Ocrelizumab Treatment Modulates B-Cell Regulating Factors in Multiple Sclerosis.

BACKGROUND AND OBJECTIVES: Antibodies to CD20 efficiently reduce new relapses in multiple sclerosis (MS), and ocrelizumab has been shown to be effective also in primary progressive MS. Although anti-CD20 treatments efficiently deplete B cells in blood, some B cells and CD20- plasma cells persist in lymphatic organs and the inflamed CNS; their survival is regulated by the B cell-activating factor (BAFF)/A proliferation-inducing ligand (APRIL) system. The administration of a soluble receptor for BAFF and APRIL, atacicept, unexpectedly worsened MS. Here, we explored the long-term effects of ocrelizumab on immune cell subsets as well as on cytokines and endogenous soluble receptors comprising the BAFF-APRIL system. METHODS: We analyzed immune cell subsets and B cell-regulating factors longitudinally for up to 2.5 years in patients with MS treated with ocrelizumab. In a second cohort, we determined B-cell regulatory factors in the CSF before and after ocrelizumab. We quantified the cytokines BAFF and APRIL along with their endogenous soluble receptors soluble B-cell maturation antigen (sBCMA) and soluble transmembrane activator and calcium-modulator and cyclophilin ligand (CAML) interactor (sTACI) using enzyme-linked immunosorbent assays (ELISAs). In addition, we established an in-house ELISA to measure sTACI-BAFF complexes. RESULTS: Ocrelizumab treatment of people with MS persistently depleted B cells and CD20+ T cells. This treatment enhanced BAFF and reduced the free endogenous soluble receptor and decoy sTACI in both serum and CSF. Levels of sTACI negatively correlated with BAFF levels. Reduction of sTACI was associated with formation of sTACI-BAFF complexes. DISCUSSION: We describe a novel effect of anti-CD20 therapy on the BAFF-APRIL system, namely reduction of sTACI. Because sTACI is a decoy for APRIL, its reduction may enhance local APRIL activity, thereby promoting regulatory IgA+ plasma cells and astrocytic interleukin (IL)-10 production. Thus, reducing sTACI might contribute to the beneficial effect of anti-CD20 as exogenous sTACI (atacicept) worsened MS. CLASSIFICATION OF EVIDENCE: This study provides Class IV evidence that endogenous sTACI in blood and CSF is decreased after ocrelizumab treatment.

Relapse-independent multiple sclerosis progression under natalizumab.

The objective of this study was to investigate confirmed progression independent of relapse activity in relapsing-remitting multiple sclerosis patients under long-term natalizumab treatment. We performed a retrospective, cross-sectional study of clinical data captured between 1994 and 2019 at two German multiple sclerosis tertiary referral centres. Data files of all relapsing-remitting multiple sclerosis patients treated with natalizumab for ≥24 months were analysed. Confirmed progression independent of relapse activity was defined as ≥12 week confirmed disability progression on a roving Expanded Disability Status Scale reference score by 1 point in patients with an Expanded Disability Status Scale score ≤3 or 0.5 in patients with an Expanded Disability Status Scale score ≥3.5 in the absence of a relapse. Cox proportional hazard models were used to analyse the probability of developing confirmed progression independent of relapse activity depending on both disease and natalizumab treatment duration. Among the 184 patients identified, 44 (24%) developed confirmed progression independent of relapse activity under natalizumab irrespective of the Expanded Disability Status Scale score at natalizumab onset. Time to confirmed progression independent of relapse activity was not affected by Expanded Disability Status Scale at natalizumab onset (categorized by Expanded Disability Status Scale score ≤3.5 versus >3.5) nor by duration of disease nor by duration of therapy. Confirmed progression independent of relapse activity occurred earlier in the disease course in patients with an earlier natalizumab therapy onset with regard to disease duration. A stepwise forward regression analysis revealed disease duration as the main factor for confirmed progression independent of relapse activity development (P = 0.005). Taken together, confirmed progression independent of relapse activity occurs in a substantial proportion of patients on long-term natalizumab treatment and independent of Expanded Disability Status Scale score at natalizumab onset. Our findings suggest that patients who are initiated on natalizumab early during disease course, usually in order to treat an aggressive clinical phenotype, have a higher risk of early confirmed progression independent of relapse activity.

Effects of Natalizumab Therapy on Intrathecal Immunoglobulin G Production Indicate Targeting of Plasmablasts.

OBJECTIVES: To evaluate the long-term effects of natalizumab (NTZ) on different features of intrathecal immunoglobulin (Ig) synthesis in patients with multiple sclerosis (MS) and to quantify the expression of α4-integrin in stages of B-cell maturation. METHODS: We combined a cross-sectional (49 NTZ-treated MS patients, mean treatment duration 5.1 years, and 47 untreated MS patients) and a longitudinal study (33 patients with MS before and during NTZ, mean treatment duration: 4.8 years), analyzing paired serum and CSF samples for IgG, IgA, and IgM levels, reactivity against selected viruses (measles virus, rubella virus, and varicella zoster virus [MRZ] reaction), and oligoclonal bands (OCBs). Banding patterns before and after therapy were directly compared by isoelectric focusing in 1 patient. In addition, we determined the expression of α4-integrin by FACS analysis on blood-derived B-cell subsets (plasmablasts, memory B cells, and naive B cells) of healthy controls. RESULTS: In serum, NTZ decreased IgM and IgG, but not IgA, levels. IgM hypogammaglobulinemia occurred in 28% of NTZ-treated patients. In CSF, NTZ treatment resulted in a strong reduction of intrathecally produced IgG and, to a lesser extent, IgA, whereas IgM indices [(Ig CSF/Serum)/(Albumin CSF/Serum)] remained largely unchanged. Reduction of the IgG index correlated with NTZ treatment duration, as did serum IgM and IgA levels. MRZ reaction was unchanged and OCB persisted. Direct comparison of OCB pattern before and after NTZ revealed the persistence of individual bands. α4-Integrin expression was highest on plasmablasts (CD19+CD38+CD27+). CONCLUSION: Our data indicate that NTZ reduces short-lived plasmablasts in the CNS compartment but has little effect on locally persisting long-lived plasma cells.

Anti-CD20 therapies and pregnancy in neuroimmunologic disorders: A cohort study from Germany.

OBJECTIVE: To report pregnancy outcomes and disease activity (DA) in women with MS, neuromyelitis optica spectrum disorders (NMOSDs), and other neuroimmunologic diseases (ONID) after treatment with rituximab (RTX)/ocrelizumab (OCR) 12 months before or during pregnancy. METHODS: Data were collected in the German MS and pregnancy registry and centers from the Neuromyelitis Optica Study Group. Sixty-eight known outcomes of 88 pregnancies from 81 women (64 MS, 10 NMOSD, and 7 ONID) were included and stratified in 3 exposure groups: >6M-group = RTX/OCR >6 but ≤12 months before the last menstrual period (LMP) (n = 8); <6M group = RTX/OCR <6 months before the LMP (n = 47); preg group = RTX/OCR after the LMP (n = 13). RESULTS: Pregnancy outcomes were similar between groups, but significantly more preterm births (9.8% vs 45%) occurred after exposure during pregnancy. Overall, 2 major congenital abnormalities (3.3%), both in the preg group, were observed. Three women had severe infections during pregnancy. All women with MS (35) and 12/13 women with NMOSD, RTX/OCR exposure before the LMP and known pregnancy outcomes after gestational week 22 were relapse free during pregnancy. Five of 29 (17.2%) women with relapsing-remitting MS (RRMS) and 1 of 12 (8.3%) with NMOSD and at least 6 months postpartum follow-up experienced a relapse postpartum. Duration of RTX/OCR and early retreatment but not detection of B-cells were possible predictors for postpartum relapses in patients with RRMS/NMOSD. CONCLUSIONS: Although RTX/OCR might be an interesting option for women with RRMS/NMOSD who plan to become pregnant to control DA, more data on pregnancy outcomes and rare risks are needed.

Neurological phenotypes in patients with NLRP3-, MEFV-, and TNFRSF1A low-penetrance variants.

BACKGROUND: Neurological manifestations and the co-occurrence of multiple sclerosis (MS) have been reported in patients with autoinflammatory diseases (AID) and variants of the NLRP3-, MEFV-, or TNFRSF1A gene. However, type and frequency of neurological involvement are widely undetermined. METHODS: We assessed clinical characteristics of 151 (108 with MS) patients carrying NLRP3-, MEFV- and TNFRSF1A low-penetrance variants  from the Institute of Clinical Neuroimmunology. We evaluated demographic, genetic, and clinical features with a focus on central nervous system (CNS) involvement including magnetic resonance imaging (MRI) results and cerebrospinal fluid (CSF) data. The disease course of AID patients with MS was compared to a matched MS control group without mutations. RESULTS: The genetic distribution comprised 36 patients (23%) with NLRP3- and 66 patients (43%) with TNFRSF1A low-penetrance variants as well as 53 (34%) patients carrying pathogenic mutations or low-penetrance variants in the MEFV gene. MS patients displayed most frequently the R92Q TNFRSF1A variant (n = 51; 46%) followed by the Q703K NLRP3 variant (n = 15; 14%) and the E148Q substitution (n = 9; 8%) in the MEFV gene. The disease course of MS was not influenced by the genetic variants and did not differ from MS patients (n = 51) without mutations. AID patients without MS most frequently harbored MEFV mutations (n = 19, 43%) followed by NLRP3- (n = 17, 39%) and TNFRSF1A (n = 8, 18%) low-penetrance variants. Sixteen (36%) of them suffered from severe CNS involvement predominantly recurrent aseptic meningoencephalitis and optic neuritis accompanied by abnormal MRI and CSF results. Severe CNS inflammation was associated with the Q703K allele. Headache was a highly prevalent neurological symptom (up to 74%), irrespective of the underlying genetic variation. The NLRP3 cohort without MS more frequently exhibited affections of the cranial nerves (CN) (p = 0.0228) and motor symptoms (p = 0.0455). Elevated acute-phase reactants were detected in all patients, and fever episodes were present in up to 50%. Arthralgias were the most frequently identified constitutional symptom among all subgroups. CONCLUSIONS: Our data highlight the high prevalence of neurological manifestations, including concomitant MS, among NLRP3-, MEFV-, and TNFRSF1A low-penetrance variants. In particular, patients carrying the Q703K NLRP3 variant are at risk for severe CNS inflammation and CN affection.

Fingolimod Profoundly Reduces Frequencies and Alters Subset Composition of Circulating T Follicular Helper Cells in Multiple Sclerosis Patients.

Fingolimod is an effective treatment for relapsing-remitting multiple sclerosis. It is well established that fingolimod, a modulator of the sphingosine-1-phosphate pathway, restrains the egress of CCR7+ lymphocytes from lymphatic tissues into the blood, thus resulting in reduced lymphocyte counts in peripheral blood. CXCR5+ T follicular helper (Tfh) cells provide help to B cells, are essential for the generation of potent Ab responses, and have been shown to be critically involved in the pathogenesis of several autoimmune diseases. Besides lymphoid tissue-resident Tfh cells, CXCR5+ circulating Tfh (cTfh) cells have been described in the blood, their numbers correlating with the magnitude of Tfh cells in lymphoid tissues. Although the effect of fingolimod on circulating lymphocyte subsets has been established, its effect on cTfh cells remains poorly understood. In this study, we found that although fingolimod strongly and disproportionally reduced cTfh cell frequencies, frequencies of activated cTfh cells were increased, and the composition of the cTfh cell pool was skewed toward a cTfh1 cell phenotype. The circulating T follicular regulatory cell subset and CXCR5+ CD8+ T cell frequencies were also strongly and disproportionally decreased after fingolimod treatment. In contrast, relative frequencies of CXCR5- memory Th cells as well as regulatory T and B cells were increased. In summary, these data provide new insights into fingolimod-induced compositional changes of lymphocyte populations in the blood, in particular cTfh cells, and thus contribute to a better understanding of the mechanism of action of fingolimod in multiple sclerosis patients.

Recurrence of disease activity during pregnancy after cessation of fingolimod in multiple sclerosis.

BACKGROUND: Fingolimod is an effective treatment for active relapsing-remitting multiple sclerosis (MS). Discontinuation of therapy may be followed by recurrence of disease activity. Thus, female MS patients may be at risk of relapse during pregnancy after stopping fingolimod. OBJECTIVES AND METHODS: To report the disease course during pregnancy of five women who interrupted therapy with fingolimod for pregnancy. RESULTS: All patients experienced relapses during pregnancy and/or postpartum after stopping fingolimod. CONCLUSION: The risk of recurrence of disease activity during pregnancy after stopping fingolimod may be substantial. This should be considered and discussed with MS patients who are planning to become pregnant.

Features of Human CD3+CD20+ T Cells.

Monoclonal Abs against CD20 reduce the number of relapses in multiple sclerosis (MS); commonly this effect is solely attributed to depletion of B cells. Recently, however, a subset of CD3(+)CD20(+) T cells has been described that is also targeted by the anti-CD20 mAb rituximab. Because the existence of cells coexpressing CD3 and CD20 is controversial and features of this subpopulation are poorly understood, we studied this issue in detail. In this study, we confirm that 3-5% of circulating human T cells display CD20 on their surface and transcribe both CD3 and CD20. We report that these CD3(+)CD20(+) T cells pervade thymus, bone marrow, and secondary lymphatic organs. They are found in the cerebrospinal fluid even in the absence of inflammation; in the cerebrospinal fluid of MS patients they occur at a frequency similar to B cells. Phenotypically, these T cells are enriched in CD8(+) and CD45RO(+) memory cells and in CCR7(-) cells. Functionally, they show a higher frequency of IL-4-, IL-17-, IFN-γ-, and TNF-α-producing cells compared with T cells lacking CD20. CD20-expressing T cells respond variably to immunomodulatory treatments given to MS patients: they are reduced by fingolimod, alemtuzumab, and dimethyl fumarate, whereas natalizumab disproportionally increases them in the blood. After depletion by rituximab, they show earlier and higher repopulation than CD20(+) B cells. Taken together, human CD3(+)CD20(+) T cells pervade lymphatic organs and the cerebrospinal fluid, have a strong ability to produce different cytokines, and respond to MS disease modifying drugs.

PML risk stratification using anti-JCV antibody index and L-selectin.

BACKGROUND: Natalizumab treatment is associated with progressive multifocal leukoencephalopathy (PML) development. Treatment duration, prior immunosuppressant use, and JCV serostatus are currently used for risk stratification, but PML incidence stays high. Anti-JCV antibody index and L-selectin (CD62L) have been proposed as additional risk stratification parameters. OBJECTIVE: This study aimed at verifying and integrating both parameters into one algorithm for risk stratification. METHODS: Multicentric, international cohorts of natalizumab-treated MS patients were assessed for JCV index (1921 control patients and nine pre-PML patients) and CD62L (1410 control patients and 17 pre-PML patients). RESULTS: CD62L values correlate with JCV serostatus, as well as JCV index values. Low CD62L in natalizumab-treated patients was confirmed and validated as a biomarker for PML risk with the risk factor "CD62L low" increasing a patient's relative risk 55-fold (p < 0.0001). Validation efforts established 86% sensitivity/91% specificity for CD62L and 100% sensitivity/59% specificity for JCV index as predictors of PML. Using both parameters identified 1.9% of natalizumab-treated patients in the reference center as the risk group. CONCLUSIONS: Both JCV index and CD62L have merit for risk stratification and share a potential biological relationship with implications for general PML etiology. A risk algorithm incorporating both biomarkers could strongly reduce PML incidence.

Natalizumab use during the third trimester of pregnancy.

IMPORTANCE: Natalizumab reduces multiple sclerosis relapses very effectively; however, severe disease activity may return once natalizumab treatment is withdrawn, as recommended during pregnancy. Sometimes restarting natalizumab treatment may be the best option for the mother, but the consequences for the infant are unknown. Except for a few single case reports, to our knowledge, comprehensive data about third-trimester natalizumab exposure are scant. OBSERVATIONS: In a case series of 12 women with 13 pregnancies and highly active multiple sclerosis who were treated with natalizumab during their third trimester of pregnancy, we assessed the clinical and laboratory effects on the newborns. We observed mild to moderate hematologic alterations in 10 of 13 infants including thrombocytopenia and anemia. In a subsample of 5 mother-child pairs, we analyzed natalizumab levels in the umbilical cord blood. Natalizumab was detectable in all 5 newborns. CONCLUSION AND RELEVANCE: Natalizumab can be a therapeutic option in patients with highly active multiple sclerosis during pregnancy. We recommend that a pediatrician be available at the time of delivery to evaluate for potential complications of anemia and thrombocytopenia in newborns exposed to natalizumab during the third trimester.

Predictors for multiple sclerosis relapses after switching from natalizumab to fingolimod.

BACKGROUND: Risks of natalizumab (NAT) therapy have to be weighed against disease recurrence after stopping NAT. OBJECTIVES: The objective of this paper is to identify risk factors for recurrence of relapses after switching from NAT to fingolimod (FTY) in relapsing-remitting multiple sclerosis (RRMS). METHODS: Patients (n = 33) were treated with NAT for ≥1 year, and then switched to FTY within 24 weeks (mean follow-up on FTY 81.1 (SD 26.5) weeks). Annual relapse rates (ARR) and Expanded Disability Status Scale scores (EDSS) were assessed. Descriptive statistics, univariate logistic regression analysis, and receiver operating characteristic curves were conducted. RESULTS: Overall, 20 patients (61%) had relapses after discontinuation of NAT and 16 (48%) during FTY therapy. The maximum incidence of relapses occurred between weeks 13-24 post-NAT. The last EDSS during the switching period predicted relapses during subsequent FTY therapy. EDSS >3 separated most powerfully between the groups (sensitivity 64%, specificity 88%) and significantly predicted relapses (relative risk 3.27, 95% CI: 1.5-7.3). Seventy-five percent of patients with EDSS ≤ 3 remained free of relapses, compared to 18% of patients with EDSS >3. CONCLUSIONS: There was an increase of the ARR in the first year after switching from NAT to FTY. Last EDSS during the switching period was a predictor of relapses during FTY.

L-selectin is a possible biomarker for individual PML risk in natalizumab-treated MS patients.

OBJECTIVE: To find biomarkers identifying patients at risk for the development of progressive multifocal leukoencephalopathy (PML) during natalizumab treatment. METHODS: Patients were recruited from 10 European and US cohorts. Of 289 patients with multiple sclerosis (MS), 224 had been treated with natalizumab (18-80 months), 21 received other immune-modulatory treatments, and 28 were untreated. We had access to samples from 16 natalizumab PML patients. Eight of these patients had given blood before the diagnosis of PML. We also analyzed non-natalizumab-treated patients who developed PML (n = 10) and age- and sex-matched healthy donors (n = 31). All flow cytometric assessments were done on previously cryopreserved, viable peripheral blood mononuclear cells. RESULTS: The percentage of l-selectin-expressing CD4+ T cells was significantly lower in patients treated long-term with natalizumab (40.2%) when compared with patients not receiving natalizumab treatment (47.2%; p = 0.016) or healthy controls (61.0%; p < 0.0001). An unusually low percentage (9-fold lower; 4.6%) was highly correlated with the risk of developing PML in the patient group with available pre-PML samples when compared with non-PML natalizumab-treated patients (p ≤ 0.0001). Samples were gathered between 4 and 26 months before PML diagnosis. CONCLUSIONS: The cell-based assessment of the percentage of l-selectin-expressing CD4 T cells could provide an urgently needed biomarker for individual PML risk assessment.

Fingolimod reduces recurrence of disease activity after natalizumab withdrawal in multiple sclerosis.

After discontinuation of natalizumab (NAT), multiple sclerosis (MS) disease activity often recurs. We assessed the recurrence of clinical disease activity during the first year after switching from NAT to fingolimod (FTY) in patients with relapsing-remitting MS. The number of relapses and the annualized relapse rate (ARR) before, during and after NAT discontinuation were determined and compared between 26 MS patients who switched to FTY within 24 weeks, and 10 MS patients who remained without disease modifying therapy (therapy free group = TFG). Median follow-up post-NAT discontinuation was 55.1 weeks. In a subgroup (n = 20), the occurrence of contrast-enhancing-lesions (Gd+) on magnetic resonance imaging (MRI) was determined. Eleven patients (42 %) in the FTY group and seven patients (70 %) in the TFG had one or more relapses after cessation of NAT during follow-up (p < 0.05). One of the 11 (9 %) patients in the FTY group and 6/9 (67 %) patients in the TFG showed Gd+ lesions during follow-up (p < 0.05). Patients who switched to FTY ≤ 12 weeks after NAT discontinuation (n = 9) showed a trend for a lower post-NAT ARR compared to patients who started FTY therapy >12 weeks after NAT was stopped (n = 17). Most relapses in the FTY group occurred just before or within 8 weeks after starting FTY. Our observation suggests that initiation of FTY treatment after NAT discontinuation reduces the recurrence of disease activity compared to withdrawal without further immunomodulatory treatment. In the FTY group the ARR tended to depend on the time interval between discontinuation of NAT and initiation of FTY.

Rebound of disease activity after withdrawal of fingolimod (FTY720) treatment.

BACKGROUND: The oral sphingosine-1-phosphate receptor modulator fingolimod (FTY720) was recently approved for the treatment of relapsing-remitting multiple sclerosis. To date, data about a possible recurrence of disease activity after discontinuation of fingolimod treatment are scarce. OBJECTIVE: To describe a patient who discontinued fingolimod treatment after a local malignant melanoma was diagnosed. Three months after cessation, he had a striking rebound of multiple sclerosis activity. DESIGN: Case report and review of literature. SETTING: Institute of Clinical Neuroimmunology, Ludwig-Maximilians-University, Munich, Germany. PATIENT: A 45-year-old man diagnosed as having relapsing-remitting multiple sclerosis. MAIN OUTCOME MEASURES: Multiple sclerosis disease activity including annual relapse rate, Expanded Disability Status Scale score, and number of gadolinium-enhancing lesions on magnetic resonance imaging before, during, and after treatment with fingolimod. RESULTS: Three months after discontinuation of treatment with fingolimod, the patient experienced a severe relapse, with Expanded Disability Status Scale score progression from 2.5 to 4.5. On brain and spinal magnetic resonance imaging, he showed a rebound of disease activity, with a drastic increase of gadolinium-enhancing lesions (>20). CONCLUSIONS: Two aspects relevant to any newly approved multiple sclerosis treatment with immunomodulatory properties are highlighted with this case: first, possible rebound of disease activity after discontinuation; second, the occurrence of a tumor as a possible treatment-related complication.

De-escalation from natalizumab in multiple sclerosis: recurrence of disease activity despite switching to glatiramer acetate.

Natalizumab (NAT) is an effective therapy for relapsing-remitting multiple sclerosis (MS), but is associated with an increased risk of progressive multifocal leucoencephalopathy after 2 years therapy. Thus, NAT treated patients often decide to stop NAT therapy after 2 years. Reports on recurrence of disease activity after NAT discontinuation are controversial. We studied disease activity in 13 MS patients who stopped NAT therapy and either remained without disease modifying therapy (no DMT, n = 6), or switched to glatiramer acetate (GLAT, n = 7). Annual relapse rate (ARR), expanded disability status scale (EDSS), and number of patients with contrast-enhancing-lesions (Gd+) on MRI before, during and within 1 year after NAT were determined. We observed recurrence of disease activity in both groups (5/7 GLAT treated patients and 6/6 patients without DMT) within 12 months after cessation of NAT (mean time to first relapse was 5.5 months for all patients). One of the GLAT treated patients and three patients without DMT had severe relapses with sustained EDSS worsening. No differences in ARR, EDSS and MRI parameters were seen between both groups. Patients with relapses after NAT therapy, however, tended to show higher disease activity (EDSS, ARR) before initiation of NAT therapy compared to patients without relapses. Duration of NAT treatment was not associated with higher disease activity after NAT discontinuation. In this observation the majority of patients showed reappearance of disease activity after discontinuation of NAT regardless of whether they switched to GLAT or remained without DMT. Further treatment strategies are warranted for patients who discontinue NAT therapy.