TITLE: MAIT Cell Adoptive Transfer
AUTHORS: Timothy S C Hinks, Bonnie van Wilgenburg, Huimeng Wang, Liyen Loh, Marios Koutsakos, Katherine Kedzierska, Alexandra J. Corbett, Zhenjun Chen

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is part 3.2 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols.</div><br/><div class = "text-block"><span style = "font-weight:bold;">Collection Abstract: </span><span>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.</span></div></div>

[STEPS]
?. 7 days or more after intranasal infection with S. Typhimurium, MAIT cells can be harvested (see Note 9).
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  – 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.
8 mL
[ice-cold RPMI]
?. 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 , at –, for .
37 °C
33
33
?. During this time prepare Percoll gradients and antibody cocktails (see Table 1).
?. After  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 .
[sterile FACS buffer]
?. Centrifuge at  for  to pellet the cells. Pour off supernatant (SN).
Centrifuge: 400 34
?. Resuspend cells in . Underneath this layer use a transfer pipette to layer  (see Note 12). Centrifuge this gradient at  with the centrifuge brake OFF. Lymphocytes and other immune cells will form a visible interphase layer between the 40% and 70% Percoll post centrifugation.
[40% Percoll]
[70% Percoll]
Centrifuge: 800 34, 20 min
?. 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  for  at , then washed once with .
[TAC]
37 °C
[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 . Centrifuge at .
50 µl
Centrifuge: 400 34, 5 min
?. Pour off supernatant and resuspend in , transferring to a 10 mL Falcon tube. Centrifuge at .
[FACS buffer]
Centrifuge: 400 34, 5 min
?. Resuspend all lung cells in .
[FACS buffer]
?. Block non-specific tetramer binding by adding , containing MR1-6- FP tetramer [8, 16] (no fluorochrome, 1:100). Incubate at  on a roller or bench rocker for .
[2.4G2 (anti CD16/32) cell culture supernatant]
0 Room temperature
?. For lungs from 5 mice, add  (Table 1).
[staining cocktail]
?. Cover in aluminum foil to protect fluorochromes from light and shake on roller for .
0 Room temperature
?. Wash with . Centrifuge at . Pour off supernatant.
[FACS wash]
Centrifuge: 400 34, 5 min
?. Wash again with . Centrifuge at . Pour off supernatant.
[FACS wash]
Centrifuge: 400 34, 5 min
?. Resuspend cells in  and filter through 40 μm filter into non-pyrogenic FACS tubes.
[FACS wash]
?. Sort live MAIT cells (defined as CD3+CD45+MR1-5-OP-RU tetramer+ cells) (Fig. 1) into  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  for injection to each mouse.
[ FCS]
200 µl
?. Inject 105 cells into the tail vein of recipient mice using cells suspended in  in a 1-mL syringe with a 26-G cannula after warming the mice for – with appropriate monitoring.
[PBS]
?. To deplete residual non-MAIT T cells (see Note 13), inject recipient mice on days 2 and days 5 or 6 with  each of purified anti-CD4 (GK1.5) and anti-CD8 (53.762) monoclonal antibodies i.v.
0.1 mg
?. Rest mice for a total of 2 weeks post adoptive transfer to allow MAIT cell populations to settle in the host.