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

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-γ-producing T cells and is released through a secretory mechanism activated by TCR engagement and regulated by neutral sphingomyelinase. 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.

Oligoclonal M bands unveil occult inflammation in multiple sclerosis.

INTRODUCTION: Recent works demonstrate that patients with multiple sclerosis (pwMS) and oligoclonal M bands (OCMB) in cerebrospinal fluid (CSF) are at higher risk of conversion to secondary progressive course, suggesting a distinct pathophysiology pathway in these patients. OBJECTIVES: To analyze the relationship of serum neurofilament light chain (s-NFL) in absence of inflammatory activity in people with multiple sclerosis (pwMS) according to the presence of OCMB versus healthy controls (HC), and the effect of aging. METHODS: Two cohorts of HC were compared to a cohort of pwMS without clinical or radiological signs of acute inflammation. Lack of inflammation was defined as the absence of relapses or gadolinium-enhancing lesions (GEL) brain in an MRI performed within three months before and after s-NFL determination. S-NFL was measured with SIMOa technology. OCMB in the cerebrospinal fluid (CSF) were analyzed with isoelectric focusing and immunoblotting. RESULTS: 254 people were studied: 124 healthy voluntary controls and 130 pwMS. Despite the absence of inflammatory activity, pwMS and OCMB showed higher levels of s-NFL compared to those without OCMB and HC (11.4 pg/mL, 8.9 pg/mL and 9.0 pg/mL, respectively). A positive and exponential correlation between age and s-NFL was observed, with highest increases among pwMS and OCMB in the CSF. DISCUSSION: In absence of overt inflammatory activity, pwMS and OCMB exhibit higher s-NFL levels, and a greater age-related increase. Thus, OCMB may portray an underlying inflammatory process not detected by conventional MRI studies and may explain the poorer prognosis of these patients.

Stabilization of leukocytes from cerebrospinal fluid for central immunophenotypic evaluation in multicenter clinical trials.

Analysis of cerebrospinal fluid (CSF) represents a valuable window into the pathogenesis of neuroinflammatory diseases, such as multiple sclerosis (MS). However, analysis of the cellular fraction of CSF is often neglected because CSF cells die rapidly ex vivo. Immunophenotyping of CSF cells in multicenter clinical trials requires sample preservation and shipping to a centralized lab. Yet, there is no consensus on the best method to preserve intact CSF cells and no detailed evaluation of subset-specific cell loss. We used flow cytometry to compare major leukocyte populations in fresh CSF (processed within 2 h) to cells fixed for 48 h with TransFix-EDTA® or cryopreserved and thawed after 96 h. We observed a statistically significant loss of total mononuclear cells, total T cells, CD3+ CD8- T cells, and CD3+ CD8+ T cells after cryopreservation compared to fresh or fixed (p < 0.001), with no significant difference between fresh and fixed. Thus, our results demonstrate that TransFix-EDTA® was superior to cryopreservation for preserving intact CSF T cells. Surprisingly, neither cryopreservation nor fixation had a significant effect on recovery of low frequency cell subsets in CSF, including B cells, NK cells, NKT-like cells, CD14+ monocytes, or CD123+ DCs, versus fresh CSF. To determine the effect of prolonged fixation on cell recovery, we analyzed major CSF cell subsets by flow cytometry after 24, 48, or 72 h of fixation with TransFix-EDTA®. We observed a consistent and progressive loss in the absolute counts of all subsets over time, although this effect was not statistically significant. We conclude that for immunophenotyping of major CSF cell subsets by flow cytometry, fixation with TransFix-EDTA®, shipment to a central lab, and analysis within 48 h is a feasible method to ensure stability of both absolute cell number and relative frequency. This method is a valuable alternative to fresh CSF analysis and can be implemented in multicenter clinical trials.

Baseline Inflammatory Status Reveals Dichotomic Immune Mechanisms Involved In Primary-Progressive Multiple Sclerosis Pathology.

Objective: To ascertain the role of inflammation in the response to ocrelizumab in primary-progressive multiple sclerosis (PPMS). Methods: Multicenter prospective study including 69 patients with PPMS who initiated ocrelizumab treatment, classified according to baseline presence [Gd+, n=16] or absence [Gd-, n=53] of gadolinium-enhancing lesions in brain MRI. Ten Gd+ (62.5%) and 41 Gd- patients (77.4%) showed non-evidence of disease activity (NEDA) defined as no disability progression or new MRI lesions after 1 year of treatment. Blood immune cell subsets were characterized by flow cytometry, serum immunoglobulins by nephelometry, and serum neurofilament light-chains (sNfL) by SIMOA. Statistical analyses were corrected with the Bonferroni formula. Results: More than 60% of patients reached NEDA after a year of treatment, regardless of their baseline characteristics. In Gd+ patients, it associated with a low repopulation rate of inflammatory B cells accompanied by a reduction of sNfL values 6 months after their first ocrelizumab dose. Patients in Gd- group also had low B cell numbers and sNfL values 6 months after initiating treatment, independent of their treatment response. In these patients, NEDA status was associated with a tolerogenic remodeling of the T and innate immune cell compartments, and with a clear increase of serum IgA levels. Conclusion: Baseline inflammation influences which immunological pathways predominate in patients with PPMS. Inflammatory B cells played a pivotal role in the Gd+ group and inflammatory T and innate immune cells in Gd- patients. B cell depletion can modulate both mechanisms.

Effect of Ocrelizumab in Blood Leukocytes of Patients With Primary Progressive MS.

OBJECTIVE: To analyze the changes induced by ocrelizumab in blood immune cells of patients with primary progressive MS (PPMS). METHODS: In this multicenter prospective study including 53 patients with PPMS who initiated ocrelizumab treatment, we determined effector, memory, and regulatory cells by flow cytometry at baseline and after 6 months of therapy. Wilcoxon matched paired tests were used to assess differences between baseline and 6 months' results. p Values were corrected using the Bonferroni test. RESULTS: Ocrelizumab reduced the numbers of naive and memory B cells (p < 0.0001) and those of B cells producing interleukin (IL)-6, IL-10, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNFα) (p < 0.0001 in all cases). By contrast, the proportions of plasmablasts and B cells producing GM-CSF and TNFα increased significantly, suggesting the need for treatment continuation. We also observed a decrease in CD20+ T-cell numbers (p < 0.0001) and percentages (p < 0.0001), and a clear remodeling of the T-cell compartment characterized by relative increases of the naive/effector ratios in CD4+ (p = 0.002) and CD8+ (p = 0.002) T cells and relative decreases of CD4+ (p = 0.03) and CD8+ (p = 0.004) T cells producing interferon-gamma. Total monocyte numbers increased (p = 0.002), but no changes were observed in those producing inflammatory cytokines. The immunologic variations were associated with a reduction of serum neurofilament light chain (sNfL) levels (p = 0.008). The reduction was observed in patients with Gd-enhanced lesions at baseline and in Gd- patients with baseline sNfL >10 pg/mL. CONCLUSIONS: In PPMS, effector B-cell depletion changed T-cell response toward a low inflammatory profile, resulting in decreased sNfL levels.