Mouse cd4 t cell isolation kit

Naïve CD4⁺ T cells were purified from splenocytes using mouse CD4⁺ T cell isolation kit (Stemcell Technologies), and further sorted by FACS Aria II (BD Biosciences). 24-well plates were coated with 40 μg/ml anti-hamster antibody (MP Biomedical) at 37°C for 4 hours. For iTreg differentiation, naïve CD4⁺ T cells were cultured in T cell media IMDM (Sigma-Aldrich) supplemented with soluble 0.25 μg/ml anti-mouse CD3 (145–2C11), 1 μg/ml anti-mouse CD28 (37.51), 2 μg/ml anti-mouse IL-4 (11B11), 2 μg/ml anti-mouse IFN-γ (XMG1.2) and 5 ng/ml TGF-β, with (Th17) or without (iTreg) 20 ng/ml IL-6, for 3.5 days. For human cells, naïve T cells were enriched using the human CD4⁺ T cell isolation kit (Stemcell) from human PBMC cells and further sorted (CD3⁺CD4⁺CD45RA⁺CD25⁻). For iTreg differentiation, the sorted naïve human CD4⁺ T cells were seeded into 48-well plates precoated with 1 μg/mL anti-CD3 antibody (clone HIT3a), and cultured in RPMI media (Sigma-Aldrich) supplemented with IL-2 (10 ng/ml), soluble anti-CD28 (1 μg/ml; clone CD28.2) and 5 ng/ml TGF-β for 6 days. In some experiments, FICZ was added at a concentration of 200 nM as indicated in the text.

Cells were purified either from fresh naive splenocytes or following culture of splenocytes with Us/o + CBD. For cultured cells, splenocytes (6 × 10⁶ cells/ml, 6 ml/well) were seeded in a 6-well flat bottom plate and treated with CBD (10 μM) for 30 min followed by Us/o stimulation for 5 days. CD4⁺CD25⁺ Tregs were isolated using the Mouse CD25 Regulatory T cell Positive Selection Kit (Stemcell, Vancouver, BC, Canada) and CD4⁺CD25⁻ T cells were obtained using Mouse CD4⁺ T Cell Isolation Kit (Stemcell) according to the manufacturer's protocol. Briefly, CD4⁺CD25⁺ cells were initially purified by positive selection, followed by purification of the CD4⁺CD25⁻ T cells from the decanted cells. The purity of CD4⁺CD25⁺ and CD4⁺CD25⁻ T cells was consistently higher than 80% as assessed by immunofluorescence staining for viability, CD4 and CD25.

For the isolation of CD4⁺ T cells from cLN, cLNs were harvested from ligature mice (day 14) and the mouse CD4⁺ T cell Isolation Kit (STEMCELL Technologies) was used to isolate cells from single cell suspensions prepared from the cLNs. For the preparation of the bone marrow–derived dendritic cells (BMDMs), murine bone marrow cells were cultured in the RPMI/10% FBS containing recombinant murine 10 ng/mL GM-CSF (PeproTech, Rocky Hill, NJ) for 6 days. Loosely attached DC cells were then collected using the biotin selection kit (STEMCELL Technologies) and enriched with biotinylated CD11c monoclonal antibody (Thermo Fisher Scientific, Waltham, MA). BMDCs were pulsed with heat-denatured bacterial antigens for 18 h at 37°C. Antigen-pulsed BMDCs and cLN-derived CD4⁺T cells were co-cultured for 24 h at 37°C. Cytokines in supernatants were quantified by ELISA.

To deplete CD8 T Cells, 200ug/mouse anti-CD8a antibodies (clone: 53-6.7; InVivoMab) were diluted in PBS, and injected intraperitoneally every three days, 10 days prior to tumor initiation until the end point of experiments. For adoptive transfer, naïve CD8⁺ T cells, CD4⁺ T cells and/or B cells were separated from spleens by negative selection with EasySep Mouse CD8⁺ T Cell Isolation Kit, Mouse CD4⁺ T Cell Isolation Kit and/or Mouse B Cell Isolation Kit, respectively (STEMCELL technologies). For rescue experiments, 1 x 10⁶ SW_HEL or WT C57BL/6 B cells were transferred into age- and sex-matched uMT mice, and 1 x 10⁵ SMARTA or OT-II T cells were transferred into age- and sex-matched CD4-cre Bcl6^fl/fl mice, with 2 x 10⁵ KP-HELLO s.c. implanted one day later. For co-transfer experiments, CD45.1/CD45.2 or Thy1.1/Thy1.2 allotype-marked P14 (5 x 10⁵/mouse), SMARTA (1 x 10⁵/mouse) and SW_HEL (1 x 10⁵/mouse) cells were co-transferred into naïve CD45.2/CD45.2 Thy1,2/Thy1.2 mice one day prior to tumor initiation.