Esophageal Cancer Responsive to the Combination of Immune Cell Therapy and Low-dose Nivolumab

Two Case Reports

Rishu Takimoto; Takashi Kamigaki; Takuji Gotoda; Toshimi Takahashi; Sachiko Okada; Hiroshi Ibe; Eri Oguma; Shigenori Goto


J Med Case Reports. 2021;15(191) 

In This Article

Patients and Methods


This study was conducted from September 2017 to March 2019. The patients were administered ACT twice at a 2–3-week interval followed by 0.3–0.6 mg/kg nivolumab and ACT four times at 2-week intervals. The institutional review board of the hospital approved the study, and written informed consent was obtained from the patients (clinical study number, UMIN000028756: an exploratory clinical trial on the safety of combination therapy with effector cell therapy and immune checkpoint inhibitors for patients with malignant tumor).

Preparation for ACT

Activated lymphocytes were generated as previously described.[14] In brief, peripheral blood mononuclear cells (PBMCs) were isolated from the patients' peripheral blood using a Vacutainer (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). The PBMCs were activated in a culture flask with an immobilized monoclonal antibody to CD3 (Jansen-Kyowa, Tokyo, Japan) in HyMedium 930 (Kohjin Bio, Saitama, Japan) containing 1% autologous serum. The PBMCs were then cultured for 14 days with 700 IU/ml IL-2 (Proleukin®; Chiron, Amsterdam, Netherlands), after which 3–10 × 109 cells were harvested and suspended in 50 ml normal saline for intravenous injection. To prepare a dendritic cell (DC) vaccine, PBMCs were collected from the patients by leukapheresis and allowed to adhere to a plastic culture flask. The adherent cell fraction was used for DC culture for 6 days in a medium supplemented with 50 ng/ml IL-4 (Primmune Corp., Osaka, Japan) and 50 ng/ml granulocyte macrophage colony-stimulating factor (GM-CSF) (Primmune Corp.) to generate immature DCs. The DCs were pulsed with antigenic tumor-specific peptides or an autologous tumor lysate and allowed to mature for 24 h. After the culture, 1–10 × 10[6] mature DCs were harvested and suspended in 1 ml normal saline for subcutaneous injection, then cryopreserved until the day of administration.

Flow Cytometry of PBMCs

Heparinized whole blood was collected from the patients. The phenotype of PBMCs was analyzed by whole-blood staining with OptiLyse C lysis solution.[16] Absolute cell number was determined using Flow-Count™ fluorospheres as internal standard beads. OptiLyse C, Flow-Count beads, and monoclonal antibodies (mAbs) against CD3, CD4, CD8, CD14, CD16, CD19, CD45, CD56, TCR pan αβ, TCR pan γδ, and TCR Vγ9 were purchased from Beckman Coulter (Brea, CA, USA). Lymphoprep™ (Axis-Shield PoC AS, Oslo, Norway) was used with gradient centrifugation to isolate the PBMCs. For Foxp3 staining, the PBMCs were fixed and permeabilized using a fixation/permeabilization kit (BioLegend, San Diego, CA, USA) in accordance with the manufacturer's protocol, and Foxp3 was stained with anti-Foxp3 mAb (clone 259D, BioLegend). For intracellular cytokine production assay, the PBMCs were suspended in a conditioned medium supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Grand Island, NY, USA) containing phorbol 12-myristate 13-acetate (Sigma-Aldrich, St. Louis, MO, USA), ionomycin (Sigma-Aldrich), and brefeldin A (Sigma-Aldrich). The cells were incubated at 37 °C in a humidified atmosphere with 5% CO2 for 4 h for the IFN-γ/IL-4 assay. After the activated cells were fixed and permeabilized, intracellular cytokines were stained with an anti-IFN- γ or anti-IL-4 (Beckman Coulter) antibody. A Cytomics FC500 or a Gallios flow cytometer (Beckman Coulter) was used for data acquisition, and the data were analyzed using CXP or Kaluza software (Beckman Coulter).


To measure the PD-L1 expression level in cancer tissue, PD-L1 IHC 22C3 pharmDx (Agilent Technologies, Santa Clara, CA, USA) was used in accordance with the manufacturer's instructions.

Microsatellite Instability

Microsatellite instability (MSI) was determined using a modified version of the pentaplex polymerase chain reaction (PCR) assay as described by Buhard et al.[25] using five markers (NR-21, BAT-26, BAT-25, NR-24, and MONO-27) and analyzed using an ABI PRISM 3100 genetic analyzer (Applied Biosystems, Foster City, CA, USA) in accordance with the manufacturer's instructions.