Analyzing human CD4⁺ T cells activated in response to macrophages infected with Mycobacterium tuberculosis

Summary

M1- and M2-like macrophages infected with Mycobacterium tuberculosis (Mtb) have been found to differ in their capacity to elicit memory CD4⁺ T cell activation. Here, we present a protocol to quantify and isolate the subset of human memory CD4⁺ T cells activated in response to autologous monocyte-derived macrophages (MDMs) infected with virulent Mtb. We describe steps for CD14⁺ monocyte isolation, generating MDMs, culturing Mtb and infection of macrophages, and identifying activated CD4⁺ T cells by flow cytometry.

For complete details on the use and execution of this protocol, please refer to Gail et al.¹

Graphical abstract

Highlights

• •Ex vivo differentiation and infection of human monocyte-derived macrophages
• •Co-culture of CD4⁺ T cells and macrophages infected with Mycobacterium tuberculosis
• •Unbiased assessment of rare CD4⁺ T cell activation based on activation-induced markers
• •Identification and sorting of human CD4⁺ T cells activated by infected macrophages

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

M1- and M2-like macrophages infected with Mycobacterium tuberculosis (Mtb) have been found to differ in their capacity to elicit memory CD4⁺ T cell activation. Here, we present a protocol to quantify and isolate the subset of human memory CD4⁺ T cells activated in response to autologous monocyte-derived macrophages (MDMs) infected with virulent Mtb. We describe steps for CD14⁺ monocyte isolation, generating MDMs, culturing Mtb and infection of macrophages, and identifying activated CD4⁺ T cells by flow cytometry.

Before you begin

This protocol describes the differentiation of human monocytes into M1 or M2-like macrophages over 6 days, subsequent infection with Mtb or loading with Mtb antigens, and co-culture of these MDMs with autologous CD4⁺ T cells to quantify the subset that become activated. Compared to simple and widely-used methods of stimulating T cells by adding Mtb peptides directly to PBMCs, a key advantage of this assay is the ability to identify T cells that directly recognize infected macrophages, the niche cell for Mtb. In the same assay, CD4⁺ T cell responses to infected MDMs can be compared to those loading with Mtb antigens including whole cell Mtb lysate, gamma-irradiated Mtb, or peptides.

To perform the following assay, you must first obtain peripheral blood mononuclear cells (PBMCs) from individuals expected to have circulating Mtb antigen-specific T cells based on a prior history of tuberculosis (TB), a diagnosis of latent Mtb infection (LTBI), or from individuals who have received a TB vaccine. You must also obtain an Mtb stock, cultured to mid-log phase, aliquoted, and stored under biosafety level-3 (BSL-3) conditions. We recently published data using this protocol to quantify the memory (CD45RA^Lo) CD4⁺ T cell response to Mtb-infected M1 or M2-like macrophages, compared to macrophages loaded with whole cell Mtb lysate or irradiated Mtb.¹

Institutional permissions

All protocols involving human subjects were approved through the Institutional Review Board of University Hospitals Cleveland Medical Center, and informed consent was obtained for all participants. All procedures using Mycobacterium tuberculosis (Mtb) were approved by the BSL-3 Advisory Committee of Case Western Reserve University. Protocols involving human subjects must be approved by an Institutional Review Board. Experiments with live Mtb must be performed in a BSL-3 facility. Protocols to handle and inactivate virulent bacteria must be approved by the institution’s biosafety committee.

Prepare buffers and media

Timing: 1 h

• 1.Prepare complete RPMI (cRPMI) cell culture media for culturing human macrophages and T cells.
• 2.Prepare Mtb culture medium and plates. Middlebrook 7H9 + OADC + glycerol is used for liquid culture, and Middlebrook 7H10 agarose plates are used for plating bacterial colony forming units (CFU).

Start bacterial and cell cultures

Timing: 6–7 days before infecting macrophages

• 3.Start Mtb culture from frozen stock and expand to obtain sufficient bacterial culture at mid-log phase growth (OD₆₀₀ 0.3–0.9) for infection of macrophages.
• 4.Perform CD14 selection of monocytes from peripheral blood mononuclear cells (PBMCs) and incubate with cRPMI containing GM-CSF to initiate macrophage differentiation.

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER
Antibodies
Purified NA/LE mouse anti-human HLA-DR (L243) (concentration used 25 μg/mL)	BD Pharmingen	Cat# 555809; RRID: AB_396143
Purified NA/LE mouse anti-human HLA-DR, DP, DQ (Tü39) (concentration used 25 μg/mL)	BD Pharmingen	Cat# 555556; RRID: AB_395938
Ultra-LEAF purified anti-human CD40 (W17212H) (concentration used 1 μg/mL)	BioLegend	Cat# 668103; RRID: AB_2814510
BUV395 mouse anti-human CD69 (FN50) (dilution used 1:50)	BD Biosciences	Cat# 564364; RRID: AB_2738770
BV650 mouse anti-human CD45RA (HI100) (dilution used 1:50)	BioLegend	Cat# 304136; RRID: AB_2562653
BV711 mouse anti-human PD-L1 (29E.2A3) (dilution used 1:50)	BioLegend	Cat# 329722; RRID: AB_2565764
BV785 mouse anti-human CD4 (RPA-T4) (dilution used 1:100)	BioLegend	Cat# 300554; RRID: AB_2564382
BB515 mouse anti-human CD25 (M-A251) (dilution used 1:25)	BD Biosciences	Cat# 565096; RRID: AB_2739065
PE-Dazzle-594 mouse anti-human CD40L/CD154 (24-31) (dilution used 1:20)	BioLegend	Cat# 310840; RRID: AB_2566245
BUV737 mouse anti-human CD3 (UCHT1) (dilution used 1:100)	BD Biosciences	Cat# 612750; RRID: AB_2870081
FITC mouse anti-human CD4 (RPA-T4) (dilution used 1:50)	BioLegend	Cat# 300538; RRID: AB_314074
PE mouse anti-human CD25 (BC96) (dilution used 1:50)	BioLegend	Cat# 302606; RRID: AB_314275
BV421 mouse anti-human CD69 (FN50) (dilution used 1:20)	BD Biosciences	Cat# 562883; RRID: AB_2737863
BV605 mouse anti-human CD8a (RPA-T8) (dilution used 1:50)	BioLegend	Cat# 301040; RRID: AB_2563185
APC mouse anti-human CD137 (4B4-1) (dilution used 1:20)	BD Biosciences	Cat# 561702; RRID: AB_10897995
APC-Fire750 mouse anti-human CD45RA (HI100) (dilution used 1:50)	BioLegend	Cat# 304152; RRID: AB_2616715
APC-Fire 750 mouse anti-human CD8a (RPA-T8) (dilution used 1:100)	BioLegend	Cat# 301066; RRID: AB_2629695
Human TruStain FcX (dilution used 1:50)	BioLegend	Cat# 422302; RRID: AB_2818986
Bacterial and virus strains
Mycobacterium tuberculosis strain H37RV	BEI Resources	Cat# NR-123
Biological samples
PBMCs isolated from freshly collected whole blood from healthy volunteers with latent Mtb infection	University Hospitals IRB-approved protocol	N/A
Chemicals, peptides, and recombinant proteins
Recombinant human GM-CSF protein	PeproTech	Cat# 300-03-50 ug
Recombinant human M-CSF	PeproTech	Cat# 300-25-50 ug
Critical commercial assays
Human Memory CD4+ T Cell Isolation Kit	Miltenyi Biotec	Cat# 130-091-893
Human IFNg Secretion Assay (PE) (dilution used 1:10)	Miltenyi Biotec	Cat# 130-090-762
Human CD14 microbeads	Miltenyi Biotec	Cat# 130-050-201
Human CD4+ T Cell Isolation Kit	Miltenyi Biotec	Cat# 130-096-533
LIVE/DEAD Fix Aqua-400 (dilution used 1:1,000)	Life Technologies	Cat# L34966
7-AAD (7-aminoactinomycin D) (dilution used 1:100)	Thermo Fisher Scientific	Cat# A1310
Other
7H9 Broth dehydrated culture media	BD Diagnostics	Cat# 271310
Middlebrook 7H10 agar	BD Diagnostics	Cat# 262710
Middlebrook OADC	BD Diagnostics	Cat# 212351
RPMI 1640 medium	Thermo Fisher Scientific	Cat# 11875119
Fetal bovine serum	Thermo Fisher Scientific	Cat# 16000069
Ficoll-Paque PLUS	Cytiva Life Sciences	Cat# 17144003
HEPES buffer 1 M	Gibco	Cat# 15630080
MEM non-essential amino acids (100x)	Gibco	Cat# 11140050
Sodium pyruvate (100 mM)	Gibco	Cat# 11360070
2-Mercaptoethanol	Gibco	Cat# 21985023
L-glutamine (200 mM)	Gibco	Cat# 25030081
Glycerin (Glycerol), 50% (v/v)	Ricca Chemical	Cat# 329032
Tween 80	MP Biomedicals	Cat# 0210317080
Corning Costar flat bottom 24-well plates	Thermo Fisher Scientific	Cat# 0720084
Corning Costar 96-well cell culture plate	Thermo Fisher Scientific	Cat# 0720090
AutoMACs running buffer	Miltenyi Biotec	Cat# 130091221
AutoMACs rinsing solution	Miltenyi Biotec	Cat# 130091222
MACS BSA stock solution	Miltenyi Biotec	Cat# 130091376
4% Paraformaldehyde solution in PBS	Thermo Scientific Chemicals	Cat# J61899AK
Staphylococcal Enterotoxin B (SEB)	Toxin Technology	Cat# BT 202
Dimethyl sulfoxide (DMSO)	MilliporeSigma	Cat# D2650

Materials and equipment

The following is a list of reagents to make in preparation of the experiment.

Complete RPMI Media (cRPMI)

Reagent	Final concentration	Amount
RPMI 1640		430 mL
Fetal Bovine Serum	10%	50 mL
200 mM/mL L-Glutamine	2 mM	5 mL
100x MEM NEAA	1x	5 mL
100x Na Pyruvate	1 mM	5 mL
1 M HEPES Buffer	0.01 M	5 mL
1000x 2-mercaptoethanol	55 μM	0.5 mL
Sterile 2 M NaOH	2 mM	0.5 mL
Total		500 mL

Store at 4°C and use for up to 1 month.

Rinse Buffer

Reagent	Final concentration	Amount
AutoMACS Rinsing Solution	N/A	1450 mL
MACS BSA Stock Solution (containing 10% BSA in PBS)	0.5% BSA	75 mL
Total		1525 mL

After preparing, store at 4°C for up to 3 months.

Middlebrook 7H9 Broth

Reagent	Final concentration	Amount
Middlebrook 7H9	4.7 mg/mL	4.7 g
Glycerin (50% glycerol)	0.2% glycerol	4 mL
Tween 80	0.5%	0.5 mL
Sterile, deionized Water	N/A	900 mL
Middlebrook OADC	10%	100 mL
Total		1000 mL

Add 7H9, Glycerin, Tween 80, water and autoclave. Add OADC after cooling.

Store at 4°C and use for up to 1 month.

Middlebrook 7H10 Agarose

Reagent	Final concentration	Amount
Middlebrook 7H10	19 mg/mL	19 g
Glycerin (50% glycerol)	0.5% glycerol	10 mL
Milli-Q Water	N/A	900 mL
Middlebrook OADC	10%	100 mL
Total		1000 mL

Add 7H9, Glycerin, water and autoclave. Add OADC after cooling. Pour 20 mL of warm agarose into 100 mm plates. Store at 4°C and use for up to 1 month.

CFU Dilution Buffer

Reagent	Final concentration	Amount
Phosphate Buffered Saline	N/A	500 mL
Tween-80	0.02%	0.1 mL
Total		500 mL

Sterile filter and store at 22°C for up to 1 year.

CFU Lysis Buffer

Reagent	Final concentration	Amount
Phosphate Buffered Saline	N/A	100 mL
Triton-X 100	1%	1 mL
Total		500 mL

Sterile filter and store at 22°C for up to 1 year.

2X GM-CSF Macrophage Differentiation Media

Reagent	Final concentration	Amount
Recombinant Human GM-CSF (100 μg/mL Stock)	50 ng/mL	6 μL
Sterile cRPMI	N/A	12 mL
Total		12 mL

Prepare 2X GM-CSF Macrophage Differentiation Media fresh, store at 4°C, and use the same day.

Freezing Media

Reagent	Final concentration	Amount
DMSO	10%	1 mL
FBS	90%	9 mL
Total		10 mL

Prepare Freezing Media fresh, store at 4°C, and use the same day.

2X CD40 Blocking Media

Reagent	Final concentration	Amount
Ultra-LEAF purified anti-Human CD40 (2.32 mg/mL)	2.32 μg/mL	12 μL
Sterile cRPMI	N/A	12 mL
Total		12 mL

Prepare CD40 Blocking Culture media fresh, store at 4°C, and use the same day.

1% Paraformaldehyde (PFA) in PBS

Reagent	Final concentration	Amount
4% Paraformaldehyde	1%	1 mL
Phosphate-buffered saline	N/A	3 mL
Total		4 mL

Prepare and use diluted paraformaldehyde the same day or freeze as aliquots at -20°C.

Step-by-step method details

CD14⁺ monocyte isolation from PBMCs

Timing: approximately 1 h

This step describes the procedure to separate CD14⁺ monocytes from PBMCs that were freshly isolated from whole blood. The step will generate aliquots of CD14⁺ monocytes for MDM differentiation and aliquots of CD14^- cells for subsequent T cell isolation.

• 1.
  Isolate PBMCs from fresh whole blood using Ficoll gradient centrifugation according to the manufacturer’s instructions.

  • a.
    Count PBMCs using Trypan Blue.
  • b.
    Centrifuge cells for 10 min at 400 × g at 10°C.
• 2.
  Resuspend cells in 80 μL of Rinse Buffer per 10⁷ cells.

  • a.
    To ensure the correct volume, estimate residual volume before resuspending.

Note: For example, if 100 × 10⁶ cells are counted and there is an estimated 100 μL of residual Rinse Buffer with cell pellet, only add ∼700 μL of Rinse Buffer for a final volume of 800 μL.

• 3.Add 20 μL (per 10⁷ cells) of Miltenyi human anti-CD14 microbeads and elute CD14+ monocytes by positive selection according to the manufacturer’s instructions.
• 4.
  Count 10 μL from each of the positive (CD14⁺) and negative (CD14^-) fractions immediately after separation.

  • a.
    After tubes containing each of the positive (CD14⁺) and negative (CD14^-) fractions are inverted (to ensure cells are resuspended), dilute 1:10 in Trypan blue.
  • b.
    Count using a hemocytometer or automated cell counter.
  • c.
    Multiply by the appropriate dilution factor (and number of mL of Rinse Buffer containing cells) to obtain cell count.
• 5.
  Centrifuge each fraction at 350 × g for 10 min at 10°C,

  • a.
    Aspirate supernatant.
  • b.
    If CD14⁺ cells are to be plated immediately, proceed to step 11 for these cells.
  • c.
    T cells from the freshly-drawn CD14^- PBMC fraction will be used in ∼7 days and should likely be cryopreserved until this time (step 6).
• 6.
  During centrifugation, prepare the freezing media.

  • a.
    Prepare a fresh solution of 10% DMSO and 90% FBS, kept cold.
• 7.
  Critical Step: Resuspend each fractions in freezing media and freeze each in 1 mL aliquots in separate cryovials. Once resuspended, rapidly transfer the tubes to a freezing container and place at -80°C.

  • a.
    CD14⁺ PBMCs are typically frozen at 4-5 × 10⁶ cells/mL, while CD14^- PBMCs are typically frozen at 10-20 × 10⁶ cells/vial, and should be based on the cell numbers needed for experiments.
  • b.
    Quickly seal cryovials and add to a Nalgene Mr. Frosty freezing container (or equivalent), pre-cooled to 4°C, seal and then transfer it to the -80°C freezer to freeze for at least 6 h.

CRITICAL: While the DMSO in freezing media is necessary to prevent cell membrane crystallization during freezing, it can be toxic to cells at ambient temperatures. Therefore work quickly once cells are resuspended in Freezing Media.

• 8.The following day, move all cryovials from -80°C to a Liquid nitrogen freezer.

Generating monocyte-derived macrophages (MDMs)

Timing: approximately 1 h, then 6 days in culture

This step describes differentiation of MDMs from CD14⁺ monocytes. Infection with Mtb can occur on or after day 6 of differentiation.

• 9.
  If thawing CD14⁺monocytes from frozen: Rapidly thaw a 1 mL aliquot of cryopreserved CD14+ monocytes over 1–2 min in a 37°C water bath.

  • a.
    Immediately pipet cells into 20 mL pre-warmed cRPMI in a 50 mL conical tube.
  • b.
    Wash the inside cryotube 5 times with 1 mL of pre-warmed cRPMI, to collect remaining cells.
  • c.
    Centrifuge cells at 350 × g for 10 min at 22°C.
  • d.
    Aspirate supernatant.
  • e.
    Resuspend cells in 10 mL of pre-warmed cRPMI.
  • f.
    Rest the thawed monocytes in the incubator at 37°C with 5% CO₂ for at least 3 h (up to 24 h is acceptable).

CRITICAL: The DMSO in Freezing Media can be toxic to cells at ambient temperatures. Therefore work quickly to dilute with cRPMI once cells are thawed.

• 10.
  Using fresh CD14⁺ monocytes:

  • a.
    Count cells using trypan blue and a hemocytometer or automated cell counter.
  • b.
    Centrifuge at 350 × g for 10 min at 22°C.
• 11.
  Dilute cells in cRPMI to a concentration of 250,000 cells per 500 μL cRPMI (for plating in 24-well flat-bottom plates).

  • a.
    Plate 500 μL of cells per well in tissue culture treated 24-well flat-bottom plates. Figure 1 depicts the suggested layout for plating MDMs for each subsequent experimental condition.

Note: An alternative is to plate 100 μL of cells per well in a 96-well flat-bottom plate for experiments where greater numbers of conditions (and fewer cells) are needed.

• 12.
  Incubate monocytes with GM-CSF Macrophage Differentiation Media.

  • a.
    Add GM-CSF at a concentration of 50 ng/mL to cRPMI to create 2X GM-CSF Macrophage Differentiation Media.
  • b.
    Add 500 μL of GM-CSF Macrophage Differentiation Media to the 500 μL of monocytes in each well in the 24-well plate, creating a 1X final concentration of 25 ng/mL GM-CSF to differentiate M1-like MDMs.^1,2,3

Note: Alternatively, M-CSF enriched differentiation media can be used at a 1X concentration of 25 ng/mL to generate M2-like MDMs, as described.^1,2,3

Note: A final (1X) concentration of GM-CSF (or M-CSF) of 50 ng/mL was found to be equivalent to 25 ng/mL¹.

• 13.Incubate monocytes in macrophage differentiation media at 37°C with 5% CO₂ for 3 days.
• 14.
  On Day 3, remove half the volume of media from each well.

  • a.
    Again add 500 μL of cRPMI containing a 2X (50 ng/mL) working concentration of GM-CSF (or M-CSF) to each well in a 24-well plate.
  • b.
    Incubate for 3 more days.
• 15.
  On Day 6, cells have fully differentiated into MDMs and are ready for further experiments.

  • a.
    Remove the media from each well and replace with plain cRPMI.
  • b.
    Removed media can be saved for cytokine or other analysis if desired.

Figure 1.

Layout of experimental conditions

Graphical layout shows the conditions used in both flow cytometry analysis and sorting experiments. Unless noted, 2-3 wells are used for each condition. Wells containing Mtb-infected macrophages are encompassed by a solid line. Wells containing macrophages left uninfected are encompassed by a dashed line. CD4⁺ T cells and α-CD40 mAb are added to all wells 24 h post-infection, except for the 4 wells used for Day 1 CFU (green). T cells are typically harvested from each well for antibody staining and flow cytometry 16 h later. Created with BioRender.com.

Culturing Mtb and infection of macrophages

Timing: A total of 6–7 days, macrophage infection procedure requires ∼6 h

This step should be initiated the same day as (or one day prior to) plating monocytes for the generation of MDMs to have the bacterial culture prepared for infection on day 6. Ensure that on the day of macrophage infection the bacterial culture is at mid-log phase growth.

• 16.
  Thaw a ∼0.5–1 mL stock aliquot of Mtb strain H37Rv (or other Mtb strain).

  • a.
    Add stock Mtb culture to 9 mL of OADC-enriched 7H9 media in a 125 mL filter-top flask.
  • b.
    Incubate in a shaking bacterial incubator at 37 °C at 100 rpm.
• 17.Two days later (i.e., Day 2), add ∼15 mL of 7H9 media to the culture (to total ∼25 mL), and continue incubating for 2–3 days.

Note: If bacterial culture is not yet opaque, wait an additional 1–2 days before adding more 7H9 media to the culture.

Note: An alternative is to check the optical density at 600 nm (OD₆₀₀) with aseptic technique using a spectrophotometer and add media when OD₆₀₀ is $\ge$ 0.2.

• 18.2–3 days later, check the OD₆₀₀ of the culture and if $\ge$ 0.2, add ∼50 mL of 7H9 media to bring the total volume to ∼75 mL.

Note: In liquid culture, Mtb has a doubling period of approximately 20–24 h. Use the OD₆₀₀ and the date you intend to use the Mtb culture to infect macrophages as a guide to modify the amount of 7H9 to add. This protocol assumes Mtb infection of macrophages will occur on Day 6 or 7.

• 19.
  On the date of Mtb infection of MDMs, check the OD₆₀₀.

  • a.
    Proceed with the infection protocol if Mtb culture is at mid-log growth phase (OD₆₀₀ of ∼0.3–0.9).
  • b.
    For OD₆₀₀ < 0.2, wait an additional day before proceeding with infection.
  • c.
    For OD₆₀₀ > 1, transfer 25 mL of the culture to a new flask and dilute 3-fold by adding 50 mL of 7H9, and wait an additional day before infecting to ensure the culture is at mid-log phase growth.

Note: The entire Mtb culture is used and diluted to retain the bacterial population represented in the stock culture. Passaging small aliquots of the working culture is usually avoided to minimize lab adaptation of a subpopulation and loss of virulence.

• 20.
  To prepare the bacteria for infection of MDMs, first split the total volume of bacterial culture equally between two 50 mL conical tubes.

  • a.
    Centrifuge at 3000 × g for 10 min at 22°C.
  • b.
    Decant the supernatants.
  • c.
    Gently vortex the pellets.
• 21.
  Resuspend bacterial pellets in a total of 50 mL RPMI1640, each to wash.

  • a.
    Initially resuspend each pellet by adding 1 mL of RPMI1640 and pipetting up and down.
  • b.
    Then add the remaining 49 mL of RPMI1640 to top-up each tube before centrifugation.
• 22.If the bacterial culture was observed to be clumpy, or there is difficulty filtering through a 5 μm filter in Step 26, pulse sonication of the bacterial suspensions (in 1 mL of RPMI in sealed conical tubes) for 10–30 s, using a bath sonicator, can facilitate dispersion of bacterial clumps.

Note: If used, sonication should be brief (< 30 s) as mycobacteria are sensitive to the heat that is generated during sonication. The effect of different sonication settings on Mtb growth should first be tested with your bacterial stock and sonicator.

• 23.
  Centrifuge at 3000 × g for 10 min at 22°C.

  • a.
    Decant the supernatants.
  • b.
    Gently vortex the pellets.
• 24.
  Resuspend each pellet in 1 mL cRPMI and combine into a single tube.

  • a.
    Add 8 mL of cRPMI to the combined bacterial suspensions for a total of 10 mL.
• 25.
  Prepare the syringe filters for bacterial filtration.

  • a.
    Remove the plungers from two 10 mL syringes.
  • b.
    Attach the Luer-lock tip of the syringes to two 5 μm syringe filters.
  • c.
    Pre-wet each filter by pipetting 0.5 mL of cRPMI to each syringe.

Note: Use sterile technique when handling the rubber portion of each plunger, along with the syringes, as they will come into contact with the bacterial suspension.

• 26.
  Filter the 5 mL of the cRPMI containing bacteria through each syringe filter and into a single tube.

  • a.
    Allow the bacterial suspension to flow through each syringe filter by gravity filtration.
  • b.
    Insert and gently press on the plunger to filter the remaining volume, but do not force the plunger once resistance is met.

    CRITICAL: Do not press too firmly or rapidly as bacterial clumps may pass through the filter. It is better to leave a small amount of residual bacterial suspension in each syringe than to risk shearing the bacteria or forcing bacterial clumps through the filter.

  • c.
    Determine the volume of filtered bacteria, which now represent a single-cell suspension of Mtb.
• 27.
  Determine concentration of the single-cell bacterial suspension using the OD₆₀₀.

  • a.
    After filtering bacteria with 5 μm syringe filter, measure the OD₆₀₀ of the filtered bacteria (using cRPMI to blank the spectrophotometer).

    Note: Despite concentrating ∼75 mL of Mtb culture in 7H9 into ∼10 mL of Mtb culture in cRPMI, the OD₆₀₀ of filtered bacteria is usually lower due to loss of bacterial clumps in the filter.

  • b.
    Estimate the bacterial concentration assuming OD₆₀₀ of 1.0 = ∼10⁸ bacteria/mL. Interpolate the number of bacteria/mL cRPMI (e.g., OD₆₀₀ of 0.25 is ∼2.5 × 10⁷/mL).

    Note: This estimate can be refined by plating serial dilution of the Mtb inoculum for each bacterial stock used, and later adjusting this assumption using the O.D. after counting CFUs of the inoculum.

• 28.Dilute the bacteria in cRPMI so that the final concentration will result in the desired number of bacteria per well.

CRITICAL: We infect cells in a small volume to increase the interaction between bacteria and macrophages in a well using a total infection volume of 250 μL/well for wells in a 24-well plate, or 50 μL/well for 96-well plates.

Note: For example, for an intended multiplicity of infection (MOI) of 5 bacteria/macrophage, dilute the bacterial suspension to 5 × 10⁶/mL. For MOI of 1, dilute to or 1 × 10⁵/mL since macrophages are plated 250,000/well (24-well plate) or 50,000/well (96-well plate).

• 29.Remove all media from each macrophage well and add 250 μL of the diluted bacterial suspension to each well in a 24-well plate (or 50 μL to each well in a 96-well plate).

Note: Infection and other experimental conditions are shown in Figure 1. Wells containing Mtb-infected macrophages are encompassed by a solid line. Those left uninfected are encompassed by a dashed line.

• 30.Incubate the 24 (or 96) well plates at 37°C with 5% CO₂ for at 4 h.
• 31.After 4 h, wash wells 3 times with sterile RPMI1640 and then 1 time with cRPMI.
• 32.Add 1 mL of cRPMI to each well and place at 37°C with 5% CO₂ for ∼24 h.
• 33.After the washout of bacteria, add Mtb (H37Rv) whole cell lysate diluted to 10 μg/mL in cRPMI, and/or SEB as needed to selected subset of macrophage wells.

Note: Ultimately, examining T cell responses to positive and negative control wells can be compared with responses to Mtb-infected macrophages.

• 34.Prepare to enumerate bacterial colony-forming units (CFUs) one day after Mtb infection from 4 wells of infected macrophages.

Enumerating bacterial CFUs to determine bacterial load

Timing: 1–2 h, then await colony growth for 3 weeks

This step estimates the bacterial load (actual MOI) 1 day after Mtb infection of MDMs and will require 3 weeks of culture growth on agarose plates.

• 35.The day after infecting macrophages with Mtb, remove all media from each of the 4 wells of infected macrophages designated for enumerating CFU and discard.

Note: If desired, supernatants can be saved and stored at -80°C for subsequent cytokine profiling.

• 36.
  Add 500 μL of CFU Lysis buffer to each well in a 24-well plate.

  Note: If cells are plated in 96-well plates, instead add 100 μL of Cell Lysis buffer.

  • a.
    Wait 5 min for cell lysis (confirmed by observing under the microscope).
• 37.
  Perform 10-fold serial dilutions using 100 μL of cell lysate containing the bacteria and 900 μL of CFU Dilution Buffer.

  • a.
    Plate 100 μL of dilutions 2, 3, and 4 on Middlebrook 7H10 agarose plates.
  • b.
    Spread the liquid on the plate using a T or L-shaped spreader using aseptic technique, beginning with plating the most dilute (dilution 4), moving to the most concentrated (dilution 2) samples.
• 38.Incubate plates up-side down, stacked in translucent biohazard bags, taped shut, and placed in secondary containment in an incubator at 37°C for approximately 3 weeks. Colonies can usually be counted at 18–21 d after plating.

Note: Colony size can be periodically checked to determine when they are ready to count by observation through the translucent bags without opening them.

CD4⁺ T cell / Mtb-infected macrophage co-culture

Timing: 2 h preparation, then 16 h incubation

This step explains the procedure for isolating CD4⁺ T cells by immunomagnetic negative selection and the conditions for the co-culture with Mtb-infected MDMs.

• 39.
  On the day of Mtb infection of macrophages, thaw a 1 mL aliquot of the cryopreserved CD14-negative PBMCs rapidly over 1–2 min in 37°C water bath.

  • a.
    Immediately pipet cells into 20 mL pre-warmed cRPMI in a 50 mL conical tube.
  • b.
    Wash cryotube 5 times with 1 mL of pre-warmed cRPMI, to collect remaining cells.
  • c.
    Centrifuge cells at 350 × g for 10 min at 22°C.
  • d.
    Aspirate supernatant.
  • e.
    Resuspend cells in 10 mL of pre-warmed cRPMI.

CRITICAL: The DMSO in Freezing Media can be toxic to cells at ambient temperatures. Therefore work quickly to dilute with cRPMI once cells are thawed.

• 40.Rest the thawed PBMCs in incubator at 37°C with 5% CO₂ for at least 3 h (or up to 24 h) prior to CD4⁺ T cell isolation.
• 41.
  Prepare to perform immunomagnetic negative selection of CD4⁺ T cells.

  • a.
    Centrifuge cells at 350 × g for 10 min at 10°C.
  • b.
    Aspirate the supernatant.
  • c.
    Resuspend the pellet in 40 μL of Rinse Buffer per 10⁷ cells.
• 42.
  Perform an immunomagnetic negative selection to isolate either:

  • a.
    Total CD4⁺ T cells using the Miltenyi human CD4+ T cell isolation kit, or.
  • b.
    Memory CD4⁺ T cells using human memory CD4+ T cell isolation kit.
• 43.
  Once negative selection is performed, perform a cell count using 10 μL of cells from the negative fraction (containing the CD4+ T cells of interest) and trypan blue.

  • a.
    After removing 10 μL of the cell suspension to count, centrifuge each fraction of cells at 350 × g for 10 min at 10°C.
  • b.
    Aspirate the supernatant.

Note: Counting cells during centrifugation saves time.

• 44.
  Resuspend CD4⁺ T cells in cRPMI. Prepare to add a ratio of 2–4 T cells per macrophage for each well.

  • a.
    Depending on the number of CD4⁺ T cells isolated, or conditions desired (Figure 1), resuspend ∼2-4 × 10⁶ CD4⁺ T cells per mL of cRPMI.

Note: For macrophages in 24-well plates, plan to add 250 μL of T cells in cRPMI to each well. For macrophages in 96-well plates, plan to add 50 μL T cells in cRPMI to each well.

• 45.
  If using CD40L to detect CD4⁺ T cell activation by flow cytometry, create a solution of 2X CD40 Blocking Media by adding anti-CD40 mAb (2X = 2 μg/mL) to cRPMI.

  CRITICAL: During co-culture, anti-CD40 mAb will block the CD40L-CD40 interaction, which normally results in internalization of CD40L on T cells, preventing its detection. CD40 blockade enhances the detection of CD40L expression on activated T cells in the assay.

  • a.
    Prepare enough 2X CD40 Blocking Media for all conditions to add 250 μL per well (for 24-well plates) or 50 μL per well (for 96-well plates).

    Note: An alternative is to instead use fluorescently-labeled anti-CD40L mAb to identify CD40L expressed on T cells that may become internalized after ligation with CD40, which is upregulated during Mtb infection of MDMs.⁴ This approach is taken during cell sorting assays when downstream analyses of functional CD4⁺ T cells will later be performed.

  • b.
    For control wells that will receive anti-MHC-II mAb blockade, aliquot enough CD40 Blocking Media for the number of desired control wells and add a combination of anti-HLA-DR (L243) and anti-HLA-DR/DQ/DP (Nu39) (for each, 2X = 50 μg/mL).
• 46.Remove supernatant from MDM wells (2-3 at a time using aseptic technique).
• 47.To each well, add 250 μL (for MDMs in 24-well plates) or 50 μL (for 96-well plates) of CD40 Blocking Media (± anti-HLA-DR/DQ/DP mAb blockade for anti-MHC-II mAb blockade controls).
• 48.
  To include a positive control using staphylococcal enterotoxin B (SEB), add a 2X concentration of SEB (2X = 2 μg/mL) to a separate aliquot of anti-CD40 Blocking Media, enough for ∼2 wells. This will result in a final concentration of 1 μg/mL after T cells are added.

  • a.
    If the numbers of T cells or macrophages from clinical samples are limited, an alternative is to add SEB at 1 μg/mL directly to a separate aliquot of 1–2 million PBMCs in 1 mL of cRPMI that also contain anti-CD40 mAb (1 μg/mL ) to serve as a positive control for T cell stimulation.
  • b.
    T cells / PBMCs will be SEB-stimulated for 16–18 h and harvested at the same time as those from the other experimental conditions to assess T cell activation.
• 49.
  Add the T cell suspension to each well containing MDMs.

  • a.
    Add 250 μL (for MDMs in 24-well plates) or 50 μL (for 96-well plates) of the CD4⁺ T cell suspension in cRPMI (after gently pipetting up and down or inverting the tube to resuspend).

CRITICAL: Each well should now contain a total of 500 μL (or 100 μL for 96-well plates) and a final 1X concentration of anti-CD40 mAb of 1 μg/mL. Wells receiving anti-MHC-II blockade should additionally contain anti-HLA-DR/DQ/DP mAbs at a final 1X concentrations of 25 μg/mL, each.

Note: A layout of experimental conditions is shown in Figure 1

• 50.Incubate the co-culture of T cells and macrophages (± Mtb infection) for 16–18 h in a tissue culture incubator set at 37°C with 5% CO₂.

Identification of activated CD4⁺ T cells by flow cytometry

Timing: 2–4 h

This step explains the procedure for harvesting, staining, and assessing activation of CD4⁺ T cells following co-culture with Mtb-infected MDMs. The following steps can be used either to perform flow cytometry analysis with the IFNγ secretion assay (OPTION 1), flow cytometry analysis without the IFNγ secretion assay (OPTION 2), or flow sorting of activated CD4⁺ T cells for use in downstream assays (OPTION 3).

• 51.
  Prepare a master mix of fluorescence-conjugated mAbs in AutoMACS running buffer that will be used to stain T cells for flow cytometry or cell sorting.

  • a.
    Use Tables 1 and 2 as a guide to choose markers, antibody dilutions, and the volume of master mix needed to stain all samples.
  • b.
    Include Human TruStain FcX (anti-Fc receptor blockade) at a dilution of 1:50 to block non-specific binding of the Fc receptors.
  • c.
    Prepare the antibody master mix fresh and store at 4°C protected from light.

    Note: We typically stain each sample for flow cytometry analysis in a volume of 50 μL in 96-well round bottom plates, whereas samples used for cell sorting are typically stained in a volume of 100 μL in 5 mL FACS tubes, due to greater cell numbers.

  • d.
    If performing flow cytometry analysis with the IFNγ secretion assay (OPTION 1): Set 50 mL of cRPMI in a conical tube with the cap loosened to pre-warm in a tissue culture incubator set at 37°C with 5% CO₂ (for use in the Miltenyi IFNγ secretion assay).
• 52.
  After the 16–18 h co-culture, resuspend T cells by pipetting up and down, with a P1000 for cells in 24-well plates.

  Note: For harvesting T cells from a 96-well plate, pipet 3 wells at a time, up and down, using a multichannel P200.

  • a.
    Transfer the resuspended T cells from each well EITHER to separate 1.5 mL Eppendorf tubes (if performing flow cytometry analysis with IFNγ secretion assay – OPTION 1), OR to 5 mL FACS tubes (if performing flow analysis without IFNγ secretion assay or flow sorting – OPTIONS 2 and 3).
  • b.
    Add another 1 mL of cold D-PBS to each well in a 24-well plate (or add 200 μL of cold D-PBS to wells in a 96-well plate).
  • c.
    Pipet up and down to wash and resuspend the remaining T cells and transfer to each of the same tubes.
• 53.
  Centrifuge the harvested T cells.

  • a.
    For cells in Eppendorf tubes, centrifuge at 500 × g for 5 min at 10°C in a microcentrifuge.
  • b.
    For cells in FACS tubes, centrifuge at 350 × g for 10 min.
  • c.
    Aspirate the supernatant.
  • d.
    Proceed EITHER with:

    • i.
      OPTION 1: The workflow for flow cytometry analysis with IFNγ secretion assay (proceed to step 54).
    • ii.
      OPTION 2: The workflow for flow cytometry analysis without IFNγ secretion assay and (proceed to step 54).
    • iii.
      OPTION 3: The workflow for flow sorting of activated T cells (skip to step 60).
• 54.
  To perform Live-Dead viability staining of samples for flow cytometry analysis:

  • a.
    Resuspend pellets in 50 μL of Live-Dead Aqua working solution, diluted 1:1000 in PBS and incubate for 15 min at 4°C in dark.
  • b.
    If not performing the IFNγ secretion assay (OPTION 2), skip to step 56.
• 55.
  If performing the IFNγ secretion assay for flow cytometry analysis (OPTION 1):

  • a.
    From the Miltenyi IFNγ Secretion assay kit, create a solution containing 2X IFNγ catch reagent (in Rinse Buffer) by combining 40 μL Rinse Buffer, 10 μL IFN-γ catch reagent, and 1 μL Human TruStain Fcx (Fc Block) for each sample to be stained for flow cytometry.
  • b.
    Add 50 μL of this 2X IFNγ catch reagent solution directly to the 50 μL of T cells in Live-Dead/PBS solution (for a final volume of 100 μL) and incubate at 22°C for 5 min.
  • c.
    Add 750 μL of the pre-warmed cRPMI (from step 51d) to each tube.
  • d.
    Place Eppendorf tubes in an angled tube rotator and incubate at 37°C while rotating for 45 min.
  • e.
    Remove from incubator and add 700 μL of cold AutoMACS running buffer.
  • f.
    Place tubes on ice or at 4°C for 10 min to cool cells and stop the reaction.
  • g.
    Centrifuge tubes at 500 × g for 5 min at 10°C in microcentrifuge, aspirate supernatant.

CRITICAL: Incubating cells with cold AutoMACS running buffer prior to centrifugation is important to reduce non-specific IFNγ staining.

• 56.
  Stain and prepare T cells for flow cytometry analysis (OPTIONS 1 and 2):

  • a.
    Resuspend cells in 200 μL of cold AutoMACS running buffer and transfer to wells in a 96-well round-bottom plate.
  • b.
    Once all samples are in the plate, centrifuge at 700 × g at 10°C for 2 min.
  • c.
    Flick the plate to remove supernatant.
  • d.
    Add 50 μL of an antibody master mix (prepared in step 51) containing AutoMACS Running Buffer, a 1:50 dilution of Fc block (Human TruStain FcX) and the fluorescently-labeled antibodies needed for immunostaining T cells and analysis by flow cytometry (listed in Table 1).

    Note: Include single-stain compensation controls for each channel using PBMCs.

  • e.
    Pipet up and down using a multichannel P200 to mix.
  • f.
    Incubate at 4°C for 20 min, protected from light.
  • g.
    Add 150 μL of AutoMACS running buffer to wash cells.
  • h.
    Centrifuge at 700 × g at 10°C for 2 min.
  • i.
    Flick the plate to remove supernatant.
  • j.
    Resuspend cells in 200 μL AutoMACS running buffer to wash.
  • k.
    Centrifuge at 700 × g at 10°C for 2 min.
  • l.
    Flick the plate to remove supernatant.
• 57.Resuspend cells in 100 μL of a 1% Paraformaldehyde (PFA) Solution for at least 30 min for fixation of fluorescent antibody staining and to inactivate Mtb.

Note: Longer incubation times in 1% PFA may be required by local biosafety committee-approved protocols for inactivating Mtb and removing samples from BSL-3 conditions. All regulations at your institution should be followed.

• 58.
  Wash PFA from cells and prepare to run flow cytometry:

  • a.
    Add 100 μL of AutoMACS Running Buffer to each well to initiate washing of PFA from each sample in the 96-well round bottom plate.
  • b.
    Centrifuge plate at 700 × g for 2 min.
  • c.
    Flick the plate to remove supernatant.
  • d.
    Resuspend cells in 200 μL AutoMACS running buffer, and store at 4°C, protected from light, until ready to run flow cytometry.
• 59.
  Using flow cytometry, enumerate the CD4⁺ T cells that co-express at least 2 activation induced markers (AIMs). Gating strategy and representative data are show below (Figure 2).

  • a.
    Perform flow cytometry on all 200 μL of each sample since the proportion of T cells that will be Mtb-specific will be a rare population.
  • b.
    Co-expression of the following AIMs are commonly used to identify activated T cells: CD69 and CD40L, CD69 and CD25, CD69 and CD137, or CD274 and CD40L (Figure 2).
  • c.
    T cell production of IFNγ can be used alone or together with CD69 as in Figure 2.
  • d.
    If desired, use of a 3rd activation marker to define activated T cells can either be used together with two other AIMs in an “and” or “or” fashion by Boolean gating.
  • e.
    Controls such as anti-MHC-II mAb blockade of Mtb-infected macrophages, or T cell co-culture with non-infected macrophages, can aid in setting gaits to identify the T cell population activated in response to infected macrophages (Figure 2).
• 60.
  For experiments that require flow-sorting of live T cells for downstream assays (OPTION 3), the workflow is similar to that for flow cytometry (without IFNγ secretion assay).

  • a.
    Resuspend cells in FACS tubes in 100 μL of the antibody master mix (prepared in step 51) containing AutoMACS Running Buffer, a 1:50 dilution of Fc block (Human TruStain FcX) and the fluorescently-labeled antibodies needed for immunostaining T cells and flow sorting (listed in Table 2). Include single-stain compensation controls for each channel using PBMCs.
  • b.
    Stain for 20 min at 4°C, protected from light.
  • c.
    Add 2 mL AutoMACS Running Buffer to each tube to wash cells.
  • d.
    Centrifuge at 350 × g for 10 min and aspirate the supernatant.
  • e.
    Resuspend cells in 200 μL of AutoMACS Running Buffer and filter cells through the strainer of a 40 μm filter-top tube.
  • f.
    Wash the original tube and the filter with an additional 200 μL to dilute cells.

    CRITICAL: Leave samples on ice, protected from light before and after cell sorting.

    Note: At this point approximately 4 × 10⁶ cells (range: 2–6×10⁶) pooled from 2‒6 wells for each condition are resuspended in 400 μL of AutoMACS Running Buffer and are ready for cell sorting.

  • g.
    Approximately 2 min before sorting each sample, add 4 μL of 7-AAD (or equivalent viability dye) to the sample.
  • h.
    Prepare polypropylene collection tubes containing 1 mL of desired buffer for collecting sorted cells.
  • i.
    After setting up the sorter according to manufacturer’s instructions (including setting voltages for each channel, running compensation controls, and performing compensation for each channel), record events for a small amount of sample so that the desired gates for each population can be set (Figure 3).
• 61.
  Sort cells using flow cytometry and gate the populations of CD4⁺ T cells that co-express AIMs, as described in step 58, using a gating strategy similar to that shown in Figure 3.

  Note: Co-expression of CD69 and CD40L or CD137 is commonly used to identify CD4⁺ T cells activated in response to Mtb antigen presentation.^1,4,5,6

  • a.
    If desired, use of a 3rd activation marker to define activated T cells can either be used together with two other AIMs in an “and” or “or” fashion by Boolean gating.

    Note: Staining cells with anti-CD3 mAb is not performed for T cells that will be flow-sorted and used for downstream functional assays due to concern that CD3 ligation will alter the natural activation and function after co-culture with Mtb-infected macrophages.

    CRITICAL: Controls such as anti-MHC-II mAb blockade of Mtb-infected macrophages can aid in setting gates for sorting the T cells activated in response to Mtb-infected macrophages (Figure 3).

  • b.
    Depending on the number of cells sorted, downstream assays such as generating T cell clones, or performing transcriptomics and TCR sequencing, in bulk or at the single-cell level, can be used to ascribe function and antigen specificities to these T cells.

Table 1.

Antibody panel for flow cytometry analysis

Fluorophore	Antibody target	Final dilution
BUV 395	CD69	1:50
BUV 737	CD3	1:100
BV510	Live-Dead Aqua	1:1000
BV650	CD45RA	1:50
BV711	CD274	1:50
BV785	CD4	1:100
BB515	CD25	1:25
PE	IFNγ	1:10
PE-Dazzle-594	CD40L	1:20
APC	CD137	1:20
APC Fire 750	CD8	1:100

Table 2.

Antibody panel for flow sorting of live T cells

Channel	Fluorophore	Target	Final dilution
FL1	FITC	CD4	1:50
FL2	PE	CD25	1:50
FL3	PE-Dazzle 594	CD40L	1:20
FL4	PE-Cy7	7-AAD	1:100
FL6	BV421	CD69	1:20
FL9	BV605	CD8	1:50
FL10	APC	CD137	1:20
FL12	APC Fire 750	CD45RA	1:50

Figure 2.

Gating strategy for flow cytometry analysis

Representative flow plots show the recommended gating strategy to identify memory CD4⁺ T cells activated in response to Mtb-infected macrophages. Memory CD4⁺ T cells were pre-enriched from PBMCs by immunomagnetic negative selection for the CD45RA^Lo CD4⁺ fraction prior to co-culture with infected MDMs. After gating on lymphocytes (SSC Area vs. FSC Area) and singlets (FSC Height vs. FSC Area), live CD3⁺ T cells (Live-Dead^Lo CD3⁺) were identified, followed by gating on the CD4⁺ CD45RA^Lo fraction. Those activated in response to Mtb infection were enumerated based on co-expression of two AIMs by flow cytometry, including: CD69 and CD25, CD69 and CD40L, CD69 and IFNγ, or CD274 and CD40L. The proportion of T cells activated in response to infected macrophages (first column) is assessed by comparing co-expression of AIMs to negative controls, including α-MHC-II mAb blockade of infected macrophages (second column) or T cells co-cultured with non-infected macrophages (third column).

Figure 3.

Gating strategy for flow sorting

Representative flow plots show the strategy of gating for flow sorting of CD4⁺ T cells activated in response to Mtb-infected MDMs. Total CD4⁺ T cells were pre-enriched from PBMCs by immunomagnetic negative selection prior to co-culture with infected MDMs. After gating on lymphocytes (SSC Area vs. FSC Area) and singlets (FSC Height vs. FSC Area), live cells (7-AAD^Lo) and CD4⁺ T cells (CD4⁺ CD8^-) were identified. CD4⁺ T cells activated in response to Mtb infected macrophages were sorted based on co-expression of two AIMs (highlighted in red), including CD69 and CD40L or CD69 and CD137. Co-expression of either combination, or both, can be used to sort activated T cells, represented by red gates. Treatment of infected macrophages with α-MHC-II mAb blockade serves as a negative control for antigen-specific T cell activation, represented by black gaits.

Expected outcomes

This protocol facilitates identification of the subset of human CD4⁺ T cells that become activated in response to Mtb-infected macrophages. Approximately 10% of total PBMCs will be CD14⁺ monocytes, and approximately 20% of PBMCs are CD4⁺ T cells, of which typically half have a memory (CD45RA^Lo) phenotype. Activated CD4⁺ T cells are quantified using co-expression of at least two AIMs after a co-culture with autologous, Mtb-infected MDMs, as described previously.^1,4,5,6 Gating strategies we use for flow cytometry analysis (Figure 2) and flow sorting (Figure 3) are shown. Typically, ∼0.2–1% of the CD45RA^Lo memory CD4⁺ T cell population (Figure 2) and ∼0.1–0.5% of total CD4⁺ T cells (Figure 3) are observed to co-expresses AIMs in response to Mtb-infected MDMs. The use of anti-HLA-DR/DQ/DP mAbs to block the TCR-pMHC-II interaction is used to identify background, non-specific T cell activation which can be compared to or subtracted from the response to infected MDMs. The proportion of non-specifically activated CD4⁺ T cells varies, but can represent as much as 50% of the response to Mtb-infected or lysate-treated MDMs.

Since CD40 expression increases on MDMs upon Mtb infection,¹ treatment of the T cell-macrophage co-culture with anti-CD40 blocking mAb is necessary for the use of CD40L as an activation marker in this assay, preventing CD40-CD40L binding which leads to the internalization of CD40L. Alternatively, fluorescently-labeled CD40L mAb can be included through T cell-macrophage co-culture to detect CD40L that becomes internalized.⁴ The production of pro-inflammatory cytokines such as IFNγ can also be used to detect CD4⁺ T cell activation in response to Mtb-infected MDMs and can be compared with AIM expression. Use of the IFNγ secretion assay is an alternative to traditional intracellular cytokine staining (ICS), and is preferred in this protocol because it avoids the effects of brefeldin-A (used in ICS) on the Mtb-infected macrophage during co-culture with T cells. Gail el al. describes the complete details on the expected differences of memory CD4⁺ T cell activation in response to M1 or M2-like Mtb-infected macrophages using this protocol.¹

Finally, flow-sorted CD4⁺ T cells activated in response to Mtb-infected macrophages (± anti-MHC-II mAb blockade) can be used to perform TCR sequencing to further refine estimates of those that are specific for Mtb antigens processed and presented by the infected macrophage. Mtb-specific CD4⁺ T cells can then be identified by focusing on those that contain TCRs that were clonally-expanded in vivo, and are now identified by expression of AIMs in response to infected macrophages. TCR clonotypes from memory CD4⁺ T cells that were non-specifically activated (i.e., in the presence of anti-MHC-II mAb blockade) can be used to focus the analysis. TCR clonotypes linked to responses to infected macrophages can be evaluated in downstream screening assays to identify their antigen specificity.

Quantification and statistical analysis

Flow cytometry data (fcs files) were generated using the FACSDiva software on the BD LSRFortessa X-20 flow cytometer, or using the Sony Cell Sorter Software on the Sony MA900. Data were analyzed using the gating strategies outlined in Figures 2 and 3 using FlowJo (BD). Co-expression of 2 AIMs was used to determine the proportion of CD4⁺ T cells activated in response to Mtb-infected macrophages, and was compared to anti-MHC-II mAb blockade controls to estimate non-specific activation.

Limitations

Despite its advantages, this protocol has limitations. First, it is labor intensive and expensive compared to traditional peptide or protein stimulation using total PBMCs since it requires monocyte and T cell separations, macrophage differentiation, and Mtb infection. This protocol requires cultivation of Mtb under BSL-3 conditions. Attenuated mycobacterial strains, such as Mycobacterium bovis-BCG, may be substituted if BSL-3 work is not possible or practical. Drawbacks to the use of attenuated strains such as BCG include the lack of certain Mtb antigens (e.g., CFP10 and ESAT-6) and virulence factors which may affect T cell activation. This protocol has been optimized for the short-term co-culture of human T cells with Mtb-infected macrophages to induce the expression of AIMs (or IFNγ) by Mtb-specific CD4⁺ T cells, facilitating downstream use of live T cells in functional assays. However, the 16 h stimulation does not provide enough time for T cell proliferation, which cannot be used as a readout for Mtb-specific T cell responses. Similarly, a 16 h stimulation may not be optimal timing for the quantification of cytokine production in cell culture supernatants by ELISA. Although we have focused on CD4⁺ T cells, direct recognition by CD8⁺ or non-conventional T cells could also be evaluated after optimizing the timing and conditions of co-culture with Mtb-infected macrophages.

While the ability to isolate viable, Mtb-reactive T cells in a manner that is agnostic of effector functions is critical to perform downstream functional assays, a limitation inherent to AIMs (or IFNγ) is non-specific memory T cell activation mediated by inflammatory cytokines. Requiring the co-expression of 2 or more AIMs, and subtraction of background activation (using anti-MHC-II mAbs), can mitigate but does not completely resolve this limitation. Shorter incubation periods (e.g., 4–6 h) can also reduce background but are only compatible with certain AIMs (e.g., CD69 and CD40L), and some downstream assays such as transcriptomics may depend on a longer stimulation period to detect transcriptional changes. A refined estimate of Mtb-specific CD4⁺ T cells activated in response to infected macrophages can be achieved through TCR sequencing of flow-sorted, activated T cells, focusing on the proportion of TCRs found to be clonally expanded (2 or more TCR copies) and by excluding TCRs also found when anti-MHC-II mAb blockade is added.

A final limitation is that not all Mtb-specific T cells are necessarily captured with the use of certain AIMs. The activation markers chosen should be tailored to the goals of each experiment. For example, the CD40-CD40L interaction was shown to be necessary for CD4⁺ T cell IL-17 production in response to Mtb.⁷ Therefore, the use of anti-CD40 mAb blockade for optimal CD40L detection is not recommended when downstream assays focus on T cell effector functions.

Troubleshooting

Problem 1

Contamination of MDM cultures with bacteria or mold at steps 14 or 15.

Potential solution

• •Discard samples that are contaminated and sterilize the contaminated wells with ethanol or detergent if attempting to salvage neighboring MDM wells. Ensure the incubator and other surfaces where cells are handled are sterile before replating cells.
• •If uncertain whether PBMCs were originally prepared with aseptic technique, include penicillin, streptomycin, and amphotericin B (or equivalent antimicrobials) in the cRPMI during cell prep and MDM differentiation. However, before Mtb infection of MDMs, wash cells 3‒4 times with cRPMI that does not contain antibiotics since the presence of streptomycin will prevent mycobacterial infection and growth.

Problem 2

Lower than expected CD4⁺ T cell activation after co-culture with infected macrophages at steps 59 or 61.

Potential solution

• •Check cell count and viability of monocytes and CD4⁺ T cells prior to culture.
• •Ensure T cells only undergo one cycle of cryopreservation. Re-freezing lowers viability and could impair T cell function.
• •Check the plated MDMs under microscope prior to Mtb infection to verify that all wells have similar cell density. Only perform Mtb infection of MDM wells that contain the expected cell numbers and distributions. Too few, sparsely distributed MDMs result in fewer APCs with which to activate CD4⁺ T cells.
• •Perform Day 1 CFU after Mtb infection on 3‒4 wells to check the actual MOI, indicating the level of infection. Poor infection of MDMs similarly reduces the potential for T cell activation.
• •When staining T cells after co-culture, ensure that antibody titrations and flow cytometer voltage settings are optimized for the detection of AIM expression.

Problem 3

Higher than expected T cell activation at steps 59 or 61.

Potential solution

• •Certain T cell subsets, such as tissue resident memory T cells in broncho-alveolar lavage (BAL) samples, can constitutively express CD69. Therefore, activation markers should be carefully selected, and the use of two or more AIMs is recommended for specificity.
• •Base gating of T cell AIM expression on controls conditions where T cells are exposed to Mtb-infected macrophages, such as flow minus one (FMO) or isotype controls for activation markers, or by using an anti-MHC-II mAb blockade control. Non-infected macrophages co-cultured with T cells can also serve as a point of reference, but AIM expression is usually even lower for these controls and could lead to over-generous gating.
• •Ensure the concentrations of fluorescence-conjugated antibodies for AIMs is optimal and be vigilant of spectral overlap from channels closely related to those containing the AIMs when designing antibody staining panels for flow cytometry.

Problem 4

Spectral compensation and difficulty distinguishing cell populations by flow cytometry in steps 59 or 61.

Potential solution

• •Ensure antibody titrations and flow cytometer voltage settings are optimized. Be vigilant of spectral overlap from channels closely related to those containing the AIMs when designing antibody staining panels for flow cytometry.
• •Optimize flow cytometer voltage settings for each channel by running a small amount of experimental sample and individual single-stain controls with the goal of maximizing positive signal while minimizing negative or background signal.
• •Instead of compensation beads, use PBMCs or T cells for single-stain controls to compensate the spectral overlap for each channel. Using cells with a size and shape that is the same as those used in your experiment reduces or eliminates the need for manual adjustments to automated compensation matrix calculations, reducing bias.
• •Ensure that the PBMC samples used for compensation are prepared in the same way as your experimental samples.
• •Perform a new compensation with every experiment.
• •Choose antibody targets for single-stain controls that are abundant and expressed at the same density or greater than the targets used for experimental samples in the master mix. CD4, CD8, and CD45 typically exhibit abundant expression on T cells. If the cells used for single-stain controls do not contain a portion that lack expression of the marker used (for comparison), spike-in a similar portion of unstained PBMCs to use as a negative population.

Problem 5

Actual MOI is lower than expected when counting CFU in step 38.

Potential solution

• •While it is not unusual to obtain actual MOI of approximately half of the intended MOI, drastic or inconsistent differences between intended and actual MOIs could lead to poor T cell activation responses.
• •For unexpected or inconsistent low actual MOI based on colony counts, ensure all Mtb growth media and agarose reagents were prepared recently. Use freshly made and non-expired 7H10 plates, and use 7H9 liquid media that was prepared within 30 days. Ensure media contained all components. Also ensure adequate macrophage viability and distribution prior to infection.
• •Mold contamination of 7H10 plates during incubation can impair colony counts. Mtb overgrowth can also make it difficult to distinguish individual colonies, resulting in a lower colony count and thus an artificially lower actual MOI.
• •If the actual MOI is consistently too low despite optimizing regent preparation, avoiding contamination, and accurate colony counts, you may have to adjust the calculation for bacterial concentration for intended MOI based on the OD₆₀₀ of the bacterial suspension in cRPMI in step 27. For example, if your initial estimate assumed an OD₆₀₀ of 0.25 equals 2.5 × 10⁷ CFU/mL, but colony counts yield only 20% of your intended MOI, then consider adjusting your calculation such that an OD₆₀₀ of 0.25 instead equals 0.5 × 10⁷ CFU/mL. Performing additional experiments to enumerate CFUs for Mtb-infected macrophages (prior to including the T cell activation assay) will allow you to refining this calculation using your lab equipment.

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Stephen Carpenter (sxc1507@case.edu).

Technical contact

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Daniel Gail (dxg468@case.edu).

Materials availability

This study did not generate new reagents.

Data and code availability

• •The protocol contains all data generated/analyzed during the study.
• •This paper did not generate original code.
• •Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.