Methods to Assess Beta Cell Death Mediated by Cytotoxic T Lymphocytes

Summary

Cell-mediated lymphocytotoxicity (CML) assays can be used to test autoreactive responses and study mechanisms of cell death in vitro. However, using live-cell confocal microscopic imaging techniques with fluorescent dyes, the type and kinetics of cell death as well as the pathways utilized can be studied in greater detail.

Abstract

Type 1 diabetes (T1D) is a T cell mediated autoimmune disease. During the pathogenesis, patients become progressively more insulinopenic as insulin production is lost, presumably this results from the destruction of pancreatic beta cells by T cells. Understanding the mechanisms of beta cell death during the development of T1D will provide insights to generate an effective cure for this disease. Cell-mediated lymphocytotoxicity (CML) assays have historically used the radionuclide Chromium 51 (⁵¹Cr) to label target cells. These targets are then exposed to effector cells and the release of ⁵¹Cr from target cells is read as an indication of lymphocyte-mediated cell death. Inhibitors of cell death result in decreased release of ⁵¹Cr.

As effector cells, we used an activated autoreactive clonal population of CD8⁺ Cytotoxic T lymphocytes (CTL) isolated from a mouse stock transgenic for both the alpha and beta chains of the AI4 T cell receptor (TCR). Activated AI4 T cells were co-cultured with ⁵¹Cr labeled target NIT cells for 16 hours, release of ⁵¹Cr was recorded to calculate specific lysis

Mitochondria participate in many important physiological events, such as energy production, regulation of signaling transduction, and apoptosis. The study of beta cell mitochondrial functional changes during the development of T1D is a novel area of research. Using the mitochondrial membrane potential dye Tetramethyl Rhodamine Methyl Ester (TMRM) and confocal microscopic live cell imaging, we monitored mitochondrial membrane potential over time in the beta cell line NIT-1. For imaging studies, effector AI4 T cells were labeled with the fluorescent nuclear staining dye Picogreen. NIT-1 cells and T cells were co-cultured in chambered coverglass and mounted on the microscope stage equipped with a live cell chamber, controlled at 37°C, with 5% CO₂, and humidified. During these experiments images were taken of each cluster every 3 minutes for 400 minutes.

Over a course of 400 minutes, we observed the dissipation of mitochondrial membrane potential in NIT-1 cell clusters where AI4 T cells were attached. In the simultaneous control experiment where NIT-1 cells were co-cultured with MHC mis-matched human lymphocyte Jurkat cells, mitochondrial membrane potential remained intact. This technique can be used to observe real-time changes in mitochondrial membrane potential in cells under attack of cytotoxic lymphocytes, cytokines, or other cytotoxic reagents.

Protocol

1. Preparation of cells

1. Target cell culture: Culture of the mouse beta cell lines, NIT-1 and NIT-4, was a previously described^1,2 in DMEM supplemented with 10% FBS, 0.02%BSA, Non-essential Amino Acid, 15mM HEPES, 16.5mM Glocose and Penicillin/Streptomycin. For ⁵¹Cr labeling, seed cells in a flat-bottom 96-well culture plate at 5X10⁴ cells/well in 200 μl culture medium. For microscopy experiments, seed cells in a 8-well Lab-Tak II chambered coverglass at 5x10⁴ per chamber in 500 μl culture medium, and allow to grow for 48 hours prior to using in the cytotoxicity experiments.
2. Control T cell line cult....

Discussion

Cytotoxic T cell mediated beta cell death is the main pathophysiology of T1D ⁵. CML assays using ⁵¹Cr release allow us to study the degree of effector-target response ⁶. However, the detailed process and pathways of T cell-mediated beta cell death is still not fully understood. Since mitochondria are critical for beta cell function and death⁷, we focused on mitochondrial changes during the visual CML. Mitochondrial membrane potential dissipation is an early and irreversible.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cr-51	PerkinElmer, Inc.	NEZ030500IMC
PBS	Cellgro	21-040-60
Tetramethyl Rhodamine Methyl Ester (TMRM)	Invitrogen	T668
Picogreen	Invitrogen	P7581
AI4 mimotope	mimotopes.com	amino acid sequence: YFIENYLEL
IL-2	R&D Systems	485-MI
DMEM	Invitrogen	11885
FBS	Hyclone	SH30071.03
Bovine Serum Albumin	Sigma-Aldrich	A8412
HEPES	Cellgro	25-060-Cl
MEM Non-Essential Amino Acids	GIBCO, by Life Technologies	11140
Penicillin/Streptomycin	Gemini Bio Products	400-109
Phenol red-free DMEM	Sigma-Aldrich	D5303
CO2 Incubator	Thermo Fisher Scientific, Inc.	HeraCell 150i	Kept sterile for cell culture
Biological safety cabinet	Thermo Fisher Scientific, Inc.	Forma 1440
Disposable culture tubes, 6x50 mm Lime Glass	Fisher Scientific	14-958-A
Gamma Counter	PerkinElmer, Inc.	Wizard 1470 Automatic Gamma Counter
Confocal microscope	Carl Zeiss, Inc.	LSM 510 Meta Confocal	With motorized stage and live cell chamber
Pipette (1ml, 200μl, 20μl,10μl, 2μl)	Eppendorf
Tubes (Amber)	Fisher Scientific	50819772
Centrifuge	Sorvall, Thermo Scientific	75004377
Hemacytometer	Fisher Scientific	267110
Upright Microscope	Carl Zeiss, Inc.	Axioskop40
NOD.Cg-Rag1tm1Mom Tg(TcraAI4)1Dvs/DvsJ Mice	Jackson Laboratory	004347
NOD.Cg-Rag1tm1Mom Tg(TcrbAI4)1Dvs/DvsJ Mice	Jackson Laboratory	004348
NOD-AI4α/ β F1 mice	N/A	N/A	Bred in UF animal facility