Soluble CD27 is an intrathecal biomarker of T-cell-mediated lesion activity in multiple sclerosis

doi:10.21203/rs.3.rs-3495160/v1

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Research Article

Soluble CD27 is an intrathecal biomarker of T-cell-mediated lesion activity in multiple sclerosis

https://doi.org/10.21203/rs.3.rs-3495160/v1

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Journal Publication

published 12 Apr, 2024

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Objective: Soluble CD27 is a promising cerebrospinal fluid inflammatory biomarker in multiple sclerosis. In this study, we investigate relevant immune and neuro-pathological features of soluble CD27 in multiple sclerosis.

Methods: Protein levels of soluble CD27 were correlated to inflammatory cell subpopulations and inflammatory cytokines and chemokines detected in cerebrospinal fluid of 137 patients with multiple sclerosis and 47 patients with inflammatory and non-inflammatory neurological disease from three independent cohorts. Production of soluble CD27 was investigated in cell cultures of activated T and B cells and CD27-knockout T cells. In a study including matched cerebrospinal fluid and post-mortem brain tissues of patients with multiple sclerosis and control cases, levels of soluble CD27 were correlated with perivascular and meningeal infiltrates and with neuropathological features.

Results: We demonstrate that soluble CD27 favours the differentiation of interferon-g-producing T cells and is released through an exocytosis mechanism activated by TCR engagement. We also show that the levels of soluble CD27 correlate with the representation of inflammatory T cell subsets in the CSF of patients with relapsing-remitting multiple sclerosis and with the magnitude of perivascular and meningeal CD27+ CD4+ and CD8+ T cell infiltrates in post-mortem central nervous system tissue, defining a subgroup of patients with extensive active inflammatory lesions.

Interpretation: our results demonstrate that soluble CD27 is a biomarker of disease activity, potentially informative for personalized treatment and monitoring of treatment outcomes.

Multiple sclerosis (MS) is an immune-mediated disease of the central nervous system (CNS) characterized by inflammation, demyelination and neurodegeneration [1, 2]. Intrathecal inflammation with mild lymphocytic pleocytosis and oligoclonal immunoglobulins is routinely detectable [3, 4] and is consistent with persistent activation of B and T cells in the CNS [5–8]. Management of MS would benefit from biomarkers that reflect the pathological processes of the disease and enable disease monitoring and stratified or ideally personalized treatment in individual patients [9, 10].

Soluble CD27 (sCD27) has been detected in the cerebrospinal fluid (CSF) of patients with neuroinflammatory diseases [11–13] and described as a prognostic biomarker of MS in clinically isolated syndrome (CIS) [14] and childhood-acquired demyelinating syndromes [15]. Furthermore, sCD27 was found to be the best single biomarker of active intrathecal T-cells in progressive MS [16, 17] as well as of B cells in patients with relapsing-remitting (RR)-MS [18] and was reported to be associated with a higher risk of developing new MRI lesions when present in the CSF at the time of diagnosis [19]. The cellular source and role of sCD27 in the pathogenesis of MS remain poorly understood, and a better understanding of the immune and neuropathological processes associated with the biomarker could provide valuable insights into disease mechanisms. We hypothesised that abnormally high levels of sCD27 promote the differentiation of inflammatory T cells in patients with MS. By analysing CSF from different patient cohorts, we demonstrate that CSF levels of sCD27 are significantly higher in patients with RR-MS compared to other inflammatory and non-inflammatory neurological diseases (ODN). With studies in vitro, we show that sCD27 contributes to the differentiation of IFNg-producing-T cells and that high levels of sCD27 are released mainly by T cells upon TCR activation through an exocytosis mechanism regulated by neutral sphingomyelinase (nSMase). Furthermore, with immunopathological studies of paired CSF and brain tissues obtained from post-mortem MS cases, we demonstrate a correlation between levels of sCD27 in the CSF and the number of CD27 + T cells in perivascular and meningeal areas, most prominently found in cases with early active MS lesions and an overall high degree of lesion activity. Our results suggest that sCD27 is a biomarker of T cell-mediated inflammatory disease activity in patients with MS and explain mechanistically its association with neuropathological processes that strongly affect disease outcomes.

CSF and blood samples from participants

The National Health Service Research Ethics Committee had approved this study (IRAS ID: 204872 and FIS PI15/00513), and all subjects have provided written informed consent to participate in the study.

CSF supernatants were collected and processed according to the standard procedures [16] from 71 patients with a diagnosis of RR-MS and Secondary progressive (SP)-MS and 12 with OND from Charing Cross Hospital of London.

Matched serum and CSF samples were collected from 65 patients with RR-MS and 35 with OND in Hospital Universitario Ramon y Cajal of Madrid. The demographic and clinical data of MS and OND were reported in Supplementary Table 1.

Cellular pellets from CSF centrifugation were investigated for surface and intracellular proteins by using multiparametric Flow cytometry analysis. FACSCanto II flow cytometer (BD Biosciences) was used to acquire the samples. The different cell subsets were identified according to the combination of markers shown in Supplementary Table 2.

Enzyme-linked immunosorbent assay (ELISA) and Electro chemiluminescent Assay

The ELISA was performed to investigate protein levels of sCD27, BAFF, CXCL13, IFN-g, TNF (DuoSet Elisa development System, R&D, UK) and NFL-light (Tecan, Switzerland) according to the manufacturing protocols. The MSD U-Plex plates were customised with the cytokines (IL-6, IL-10, TNF) and chemokines (CCL19, CXCL10, CXCL12) and developed as suggested by the supply (Meso Scale diagnostics, UK).

Cell culture and protein extraction

Peripheral blood mononuclear cells (PBMCs) were isolated from fresh blood samples of 25 healthy donors as described [20].

CD4, CD8 T and B cells were isolated using magnetic beads (Miltenyi Biotech, Germany) as suggested by the supply. CD19 B cells were stimulated with Immunoglobulin M (IgM, Invitrogen), CD40L (Invitrogen), cytosine phosphate guanine (CpG) deoxynucleotide (ODN 2006) (InvivoGen) and resiquimod (R848, Sigma-Aldrich). CD4 or CD8 T cells were stimulated in 96-well plates pre-coated with anti-CD3 (clone UCHT1, Biosciences). Supernatants from CD3 T and CD19 B cell cultures were collected and analysed for inflammatory cytokines by Elisa. Pelleted cells were resuspended in cell extraction buffer (RIPA buffer with protease inhibitor cocktail and EDTA, Invitrogen, UK) and after centrifugation, the protein quantification was performed on the collected supernatant with BCA protein assay (Pierce).

CRISPR/Cas9 RNPs

The CD27 gene ablation on CD4 T cells was performed as described [21].

Purification of extracellular vesicles

Extracellular vesicles were isolated from supernatants of CD4 T cells isolated from fresh PBMC as described [22] and the total protein extraction was performed on pelleted exosomes as described above.

Western blot analysis

Proteins were separated by 4–12% SDS-PAGE (Mini protean TGX stain-free gels, Bio-Rad, UK) and transferred onto PVDF membranes. The blots were blocked with 5% non-fat dry milk and incubated with the working concentration of primary and HRP-conjugated secondary antibodies (Invitrogen) as reported in Supplementary Table 3. The membranes were developed with an enhanced chemiluminescence (ECL) detection kit (Pierce).

Post-mortem CSF and brain tissues of patients with MS and controls

Snap-frozen brain and/or paraffin-embedded 4% paraformaldehyde-fixed (FFPE) tissues and matched CSF from a total of 55 cases with a diagnosis of SPMS and 17 control cases were provided by the UK Multiple Sclerosis Society Tissue Bank (Imperial College London, UK). Demographic and clinical features of MS cases were described in Supplementary Table 4 and control cases in Supplementary Table 5. FFPE tissues from 35 out of the 55 examined post-mortem MS cases were used for neuropathological investigation. The combined CSF samples were analysed for protein levels of sCD27 and sCD21 by using the R&D system Elisa kit and analysed by Simple Plex (ProteinSimple, San Jose, CA) following the manufacturer's instructions. In addition, the protein levels of CCL19, CXCL10, CXCL12, CXCL13, IL-6, IL-10, BAFF, IFN-g and NFL were determined using customised immune-assay multiplex Luminex technology (Bio-Plex X200 System equipped with a magnetic workstation, Bio-Rad, Hercules, CA, USA), following the procedure previously optimized [23].

Immunohistochemistry

Snap-frozen 10µm sections were fixed with cold methanol, treated with a solution of 0.3% hydrogen peroxidase, blocked with a solution of 2.5% horse serum and incubated with a working solution of primary antibody and horseradish peroxidase-conjugated secondary antibody. The slides were washed, stained in haematoxylin, washed in water, and dipped in solutions with rising percentages of ethanol 70%, 80%, 100% and 100% of Xylene. The slides were coverslipped and the images were acquired by using a slide scanner Leica biosystem Aperio AT2 and the cell count was performed by ImageJ Fiji software.

Immunofluorescence

An immunofluorescence assay was performed on snap-frozen sections. The slides were blocked in a solution of 2.5% horse serum, stained with a working solution of a combination of primary antibodies (mouse anti-human CD4, goat anti-human CD27, rabbit anti-human CD8, and mouse anti-human CD3, goat anti-human CD27 and rabbit anti-human CD20), stained with anti-rabbit biotin antibody in 2.5% horse serum and incubated with a working solution of secondary fluorescence antibodies (donkey anti-mouse 555, donkey anti-goat 488 and donkey streptavidin-647). Subsequently, the sections were stained with a DAPI solution (Roche Diagnostics GmbH, Germany), mounted with antifade medium (Vectashield, Vector laboratories) and coverslipped. The images were obtained by using a Leica TCS SP8 confocal microscope. Antibodies used in immunohistochemistry and immunofluorescence are given in Supplementary Tables 6 and 7.

Neuropathological characterization of brain tissues

A semiquantitative scoring system was developed in a subgroup of 35 MS cases to have a standardised measure of the degree of demyelination and inflammation in the different compartments examined (meninges, perivascular spaces, choroid plexus). The presence and extent of demyelination and the activity of MS cases were assessed by combining immunohistochemical detection of myelin oligodendrocyte glycoprotein (MOG) and major histocompatibility complex (MHC) class II combined with Luxol fast blue (LFB) staining on serial sections, as previously described [24, 25]. The percentage area of demyelinated white matter (WM) and grey matter (GM) was measured using Qpath [26]. According to previously published procedures [27], score 0 corresponds to absent/scarce (0–5 cells), 1 corresponds to moderate (6–25 cells), 2 corresponds to high (25–50 cells), 3 (> 50 cells) corresponds to elevated inflammatory infiltrate. All semi-quantitative scoring was performed on images acquired at 20x objective (field area 556.5 µm x 312,9 µm equal to 174128,85 µm²).

R analysis

PCA and HCA were made with R Studio version 4.0.2, Factoextra (10.7) and factoMiner packages, missMDA package for missing values., Corrplot and ggplot2 packages for hierarchical clustering, To establish whether sCD27 is a predicting factor for a specific neuropathological variable, we adopted the Random Forest Recursive Feature Elimination method (RF-RFE) [28]. In detail, a 10-iteration 5-fold cross-validation protocol was adopted, and performances were measured using the Mean Squared Error. The analyses were performed with MATLAB version R2022b.

Statistical analysis

Statistical analyses were performed using Prism version 9.2 (GraphPad Software Inc.) and MATLAB. Differences between groups were compared using the Mann-Whitney test, 2way Anova multiple comparisons, Ratio paired t-test, one sample t-test, Kruskal-Wallis test, and Dunn test. Correlations were assessed using the Spearman rank correlation. Flow cytometry analyses were performed using Flow-jo software version 10.6.1 (FlowJo LLC).

CSF sCD27 correlates with inflammatory lymphocytes in MS patients

We first measured the levels of sCD27 in the CSF alongside several other variables in a discovery cohort (DC) of 65 patients with RR-MS (MS DC). In the MS DC, CSF sCD27 was significantly higher (**p < 0.001) than in a group of 35 patients with OND as shown in Fig. 1A. MS variables from this discovery cohort including clinical data, percentage of CSF mononuclear cells, levels of CSF soluble factors and levels of sCD27 in serum and CSF were examined in a heatmap correlation matrix with hierarchical clustering (HCA) (Fig. 1B). The heatmap showed 4 principal clusters: Cluster 1, inflammatory interferon (IFN)-g, Tumour necrosis factor (TNF), interleukin (IL)-17 and granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing CD4 T cells, IL-17-producing CD8 T cells, plasma blasts, levels of CSFs CD27, disease duration (DD), gender, progression index (PI) and the number of relapses within 2 years (R2Y); Cluster2, sCD27 levels in serum frequencies in CSF of CD4Treg, Natural Killer (NK)T cells, CD56^Bright NK cells, monocytes, age at onset (AO), age at the first symptom (AFS) and age at lumbar puncture (ALP); Cluster 3, inflammatory GM-CSF, IFNg, TNF-producing CD8 T cells and expanded disability status scale (EDSS) score; and Cluster 4, CD19 B cells, GM-CSF, TNF-producing CD19 B cells, immunoglobulin (Ig)M and IgG index, memory B cells and CD19 CD5+/- B cells. Levels of CSF sCD27 were positively correlated with the percentage of mononuclear cells, plasma cells, inflammatory IL-17, IFNg and TNF-producing CD4 T cells, R2Y and negatively correlated with IL-17-producing CD8 T cells and PI (Fig. 1B). The principal component analysis (PCA) showed that sCD27 constituted one of the key markers for patient clustering with IL-17-producing CD4 and CD8 T cells, CD4Treg, CD56^Bright NK cells and plasma blasts in the CSF. PCA showed also that inflammatory CD19 B cells and inflammatory CD8 T cells generated different patient clustering not associated with levels of sCD27 (Fig. 1C).

Considering the association of sCD27 with inflammatory T lymphocytes in the CSF, we carried out a detailed analysis of the CSF to investigate the potential correlation of other inflammatory factors with sCD27 for patient clustering. CSF samples from a first validation cohort (VC) of RR-MS patients (MS VC1) were tested for several pro-inflammatory and anti-inflammatory cytokines (IFNγ, TNF, IL-10, and IL-6), chemokines (CXC motif chemokine ligand 12 (CXCL12), CXCL13, CXCL10, C-C motif ligand 19 (CCL19), neurofilament light chain (NF-L) and sCD27. Amongst them, sCD27 (**p < 0.006), CXCL13 (***p < 0.001), CXCL10 (***p < 0.001), CCL19 (*p < 0.014), TNF (***p < 0.001) and IL-10 (***p < 0.001) were significantly increased in MS VC1 compared to OND (Fig. 1D). Furthermore, the HCA (Fig. 1E) showed 4 principal clusters: Cluster A, IL-6, CCL19, CXCL12. and TNF; Cluster B, IL-10, CXCL13, CXCL12; Cluster C, AO, DD and EDSS score; Cluster D, R2Y, PI, IFNg, NFL and B-cell activating factor (BAFF). Levels of sCD27 correlated positively with CXCL13. and in PCA sCD27 is one of the key markers for patients clustering with CXCL13, and CXCL10. (Fig. 1F). We then examined a subset of factors of interest in a second, distinct validation cohort of CSF from RRMS patients (MS VC2). The levels of sCD27, CXCL13 and BAFF were significantly different in MS VC2 compared to OND. sCD27 and CXCL13 were confirmed as being higher in MS (***p < 0.001), while BAFF was significantly lower (****p < 0.0001) (Fig. 1G). In the HCA, sCD27 clustered with CXCL13 and BAFF (Fig. 1H). PCA confirmed the cluster of sCD27 and CXCL13 in RRMS (Fig. 1I). Taken together, the data suggest that levels of sCD27 are correlated with inflammatory CD4 T cells and are a relevant marker for patient clustering with IL-17-producing CD4 and CD8 T cells and CXCL13. The different correlation between the levels of sCD27 and BAFF in the two validation cohorts could be associated with intrathecal B cell activity. The clustering of sCD27 and CXCL13 is confirmed in both validation cohorts.

(A) Levels of sCD27 in the CSF of MS DC compared to OND as measured by ELISA. Plots display individual levels (dots) and mean (horizontal line) of 65 patients with MS and 35 patients with other neurological disease (OND), statistically compared by Mann-Whitney test.

(B) HCA (C) and PCA of CSF analytes and features of MS DC. (D) Plots compare the CSF levels of sCD27, CXCL13, CXCL10, CCL19, TNFa and IL-10 in a MSVC1 and patients OND as measured by ELISA. Plots display results from 41 MS VC2 and 12 OND, statistically compared by Mann-Whitney test. (E) HCA and (F) PCA of CSF analytes and features of MS VC1. (G) Plots compare CSF levels of sCD27, BAFF and CXCL13 in MS VC2 and OND measured by ELISA. Plots display results from 37 patients with MS and 12 OND. Statistical analyses were performed by using Mann-Whitney test. (H) HCA and (I) PCA of CSF analytes and features of MS VC2.

CD27 cleavage is triggered by TCR activation.

To assess the producer of sCD27, sCD27 was investigated in B and T cells.

CD4 and CD8 T cells were activated with 5ug/ml of anti-CD3 that cross-links T cell receptor (TCR), for 72 hours before cell lysis for western blot analysis. The immunoblot for CD27 showed two bands related to the cleaved form at 32 kDa (sCD27) and not cleaved form at 50 kDa (Fig. 2A). The normalized levels of the two forms of CD27 showed an intracellular production of CD27 cleavage upon TCR activation in both CD4 and CD8 T cells as reported in Fig. 2B and 2C.

Protein analysis was performed in B cells isolated from 2 healthy donors and stimulated with IgM that promotes B cell survival and activation and resiquimod, a synthetic agonist of Toll-like receptors (TLRs) 7 and 8, for 72 hours, revealed the absence of bands for sCD27 and CD27 in western blot analysis (supplementary Fig. 1A).

Protein levels of sCD27 and IFNg were measured in supernatants collected at 72 and 96 hours from cultures of CD4 and CD8 T cells activated with anti-CD3. Significant levels of sCD27 were observed in the supernatants of activated CD4 T cells stimulated with 1, 5 and 10 µg/ml of anti-CD3 at 72 and 96 hours (Fig. 2D) and in activated CD8 T cells stimulated with 5 and 10 µg/ml of anti-CD3 at 96 hours (Fig. 2E) compared to untreated cells. Significant levels of IFNγ were observed in CD4 T cells activated with 1µg/ml of anti-CD3 at 72 and 96 hours and 5, 10 µg/ml of anti-CD3 at 96 hours (Fig. 2F) and in CD8 T cells activated with 1, 5 and 10 µg/ml of anti-CD3 at 72 hours and at 96 hours (Fig. 2G) compared to untreated cells.

Protein levels of sCD27 and TNF were investigated in the supernatants of B cells stimulated with IgM, CD40 ligand (CD40L) that induces the activation of B cell activation through the CD40 receptor, and two synthetic TLR agonists like CpG, a synthetic agonist of TLR-9, and R848. In this experiment, B cells released significant levels of sCD27 upon stimulation with IgM at 48 hours (supplementary Fig. 2B). Significant release of TNF was observed in B cells stimulated with R848 at 12, 24 and 48 hours (supplementary Fig. 2C).

Our results showed that CD4 and CD8 T cells produce sCD27 intracellularly and release it by a mechanism involving TCR-mediated activation.

(E) Western blot analysis of CD27 in whole cell lysate from CD4 and CD8 T cells in resting and stimulated with 5ug/ml of anti-aCD3 at 72 hours.

Quantification of CD27 and sCD27 as a percentage of control in CD4 (B) and CD8 (C) T cells by western blotting. Data were reported as mean ± SD of 4 independent experiments. Statistical analyses were performed using One sample t and Wilcoxon test. NS = not significant, *p < 0.05, **p < 0.01.

Levels of sCD27 and IFNg released from CD4 (D, E) and CD8 (F, G) T cells activated with concentrations of anti-CD3 1, 5 and 10 ug/ml at 72 and 96 hours as measured by ELISA. Statistical analyses were performed using Tukey’s multiple comparison tests two-way ANOVA (D, E) and Tukey’s multiple comparison test mixed-effects analysis (F, G). Data represent mean ± SD of 6 (D, E) and 4 independent experiments (F, G).*p < 0.05,**p < 0.01,****p < 0.0001.

TCR activation releases extracellular vesicles carrying CD27

We hypothesized that sCD27 is released through an exocytosis mechanism triggered by TCR activation. To test this hypothesis, CD4 and CD8 T cells were pre-treated with GW4869 (10 µg/ml) which inhibits nSMase, and then stimulated with 5µg/ml of anti-CD3 for 72 hours. Levels of sCD27 and IFNg were significantly reduced in CD4 (Fig. 3A) and CD8 (Fig. 3B). Furthermore, to investigate whether sCD27 is present in extracellular vesicles generated after TCR engagement, CD4 T cells were activated with 5µg/ml of anti-CD3 for 72 hours and extracellular vesicles were isolated from the medium. Proteins were then extracted from exosomes and whole cell lysates and levels of CD27, CD9 (exosome marker), and GAPDH were assessed by western blotting (Fig. 3C). After normalization, the data showed that levels of CD27 increased in T cells and exosomes upon activation (Fig. 3D), whereas levels of sCD27 were only observed in T cells upon activation (Fig. 3E). Those results demonstrated that sCD27 production and release is mediated by TCR activation and is regulated by exocytosis mechanisms.

Levels of sCD27 and IFNg released from CD4 (A) and CD8 (B) T cells in resting or stimulated with anti-CD3 when they are pretreated or not with GW4868 for 72 hours as measured by Elisa. Data represent mean ± SD of 4 independent experiments. Statistical analyses were performed using ordinary one-way ANOVA. NS = not significant, p***<0.001, p****<0.0001. (C) Immunoblot for CD27 and CD9 in the whole cell lysate (W Ctrl and W aCD3) and purified exosomes (Exo Ctrl and Exo aCD3) from purified CD4 T cells in resting and stimulated with aCD3.The same amount of protein from the exosome and whole cell lysate were loaded. Quantification of CD27 (D) and sCD27 (E) as a percentage of control in the whole cell lysate and purified exosomes in resting and stimulated with aCD3 by western blotting. The experiments were repeated 4 times independently. Data represent mean ± SD. Statistical analyses were performed using the Mann Whitney test. *p < 0.005

Knocking out CD27 reduces IFNγ-production in CD4 T cells

The analysis of CSF in the discovery cohort demonstrated a correlation between levels of sCD27 and inflammatory CD4 T cells and for this reason, we have focused on CD4 T cells. As CD27 changes after activation on CD4 T cells, to investigate whether sCD27 contributes to the differentiation of IFNg-producing cells, the CD27 gene was efficiently “knocked out” in CD4 T cells by using CRISPR-Cas9 RNPs (Fig. 4A). The gene editing caused the ablation of CD27 on 80–90% of CD4 + T cells knocked down for CD27 (KO) compared with control (WT) (Fig. 4B). Furthermore, proteins extracted from KO and WT CD4 + T cells were analyzed by western blotting (Fig. 4C) and the analysis demonstrated a significant decrease of CD27 (Fig. 4D) and significant increase of levels of granzyme b (Fig. 4E) in KO CD4 + T cells compared to WT.

Reduced IFNγ production was observed in supernatants of KO compared with WT CD4 T cells collected after 24 and 48 hours of TCR activation (10µg/ml anti-CD3) (Fig. 4F). Flow cytometric analysis of levels of intracellular transcriptional factors showed a significant reduction of levels of transcriptional factor T-bet in KO compared to WT CD4 + T cells (Fig. 4G) and similar levels of the retinoid orphan receptor (ROR)-gT in KO and WT CD4 + T cells (Fig. 4E). Our experiment demonstrated that sCD27 is relevant in the differentiation of Th1 CD4 T cells and its effect is mediated via regulation of T-bet expression.

(A) The CD27 gene was efficiently “knocked out” in CD4 T cells by using CRISPR-Cas9-single-guide RNA ribonucleoproteins (Cas9-RNP). CRISPR-cas9 ribonucleoproteins (crRNPs) were synthesized in vitro and delivered to activated CD4 T cells by nucleofection for editing. The expression of CD27 on the surface of CD4 T cells was investigated by flow cytometry and immunoblotting. (B) Flow cytometry histograms showing CD27 levels on the cell surface of purified CD4 T cells treated with non-targeting crRNPs (WT, in grey) or CD27-targeting crRNP (KO, in dark) in 4 independent experiments. (C) Immunoblots for CD27 and granzyme B in the whole cell lysate of CD4 KO and CD4 WT T cells. Quantification of CD27 (D) and (E) granzyme b as a percentage of control by western blotting. Data represent mean ± SD of 4 independent biological experiments. Statistical analyses were performed using one sample t and Wilcoxon test. *p < 0.05, ***p < 0.001. (F) Levels of sCD27 (pg/ml) from stimulated CD4 KO and CD4 WT T cells were compared at 24 hours and 48 hours as measured by ELISA Statistical analyses were performed using ratio paired t-est. *p < 0.05. Histograms reporting intracellular levels of T-bet (G) and RoR-gT (G) in CD4 KO and CD4 WT T cells as measured by flow cytometry. T-bet and RoR-gT were reported as the median of expression. Data represent mean ± SD. Statistical analyses were performed using paired t-tests of 4 independent experiments. NS not significant, *p < 0.05.

Levels of sCD27 in CSF are correlated with CD27 + T cell density in SPMS and PPMS post-mortem CNS tissues.

Our results indicated that sCD27 in the CSF of patients with MS could promote activation and differentiation of inflammatory T cells, a likely relevant effect in the disease pathophysiology. To examine this hypothesis in CNS tissue, we have analyzed the percentage of CD27 + T and B cell infiltrates and the neuropathology of post-mortem brain tissues from patients with MS and tested the associations with sCD27 in the CSF. Levels of sCD27 were analyzed in the CSFs isolated post-mortem from 27 individuals with PP-MS and SPMS and 9 controls. In parallel, the cell density of CD3, CD20 and CD27 (Fig. 5A-C) cells was evaluated in the immune infiltrates of perivascular cuffs in the WM and meninges of matched snap-frozen brain tissues. Significantly increased number of CD3+ (**p < 0.0034), CD27+ (****p < 0.0001) and CD20 + cells (*p < 0.0469) were detected in perivascular and meningeal infiltrates of MS cases compared to controls (Fig. 5D). A significant correlation was observed between CD27 and CD20 positive cells (Fig. 5E *p < 0.0086), as well as CD3-expressing cells (Fig. 5F ****P < 0.0001). The levels of CSF sCD27 correlated significantly to the number of CD27+ (Fig. 5G, ****p < 0.0001) and CD3 + T cells (Fig. 5H, ****p < 0.0001) (cells/mm2), but not with CD20 + cells (data not shown) counted in the same MS cases.

Representation of immunohistochemistry staining of CD3+(A), CD20+ (B) and CD27+ (C) in perivascular cuffs on sections of snap-frozen post-mortem brain tissues from patients with MS. The arrows indicate the positive cells for CD3, CD20 and CD27.

(D) Scatter plot reporting the cell density of CD3, CD27 and CD20 cells detected in the perivascular cuffs and meninges in 6 control (grey dots) and 27 MS cases (black dots). Data represent mean ± SD of 6 control cases, 24 MS cases for aCD3, 27 MS cases for CD27, and 23 MS cases for CD20. Statistical analyses were performed by using one-way ANOVA, Kruskal Wallis test with Dunn’s multiple comparison test. *p < 0.05, **p < 0.01, ***p < 0.001. Simple linear regression between CD27 cell density and CD20 cell density (E), and between CD27 cell density and CD3 cell density (F) detected in perivascular cuffs and meninges. P and r² squared are shown in the legends.

Simple linear correlation between levels of CSF sCD27 and CD27 cell density (G), CD20 cell density (H) and CD3 cell density (I). P and r² are reported in the plot’s legends.

To define the immune profile of CD27 + cells, 4-colour immunofluorescence was performed on post-mortem brain tissues from two patients with MS. CD27 was detected on B and T cells (Fig. 6A-H) and CD4 and CD8 T cells (Fig. 6I-R). More CD3 T cells co-express CD27 than CD20 B cells (Fig. 6S, ***p < 0.001) and more CD8 than CD4 T cells (Fig. 6T, ***p < 0.001).

Representation of immunofluorescence on sections of snap-frozen post-mortem brain tissue performed with 4 colour staining. Single labelling DAPI (A, blue), aCD3 Alexa Fluor 555 (B, purple), CD20 Alexa Fluor 647 (C, red) and CD27 Alexa Fluor 488 (D, green). Colocalization of CD27 on CD20 B cells (E-G) and CD3 T cells (F-H). Single staining DAPI (I, blue), CD4 Alexa fluor 555 (L, purple), CD8 Alexa fluor 647 (M, red) and CD27Alexa fluor 488 (N, green). Colocalization of CD27 on CD8 T (O-Q) and CD4 T cells (P-R). (S) histograms report the percentage of CD27 + cells on CD3 and CD20 cells and (T) on CD8 and CD4 T cells detected in perivascular cuffs and meninges. Data represent mean ± SD. Statistical analysis was performed by using the Mann-Whitney test. ***p < 0.001

CSF sCD27 levels are increased in post-mortem cases with active MS lesions

To examine potential neuropathological associations, the levels of sCD27 were compared across different groups, which were obtained by stratifying 30 out of 55 of the examined MS cases according to each neuropathological variable. sCD27 was found to be distributed in a significantly different way according to the degree of lesion activity, graded as defined in the Methods (Fig. 7A Kruskal-Wallis p-value 0.0104); in particular, sCD27 was significantly higher (Dunn test p-value 0.0077) in the group of subjects with the highest inflammation degree of lesion activity (degree equal to 3) than in the group with lower one (inflammation degree equal to 1). Furthermore, the sCD27 was found to be significantly increased in subjects with early active lesions (Fig. 7F Mann-Whitney U test p-value 0.0210), while the seven remaining neuropathological features did not have any influence on the levels of sCD27 (Fig. 7C-I). The principal component analysis (PCA) including 12 CSF markers, including CXCL10, CCL19, TNF, BAFF, CXCL12, soluble CD21, CXCL13, IL-10, NFL, IL-6, IFN-g and sCD27 showed sCD27 clustering together with IFN-g, and the CXCL13 and the complement receptor type 2 CD21 (Fig. 7J).

sCD27 is a predictive factor of extensive active inflammatory lesions in postmortem MS cases

For each neuropathological feature, 12 CSF markers measured on 30 post-mortem subjects were imputed to the RF- RFE algorithm. Among the 9 sets of markers returned by the RF-RFE, 2 of them included sCD27. In detail, sCD27 is the marker with the highest importance in predicting the degree of lesion activity, in a signature made up of 8 CSF markers (Fig. 7K). sCD27 is also one of the markers making up the signature of the number of early active lesions (Fig. 7L): it is the 4th in terms of importance, which is comparable to other molecules for this feature.

Histograms reported levels of CD27 in different groups defined by degree of lesion activity (A), number of meningeal follicles (B), degree of CP inflammation (C), degree of perivascular inflammation (D), degree of meningeal inflammation (E), number of early active lesions (F), number of active lesions (G), number of chronic active lesions (H), number of inactive lesions (I). The data represent mean ± SD of 30 post-mortem MS cases. Statistical analyses were performed by using one-way non-parametric ANOVA and Mann-Whitney U test (F). (J) Principal component analysis of cytokines detected in the matched CSF of 30 post-mortem MS cases. Histograms report the features stratified for importance for degree of lesion activity (K) and number of early active lesions (I) obtained from Random Forest (RF)-recursive feature elimination (RFE) algorithm.

In this study, we demonstrate that sCD27 is increased in CSF from patients with MS, is correlated with inflammatory T cells and is a key marker for the clustering of patients with intrathecal lymphocyte infiltrates and CXCL13. Our in vitro studies demonstrate that sCD27 is produced intracellularly in T cells by cross-linking of TCR and secreted by sphingomyelinase-mediated exocytosis. CD27 is released in soluble form and in micro-vesicles and promotes the differentiation of inflammatory effector, IFNg-producing T cells by modulating T-bet expression. Results from our analysis of matched CSF and brain tissues from post-mortem MS cases demonstrate that sCD27 in the CSF of patients with MS is a marker of inflammatory lesions generated by intrathecal T-cell activity.

We find a different pattern of BAFF in the CSF of MS patients associated with patient clustering. Analysis of CSF showed that lower levels of BAFF in the CSF are associated with higher production of sCD27 and in those MS patients we observe a clustering of CSF sCD27, BAFF and CXCL13 supporting a contribution of B cells to T cell activation and/or the involvement of sCD27 to B cell activation [29–31]. In MS, BAFF is produced in the CNS by astrocytes [29] located in the perivascular area and parenchyma of chronic and acute lesions and promotes the persistence and clonal expansion of BAFF-R-expressing B cells [30] and T cell activation [31]. We hypothesize that the lower levels of BAFF in the CSF are associated with the consumption by inflammatory cells in patients with intrathecal disease activity. A clustering of CSF sCD27, CXCL13 and CXCL10 is observed in MS cases with normal levels of BAFF. CXCL10 is a chemokine which is induced by IFN-g and is crucial for T cell lymphocyte trafficking to inflamed tissue [32, 33]. In previous reports, higher CSF CXCL10 in patients with MS was associated with an increased leukocyte number in the CSF [34] and demyelination [35]. In actively demyelinating lesions examined in post-mortem specimens from MS cases, CXCL10 is produced by macrophages within the lesion and by reactive astrocytes present in the surrounding parenchyma [36]. We found a clustering of sCD27, CXCL13 and soluble CD21 in the CSF of progressive post-mortem cases, suggesting a potential link between sCD27 and elevated intrathecal B cell activity in the progressive phase, possibly localised in meningeal lymphoid-like structures. Levels of CXCL13 were demonstrated to be more associated with peripheral lymphocyte trafficking in the CNS [37] and Th1-mediated inflammatory process in the CNS [9] than with production from the CNS-resident meningeal and glial cells [38]. Levels of CXCL13 and sCD27 in the CSF of patients with active RR-MS undergoing autologous haematopoietic stem cell transplantation (AHSCT) and showing a remission of disease activity over the 5-year follow-up after AHSCT decreased to levels of controls for all the period study suggesting that the long-lasting remission of disease activity in those patients can be associated with reducing the trafficking of lymphocytes in the CNS [39].

We demonstrate with in vitro experiments that T cells are the main lymphocyte producers of sCD27. Activated T cells produce intracellularly sCD27 that is released in the range of 2000–6000 ng/ml, whereas the same number of activated B cells only released up to 200 ng/ml sCD27. Moreover, our in vitro experiments with CD27 knockout T cells show a significant reduction of transcription factor T-bet, which is the master regulator of T helper 1 (Th1) differentiation and T-cell homing to inflammatory sites. CD27 knockout CD4 T cells differentiate in cytotoxic CD4 T cells with increased production of granzyme b and reduction of IFNg release. Previous reports demonstrated that sCD27 is released by metalloproteinase. We also demonstrate that the inhibition of nSMase that regulates exocytosis significantly diminished the release of IFNg and sCD27. In our experiments, protein analysis of extracellular vesicles isolated from activated T cells shows the presence of a membrane form of CD27. nSMase has been demonstrated to be activated in response to inflammatory cytokines such as TNF and IL-1b, CD95, CD40, oxidative stress [40] and pathogens [41] and regulates different cellular processes including apoptosis, cell cycle, exosome release and activity of immune cells [42]. By examining matched CSF and tissues in post-mortem cases, we identify a correlation between sCD27 and CD27 + T cells detected in inflammatory infiltrates in perivascular areas and leptomeninges. Stratification of cases according to the degree of lesion activity and type of lesions reveals a significant association of levels of sCD27 with both the presence of high degree lesion activity and of early lesions in the WM. These results suggest that levels of sCD27 are associated with lymphocyte infiltration and T-cell-associated disease activity in the CNS of patients with MS. A limitation of this observation is that since a longitudinal neuropathological evaluation is not possible, we cannot establish when these interactions develop during the disease process. One further limitation of our study includes a relatively small number of subjects, yet they were sufficient to identify several statistically robust associations that confirm and expand current knowledge.

We conclude that sCD27 is primarily produced by T cells, is clinically and mechanistically associated with pro-inflammatory cytokine-mediated CNS injury, and could be further developed as a surrogate biomarker of disease activity, potentially informative also for the monitoring of anti-inflammatory treatment effects.

Acknowledgements:

We thank Dr Margarita Dominiguez-Villar for the CRISPR-cas9 technique and the students who have helped during their Master of Science and Master of Research: Shamsideen Yusuf, Sarah Lee, and Dr Bee Eng Ong. We thank the UK MS Society Tissue Bank at Imperial College (funding from the MS Society of Great Britain, grant 007/14 to RR, RN, and PM) for the supply of post-mortem MS samples.

Ethical approval

The IRAS project ID 204872, REC reference 17/L0/0172 received approval from the Health Research Authority (HRA) and Health Care Research Wales (HCRW) ref 17/LO/0172/.

The study project of investigation FIS PI15/00513 received approval from the ethics committee of University Hospital Ramon y Cajal.

All the human post-mortem tissues were collected with written informed consent forms via a prospective donor scheme with Ethical approval by the National Research Ethics Committee: IRAS project ID 246227, REC reference 18/WA/0238.

Funding:

MTC has received support from the Elena Pecci research project and the Fondazione Careggi Onlus.

EM, JIFV, LCF, LMV and NVM were supported by Red Española de Esclerosis Múltiple (REEM) (RD16/0015/0001) and by the Grant PI15/00513, integrated into the Plan Estatal I+D+I and co-funded by ISCIII-Subdirección General de Evaluación and Fondo Europeo de Desarrollo Regional (FEDER, "Otra manera de hacer Europa").

P.A.M. acknowledges infrastructure support from the NIHR Biomedical Research Centre funding scheme to Imperial College London.

Availability of data and material

The data that supports the findings of this study are available on request from the corresponding authors.

Author contributions: MTC designed, performed experiments and data analysis, and wrote the manuscript; RM performed pathology investigation of post-mortem tissues, data analysis, contributed to the experimental investigation and wrote and edited the manuscript; IP and KS contributed to experimental plan and to reviewing it critically for important intellectual content; AM performed programming and data analysis and contributed to writing the manuscript; LFF performed post-mortem tissue staining; JIFV, NVM, LCF, EM performed experiments, provided research material for analysis and edited the manuscript, LMV provided CSF from patients with RR-MS and other neurological diseases and experimental advice; RA provided research material for experiments and contributed to the revision; NM provided experimental advice; RR provided supervision, experimental advice, post-mortem tissues from MS cases and revised of the manuscript providing critical and intellectual content; RN provided CSF from patients with RR-MS and other neurological diseases for the experiments, contributed to data analysis and edited the manuscript; PM designed experiments, interpreted the data, revised conceptually and critically the manuscript and provided funding.

Competing interests:

MTC, RM, MR, AM, IP, KS, JIFV, NVM, LCF, EM, LMV, RA, LFF, NM and RR declare

no competing interests.

RN declares to have attended paid advisory boards with Roche, Biogen, and Novartis; to have received grants from the UK MS Society; and to act as Vice-chair of the National Institute of Clinical and Health Excellence HTA committee C. PAM reports no competing interest, and discloses consulting to Magenta Therapeutics, Jasper Therapeutics and Cellerys, none related to this study.

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No competing interests reported.

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Soluble CD27 is an intrathecal biomarker of T-cell-mediated lesion activity in multiple sclerosis

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Journal Publication

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Abstract

Figures

Introduction

Materials and Methods

Statistical analysis

Results

CSF sCD27 correlates with inflammatory lymphocytes in MS patients

TCR activation releases extracellular vesicles carrying CD27

Knocking out CD27 reduces IFNγ-production in CD4 T cells

CSF sCD27 levels are increased in post-mortem cases with active MS lesions

sCD27 is a predictive factor of extensive active inflammatory lesions in postmortem MS cases

Discussion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1