Animals and reagents
C57BL/6, DsRed.T3, H2-K1/H2-D1 knockout (MHCI−/−), Eα52−68, LCMV GP61–80-specific (SMARTA) and OVA323–339-specific (OT-II) TCR transgenic mice were acquired from The Jackson Laboratory. H2-Ab1 knockout (MHCII−/−) mice were obtained from Taconic Biosciences. H2-K1/D1−/−H2-Ab1−/− (MHCI-II double knockout) mice were obtained by breeding single knockout mice to homozygosity. All mice were maintained in Ohio State University ULAR and National Institutes of Health (NIH) animal facilities in compliance with Institutional Animal Care and Use Committee standards.
Cell cultures were maintained in sterile complete Roswell Park Memorial Institute (RPMI) media. The RPMI 1640 medium was supplemented with 10% heat-inactivated fetal bovine serum, 50 U/ml penicillin, 50 µM streptomycin, 1 mM sodium pyruvate, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 50 µM 2-mercaptoethanol, and 10 mM HEPES (Thermo Fisher Scientific). For magnetic separations, cells were maintained in filtered and degassed magnetic-activated cell sorting (MACS) buffer. The MACS buffer consisted of PBS (Lonza) supplemented with 0.5% BSA (Sigma-Aldrich) and 2 mM EDTA (Sigma-Aldrich). For splenic dendritic cell (DC) isolation, Liberase TM (Blendzyme) and DNase I were purchased from Roche. 50 mg Liberase was dissolved in complete RPMI, resulting in a final concentration of 5 mg/mL. The solution was kept on ice, stirred gently every 5 minutes for 30 minutes and aliquoted into 1 mL portions. 10 mg/mL DNAse stock solution was prepared with 0.15 M NaCl and divided into 500 µL aliquots. Both enzymes were stored at -20ºC.
Samples were stained for flow cytometry using fluorescence-activated cell sorting (FACS) buffer (either PBS or Hanks’ balanced salt solution (HBSS) supplemented with 2% fetal bovine serum, 1% HEPES, and 10 mM sodium azide (Sigma-Aldrich)). For confocal microscopy, PBS supplemented with 1% BSA was used. The antibodies used for flow cytometry are as follows: anti-CD4-efluor450 (clone GK1.5), anti-B220-PE (clone RA3-6B2), anti-CD11c-AF700 (clone N418), anti-CD45.1-PE.Cy7 (clone A20, Thermo Fisher Scientific), anti-CD45.2-AF700 (clone 104), anti-CD44-BUV395 (clone IM7, BD Biosciences), anti-I-A/I-E-BV605 (clone M5/114.15.2), anti-I-A/I-E-BV785 (clone M5/114.15.2), streptavidin-BV650, streptavidin-AF647 and purified anti-CD16/32 (clone 93). The antibodies were purchased from BioLegend unless stated otherwise. All peptides used in the study (Supplementary Table 1) were obtained from the NIH Research Technologies Branch, NIAID Peptide Core Facility.
For AsH probe labeling, peptides were pre-treated with fresh Tris Carboxy Ethyl Phosphene (TCEP, Sigma-Aldrich) to protect tetracysteine residues from oxidation. 10 mM ReAsH stock solution was prepared by adding 45 µL sterile cell-culture grade DMSO (Sigma-Aldrich) to 250 µg solid ReAsH (Cayman Chemical) and 10 mM FlAsH solution was prepared by adding 150uL DMSO to 1 mg FlAsH powder (Cayman Chemical). British Anti-Lewisite (BAL, Alfa Aesar) and HBSS with Ca++ and Mg++ (Thermo Fisher Scientific) was used to wash excess FlAsH off the cells. BAL is resuspended in sterile degassed water to obtain 8 mM stock solution. FlAsH, ReAsH and BAL are highly sensitive to oxidation, therefore their activity may decline in subsequent uses. This may be averted, at least in part, by quickly purging stock solution containers with 4M Argon gas (Sigma-Aldrich, 501247), placing container in a 50mL tube, purging again with Argon before storing FlAsH and ReAsH at -20ºC, BAL at 4ºC. We do not recommend storage longer than one month or repeated use of the same reagent stock.
DC isolation, peptide loading and -AsH probe labeling
Mature splenic DCs were isolated as previously described [35] and resuspended in complete RPMI. Peptides were added into 20 mM TCEP solution at twice the desired concentration for loading DCs. The solution was vortexed thoroughly and incubated at 37ºC for 30 minutes. Meanwhile, ReAsH stock solution was diluted to 100 uM in complete RPMI and added on DC suspension to obtain 1 uM final ReAsH concentration. Cells were incubated at 37ºC for 30 minutes and washed twice with complete RPMI. ReAsH labeled DCs were mixed with equal volume of peptide-TCEP solution, incubated at 37ºC for 30 minutes, washed twice and resuspended in complete RPMI to proceed with FlAsH labeling. Flash was added to cell suspension to achieve 1 uM final concentration, incubated at 37ºC for 30 minutes and washed twice with complete RPMI. To remove excess FlAsH and reduce non-specific binding, cells were resuspended in 240 uM BAL diluted in HBSS with Ca++ and Mg++, washed twice, then washed a third time with PBS and resuspended in PBS.
DC-T cell cocultures
Freshly isolated DCs were pulsed with peptide in complete RPMI and were incubated at 37°C for 30 min followed by three washes with complete RPMI to remove unbound peptide. Pulsed DCs were then treated with -AsH as decribed above then seeded into flat-bottom 96-well plates (Corning) at a density of 5 × 104 to 1 × 105 cells per well. T cells and iTreg cells were labeled with e450 or e670 (eBioscience) according to the manufacturer’s protocol. Cells were cocultured at 37°C for for indicated periods, at a 1:10 ratio of DCs to T cells for proliferation assays and 1:1 for trogocytosis assays.
iTreg differentiation
For iTreg differentiation, Tnaive cells were isolated and 24-well sterile tissue culture plates (Corning) were coated with anti-CD3ε (BioLegend) and anti-CD28 (BioLegend) as described [36]. Naïve CD4+ T cells were resuspended in complete RPMI media supplemented with 100 IU ml − 1 recombinant human IL-2 (Peprotech) and 5 ng ml − 1 recombinant human TGF-β (Peprotech). 3 × 105 cells were added to the wells at a volume of 1 ml per well. Cells were cultured at 37°C, 5% CO2 for 3–4 days.
Trogocytosis assay
Trogocytosis assays were perfomed as described in [20]. Briefly, freshly isolated splenic DCs were labeled with 4µM PKH26 (Sigma-Aldrich) for labeling of the cell membrane as described in Puaux et al.48. DCs were then pulsed with 10 µM peptide, treated with -AsH and were cocultured with T cells for up to 18 h. Cell conjugates were dissociated by washing cells with MACS buffer with 2 mM EDTA; cell suspensions were prepared for flow cytometry.
Flow cytometry
Cells were stained in FACS buffer containing fluorochrome-conjugated antibodies for 30 minutes at 4°C, protected from light. Data acquisition was performed with BD LSRFortessa and BD LSR II cytometers (BD Biosciences). Data analysis was performed with FlowJo v10.8.1.
Adoptive transfer
Mice were anesthetized in an induction chamber with 1.5% isoflurane USP (Baxter). Pulsed DCs were adoptively transferred through the right footpad in 50uL sterile PBS. T cells labelled with e450 or e670 (Thermo Fisher Scientific) were injected intravenously in 100uL sterile PBS through the retro-orbital sinus.
Confocal microscopy
35mm dishes with a 14mm glass coverslip bottom (MatTek) were pre-treated with 10ug/ml fibronectin (Sigma-Aldrich) in PBS for 1h at room temperature and washed twice. Splenic DCs were added onto the glass coverslip in complete RPMI and pulsed with 5–10 µM peptide at 37°C for 1 hour. Excess peptide was washed off with complete RPMI before addition of CD4 T cells. Co-cultures were imaged for 3-18h using a Leica SP-8 inverted microscope (Leica Microsystems) equipped with a full range of visible lasers, two hybrid detectors, three photomultiplier detectors, and a motorized stage. 63× objective (Leica Microsystems) was used and microscope configuration was set up for 3D analysis (x, y, z) of the cellular layer. The following lasers were used: diode laser for 405 nm excitation; argon laser for 488 and 514 nm excitation; diode-pumped solid-state laser for 561 nm; and HeNe laser for 594 and 633 nm excitation. All lasers were tuned to minimal power (between 0.3% and 2%) to prevent photobleaching. z stacking of images of 10–12 µm were collected. Mosaic images of large cell culture areas (1 mm2) were generated by acquiring multiple z stacks using the Tile scan mode and were assembled into tiled images using LAS X v4.0 (Leica Microsystems).
Intravital two-photon laser-scanning microscopy of mouse popliteal lymph nodes
Mice were anesthetized in 1.5% isoflurane USP (Baxter) through a rodent nose cone followed by surgery to expose the right popliteal lymph node. Warm PBS was added to maintain lymph node moisture. Mouse body temperature was maintained with an infrared blanket (Braintree Scientific) throughout the imaging process after which they euthanized through cervical dislocation under anesthesia. Mice were imaged on a glass stage with a two-photon laser-scanning microscopy setup with a Leica SP8 inverted confocal microscope with dual multi-photon lasers, Mai Tai and InSight DS (Spectra-Physics), L 25.0 water-immersion objective, 0.95 NA (Leica Microsystems), and a 37°C incubation chamber (NIH, Division of Scientific Equipment and Instrumentation Services). The Mai Tai laser was tuned to 890 nm to excite e450and FlAsH; the InSight DS laser was tuned to 1,150 nm to excite DsRed/ PKH-26/ ReAsH and e670. For time-lapse imaging, a z stack consisting of 10–12 single planes (5 µm each over a total tissue depth of 50–60 µm) was acquired at 15 second intervals for a total period of 1–4 h.
Confocal microscopy of live lymph node sections
Live lymph node sections were imaged ex vivo using confocal microscopy. To maintain tissue architecture, popliteal lymph nodes were immediately transferred to PBS with 1% BSA and kept on ice immediately following harvest. Residual fat and connective tissue were removed using a Leica MZ6 StereoZoom microscope (Leica Microsystems). Lymph nodes were suspended in 38°C agarose in DMEM and kept over ice. Following complete agarose solidification, complete RPMI was added and blocks were cut containing one lymph node each. Lymph node blocks were cut into 200 µm sections with a Leica VT1000 S vibrating blade microtome (Leica Microsystems) at speed 5 in ice-cold PBS and frozen at -80°C. Prior to imaging, complete RPMI medium was added to tissue sections and incubated at 37°C for 2 h. Sections were held down with tissue anchors (Warner Instruments) in 35mm dishes with a 14mm glass coverslip bottom (MatTek) and were imaged with a Leica SP8 inverted five-channel confocal microscope equipped with an environmental chamber (NIH, Division of Scientific Equipment and Instrumentation Services) and a motorized stage. The microscope configuration was set up for four-dimensional analysis (x, y, z, t) of cell segregation and migration within tissue sections. A diode laser for 405 nm, an argon laser for 488 and 514 nm, a diode-pumped solid-state laser for 561 nm, and a HeNe laser for 594 and 633 nm excitation wavelengths were tuned to minimal power (between 0.3% and 2%). z-stack of images of 10–25 µm were collected. Mosaic images of whole lymph nodes were generated by acquiring multiple z stacks using a motorized stage to cover the whole lymph node area and assembled into a tiled image with the LAS X software. For time-lapse analysis of cell migration, tiled z stacks were collected over time (1–4 h).
Image analysis
Analysis of confocal and intravital two-photon images was performed using Imaris v9.2.1 and v10.0.0 (Bitplane) as previously described [35]. In brief, e450 and e670 signals were utilized for reconstructing the 3D structure of T cells and iTreg cells as surface objects. To reconstruct the 3D structure of DCs, overlapping DsRed, PKH-26, and ReAsH signals were employed. Imaris v10.0.0's built-in algorithms were used to calculate 3D volumes, track velocities, surface-to-surface colocalization parameters, and FlAsH (green) intensities of cells.
Statistical analysis
Statistical analysis was conducted on Prism v9.5.1 (GraphPad). Statistical tests and relevant P values for each figure are indicated in the figure or figure legend.