TITLE: MAIT Cell Expansion in Donor Mice
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 3.1 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><br/><div class = "text-block"><span style = "font-weight:bold;">Abstract: </span><span>MAIT cells are rare in specific pathogen-free mice [6], typically comprising about 1 x 10</span><span style = "vertical-align:super;">4</span><span> recoverable pulmonary MAIT cells in an infection-naive adult C57BL/6 mouse. Therefore, for adoptive transfer experiments, the MAIT cell population should first be expanded using intranasal infection [15] or immunization (5-OP-RU with TLR agonists) [3, 15] (see Note 5). When planning the adoptive transfer experiment, estimate that one S. Typhimurium BRD509-infected mouse will yield 1–2 x 10</span><span style = "vertical-align:super;">6</span><span> sorted MAITcells, which are enough for 10–20 recipient mice (10</span><span style = "vertical-align:super;">5</span><span> MAIT cells/RAG2</span><span style = "vertical-align:super;">–/–</span><span>γC</span><span style = "vertical-align:super;">–/– </span><span>mouse in this case). Infect donor mice 7 days earlier than the adoptive transfer.</span></div></div>

[STEPS]
?. Two days before infection streak out a plate of S. Typhimurium BRD509 (an attenuated vaccine strain [14]) on LB agar plates, containing  and incubate plates  at .
[streptomycin]
37 °C
?. The day before infection, pick a single colony under flame and inoculate to  with  and leave static at  (double contained if working with wild type/virulent SL1344 or equivalent strains) .
[LB culture medium]
[streptomycin]
37 °C
?. On the day of infection, re-inoculate into fresh  with , , or  of overnight culture, under flame. This is to ensure an optimal optical density (O.D.) reading (bacteria in log phase growth) for preparing the inoculum later (see Note 6). The doublingtime for Salmonella can vary between 0.5 and 1 h. Make the infection inoculum from culture with O.D.600nm reading between 0.2 and 0.6. Calculate the required CFU of bacteria estimating 1 O.D. = 5–10 × 108 CFU (this constant needs to be established for individual labs). Dilute with PBS to a final concentration of 2 x 107 CFU/mL, allowing 50 μL inoculum/mouse, i.e., 106 CFU/50 μL/mouse.
[pre-warmed LB culture medium]
0.5 µl
100 µl
20 µl
?. Infect mice i.n. with 106 CFU S. Typhimurium BRD509 in  under isoflurane anesthesia (see Notes 7 and 8).
[PBS]
?. Allow mice to recover and monitor mice for 7 days to allow the infection to take its course and MAIT cell frequencies to expand dramatically from 104 to 5 × 106 MAIT cells, or from  to – of all alpha-beta T cells [15] (see Note 8).