Eh12.2h7

Single cell suspensions were prepared from lymph node and gastrointestinal mucosal biopsies as previously described [10] and stored in liquid nitrogen. Cryopreserved samples of PBMC, LNMC and GMMC were thawed at 37 °C in RPMI 1640 containing 10% FBS and benzonase (Millipore) at 50 U/mL. Cells were re-suspended in 1X PBS containing Aqua LIVE/DEAD Fixable Dead Cell Stain (Life Technologies) for 20 min at RT in the dark. Cells were washed and re-suspended in wash buffer containing the following fluorescently conjugated antibodies at the indicated dilutions: CD45 (D058-1283, BD #563861,1:80), CD28 (CD28.2, BD #5622596, 1:200), CD4 (L200, BD #563913, 1:100), CCR7 (150503, BD # 561271, 1:40), CD95 (DX2, BD #555674, 1:100), CD3 (SP34-2, BD # 557917, 1:40), and CD8 (SK1, BD #560179, 1:80), CXCR5 (MU5UBEE, Thermo-Fisher # 25-9185-42, 1:30), and PD-1 (EH12.2H7, Biolegend # 329919, 1:100). Cells were sorted in purity mode, using a BD FACSAria II. Sorted CD4+ T cells were FSC singlets, live, CD45+CD3+CD4+CD8− lymphocytes and subsets were defined as follows. For GMMC: naïve (CD28+CD95−) and memory (CD95+). For LNMC: naive (CD95−CD28+), T follicular helper cells (Tfh, CD28+CD95+CXCR5+PD-1^high), central memory (CD95+CD28+, non-Tfh), effector and effector memory (CD95+CD28−). CXCR5+ events were determined by a CXCR5 FMO antibody stained sample.

Peripheral blood samples to measure CD8 T lymphocyte activation were obtained at baseline and at defined times during the course of the study. Cryopreserved PBMC samples from pre-treatment and post-treatment time points were thawed and stained with master mix of antibodies for surface stains including CD8 (ebioscience, San Diego, CA; RPA-T8), PD-1 (Biolegend, San Diego, CA, USA; EH12.2H7), CD45RA (Biolegend; HI100), CD27(BD, San Diego, CA, USA; L128) and intracellular stains for CTLA4 (BD; BNI3), Eomes (ebioscience; WD1928), Tbet (biolegend; 4B10) and Ki67(BD; B56). Permeabilisation was performed using the Foxp3 Fixation/Permeabilisation Concentrate and Diluent kit (eBioscience). Cells were resuspended in 1% paraformaldehyde until acquisition on a BD Biosciences LSR II cytometer and analysed using FlowJo (Tree Star, San Diego, CA, USA).

Surface and intracellular staining were performed as described previously (38 (link)). For the surface staining of human cells, specific antibodies against human CD3 (OKT3; BioLegend), CD4 (OKT4; BioLegend), CD8 (BW135/80; Miltenyi Biotec), CD45RO (UCHL1; BioLegend), CCR7 (G043H7; BioLegend), CD28 (CD28.2; BioLegend), and PD-1 (EH12.2 H7; BioLegend) were used. For intracellular staining, antibodies against human GzmA (CB9; BioLegend), GzmB (QA16A02; BioLegend), and perforin (B-D48; BioLegend) were used. Dead cells were determined by Fixable Viability Dye (Thermo Fisher) staining and excluded from analysis. The numbers of CD3⁺ CD4⁺ and CD3⁺ CD8⁺ T cells in the blood were calculated from lymphocyte counts measured in a certified clinical laboratory for every patient.
Data were acquired on an LSR II flow cytometer (Becton, Dickinson) from 250,000 to 300,000 lymphocyte-gated events per sample. Analyses were done using FACSDiva software (Becton, Dickinson) and FlowJo software (Becton, Dickinson).

Stainings were performed in 50 µL in 96-well plates. Live/death staining was performed with fixable eFluor506 viability dye (1:2000, 65-0866-14, ThermoFisher) in PBS (30 min, RT). Antibody stainings were performed in PBA (PBS + 5% FBS + 0.01% NaN₃) (30 min, 4°C). DC purity was assessed by antibodies against CD141 (1:10, APC, AD5-14H12, 130-113-314, Miltenyi) and XCR1 (1:10, PE, S15046E, 372604, BioLegend), and T cell phenotype was assessed by antibodies against CD25 (1:100, AF488, BC96, 302616, BioLegend), CD127 (1:100, PE/Cy7, A019D5, 351320, BioLegend), CD137 (1:100, PE, 4B4-1, 555956, BD Pharmigen), CD279 (1:100, BV421, EH12.2H7, 329920, BioLegend), and TIGIT (1:100, BB700, 741182, 747846, BioLegend). Cells were washed 2× with PBA before acquisition on a FACSLyric (BD).

Female NSG mice age 6-8 weeks were purchased from Jackson Laboratory, Bar Harbor, ME, USA. 3x10⁶ SKOV3- MUC16^ecto/-Luc, or OVCAR3 tumor cells were injected intraperitoneally (i.p.) on D0, and animals were untreated, treated with T-cells intravenously (i.v) alone or treated with a combination of T-cells and 5μg MUC16^ecto- BiTEDs on day 7. Animals in the BiTEDs treatment group received additional 5μg MUC16^ecto- BiTEDs treatment on days 9, 11, 14, 16, and 18 for a total of 6 treatments over 2 weeks. Tumor-bearing mice were injected intraperitoneally with D-Luciferin (Goldbio Technology) (150 mg/kg) and after 10 min were imaged under isofluorane anesthesia. Bioluminescent imaging was achieved using the Caliper IVIS imaging system and analyzed with Living Image 4.0 software (PerkinElmer). Image acquisition was achieved using a 25 cm field of view, medium binning level and 60s exposure time. Animals treated with αPD-1 blocking antibody (BioLegend EH12.2H7) received 250μg injected i.p on days 7, 14, 21 and 28 (weekly injections x 4 weeks) after tumor inoculation (day 0). Animals treated with αVEGF blocking antibody (Invivo Gen, hvegf-mab1) received 5 mg/kg i.p injections on days 7, 11, 14, 18, and 21 after tumor inoculation. All mice were monitored for survival and were euthanized when showing signs of distress.