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Keywords:

  • B10 cells;
  • MG;
  • FACS;
  • MGFA;
  • Rituximab

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Introduction: A subset of regulatory B cells in humans and mice has been defined functionally by their ability to produce interleukin (IL)-10. We characterized IL-10-producing B (B10) cells in myasthenia gravis (MG) patients and correlated them with disease activity and responsiveness to rituximab therapy. Methods: Frequencies of B10 cells from MG patients and healthy controls were monitored by fluorescence-activated cell sorting (FACS). Results: MG patients had fewer B10 cells than controls, which was associated with more severe disease status. Moreover, patients who responded well to rituximab therapy exhibited rapid repopulation of B10 cells, whereas in patients who did not respond well to rituximab, B10 cell repopulation was delayed. The kinetics of B10 cells were related to the responsiveness to rituximab in MG. Conclusions: We have characterized a specific subset of B10 cells in MG patients which may serve as a marker for disease activity and responsiveness to immune therapy. Muscle Nerve 49:487–494, 2014

Abbreviations
AChR

acetylcholine receptor

APC

allophycocyanin

FACS

fluorescence-activated cell sorting

FITC

fluorescein isothiocyanate

IL-10

interleukin-10

B10 cells

IL-10-producing B cells

IRB

institutional review board

MG

myasthenia gravis

MGFA

Myasthenia Gravis Association of America

MuSK

muscle specific kinase

PBMCs

peripheral blood mononuclear cells

PE

phycoerythrin

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction in which autoantibodies to the acetylcholine receptor (AChR) or muscle specific kinase (MuSK) impair neuromuscular transmission and cause muscle weakness. Anti-AChR or MuSK autoantibodies are produced by B cells with the help of T cells and other lymphocytes. Although a role for the thymus in the pathophysiology of the disease has been proposed, the etiology of MG remains unknown. Even in MG patients with thymic abnormality, there are many missing links between sensitization of antigen presenting cells and initiation of a cascade of pathogenic humoral autoimmunity.

Among several mechanisms that are capable of controlling immune responses and preventing autoimmunity, immune suppression conferred by a group of “regulatory” cells has gained significant attention in the past decade.[1] A subset of regulatory B cells has been defined functionally in humans and mice by their ability to express interleukin (IL)-10.[2-4] In contrast to the conventional role of effector B cells, i.e., antibody production and priming of the immune response, regulatory B cells suppress inflammatory and autoimmune responses by means of their capacity to produce IL-10.[5-7] The frequencies and functional properties of IL-10-producing B cells (referred to as B10 cells hereafter) have been reported in patients with autoimmune and inflammatory diseases, including rheumatoid arthritis, Graves disease, and systemic lupus erythematosus.[5, 8-12] With notable exceptions, most studies have found that B10 cells exhibit reduced frequency and impaired function in these disorders.[8-10] However, this novel B cell subpopulation has not been characterized in MG patients.

B cell targeted therapy, achieved by a chimeric murine/human monoclonal antibody, rituximab, has several advantages in controlling the symptoms of MG, particularly in patients who are refractory to conventional therapies.[13-19] Rituximab targets the CD20 antigen and efficiently depletes pre-B cells and mature B cells, all of which express CD20.[20] After B cell depletion, repopulation follows subsequently. Therefore, rituximab therapy provides a unique opportunity to study the kinetics of repopulation of subsets of B cells in MG.

Here, we investigated the presence and dynamics of B10 cells in patients with MG compared with healthy subjects. We sought a correlation between clinical severity as rated by Myasthenia Gravis Association of America (MGFA) status and the frequency of B10 cells. As patients underwent therapy with rituximab, we studied the clinical response to treatment and looked for an association with different patterns of B10 cell populations. The goal was to identify a possible marker for disease activity and responsiveness to immune therapy in MG.

MATERIALS AND METHODS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Human Subjects

A total of 48 consecutive patients seen at the clinics of Barrow Neurological Institute and Tianjin Medical University General Hospital who met diagnostic criteria were recruited for the study. Men and women over age 18 years with antibodies to either acetylcholine receptors (AChRs) or muscle specific receptor tyrosine (MuSK) were included. Twenty healthy subjects were recruited as controls and were matched with the MG patients for age, gender, and demography. All subjects signed informed written consent before they were enrolled in the study. The use of human subjects and the prospective follow-up with collection of blood samples for detailed immunologic assessments for this study were approved by the institutional review board (IRB) in both sites.

Rituximab Therapy and Outcome Assessment

Among the 48 eligible MG patients enrolled, 22 who were refractory to conventional therapies (Table 1) were offered rituximab therapy for up to 1.5 years. Refractory was defined as having an unsatisfactory response to 2 immunomodulatory agents, at least 1 of which was prednisone/prednisolone. Each patient received 4 infusions of IV rituximab at a dosage of 375 mg/m2, administered once per week.[13] Peripheral blood samples were obtained at baseline, 3–4, 8–9, and 15–16 months thereafter to evaluate lymphocyte subsets, including B10 cells. Reinfusion of rituximab with a single dose of 375 mg/m2 was initiated when circulating CD19+ B cells exceeded 1%. Because rituximab was provided as part of routine care on an off-label basis, IRB approval was not obtained. Nevertheless, patients were informed of potential adverse events, and risks and benefits were discussed in detail. All questions were answered before initiation of rituximab.

Table 1. Characteristics of MG patients who received rituximab.
Patient noGenderAge (year)Disease duration (years)Follow-up (month)All previous therapies
  1. AZA = azathioprine; CYC A = cyclosporine; IVIg = intravenous immunoglobulin; MG, myasthenia gravis; PE, phycoerythrin; Pred = prednisone/prednisolone; RTX = rituximab.

1W21312Pred, AZA, IVIg
2W38615Pred, IVIg, CYC A
3M60518Pred, AZA
4M33716Pred, IVIg
5W39425Pred, PE, AZA
6W621511Pred, IVIg, PE
7W45713Pred, AZA
8M371214Pred, IVIg, PE
9M42523Pred, PE, AZA
10W43519Pred, IVIg, thymectomy
11M40418Pred, thymectomy, AZA
12W29413Pred, CYC A
13M26318Pred, IVIg
14W54921Pred, IVIg, CYC A
15W59218Pred, IVIg, CYC A
16W47621Pred, IVIG, AZA
17W52719Pred, IVIg, PE, AZA
18W45617Prez, PE, AZA
19W56812Pred, IVIg, CYC A, PE
20M28515Pred, CYC A, IVIg
21W33318Pred, PE, IVIg, AZA, CYC A
22W42618Pred, AZA, IVIg, PE

Laboratory studies included complete blood count, hepatic and renal functions, tuberculin skin test, and electrocardiogram monitoring. Patients were followed up prospectively and periodically in the clinic at 1.5 years following initiation of rituximab therapy. The baseline and end point severity-adjusted analyses (responder analysis) and MGFA status were obtained and compared.

FACS Immunofluorescence Analysis of B cells

Peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation on a Ficoll-Hypaque density gradient. Single cell suspensions were washed, and cell numbers were counted by a hemocytometer. A total of 1 × 106 PBMCs were stained with the following anti-human primary mAbs (BD Pharmingen, San Diego, CA): CD19 (H1B19), CD5 (L17F12), CD1d (CD1d42), CD24 (ML5), CD27 (M-T271), and CD38 (HB7). They were conjugated with 1 of the following fluorescent tags: fluorescein isothiocyanate (FITC), phycoerythrin (PE), PeCy5, or allophycocyanin (APC). Multicolor flow cytometry was performed with a FACSAria™ flow cytometer (Becton Dickenson) on viable cells determined by forward-scatter and side-scatter signals. At least 30,000 events were acquired for each sample, and data were analyzed using Diva™ software.

Quantify IL-10 Producing B Cell (B10) Subpopulation

Intracellular IL-10 production was measured by FACS staining as described previously. Briefly, 2 × 106 PBMCs/ml were cultured in 24-well plates with medium in the presence of LPS (5 μg/ml; Escherichia coli 0127:B8, sigma), and/or CD40L (1 μg/ml, BD) for 2 days and then stimulated with 20 ng/ml PMA, 1 μg/ml ionomycin (Sigma-Aldrich), and 1μl/ml GolgiStop (BD Bioscience) for 5 h before staining. Cells were surface-stained with anti-CD19, anti-CD5, and/or CD1d conjugated with FITC, PE, or PeCy5 and fixed and permeabilized with Cytofix/Cytoperm kit (BD Bioscience). Then intracellular cytokines were stained with anti-IL-10 mAb conjugated with APC (JES3-19F1). Samples were read using a FACSAria™ flow cytometer, and data were analyzed using Diva™ software.

Statistical Analysis

Data are presented as means ± SEM, and analysis was performed with the aid of GraphPad Prism software (San Diego, California). Two-tailed unpaired Student t-test or ANOVA were used as appropriate for all other comparisons among groups (Student t-test: 2 groups; ANOVA: multiple groups). Significance was defined as P < 0.05 and expressed in the individual figures as *P < 0.05, **P < 0.01. P > 0.05: not statistically significant.

RESULTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

MG Patients Have Reduced Frequency and Functional Defects in B10 cells

Before rituximab treatment, we first quantified the total population of CD19+ B cells and found that the percentage and absolute numbers were similar between MG patients and healthy subjects (MG vs. control per 106 PBMC: 10.5 ± 0.6% vs. 11.1 ± 0.8%, P > 0.05; cell number, 2791 ± 1210 vs. 2201 ± 597, P > 0.05; Fig. 1A). However, compared with healthy subjects, we found that the percentage and cell number of CD19+ B cells that produce IL-10 were significantly lower in patients with MG (MG vs. control per 106 PBMC: 1.4 ± 0.1% vs. 2.6 ± 0.3%, P < 0.05; Fig. 1B,C. cell number: 46.6 ± 9.7 vs. 7.6 ± 2.7, P < 0.01; Fig. 1D). Therefore, IL-10-producing B cells were less frequent in MG.

image

Figure 1. Characterization of IL-10+ B cells in patients with MG. Peripheral blood mononuclear cells (PBMCs) were obtained from MG patients (n = 42) and healthy control subjects (n = 20). Cells were stained for CD19 and IL-10, and expression was determined by flow cytometry. Representative dot plots were gated on lymphocytes. Each symbol indicates an individual subject, and small horizontal lines indicate the mean. (A) Representative FACS dot plots of CD19+ cells, frequencies, and numbers of CD19+ cells. (B) Representative plots of CD19+IL-10+ cells in MG and controls. (C) The frequencies of CD19+IL-10+ cells from PBMCs of MG patients and controls. (D) The numbers of CD19+IL-10+ cells from PBMCs of MG patients and controls. P value; Student t-test. Error bars represent the means ± SEM.

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To further characterize the IL-10+ B cell subpopulation, we determined their surface expression of CD1d and CD5, the 2 markers that are believed to be co-expressed by “regulatory” B cells.[6] Here, we found that the frequencies of CD5+CD1d+ cells and CD19+CD5+CD1d+ cells were lower in MG patients than healthy controls (Fig. 2A,B). Upon determination of production of IL-10, we found that the majority of B cells that produced IL-10 both in healthy subjects and MG patients were the CD5+CD1d+ phenotypic subset, but not the CD5CD1d+ or the CD5+CD1d B cells (Fig. 2A, and data not shown). The percentage of CD5+CD1d+CD19+ B cells was lower in MG patients (MG vs. control per 106 PBMC: 1.5 ± 0.3% vs. 2.7 ± 0.4%; P < 0.05; Fig. 2B). A markedly decreased number of CD5+CD1d+CD19+ B cells was also seen in MG patients when compared with healthy controls (MG vs. control per 106 PBMC: 0.3 × 104 ± 0.1 × 104 vs. 1.4 × 104 ± 0.3 × 104; P < 0.05; Fig. 2C). There was a significant decrease of IL-10 production by CD5+CD1d+ CD19+ B cells in MG patients when compared with those of healthy controls (MG vs. control per 106 PBMC: 1.4 ± 0.2% vs. 2.3±0.3%, P < 0.05; Fig. 2D). Therefore IL-10-producing B cells were defective in MG.

image

Figure 2. Co-expression of CD5 and CD1d on B10 cells in patients with MG and healthy controls. PBMCs were obtained from patients with MG (n = 42) and healthy controls (n = 20). (A) Flow cytometry of PBMCs representative plots were first gated on CD5+CD1d+ lymphocytes and then gated on the sub-cell populations CD19+ cells; the number in plots correspond to the percentages of the indicated subset cell populations. (B) Scatter plot of frequencies of CD5+CD1d+CD19+ cells in PBMCs of MG patients and controls. Each symbol indicates an individual subject, and small horizontal lines indicate the mean. (C) The numbers of CD5+CD1d+CD19+ cells in PBMCs of MG patients and controls. (D) The percentages of CD19+IL-10+ cells gated on CD5+CD1d+ cells in PBMCs of MG patients and controls. P-value: Student t-test. Error bars represent the means ± SEM.

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It has been reported that regulatory B cells are also characterized by expression of 2 other cell surface antigens, CD24 and CD38.[8] We found that the percentage of a subset of B cells (CD24+CD38+ CD19+ cell population) was reduced marginally in MG patients when compared with healthy subjects (MG vs. Control per 106 PBMC: 2.1 ± 0.5% vs. 2.8 ± 0.7%; P > 0.05; Fig. 3B), and the absolute number of this subset was reduced significantly in the MG patients (MG vs. control /per 106 PBMC: 1113±76 vs. 1602 ± 451; P < 0.05; Fig. 3C). Furthermore, after cross-linking B cell receptor and stimulating with LPS, the CD24+CD38+CD19+ B cells are the predominant subset of B cells to produce IL-10 in both MG patients and healthy controls (between 30% and 68%); the cultured CD24+CD38+CD19+ B cells from MG patients produced less IL-10 than control compartments (MG vs. control per 106 PBMC: %, 40.7 ± 8.1 vs. 60.7 ± 9.9, P < 0.05; Fig. 3D).

image

Figure 3. Co-expression of CD24 and CD38 on B10 cells in patients with MG and healthy controls. PBMCs were obtained from patients with MG (n = 42) and healthy controls (n = 20). (A) Flow cytometry of PBMC representative plots were first gated on CD24+CD38+ lymphocytes and then gated on the sub-cell populations CD19+IL-10+ cells; the numbers in plots correspond to the percentages of the indicated subset cell populations. (B) The percentages of CD24+CD38+CD19+ cells in PBMCs of MG patients and controls. (C) The numbers of CD24+CD38+CD19+ cells in PBMCs of MG patients and controls. (D) The percentages of CD19+IL-10+ cells gated on CD24+CD38+ cells in PBMCs of MG patients and controls. P-value: Student t-test. Error bars represent the means ± SEM.

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Frequencies of B10 Cells Correlate with Clinical Stages of MG

The reduction of B10 cells in patients with MG suggested that this subpopulation of B cells might be involved in the pathogenesis of disease. We therefore determined the frequencies of these cells in MG patients with different stages of disease, as defined by MGFA disease status. We found that patients with severe disease tend to exhibit lower frequencies of IL-10+ B cells overall, and that frequencies of IL-10+ B cells in patients with stage I or II disease were higher than those with stage III and IV disease (P < 0.05; Fig. 4A). However, differences among I, IIa, IIb, and between III and IV were not evident (P > 0.05; Fig. 4A). In addition, although there was no significant difference, a trend of higher frequencies of B10 cells was seen in patients with anti-AChR antibodies than those with anti-MuSK antibodies (P > 0.05; Fig. 4B).

image

Figure 4. Relationship between B10 cells and clinical status of MG patients stratified by MGFA clinical classification or serological serum antibodies. (A) Frequencies of CD19+IL-10+ cells in MG patients with MGFA classification I to IV (n = 48). (B) Frequencies of CD19+IL-10+ cells in MG patients with positive AChR antibodies (n = 20) or positive MuSK antibodies (n = 10). Each symbol indicates an individual subject, and small horizontal lines indicate the mean. P value: Student t-test.

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Differential Responsiveness to Rituximab Therapy in MG

In our cohort, muscle weakness and other related symptoms in 22 patients were not well controlled and were considered “refractory” to conventional therapies (Table 1). These patients were offered repeated doses of rituximab (375 mg/m2) during a 1.5-year follow-up period. Moderate or dramatic improvement was observed in 16 patients within the first 14–56 h of rituximab infusion. This was reflected by disappearance of ptosis, ophthalmoparesis, or respiratory weakness in some patients, and overall improvement in MGFA disease severity status with complete remission or minimal manifestations (Table 2). During the course of rituximab therapy, 9 patients were able to completely cease steroid therapy and other medications, while the remaining patients were able to reduce the dosages and frequencies of medications (Table 2).

Table 2. Differential responsiveness to rituximab therapy in MG patients.
Patient no.Auto-antibodyMaximum disease severity (MGFA)Postintervention status (MGFA)ReinfusionOther therapy changes post RTX
  1. AChR = acetylcholine receptor; AZA, azathioprine; CR/MM = complete remission or minimal manifestations; CYC A = cyclosporine; MGFA = Myasthenia Gravis Association of America; MuSK = muscle specific tyrosine kinase; Pred, prednisone/prednisolone; RTX, rituximab.

1AChRIIIaCR/MM1None
2AChRIIIaCR/MM1Pred 20 mg
3MuSKIIIaCR/MM0None
4AChRIIIaCR/MM1None
5AChRIIIaCR/MM0None
6AChRIIIaCR/MM2Pred 20 mg
7MuSKIIIbCR/MM1Pred 20 mg
8AChRIIIbCR/MM2Pred 20 mg
9MuSKIIIbCR/MM2None
10AChRIVaCR/MM1Pred 20 mg
11AChRIVbCR/MM1AZA 50 mg/d
12MuSKIVbCR/MM0None
13AChRIVbCR/MM0None
14MuSKIVbCR/MM0None
15MuSKIVbCR/MM0None
16AChRIVaCR/MM1None
17AChRIIIbUnchanged3Pred 20 mg, AZA 125 mg/d
18MuSKIVaUnchanged3Pred 20 mg, AZA 125 mg/d
19AChRIVbUnchanged3Pred 20 mg
20AChRIVbUnchanged3CYC A 200 mg/d
21AChRVUnchanged4Pred 20 mg, AZA 50 mg
22AChRIVbWorse4AZA 125 mg/d

In contrast, 6 of 22 patients did not respond to rituximab treatment as indicated by the above measurements, and 1 experienced worsened symptoms (Table 2). Rituximab was discontinued within the 1.5-year period in some patients. Instead, corticosteroid or other modalities, including azathioprine, plasma exchange, and cyclosporine A, were increased in dosages and/or frequencies to control the disease (Table 2). In a separate analysis, a majority (6 of 7, 86%) of MG patients with anti-MuSK antibodies exhibited a better response to rituximab therapy than MG patients with anti-AChR antibodies (10 of 15, 67%).

Responsiveness to Rituximab Therapy Is Associated with Repopulation of B10 Cells in MG

We investigated whether responsiveness to rituximab in MG could be linked to kinetics of B cell repopulation after cell depletion. For this purpose, we quantified IL-10 nonproducing B cells and B10 cells at various time points during the therapy. For rituximab responders and nonresponders, we did not find a significant difference for recovery of IL-10 nonproducing B cells (effector B cell) between the 2 groups for any time point (Fig. 5A). In contrast, although there were no significant differences for B10 cells at baseline, we found that repopulation of B10 cells was delayed in rituximab nonresponders (for months 8–9, P < 0.05, Fig. 5B), and such delay was more apparent at the late follow up period (for months 15, 16, P < 0.01; Fig. 5B). Recovery of the B10 cell population appeared to be faster in patients with anti-MuSK antibody than those with anti-AChR antibody, without reaching statistical significance (P values are 0.077 or 0.072 for the third [months 8–9] and fourth [months 15–16] time points, respectively). It is interesting to note that this group exhibited a better response to rituximab than patients with anti-AChR antibody positivity (Table 2). Collectively, our results suggest that responsiveness to rituximab therapy is not associated with repopulation of effector B cells, but with B10 cells specifically.

image

Figure 5. Repopulation of effector and B10 cells in MG patients that received rituximab. PBMCs were obtained from 22 MG patients (Patient-1 to Patient-22) receiving Rituximab therapy at 4 time points: baseline (before treatment), after treatment 3–4, 8–9, or 15–16 months. (A) Percentages of CD19+IL-10 cells. (B) Percentages of CD19+IL-10+ cells. Each symbol indicates an individual subject. P value: Student t-test.

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DISCUSSION

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

Immune dysregulation and failure of regulatory cells are believed to be among the causes of emergence of autoimmunity.[1] Indeed, deficiency of regulatory T cells has been reported in many autoimmune diseases such as type 1 diabetes, multiple sclerosis, and MG.[1, 21] We found that a subset of regulatory B (B10) cells are highly relevant to the pathogenesis of MG. Compared with healthy subjects, the frequencies of B10 cells, as well as expression of several surrogate markers, i.e., CD1d, CD5, CD24, CD38, are reduced significantly. Such reduction could be secondary to the pathogenesis of MG or could be a primary reason leading to activation of auto-reactive T and B cells. To differentiate these possibilities and use a more functional characterization of B10 cells in MG, including determining their capacity in in vitro expansion and suppression of effector T, B, and antigen presenting cells, their antigen specificity and presence or absence in thymus abnormality tests should be carried out. In turn, the outcomes of these assessments can shed light on the pathogenesis of B10 deficiency in MG patients.

Our finding that frequencies of B10 cells were decreased in MG patients mirrors several previous studies in patients with systemic lupus erythematosus and Graves disease.[8-10] In contrast, a recent report suggests that B10 cells are present in most autoimmune disease patients, and their frequencies are increased.[5] Stages of each autoimmune condition as well as the timing of immune therapies and B cell analysis may contribute to the diverse phenotypes of B10 cells in autoimmune diseases, and this clearly warrants further investigation.

No matter what mechanisms lead to B10 cell deficiency in MG, this small cell population appears useful for clinical practice. Our results show that B10 frequencies are lower in moderate to severe MG compared with mild MG patients (Fig. 4). Furthermore, patients with anti-MuSK antibody also exhibited a tendency for lower B10 cell frequency. Our results may suggest a role of B10 cells as a surrogate marker for disease phenotype and severity. Its usefulness will be dependent upon whether deficiency of B10 cells occurs before progression of disease and how early such phenotypes can be recognized. Thus, these findings can guide how aggressive the therapy should be to halt the progression of disease.

The utility of B10 cells in MG is perhaps more evident for monitoring rituximab therapy in MG. In examining the kinetics of effector B cell and B10 cell recovery, we found that the majority of patients who responded well have rapid repopulation of B10 cells, whereas none of the patients who do not respond well to rituximab demonstrated a timely recovery of B10 cells. We postulate that the delay in B10 cell recovery could unleash the propagation of B effector cells that abrogate the effect of rituximab.

It has been reported that B cell depletion with rituximab treatment has long-lasting effects on MuSK MG.[22] The outcome in our cohort is consistent with this finding. Interestingly, MuSK MG patients also had a tendency for faster B10 cell repopulation during the course of rituximab therapy. However, the small number of MuSK MG patients enrolled in this study precludes statistical analysis.

Nonresponders to rituximab therapy have been identified in autoimmune diseases such as neuromyelitis optica,[23] inflammatory bowel disease,[24] and systemic lupus erythematosus.[25] Compared with previous studies on other autoimmune diseases, it is noteworthy that the rate of nonresponsiveness is similar to that seen in our cohort (∼15%). It is also remarkable that all these conditions are B cell-mediated. These findings certainly warrant further investigation into whether nonresponsiveness is associated with a delay in B10 cell repopulation after B cell ablation therapy.

ACKNOWLEDGMENTS

  1. Top of page
  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES

We thank our patients for participating in this study, and Dr. R. Liu and Ms. A. Kousari for laboratory and editorial assistance.

REFERENCES

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  2. ABSTRACT
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. ACKNOWLEDGMENTS
  7. REFERENCES
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