Study of MAIT Cell Activation in Viral Infections In Vivo

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 MAITcells 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.


Introduction
MAIT cells are relatively recently described innate-like lymphocytes, with similarities to the invariant natural killer T (iNKT) and γδ T cell subsets [1][2][3][4]. They are the most abundant innate-like population in the lungs in humans [5] though relatively rare in specific pathogen-free mice [6] and show a striking evolutionary conservation between diverse species of mammals [7]. MAIT cells express a semi-invariant T cell receptor (TCR), which recognizes microbially derived small molecule intermediates from the riboflavin biosynthetic pathway [1,4,8,9]. These molecular intermediates exist only in microbes but not in mammals, and therefore constitute a signature of microbial infection. This property implicates MAIT cells in anti-bacterial host defense, and potentially also in other roles such as tissue repair [3]. However, in addition to their TCR-dependent functions, they can be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon [10][11][12]. Emerging data suggest that they are expanded and activated by a range of human viral infections including dengue, hepatitis C, and influenza virus [11,13]. It was not clear from observational human studies whether this would lead to enhanced immune protection, or, conversely, contribute to immunopathology. To address this question, we conducted experimental influenza A virus challenge in vivo in mice and demonstrated that MAIT cells could contribute to a protective anti-viral response [12].
Here we describe the methods used to investigate these antiviral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe (1) how pulmonary MAIT cells can be expanded using intranasal (i.n.) bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists as well as protocols for (2) adoptive transfer of MAIT cells, (3) viral preparation and infection of mice, (4) lung homogenization, (5) surface and intracellular cytokine staining to determine MAIT cell activation, and (6) plaque assays. Tables 1-4. 2. Collagenase medium: Roswell Park Memorial Institute medium (RPMI) containing 3 mg/mL collagenase III, 5 μg/ mL DNase, and 2% fetal calf serum (FCS). Aliquots can be frozen at À20 C.

Antibodies are specified in
3. Fluorescence activated flow cytometry (FACS) buffer: phosphate buffered saline (PBS), 2 mM EDTA, 0.5% bovine serum albumin (BSA). From a 500 mL bottle of PBS, add 40 mL to a 50 mL falcon containing 2.5 g BSA powder, vortex hard, then filter sterilize back into PBS bottle using a syringe through a 0.22-μm filter. Do not add azide as will be toxic to the cells. 4. Percoll (Density 1.13 g/mL) 40% and 70% solutions, pre-warmed to room temperature for each use. 5. RPMI with pen/strep: RPMI containing 100 μg/mL streptomycin and 100 U/mL penicillin.
6. Tris-based Ammonium Chloride (TAC)-HCl, pH 7.5 hypotonic red blood cell lysis buffer: 0.14 M NH 4 Cl, 0.017 mM Tris (pH 7.5), then adjust pH to 7.2 with HCl (2 M). The solution is filter (0.22 μm) sterilized and kept at room temperature.   Remove 100 mL of the water but keep for later use. Add two 14 g packets of L-15 powdered media (kept at 40 C). Add magnetic flea and stir for 4 h or more to ensure the powder is completely dissolved. Adjust pH to 6.8 using 1 M HCl. Then add the following to the medium.
(d) Make up the volume to 1 L (using the 100 mL previously removed) and filter sterilize. Store at 4 C. To reduce precipitation, aliquot into 50 mL tubes for storage.

Methods
Personal protective equipment (PPE) should be worn at all times (gloves, lab coat, & eye protection) (see Notes 3 and 4).
1. Two days before infection streak out a plate of S. Typhimurium BRD509 (an attenuated vaccine strain [14]) on LB agar plates, containing 50 μg/mL streptomycin and incubate plates overnight at 37 C.
2. The day before infection, pick a single colony under flame and inoculate to 10 mL LB culture medium with 50 μg/mL streptomycin and leave static at 37 C (double contained if working with wild type/virulent SL1344 or equivalent strains) overnight.
3. On the day of infection, re-inoculate into fresh 10 mL pre-warmed LB culture medium with 0.5, 100, or 20 μL 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). 5. 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 10 4 to 5 Â 10 6 MAIT cells, or from 1% to 20-50% of all alpha-beta T cells [15] (see Note 8).

MAIT Cell
Adoptive Transfer 1. 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.
3. Mice should be euthanized (e.g., using a rising concentration of CO 2 with a second method to confirm death).
4. Open the diaphragm by cutting the rib cage to expose both the heart and lungs. Gently perfuse the right ventricle with 8-10 mL of 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.

5.
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.
7. 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 100-180 rpm, for 90 min.
8. During this time prepare Percoll gradients and antibody cocktails (see Table 1).
9. After 90 min 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 of sterile FACS buffer.
10. Centrifuge at 400 Â g for 5 min to pellet the cells. Pour off supernatant (SN).
Centrifuge this gradient at 800 Â g for 20 min at room temperature with the centrifuge brake OFF. Lymphocytes and other immune cells will form a visible interphase layer between the 40% and 70% Percoll post centrifugation.
12. 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 5 min at 37 C, then washed once with 5 mL FACS buffer.
13. 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 mL. Centrifuge at 400 Â g for 5 min.
14. Pour off supernatant and resuspend in 5 mL FACS buffer, transferring to a 10 mL Falcon tube. Centrifuge at 400 Â g for 5 min.  6. Allow mice to recover and monitor mice for until recovery (typically 10 days) (see Note 15).

Lung Homogenization
1. Collect the lungs into 2 mL of RPMI supplemented with penicillin/streptomycin.

5.
Centrifuge the samples at 1000 Â g for 7 min.
6. Using a 1 mL pipette, carefully draw up approximately 1 mL (a little bit more is good) of supernatant, avoiding the pellet and fatty residue on top. Divide this volume into two 1.5 mL Eppendorf tubes. Store at À80 C for subsequent plaque assays.

MAIT Cell Intracellular Cytokine Staining
To analyze MAIT cell frequencies and function during viral infection.
2. Mice should be euthanized (e.g., using a rising concentration of CO 2 with a second method to confirm death).
3. Open the diaphragm by cutting the rib cage to expose both the heart and lungs. Gently perfuse the right ventricle with 8-10 mL of ice-cold RPMI to remove circulating blood. Perfuse using a 10-mL syringe and a 26-G needle. Proper perfusion will result in lung inflation and a color change to pink/ white.

4.
Remove lungs (see Note 18) using scissors to cut through the hilum and place into a 24-well plate containing ice-cold RPMI to transfer organs to the laboratory.

Resuspend cells in 100 μL FACS buffer (see Note 25).
To enable estimation of absolute cell numbers, add a known number of calibration beads.
(a) Vortex calibration beads hard. Dilute (1:10) counting beads in PBS before using. To each sample 25 μL of these diluted beads was added, and an additional 10 μL of beads were saved to be counted with a hemocytometer, giving a count of X in a large square, i.e., X Â 10 4 beads/ mL (which is X Â 10 beads/μL, or X Â 10 Â 25 beads/ sample). Typically add a total of 25,000 beads per sample.
(b) When samples have been acquired on flow cytometer, these calibration beads can be detected using their FSC/SSC profile and the absolute number of cells of interest can be estimated using the following approach. 5. Add MDCK cell media to a total volume of 10 mL to the trypsinized cells, and transfer cells to a 10-mL tube.
7. Set up multiple T75 flasks with different cell densities to determine the growth pattern of MDCK cells. Generally~3-5 Â 10 5 for 3-day split.
8. Incubate at 37 C, 5% CO 2 . Amplification of MDCK cells for plaque assay: 9. Warm up MDCK cell media, trypsin-versene, and PBS at 37 C. 10. Check the confluency of MDCK cells, aspirate the medium, add 10 mL PBS, aspirate the medium, and repeat wash.
11. Discard PBS, add 2-3 mL of trypsin-versene (stored À20 C) to MDCK monolayers, and incubate at 37 C for 5 min. After 5 min tap the flasks, and incubate for longer if required (maximum 15 min).
12. In the meantime, add 40 mL of MDCK cell media to fresh T175 flasks. Set up one T175 flask of MDCK cells per~4 plates for plaque assay. Each 6-well plate assays 3 viral dilutions (as dilutions are done in duplicate).
13. Add MDCK cell media to a total volume of 10 mL to trypsinized cells, and transfer cells to a 10-mL tube.
17. Check the confluency of MDCK cells, aspirate the medium, add 10 mL of PBS, aspirate the medium, and repeat the wash.

Pool cells into one flask.
21. Count the cells, adjust the concentration to 3.3 Â 10 5 cells/ mL.
23. Incubate cells at 37 C, 5% CO 2 overnight. Aim for monolayers to be confluent in 6-well plates for assay. 29. For titration of viral stocks use dilutions from 10 À4 to 10 À6 . 10 À1 can be used as a positive control. Half-log dilutions can also be performed. For titration of mouse lung homogenates, generally: (a) Days 1-5: 10 À1 to 10 À3 .
(b) Days 6-10: neat to 10 À2 (see Note 26).  5. Pulmonary MAIT cells can be expanded using any source of 5-OP-RU and an appropriate TLR agonist [15,17]. A systematic assessment of effective TLR agonists has shown strong MAIT cell expansion 7 days after intranasal inoculation with 76 pmol 5-OP-RU on days 1, 2, and 4 in combination with a single dose of agonist on day 1 to TLR3 (high molecular weight poly I:C), TLR4 (lipopolysaccharide from E. coli), TLR2/6 (FSL-1 (Pam2CGDPKHPKSF)), or TLR9 (CpG ODN1826), but not with agonists of TLR1/2 (Pam3CSK4), TLR2, TLR5, TLR7 [3]. Each inoculum should be instilled in 50 μL PBS. However, the requirement for accurate repeated inoculations can introduce significant variability in MAIT cell expansion. A simple, less costly on reagents and time, and equally effective, if not more so, is a single intranasal inoculation with S. Typhimurium BRD509 in 50 μL PBS.

Wash
6. Growth of bacteria is estimated by measuring the culture in a spectrophotometer at 600 nm. To do so fill a cuvette with fresh LB media, place in spectrophotometer, and use this to blank. Then take 500 μL of bacteria-containing broth and measure optical density. To calculate the inoculum dose, use the estimate that an O.D. 600nm of 1 ¼ 5 Â 10 8 CFU/mL. 7. Accurate intranasal inoculation depends critically on the depth of anesthesia. Administer isoflurane and observe breathing pattern until respiratory rate has decreased to approximately 100 breaths/min and is deep and relaxed. If insufficient depth is achieved mice will sneeze. If depth of anesthesia is too great (further slowing of respiratory rate and very deep breaths), then mice tend to spontaneously breath-hold and again, volume inhaled will be unreliable. Place 50 μL of inoculum onto the left nasal opening (if user is right-handed) using a P200 pipette, gradually ejecting the 50 μL over a few breath cycles until all has been inspired.
8. Intranasal S. Typhimurium is well tolerated in immunocompetent strains such as C57BL/6 and BALB/c with less than 5% of animals showing minor signs of illness (ruffled hair) within 1-2 days after infection. These animals fully recover after days 3-5. The lethal dose of S. Typhimurium BRD509 is >2 Â 10 7 CFU/mouse (wild-type C57BL/6 adult). Caution should be used in immunocompromised strains in which pilot experiments should be performed to confirm optimal safe inocula.
9. This MAIT cell expansion is long-lived [15], so donor mice can be prepared several weeks in advance. 10. The lungs can conveniently be chopped up using the back of an upturned Petri dish. Using fine forceps lift lungs from the RPMI in which they have been transferred, gently blot off excess liquid with tissue paper and place on the Petri dish. Use a large curved scalpel blade to repeatedly chop through the lungs at multiple angles for at least 60 s each until a very fine and homogeneous texture is achieved.
11. Typically this method will yield 1.5 Â 10 6 pulmonary MAIT cells per mouse, so multiple mice may be required as donors, depending on the requirements of the experiment.
12. This will be sufficient for lungs from 8 mice.
13. If transferring cells into a Rag2 À/À γC À/À mouse then low frequencies of "contaminating" conventional CD4 + or CD8 + T cells tend to expand more rapidly than the MAIT cells and produce artifacts (not obvious for other T-cell-deficient mice, e.g., TCRα À/À or RAG2 À/À ). As many MAIT cells are doublenegative, it is possible to prevent this effect by repeated injections with T-cell-depleting anti-CD4 and anti-CD8 antibodies [17].
14. The PR8 strain of influenza virus is highly virulent in mice and only low inoculate are tolerated. The exact inoculum required for each experimental system will need to be carefully determined depending on the exact strain and batch of PR8 and the strain of mice, and local welfare and monitoring requirements. In our hands C57BL/6 mice receiving 100 PFU of A/PR/8/ 34 AF18 WCN experienced severe pneumonia in mice, characterized by parenchymal necrosis and infiltrates of macrophages, lymphocytes, and neutrophils, with 10-25% mortality due to welfare concerns or weight loss >20%.
15. Virally infected mice experience a transient viral illness with transient. Viral titers peak at day 3. Weight loss peaks at day 5-7 post infection, and there would be a significant weight gain expected by day 8 and resolving by day 10 post infection. Typically mice should be monitored and/or weighed daily for signs of ill health such as ruffled fur, hunched-up appearance, gait abnormalities, lethargy and loss of body condition for 10 days after challenge or till all the symptoms disappear and body weight returns to pre-challenge level. Monitoring can then return to twice weekly. 16. For many homogenization probes a wide tube is needed, such as the sterile, capped, round-bottom polypropylene tubes which are available.
17. The homogenizer generates a lot of heat at the probe tip. Samples should be kept on ice before and after homogenization, and the probe should be intermittently rested to cool down in ice-cold EtOH between groups of 5 or 10 samples.
Between samples or groups of samples clean the probe by running briefly in EtOH and then rinsing briefly in HBSS.
Often connective tissue will clog the probe and this can be removed with large forceps. After use the probe tip should be sterilized.
18. Only approximately 2/7 of one lung is needed for intracellular cytokine staining, so the other lung, or other sections of lung, can be saved for viral titer estimation, histology, or other assays if required.
19. To clarify terminology there are two lungs in each animal, so "one lung" refers to all the 2 or 3 lobes in a single hemithorax. Due to the presence of the heart on the left side, the left lung is smaller with only 2 lobes. 21. An alternative is to resuspend the entire pellet in 700 μL of FACS buffer and take 200 μL into 96-well plate: this should contain approximately 1-1.5 Â 10 6 cells, appropriate for staining.
22. To avoid using multiple filters, it is possible to buy large sheets of 40 μm mesh. A single rectangle can be cut which covers a whole plate. Using this, multiple cells can be pipette simultaneously with a multichannel pipette.
23. In round-bottom plates cells may clump so consider using flatbottom plate for the stimulation step, especially if doing further steps in FACS tubes rather than staining in plate format.
24. While surface markers can be measured on the intracellularly stained cells, the most accurate measurement of MAIT cell frequencies will be obtained from immediate surface staining prior to stimulation, due to activation-induced downregulation of the TCR.
25. If cells are not to be acquired immediately, then they can instead be resuspended in 100 μL of fixation buffer and stored at 4 C until required.
26. This may differ depending on virus and mouse strains.
27. The overlay media will start setting so proceed to the following steps quickly. Overlay media can be made in batches to assist with that.