Repopulation of T, B, and NK cells following alemtuzumab treatment in relapsing-remitting multiple sclerosis.

OBJECTIVE: To characterize long-term repopulation of peripheral immune cells following alemtuzumab-induced lymphopenia in relapsing-remitting MS (RRMS), with a focus on regulatory cell types, and to explore associations with clinical outcome measures. METHODS: The project was designed as a multicenter add-on longitudinal mechanistic study for RRMS patients enrolled in CARE-MS II, CARE-MS II extension at the University of Southern California and Stanford University, and an investigator-initiated study conducted at the Universities of British Columbia and Chicago. Methods involved collection of blood at baseline, prior to alemtuzumab administration, and at months 5, 11, 17, 23, 36, and 48 post-treatment. T cell, B cell, and natural killer (NK) cell subsets, chemokine receptor expression in T cells, in vitro cytokine secretion patterns, and regulatory T cell (Treg) function were assessed. Clinical outcomes, including expanded disability status score (EDSS), relapses, conventional magnetic resonance imaging (MRI) measures, and incidents of secondary autoimmunity were tracked. RESULTS: Variable shifts in lymphocyte populations occurred over time in favor of CD4+ T cells, B cells, and NK cells with surface phenotypes characteristic of regulatory subsets, accompanied by reduced ratios of effector to regulatory cell types. Evidence of increased Treg competence was observed after each treatment course. CD4+ and CD8+ T cells that express CXCR3 and CCR5 and CD8+ T cells that express CDR3 and CCR4 were also enriched after treatment, indicating heightened trafficking potential in activated T cells. Patterns of repopulation were not associated with measures of clinical efficacy or secondary autoimmunity, but exploratory analyses using a random generalized estimating equation (GEE) Poisson model provide preliminary evidence of associations between pro-inflammatory cell types and increased risk for gadolinium (Gd+) enhancing lesions, while regulatory subsets were associated with reduced risk. In addition, the risk for T2 lesions correlated with increases in CD3+CD8+CXCR3+ cells. CONCLUSIONS: Lymphocyte repopulation after alemtuzumab treatment favors regulatory subsets in the T cell, B cell, and NK cell compartments. Clinical efficacy may reflect the sum of interactions among them, leading to control of potentially pathogenic effector cell types. Several immune measures were identified as possible biomarkers of lesion activity. Future studies are necessary to more precisely define regulatory and effector subsets and their contributions to clinical efficacy and risk for secondary autoimmunity in alemtuzumab-treated patients, and to reveal new insights into mechanisms of immunopathogenesis in MS. TRIAL REGISTRATION: Parent trials for this study are registered with ClinicalTrials.gov: CARE-MS II: NCT00548405, CARE-MS II extension: NCT00930553 and ISS: NCT01307332.

The dual role of CXCL8 in human CNS stem cell function: multipotent neural stem cell death and oligodendrocyte progenitor cell chemotaxis.

Research into multiple sclerosis (MS) has shown that cells purportedly important to myelin repair within the CNS, namely neural stem cells (NSC) and oligodendrocyte progenitor cells (OPC), are recruited to active lesion sites during the course of the disease. However, over time these cells appear to become depleted or functionally blocked in and around lesions, accompanied by a failure of repair mechanisms. We have previously demonstrated elevated CXCL8 in patients with MS, and hypothesized that this chemokine may play a role in the pathology of this disease. Using NSC and OPC derived in vitro from human embryonic stem cells (hESC) we demonstrate here that CXCL8 has a dual role on stem cell biology in vitro. CXCL8 caused CXCR1-mediated death of NSC, but not OPC, whilst also acting as a potent chemoattractant for both cell types. These data support a context-dependent role for CXCL8 expression in the CNS in which it may drive recruitment of NSC and OPC to sites of inflammation, but as a side-effect could also contribute to the failure of myelin repair in MS.

The geometric and spatial constraints of the microenvironment induce oligodendrocyte differentiation.

The oligodendrocyte precursor cell (OPC) arises from the subventricular zone (SVZ) during early vertebrate development to migrate and proliferate along axon tracts before differentiating into the myelin-forming oligodendrocyte. We demonstrate that the spatial and temporal regulation of oligodendrocyte differentiation depends intimately on the axonal microenvironment and the density of precursor cells along a specified axonal area. Differentiation does not require dynamic axonal signaling, but instead is induced by packing constraints resulting from intercellular interactions. Schwann cells and even artificial beads bound to the axonal surface can mimic these constraints and promote differentiation. Together, these results describe the coordinately controlled biophysical interaction of oligodendrocyte precursors within an axonal niche leading to self-renewal and differentiation.

Immune Regulatory Cell Bias Following Alemtuzumab Treatment in Relapsing-Remitting Multiple Sclerosis.

Alemtuzumab is a highly effective treatment for relapsing-remitting multiple sclerosis. It selectively targets the CD52 antigen to induce profound lymphocyte depletion, followed by recovery of T and B cells with regulatory phenotypes. We previously showed that regulatory T cell function is restored with cellular repletion, but little is known about the functional capacity of regulatory B-cells and peripheral blood monocytes during the repletion phase. In this study (ClinicalTrials.gov ID# NCT03647722) we simultaneously analyzed the change in composition and function of both regulatory lymphocyte populations and distinct monocyte subsets in cross-sectional cohorts of MS patients prior to or 6, 12, 18, 24 or 36 months after their first course of alemtuzumab treatment. We found that the absolute number and percentage of cells with a regulatory B cell phenotype were significantly higher after treatment and were positivity correlated with regulatory T cells. In addition, B cells from treated patients secreted higher levels of IL-10 and BDNF, and inhibited the proliferation of autologous CD4+CD25- T cell targets. Though there was little change in monocytes populations overall, following the second annual course of treatment, CD14+ monocytes had a significantly increased anti-inflammatory bias in cytokine secretion patterns. These results confirmed that the immune system in alemtuzumab-treated patients is altered in favor of a regulatory milieu that involves expansion and increased functionality of multiple regulatory populations including B cells, T cells and monocytes. Here, we showed for the first time that functionally competent regulatory B cells re-appear with similar kinetics to that of regulatory T-cells, whereas the change in anti-inflammatory bias of monocytes does not occur until after the second treatment course. These findings justify future studies of all regulatory cell types following alemtuzumab treatment to reveal further insights into mechanisms of drug action, and to identify key immunological predictors of durable clinical efficacy in alemtuzumab-treated patients.

Activation of the Protective Arm of the Renin Angiotensin System in Demyelinating Disease.

The renin angiotensin system (RAS), which is classically known for blood pressure regulation, has functions beyond this. There are two axes of RAS that work to counterbalance each other and are active throughout the body, including the CNS. The pathological axis, consisting of angiotensin II (A1-8), angiotensin converting enzyme (ACE) and the angiotensin II type 1 receptor (AT1R), is upregulated in many CNS diseases, including multiple sclerosis (MS). MS is an autoimmune and neurodegenerative disease of the CNS characterized by inflammation, demyelination and axonal degeneration. Published research has described increased expression of AT1R and ACE in tissues from MS patients and in animal models of MS such as experimental autoimmune encephalomyelitis (EAE). In contrast to the pathological axis, little is known about the protective axes of RAS in MS and EAE. In other neurological conditions the protective axis, which includes A1-7, ACE2, angiotensin II type 2 receptor and Mas receptor, has been shown to have anti-inflammatory, regenerative and neuroprotective effects. Here we show, for the first time, changes in the protective arm of RAS in both EAE and MS CNS tissue. We observed a significant increase in expression of the protective arm during stages of disease stabilization in EAE, and in MS tissue showing evidence of remyelination. These data provide evidence that the protective arm of RAS, through both ligand and receptor expression, is associated with reductions in the pathological processes that occur in the earlier stages of MS and EAE, possibly slowing the neurodegenerative process and enhancing neural repair. Graphical Abstract.

Measuring apoptosis in neural stem cells.

In trauma to, and diseases of, the central nervous system (CNS), apoptotic events are frequently observed in and around areas of damage. Human embryonic stem cells (hESCs) and their progeny have been suggested as possible therapeutic agents in the treatment of CNS diseases. The success of stem cell transplantation not only depends on the capacity of these cells to retain their functionality after transplant into the CNS but also on their ability to resist the in situ environmental cues that may lead to apoptosis. Although there are many methods used to detect apoptosis, the assessment of apoptosis in adherent cultures of primary stem cells and their progeny is more limited. We describe a series of protocols we have used to assess apoptosis in these cells.

The influence of retinoic acid on the human oligodendrocyte precursor cells by RNA-sequencing.

Retinoic acid (RA), a metabolite of vitamin A, has been found to influence regeneration in the adult central nervous system (CNS). There may be an effect of RA in the recovery/repair in multiple sclerosis (MS), an autoimmune and neurodegenerative disease of the CNS. We hypothesized that RA is a regulator of the further differentiation of oligodendrocyte precursor cells (OPCs) - cells key to the remyelination process in MS. We conducted studies utilizing RNA-sequencing in human embryonic stem cell (hESC)-derived neural stem cells (NSCs) and OPCs so as to understand the role of transcriptional regulators during transition from both ESCs to NSCs and NSCs to OPCs. We identified that expression of retinoic acid receptors ß and γ (RARß and RARγ ) was significantly increased following the transition from NSCs to OPCs. We also demonstrated that long term in vitro culture of hESC-derived OPC with different isoforms of RA led to the significant up-regulation of two known transcriptional inhibitors of oligodendrocyte differentiation: Hes5 following prolonged treatment with all-trans-RA, 9-cis RA and 13-cis RA; and Id4 following treatment with 13cisRA. These results suggest that long term exposure to certain RA isoforms may impact the continued differentiation of this population.

Characterisation of UBP296: a novel, potent and selective kainate receptor antagonist.

Willardiine derivatives with an N3-benzyl substituent bearing an acidic group have been synthesized with the aim of producing selective antagonists for GLUK5-containing kainate receptors. UBP296 was found to be a potent and selective antagonist of native GLUK5-containing kainate receptors in the spinal cord, with activity residing in the S enantiomer (UBP302). In cells expressing human kainate receptor subunits, UBP296 selectively depressed glutamate-induced calcium influx in cells containing GLUK5 in homomeric or heteromeric forms. In radioligand displacement binding studies, the willardiine analogues displaced [3H]kainate binding with IC50 values >100 microM at rat GLUK6, GLUK2 or GLUK6/GLUK2. An explanation of the GLUK5 selectivity of UBP296 was obtained using homology models of the antagonist bound forms of GLUK5 and GLUK6. In rat hippocampal slices, UBP296 reversibly blocked ATPA-induced depressions of synaptic transmission at concentrations subthreshold for affecting AMPA receptor-mediated synaptic transmission directly. UBP296 also completely blocked the induction of mossy fibre LTP, in medium containing 2 mM (but not 4 mM) Ca2+. These data provide further evidence for a role for GLUK5-containing kainate receptors in mossy fibre LTP. In conclusion, UBP296 is the most potent and selective antagonist of GLUK5-containing kainate receptors so far described.

Characterisation of zinc uptake into rat cultured cerebrocortical oligodendrocyte progenitor cells.

In the mammalian brain, extracellular Zn(2+) is reported to play a neuromodulatory role and, during acute CNS injury, increased Zn(2+) release may be neurotoxic. Although several recent studies have examined possible mechanisms of neuronal Zn(2+) accumulation, little is known about oligodendroglial Zn(2+) uptake, the focus of the present study. 65Zn(2+) uptake was time-dependent and saturable (K(m)=3.2+/-1.0 microM, V(max)=697.2+/-67.3 pmoles mg protein(-1) 15 min(-1)). Neither kainate (an AMPA/kainate receptor agonist) nor nicardipine (an L-type Ca(2+) channel inhibitor) influenced 65Zn(2+) uptake, in contrast with pyrithione (a Zn(2+) ionophore). Either increasing extracellular H(+) concentration (pH 5.5) or co-application of either 100 microM Co(2+) or 100 microM Cu(2+) reduced (65)Zn(2+) uptake. However, 100 microM Fe(2+) failed to influence 65Zn(2+) uptake and 100 microM La(3+) increased 65Zn(2+) accumulation. These data are consistent with oligodendrocyte progenitor cells possessing a high-affinity Zn(2+) uptake mechanism similar to that described for the Zrt, Irt-like protein (ZIP) transporter family.

In vitro assessment of the direct effect of laquinimod on basic functions of human neural stem cells and oligodendrocyte progenitor cells.

Laquinimod is an orally active immunomodulatory small molecule that has shown clear clinical benefit in trials for relapsing-remitting multiple sclerosis and in experimental rodent models that emulate multiple sclerosis (MS). Studies in healthy mice, and in mice with experimental autoimmune encephalomyelitis, have demonstrated that laquinimod is capable of entering the central nervous system. It is therefore important to determine if laquinimod is capable of a direct influence on basic functions of neural stem cells (NSC) or oligodendrocyte progenitor cells (OPC)--cells critical for myelin repair in MS. In order to address this question, a series of experiments was conducted to determine the effect of exogenous laquinimod on viability, proliferation, migration and differentiation of human NSC and OPC in vitro. These data show, for the first time in cells of human origin, that direct, short-term interaction between laquinimod and NSC or OPC, in an isolated in vitro setting, is not detrimental to the basic cellular function of these cells.

Assessment of changes in immune measures of multiple sclerosis patients treated with laquinimod.

Laquinimod is a novel orally active agent with immunomodulatory properties that was shown to be effective in suppressing disease activity in relapsing-remitting multiple sclerosis patients. Though many mechanisms of action of laquinimod have been described, little is known about the in vivo effects of laquinimod on the functionality of circulating human peripheral blood mononuclear cell populations. We assessed both phenotypical and functional measures of PBMC in a prospective longitudinal analysis comparing laquinimod and placebo treated cohorts. We determined that there were no significant changes in the relative proportion of T-cells, B-cells, monocytes & macrophages, NK-cells, dendritic cells or FoxP3(+) CD25(hi) T-regs in laquinimod treated patients. There were also no significant differences in the proliferative response to PHA or tetanus antigen, or in the inflammatory cytokine bias of these responses. These data demonstrated that there were no significant changes in immune function of PBMC in patients receiving two years of continuous laquinimod therapy who retained a full complement of the major populations of circulating PBMC and retained their capacity to respond to immunologic stimuli.

Pyruvate limits zinc-induced rat oligodendrocyte progenitor cell death.

A growing body of evidence suggests that excessive Zn2+ release plays a key role in inducing neuronal death during central nervous system injury. However, the possible cytotoxicity of extracellular Zn2+ to oligodendrocyte lineage cells remains unknown. Employing cultures of rat oligodendrocyte progenitor cells (OPC), we report here that OPC are vulnerable to increased extracellular Zn2+ levels and that pyruvate limits Zn2+-induced OPC death. Zn2+-induced concentration-dependent (pEC50 = -4.1 +/- 0.1) OPC death, which was insensitive to both alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (Evans Blue) and l-type Ca2+ channel (nicardipine) inhibition. Neither kainate nor nicardipine influenced OPC 65Zn2+ accumulation, in contrast with the Zn2+ ionophore, pyrithione. Cytotoxic extracellular Zn2+ concentrations failed to increase OPC reactive oxygen species production and the antioxidant reagents, trolox, N,N'-diphenyl-1,4-phenylenediamine and N-tert-butyl-alpha-phenylnitrone did not afford significant protection from Zn2+ insults. The apoptotic inducer staurosporine induced the appearance of known apoptotic markers [pyknotic nuclei and caspase-3 specific (120 kDa) alpha-fodrin cleavage fragment], events not reproduced with Zn2+ insults. Zn2+ insults were also insensitive to the pan-caspase inhibitor Z-VAD-fmk. However, pyruvate afforded significant OPC protection from lethal Zn2+ insults. We conclude that cultured OPC are vulnerable to Zn2+ insults, via a nonoxidative stress and noncaspase-3-based mechanism, involving Zn2+ inhibition of OPC glycolysis.