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was then applied onto the mouse and rabbit skin for transdermal temperature-responsive OCT gel for immunofluorescence staining applications. with different antibodies, including anti-Keratin 14 and anti-ZO-1. After For mice administration, the mixed milk-like ointment was then washing the primary antibodies, the tumor slides were stained with swiped on the mouse skin with a round cover of 3.14 cm (1 cm in fluorescently labeled secondary antibodies and DAPI. After mounting. radius) for 12 h. Transparent film (3 M) was covered finally to avoid the the tumor slices were imaged by the confocal fluorescence microscope influence of mice lick. (Leica SP5). For rabbit administration, the mixed milk-like ointment was then swiped on the rabbit skin with a round cover of 50.24 cm2 (4 cm in Analysis of T cells in different organs radius) for 12 h. Transparent film (3 M) was covered finally to avoid the Spleens, tumors, skins, and lymph nodes were collected and homogenized into single-cell suspensions following the standard protocol. Briefly, they were processed through mechanical disruption before labeling, stability, and in vivo biodistribution digestion for 1 h at 37 C in an enzymatic solution with RPMI-1640 (10% IgG was labeled with si by the standard chloramine-T oxidation FBS and 1% PS), 1.5 mg/ml collagenase IV (Sigma), 1.5 mg/ml col method. Briefly, IgG, Nal2SI, and chloramine-T were mixed at 37 C for lagenase I (Sigma), 1.5 mg/ml hyaluronidase (Sigma), and 0.2 mg/ml 10 min under slight shaking. The synthesized 12-IgG was washed by DNase I (Sigma). The samples were then passed through 200-mesh 10 kDa ultrafiltration three times. To determine radiolabeling stability, nylon mesh filters to obtain single-cell suspensions. The obtained 12S-IgG was mixed with serum for 24 h at 37 C. Portions of the mixture single-cell suspensions were incubated with anti-CD16/32 for 30 min at were sampled at different time intervals and filtered by 10 kDa ultra 4 C. Then, these cells were stained with different antibodies according filtration. The radioactivities retained on the filters were detected by to the standard protocol. Wizard2 Gamma Counter (PerkinElmer to calculate radiolabeling DCs in lymph nodes and skins were stained with anti-CD11c-FITC stability. (Biolegend, Cat. 117305), anti-CD80-APC (Biolegend, Cat. 104713), and For in vivo biodistribution of 1i-IgG assay, B16F10 melanoma anti-CD86-PE (Biolegend, Cat. 105007). tumor-bearing mice were topically administrated with free i2I-IgG and T cell population in tumors and spleens were stained with anti- FCS/12-IgG ointment for different time points. Then, the major organs, CD3-FITC (Biolegend, Cat. 100203), anti-CD4-APC (Biolegend, Cat. including the liver, spleen, kidney, heart, lung, and tumor, were col- 100411), anti-CD45-PerCP (Biolegend, Cat. 103130), and anti-CD8a-PE lected and measured by Wizard2 Gamma Counter (PerkinEImer). The (Biolegend, Cat. 100707). accumulation efficacy was calculated by the formulation To further analyze helper T cells in tumors, the suspension was stained with anti-CD3-FITC (Biolegend, Cat. 100203), anti-CD4-APC %ID/g=R/Ro/m 100% (2) (Biolegend, Cat. 100411), anti-Foxp3-PE (Biolegend, Cat. 126403) anti- bodies with eBiosciencetm Foxp3/Transcription Factor Fixation/Per- Where R, was the radio intensity of the organ at the exact time point, meabilizationConcentrateand Diluent according to the m, was the mass of the organ, Ro was the radio intensity of the applied manufacturer's protocols. Briefly, after the cells were stained with ointment. CD16/CD32 and cell surface markers, they were fixed with the Foxp3 Fixation/Permeabilization working solution for 30 min at room tem- In vivo treatment for melanoma perature. Then, they were washed with permeabilization buffer at The B16F10 melanoma tumor model was established by subcutaneous 500g for 5 min and stained with CD16/CD32 in the permeabilization injection of 2 10 B16F10 cells into the right flank of each female buffer for 30 min at 4 C. Then, they were stained with anti-Foxp3-PE C57BL/6 mouse. The mice were randomly divided into five groups for another 30 min at room temperature and resuspended in the flow when their tumor volumes reached about 50 mm.Each group was cytometry staining buffer for analysis. treated with an equal dose of aPDL1 and aCTLA4 (m(ab) = 20 g each For the analysis of cytotoxic T cell lymphocytes, the suspension mouse). The tumors of mice were swiped with different FCS ointment was stained with anti-CD3-APC (Biolegend, Cat. 100206), anti-CD8a- for 12 h, and covered by Transparent film (3 M) to avoid the influence PE(Biolegend, Cat. 100707, anti-CD45-PerCP(Biolegend, Cat. of mice lick. The size of the tumor was measured by a caliper every 103130) and anti-Ki67-FITC (Biolegend, Cat. 652410) for the level of 2 days: Ki67 with the same protocol as the Foxp3. The suspension was also stained with anti-CD45-FITC (Biolegend, Cat. 157214), anti-CD8a-PE Volume = (length width2)/2 (3) (Biolegend, Cat. 100707), anti-GranzymeB-PE-Cy7 (Biolegend, Cat. 372214) and anti-CD3-APC (Biolegend, Cat. 100206) for the level of Growth curves were stopped when the first mouse in the related Granzyme B with eBioscienceT Intracellular Fixation & Permeabili- group was dead, or the first mouse's tumor size exceeded 1000 mm3 zation Buffer Set. by ethics from Soochow University Laboratory Animal Center and For the analysis of IFN- + T cells in spleens, the suspension was Soochow University Ethics Committee. In some cases, this limit was firstly incubated with a stimulation medium with RPMI-1640 (10% FBS exceeded by the last day of measurement, and the mice were imme- and 1% PS), 50 M HEPES solution (Sigma), 1 mM Sodium pyruvate diately euthanized. solution (Sigma), 50 M 2-Mercaptoethanol, 10 g/mL S1 or OVA protein, and 1 eBiosciencem Brefeldin A solution for 6 h and then Immunofluorescence staining stained with anti-CD3-FITC (Biolegend, Cat. 100203), anti-CD4-PerCP For in vitro immunofluorescence staining, HACAT cells were incubated (Biolegend, Cat. 100432), anti-CD8a-PE (Biolegend, Cat. 100707) and in a 24-well plate to form a single cell layer. Then, FCS/IgG was added anti- IFN--APC (Biolegend, Cat. 505810) with eBioscienceTM Intracel- and incubated for another 12 h. After removing FCS-IgG, the Iular Fixation & Permeabilization Buffer Set. For the analysis of IFN- HACAT cells were fixed by 4% paraformaldehyde, blocked by 5% BSA, + T cells in tumors, eBioscienceTM Cell Stimulation Cocktail (plus and stained with an anti-ZO-1 antibody. One hour later, the cells were protein transport inhibitors) (500) was added in the stimulation stained with a secondary antibody and 4, 6-diamidino-2-phenylindole medium to replace protein and Brefeldin A in the above protocol. (DAPI). After mounting, slices were imaged by a confocal fluorescence For the analysis of memory T cells, spleens was collected from microscope (Leica SP5). For in vivo immunofluorescence staining, mice 90 days after the prime-boost and stained with anti-CD3-FITC B16F10 tumors and skin were collected from mice at different time (eBioscience, Cat. I1-0031), anti-CD8-PerCP-Cy5.5 (eBioscience, Cat. points after being swiped with different ointments and embedded in a 45-0081), anti-CD44-PE (eBioscience, Cat. 12-0441), and anti-CD62L-
