Anti mica b

T cells were resuspended in complete medium with a density of 1×10⁶ cells/ml and were cultured with anti-CD28 (2 µg/ml) and IL-2 (10 U/ml) in a 24-well plate pre-coated with 5 µg/ml anti-CD3. Subsequently, T cells were co-cultured with or without 100 µl serial diluted tumor sup for 24 h. For neutralizing sNKG2DL, single and combined soluble NKG2DL neutralizing antibodies (1, 2, or 5 µg/ml anti-MICA/B, ULBP1, ULBP2, and/or ULBP3) were added to T cells and to the tumor cell supernatant co-culture system. Then, these T cells were harvested and prepared for flow cytometric analysis. Neutralized antibodies (anti-MICA/B, anti-ULBP1, anti-ULBP2, and anti-ULBP3) were purchased from R&D Systems, Inc. (cat. nos. MAB13001, MAB1380, MAB1298 and MAB1517, respectively).

In selected cases (2 mice per group) we explored by IHC the presence of CAR+CIK in the explanted tumors along with the expression of NKG2D ligands (MIC A/B; ULBPs 2,5,6) and CD44v6.
Sections from formalin fixed, paraffin-embedded samples were cut into 3-μm thick sections. The tissue slides were treated according to standard immunohistochemistry procedures. In short, the slides were permeabilized in 0.1% Triton X-100 and 0.3% Tween 20 (Sigma-Aldrich) in TBS, treated for 30 min with 1% hydrogen peroxide to quench endogenous peroxidases. The slides were incubated with individual primary antibodies overnight at 4°C inside a moist chamber. After rinsing in PBS, a secondary antibody was added. Secondary HRP-conjugate antibodies (EnVision; DakoCytomation) were used for immunohistochemistry and the reaction was visualized with DAB chromogen (DakoCytomation Liquid DAB Substrate Chromogen System, Dako). The tissues were counterstained with Mayer hematoxylin (Bio-Optica), mounted on glass slides and visualized with a BX-60 microscope (Olympus) equipped with a color Qicam Fast 1394-digital CCD camera (12 bit; QImaging). The tissues were stained with the following primary antibodies: anti–CD3 (DAKO); anti-MIC A/B (clone #159207, R&D SYSTEM, BIOTECHNE BRAND); anti-ULBP 2/5/6 (R&D SYSTEM, BIOTECHNE BRAND); anti-CD44v6 (clone #SP37, Acris, an OriGene Company).

Tissue cores (0.6 mm) from 59 adenocarcinoma (ADC), 67 squamous cell carcinoma and 14 normal adjacent human lung tissues purchased from US Biomax were fixed in 10% (vol/vol) neutral buffered formalin for construction of a tissue microarray (TMA). Sections of the TMA (4 μm) were stained for MICA/B expression as described using anti-MICA/B (R&D Systems)¹⁷ at a dilution of 1:100, for 2 h at room temperature with the Ventana automated immunostainer Discovery XT (Ventana Medical Systems, Tucson, AZ). As a negative control, non-immune mouse sera were used, omitting the MICA/B antibody during the primary antibody incubation step. The slides were read by a certified pathologist and co-author (DC) in a blinded fashion and the MICA/B protein expression levels were measured using the Allred semiquantitative scoring system.⁴¹