Degranulation Response in Cytotoxic Rat Lymphocytes Measured with a Novel CD107a Antibody

Abstract

Measuring degranulation through CD107a expression has become an advantageous tool for testing the functional capacity of cytotoxic cells. Such functional studies have been hampered in the rat by the lack of a suitable anti-rat CD107a antibody. In this study, we report a novel hybridoma generated by immunizing Armenian inbred hamsters with transfected Chinese hamster ovary cells expressing CD107a. The SIM1 clone exhibited specific reactivity with CD107a and measured degranulation from natural killer (NK) cells stimulated with target cells or mAb crosslinking of their activating receptors. Degranulation in IL-2-activated NK cells could also be measured, when using low effector to target ratios. SIM1 also stained activated CD8, but not CD4 T cells. This report characterizes the degranulation response in cytotoxic rat cells with a new antibody against rat CD107a.

Keywords: CD107a; cytotoxicity; degranulation; functional assay; lymphocyte; rat.

Figure 1

Generation of an anti-rat CD107a monoclonal antibody (SIM1). (A) Schematic vector map of the pMX expression vector with leader peptide, FLAG-tag, CD107a insert, and the transmembrane domain of CD8. (B) Stably transfected Chinese hamster ovary and BWZ.36 cells were tested for cell surface expression of FLAG-tagged CD107a using an anti-FLAG antibody (M2) (shaded histograms). Negative control is secondary antibody alone (transparent histogram). (C) BWZ cells transfected with CD107a-FLAG or irrelevant antigen (NKR-P1F-FLAG) were stained with hamster control serum, SIM1 supernatant, or an anti-FLAG antibody (M2).

Figure 2

SIM1 measures degranulation from natural killer (NK) and CD8 T cells. (A) Percent degranulating cells were measured using anti-CD107a antibodies SIM1, H4A3, and LS-C8580, with or without YAC-1 target cells as a stimulus. One representative experiment of four is shown (B). Staining of the BWZ.CD107a-FLAG cell line with SIM1, H4A3, LS-C8580, or ID48 antibody clones. BWZ.36 cells transfected with irrelevant FLAG-tagged antigen (BWZ.NKR-P1G-FLAG) was included as a negative control, (C) enriched NK cells were stimulated with plate bound anti-NKp46, anti-NKR-P1A, or mouse IgG1 as isotype control, and SIM1 was used to measure percentage degranulating cells. NKp46⁺ (Wen23) or NKR-P1A^bright (3.2.3) cells were gated on. (D) TCR⁺ cells were sorted (purity 99%) and cultured in IL-2 for 2–3 days. The cells were then stimulated with plate bound anti-CD3 and anti-CD2 or mouse IgM isotype control for anti-CD3. The lower panel shows stimulation with anti-CD3 and anti-CD2 and staining with a SIM1 isotype control (FITC hamster IgG polyclonal). T cells were sorted using R73-Alexa647 and 3.2.3-PB antibodies, gating on R73⁺/3.2.3⁻ cells. Data shown are from one representative experiment of three separate independent experiments.

Figure 3

Low effector:target ratios are important to ensure adequate sensitivity in degranulation assays with rat NK cells. (A) Degranulation of LAK cells cultured 8–10 days in IL-2 against YAC-1 targets at different effector:target ratios. (B) Cytotoxicity of IL-2 cultured LAK against YAC-1 target cells was confirmed with the chromium release assay completed on days 8–10. All graphs are cumulative of three or more separate experiments with bars showing SD.