Mfoxp3 apc

Excised tumor and spleen tissues were fixed in 4% formaldehyde overnight and then cryopreserved in 30% sucrose for 24 h. Tissue samples were embedded in Tissue-Tek OCT (Sakura Finetek, Alphen aan den Rijn, NL) and sectioned at 8 μm using a cryostat (Leica Microsystems, Diegem, BE). Sections were stained with antibodies directed against murine CD4, CD8, and FoxP3. Briefly, sections were first blocked with 0.2% (v/v) Triton X-100 (Sigma), 10% (w/v) goat serum, 5% rat serum, and 2% BSA in PBS for 1 h at room temperature. Subsequently, the primary antibodies (rat CD8a-FITC 1:500 [clone 53–6.7, BioLegend] and CD4-FITC 1:500 [clone GK1.5, BioLegend] and mFoxP3-APC 1:500 [clone FJK-16s, eBioscience] were applied to the slides for 1 h at room temperature protected from the light. After washing with PBS, the sections were mounted using Vectashield Mounting Medium (Vector Laboratories, Burlingame, CA, USA) containing DAPI to visualize the cell nuclei. The slides were imaged using a structured illumination AxioImager microscope (Zeiss, Oberkochen, GE). The results are presented in Supplemental Information.

Before flow cytometry analysis, splenocytes were lysed with ACK lysis buffer (Lonza) and washed with PBS. Isolated tumors were incubated in media with 1 mg/mL Collagenase type II from Clostridium hystoliticum (Sigma) for 1 h and later washed with PBS and counted. Prepared cells were stained with murine antibodies for 1 h at 4⁰C protected from light. The following antibodies were used in the study: Live/Dead-eFluor 506 (eBioscience), Fc block (mCD16/CD32, BioLegend), CD3-APC-Cy7 (BioLegend), CD8a-FITC (BioLegend), CD4-FITC (BioLegend), mFoxP3-APC (eBioscience). All data were collected on a FACSverse flow cytometer and analyzed using CaseViewer software (3DHISTECH Ltd., Budapest, HU).