Mouse lung MAIT cell expansion and purification protocol

Gabriel A Ascui, Eleni Phung, Alba Mendis, Mitchell Kronenberg

Published: 2022-08-24 DOI: 10.17504/protocols.io.x54v9yrpqg3e/v1

Disclaimer

The Authors declare no competing interests.

Abstract

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

MAIT cell in vivo expansion with BRD509

Salmonella enterica serotype Typhimurium BRD509 vaccine strain culture

Safety information

Work in BSL2 biosafety conditions.

1.1.

Day 0: Overnight culture of Salmonella Typhimurium BRD509 strain.

Prepare 5mL of LB media with 100µg/ml of Streptomycin.
Pick -80°C BRD509 stock and inoculate tube.
Incubate in agitation225rpm.

1.2.

Day 1: Prepare 4h culture of BRD509.

Take 1mL of overnight BRD509 culture and dilute with LB media until 5mL .
Culture for an additional 4h 0m 0s to 6h 0m 0s .
Measure OD₆₀₀ on spectrophotometer.
Calculate the amount of culture that will need to be diluted in 900µL of sterile PBS to get 10^6 CFU per mouse based on the following conversion table (input OD600):

A	B	D	E	F
A600	1	A600 of culture	0.6
CFU/mL	=6*10^8	CFU/mL	=(B2*E1)/B1
		Desired CFU/mouse	=(110^6)/E21000	uL per mouse
	Resuspend in 900uL (30uL per mouse)		=E3*30	uL for 30 mice

A600 BRD509 spreadsheet

1.3.

Day 1: Infection Mice

Anesthetize the mice with isoflurane gas.
Administer retro-pharyngeal injections of 30µL of diluted BRD509 per mouse.
Prepare dilutions of the original dose at 10^4 and 10^5 in sterile PBS.
Plate dilutions on LB agar plate with Streptomycin. Count colonies next day to calculate actual dose.

MAIT cell sorting

Prepare Lungs MAIT cells

Safety information

Work in BSL2 biosafety conditions.

2.1.

Day 6: Collect lungs

Collect lungs from infected mice and process inwith 2mL of using program 37C_m_LDK_1. This should take about 0h 30m 0s .
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 25mL .
Centrifuge at 1500rpm,4°C .
Lyse red blood cells by adding 1mL of . Incubate at Room temperature for 0h 5m 0s .
Add 14mL of HBSS 10%FBS and centrifuge at 1500rpm,4°C .
Re-suspend cells in 2mL of MACS buffer.

2.2.

MAIT cell enrichment

Add 50 ul of Rat Serum to sample.
Add the following biotin-conjugated antibodies:

A	B	C
Antigen	Stock [ ] (mg/ml)	V in 2 ml (ul)
CD11b	0.5	4
CD11c	0.5	4
Ter119	0.5	4
F4/80	0.5	4
B220	0.5	4
Ly6G	0.5	4

Biotin-conjugated antibodies

Incubate atRoom temperature for 0h 10m 0s .
Vortex for 0h 0m 30s . Add 50µL of RapidSpheres to sample.
Mix and incubate for 0h 5m 0s at Room temperature .
Add 2mL MACS Buffer.
Place tube into . Incubate for 0h 5m 0s at Room temperature .
Gently pour supernatant in a new 5 ml tube.

2.3.

Fluorescent labelling

Stain cells with PE-conjugated or mouse MR1 tetramer loaded with 5-OP-RU at 1:300 dilution for 0h 45m 0s at Room temperature . Wash with PBS 2% FBS and centrifuge at 1500rpm,4°C .

Note

Mouse MR1 tetramers were provided by the NIH Tetramer Core Facility:https://tetramer.yerkes.emory.edu/

Stain cells with at 1:500 dilution, FcBlock (2G4) at 1:500 dilution and 1:500 dilution of 1mg/mL Free Streptavidin in PBS.
Note
NOTE: Make sure the PBS has no other protein content.
Incubate at 4°C for 0h 15m 0s . Wash with PBS 2% FBS.
Stain cells with the following antibodies:

A	B	C	D	E	F	G
#	Marker	Population	Channel	Host	Clone	Dilution
2	gd TCR	DUMP	PerCP-Cy5.5	Mouse	GL3	1:400
3	IgD	DUMP	PerCP-Cy5.5	Rat	11-26c.2a	1:300
4	CD11b	DUMP	PerCP-Cy5.5	Rat	M1/70	1:300
5	CD11c	DUMP	PerCP-Cy5.5	Rat	N418	1:300
6	B220	DUMP	PerCP-Cy5.5	Rat	RA36B2	1:300
7	5-OP-RU	MAIT cells	PE	-	-	1:300
8	TCR beta	T cells	APC-eF780	Mouse	H57-597	1:300
9	Live/Dead	Yellow	BV570	Rat	MEL14	1:200
10	CD45	Lymphocytes	BV785	Rat	30.F11	1:300

MAIT cell Sorting Panel

Incubate at4°C for 0h 30m 0s .
Wash cells with PBS 2% FBS. Resuspend in 4mL .

Cell Sorting

Sort MAIT cells with 85 nm nozzle on low pressure. Sort into 500µL of sterile FBS in 5 ml FACS tubes.
Gate samples as following: Lymphocytes/Singlets/Live/DUMP-CD45+/Tetramer+TCRbint
Citation
You should obtain a 20-30% frequency of MAIT cells from the DUMP-CD45+ gate. Total number of MAIT cells should be between 20,000-50,000 per lung.

In vitro culture

Day 1: MAIT cell activation with anti-CD3/CD28

4.1.

Coat 48-well plate

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

Dilute anti-CD3e (2C11) to 5µg/ml and anti-CD28 to 2µg/ml in sterile PBS.
Add 100µL to each well of a 48-well plate.
Incubate at 37°C for 2h 0m 0s .
Wash plate with 200µL of PBS, twice.
remove all liquid from every well.

4.2.

Plate MAIT cells

Note

MAIT cell culture media: RPMI 10% FBS 1X Pen/Strep 1X Pyruvate 1X HEPES 55 uM β-mercaptoethanol.

Wash sorted cells with MAIT cell culture media.
Resuspend cells in MAIT cell at 0.5 x 10^6 cells/mL in MAIT cell culture media supplemented with the following cytokines:

A	B	C	D
Cytokine	Final [ ]	Stock [ ]	Dilution
IL-2	10 ng/ml	20 ug/ml	1:2000
IL-7	20 ng/ml	20 ug/ml	1:1000
IL-1b	10 ng/ml	100 ug/ml	1:1000
IL-23	10 ng/ml	10 ug/ml	1:1000

Cytokines

Plate 500,000 cells per well.

Day 2: Put in fresh plate.

Analyze MAIT cells through flow

Stain cells with PE-conjugated or mouse MR1 tetramer loaded with 5-OP-RU at 1:300 dilution for 0h 45m 0s at Room temperature . Wash with PBS 2% FBS and centrifuge at 1500rpm,4°C .
Stain cells with at 1:500 dilution, FcBlock (2G4) at 1:500 dilution and 1:500 dilution of 1mg/mL Free Streptavidin in PBS.
Note
NOTE: Make sure the PBS has no other protein content.
Incubate at 4°C for 0h 15m 0s . Wash with PBS 2% FBS.
Stain cells with the following antibodies:

A	B	C	D	E	F	G
#	Marker	Population	Channel	Host	Clone	Dilution
2	gd TCR	DUMP	PerCP-Cy5.5	Mouse	GL3	1:400
3	IgD	DUMP	PerCP-Cy5.5	Rat	11-26c.2a	1:300
4	CD11b	DUMP	PerCP-Cy5.5	Rat	M1/70	1:300
5	CD11c	DUMP	PerCP-Cy5.5	Rat	N418	1:300
6	B220	DUMP	PerCP-Cy5.5	Rat	RA36B2	1:300
7	5-OP-RU	MAIT cells	PE	-	-	1:300
8	TCR beta	T cells	APC-eF780	Mouse	H57-597	1:300
9	Live/Dead	Yellow	BV570	Rat	MEL14	1:200
10	CD45	Lymphocytes	BV785	Rat	30.F11	1:300

Incubate at4°C for 0h 30m 0s .
Wash cells with PBS 2% FBS. Resuspend in 4mL .
Analyze cells through flow.