Sep 22, 2021
MAIT Cell Adoptive Transfer
- Timothy S C Hinks1,
- Bonnie van Wilgenburg2,
- Huimeng Wang3,
- Liyen Loh3,
- Marios Koutsakos3,
- Katherine Kedzierska3,
- Alexandra J. Corbett3,
- Zhenjun Chen3
- 1Respiratory Medicine Unit, Nuffield Department of Medicine Experimental Medicine, University of Oxford, Oxfordshire, UK;
- 2Peter Medawar Building for Pathogen Research and Translational Gastroenterology Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK;
- 3Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Parkville, Australia
- Springer Nature Books
Protocol Citation: Timothy S C Hinks, Bonnie van Wilgenburg, Huimeng Wang, Liyen Loh, Marios Koutsakos, Katherine Kedzierska, Alexandra J. Corbett, Zhenjun Chen 2021. MAIT Cell Adoptive Transfer. protocols.io https://dx.doi.org/10.17504/protocols.io.bmgxk3xn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
Created: September 17, 2020
Last Modified: September 22, 2021
Protocol Integer ID: 42231
Keywords: Virus, MAIT cell, Flow cytometry, MR1-tetramer, Infection, Mouse ,
Abstract
This is part 3.2 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols.
Collection Abstract: MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation.
Attachments
Materials
For materials, please refer to the Guidelines section of the '"Study of MAIT Cell Activation in Viral Infections In Vivo" collection.
Safety warnings
Personal protective equipment (PPE) should be worn at all times (gloves, lab coat, & eye protection) (see Notes 3 and 4).
For hazard information and safety warnings, please refer to the SDS (Safety Data Sheet).
Before start
As MAIT cells are to be used for adoptive transfer, all procedures should be performed in a BSCII biosafety cabinet. All tools and reagents should be sterile.
7 days or more after intranasal infection with S. Typhimurium, MAIT cells can be harvested (see Note 9).
Safety information
As MAIT cells are to be used for adoptive transfer, all procedures should be performed in a BSCII biosafety cabinet. All tools and reagents should be sterile.
Prewarm collagenase media and shaking incubator to 37 °C .
Mice should be euthanized (e.g., using a rising concentration of CO2 with a second method to confirm death).
Open the diaphragm by cutting the rib cage to expose both the heart and lungs. Gently perfuse the right ventricle with 8 mL –10 mL ice-cold RPMI to dispense circulating blood. Perfuse using a 10-mL syringe and a 26-G needle. Efficient perfusion will result in lung inflation and a color change to pink/white.
Remove lungs using scissors to cut through the hilum and place into a 24-well plate containing ice-cold RPMI to transfer organs to the laboratory.
Chop lungs into fine pieces (see Note 10).
Place lung tissue into a 1-mL Eppendorf tube containing 1–2 mL/lung of pre-warmed collagenase medium. Incubate tubes on their sides in a shaking incubator at 37 °C , at 100 rpm –180 rpm , for 01:30:00 .
During this time prepare Percoll gradients and antibody cocktails (see Table 1).
After 01:30:00 pour digested tissue through a 70-μm cell strainer and force through into a Petri dish with the plunger from a 1-mL syringe. Rinse residual sample with extra FACS buffer for maximum MAIT cell yield. Cells from multiple lungs (if required) (see Note 11) are pooled into a single 50-mL Falcon tube with a total of 50 mL sterile FACS buffer .
Centrifuge at 400 x g for 00:05:00 to pellet the cells. Pour off supernatant (SN).
Resuspend cells in 20 mL 40% Percoll . Underneath this layer use a transfer pipette to layer 20 mL 70% Percoll (see Note 12). Centrifuge this gradient at 800 x g, Room temperature, 00:20:00 with the centrifuge brake OFF. Lymphocytes and other immune cells will form a visible interphase layer between the 40% and 70% Percoll post centrifugation.
During this centrifugation step, prepare single color controls. It is convenient to use part of a spleen forced through a 70-μm filter and resuspended in 5 mL TAC for 00:05:00 at 37 °C , then washed once with 5 mL FACS buffer .
Collect the interphase between 40% and 70% Percoll into a fresh 50 mL Falcon and top up with FACS buffer to a total of 50 µL . Centrifuge at 400 x g, 00:05:00 .
Pour off supernatant and resuspend in 5 mL FACS buffer , transferring to a 10 mL Falcon tube. Centrifuge at 400 x g, 00:05:00 .
Resuspend all lung cells in 750 µL FACS buffer .
Block non-specific tetramer binding by adding 7.5 µL 2.4G2 (anti CD16/32) cell culture supernatant , containing MR1-6- FP tetramer [8, 16] (no fluorochrome, 1:100). Incubate at Room temperature on a roller or bench rocker for 00:15:00 .
For lungs from 5 mice, add 750 µL staining cocktail (Table 1).
Cover in aluminum foil to protect fluorochromes from light and shake on roller for 00:30:00 Room temperature .
Wash with 10 µL FACS wash . Centrifuge at 400 x g, 00:05:00 . Pour off supernatant.
Wash again with 10 mL FACS wash . Centrifuge at 400 x g, 00:05:00 . Pour off supernatant.
Resuspend cells in 2 mL FACS wash and filter through 40 μm filter into non-pyrogenic FACS tubes.
Sort live MAIT cells (defined as CD3+CD45+MR1-5-OP-RU tetramer+ cells) (Fig. 1) into 3 mL FCS in 15 mL Falcon tube. For detailed gating strategy, refer to [17]. Wash cells and adjust cell concentration to 5 × 105 cell/mL, allowing 105 in 200 µL for injection to each mouse.
Fig. 1 Flow cytometry gating strategy for MR1-5-OP-RU-tetramer+ MAIT cells
Inject 105 cells into the tail vein of recipient mice using cells suspended in 200 µL PBS in a 1-mL syringe with a 26-G cannula after warming the mice for 00:05:00 –00:15:00 with appropriate monitoring.
To deplete residual non-MAIT T cells (see Note 13), inject recipient mice on days 2 and days 5 or 6 with 0.1 mg each of purified anti-CD4 (GK1.5) and anti-CD8 (53.762) monoclonal antibodies i.v.
Rest mice for a total of 2 weeks post adoptive transfer to allow MAIT cell populations to settle in the host.