immunology.sciencemag.org/cgi/content/full/5/48/eabb1817/DC1

Supplementary Materials for

T cell engagement of cross-presenting microglia protects the brain from a nasal

virus infection

E. Ashley Moseman*, Alexa C. Blanchard, Debasis Nayak, Dorian B. McGavern*

*Corresponding author. Email: mcgavernd@mail.nih.gov (D.B.M.); ashley.moseman@duke.edu (E.A.M.)

Published 5 June 2020, Sci. Immunol. 5, eabb1817 (2020)

DOI: 10.1126/sciimmunol.abb1817

The PDF file includes:

Supplementary Materials and Methods Fig. S1. Diagrams of sectioning planes through the mouse head. Fig. S2. Parenchymal cells within the OB and brain survive VSV infection after intranasal inoculation. Fig. S3. Virus-specific CTLs flux calcium for longer durations while undergoing stable interactions. Fig. S4. Generation of floxed β2M transgenic mice. Fig. S5. VSV does not infect microglia and CTB traffics from OSNs to OB glomeruli. Fig. S6. Quantification of microglia and infiltrating cells in the OB using different microglia depletion methods. Legends for movies S1 to S4 References (102, 103)

Other Supplementary Material for this manuscript includes the following: (available at immunology.sciencemag.org/cgi/content/full/5/48/eabb1817/DC1)

Movie S1 (.mp4 format). Virus-specific CTLs rapidly migrate across VSV infected axon tracts within the infected outer nerve layer. Movie S2 (.mp4 format). Virus-specific CTLs survey the infected glomerular layer with the OB. Movie S3 (.mp4 format). Antiviral CTLs engage cognate antigen and flux calcium within the infected OB. Movie S4 (.mp4 format). Antiviral CTLs flux calcium upon contact with microglia.

Supplementary Materials and Methods

In vivo depletion studies. Antibodies used for cell-specific depletions were purchased

from BioXCell. C57BL6/J mice were injected with 300 µg of anti-CD8α (YTS169), 600

µg of anti-CD4 (GK1.5) or 600 µg of isotype Rat IgG2b control antibodies. All antibodies

were administered intravenously one day prior to infection. For microglia depletions

CX3CR1-Cre-ER x Rosa Stopfl/fl DTR mice (>6 weeks of age) were provided tamoxifen

chow (Envigo) for 3 weeks to induce diphtheria toxin receptor (DTR) expression on

CX3CR1-expressing cells. Mice were returned to regular chow for >60 days to allow for

complete monocyte turnover. Mice were then infected with VSV-OVA and administered

1 ug diphtheria toxin (DT) i.p. for three days prior to sacrificing to deplete microglia.

Percent of microglial depletion ranged from 60-90%. Alternatively, C57BL6/J mice were

provided chow containing 300 mg/kg PLX3397 for 30-40 days. PLX3397 was purchased

from Adooq Biosciences and formulated in NIH-31 chow by Envigo.

Adoptive lymphocyte transfer. For imaging and flow cytometric studies, blood was

isolated from OT-I or P14 mice, checked for transgene positivity, and counted to

determine cell numbers for transfer. Three thousand mCerulean+ CD8+ OT-I T cells or

mTomato+ GCaMP6s+ CD8+ OT-I T-cells were transferred from the transgenic host one

day prior to infection. To transfer P14 LCMV-specific T cells into VSV-infected mice,

10,000 OFP+ P14 or mTomato+GCaMP6s+ P14 T cells were adoptively transferred to a

wild type C57BL6/J mouse prior to i.p. infection with 2 x 105 PFU LCMV Armstrong. At

day 7 post-LCMV infection, spleens from infected animals were dissociated and 2.5x107

splenocytes containing the expanded P14 T cells were transferred i.v. to a day 5 VSV-

infected host prior to day 6 imaging.

Flow cytometry and cell isolation. Lymphoid organs were dissociated into single cell

suspensions, through a 100-µm filter mesh in PBS containing 1% FBS and 5 µM EDTA,

blocked in 1.5 μg/ml anti-CD16/32 (2.4G2) and 15 μg/ml mouse IgG (Jackson

ImmunoResearch). Brain samples of saline perfused animals were isolated, dissected

with scissors, and digested in 1 mg/mL Collagenase-D and 0.25 mg/mL DNAse in RPMI

Complete Medium (Gibco) for 30 minutes at 37 degrees Celsius with frequent trituration

in a 5 mL conical tube. Cells were then washed, spun down at 1500 x g for 3 minutes,

and resuspended in 3 mL of a 37% isotonic Percoll solution. The samples were spun at

1500 x g for 15 minutes, and the supernatant was removed. Pelleted cells were washed

with cold RPMI, spun down, and resuspended in Fc block solution for 10 minutes. A 2x

antibody cocktail was added directly to cells for 20 minutes before a final wash and

resuspension in FACS buffer.

Antibodies used to label microglia and hematopoietic cells were from BioLegend

unless otherwise noted: anti-Thy1.2 (AL700, 30-H12), CD45 (PE; 30-F11), CD45.2 (PE,

BV650, and APC; 104), anti-CD4 (BV785; RM4-5), anti-CD8 (BV421 and APC-Cy7; 53-

6.7), anti-TCR-beta (AL647; H57-597), anti-Ly6C (APC-Cy7; HK1.4), anti-Ly6G (BV510;

1A8), anti-CD11b (PE-Cy7 and APC-Cy7; M1/70), anti-CD40 (PE-Cy7; 3/23), Rat IgG2a

κ Isotype Ctrl (PE/Cy7; RTK2758), anti-CD11c (BV605 and BV785, N418), anti-CD80

(PE; 16-10A1), Armenian Hamster IgG Isotype Ctrl (PE; HTK888), anti-KbDb (MHC I;

AL647; 28-8-6), mouse IgG2a, κ Isotype Ctrl (AL647; MOPC-173), anti-IA/IE (MHCII;

PB; M5/114.15.2), and rat IgG2b, κ Isotype Ctrl (PB; RTK4530). Anti-CD86 (FITC; GL1)

and rat IgG2a, κ Isotype (FITC; R35-95) were obtained from BDPharmingen. CD45.1

(APC; A20) and NK1.1 (PE; PK126) were obtained from eBioscience. 7-AAD and DAPI

(BD Pharmingen) were used to identify and exclude dead cells. Twenty-five microliters

of Flow Cytometry Absolute Count Standard™ (Bangs Laboratories) was added to each

sample prior to acquisition to allow calculation of cells/sample. Samples were acquired

on an LSR II digital flow cytometer (BD Biosciences), and data was analyzed using the

FlowJo software (Tree Star).

Immunohistochemistry. Mice were perfused intracardially with 2.5% neutral buffered

formalin (NBF) and skin around the head and nose was removed before fixing whole

heads in 2.5% NBF overnight at 4C. Heads were then moved to a 0.5 M EDTA solution

for 5-7 days at 4C to decalcify bone around the olfactory regions. Once decalcified,

heads were embedded intact for axial sections, or cut sagittally or coronally and

embedded in tissue freezing medium (Triangle Biomedical Sciences). Cryosections

were cut using a Leica CM1850 cryostat at a thickness of 20-30 µm after which sections

were washed and stained in PBS containing 5 μM EDTA and 2% FBS. Sections were

stained with a combination of DAPI (1 µg/ml) and the following antibodies: CD45.2 PE

(0.4 µg/ml; clone 104; Biolegend), DyLight 488 conjugated polyclonal anti-GFP (1 µg/ml;

Rockland) and rabbit anti-Iba-1 (0.4 µg/ml; Wako). For Iba-1 staining, a secondary anti-

rabbit Alexa Fluor 647 antibody (Jackson Immunoresearch) was used. Primary

antibodies were incubated on cryosections overnight at 4C, and secondary anti-rabbit

staining was done for 2 hrs at 4C. After staining, sections were washed 4-5 times in

staining buffer, and 2-3 drops of FluorSave Reagent (Calbiochem) were added to each

section before addition of a coverslip. Images were acquired using an Olympus FV1200

laser scanning confocal microscope equipped 4 detectors, 6 laser lines (405, 458, 488,

515, 559, and 635nm) and 5 objectives (4x/0.16 NA, 10x/0.4 NA, 20x/0.75 NA, 40x/0.95

NA, and chromatic aberration corrected 60x/1.4 NA).

Quantification of viral escape by immunohistochemistry. To measure viral escape,

mice were infected with VSV-GFP and euthanized on day 8 post-infection. Consecutive

sagittal sections (Fig. S1B) were obtained for the first 1000 µm lateral from the midline

for both hemispheres of the decalcified heads. Slides were screened using

epifluorescence microscopy, and slides with escape (VSV-GFP signal outside of

olfactory bulb glomeruli) were selected for further antibody staining, imaging, and

analysis. Selected slides were stained with goat anti-GFP, anti-CD45.2 PE, and DAPI.

After tiling images of the entire brain, the slide with maximal VSV-GFP signal for each

animal was chosen for analysis. Using Imaris software (Bitplane) version 7.6.1, a

uniform threshold was set for each channel and applied to all samples. A custom

surface was manually drawn around all regions containing VSV-GFP signal. Hindbrain

regions are categorized as the regions rostral to the midbrain (not including the

cerebellum); forebrain regions are caudal to the midbrain and include the olfactory bulb

region interior to the glomerular layer. Virus was quantified as the sum intensity of VSV-

eGFP signal per unit area of the particular brain region.

Generation of Bone Marrow Chimeras. H2KbDb-/- mice and CX3CR1gfp/+ mice (>6

weeks) and their age and sex-matched controls received 950 RADs in a cesium 137

irradiator. Mice were reconstituted with bone marrow from CD45.1+ congenic donors.

Six to eight weeks following irradiation, mice were infected and used for

experimentation.

Skull thinning and two-photon microscopy. For imaging experiments, mice were

anesthetized with ketamine (85 mg/kg), xylazine (13 mg/kg), and acepromazine (2

mg/kg) in PBS and maintained at a core temperature of 37°C. Two-photon imaging

through a surgically thinned skull was adapted from methods described previously (38,

39). The skull above the olfactory bulb (rostral part of the frontal bone) was thinned to

allow imaging of this brain structure. 3D time lapses were captured using a Leica SP8

two-photon microscope equipped with an 8,000-Hz resonant scanner, a 25× color

corrected water-dipping objective (1.0 NA), a quad HyD external detector array, a Mai

Tai HP DeepSee Laser (Spectra-Physics) tuned to 905nm and an Insight DS laser

(Spectra-Physics) tuned to 1050nm. The following dichroic mirrors were used for most

imaging studies: 458nm-LP, 484nm-LP or 509nm-LP, 562nm-LP or 624nm-LP. For non-

GCaMP imaging, stacks were acquired at 10-30 sec intervals. For GCaMP6s movies,

stacks consisted of 7 planes (3 μm step size) and were acquired at 5.47 sec intervals.

Intravital imaging analysis. Movies were imported into Imaris 8.0.0 (Bitplane) and the

equidistant time interval was entered for accurate tracking (5.47 seconds). The spot

feature in Imaris was used to track fluorescent OT-I and P14 T cells. Position data was

exported to Excel and imported into a T cell analyzer (version 1.7.0 software written by

Dr. John Dempster, University of Strathclyde, Glasgow, Scotland) (102) to analyze

average track velocities, arrest coefficients, and motility coefficients (Fig. 4C-D). To

measure the percent of OT-I GCaMP6s+ and P14 GCaMP6s+ T cells fluxing calcium,

the percentage of flashing vs. non-flashing cells was calculated at 6 equidistant time

points for each time lapse (Fig. 5B, 5H, 7F). To analyze OT-I GCaMP6s+ mTomato+ T

cell fluxing rates and velocities, mTomato+ T cells were randomly selected from each

quadrant of the entire time lapse. The path of each T cell was tracked manually for the

entire flashing period. The flashing period was defined as the interval spanning three

time points before the first calcium flux to three time points after the final calcium flux.

Manual tracking was performed to ensure all GCaMP6s+ calcium signal was collected.

Position and mean GCaMP6s intensity data were exported into Excel. Calcium flux

activity was calculated as a ratio of the GCaMP6s mean intensity divided by mTomato

mean intensity. This was done to account for changes in cell brightness throughout the

time lapse. A GCaMP6:mTomato mean intensity ratio of greater than 0.55 was

considered a calcium flux event. Non-fluxing periods were a minimum of five time points

and had a GCaMP6s:mTomato ratio of less than 0.55. The instantaneous velocities

across all fluxing and non-fluxing time intervals were averaged for each T cell and used

to generate a single fluxing and non-fluxing data point shown in Figure 5C. In Figure

5G, OT-I GCaMP6s+ mTomato+ T cells were tracked for the entirety of each time lapse

(as described above) and the number of flashes during the tracked period was counted.

A mean velocity threshold of 2 um/min was used to define stable vs. kinetic T cell

interactions in Figures 5F,G. To calculate the percent of antigen-specific interactions

between OT-I GCaMP6s+ mTomato+ T cells and microglia, the number of T cell calcium

flux events occurring in contact with CX3CR1gfp/+ microglia or off target was calculated

at seven equidistant time points throughout each time lapse. The ratio of “on” vs. “off”

contact flux events was calculated at each time point and averaged (Fig. 6C).

Uptake of OSN material. Fifteen micrograms of cholera toxin subunit B (CTB) diluted in

PBS (Recombinant, Alexa Fluor 647, Thermofisher Scientific) was administered

intranasally 10 hours prior to infection at a concentration of 1 mg/mL (7.5 µL per nostril,

15 µL per mouse) to label olfactory sensory neurons. Mice were then infected with VSV-

eGFP, VSV-PeGFP, or VSV-OVA and saline perfused at day 6 for flow cytometric and

immunohistochemical analysis. Uptake measured flow cytometrically was represented

as the percent of each cell population containing CTB-647+ material. For

immunohistochemistry analysis, three groups of mice were analyzed: naïve with CTB

alone, VSV-GFP infected without CTB, and VSV-GFP with CTB. Five large regions

containing both VSV-GFP and CTB-647, each with 3-5 individual 40x tiled regions were

analyzed per mouse and averaged. Using Imaris 7.6.1, surfaces were created for

microglia around the glomerular layer alone (where there is viral exposure) and masks

were generated for CTB and VSV-GFP signal outside the microglial surfaces. Surfaces

were generated for all CTB-647+ signal within the microglia and masked against VSV-

GFP to eliminate autofluorescent signals. The number of microglia containing CTB-647

only was calculated for each experimental group. The same acquisition procedure was

used to analyze VSV-PeGFP uptake. However, after microglial surface creation,

surfaces were instead created around PeGFP signal within the microglia and masked

against CTB-647 to eliminate autofluorescence caused by infection. The number of

microglia containing PeGFP signal only was then calculated for each group.

Clarity. Stopfl/fl TdTomato mice were infected with 4 x 105 VSV-iCre intranasally. After

day 50 post-infection, mice were sacrificed, and the olfactory bulbs were placed in 4%

PFA overnight. Clearing of brain tissue followed protocols described previously (103).

After two weeks in clearing reagent, whole mounted olfactory bulbs were imaged via

confocal microscopy.

Statistical Analysis. Statistical significance (P < 0.05) was determined using

Student’s t test (two groups, parametric data), ranked sum test (two groups, non-

parametric data), one-way analysis of variance (ANOVA; more than two groups with

parametric data), or a one-way ANOVA on ranks (more than two groups with non-

parametric data). Statistical differences between survival curves were determined used

log-rank test. The correlation coefficient and p value shown in Figure 7G were

calculated using a Pearson’s product-moment correlation test. All statistical analyses

were performed using GraphPad Prism 6.0 or SigmaPlot 11.0.

Fig. S1. Diagrams of sectioning planes through the mouse head.

Fig. S2. Parenchymal cells within the OB and brain survive VSV infection after

intranasal inoculation. A) Representative micrographs from VSV-iCre infected Stopfl/fl

tdTomato mice captured in the olfactory bulb at least 40 days post-infection. Cells that

were VSV-iCre infected but cleared the virus are permanently marked with Tdtomato

expression (red). DAPI was used to stain nuclei (blue) and reveal the olfactory bulb

anatomy. B) Representative sagittal brain section from a VSV-iCre infected Stopfl/fl

tdTomato mouse shows the distribution of previously infected cells in the olfactory bulb

(OB) and a few caudal brain regions (white arrowheads).

Fig. S3. Virus-specific CTLs flux calcium for longer durations while undergoing

stable interactions. As in Figure 5, OT-I interaction behaviors were identified as kinetic

(velocity of >2µm/min) or stable (velocity of <2µm/min). Sum calcium flux durations (left

panel) and maximum calcium flux durations (right panel) plotted against velocity are

shown for OT-I mTomato+ GCaMP6s+ T cells in VSV-OVA infected OB at day 6.

Antiviral CTLs generally fluxed calcium for longer time periods when engaged in stable

vs. kinetic interactions.

Fig. S4. Generation of floxed β2M transgenic mice. A) Lox P sites were inserted

flanking exons 2 and 3 of the endogenous beta 2 microglobulin (β2M) locus. B)

Genomic DNA was isolated from olfactory epithelial tissue of floxed β2M mice infected

intranasally with VSV-iCre either 37 days or 48 hours prior to harvest. iCre expression

results in removal of exons 2 and 3 from infected cells and generates a smaller band

(286 bp). Not all cells in the olfactory epithelium are infected by VSV and thus retain the

original conditional β2M allele (1906 bp PCR product).

Fig. S5. VSV does not infect microglia and CTB traffics from OSNs to OB

glomeruli. A,B) C57BL/6 or Stopfl/fl Tdtomato mice were infected intranasally with VSV-

eGFP or VSV-iCre, respectively, and the expression of VSV-eGFP (A) or TdTomato (B)

was analyzed within olfactory bulb microglia (defined as Thy1.2- CD11b+, Ly6C/G-,

CD45int) or nonhematopoietic cells (CD45-) at the denoted time points post-infection

(n=4 mice per group). C) OMP-GFP mice received an intranasal inoculation of cholera

toxin B (CTB) 10 hrs prior to harvest. A representative confocal micrograph

demonstrates that CTB (red) traffics along the OSNs (green) to termini in olfactory bulb

glomeruli (GL). DAPI was used to label cell nuclei (blue). The individual color channels

are shown in grayscale for clarity. (ONL, outer nerve layer)

Fig. S6. Quantification of microglia and infiltrating cells in the OB using different

microglia depletion methods. A,B) At day 6 post VSV-OVA infection, the number

microglia (A; CD45int Thy1.2- CD11b+ Ly6C- Ly6G-; *p=0.017) and infiltrating

monocytes (B; CD45hi Thy1.2- CD11b+ Ly6G- Ly6C+) were quantified in the OB of wild

type (WT) control vs. CX3CR1-CreER x ROSA Stopfl/fl DTR microglia depleted mice.

Colored horizontal lines represent mean+SD. Data are pooled from seven independent

experiments (n=10 WT mice and n=13 CX3CR1-CreER x ROSA Stopfl/fl DTR mice per

group). C-E) C57BL6/J mice received control or PLX3397 containing chow for one

month. Mice then received an adoptive transfer of naïve mTomato+ OT-I T cells and

were intranasally infected with VSV-OVA. At day 7 post-infection, the number of

microglia (A; CD45int, CD11b+, CD39+, Ly6C-; ****p<0.0001), OT-I T cells (B; CD45hi,

mTomato+, CD8+, CD4-, CD11b-), and CD8+ T cells (C; CD45hi, CD8+, CD4-, CD11b-)

in the OB was determined flow cytometrically. Colored horizontal lines represent

mean+SD. Data are pooled from two independent experiments (n=10 mice per group).

NS, not significant.

Movie Legends

Movie S1. Virus-specific CTLs rapidly migrate across VSV infected axon tracts

within the infected outer nerve layer. Representative 3D reconstructed time-lapse(s)

of olfactory bulb imaging windows from mice infected with a mix of VSV-eGFP and

VSV-OVA (green). Mice received an adoptive transfer of mCerulean expressing OT-I T

cells (cyan) the day prior to infection. Antiviral CTL behaviors within infected olfactory

sensory nerve axons leading into the OB were visualized ten days after intranasal

infection. Immediately prior to imaging, mice were given an intravenous injection Evans

Blue to label vasculature (red).

Movie S2. Virus-specific CTLs survey the infected glomerular layer with the OB.

Representative 3D reconstructed time-lapse of the olfactory bulb of a mouse infected

with a mix of VSV-eGFP (cyan) and VSV-OVA. Mice received an adoptive transfer of

mCerulean expressing OT-I T cells (blue) the day prior to infection. Antiviral CTL

dynamics within and surrounding infected olfactory glomeruli were visualized six days

after intranasal infection.

Movie S3. Antiviral CTLs engage cognate antigen and flux calcium within the

infected OB. Representative time-lapse of 3D reconstruction from VSV-OVA infected

olfactory bulb. OT-I T cells expressing constitutive GCaMP6s (green) and membrane

targeted tdTomato (red) proteins were transferred into mice the day prior to infection.

Olfactory bulbs were imaged 6 days after intranasal infection. Immediately prior to

imaging, mice were given an intravenous injection Evans Blue to label vasculature

(blue).

Movie S4. Antiviral CTLs flux calcium upon contact with microglia. Representative

time-lapse of 3D reconstruction from VSV-OVA infected olfactory bulb. OT-I T cells

expressing constitutive GCaMP6s (green) and membrane targeted tdTomato (red)

proteins were transferred into CX3CR1gfp/+ mice containing wildtype bone marrow the

day prior to infection. Olfactory bulbs were imaged 6 days after intranasal infection.