Pcl neo mova

Jurkat T (TIB-152), HEK293T (CRL-1573), and B16F10 (CRL-6475) cell lines were purchased from ATCC. Adult leukemia cell lines, MT2, and MT4 were purchased from CellBank Australia (Westmead, NSW, Australia). The retroviral ecotrophic packaging cell line Platinum-E was purchased from Cell Biolabs (San Diego, CA, USA). Cells were maintained in RPMI-1640 or Dulbecco’s modified Eagle medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (FBS; Invitrogen). A stable B16F10 cell line expressing membrane-bound OVA (B16F10-OVA) was produced by transient transfection with pCL-neo-mOVA (Addgene, Cambridge, MA) using Lipofectamine 2000 reagent (Invitrogen) and selected with G418 (InvivoGen; San Diego, CA, USA). Naïve CD3⁺ T cells were purified from mouse spleen and lymph nodes by negative selection using a T-cell enrichment column (R&D Systems). Naïve CD4⁺ and CD8⁺ T cells and CD11C⁺ dendritic cells (DCs) were purified from mouse spleen and lymph nodes by negative selection using an EasySep magnetic separation system (Stemcell Technologies; Vancouver, Canada). To generate mouse T-cell blasts, isolated T cells were incubated in 2 µg/ml anti-CD3/28-coated culture plates with 100 U/ml rIL-2 for 48 h and cultured for an additional 3 days with 100 U/ml rIL-2. The purity of each population was confirmed to be more than 95% by flow cytometry.

B16F10 (CRL-6475) cell lines were purchased from ATCC. A stable B16F10 cell line expressing membrane-bound OVA (OVA⁺B16F10) was produced by transient transfection with pCL-neo-mOVA (Addgene, Cambridge, MA) using Lipofectamine 2000 reagent (Invitrogen) and selection with G418 (InvivoGen, San Diego, CA, USA). For BMDCs cultures, 5 × 10⁶ BM cells were cultured in 10 mL of RPMI supplemented with 20 ng/mL recombinant murine GM-CSF for 7 to 9 days. GM-CSF was added every 3 days. To generate cDC1s, 3 × 10⁶ BM cells were incubated in 3 mL of RPMI supplemented with 200 ng/ mL Flt3-L for 9 days. Flt3-L was added every 2 days and cDC1s (CD11c⁺B220⁻) were isolated by anti-B220 positive selection beads to exclude plasmacytoid DCs (CD11C⁺B220⁺) for further experiments. However, unless otherwise indicated (for Additional file 1: Figs. S1 and 2), we used GM-CSF-induced BMDCs for most of the experiments. Naive CD4⁺ T cells were purified from the mouse spleen and LNs by negative selection using an EasySep magnetic separation system (Stemcell Technologies, Vancouver, Canada). To generate mouse T cell blasts, OTII CD4⁺ T cells were incubated in 2 µg/mL anti-CD3/28-coated culture plates with 100 U/mL rIL-2 for 48 h and cultured further for 3 days with 100 U/mL rIL-2.