Central and peripheral myeloid-derived suppressor cell-like cells are closely related to the clinical severity of multiple sclerosis

Human tissue

Snap-frozen tissue (MS) was supplied by UK MS Tissue Bank and formalin-fixed paraffin-embedded (FFPE) MS tissue (MSD) was provided by Prof. Denise Fitzgerald from the Dame Ingrid V. Allen tissue collection (Belfast, UK). Post-mortem MS cortical snap-frozen tissue (n = 20 secondary progressive MS-SPMS; n = 13 PPMS) and six matched controls (Ct) were analyzed (Table1). Clinical course of MS patients were classified as long or short according to the median of the disease duration [SPMS: 16 years (y), inter quartile range-IQR (13.5–34.5); PPMS: 17 y (11–28.5)].

Table 1 Demographic and clinical characteristics of MS patients and control subjectsImmunohistochemistry and eriochrome cyanine for myelin staining

Cryosections (10 µm, Leica) from snap-frozen tissue were fixed in 4% paraformaldehyde (PFA: Sigma-Aldrich) for 1 h. Immunohistochemistry (IHC) or immunofluorescence (IF) staining was performed as described previously [4] by incubating with the following primary antibodies: anti-CD11b (1:100; Abcam, ab133357); anti-CD3 (1:200; Agilent, A0454); anti-GFAP (1:500; Merck, MAB3402, GA5 clone); anti-TMEM119 (1:50; Sigma-Aldrich, HPA0518870); anti-CD33 (1:50; Merck, 133 M-14, PWS44 clone), anti-CD14 (1:25; R&D, BAF383), anti-CD84 (1:200; Invitrogen, PA5-64444), anti-CD15 (1:25; Agilent, ISO62, carb-3 clone), and anti-human leukocyte antigen (HLA)-DR (1:200 for IHC, 1:100 for IF; Agilent, M0746, TAL.1B5 clone). Antigen retrieval with citrate buffer 0.1 M pH 6.0 at 90ºC for 10 min was performed for CD33 labeling. Detection of TMEM119 and CD33 was visualized using TSA signal amplification system (Tyramide SuperBoost™ kit, Invitrogen for TMEM119 and TSA Plus biotin kit, AkoyaBio for CD33). Appropriate fluorescent-tagged (1:1000, Invitrogen) or biotinylated (1:200; Vector Labs) secondary antibodies were used. The IHC reaction was developed using the Vectastain Elite ABC reagent (Vector Labs) and the peroxidase reaction product was visualized with 0.05% 3, 3´-diaminobenzidine (DAB, Sigma-Aldrich) and 0.003% H2O2 in 0.1 M Tris–HCl, pH 7.6. The reaction was monitored under the microscope and terminated by rinsing the slides with PB. Fluorescent Hoechst 33342 staining (10 µg/ml: Sigma-Aldrich) was used for cell nuclei detection. Negative stain controls were included and the absence of the appropriate antibodies yielded no signal.

To visualize myelin after HLA-DR immunostaining in tissue from snap-frozen brain blocks, eriochrome cyanine (EC) staining was carried out as described. The sections were air-dried overnight at RT and for 2 h at 37 °C in a slide warmer. The sections were then placed in fresh acetone for 5 min and air-dried for 30 min, before they were stained in 0.5% EC for 1 h and differentiated in 5% iron alum and borax-ferricyanide for 10 and 5 min, respectively (briefly rinsing the sections in tap water between each step). After washing with abundant water, correct differentiation was assessed under the microscope whereby the myelinated areas were stained blue and the demyelinated areas appeared white-yellowish. The stained sections were dehydrated and mounted for preservation at RT.

Triple chromogenic immunohistochemistry on FFPE sections (from MSD tissue blocks) was performed with a triple stain IHC kit (DAB, AP/Red & HRP/Green) from Abcam (ab183286) which allowed red–green colocalisation using a modified protocol. FFPE sections were dewaxed in clearene and rehydrated through a graded series of alcohols. Heat-induced epitope retrieval was conducted in a steamer for 1 h, while slides were incubated in sodium citrate buffer (pH 6.0). Endogenous peroxidases were blocked with 0.3% H2O2 in methanol before blocking with 5% normal goat serum in Tris-buffered saline (TBS, pH 7.4). Slides were then incubated with anti-CD15 (1:50; Agilent, ISO62, carb-3 clone) in blocking solution overnight. Slides were washed in TBS before EnVision anti-mouse HRP secondary antibody (Agilent) was applied for 30 min at RT. Antibody binding was visualized with DAB (Immpact DAB; Vector) as chromogen. To prevent cross-reactivity, slides were heated to 80 °C in supplied antibody blocker solution and then incubated with Blocker A and B according to the manufacturer’s protocol. Slides were washed in TBS before incubation with anti-HLA-DR antibody (1:200; Agilent, M0746, TAL.1B5 clone) and anti-CD14 (1:50; R&D, BAF383) in blocking solution overnight at 4 °C. After washing, tissue sections were incubated with kit-supplied anti-mouse AP secondary antibody for 30 min at RT. Followed further washing, sections were incubated with an ABC peroxidase-linked reporter system (Vector Laboratories) for 30 min at RT. Detection of HLA-DR was visualized with Permanent Red chromogen and counterstained with Gill’s haematoxylin No. 2 (GHS232; Sigma) diluted 1:10. Slides were briefly washed and detection of CD14 was visualized by applying Emerald Green chromogen for 5 min at RT. Slides were rapidly cleared and mounted with supplied mounting medium according to the manufacturer’s instructions. Negative and single stain controls were included, and in all instances, the absence of the relevant antibodies yielded no signal.

Classification of MS lesions

MS lesions were classified according to demyelination and cellular distribution as described [4, 18, 22]. Briefly, active lesions (AL) are demyelinated areas loaded by dense infiltration of macrophage/microglial cells. T cells were observed both perivascularly and dispersed in the lesion and astrogliosis were confirmed by GFAP upregulation. Mixed active/inactive lesions (AIL) are demyelinated areas with a hypocellular lesion center (cAIL) surrounded by a lesion rim (rAIL) enriched with macrophage/microglial cells. Moderate T-cell infiltration was observed in the center of AILs and the presence of hypertrophied astrocytes was also found. Inactive lesions (IL) contained very few macrophage/microglial cells and T cells within the demyelinating lesion. Gliotic scar formed by astrocytes was found in the center of the ILs.

Cell counting in human tissue

The IHC staining for HLA-DR, CD14, and CD15 was used for the quantification of M-MDSCs in serial sections of each case and lesion. To measure HLA-DR fluorescence intensity, photomicrographs of the MS lesions were acquired as a mosaic of 20X magnification images captured on a confocal microscope equipped with a resonant scanning system (SP5: Leica), quantifying the fluorescence intensity of the cells using the ImageJ software. A low level of HLA-DR fluorescence was established in a blinded manner by measuring HLA-DR staining in 100 CD14+ CD15− cells in each patient, among which there were 25 cells with no HLA-DR immunostaining, 25 cells with very faint staining (HLA-DRlow), and 50 cells with mild-to-strong immunostaining (HLA-DRint/high: Suppl. Fig. 1a). When the maximum fluorescence intensity was quantified in each HLA-DR cell from each patient, a receiver-operating characteristic (ROC) curve analysis was performed to find the optimal cut-off to accurately classify HLA-DRlow cells avoiding false-positive cells, i.e., cells that were classified as HLA-DRlow by the blinded observer but with a fluorescence intensity above the cut-off value. Once this threshold was calculated for each patient, only those cells with HLA-DR fluorescence intensity below the cut-off value were considered to quantify the M-MDSC density (Suppl. Fig. 1b). Due to the variation of HLA-DR staining between patients, quantification and cut-off value determination were carried out on an individualized basis for each patient. The density of M-MDSCs was obtained by manually counting of HLA-DR−/low CD14+ CD15− cells within the MS lesions, using 5–15 fields of the area of interest at a magnification of 20X, depending on the size of the lesions (SP5: Leica).

Color deconvolution was performed to quantify M-MDSCs (HLA-DR−/low CD14+ CD15− cells) in the MS lesions from FFPE tissue from the Dame Ingrid V. Allen tissue collection (Belfast, UK). The color deconvolution plugin for Fiji implements stain separation with Ruifrok and Johnston’s method previously described [38]. The plugin allows us to transform each single staining from the multiple immunolabelling into a separate channel to analyze the pictures and quantified the density of MDSCs in different MS lesions as described above (Suppl. Fig. 1).

Induction of EAE

EAE was induced 6-week-old C57/BL6 mice from both sexes (Janvier Labs) by immunization with 200 µg of Myelin Oligodendrocyte Glycoprotein (MOG35-55) peptide (GenScript) as previously described [31]. EAE was scored clinically on a daily basis in a double-blind manner [29, 31]. All animal manipulations were approved by the institutional ethical committees (Comité Ético de Experimentación Animal, Hospital Nacional de Paraplejicos-HNP), and all the experiments were performed in compliance with the European guidelines for animal research (European Communities Council Directive 2010/63/EU, 90/219/EEC, Regulation No. 1946/2003).

The clinical parameters analyzed were defined as: (i) the severity index (SI), quantified as the ratio between the maximal clinical score at peak and the disease duration (i.e., days elapsed from the onset to the peak of the disease [27]; (ii) the accumulated clinical score was considered as the sum of the individual clinical scores from the day of onset or the peak of the disease, until the end of the clinical evaluation; (iii) the percentage of recovery was determined as the following percentage: (the maximal clinical score at peak − the residual score in the plateau phase) × 100/maximal clinical score at peak; and (iv) the recovery index, as the absolute score recovered from the peak to the plateau phase/days elapsed from the peak to the end of the remission phase.

Flow cytometry of peripheral blood cells from EAE mice

Blood was collected in 2% EDTA tubes from the orbital vein of 16 isoflurane-anesthetized mice at disease onset (clinical score ≥ 0.5) and at the peak of the EAE. After blocking Fc receptors, cells were labeled with the following antibodies: anti-Ly-6C FITC (10 µg/mL, AL-21 clone), anti-Ly-6G PE (4 µg/ml, 1A8 clone), and anti-CD11b PerCP-Cy5.5 (4 µg/ml, M1/70 clone) all from BD Biosciences; anti-MHC-II PE-Cy7 (4 µg/ml, M5/114.15.2 clone), anti-CD11c APC (4 µg/ml, N418 clone), and anti-F4/80 eFluor450 (4 µg/ml, BM8 clone) from eBioscience. Analysis was performed in an FACS Canto II cytometer (BD Biosciences) and data analysis was assessed using FlowJo 10.6.2 software (FlowJo, LLC-BD Biosciences).

Immunosuppression assay with circulating Ly-6Chi cells of EAE mice

Splenocytes were obtained from MOG-immunized female C57BL/6 mice at the onset of clinical score (0.5–1.5, Suppl. Fig. 2a), as described previously [31, 32]. Splenocytes were exposed to 5 µM Tag-it Violet™ Proliferation and Cell Tracking Dye (Biolegend) diluted in PBS supplemented with 0.1% BSA at 37 °C for 20 min. After washing, 2 × 105 splenocytes were plated in EX-VIVO (Lonza) culture medium supplemented with 1% HEPES and 6 µM 2-ME (Sigma) in U-bottom 96-well plates and stimulated for 24 h with 5 µg/mL MOG. Then, Ly-6Chi-cells (CD11b+ Ly-6Chi Ly-6G−/low -cells) were sorted by FACS Aria Ilu from the whole blood of different EAE mice at disease onset (clinical score 0.5–1.5) and 5 × 104 cells were plated in co-culture with the stimulated splenocytes (4:1, splenocytes:Ly-6Chi-cells). After 48 h, cells were harvested and stained with anti-CD11b PerCP-Cy5.5 (4 µg/ml, M1/70 clone), anti-CD3 APC (4 µg/ml; 145-2C11 clone), anti-CD4-PE (2 µg/ml; RM4-5 clone), and anti-CD8 FITC (5 µg/ml, 53–6.7 clone).

To analyze the T-cell proliferation without the suppressive effect from endogenous Ly-6Chi cells, splenocytes from MOG-immunized EAE mice at the onset of the clinical score were depleted of Ly-6Chi cells by cell sorting in a FACS Aria IIu (Suppl. Fig. 2b). After that, Ly-6Chi depleted-splenocytes were labeled with Tag-it Violet™ Proliferation and Cell Tracking Dye and then MOG-stimulated as abovementioned. After 24 h, 5 × 104 sorted Ly-6Chi cells from the whole blood of other EAE mice at disease onset (clinical score ≥ 0.5) were added (4:1; depleted-splenocytes:Ly-6Chi cells). After 72 h, cells were harvested and stained as previously described (see above).

Analysis was performed in an FACS Canto II cytometer (BD Biosciences) and data analysis was assessed using FlowJo 10.6.2 software (FlowJo, LLC-BD Biosciences).The proliferation index was calculated as the ratio of the percentage of stimulated divided cells with respect to control divided cells.

Tissue extraction and histological analysis of EAE tissue

Ten female mice with EAE from a second cohort of animals were used for histological analysis. Peripheral blood was also collected from all the mice at the onset of the clinical signs and all the animals were sacrificed at the peak of the clinical course, when they were perfused transcardially with 4% PFA. The spinal cord of the mice was dissected out and post-fixed for 4 h at RT in the same fixative. After immersion in 30% (w/v) sucrose in PB for 12 h, coronal cryostat sections (20 μm thick: Leica) were thaw-mounted on Superfrost® Plus slides.

The same EC staining for myelin visualization was carried out as that used for the histopathology of human samples with the following modifications: the tissue was stained in 0.5% EC for 30 min, and differentiated in 5% iron alum and borax-ferricyanide for 10 and 5 min, respectively.

Axonal damage was analyzed by staining the non-phosphorylated form of the neurofilament protein (SMI-32) in spinal cord sections from mice with EAE at the peak. Immunohistochemistry was performed by incubating the sections overnight at 4 °C with SMI-32 antibody (1:200, Covance). After rinsing, the sections were then incubated for 1 h at RT with the corresponding fluorescent secondary antibody (1:1000; Invitrogen). The cell nuclei were then stained with Hoechst 33342 (10 µg/ml: Sigma-Aldrich), and the sections were mounted in Fluoromount-G (Southern Biotech).

Image acquisition and analysis of murine tissue

In all cases, three sections from each thoracic spinal cord (separated by 420 μm) were selected from 10 EAE mice in the histological cohort. To measure demyelination, the EC stained spinal cord sections were analyzed on a stereological Olympus BX61 microscope, using a DP71 camera (Olympus) and VisionPharm software for anatomical mapping. Superimages were acquired at a magnification of 10× using the mosaic tool and analyzed with the Image J software, expressing the results as the percentage of white matter area with no signs of blue staining as well as the total demyelinated area.

To quantify axonal damage, mosaic images from the whole spinal cord of each animal were obtained on a DMI6000B microscope (Leica). The area of axonal damage relative to the total area or the infiltrated area was analyzed with an ad-hoc plugin designed by the Microscopy and Image Analysis Service at the HNP. Briefly, after selecting the appropriate area (the infiltrated area relative to the whole section or to the whole white matter area), a threshold for immunofluorescence was established and SMI-32 immunostaining was assessed, presenting the result as an area (μm2).

MS patient cohort for M-MDSC blood analysis

All patients were diagnosed MS according to the revised 2017 McDonald criteria. The cohort included 47 untreated RRMS patients who had not received corticosteroids in the last 6 months and who experienced their first relapse up to 1 year before blood sampling (Suppl. Table 2). All MS patients were recruited at the Department of Neurology at Hospital Universitario Virgen de la Salud (Toledo, Spain) or at Hospital General Universitario Gregorio Marañón (Madrid, Spain). Peripheral blood samples were also obtained from matched healthy volunteers recruited in the HNP. The study was approved by the Comité Ético de Investigación Clínica con Medicamentos (#349) of the Complejo Hospitalario de Toledo and informed written consent was obtained from all participants in accordance with the Helsinki declaration.

Flow cytometry of human peripheral blood mononuclear cells

Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation (GE-171440-02, Merck). Separated cells were subsequently collected from the interphase, washed with isolation buffer (2.23 g/l d-glucose, 2.2 g/L sodium citrate, 0.8 g/L citric acid, 0.5% BSA in PBS), and further centrifuged at 500 g for 10 min at RT. The cell pellet was resuspended in FBS, counted, and aliquoted 1:1 in FBS with 20% dimethyl sulfoxide (DMSO, Sigma-Aldrich). The samples were then stored in liquid nitrogen at − 160 °C until use.

Freshly thawed PBMCs (1 × 106) were washed with RPMI and stained with Zombie NIR Dye (Biolegend) for living cell identification following the manufacturer’s instructions. Then, Fc receptors were blocked with beriglobin (50 μg/ml; CSL Behring) for 10 min at 4 °C. The antibody panel for MDSC analysis was made up of anti-CD15 FITC (1.25 µl/test, HI98 clone), anti-CD14 PerCP-CyTM5.5 (0.5 µl/test, Mφ29 clone), anti-CD11b PE-Cy7 (0.5 µl/test, ICRF44 clone), anti-CD33 APC (1.25 µl/test, WM53 clone), and anti-HLA-DR BV421 (0.5 µl/test, G46-6 clone, all from BD Biosciences). PBMCs were fixed with 0.1% PFA and then were analyzed as described above for murine blood cells.

Statistical analysis

Data were expressed as mean ± SEM and analyzed with SigmaPlot version 11.0 (Systat Software). To compare between different MS lesion types, a one-way ANOVA test was performed or its corresponding ANOVA on ranks, followed by the Tukey or Dunn post hoc tests, respectively. Student’s t test was used to perform two-by-two comparisons (Mann–Whitney U test for non-parametric data). Paired t test was used for comparison in the in vitro analysis of immunosuppressive activity. Shapiro–Wilk normality tests were performed on human MS tissue samples. Pearson or Spearman tests were used for correlations as appropriate. The ROC curve was quantified using the area under the curve (AUC). The minimal statistical significance was set at p < 0.05 and represented as: *, #p < 0.05; **, ##p < 0.01; ***, ###p < 0.001.

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