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B cells are implicated in the pathogenesis of multiple sclerosis (MS). A beneficial effect of B cell depletion using rituximab has been shown, but the complete mechanism of action for this drug is unclear.
To determine the relationship between T cells, B cells, and changes in CSF chemokines with rituximab, a monoclonal antibody that targets CD20.
Phase II trial of rituximab as an add-on therapy.
The John L. Multiple Sclerosis Center, Washington University, St. Louis, Missouri.
Thirty relapsing-remitting MS subjects with clinical and MRI activity despite treatment with an immunomodulatory drug received four weekly doses of 375mg/m2 rituximab.
Lumbar puncture was performed before and after rituximab infusions in 26 subjects. CSF B and T lymphocytes were enumerated by flow cytometry, and chemoattractants were measured by ELISA.
After rituximab administration, CSF B cells were decreased or undetectable in all subjects and CSF T cells were reduced in 81% of subjects. The mean reduction in CSF cellularity was 95% for B cells and 50% for T cells. After rituximab infusion, CSF CXCL13 and CCL19 decreased (P= 0.002, P=0.03, respectively). The proportional decline in CSF T cells correlated with the proportional decrease in CXCL13 (r=0.45;P=0.03), suggesting a possible relationship. CSF IgG index, IgG concentration, and oligoclonal band number were unchanged following treatment.
B cells are critical for T cell trafficking into the CNS in MS patients, and may alter T cell trafficking by influencing chemokine production within the CNS.
Multiple Sclerosis (MS) is a CNS disorder that affects 2.5 million people worldwide. The pathogenesis of MS is still not fully understood. MS had been thought to be mediated primarily by autoreactive T cells, but recent findings have indicated a critical role of B cells. 1, 2 Abnormal B cell activity is a prominent feature in MS. B cells and numerous plasma cells can be observed in active 3, 4 and chronic 5 MS lesions. Intrathecal synthesis of immunoglobulins (Ig) is a diagnostic feature of the disease. 6 B cells could also play a role through antibody independent mechanisms that include antigen presentation, costimulation, cytokine and chemokine production. 7
A recent Phase II trial of B cell depletion using rituximab, a monoclonal antibody targetting CD20, demonstrated decreased clinical and imaging activity in relapsing-remitting MS (RRMS) subjects when used as monotherapy. 8 In the present Phase II study, rituximab was used as an add-on therapy to deplete B cells in RRMS subjects with breakthrough inflammatory activity despite standard immunomodulatory drug (IMD) therapy. In an earlier report we demonstrated a reduction in CSF T cells as well as B cells in the first 15 subjects treated. 9 The completed clinical trial included 26 subjects who had undergone pre- and post-rituximab lumbar punctures. A significant reduction of CSF T cells as well as CSF B cells was observed, an unexpected finding because rituximab specifically targets only B cells. One possible reason for the T cell decline is through a reduced production of chemoattractant factors in the CNS, either directly or indirectly by B cells. Thus, levels of 17 candidate chemokines and chemoattractant factors, selected because they are either produced by B cells or known to be elevated in the CSF in MS, were compared in CSF before and after rituximab treatment. CXCL13 and CCL19, two chemokines which are involved in the organization of lymphoid follicles 10–12, declined significantly following treatment.
This Phase II trial was designed to study rituximab as add-on therapy in RRMS patients with ongoing disease activity despite therapy with an FDA-approved medication. The study was approved by the Washington University Human Research Protection Office. All subjects provided informed consent. Inclusion criteria included: RRMS with an expanded disability status score (EDSS) ≤6.5, exacerbation within 18 months despite receiving ≥ 6 months of IMD, and at least one gadolinium-enhancing lesion on any of three pre-treatment monthly brain MRIs. Prior treatment with an immunosuppressive agent, other than periodic corticosteroids, was exclusionary. Patients had to be off corticosteroids for 30 days prior to initial screening. Rituximab was administered at 375 mg/m2 weekly for four doses. Subjects continued to take their IMD (Table 1). The Multiple Sclerosis Functional Composite (MSFC) 13 was performed at each visit. Three or more “practice” MSFCs were performed prior to assessing baseline MSFC. Twenty-six subjects underwent CSF and blood sampling one week prior to and 24 or more weeks following the initial dose of rituximab (Figure 1). CSF was assessed for IgG concentration, number of oligoclonal bands, IgG synthesis rate 14, and IgG Index (normal <0.68) by the hospital laboratory.
Rituximab is a chimeric murine/human IgG1 kappa monoclonal antibody that targets CD20, a transmembrane phosphoprotein expressed only by pre-B and mature B cells. 15 Rituximab lyses B cells via complement 16 and antibody-dependent cellular cytotoxicity. 15, 17
CSF cells were examined by flow cytometry (Becton Dickinson Biosciences FACSCalibur) within 5 hours of lumbar puncture (LP). After aliquotting CSF for immunoglobulin analyses, the remainder was centrifuged, and supernatants were frozen at −80°C for chemokine analyses. T and B cells were identified with PerCP-labeled antihuman CD3 and with APC-labeled anti human CD19 (both from BD Pharmingen, San Jose, CA), respectively. CSF cells were also stained for CD80, CD86, and CD138, and in some cases for CD25, CD38, CD27 or CXCR5 (BD Pharmingen). Isotype-matched Abs served as controls. Data were analyzed with CellQuest (Becton-Dickinson, Franklin Lakes, NJ). For peripheral blood, white blood cells were isolated by Ficoll-Paque (Amersham Biosciences, Piscataway, NJ) density gradient and stained as described for CSF cells.
CSF aliquots were assayed in duplicate (kits from R&D except C3a and C5a from Becton Dickinson). Intra-assay coefficients of variation ranged from 0.1% for CXCL13 to 4.4 % for CXCL12 ELISAs.
Non-parametric Wilcoxon matched pairs tests were used to compare numbers of B and T cells, proportion of CSF B cells expressing phenotypic markers and levels of CXCL13, CCL19 and other chemokines at baseline and after treatment. Spearman rank correlations were used to examine relationship between CSF T and B cell numbers and chemokine levels, to address relationships between proportional changes in T cell numbers with changes in CXCL13 and CCL19, and to test for associations of changes in B cell and T cells counts in CSF with changes in MSFC. P values were adjusted for multiple comparisons using the stepdown Bonferroni approach.
Thirty subjects (22 females, 8 males) received four doses of rituximab (Table 1 and Figure 1). Twenty-six of these underwent lumbar puncture (LP) before and after treatment. The post-treatment LP was 24 to 30 weeks after the first rituximab infusion, except in three subjects where it was delayed due to scheduling issues (33, 35 or 38 weeks).
Nineteen of the 26 subjects undergoing LP had undetectable B cells in the blood at time of the second LP; the other seven had B cells comprising 1% to 11% of circulating mononuclear cells. The highest percentages were in subjects that delayed post-treatment LP. CSF B cells decreased after treatment in 20 subjects (Figure 2A; P<0.0001 by Wilcoxon matched pairs test). In the remaining 6 subjects, B cells were undetectable in CSF prior to or after rituximab infusion. In no case did B cells increase in CSF after treatment.
CSF T cells declined in 21 of the 26 subjects after treatment (P=0.0001, Wilcoxon matched pairs) (Figure 2B, left panel) and remained unchanged in one subject. CSF T cells increased in four subjects post-treatment, two of whom were among the 6 subjects with no detectable CSF B cells pre-treatment. Overall, CSF T cells declined by more than 50% compared to pretreatment measurements. CD3+ T cells in the blood also declined by a mean reduction of 12% after rituximab (P=0.0008; Figure 2B, right panel), a finding that has not previously been reported. In individual subjects there was no correlation between T cell numbers in blood and those in CSF before or after rituximab.
No differences were noted in CSF B and T cell numbers pre- or post-treatment between subjects taking beta-interferon versus glatiramer acetate. Baseline CSF T cell and B cell numbers did not correlate with pre-treatment MSFC or any of its individual component tests. No correlations between the percent change in T cells and the percent change in MSFC or any of its individual component tests were found.
CSF B cell expression of CD80, CD86, CD25, CD38, and CD138 was examined by flow cytometry (Table 2). Although the number of CSF B cells was greatly decreased post-treatment, the proportion of CSF B cells that also expressed the costimulatory molecules CD80 and CD86 was significantly increased (P=0.01 for both). No other significant differences were seen in CSF B cells following treatment. No significant changes in expression of CD25, CD27, CD38, CXCR5 and CD45RO were observed in CSF T cells. CSF immunoglobulin levels, including IgG Index, IgG concentration, IgG synthesis rate and oligoclonal bands did not change significantly after treatment. 9
The decline in CSF T cells was unexpected, as rituximab targets CD20, which is restricted to B cells. We hypothesized that B cells in the CNS might produce or influence production of T cell chemo-attractants affecting T cell trafficking into the CNS.
Therefore, we measured 17 candidate chemokines and chemoattractant factors in CSF. Nine of these, CCL2, CCL4, CCL19, CXCL10, CXCL12, CXCL13, CXCL16, IL-16, and C3a, were present at sufficient levels in CSF for accurate measurement (Table 3). The other eight factors were either undetectable or detected at too low levels in CSF to be reliable. These were: CCL3, CCL5, CCL21, CCL22, CXCL9, CXCL11, lymphotoxin α and C5a. Two of the nine detectable chemokines declined significantly in the CSF following rituximab therapy, CXCL13 (P=0.002), and CCL19 (P=0.03) (Table 3). The other seven chemokines that could be measured did not change significantly when comparing CSF samples obtained pre versus post-treatment.
Of 23 paired pre-and post-treatment samples tested, CSF CXCL13 declined after treatment in all but one pair (P=0.002, Figure 3A). That subject was notable for no decline in CSF T cell numbers post-treatment as well. CXCL13 was assayed in the serum in 25 paired samples before and after rituximab. CXCL13 also declined significantly overall in the blood (P<0.0001), with a decrease in 21 subjects, an increase in two and no change in two (Figure 3B). No correlation between changes in CXCL13 serum and CSF levels were found for paired samples from individual subjects (r=0.32, P=0.14) and no difference in CXCL13 levels were noted pre or post-treatment in subjects taking beta interferons versus glatiramer acetate.
CCL19 was tested in 26 sets of paired CSF and 13 sets of paired serum samples obtained before and after rituximab. CSF and serum CCL19 each declined overall post-rituximab (P=0.03 and P=0.008, respectively). CSF CCL19 levels after treatment with rituximab declined by at least 10% in 17 subjects, increased by at least 10% in four subjects and were unchanged in five (Figure 3C). Serum CCL19 declined after treatment in nine subjects, increased in one and remained stable in three (Figure 3D). Changes in CCL19 serum and CSF levels were not correlated in individual subjects (r= 0.32, p=0.36). When comparing subjects taking beta-interferon versus glatiramer acetate, no difference in CCL19 levels were noted pre or post-treatment.
We reasoned that if the decline in either of these two chemokines was related to the decline in T cells, then the proportional decreases would be correlated. Thus, the percent change in CD3+ T cells numbers in CSF was determined for each subject, as was the percent change in CXCL13 and CCL19. The proportional change in CSF T cells was mildly correlated with the proportional change in CXCL13, suggesting that this chemokine was related to the decline in T cell numbers (r=0.45; P=0.03 Spearman correlation) (Figure 4). There was no significant correlation between percent change in CSF T cells and percent change in CSF CCL19 (r=0.30, P=0.17). Interestingly, there was a negative correlation between the change in T cell numbers and the change in CSF MCP-1 (r = −0.46, P= 0.03). No relationships between proportional change in T cell numbers and changes in the other chemokines were identified.
Because initial B cell numbers in CSF prior to treatment were small, the percent change in B cells could not be precisely determined and no correlations of percent change in CSF B cells with changes in chemokine levels or T cell numbers were considered interpretable.
Until recently, evidence linking B cells and their products to MS pathogenesis has mainly consisted of observational studies. The availability of rituximab to specifically deplete B cells has provided a means to examine the pathogenic role of B cells in MS. Two studies published in 2008 indicated that B cell depletion with rituximab resulted in diminished clinical and MRI activity in RRMS patients. 8, 18 In the present trial, 30 subjects were B cell depleted with rituximab while continuing to take their IMD. B cells in CSF and peripheral blood were eliminated. Unexpectedly, a decline in CSF and blood T cells also occurred. The decline in T cells as well as B cells might be a mechanism through which rituximab benefits RRMS.
Previously, B cells have been implicated in MS through their ability to produce antibodies. Correlations of high levels of CSF antibodies with poor outcomes in MS have been shown. 19 In the present study, no changes in CSF IgG or oligoclonal band number were noted after B cell depletion, suggesting that the effectiveness of rituximab is independent of CSF IgG levels. Thus, the present study not only supports the notion that B cells are critical to development of inflammatory lesions in relapsing MS, but also that the role of B cells is not limited to antibody production. Indeed, B cells have several other functions which may be important, including antigen presentation 20 and cytokine and chemokine production. 21
After observing that T cells declined in CSF of subjects treated with rituximab, we measured CSF levels of several chemokines and chemoattractant factors that have been implicated in T cell trafficking in MS. CXCL13, a chemoattractant factor for both B cells and activated T cells and which is involved in the organization of lymphoid follicles 10–12, declined significantly following treatment. Of interest, there was a correlation between the degrees of reduction in CSF T cells and CXCL13, suggesting an association between these two changes. B cells are not known to produce CXCL13, so B cell depletion alone would not explain the decreased levels. Follicular dendritic cells (DCs) are an established source of CXCL13 within human lymphoid tissues. 22 Published observations indicate that the cellular source of CXCL13 in the CNS is likely to be DCs or monocytes/macrophages. In immunohistochemical studies of autopsied active MS lesions, CXCL13 was observed within inflammatory cells of macrophage morphology. 23 Furthermore, monocyte/macrophages are the source of CXCL13 in EAE. 24 Notably, mice lacking CXCL13 develop less severe experimental autoimmune encephalomyelitis (EAE) with less CNS inflammation, indicating an important role for this chemokine in EAE pathogenesis. 24 T cells themselves might be a source of CXCL13. Recent human studies have detected the expression of CXCL13 by antigen-experienced helper T cells within synovial follicles and by Th17 clones. 25, 26 Thus, a reduction in trafficking of T cells to the CNS might amplify the decline in CSF CXCL13.
The receptor for CXCL13 is CXCR5, expressed by most B cells, subsets of CD4+ T cells and some DCs. Studies in MS subjects 23 demonstrated that about 20–30% of blood and CSF CD4+ T cells were CXCR5+, whereas virtually all B cells in blood and the majority of CSF B cells were CXCR5+. Thus, the decline in CXCL13 would be expected to affect CNS trafficking of a significant proportion of T cells and most B cells.
CCL19 levels were also decreased in the CSF and serum of MS patients after treatment with rituximab. CCL19 is made by macrophages and DCs in T cell zones of secondary lymphoid organs where it is critically involved in the migration of lymphocytes and mature DCs 27 CCL19 is constitutively expressed in the CNS 28–30 and may be critical for CNS immune surveillance. CSF CCL19 levels were increased in MS and other CNS inflammatory diseases compared to controls 29, 30, and CCL19 RNA was elevated in active and inactive MS lesions. 29
Expression of CXCL13 and CCL19 in MS CNS would be enhanced by the B cell derived factor, lymphotoxin alpha (LT-α) 10, 31 which is a key inducer of both CCL19 32 and CXCL13. 33 In other autoimmune diseases CXCL13 promotes B cell expression of LT-α, which promotes follicular DC development and further expression of CXCL13, in a positive feedback loop. 10 We speculate that greatly decreased levels of LT-α would result from B cell depletion in CNS, leading to decreased levels of CXCL13 and CCL19. However, we were unable to reliably measure LT-α in the CSF so this hypothesis remains to be tested.
Reductions in CCL19 and CXCL13 levels following therapy with rituximab were notable because of recent renewed interest in lymph node like structures in MS CNS. Both CXCL13 and CCL19 have been implicated in the development of ectopic lymphoid follicles34. Ectopic lymphoid-like structures have been seen in some MS plaques 35, and the endothelium in MS lesions sometimes resembles that of secondary lymphoid organs. Follicle-like structures, described in the meninges of some secondary progressive MS cases 34, 36, have been associated with an aggressive clinical course. 36 CXCL13 is of special importance because its expression has been detected in intrameningeal follicles in MS 34, 36 and in active MS lesions. 23 Together, these findings suggest a CNS microenvironment in MS that would attract B cells and promote their expansion, maturation and production of immunoglobulins and cytokines/chemokines, as well as recruit T cells.
Although the present studies indicate chemokine modulation as an important function of B cells in MS, this is unlikely to be the only role of B cells in MS pathogenesis. B cells can function as potent antigen presenting cells (APCs) to T cells, especially in situations of low antigen density. 37 Loss of B cells as essential APCs in the periphery would result in decreased activation of T cells and decreased trafficking to the CNS. However, if this was the only mechanism of B cell depletion leading to diminished immune cell trafficking, we would have expected a generalized decrease in inflammatory chemokines in CSF, not selectively CXCL13 and CCL19.
These studies provide further support for a critical role of B cells in MS, and yield additional insight into the nature of that role. Depletion of B cells by rituximab reduced not only CSF B cells, but also T cells. This effect of B cells may be indirect, involving the reduced CNS production of chemoattractant factors, such as CXCL13, by non-B cells. B cells may also play a role as APCs to auto reactive T cells. In this current era of increasing numbers of monoclonal antibody therapies, the present study underscores that even when targeting a single molecule restricted to a single cell type, there may be important downstream effects which may relate to its mechanism of action.
Funding/Support: This work was supported by the National Multiple Sclerosis Society USA (RG 3292); National Institutes of Health (K24RR017100; 5UL1 RR024992); the Barnes-Jewish Hospital Foundation, Genentech, Inc. and Biogen-Idec. AHC was supported in part by the Manny and Rosalyn Rosenthal-Dr. John L. Trotter Chair in Neuroimmunology. LP was supported by a post-doctoral fellowship from the National MS Society (FG 1665-A-1) and in early studies by Fondazione Italiana Sclerosi Multipla (FISM; 2004/B/4). RTN was funded by K23NS052430-01A1 and K12RR02324902.
Author Contributions: Dr Anne Cross had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Laura Piccio, Robert Naismith, Jeri Lyons and Anne Cross. Acquisition of data: Laura Piccio, Robert Naismith, Becky Parks. Analysis and interpretation of data: Laura Piccio, Kathryn Trinkaus, Anne Cross. Drafting of the manuscript: Laura Piccio, Anne Cross. Critical Revision of the manuscript for important intellectual content: Laura Piccio, Robert Naismith, Kathryn Trinkaus, Robyn Klein, Becky Parks, Jeri Lyons and Anne Cross. Statistical analysis: Kathryn Trinkaus and Anne Cross. Obtained funding: Anne Cross. Administrative, technical, and material support: Laura Piccio, Kathryn Trinkaus, Robyn Klein, Becky Parks, Jeri Lyons and Anne Cross. Study supervision: Laura Piccio, Robert Naismith, Anne Cross.
Financial Disclosure: Dr. Cross serves on the Research Programs Advisory Committee and the National Clinical Advisory Board of the National MS Society of the National MS Society, scientific advisory board for Lilly and BioMS; has received speaker honoraria for non-industry-sponsored activities; serves on the speakers' bureaus of BayerHealthcare (formerly Berlex), Genentech, Inc., Biogen Idec, and Teva Neuroscience; and receives research support from the NIH [NINDS #PO1 NS059560- 01 (Overall PI, PI of Project 3 and Core A), NINDS #UO1 NS45719-01A1 (Co-investigator), #RO1 NS047592 (Coinvestigator), and NINDS/National MS Society #RO1 NS 051591/NMSS RG 3915-A-15 (PI)], and from the National MS Society. She is on the editorial boards of Brain Pathology and Journal of Neuroimmunology, and received an honorarium from the AAN for editing and co-writing two chapters in CONTINUUM (Lippincott Williams & Wilkins, 2007). Dr. Cross is Washington University site PI for clinical trials sponsored by Acorda Therapeutics and Sanofi-Aventis.
Dr. Naismith has served on speakers’ bureaus and as consultant for Bayer Healthcare, Biogen Idec, Elan Pharmaceuticals, and Teva Neurosciences; and receives research support from Acorda Therapeutics (Site PI), and the NIH [#K23NS052430-01A1(PI) and #K12RR02324902(PI)]; and received an honorarium from the AAN for editing and writing one chapter in CONTINUUM (Lippincott Williams & Wilkins, 2007).
Dr. Klein serves on the Research Committee of the National MS Society and receives research support through the Washington University/Pfizer Biomedical Program, the National MS Society (RG3982), the DANA Foundation and the NIH (NINDS #PO1 NS059560- 01 (PI of Project 2 and Core B).
Dr. Parks has served as a consultant and/or on speaker's bureaus for Bayer Healthcare, Biogen Idec, EMD Serono and Teva Neuroscience.
Dr Piccio, Dr Trinkaus and Dr Lyons have nothing to disclose.
Additional contributions: we thank Robert Mikesell, Michael Ramsbottom and Dr. Neville Rapp for excellent technical assistance, Joanne Lauber, Cathie Martinez, Monica Fairbairn and Nhial Tutlam for study subject coordination and MSFC testing, Drs. John H. Russell and Sheng-Kwei (Victor) Song for helpful discussions, and our patients for their participation. The paper is dedicated to the memory of this MS Center’s founder, Dr. John L. Trotter (1943–2001).