Mice
LFA-1 knockout mice (LFA-1−/−) were purchased from The Jackson Laboratory (B6.129S7-Itgaltm1Bll/J, number:005257. C57BL/6J mice were obtained from the Guangdong Medical Laboratory Animal Center, Guangzhou, China. The LFA-1 genotype was identified using polymerase chain reaction (PCR) with specific primers (Supplementary Table 1 and S. Figure 1). Mice were housed under specific pathogen-free conditions in the Animal Center of Guangdong Pharmaceutical University. The mice were kept at standard room temperature and humidity, alternating light and dark (12 h:12 h). We raised and managed the mice strictly according to the standards of the animal experimental center of Guangdong Pharmaceutical University (license: SCYK(YUE)2017 − 0125). All animals experiment protocols complied with Guangdong Pharmaceutical University and were approved by the Animal Ethics Committee of Guangdong Pharmaceutical University (Animal Ethics Approval Number: gdpulacspf 2021002).
Cell Culture
B16-F10 melanoma cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) were cultured in Dulbecco's Modification of Eagle's Medium (DMEM) containing 10% fetal bovine serum (FBS; GIBCO) and 1% penicillin-streptomycin. The cell was incubated in a humidified chamber containing 5% CO2 at 37°C, and it was confirmed with no mycoplasma contamination by PCR detection.
H&E and IHC
Thymus and spleen tissue sections (2–3 µm) were prepared by 4% Formalin-fixed, paraffin-embedded. After deparaffinization, sections were stained with hematoxylin for 3 minutes and eosin for 30 seconds and were then dried and sealed with neutral resin. IHC staining was performed as described on the Abcam website. Antigen retrieval was performed in citrate buffer (pH 6.0) at high temperature and pressure for 15 minutes. Next, the tissue sections were incubated overnight at 4°C with primary antibodies, including anti-CD11a (1:100, Abcam, ab186873, USA), anti-CD3 (1:100, Abcam, ab16669, USA), anti-CD4 (1:100, CST 25229s) and anti-CD8 (1:100, CST 98941T). Then, the sections were treated with secondary antibodies (1:200, ZSGB-BIO, ZB-2301) at 37°C for one hour and color was developed with DAB chromogenic solution. The sections were counterstained with hematoxylin, dehydrated in graded alcohols, cleared in xylene, and sealed with neutral resin. Digital imaging was performed using a microscope (Olympus CX31, New York, USA).
qPCR
Total RNA was extracted from mouse thymus with TRIzol reagent (Invitrogen, USA) according to the instructions. All reverse transcription reactions were set up using an EVO M-MLV RT Kit (Accurate Biology AG, China). Real-time PCR was performed using the SYBR® Green Premix Pro Taq HS qPCR Kit (AG11701, Accurate Biology AG) on a LightCycler 96 real-time fluorescent quantitative PCR detection system (Roche). Data were normalized to the endogenous control GAPDH, and fold changes were calculated using relative quantification (2^-∆∆Ct).
Flow Cytometry
Single-cell suspensions of mouse thymus and spleen were prepared, and red blood cells in the spleen were lysed using red blood cell lysis buffer. Each sample was stained with specific antibodies in various channels, including FITC channel anti-mouse CD44 antibody (BioLegend, 103005, USA), APC channel anti-mouse CD25 antibody (BioLegend,101909, USA), PerCP-Cyanine5.5 anti-mouse CD3 antibody (BioLegend,100218, USA), PE channel anti-mouse CD4 antibody (BioLegend, 100408, USA), and Brilliant Violet 421TM anti-mouse CD8a antibody (BioLegend, 100738, USA). Samples were incubated at 4°C for one hour, washed twice with PBS, and then analyzed on an LSRII flow cytometer (BD Biosciences).
Western Blotting
Thymus tissues were lysed in a lysis buffer containing 1% protease and phosphatase inhibitor on ice and centrifuged, and the supernatant was collected as the protein sample. Protein quantification was performed using the BCA protein assay. Equal amounts of protein were loaded, and after electrophoresis, transfer, and blocking, the membranes were incubated with primary antibodies overnight at 4°C, followed by secondary antibody incubation. The primary antibodies: anti-CD4(1:100, CST 25229s), anti-CD8(1:100, CST 98941T), anti-Erk (CST,4695T), p-Erk (CST,4370T), SAPK/JNK(CST,9252T), p-SAPK/JNK(CST,4668T), p38(CST,8690), P-P38(CST,8203T), GAPDH(CST,5174S).
Mice model of LFA-1 Inhibitor BIRT377
BIRT377 (Tocris; Cat# 4776) in anhydrous ethanol (22.156 mg/mL) was stored at 2–8 ℃. The vehicle was sterile water containing ethanol. On the day of injection, the vehicle was heated in bath at 37 ℃, and BIRT377 concentration is 500 ng/20 µL for i.v. injection[12]. Animals were injected within one hour of BIRT377 dilution. BIRT377 (20 µL per mouse) was injected through the tail vein into mice in the experimental group (n = 6), and mice in the control group (n = 6) were injected with 20 µL of vehicle. Three times a week for four weeks, the mice were euthanized, and thymus tissues were then excised for subsequent analysis.
Tail vein metastasis model
B16F10 melanoma cells (2×10^6 cells per mouse) were injected subcutaneously into LFA-1−/− mice (n = 6) and LFA-1+/+ mice (n = 6) through the tail vein. Mouse body weight was recorded once a day. After 2 weeks, these mice were sacrificed, and their thymus tissues were harvested and preserved for further analysis.
RNA sequencing
The total RNA of thymus of LFA-1−/− mice (n = 4) and LFA-1+/+ mice (n = 4) were extracted, and cDNA libraries were constructed, which was sequenced on the Illumina sequencing platform. The DESeq algorithm was utilized to filter the differentially expressed genes.
Statistical analysis
Unpaired tests were performed on the statistical data using GraphPad Prism 9 software, p value of less than 0.05 was considered statistically significant. Data are shown as the mean ± standard deviation (SD) values.