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. 2020 May 19;11:775. doi: 10.3389/fimmu.2020.00775

Figure 8.

Figure 8

The early production of IFNγ and TNF by CD4+ and CD8+ TRM cells is associated with the induction of the local skin memory response. (A) The mRNA levels of the indicated genes in whole naïve and healed ear extracts of BALB/c mice at 0, 1.5, 3, and 6 h after the re-challenge with 1% TNCB (on day 35 after the 1st challenge) as assessed by qRT-PCR (n = 4 each time). (B) mRNA levels in the whole ear extract of DO11.10 mice at 6 h after the re-challenge with 1% TNCB (on day 37 after the 1st challenge) (n = 4) were compared with those of BALB/c mice [data at 6 h in (A)]. (C,D) IFNγ, TNF, and IL-4 production in CD4+ and CD8+ TRM cells (on day 39 after the 1st challenge) as assessed by flow cytometry. No stimulation: naïve and healed ears underwent enzymatic digestion (3.5 h) with brefeldin A. 1% TNCB stimulation: ears (re-challenged 3.5 h before) underwent digestion with brefeldin A (3.5 h). Ionomycin + PMA stimulation: ears underwent enzymatic digestion (4.0 h) with brefeldin A, ionomycin, and PMA. Signals in orange circles were mostly a non-specific background, as shown in Supplemental Figure 7B. (E) 1st contact hypersensitivity-healed BALB/c mice were injected (i.p. or i.v.) with anti-IFNγ + anti-TNF neutralizing mAbs, or control Rat IgG1 mAb or PBS, simultaneously with the re-challenge with 0.1% TNCB on day 35. Ear swelling at 24 h after the re-challenge is shown. Each experiment was performed once. (F) Ear swelling in BALB/c mice at 24 h after the re-challenge with 1 or 0.1% TNCB (on day 36 after the 1st challenge). The mice were injected and topically treated with dimethyl sulfoxide (control) or Rux + BAY 2 h before the re-challenge. Injection: Rux (8 mg/kg) + BAY (6 mg/kg). Topical application: 0.5% Rux + 0.3% BAY. (G) The numbers of Gr-1+ cells along the cartilage in BALB/c ear skin sections at 4 h after the re-challenge (on day 36 after the 1st challenge). The mice were injected with Rux and/or BAY (20 mg/kg each) at 1 h before the re-challenge (n = 4–5 each group). All graphs represent means ± SE. *P < 0.05 (two-tailed paired or unpaired t-test). The asterisks in/on each bar indicate significant differences from each ear of the wild-type (B) or control treatment (E–G).