TITLE: Mouse lung MAIT cell expansion and purification protocol
AUTHORS: Gabriel A Ascui, Eleni Phung, Alba Mendis, Mitchell Kronenberg

[DESCRIPTION]
Mucosal Associated Invariant T (MAIT) cells are unconventional T cells present abundantly in human tissues. MAIT cells interact with antigens presented by MR1, an MHC class I protein, to fight microbial infections. They also reportedly activate through cytokines to combat viruses. While MAIT cells exhibit the innate-like characteristic of rapid responses to infections, they also display adaptive-like qualities such as an effector-memory phenotype. Defining the role of MAIT cells in the immune system is essential to understand lung immune responses against bacterial infections. The following protocol outlines the expansion of MAIT cells via a cultured Salmonella Typhimurium BRD509 vaccine strain. Six days after infecting mice retro-pharyngeally with BRD509, MAIT cells were extracted from the lungs. The cells were then enriched and labeled with fluorescence for sorting. Once sorted, MAIT cells were plated and activated with anti-CD3/CD28. The cells were then analyzed with flow cytometry.

[STEPS]
SECTION: MAIT cell in vivo expansion with BRD509
1. Salmonella enterica serotype Typhimurium BRD509 vaccine strain culture

 

SECTION: MAIT cell in vivo expansion with BRD509
1.1. Day 0: Overnight culture of Salmonella Typhimurium BRD509 strain.

Prepare 5 mL of LB media with 100 µg/ml of Streptomycin. 
Pick -80 °C BRD509 stock and inoculate tube. 
Incubate   in agitation .


SECTION: MAIT cell in vivo expansion with BRD509
1.2. Day 1: Prepare 4h culture of BRD509. 

Take 1 mL  of overnight BRD509 culture and dilute with LB media until 5 mL .
Culture for an additional 240 min to 360 min .
Measure OD600 on spectrophotometer. 
Calculate the amount of culture that will need to be diluted in 900 µL of sterile PBS to get 10^6 CFU per mouse based on the following conversion table (input OD600): 


 

SECTION: MAIT cell sorting
2. Prepare Lungs MAIT cells
 

SECTION: MAIT cell sorting
3. Cell Sorting

Sort MAIT cells with 85 nm nozzle on low pressure. Sort into 500 µL of sterile FBS in 5 ml FACS tubes. 
Gate samples as following: Lymphocytes/Singlets/Live/DUMP-CD45+/Tetramer+TCRbint
 


SECTION: MAIT cell in vivo expansion with BRD509
1.3. Day 1: Infection Mice 

1. Anesthetize the mice with isoflurane gas.
2. Administer retro-pharyngeal injections of 30 µL of diluted BRD509 per mouse. 
3. Prepare dilutions of the original dose at 10^4 and 10^5 in sterile PBS. 
4. Plate dilutions on LB agar plate with Streptomycin. Count colonies next day to calculate actual dose. 
SECTION: MAIT cell sorting
2.1. Day 6: Collect lungs

1. Collect lungs from infected mice and process inwwith 2 mL of  using program 37C_m_LDK_1. This should take about 30 min  .
2. Filter single cell suspension through   on 50 mL conical tube. Mash any remaining bits of tissue through the strainer with a syringe plunger. Wash strainer with HBSS with 10% FBS until 25 mL .
3. Centrifuge at 1500 rpm, 5 min, 4 °C .
4. Lyse red blood cells by adding 1 mL  of   . Incubate at Room temperature for 5 min .
5. Add 14 mL of HBSS 10%FBS and centrifuge at 1500 rpm, 5 min, 4 °C .
6. Re-suspend cells in 2 mL of MACS buffer.
SECTION: MAIT cell sorting
2.3. Fluorescent labelling

Stain cells with PE-conjugated or mouse MR1 tetramer loaded with 5-OP-RU at 1:300 dilution for 45 min at Room temperature . Wash with PBS 2% FBS and centrifuge at 1500 rpm, 5 min, 4 °C .


 


Stain cells with   at 1:500 dilution, FcBlock (2G4) at 1:500 dilution and 1:500 dilution of 1 mg/mL Free Streptavidin in PBS. 
 

3. Incubate at 4 °C for 15 min . Wash with PBS 2% FBS.
4. Stain cells with the following antibodies:

 
     #      Marker      Population      Channel      Host      Clone      Location      Dilution        1      Cas9      Transgenic      eGFP      -      -      -      -        2      gd TCR      DUMP      PerCP-Cy5.5      Mouse      GL3      13E08      1:400        3      IgD      DUMP      PerCP-Cy5.5      Rat      11-26c.2a      03E05      1:300        4      CD11b      DUMP      PerCP-Cy5.5      Rat      M1/70      03C07      1:300        5      CD11c      DUMP      PerCP-Cy5.5      Rat      N418      15A09      1:300        6      B220      DUMP      PerCP-Cy5.5      Rat      RA36B2      03D03      1:300        7      5-OP-RU      MAIT cells      PE      -      -      MAIT box      1:300        8      TCR beta      T cells      APC-eF780      Mouse      H57-597      03J02      1:300        9      Live/Dead      Yellow      BV570      Rat      MEL14      04F03      1:200        10      CD45      Lymphocytes      BV785      Rat      30.F11      12H03      1:300   
 

4. Incubate at4 °C  for 30 min .
5. Wash cells with PBS 2% FBS. Resuspend in 4 mL . 
SECTION: MAIT cell sorting
2.2. MAIT cell enrichment

Add 50 ul of Rat Serum to sample. 
Add the following biotin-conjugated antibodies:
 
     Antigen      Stock [ ] (mg/ml)      V in 2 ml (ul)      Location        CD11b      0.5      4      06B07        CD11c      0.5      4      06B08        Ter119      0.5       4      NKT Enrichment Box        F4/80      0.5       4      06D06        B220      0.5       4      NKT Enrichment Box   
 
3. Incubate atRoom temperature  for 10 min .
4. Vortex    for 30 s . Add 50 µL  of RapidSpheres to sample. 
5. Mix and incubate for 5 min at Room temperature .
6. Add 2 mL MACS Buffer. 
7. Place tube into   . Incubate for 5 min at Room temperature . 
8. Gently pour supernatant in a new 5 ml tube. 
SECTION: In vitro culture
4. 
Day 1: MAIT cell activation with anti-CD3/CD28
SECTION: In vitro culture
5. Day 2: Put in fresh plate.
SECTION: In vitro culture
4.1. Coat 48-well plate

While MAIT cells are been sorted, prepare anti-CD3e + anti-CD28 coated plates.

1. Dilute anti-CD3e (2C11) to 5 µg/ml  and anti-CD28 to 2 µg/ml  in sterile PBS. 
2. Add 100 µL to each well of a 48-well plate. 
3. Incubate at 37 °C for 120 min . 
4. Wash plate with 200 µL of PBS, twice. 
5. remove all liquid from every well. 

SECTION: In vitro culture
4.2. Plate MAIT cells


 


1. Wash sorted cells with MAIT cell culture media. 
2. Resuspend cells in MAIT cell at 0.5 x 10^6 cells/mL in MAIT cell culture media supplemented with the following cytokines:
 
     Cytokine      Final [   ]       Stock [   ]      Dilution      Location      Volume        IL-2      10 ng/ml      20 ug/ml      1:2000      Box 3               IL-7      20 ng/ml      20 ug/ml      1:1000      Box 3               IL-1b      10 ng/ml      100 ug/ml      1:1000      Box 7               IL-23      10 ng/ml      10 ug/ml      1:1000      Box 5          

3. Plate 500,000 cells per well. 
SECTION: In vitro culture
6. Analyze MAIT cells through flow

Stain cells with PE-conjugated or mouse MR1 tetramer loaded with 5-OP-RU at 1:300 dilution for 45 min at Room temperature . Wash with PBS 2% FBS and centrifuge at 1500 rpm, 5 min, 4 °C .
Stain cells with   at 1:500 dilution, FcBlock (2G4) at 1:500 dilution and 1:500 dilution of 1 mg/mL Free Streptavidin in PBS. 
 

3. Incubate at 4 °C for 15 min . Wash with PBS 2% FBS.
4. Stain cells with the following antibodies:

 

4. Incubate at4 °C  for 30 min .
5. Wash cells with PBS 2% FBS. Resuspend in 4 mL . 
6. Analyze cells through flow.