Figure 1.
Rationale for intradermal (ID) vaccine delivery.
(A) ID delivery of H1 VLPs to excised human skin using the Mantoux method. A 26G hypodermic needle was inserted laterally into the skin followed by injection of H1 VLP suspension (10µg VLP). A characteristic wheal, typical of ID injection, is clearly visible. (B) IHC stained (CD207) human epidermal sheet showing the extensive network characteristic of LCs. (C) A single LC displaying the typical dendritic cell nature of this cell type. Dendrites emanating from the LC body can clearly be seen infiltrating between individual keratinocytes.
Figure 2.
Changes in LC numbers and morphology in epidermal sheets.
Human epidermal sheets were isolated from untreated cultured skin and cultured skin treated by ID injection of PBS (control) or H1 VLPs. (A) Epidermal sheets were stained for CD207 and positively stained cells were counted. Data presented as the percentage of CD207+ve cells (LCs) relative to cell numbers in untreated skin at each timepoint. Skin treated by ID delivery of PBS (grey bars); Skin treated by ID delivery of H1 VLPs (white bars); data presented as mean ± SD (n = 4), significance was determined relative to untreated skin at each timepoint (***p<0.001). (B) Representative images of LCs (CD207+ve cells) taken from each time point and treatment (bar = 20µm). (C) LCs displaying a “hyper-dendritic “cell morphology. (D) LCs displaying “rounded” cell morphology. (E) Both hyper-dendritic and rounded LCs observed in the same optical plane of the epidermis (bar = 10µm all cases).
Figure 3.
Number of dendrites per LC in epidermal sheets.
(A) Microscopic examination revealed each LC to have a distinct cell body from which dendrites protruded (bar = 10µm). Two individual LCs designated (i) and (ii) with 4 and 5 dendrites per cell, indicated by the white and red arrows, respectively. Percentage frequency of dendrites per LC in (B) pre-cultured epidermal sheets and post-cultured epidermal sheets at 24 (C) and 48 (D) hours; untreated skin (black bars), skin treated with H1 VLPs delivered by ID injection (white bars). Data presented as mean ± SEM (n = 8).
Figure 4.
Area of individual LCs in epidermal sheets.
The mean individual LC area (n = 100) was determined using ImageJ software following culturing of blank skin (black bars) and skin treated with H1 VLP delivered by ID injection (white bars). Data presented as mean ± SD (n = 4); significance was determined relative to blank skin at corresponding time points (*p<0.05).
Figure 5.
Relative area and spatial distribution of LCs in histological sections.
(A) The relative area of CD207+ve cells as a percentage of total epidermal area in skin sections was determined using ImageJ software. Data presented as mean ± SD (n = 4) significance was determined relative to blank skin at each corresponding time point (*p<0.05, **p<0.01, ***p<0.001). Skin sections showing spatial distribution of LCs in histological skin sections: untreated skin at 0 hours (B), untreated skin at 24 hours (C) and skin treated with H1 VLPs delivered by ID injection at 24 hours (D). Bar = 50µm in all cases.
Figure 6.
LCs localization to epidermal basement membrane.
(A) IHC stained (CD207) histological section showing a single LC extending an exploratory dendrite toward the basement membrane; dashed line indicates the epidermal/dermal interface (bar = 20 µm). (B) The mean distance of LCs from the basement membrane in untreated and VLP-treated skin samples. (C) Representative examples of CD207+ve cells located in the skin dermis (red arrows) 24 hours after ID injection of H1 VLPs (bar 50 µm).
Figure 7.
Schematic depicting the proposed sequence of events leading to LC migration from skin epidermis.
In normal skin LCs display typical DC morphology, i.e. contain many dendritic protrusions. Upon activation LCs detach from surrounding keratinocytes and retract dendrites assuming a “rounded” morphology. A point of attachment is made from which the LC subsequently moves, potentially in a chemotactic manner, toward the basement membrane. Cells elongate to enable downward vertical movement.