key: cord-0427583-atvew6em
authors: Kay, Alasdair G; Fox, James M; Hewitson, James; Stone, Andrew; Robertson, Sophie; James, Sally; Wang, Xiao-nong; Kapasa, Elizabeth; Yang, Xuebin; Genever, Paul G
title: Identification of CD317-Positive Pro-inflammatory Immune Stromal Cells in Human Mesenchymal Stromal Cell Preparations
date: 2022-02-10
journal: bioRxiv
DOI: 10.1101/2022.02.10.479972
sha: 7ac2c71abbaa1ebaccf8148ec24cffc0e8f31597
doc_id: 427583
cord_uid: atvew6em

Mesenchymal stromal cell (MSC) heterogeneity clouds biological understanding and hampers their clinical development. In MSC cultures most commonly used in research and therapy, we have identified an MSC subtype characterised by CD317 expression (CD317pos (29.77±3.00% of the total MSC population), comprising CD317dim (28.10±4.60%) and CD317bright (1.67±0.58%) MSCs) and a constitutive interferon signature linked to human disease. We demonstrate that CD317pos MSCs induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas CD317neg MSCs had no effect. Only CD317neg MSCs were able to suppress proliferative cycles of activated human T cells in vitro, whilst CD317pos MSCs increased polarisation towards pro-inflammatory Th1 cells and CD317neg cell lines did not. Using an in vivo peritonitis model, we found that CD317neg and CD317pos MSCs suppressed leukocyte recruitment but only CD317neg MSCs suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in immunocompromised mice we were able to observe tissue generation and blood vessel formation with CD317neg MSC lines, but not CD317pos MSC lines. Our evidence is consistent with the identification of an immune stromal cell, which is likely to contribute to specific physiological and pathological functions and influence clinical outcome of therapeutic MSCs.

Mesenchymal stromal cell (MSC) heterogeneity clouds biological understanding and hampers their 22 clinical development. In MSC cultures most commonly used in research and therapy, we have 23 identified an MSC subtype characterised by CD317 expression (CD317 pos (29.77±3.00% of the total 24 MSC population), comprising CD317 dim (28.10±4.60%) and CD317 bright (1.67±0.58%) MSCs) and a 25 constitutive interferon signature linked to human disease. We demonstrate that CD317 pos MSCs 26 induced cutaneous tissue damage when applied a skin explant model of inflammation, whereas 27 CD317 neg MSCs had no effect. Only CD317 neg MSCs were able to suppress proliferative cycles of 28 activated human T cells in vitro, whilst CD317 pos MSCs increased polarisation towards pro-29 inflammatory Th1 cells and CD317 neg cell lines did not. Using an in vivo peritonitis model, we found 30 that CD317 neg and CD317 pos MSCs suppressed leukocyte recruitment but only CD317 neg MSCs 31 suppressed macrophage numbers. Using MSC-loaded scaffolds implanted subcutaneously in 32 immunocompromised mice we were able to observe tissue generation and blood vessel formation 33 with CD317 neg MSC lines, but not CD317 pos MSC lines. Our evidence is consistent with the 34

Whitney T-test p=0.051, n=52) or correlation between donor age and CD317 expression (mean age: 138 69.75±1.29 years; range 45-88; Pearson correlation p=0.141, n=52),) ( Figure 1D, 1E ). There was, 139 however, a significant negative correlation between CD317 expression and BMI (mean 28.06±0.78; 140 range 17-44; Spearman correlation p<0.05, n=52) ( Figure 1F) . 141 We previously demonstrated that the hTERT immortalised MSC lines display typical (ISCT) surface 142 marker profiles 60 . Here, we also examined surface markers commonly associated with human 143 stromal progenitor cells or subsets, including CD146, CD271 and CD164, within CD317 neg and 144 to those seen in the whole population: CD146 pos (24.21±3.23%), CD271 pos (7.78±1.35%) and CD164 pos 149 (97.18±0.66%) (n=27) ( Figure 1H ). These findings demonstrate that expression of these markers is 150 independent of CD317 positivity and that CD164 identifies virtually all CD317 neg and CD317 pos MSCs. 151

Comparative gene expression analysis has previously demonstrated a correlation between murine 152 peri-sinusoidal stromal cells and CD317 pos MSCs 62 . LEPR has been shown to mark peri-sinusoidal 153 stromal cells in mouse tissue 63 . Here we investigated CD317 pos /LEPR pos stromal cells in mouse bone 154 marrow to identify the in vivo location of this subpopulation. CD317 expression was detected 155 throughout the bone marrow with low frequency colocalisation of CD317 with LEPR restricted to 156 peri-sinusoidal regions adjacent to CD31-positive endothelial cells ( Figure 1I ). Figure S2B ). The 10 most significantly 220 upregulated genes in the CD317 pos group were immune-related and/or interferon-regulated, 221 including OAS1, OASL, RSAD2 and CD317 (BST2) ( Figure S2C ). IFN signalling and elevated signatures are associated with different human disease states 65 . When comparing the upregulated 223 Y102/Y202 gene sets with six publicly available transcriptomic databases for autoimmune and 224 related disorders (Table S2) , we identified a significant association between DEGs and GO terms that 225 were enriched in Y102/Y202 MSC lines and psoriasis, eczema and, to a lesser extent, rheumatoid 226 arthritis and osteoporosis (Table S3 ). Similar observations were made when comparing enriched 227 signalling pathways across Y102/Y202 and disease datasets (Table S4) . 228 Therefore, a resident MSC subtype can be identified as CD317 pos ICAM-1 hi CXCL10 hi with apparent 229 constitutive interferon signalling, which is likely to contribute to specific physiological and pathological 230 immune functions. Considering the stark differences in immune profiles of CD317 neg and CD317 pos MSCs, we tested their 295 effects in different inflammatory models. Prior to in vitro and in vivo testing, we confirmed the 296 representative CD317 neg and CD317 pos MSCs (Y201, Y202) were not affected by viral contamination 297 as a potential origin or contributor to constitutive IFN-γ expression. All cell samples were tested in 298 triplicate and returned negative results for molecular diagnostics of infectious diseases (Human 299

Comprehensive CLEAR Panel, Charles River) using PCR for RNA representing a panel of 26 virions. 300

Initially, we investigated the potential pro-inflammatory property of CD317 neg Y201 and CD317 pos 301 Y202 MSCs in a skin explant model, which is an in vitro tool to detect the presence of cutaneous 302 tissue damage following a pro-inflammatory insult 73,74 . CD317 neg Y201 and CD317 pos Y202 MSCs were 303 primed with IFN-γ or TNF-α and co-cultured in vitro with skin explants. 304

In this assessment, no tissue damage was observed after skin co-incubation with CD317 neg Y201 cells 305 in all conditions tested ( Figure Immortalised MSC lines and primary bone marrow derived human MSCs MSC lines immortalised with human telomerase reverse transcriptase (hTERT) were maintained in 472 culture as previously described 60 . Clonal hTERT-MSCs included the CD317 pos Y202 and Y102 lines, and 473 the CD317 neg Y201 and Y101 lines. Low-passage (p1-p5) primary MSCs were isolated from femoral 474 heads, obtained with informed consent during routine hip replacement or as explant cultures from 475 human tibial plateaux after routine knee replacement 60 . Primary MSCs were also established from 476 bone marrow aspirates purchased from Lonza. Cells were cultured at 37°C in 5% CO2 humidified 477 atmosphere incubaters using DMEM (Gibco) culture medium supplemented with 10% foetal bovine 478 serum and 1% penicillin-streptomycin. Cells were routinely passaged at 80% confluence. 479 Transwell cell migration assays 531

Migration assays were performed in transwell polycarbonate membrane cell culture inserts with a 532 5µm pore (Corning, Sigma-Aldrich) using 1.25x10 5 hTERT and primary MSCs, and monocyte-like THP-533 1 and T cell-like HUT-78 (ECACC 88041901) cells in 6 well plates with 1.5 ml of serum-free DMEM. 534

After 24 hours, 600 µl of supernatant or DMEM was added in duplicate to the wells of the transwell 535 plates. Polycarbonate filters were carefully placed above supernatant and 2.5x10 5 of the appropriate 536 cells in 100 µl serum-free RPMI-1640 were applied to the top of the filter and incubated for 5 hours 537 before removing transwells. Migrated cells were assessed by flow cytometry. The percentage cells 538 undergoing migration towards stimuli was calculated. For CCR2 testing, 500 nM CCR2 inhibitor was 539 used (Teijin compound 1) in supernatant. Inhibition of migration was calculated as a percentage of 540 cell total. 541

A bioinformatics comparison of the hTERT MSC lines gene expression data with publicly available 543 transcriptomic data from a range of autoimmune and related disorders was undertaken to identify 544 disease states that correlated with upregulated GO terms associated with the CD317 pos Y102 and 545 Y202 clonal MSC lines 60 . Cross-platform validation was performed using Python and GeneSpring 546 software was used to analyse outcomes. Differentially expressed genes were identified as greater 547 than 2-fold upregulation in disease state compared to healthy controls, and GeneSpring was used to 548 identify significance (p<0.05) in GO term occurrence. The 10 most upregulated GO terms were 549 identified and comparisons made between autoimmune disease states and hTERT immortalised MSC 550

Quantitative polymerase chain reaction (qPCR) 552 RNA was isolated from cells using TRIzol for cell lysis and Machery-Nagel RNA Nucleospin II kit for 553 RNA isolation, with RNA converted to cDNA for gene expression analyses using Superscript IV 554 reverse transcriptase enzymes (Invitrogen). Specific primers for gene expression analyses were 555 designed and optimised and are described in Table S5 . Gene expression analyses were performed as 556 previously described 60 . Gene expression of eight IFN-γ regulated genes, namely Ly6E, HERC5, IFI44L, 557 ISG15, Mx1, Mx2, EPSTI1 and RSAD2 were amplified in qPCR and fold changes were calculated 558 relative to the expression of the housekeeping gene RPS27a and relative to the Y201 cell line or 559 CD317 neg cells. The ΔΔCT fold changes were log2-transformed and averaged to calculate IFN-γ scores, 560 as previously described 64,100 . 561

To detect secreted proteins, supernatants from 100,000 cells incubated in 2.5 ml of serum free 563 DMEM for 24 hours was analysed for secreted proteins by enzyme-linked immunosorbent assays 564 T cell activation assay to assess MSC immunomodulation for deactivation and suppression of T cell 577 proliferation 578

Co-culture of primary human tonsil T cells with hTERT MSC lines was used to assess the potential 579 immunomodulatory impact of CD317 neg (Y101, Y201) and CD317 pos (Y102, Y202) cell lines on T cell 580 proliferation and T helper differentiation. Continual proliferative capacity was used as a measure of 581 T cell deactivation. hTERT MSC lines or CD317-sorted primary MSCs were seeded at a ratio of 1:10 582 with T cells with 1.0x10 4 MSCs seeded into a 96-well U bottomed plate and cultured for 24 hours at 583 37°C, 5% CO2. Primary human MSC were sorted for CD317 expression and co-cultured with 584 commercially sourced cryopreserved CD4+ human cord blood T cells (Stem Cell Technologies). 585

For assessment of proliferation, T cells were stained for 15 minutes at 37°C using 1 uM VPD450 586

Violet proliferation dye (eBioscience, Inc.). Unstained cells were used as a control. T cells were 587 activated using anti-CD3ε/CD28 Dynabeads (Thermo Fisher) at a bead-to-cell ratio of 1:1 then 588 seeded onto the MSC at a density of 1.0x10 5 /well (ratio 10:1) in 200 μl RPMI-1640 with 10% FBS, 589 0.05 μg/mL IL-2 (Peprotech, Inc) or seeded alone (no MSCs) as a control. Plates were cultured for 5 590 days at 37°C. T cell proliferation was assessed following removal of Dynabeads with the DynaMag-2 591 as per manufacturer's recommendations. Plates were cultured for 5 days at 37°C. T cell proliferation 592

was assessed with flow cytometry, with reduction in signal intensity visualised for repeated 593 proliferation peaks. Proliferation was assessed through VPD450 dilution (diminished staining 594 intensity) described through a proliferative index (PI) calculated from the fluorescence intensity at 595 each cell division as described previously 70 . Proliferative cycles undertaken were calculated on 50% 596 fluorescence intensity reduction peaks, measuring from fluorescence intensity of the first division 597 and the final division detected. 598

T cell activation assay to assess MSC immunomodulation to direct effector T cell polarisation 599

For assessment of T helper differentiation, T cells were activated and cultured with hTERT MSC 600 monolayers, as described above. The following reagents and antibodies for reactivation, transport 601 inhibition and staining were sourced from eBioscience. Following 5 days of culture, T cells were re-602 stimulated using a combination of phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) (Sigma Aldrich) 603

and Ionomycin (1 μg/ml) (Invitrogen) and intracellular cytokines retained using transport inhibitor 604 cocktail with 10 μg/ml brefeldin A and 2 μM Monensin (Invitrogen). Cells were cultured for 4 hours 605 at 37°C then stained for surface marker CD4. Intracellular staining for helper T cells was undertaken 606 for anti-human IFN-γ (Th1), IL-4 (Th2) or IL17a (Th17) or CD4 and CD25 then 607 fixation/permeabilisation and staining for nuclear protein FOXP3 for regulatory T cells. All cells were 608 measured using the CyAn ADP or Cytoflex LX flow cytometer and analysed with FCS Express 7. 609

Comparisons were drawn for percentage of T helper differentiation within the CD4+ cell population 610 and signal intensity (Median) for each antibody tested. 611

In vitro human skin explant model to assess cutaneous tissue damage 612

The human skin explant assay is an in vitro model previously used for evaluation of tissue damage 613 induced by T cell or pro-inflammatory cytokine mediated immmunopathological responses 103,104 . We In vivo assay to assess tissue forming capacity of hTERT MSC lines 658

All procedures used were approved by the University of Leeds Ethics Committee and under the UK 659 Home Office Project License (PPL:70/8549). The tissue-forming capacity of CD317 neg and CD317 pos 660 hTERT cell lines CD317 neg Y201 and CD317 pos Y202 was assessed in CD1 nude mice (Charles River) 661 aged 8-10 weeks in an in vivo transplantation assay 108 . 2.0 x 10 6 MSC cell suspension in 1 ml medium 662 was added to 40 mg hydroxyapatite (HA) synthetic bone particles (Zimmer Biomet) of 250-1000 μm 663 size and rotated at approximately 25 rpm at 37°C for 100 minutes to allow cells to attach. HA particles were bound using fibrin glue comprising 30 μl thrombin (400 I.U./ml in DMEM medium) 665 mixed 1:1 with fibrinogen (115 mg/ml in 0.85% saline solution). Implants were delivered 666 subcutaneously into immunocompromised nude mice with two constructs placed into each mouse. 667

Transplants were harvested at 3 and 8 weeks, fixed in 4% PFA, decalcified for 7 days in 10% EDTA then 668 stored overnight in 70% ethanol prior to paraffin embedding, sectioning and staining with H&E, Alcian 669

Blue and Syrius Red (Thermo Fisher). 670

Statistical analysis 671 Data were tested for equal variance and normality using D'Agostino & Pearson omnibus normality 672 test. Differences between groups were compared using two-tailed 1-way ANOVA for parametric data 673 or Kruskall-Wallis for non-parametric testing. For two factor analysis, data was analysed with a two-674 tailed 2-way ANOVA. Bonferroni post-hoc testing was conducted to compare between groups. All Requests and correspondence should be addressed to Professor Paul Genever. 709 710

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