspensions from all saline- and GM-CSF-treated melanoma patients. Myeloid DCs were gated by CD1a positivity and high side scatter levels.