Differential effects of anti-CD20 therapy on CD4 and CD8 T cells and implication of CD20-expressing CD8 T cells in MS disease activity.

A small proportion of multiple sclerosis (MS) patients develop new disease activity soon after starting anti-CD20 therapy. This activity does not recur with further dosing, possibly reflecting deeper depletion of CD20-expressing cells with repeat infusions. We assessed cellular immune profiles and their association with transient disease activity following anti-CD20 initiation as a window into relapsing disease biology. Peripheral blood mononuclear cells from independent discovery and validation cohorts of MS patients initiating ocrelizumab were assessed for phenotypic and functional profiles using multiparametric flow cytometry. Pretreatment CD20-expressing T cells, especially CD20dimCD8+ T cells with a highly inflammatory and central nervous system (CNS)-homing phenotype, were significantly inversely correlated with pretreatment MRI gadolinium-lesion counts, and also predictive of early disease activity observed after anti-CD20 initiation. Direct removal of pretreatment proinflammatory CD20dimCD8+ T cells had a greater contribution to treatment-associated changes in the CD8+ T cell pool than was the case for CD4+ T cells. Early disease activity following anti-CD20 initiation was not associated with reconstituting CD20dimCD8+ T cells, which were less proinflammatory compared with pretreatment. Similarly, this disease activity did not correlate with early reconstituting B cells, which were predominantly transitional CD19+CD24highCD38high with a more anti-inflammatory profile. We provide insights into the mode-of-action of anti-CD20 and highlight a potential role for CD20dimCD8+ T cells in MS relapse biology; their strong inverse correlation with both pretreatment and early posttreatment disease activity suggests that CD20-expressing CD8+ T cells leaving the circulation (possibly to the CNS) play a particularly early role in the immune cascades involved in relapse development.

Pro-inflammatory adiponectin in pediatric-onset multiple sclerosis.

BACKGROUND: Being obese is associated with both increased risk of developing multiple sclerosis (MS) and greater MS disease activity. OBJECTIVES: The objective of this study is to investigate levels and potential pathophysiologic contribution of serum adipose-hormones (adipokines) in pediatric-onset MS. METHODS: Following a Luminex adipokine screen, adiponectin (APN) and its isoforms were quantified by enzyme-linked immunosorbent assay (ELISA) in 169 children with incident acquired demyelinating syndromes (ADS), prospectively ascertained as having either MS or other forms of inflammatory central nervous system (CNS) demyelination. The effect of recombinant APN and APN-containing sera was assessed on functional responses of normal human peripheral blood myeloid and T cells and on human CNS-derived microglia. RESULTS: Compared to other cohorts, children with MS harbored higher serum APN levels, principally driven by higher levels of the low-molecular-weight isoform. Recombinant APN and pediatric MS serum-induced APN-dependent pro-inflammatory activation of CD14+ monocytes and of activated CD4+ and CD8+ T cells (both directly and indirectly through myeloid cells). APN induced human microglia activation while inhibiting their expression of molecules associated with quiescence. CONCLUSIONS: Elevated APN levels in children with MS may contribute to enhanced pro-inflammatory states of innate and adaptive peripheral immune responses and breach CNS-resident microglia quiescence, providing a plausible and potentially targetable mechanism by which APN contributes to MS disease activity.

Abnormal effector and regulatory T cell subsets in paediatric-onset multiple sclerosis.

Elucidation of distinct T-cell subsets involved in multiple sclerosis immune-pathophysiology continues to be of considerable interest since an ultimate goal is to more selectively target the aberrant immune response operating in individual patients. While abnormalities of both effector (Teff) and regulatory (Treg) T cells have been reported in patients with multiple sclerosis, prior studies have mostly assessed average abnormalities in either limb of the immune response, rather than both at the same time, which limits the ability to evaluate the balance between effectors and regulators operating in the same patient. Assessing both phenotypic and functional responses of Teffs and Tregs has also proven important. In studies of adults with multiple sclerosis, in whom biological disease onset likely started many years prior to the immune assessments, an added challenge for any reported abnormality is whether the abnormality indeed contributes to the disease (and hence of interest to target therapeutically) or merely develops consequent to inflammatory injury (in which case efforts to develop targeted therapies are unlikely to be beneficial). Paediatric-onset multiple sclerosis, though rare, offers a unique window into early disease mechanisms. Here, we carried out a comprehensive integrated study, simultaneously assessing phenotype and functional responses of both effector and regulatory T cells in the same children with multiple sclerosis, monophasic inflammatory CNS disorders, and healthy controls, recruited as part of the multicentre prospective Canadian Pediatric Demyelinating Disease Study (CPDDS). Stringent standard operating procedures were developed and uniformly applied to procure, process and subsequently analyse peripheral blood cells using rigorously applied multi-parametric flow cytometry panels and miniaturized functional assays validated for use with cryopreserved cells. We found abnormally increased frequencies and exaggerated pro-inflammatory responses of CD8+CD161highTCR-Vα7.2+ MAIT T cells and CD4+CCR2+CCR5+ Teffs in paediatric-onset multiple sclerosis, compared to both control groups. CD4+CD25hiCD127lowFOXP3+ Tregs of children with multiple sclerosis exhibited deficient suppressive capacity, including diminished capacity to suppress disease-implicated Teffs. In turn, the implicated Teffs of multiple sclerosis patients were relatively resistant to suppression by normal Tregs. An abnormal Teff/Treg ratio at the individual child level best distinguished multiple sclerosis children from controls. We implicate abnormalities in both frequencies and functional responses of distinct pro-inflammatory CD4 and CD8 T cell subsets, as well as Treg function, in paediatric-onset multiple sclerosis, and suggest that mechanisms contributing to early multiple sclerosis development differ across individuals, reflecting an excess abnormality in either Teff or Treg limbs of the T cell response, or a combination of lesser abnormalities in both limbs.

Comprehensive immunophenotyping of cerebrospinal fluid cells in patients with neuroimmunological diseases.

We performed unbiased, comprehensive immunophenotyping of cerebrospinal fluid (CSF) and blood leukocytes in 221 subjects referred for the diagnostic work-up of neuroimmunological disorders to obtain insight about disease-specific phenotypes of intrathecal immune responses. Quantification of 14 different immune cell subsets, coupled with the assessment of their activation status, revealed physiological differences between intrathecal and systemic immunity, irrespective of final diagnosis. Our data are consistent with a model where the CNS shapes intrathecal immune responses to provide effective protection against persistent viral infections, especially by memory T cells, plasmacytoid dendritic cells, and CD56(bright) NK cells. Our data also argue that CSF immune cells do not simply reflect cells recruited from the periphery. Instead, they represent a mixture of cells that are recruited from the blood, have been activated intrathecally and leave the CNS after performing effector functions. Diagnosis-specific differences provide mechanistic insight into the disease process in the defined subtypes of multiple sclerosis (MS), neonatal onset multisystem inflammatory disease, and Aicardi-Goutières syndrome. This analysis also determined that secondary-progressive MS patients are immunologically closer to relapsing-remitting patients as compared with patients with primary-progressive MS. Because CSF immunophenotyping captures the biology of the intrathecal inflammatory processes, it has the potential to guide optimal selection of immunomodulatory therapies in individual patients and monitor their efficacy. Our study adds to the increasing number of publications that demonstrate poor correlation between systemic and intrathecal inflammatory biomarkers in patients with neuroimmunological diseases and stresses the importance of studying immune responses directly in the intrathecal compartment.

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.

Cellular and humoral immune responses following SARS-CoV-2 mRNA vaccination in patients with multiple sclerosis on anti-CD20 therapy.

SARS-CoV-2 messenger RNA vaccination in healthy individuals generates immune protection against COVID-19. However, little is known about SARS-CoV-2 mRNA vaccine-induced responses in immunosuppressed patients. We investigated induction of antigen-specific antibody, B cell and T cell responses longitudinally in patients with multiple sclerosis (MS) on anti-CD20 antibody monotherapy (n = 20) compared with healthy controls (n = 10) after BNT162b2 or mRNA-1273 mRNA vaccination. Treatment with anti-CD20 monoclonal antibody (aCD20) significantly reduced spike-specific and receptor-binding domain (RBD)-specific antibody and memory B cell responses in most patients, an effect ameliorated with longer duration from last aCD20 treatment and extent of B cell reconstitution. By contrast, all patients with MS treated with aCD20 generated antigen-specific CD4 and CD8 T cell responses after vaccination. Treatment with aCD20 skewed responses, compromising circulating follicular helper T (TFH) cell responses and augmenting CD8 T cell induction, while preserving type 1 helper T (TH1) cell priming. Patients with MS treated with aCD20 lacking anti-RBD IgG had the most severe defect in circulating TFH responses and more robust CD8 T cell responses. These data define the nature of the SARS-CoV-2 vaccine-induced immune landscape in aCD20-treated patients and provide insights into coordinated mRNA vaccine-induced immune responses in humans. Our findings have implications for clinical decision-making and public health policy for immunosuppressed patients including those treated with aCD20.

A complex role of herpes viruses in the disease process of multiple sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disorder of the central nervous system (CNS). Neither the antigenic target(s) nor the cell population(s) responsible for CNS tissue destruction in MS have been fully defined. The objective of this study was to simultaneously determine the antigen (Ag)-specificity and phenotype of un-manipulated intrathecal CD4+ and CD8+ T cells of patients with relapsing-remitting and progressive MS compared to subjects with other inflammatory neurological diseases. We applied a novel Ag-recognition assay based on co-cultures of freshly obtained cerebrospinal fluid T cells and autologous dendritic cells pre-loaded with complex candidate Ag's. We observed comparably low T cell responses to complex auto-Ag's including human myelin, brain homogenate, and cell lysates of apoptotically modified oligodendroglial and neuronal cells in all cohorts and both compartments. Conversely, we detected a strong intrathecal enrichment of Epstein-Barr virus- and human herpes virus 6-specific (but not cytomegalovirus-specific) reactivities of the Th1-phenotype throughout all patients. Qualitatively, the intrathecal enrichment of herpes virus reactivities was more pronounced in MS patients. This enrichment was completely reversed by long-term treatment with the IL-2 modulating antibody daclizumab, which strongly inhibits MS disease activity. Finally, we observed a striking discrepancy between diminished intrathecal T cell proliferation and enhanced cytokine production of herpes virus-specific T cells among progressive MS patients, consistent with the phenotype of terminally differentiated cells. The data suggest that intrathecal administration of novel therapeutic agents targeting immune cells outside of the proliferation cycle may be necessary to effectively eliminate intrathecal inflammation in progressive MS.

The role of the neuropeptide somatostatin on methamphetamine and glutamate-induced neurotoxicity in the striatum of mice.

A large body of evidence shows that methamphetamine (METH) causes sustained damage to the brain in animal models and human METH users. In chronic users there are indications of cognitive and motor deficits. Striatal neuropeptides are in a position to modulate the neurochemical effects of METH and consequently striatal neural damage. Somatostatin (SST) is an intrinsic striatal neuropeptide that has been shown to inhibit glutamate transmission; glutamate is integral to METH toxicity and contributes to nitric oxide (NO) synthesis. We hypothesize that SST will protect from METH by inhibition of NO synthesis and thus reducing oxidative stress. To this end, the SST analogue octreotide (OCT) was microinjected into the striatum prior to a systemic injection of METH (30mg/kg). We then assessed 3-nitrotyrosine (3-NT), an indirect index of NO production, tyrosine hydroxylase (TH) protein levels (dopamine terminal marker) and Fluoro-Jade C positive cells (degenerating cells). The SST agonist OCT dose dependently attenuated the METH-induced accumulation of striatal 3-NT. Moreover, pretreatment with OCT effectively mitigated cell death but failed to protect dopamine terminals. Next we co-infused OCT and NMDA and measured 3-NT and Fluoro-Jade C staining. Treatment with OCT had no effect on these parameters. The data demonstrate that SST attenuates the METH-induced production of NO protecting the striatum from the METH-induced cell loss. However, SST failed to prevent the toxicity of the dopamine terminals suggesting that pre- and post-synaptic striatal damage occur via independent mechanisms.

Role of neurokinin-1 and dopamine receptors on the striatal methamphetamine-induced proliferation of new cells in mice.

A neurotoxic dose of methamphetamine (METH) induces the loss of some striatal neurons. Interestingly, the METH-induced apoptosis in the striatum is immediately followed by the generation of new cells (cytogenesis). In the present study, we investigated the role of the neurokinin-1, dopamine D1 and D2 receptors on the METH-induced cytogenesis. To that end, male mice were given a single injection (30 mg/kg, ip) or a binge of METH (10mg/kg, 4× at two-hour intervals, ip). BrdU (100mg/kg, ip) was given 36 h after the last injection of METH. Newly generated cells were detected by immunohistochemistry and cell counts were performed using unbiased computerized stereology. Either single or binge exposure to METH resulted in the generation of new cells. The single optimized dose was used for subsequent mechanistic studies. Pretreatment with the dopamine D1 receptor antagonist SCH23390 (0.1mg/kg, ip) 30 min prior to METH abrogated the METH-induced striatal cytogenesis. Pretreatment with the dopamine D2 receptor antagonist raclopride (1mg/kg, ip) failed to affect this phenomenon. Finally, pretreatment with the neurokinin-1 receptor antagonist WIN 51,708 (5mg/kg, ip) 30 min prior to METH abrogated the METH-induced cytogenesis. In conclusion, neurokinin-1 and dopamine D1 receptors are required for the METH-induced striatal cytogenesis while the D2 receptor is without effect.