PD-L1 on mast cells suppresses effector CD8⁺ T-cell activation in the skin in murine contact hypersensitivity

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573.e3 HIRANO ET AL

FIG E1. PD-L1–deficient (Pdl1–/–) mice exhibit equivalent ear swelling

responses in irritant dermatitis. Ear thickness changes 24 hours after the

application of 0.3% DNFB (left) and 6 hours after the application of 0.1%

phorbol 12–myristate 13–acetate (right) in WT and Pdl1–/– mice. Data are

representative of 2 independent experiments. Results are expressed as

the means 6 SDs. PMA, Phorbol 12–myristate 13–acetate; NS, not

significant.

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FIG E2. No significant difference was observed in the number or surface markers of Treg cells in the skin

under the blockade of PD-1/PD-L1 signaling. Numbers (A) and surface markers (B) of Treg cells in the ear

skin, as analyzed by flow cytometry 24 hours after the elicitation in mice treated with anti–PD-L1 mAb

(300 mg/mouse) or its isotype-matched control antibody. Results are expressed as the means 6 SDs. Data

are representative of 2 independent experiments. CTLA-4, cytotoxic T-lymphocyte protein-4; MFI, mean

fluorescence intensity; NS, not significant.

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FIG E3. Lack of PD-1/PD-L1 signaling does not affect the numbers or expression of activation markers in skin

MCs during the elicitation phase of CHS. A, The number of MCs in the ear skin, as analyzed by flow

cytometry 24 hours after the elicitation of CHS in WT and Pdl1–/– mice. B, Representative toluidine blue

staining of the ear skin 24 hours after elicitation. (Right) High magnification of degranulated MCs.

C, Activation markers of MCs in WT and Pdl1–/– mice 24 hours after elicitation, as analyzed by flow

cytometry. D-F, Ear swelling (D), activation markers of MCs (E), and representative toluidine blue staining

of the ear skin (F) 1 hour after the elicitation of CHS. Mice were treated with anti–PD-L1 mAb (300 mg/mouse)

or its isotype-matched control antibody 6 hours before the elicitation. The horizontal and vertical sections

are shown for each sample in (F). Results are expressed as means 6 SDs. Data are representative of 3 (A) or

2 (C-E) independent experiments. MFI, Mean fluorescence intensity; NS, not significant.

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FIG E4. Human MC lines constitutively express PD-L1. Flow cytometric

analysis of PD-L1 expression on LAD2 cells and LUVA cells. Gray peak

represents a control stained with isotype IgG.

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TABLE E1. PD-L1 expression in human skin MC samples from the public database Functional Annotation of the Mammalian

Genome 5 (FANTOM5)

Sample ID

Mast cell,

Mast cell,

Mast cell,

Mast cell,

Average

donor3.CNhs12593.11566-120D9

donor2.CNhs12594.11565-120D8

donor4.CNhs12592.11567-120E1

donor1.CNhs12566.11563-120D6

ID, Identifier; TPM, tags per million; TSS, transcriptional start site.

p1@CD274 (TSS) (TPM)

Rank (among a total of 847)

129.56

89.15

27.77

10.83

64.33

16

66

179

67.75

...