Animal model
Experiments were performed using male C57BL/6 wild-type (WT) mice (The Jackson Laboratory [strain C57BL/6J; stock number 000664; http://jaxmice.jax.org/). All animals were bred on location, handled regularly and group housed (max. 5) under standard laboratory conditions (12-hour light/dark cycle, 21°C, 50% humidity) in the presence of cage enrichment (nesting material). Water and food were available ad libitum. Experiments were reviewed and approved by the Animal Ethics Committee of the Central Authority for Scientific Experiments on Animals of the Netherlands (CCD, approval protocol AVD115002016659), and acting in accordance with the Directive of the European Parliament and of the Council of the European Union of September 22nd 2010 (2010/63/EU).
Plasma collection and injection
Mouse plasma was collected from WT mice (average age 20 months +/- 2 months). Mice were euthanized using Euthanimal, then plasma was collected by intracardial bleed at time of sacrifice. Plasma was prepared from blood collected with edetic acid followed by centrifugation at 1000g for 10 min at 4°C (brake 1). Plasma aliquots were stored at −80°C until further use. Prior to administration, plasma was pooled and dialyzed using 3.5-kDa Slide-A-Lyzer Dialysis Cassettes (Thermo Scientific) in PBS to remove edetic acid (2x 2 hour at RT, and 1x o/n at 4°C in 4 L PBS). WT mice were systemically treated with 8 plasma injections (150 µL), or with PBS injections as control, intravenously via the tail vein over a period of 28 days, starting at 5 months of age (± 1 week). Mice were sacrificed the day after the last plasma injection for immune cell isolations. Mice used in immunohistochemical analyses received after the last day of plasma treatment, intraperitoneal injections of BrdU (150mg/kg in PBS; Thermo Fisher) for 3 consecutive days and were directly sacrificed the day after the last BrdU injection.
Immune cell isolations from brain, spleen and blood
Tissue collection
Mice were first euthanized using Euthanimal. Blood was collected by intracardial bleed at time of sacrifice with a needle containing edetic acid via the right ventricle. Trans-cardinal perfusion was performed through the left ventricle with a peristaltic pump using PBS (no Ca2+ or Mg2+) for 5 min (3 mL/min). After perfusion, mice were decapitated and the brain and spleen were removed. For the 6-month-old “young-adult” and 20 month-old “aged” mice, total brain was used for immune cell isolation and subsequent CyTOF analysis. For the plasma or PBS-treated animals, cortical and hippocampal brain structures were removed prior isolation. Finally, tissues were stored in in RPMI (RPMI 1640, Thermo Fisher) on ice until further processing.
Spleen and brain dissection and digestion
Spleens and brains were transferred to a 24-well plate and thoroughly dissected with sharp scissors in 1 mL of digestive mix (2 U/mL Liberase TL, #5401020001). The 1 mL cell suspension was transferred to 12-mL round bottom polypropylene tubes containing 1 mL of 2 U/mL Liberase TL. A magnetic stirrer was added to each tube and the suspensions were incubated in a 37ºC stove on a magnetic stir plate at 200 RPM for 20 min (spleen) and 50 min (brain). Then, 10 mL of ice-cold RPHE (RPMI 1640, 10% FCS, 10 mM EDTA, 20 mM HEPES, 50 µm 2-mercaptoethanol) was added to each tube and the tubes were put in a glass bucket containing ice on top of a stir plate and stirred for 10 min at 500 RPM. Next, the cell suspension was filtered through a 70 µm cell strainer into a 50-mL canonical tube and 30 mL of RP10 (RPMI 1640, 10% FCS, 1% penicillin/streptomycin, 1% glutamine) was added.
Spleen processing
Splenocytes were then centrifuged for 7 min at 1500 RPM, brake 5. The pellet was resuspended in 5 mL ACK red blood cell lysis buffer (Thermo Fisher), transferred to a 15 mL tube and incubated for 5 min on room temperature (RT) with occasional shaking. Next, 8 mL of RP10 was added and samples were centrifuged for 7 min at 1500 RPM, brake 5. Supernatant was carefully removed. Cell pellets were suspended in 1 mL RP10 for counting followed by cell viability staining.
Brain processing
Myelin was separated and removed from the brain using a density gradient. The pellet was resuspended in 70% Percoll, and 30% Percoll was carefully layered on top. Following centrifugation at 900g for 30 min at 22ºC, immune cells in the 70-30% interphase were collected into 10 mL of RP10 buffer. Cell pellets were suspended in 1 mL RP10 for counting followed by cell viability staining.
Blood processing
Blood samples were centrifuged at 300g for 10 min (9 acc/6 dec) at 4ºC. Plasma was removed from the upper layer and remaining blood samples were transferred to a 15 mL tube and 5 mL ACK red blood cell lysis buffer (Thermo Fisher) was added. Samples were incubated for 5 min on RT with occasional shaking. Next, 8 mL of RP10 was added and samples were centrifuged for 7 min at 1500 RPM, brake 5. Supernatant was carefully removed and samples underwent another round of red blood cell lysis. Then, 10 mL of RP10 was added to the samples and centrifuged 7 min at 1500 RPM, brake 5. Cell pellets were suspended in 1 mL RP10 for counting followed by cell viability staining.
CyTOF viability staining, fixation and freezing of immune cells
Isolated immune cells were washed with HBSS-/- (without Mg+2, Ca+2 and phenol red). Cells were distributed in wells of a 96-well V-bottom plate, washed with Maxpar PBS (Fluidigm, #201058) and stained with the viability marker Cell-ID™ Cisplatin-198Pt (Fluidigm, #201198) for 5 min at 37°C according to the manufacturer’s instructions. Cells were then washed with RP10 and fixed with Maxpar Fix I Buffer (Fluidigm, #201065) for 10 min at RT. Fixed cells were centrifuged at 800g for 7 min at 4°C. Cell pellets were resuspended in 10% DMSO/FCS and put in a Mr Frosty Freezing Container at -80°C for 24 h. Samples were stored at -80°C until staining and sample acquisition.
CyTOF antibody labeling and titration
Antibody labelling with the indicated metal tag was performed using the MaxPAR antibody conjugation kit (Fluidigm) according to the manufacturer’s instructions. Purification of the bound antibody was performed with high-performance liquid chromatography (Thermo Fisher) and subsequently concentrated by filtering with a 10-kDa filter (Merck Millipore) in a swing-out bucket at 4000 RPM for 15 min. The end volume was determined and an equal volume of antibody stabilizer buffer (Fluidigm; supplemented with 0.05% sodium azide) was added before the antibodies were stored at 4°C. All antibodies used in this study were titrated using both fixed and unfixed thawed immune cells and the most optimal concentrations with the least spillover were chosen.
CyTOF staining
Reagents were cooled on ice, centrifugation steps were performed at 800g for 7 min at 4°C (9 acc/7 dec) and incubations were performed at RT. Samples were thawed rapidly and washed twice with 8 mL of Maxpar PBS (Fluidigm) and transferred to a 96 well V-bottom plate. After centrifugation, cells were washed twice with 150 μL of 1X Barcode Perm Buffer (Fluidigm, #201057) and incubated with the appropriate palladium barcodes (Fluidigm, #201060) in 1X Barcode Perm Buffer for 30 min. After centrifugation, samples were washed twice with 150 μL of cell staining buffer (CSB) (Fluidigm, #201068) and the cells from all samples were pooled. Cells in this combined sample were counted and after centrifugation incubated with TruStain FcX anti-mouse CD16/32 (BioLegend, #101320) diluted in CSB (1:100) for 10 min. Surface antibody cocktails (Table 1) were prepared fresh in CSB and added to the combined sample in a cell to antibody-cocktail ratio of 3 x 106 cells per 100 uL. This was followed by a 30 min incubation. After the incubation cells were washed twice with CSB, followed by a wash with Maxpar PBS. Then, cells were fixated with 1 mL of freshly made 1.6% PFA (Thermo Fisher, #28906) in Maxpar PBS for 10 min. After centrifugation, cells were permeabilized with 1 mL of FoxP3 Fix/Perm working solution (eBioscience, #00-5523) for 30 min, followed by two washes with 1X Permeabilisation Buffer (eBioscience, #00-5523). Then, a freshly prepared nuclear antibody cocktail (Table 1) in 1X Permeabilisation Buffer was added in a cell to antibody-cocktail ratio of 3 x 106 cells per 100 uL and incubated for 45 min. Cells were then washed three times with 1X Permeabilisation Buffer and fixated with 1 mL of freshly made 1.6% PFA in Maxpar PBS for 10 min. After centrifugation, nucleated cells were stained with Maxpar Intercalator (Fluidigm, #201192B) diluted 1:4000 in Maxpar Fix and Perm Buffer (Fluidigm, #201067) and incubated overnight at 4°C, until sample acquisition.
Table 1: Antibodies for CyTOF
Metal
|
Target
|
Clone
|
Concentration (uL) per 100 uL
|
Cell marker or function
|
Cocktail
|
141Pr
|
Ly-6G/C (Gr-1)
|
RB6-8C5
|
0.05
|
neutrophils
|
surface
|
142Nd
|
KLRG1
|
2F1/KLRG1
|
1
|
exhausted T cells
|
surface
|
143Nd
|
CD11a/CD18
|
H155-78
|
0.5
|
integrin
|
surface
|
144Nd
|
MHC II
|
M5/114.15.2
|
0.03
|
APCs
|
surface
|
145Nd
|
CD4
|
RM45
|
0.5
|
CD4+ T cells
|
surface
|
146Nd
|
CD138
|
281-2
|
0.5
|
plasma cells
|
surface
|
147Sm
|
CD36
|
72-1
|
1
|
fatty acid metabolism
|
surface
|
148Nd
|
CD11b
|
M1/70
|
0.05
|
myeloid cells
|
surface
|
149Sm
|
CD19
|
6D5
|
0.5
|
B cells
|
surface
|
150Nd
|
Ly-6C
|
HK1.4
|
0.1
|
monocytes, pDCs, CD8+ T cells
|
surface
|
151Eu
|
CD64
|
X54-5/7.1
|
1
|
monocytes, macrophages
|
surface
|
152Sm
|
CD3e
|
145-2C11
|
0.5
|
T cells, NKT cells
|
surface
|
153Eu
|
CD38
|
90
|
0.05
|
B cells & activated T cells
|
surface
|
154Sm
|
TER-119 (Glycophorin A)
|
TER-119
|
0.5
|
Red blood cells
|
surface
|
155Gd
|
CD274 (PD-L1)
|
10F.9G2
|
0.1
|
immune checkpoint
|
surface
|
156Gd
|
F4/80
|
BM8
|
1
|
macrophages, eosinophils
|
surface
|
158Gd
|
FoxP3
|
FJK-16s
|
2
|
regulatory T cells
|
nuclear
|
159Tb
|
CD279 (PD-1)
|
J43
|
0.1
|
immune checkpoint
|
surface
|
160Gd
|
CD5
|
53-7.3
|
0.05
|
B & T cells
|
surface
|
161Dy
|
Tbet
|
4B10
|
1.5
|
T cells (marks Th1), NKT cells, B cells (subset)
|
nuclear
|
162Dy
|
TCRgd
|
GL3
|
0.2
|
T cells with TCR γ/δ
|
surface
|
163Dy
|
BCL6
|
K112-91
|
2
|
Tfh cells, GC B cells
|
nuclear
|
164Dy
|
CX3CR1
|
SA011F11
|
0.1
|
monocytes, macrophages, T cells
|
surface
|
165Ho
|
NK1.1
|
PK136
|
0.5
|
NK cells, NKT cells
|
surface
|
166Er
|
TIGIT
|
1G9
|
7
|
immune checkpoint
|
surface
|
167Er
|
Gata3
|
TWAJ
|
2
|
T cells (marks Th2)
|
nuclear
|
168Er
|
CD8a
|
53-6.7
|
0.5
|
CD8 T cells
|
surface
|
169Tm
|
TCRb
|
H57-597
|
0.5
|
T cells with TCR α/β
|
surface
|
170Er
|
CD45R (B220)
|
RA3-6B2
|
0.1
|
B cells
|
surface
|
171Yb
|
CD44
|
IM7
|
0.05
|
effector & memory T cells
|
surface
|
172Yb
|
Ki-67
|
B56
|
1
|
cell proliferation
|
nuclear
|
173Yb
|
CD103
|
2E7
|
1
|
T cells (marks Trm), DCs
|
surface
|
174Yb
|
CD223 (LAG3)
|
C9B7W
|
1
|
immune checkpoint
|
surface
|
175Lu
|
CD127 (IL-7Ra)
|
A7R34
|
2
|
naive & memory T cells
|
surface
|
176Yb
|
CD278 (ICOS)
|
7E17G9
|
1
|
immune checkpoint
|
surface
|
209Bi
|
CD11c
|
N418
|
0.05
|
DCs and monocytes
|
surface
|
89Y
|
CD45
|
30-F11
|
1
|
immune cells
|
surface
|
CyTOF sample acquisition
Cells in Maxpar Intercalator solution were washed twice with CSB and divided over approximately 1 x 106 cells per tube, followed by washing with Milli-Q water right before acquisition. Samples were filtered and calibration beads (Fluidigm, #201078) were added to the suspension to 15% of the final volume. Cells were acquired on a Helios™ (Fluidigm), with an event rate of 250 – 350 events per second in Milli-Q water. Runs took approximately 30 – 45 min. During the day, tuning of the machine was performed during start-up and after 4 hours of sample acquisition. Within each barcoded set of samples, one reference sample was included to correct for differences in staining intensity between barcodes due to technical variation in the staining protocol or daily changes in instrument functioning, as will be discussed later.
Generation of the CyTOF reference sample
The reference sample contained a mixture of immune cells derived from the mouse spleen, blood, brain, liver, bone-marrow and tumor biopsies. Part of the spleen and bone-marrow-derived immune cells were stimulated with 30 ng/mL PMA and 1 µg/mL ionomycin (both Sigma-Aldrich) for 120 min at 37°C to induce expression of transcription factors that were included in the CyTOF panel. All cells were combined, stained for viability as described earlier, fixated and stored in aliquots at -80°C until further use.
CyTOF data analysis
Acquired samples were randomized using Gaussian negative half zero randomization in CyTOF Software version 6.7. The FCS files were normalized using bead normalization, concatenated and debarcoded using the CyTOF Software version 6.7. Batch alignment for blood and spleen samples was then performed using CytoNorm49 for each tissue and experiment (e.g. young/old and PBS/plasma-injected) separately. This batch alignment however induced noise in the staining of the brain samples and so the decision was made to work with the ‘raw’ data there instead. The processed FCS files were then uploaded into OMIQ data analysis software (http://www.omiq.ai) and data analysis was performed. First, normalization beads, cell debris and cell doublets were removed from the data using the DNA staining, bead intensity and Gaussian parameters. Next, live cells showing negative reactivity for viability marker Cell-ID™ Cisplatin-198Pt and dim-to-positive reactivity for CD45-89Y were selected and used for further processing steps. Data were visualized in UMAPs and tSNEs and cells were appointed to clusters by Phenotyping by Accelerated Refined Community21 (PARC) and PhenoGraph50 algorithms. Visual inspection of 1) PARC- and PhenoGraph-derived clusters overlaid on corresponding UMAPs and tSNEs derived from the same sample, and 2) clustered heatmaps that compare median marker expression between clusters, guided the manual merging of clusters and their annotation into biologically relevant immune cell subsets. After the ‘general’ immune analysis, T cell clusters (and for some instances myeloid clusters as well) were selected, merged and subsampled after which the cluster analysis was repeated to ensure optimal resolution of the underlying data. Counts per immune cell subset were then exported and further analyzed in Graphpad Prism 8.2.1.
Immunofluorescence
After perfusion, mice were decapitated and brains were isolated for post-fixation overnight in 4% PFA (4°C) and subsequent incubation for 48 hours in 30% sucrose in PBS solution (4°C) and stored at -80°C. In addition, per mouse, 20 μm thick coronal sections were collected and mounted on Superfrost glass slides (Menzel-glaser, Germany) stored at -20C. For the staining, antigen retrieval was performed at 95°C for 30 min in sodium citrate (pH 6). Sections were incubated with either neutralized donkey serum, neutralized goat serum or neutralized rabbit serum for 30 min at RT. Sections were incubated overnight with primary antibodies in PBS (Table 2) at 4oC. After incubation, the sections were stained with the appropriate secondary antibodies in PBS. Last, sections were counterstained with DAPI 1:10000 (DAKO) for 10 min.
Table 2: Antibodies for immunofluorescence
Antigen
|
Species
|
Dilution
|
Manufacturer
|
Cat. Number
|
CD45
|
Rat
|
1:200
|
BD Pharmingen
|
553076
|
CD4
|
Rabbit
|
1:250
|
Abcam
|
Ab217344
|
CD8
|
Rabbit
|
1:250
|
Abcam
|
Ab183685
|
Collagen IV / Coll-IV
|
Rabbit
|
1:200
|
Abcam
|
Ab6581
|
VCAM-1 / CD106
|
Rat
|
1:100
|
In house generated
|
ICAM-1 / CD54
|
Rat
|
1:100
|
Biolegend
|
116101
|
PECAM-1 / CD31
|
Mouse
|
1:200
|
Abcam
|
Ab24590
|
Fibrinogen
|
Rabbit
|
1:200
|
DAKO
|
F0111
|
BrdU
|
Mouse
|
1:100
|
Invitrogen
|
14-5071-82
|
IBA1
|
Goat
|
1:500
|
Abcam
|
ab5076
|
P2Y12
|
Rabbit
|
1:500
|
AnaSpec
|
55043A
|
Lamp1
|
Rat
|
1:100
|
Biolegend
|
|
HuC/D
|
Mouse
|
1:100
|
ThermoFisher Scientific
|
A21271
|
Homer1
|
Rabbit
|
1:500
|
Synaptic Systems
|
160 002
|
Microscopy and data analysis
T cell infiltration in the brain
Per mouse, minimal one complete coronal brain section was imaged using a wide-field microscope (Nikon TI2) at a 1.39 µm/pixel resolution. The CD45+ CD4+ and CD45+ CD8+ T cells were manually counted by a blinded observer and normalized to the total slide area. T cells inside the vessel and the meninges, visualized with Coll-IV, were excluded from the analysis.
Expression of cell adhesion molecules on the brain vasculature
Per mouse, 4 confocal images were obtained on a Leica TCS SP8 HyD confocal microscope using a 0.63 µm/pixel resolution, a z-stepsize of 0.33 µm and a total of 10 z-steps. The 3 μm depth z-stack was compressed into one 2D-image using a maximum projection. The percentage of positive vessels was determined using NIS Elements (Nikon) by dividing the total number of Coll-IV+ and ICAM-1+ / VCAM-1+ / PECAM-1+ vessels by the total number of Coll-IV+ vessels. Thresholds were manually set per ROI by a blinded observer. Vessel density was determined by measuring and dividing the total Coll-IV+ area by the total measured tissue area.
Blood vessel permeability
Per mouse, 4 confocal images were obtained on a Leica TCS SP8 HyD confocal microscope using a 0.18 µm/pixel resolution, a z-stepsize of 0.33 µm and a total of 10 z-steps. The 3 μm depth z-stack was compressed into one 2D-image using a maximum projection. Brain vessel permeability was determined by studying the area of extravascular fibrinogen. First, using NIS Elements (Nikon), a threshold was set manually per ROI by a blinded observer for both Coll-IV and fibrinogen. Per image, only the fibrinogen+ Coll-IV- area outside the vessels was measured and normalized to the total amount of Coll-IV+ vessel area.
Microglial phenotype, number and proliferation
Per mouse, two widefield images were acquired on a Vectra 3.0 spectral imaging system (PerkinElmer) using a resolution of 0.497 µm/pixel. Sections labeled for IBA1 together with P2Y12 and BrdU were analyzed using NIS Elements (Nikon). In short, mean fluorescence intensity of P2Y12 in microglia was quantified by measuring the total fluorescence intensity of P2Y12 within the IBA1+ mask. Microglia density and proliferation was determined by manually counting the number of IBA1+ and IBA1+ BrdU+ cells per area by a blinded observer.
Microglial morphology
Per mouse, 10 confocal images were obtained on a Leica TCS SP8 HyD confocal microscope using a 0.09 µm/pixel resolution, a z-stepsize of 0.3 µm and a total of 11 z-steps. The 3 µm depth z-stack was compressed into one 2D-image using a maximum projection. Each z-stack contained one IBA1+ cell located in the stratum radiatum of the hippocampus. IBA1+ microglia were manually traced using FIJI (NIH). Traced microglia were analyzed using the Sholl Analysis Plugin51 with a 0.2 µm step size from the cell soma. Soma surface area of the traced microglia was measured using the freehand selection tool in FIJI.
Neuronal cell count
Per mouse, two widefield images of HuC/D stained sections were acquired on a Vectra 3.0 spectral imaging system (PerkinElmer) using a resolution of 0.497 µm/pixel. Cortical neuronal density was determined by manually counting the number of HuC/D+ cells per cortical area by a blinded observer.
Synapse density and pruning
Per mouse, 10 confocal images each containing one IBA1+ cell using a 0.09 µm/pixel resolution, a z-stepsize of 0.3 µm and a total of 11 z-steps, were acquired from the stratum radiatum area of the hippocampus. To measure synaptic density, Homer1+ spots were created using NIS Elements (Nikon) with a spot diameter of 0.5 μm in a manually selected area lacking IBA1+ cellular processes or cell nuclei. The density of Homer1+ spots was then calculated by dividing the number of spots by the total volume of the selected region. To investigate synaptic pruning by microglia, manual thresholds were set for Lamp1 and IBA1 to obtain only Lamp1+ IBA1+ volumes. Next Homer1+ spots located within the Lamp1+ IBA1+ volumes were counted. Lysosomal size was calculated by dividing the total Lamp1+ IBA1+ volume by the total IBA1+ volume.
Statistics
Graphpad Prism 8.2.1 was used for all statistical tests. Shapiro–Wilk and F tests were performed to test for normality and equality of variances, respectively, after which appropriate tests were selected. The unpaired two-tailed Student’s t test with or without Welch’s correction for unequal variances for normally distributed data, or the Mann–Whitney test for non-normally distributed data. To examine the strength of association between two variables, either Pearson or Spearman correlation was used depending on the normality of the data. Data were judged to be statistically significant when P < 0.05 and, if significant, reported in the figures using the significance levels indicated in the figure legends.