Lang-DTR mice were untreated or treated with diphtheria toxin 7 days prior to treatment to selectively deplete epidermal LC, while allowing dermal DC to repopulate. Mice were then irradiated with UV for 3 consecutive days, and 24 hours after the last UV treatment, immunized with 25 μl of 0.3% DNFB on the shaved back. 5 days after immunization, mice were challenged with 5 μl of 0.15% DNFB on the ear. Ear thickness was measured before and 24 hour after challenge. The immune response= ear swelling (after challenge-before challenge).

(A) The individual mice and chimeras were irradiated with UV, transferred with 2.5 × 10⁵ CFSE labeled CD44^lo OT-I CD8 T cells and then immunized with OVA protein. OT-I CD8 T cell expansion was calculated 6 days later. (B) As in (A) but all of the bone marrow chimeric mice were treated with diphtheria toxin 96 and 24 hours before immunization. One group of mice was irradiated with UV and followed with OT-I CD8 T cells transfer and immunization. The graph shows OT-I CD8 T cell expansion 6 days after immunization. (C) Representative histograms show CFSE dilution of CD8 T cells from 3 experiments. n=3 mice/group

(A) 2.5 × 10⁵ CFSE labeled CD44^lo OT-I CD8 T cells were adoptively transferred into bone marrow chimeric mice whose ability to present OVA protein was limited either to radioresistant epidermal LC (bm1 into B6) or to bone marrow derived DC (B6 into bm1). Representative plot from 3 experiments shows CFSE dilution 6 days after immunization. (B) As in (A) except that bone marrow chimeras of Lang-DTR into B6 mice and B6 into Lang-DTR mice were treated with two dose of 1 μg diphtheria toxin i.p. at 96 and 24 hours before immunization. The bar graph shows OT-I CD8 T cell expansion. (C) Histogram shows the OT-I CD8 T cell division 6 days after immunization.

(A) Groups of mice were irradiated consecutively for 3 days with 45 mJ/cm² UV at each time point, then immunized and challenged according to the experimental scheme shown. DNFB immunization was performed on the flank, while challenge was on the ear. Ear thickness was measured 24 hours later. Error bars show the standard deviation, n=3, and is representative of at least 4 independent experiments. (B) As in (A), but mice were intravenously injected with 2.5 × 10⁵ CFSE labeled CD44^lo (naïve) OT-I.PL CD8 T cells and 24 hours later epicutaneously immunized with OVA protein. Mice were then harvested 6 days after immunization. Total OT-I CD8 T cell numbers from spleen and subcutaneous lymph nodes were determined using flow cytometric analysis. Data is expressed as the mean ± SD. n=18–24 mice/group from 8 independent experiments. The CFSE histogram indicated the division of OT-I CD8 T cells.

(A) Lang-EGFP transgenic mice were used to identify epidermal LC, in which all of epidermal LC were EGFP+ cells. One side of the mouse was irradiated with UV as described in Figure 1, with the other side protected from irradiation. 24 hours after the last irradiation, epidermal suspension from both sides was prepared separately and EGFP+ epidermal LC and Gr1+ cells were gated in the CD45+ hematopoietic cells gate. (B) The bar graph shows the calculated percentage of epidermal LC for 4 mice/group (mean ± SD). The “DT+” control was the Lang-EGFP-DTR mice treated with two dose of 1 μg diphtheria toxin i.p. at 96 and 24 hours before harvest. The data are representative of 2 experiments. (C) Representative histogram from 3 experiments is shown for the phenotype of epidermal LC. (D, E) Mice were transferred with 2 × 10⁶ CFSE labeled CD44 low OT-I CD8 T cells flowed by OVA protein immunization on the UV irradiated side of the flank. OT-I cells both from the draining lymph nodes and non-draining nodes were collected 48 (D) and 72 hours (E) after immunization. The plot shows CFSE dilution by CD69 at the early time point (D), or CD44 at the later time point (E). Graphs show the calculated percentage of activated OT-I CD8 T cells.