Macs system

CD4⁺ and CD8⁺ T cells were purified from the spleens of the mice immunized with MMC-treated MC38 CRC cells with 40 μg of rGRA6Nt adjuvant at 2 weeks after the second immunization using anti-CD4 and anti-CD8 antibody-coated micromagnetic beads using MACS system (Miltenyi Biotech, Gaithersburg, MD, USA) [22 (link),30 (link)]. We routinely purify these T cells using this method, and the purity of these CD4⁺ and CD8⁺ T cells with this purification method is consistently >95%. The purified T cells were suspended in RPMI1640 medium (MilliporeSigma) containing 10% FBS (Cytiva, Vancouver, BC, USA) and 2 mM L-glutamine and cultured in 96-well culture plates at 5 × 10⁵ cells/well with and without MMC-treated MC38 CRC cells at 1 × 10⁵ cells/well for 72 h. As a control, CD4⁺ and CD8⁺ T cells from unimmunized mice were cultured with and without MC38 cells in the same manner. The concentration of GzmB and IFN-γ in their culture supernatants were measured using commercial kits from R&D Biosystems (Mineapolis, MN, USA) and Meso Scale Discovery (Rocksville, MD, USA), respectively, by following manufactures’ manuals.

Immunomodulation assays were performed as previously described [14 (link)]. Briefly, peripheral blood mononuclear cells (PBMC) were obtained by Ficoll-Hypaque gradient centrifugation of peripheral blood from healthy donors with informed consent. Purification of T lymphocytes (>95% purity) was performed by positive selection using the MACS system (Miltenyi Biotec). A cocktail of phytohemagglutinin (PHA, 5 μg/mL; Remel Europe, Kent, UK) and interleukin-2 (IL-2, 20 U/mL; Biotest AG, Dreieich, Germany) was used to activate T lymphocytes. Activated T cells and stimulated or unstimulated FSK-MSCs were plated in cocultures at both cell ratios: 1/80 and 1/5 for 5 days.

PBMCs were separated according to the method of Miyasaka and Trnka [28 (link)], and CD4⁺ T-cells were purified using the MACS System (Miltenyi Biotech, Inc., Auburn, CA, USA). Briefly, PBMCs were incubated with the ILA11A monoclonal antibody (mouse anti-bovine CD4; VMRD, Inc., Pullman, WA, USA) and captured with anti-mouse IgG monoclonal antibodies conjugated to magnetic beads. Magnetic bead-bound cells were then separated on a MACS LS column (Miltenyi Biotech, Inc.). The purity of CD4⁺ T-cells was 85–89%.

Cells were separately obtained from the bone marrow and spleen. Following cell isolation, red blood cells were lysed using red blood cell lysis buffer (Hybri-Max, Sigma-Aldrich Chemical Co.). The cells were resuspended in 100 μl of a 1% FBS solution in PBS and incubated with anti-CD11b (FITC-conjugated), anti-Gr1 (APC-conjugated), anti-Ly-6G (APC-conjugated) and anti-Ly-6C (FITC-conjugated) antibodies. All antibodies used were obtained from the MACS system (Miltenyi Biotec, Germany). The cell pellets were resuspended in 400 μl of a 1% FBS solution in PBS and analyzed by flow cytometry (Navios analyzer, Beckman Coulter, Inc., FL, USA). To separate MDSCs, the cells were labeled with fluorescent antibodies against CD11b and Gr1 and sorted on a flow cytometer/sorter (FACSAria cell sorter, BD Biosciences, USA).

Human CD34+ cells for humanizing NOD.SCIDγc−/− mice were immunomagnetically isolated according to manufacturer’s instructions (Miltenyi MACS system). Briefly, RBC were depleted by Ficoll density centrifugation, CD34+ cells were enriched using CD34+ CliniMACS reagent and autoMACS (both Miltenyi Biotec GmbH, Bergisch–Gladbach, Germany). Purity of CD34+ cells was 90.9% and the residual T-cell frequency was 0.18%.
The murine NIH3T3 fibroblast cell line was engineered to express a luciferase construct under the CXCL12 promoter alone or together with a BMAL1 expression cassette under regulation of a CMV-promoter (details see below). For analysis of the murine hematopoietic compartment, blood draws were done on awake mice from the facial vein. BM and spleen were harvested after cervical dislocation. BM cells were recovered by flushing femurs or tibias with bovine serum albumin-substituted phosphate-buffered saline (PBS–BSA 0.5%) and splenocytes were isolated by gentle blunt extrusion from the capsule and resuspension in PBS–BSA (0.5%). For most of the in vitro studies (migration, flow cytometry, colony assay), cells were washed and erythrocytes were lysed with ammonium chloride lysis buffer (Sigma–Aldrich, St Louis, MO, USA; or BD Biosciences, San Jose, CA, USA) prior to the assay performance.