Imdm medium

The sequences encoding the chimeric TCRs were synthetized by GeneArt. A Kozak consensus sequence (accgcc) was inserted 5′ to the ATG to optimize the translational starting site, and a STOP codon was inserted at the 3′ of the Cβ. The cDNAs of the α and β TCRs were linked by the 2A peptide and subcloned into the LV pHRSIN-Bx-IRES-EmGFP (kindly provided by Dr. V. Cerundolo, University of Oxford), in which the TCR-coding genes can be followed by IRES and the GFP-coding sequence. The cDNA encoding CD1c was cloned by PCR with specific primers (listed in Supplementary Table 3) and inserted into the LV pHRSIN-Bx-IRES-EmGFP. The production of the lentiviral vectors was performed as described previously^{52 (link)} by the transient transfection of 293T cell line (ATCC) using a second-generation lentiviral vector system. Subconfluent 293T, seeded in a 15 cm dish in IMDM medium (Lonza) supplemented with 10% heat-inactivated FBS (EuroClone), 10U/ml penicillin and streptomycin (Lonza), were transfected by Ca₂PO₄ method with the packaging plasmids (12 μg pMD2.VSV-G, 16.25 μg pCMVΔR8.74, 6.25 μg pRSV-rev) and 32 μg of transfer vector plasmid. After 16 h medium was replaced and 30 h later the supernatant containing the LV were collected and concentrate by ultracentrifugation at 20,000 rpm for 2 h.

Human PBMC were obtained by density gradient centrifugation and human B-cells were negatively sorted from PBMC using EasySep Human B-cell Isolation Kit (StemCell) according to the manufacturer’s instructions. Purified B-lymphocytes were seeded at 1 × 10⁶ cells/ml in IMDM medium (Lonza) supplemented with 20% fetal bovine serum (Deutscher), 1% penicillin/streptomycin (Gibco) and stimulated for 4 days with 100 ng/ml human recombinant CD40L (Enzo Life Sciences) alone or with 50 ng/ml recombinant human IL-4 (Peprotech) or 2 μg/ml Gardiquimod (InvivoGen) or 2.5 μg/ml CpG oligodeoxynucleotide 2006 (InvivoGen) or 100 ng/ml IL-21 (R&D Systems) or 100ng/ml INFγ (R&D Systems) or 50 ng/ml Pam3CSK4 (InvivoGen) or 0.5 μg/ml goat anti-human kappa (Southern Biotech).

As mentioned in the previous section, a fraction of the tumor samples was snap-frozen, another part was cut into small fragments and digested using 1 mg/mL collagenase D (Roche, Basel, Switzerland) and 50 µg/mL DNAse I (Roche) in IMDM medium (Lonza BioWhittaker, Breda, The Netherlands) supplemented with 2 mM GlutaMAX (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 20% fetal bovine serum (Sigma-Aldrich, Saint Louis, Missouri, USA), 1% penicillin/streptomycin (Thermo Fisher Scientific), 1% Fungizone (Thermo Fisher Scientific), 0.1% ciprofloxacin (provided by the Leiden University Medical Center (LUMC) pharmacy), and 0.1% gentamicin (Sigma-Aldrich). Tissue fragments were incubated for 30 min at 37°C interrupted by three mechanical dissociations on a gentleMACS Dissociator (Miltenyi Biotec, Bergisch Gladback, Germany) in gentleMACS C tubes (Miltenyi Biotec), and subsequently processed through a 70 µm strainer (Miltenyi Biotec). In parallel, the lymph node tissue and spleen samples were processed in the same way. Single cell digests and remaining tumor fragments were cryopreserved for analysis and culturing at later stages.
Blood samples were obtained at their respective time points. Peripheral blood mononuclear cells (PBMC) were isolated from patients’ heparinized venous blood by Ficoll-Amidotrizoate (provided by the LUMC pharmacy) gradient centrifugation.

Lineage-negative cells were isolated from WT and Ackr2^−/− BM using LS columns and the Mouse Lineage Cell Depletion Kit (Miltenyi Biotec), according to the manufacturer’s instructions. Negative fraction was stained with Streptavidin-PB, Sca-1, cKit, CD34, and FcγRII/III antibodies and sorted. LSK were seed (1 × 10³/well) in rounded-bottom 96-well plate in IMDM medium (Lonza) supplemented with 10% FCS (Sigma), 1% l-glutamine (Lonza), 20 ng/ml stem cell factor (SCF) (Peprotech), 10 ng/ml interleukin (IL)-6 (Peprotech), and 10 ng/ml IL-3 (Peprotech), as previously described^{54 (link)}. Cells were harvested 3 and 6 days after seeding, stained, and analyzed by flow cytometry.

BxPC-3, Hs766T, Capan-1, MIA PaCa-2, and THP-1 (human monocytic cell line) cell lines were purchased from the American Type Culture Collection. Paca-44, PT45, HPAF and A8184 cell lines were kindly provided by Dr. Piemonti (San Raffaele Scientific Institute). Cell lines were cultured in IMDM 10% fetal bovine serum (FBS) (Lonza) and, in the case of THP-1, β-mercaptoethanol (50 mM) (Sigma). Primary cultures of tumor cells (PCC#353 and PCC#406) and CAFs were established from tumor samples collected at surgery, as described in [10 (link)]. Briefly, tumor pieces were put in culture in IMDM medium (Lonza) plus 10% FBS and CAFs obtained by outgrowth. Alternatively, to obtain distinct cell populations after few passages tumor cells and CAFs were separated with anti-fibroblast Ab-coated beads (Miltenyi Biotec). Primary tumor cells and CAFs were characterized by western blot (WB) for expression of pan-cytokeratin and α-SMA, respectively, as shown in [10 (link)]. Cell lines were periodically tested for Mycoplasma contamination using the MycoAlert™ Mycoplasma Detection kit (Lonza).