Down-regulation of CD40 and CD80 on B cells in patients with life-threatening systemic lupus erythematosus after successful treatment with rituximab

M. Tokunaga, K. Fujii, K. Saito, S. Nakayamada, S. Tsujimura, M. Nawata and Y. Tanaka

First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Japan, 1-1 Iseigaoka, Kitakyushu 807-8555, Japan.

Correspondence to: Y. Tanaka, First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Kitakyushu 807-8555, Japan. E-mail: tanaka{at}med.uoeh-u.ac.jp


    Abstract
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Objectives. Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by autoreactive T cells and polyclonally activated B cells that produce autoantibodies. Five SLE patients who failed to respond to conventional immunosuppressants were treated with anti-CD20 antibody (rituximab) and their clinical manifestations and laboratory data were evaluated, including phenotypic analysis of B cells.

Methods. Rituximab (375 mg/m2) was administered weekly for 2 weeks in five SLE patients who developed severe manifestations despite intensive treatment.

Results. Rituximab resulted in rapid improvement (within several days) in clinical manifestations such as consciousness disorder, seizures, progressive sensory disorder, haemolytic crisis, cardiac function and laboratory data. The effects lasted 20 months in one patient; other patients were in remission for more than 6 months. Flow cytometric analysis revealed down-regulation of CD40 and CD80 expression on CD19-positive B cells 1 week after infusion of rituximab, and such down-regulation was seen for more than 7 months in two patients.

Conclusions. Our pilot study provides sufficient evidence of excellent tolerability and high efficacy of rituximab therapy in refractory SLE. Rituximab not only reduced B-cell number and IgG levels but down-regulated CD40 and CD80 on B cells, suggesting possible disturbance of T-cell activation through these costimulatory molecules. Reduction of both quantity and quality of B cells suggests that rituximab could improve the disease course in patients with refractory SLE.

KEY WORDS: Rituximab, Anti-CD20 antibody, Systemic lupus erythematosus, Central nervous system lupus, Autoimmune haemolytic anaemia, CD40, CD80


    Introduction
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Systemic lupus erythematosus (SLE) is an autoimmune disease thought to involve disturbances in T- and B-cell functions. Immune complexes consisting of antigens and autoantibodies secreted from activated B cells cause severe inflammation on various tissues and organs. To control this inflammation, immunosuppressants such as corticosteroids, cyclosporin A (CsA) and cyclophosphamide (CY) are widely used. However, we have experience of patients with SLE who are refractory to these conventional treatments, and innovative approaches need to be developed.

CD20 is a surface molecule specific for B cells, and is expressed in most stages of B cells. Rituximab (Rituxan®; Genentech, South San Francisco, CA, USA) is a chimeric monoclonal antibody specific for human CD20, consisting of human immunoglobulin (Ig) G1{kappa} constant regions and mouse variable regions. Rituximab is known to deplete B cells by complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity [1]. This antibody has already been used and has demonstrated high effectiveness in the treatment of B-cell lymphomas. Recently, the potential efficacy of B-cell depletion therapy with rituximab has been reported in several autoimmune diseases, such as idiopathic thrombocytopenic purpura, autoimmune haemolytic anaemia (AIHA), cold-agglutinin disease, Wegener's granulomatosis, myasthenia gravis and idiopathic membranous nephropathy [2–10]. We also reported that rituximab induced rapid recovery of life-threatening refractory SLE with renal and CNS involvement [11]. However, the underlying mechanism of the efficacy of rituximab for the long-term remission remains unknown.

The CD40/CD40L and CD28/CD80–CD86 pathways on lymphocytes are known to be up-regulated in active SLE patients and their relevance to the pathogenesis of SLE has been thoroughly investigated [12–22]. In this study, we administrated rituximab to five patients with refractory SLE and investigated the association between clinical improvement and phenotypic alteration of B cells. We found long-term effects of rituximab on down-regulation of CD40 and CD80 on B lymphocytes, suggesting that rituximab maintains long-term remission of SLE by correcting B-cell aberration. This is the first report that proposes an alternative mechanism of action of rituximab on autoimmune disease with regard to the regulation of the surface molecules on lymphocytes.


    Patients and methods
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Patients
The study subjects were five patients with SLE who fulfilled the diagnostic criteria of the American College of Rheumatology. They represented all patients admitted to our department between 2000–2003 who had high disease activity and who failed to respond to conventional immunosuppressants, including steroid pulse therapy, intravenous CY pulse therapy (IV-CY), CsA, plasma exchange therapy (PE) and immunoadsorption (IA), over an average period of 40 months (range 3–135 months). The clinical characteristics and previous therapies of five patients are summarized in Table 1.


View this table:
[in this window]
[in a new window]
 
TABLE 1. Characteristics of five patients with SLE at study entry

 
Treatment protocol
Patients were treated with weekly infusions of rituximab (375 mg/m2 of body surface area, as in treatment of B-cell lymphoma) for 2 weeks. Blood pressure measurement and electrocardiography were performed regularly during rituximab infusion (3.5 h) as measures of infusion reaction. The possible actions and adverse effects of rituximab were explained in detail and informed consent was obtained from all patients (and the parents of patient 1) who enrolled in the study, according to the Declaration of Helsinki. The design of the work was approved by the ethics committee of our university.

Assessment
Clinical and laboratory assessments were performed before treatment and weekly for 1 month after the initial infusion. Patients were evaluated for clinical manifestations of SLE and any adverse effects of therapy. Laboratory measurements included full blood count, erythrocyte sedimentation rate (ESR), renal and liver serum function tests, urinary protein, serum complement, serum Ig levels, anti-double-stranded (ds) DNA levels, and CD markers on lymphocytes using flow cytometry. The SLE Disease Activity Index (SLEDAI) [23] was used for individual organ system assessment. Treatment efficacy was evaluated on the basis of improvement in both clinical and laboratory indices of active disease.

Flow cytometry
Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Lymphocyte Separation Medium (LSM; ICN/Cappel Pharmaceuticals, Aurora, OH, USA), and then washed twice with phosphate-buffered saline (PBS). PBMCs (2 x 105) were resuspended in blocking buffer [0.25% human globulin/0.5% human albumin (Yoshitomi, Osaka, Japan)/0.1% NaN3 (Sigma Aldrich) in PBS] in 96-well plates for 15 min at 4°C. Fluorescein isothiocyanate-conjugated monoclonal antibodies (mAb) against human CD40, CD86 (PharMingen, San Diego, CA, USA) and CD80 (Chemicon Europe, Chandlers Ford, UK), CyChrome-conjugated mAb against human CD19 (PharMingen) was added to PBMC in 100 µl FACS medium (0.5% human album/0.1% NaN3 in PBS) for 30 min at 4°C. Cells were washed three times in FACS medium and analysed with FACScan and CellQuest software (Becton-Dickinson, San Jose, CA, USA). Quantification of the cell surface antigens on one cell was performed using Qifkit (Dako Japan, Kyoto, Japan). Treatment efficacy was tested statistically by Student's t-test. A P value less than 0.05 denoted a statistically significant difference.


    Results
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
Clinical outcome
The clinical condition significantly improved in each patient after the administration of rituximab (Table 2). Among five patients, cases 1 and 2 showed marked decreases of SLEDAI on day 28 (Fig. 1). SLEDAI decreased from a median of 24.4 (range, 2–49) at baseline to a median of 10.2 (range 0–16) after 28 days.


View this table:
[in this window]
[in a new window]
 
TABLE 2. Clinical outcomes after anti-CD20 antibody treatments

 


View larger version (18K):
[in this window]
[in a new window]
 
FIG. 1. SLEDAI scores of five patients at baseline and at day 28 after the initial treatment with rituximab.

 
Case 1 was diagnosed with SLE in 1991. She had had many episodes of refractory lupus nephritis since 1995. The disease relapsed with leucocytopenia, increased ESR and anti-dsDNA level, a low level of complement, and nephrotic syndrome (urinary protein >8 g/day) in May 2002. Steroid pulse therapy, IV-CY and IA therapy with high-dose oral prednisolone failed to improve the clinical symptoms and signs. In addition, consciousness disorder appeared in June and progressed rapidly. Despite PE six times, her consciousness level worsened to a Glasgow Coma Scale (GCS) score of 7–11. After the first administration of rituximab, she became alert (GCS score of 15) on day 5. Proteinuria improved within 7 months and serum albumin level normalized 10 months later (Fig. 2).



View larger version (27K):
[in this window]
[in a new window]
 
FIG. 2. Case 1. Clinical course and response to treatment. The patient was treated with immunosuppressive therapies, including PE (arrows), IA (open arrows), IV-CY (open triangles), m-PSL pulse therapy (closed triangles), betamethasone (open squares), CsA (striped squares), azathioprine (AZA; dotted squares) and methotrexate (MTX; closed squares). Rituximab was administrated at 375 mg/m2 once weekly (indicated by dotted arrows).

 
Case 2 was diagnosed with SLE with renal dysfunction, pancytopenia, positive anti-dsDNA and consciousness disorder (GCS score of 3) in April 2003. Consciousness disorder and renal dysfunction progressed and abnormal data persisted despite various therapies, which included oral steroid, IV-CY, IA and PE. Finally, she received rituximab in July 2003; her consciousness improved (GCS score of 14) on day 2 and convulsion and proteinuria disappeared completely. She showed marked and rapid responses to rituximab treatment.

Case 3 was treated for SLE with oral steroid since 1978. She developed AIHA after surgery for carcinoma of the uterine cervix in 2001. IV-CY seven times followed by oral steroid improved AIHA, but she could not continue the treatment because of fungal infection. AIHA deteriorated to a haemoglobin (Hb) level of about 7–8 g/dl and a reticulocyte count of about 200% with 2 mg of betamethasone. This steroid dosage could not control haemolytic attacks, and Hb dropped to about 5 g/dl and thrombosis was observed due to red blood cell (RBC) coagulation. Accordingly, all treatment was stopped and she was started on rituximab in April 2003. RBC coagulation disappeared 3 months later and her Hb level recovered up to 10–11 g/dl without blood transfusion.

In case 4, SLE had started with malar rush and arthritis in 2002. In September 2003 the clinical condition worsened with sensory deficit on the area extending from the left axilla to the left precardia, severe headache (IgG index 0.92), myocardial dysfunction due to SLE (ejection fraction, 44% on ultrasonic cardiography), leucocytopenia, thrombocytopenia, increased ESR and anti-dsDNA, and low-level complement. Since these manifestations worsened rapidly despite the combination therapy with high doses of oral steroids and CsA for a month, rituximab was administered in November 2003. This treatment gradually reduced the sensory deficit and headache, and ejection fraction in ultrasonic cardiography increased to 72.1%.

Case 5 developed arthritis, photosensitivity, dryness of the mouth, positive anti-dsDNA and lymphocytopenia, and was diagnosed with SLE and Sjögren's syndrome in 2000. Sensory disorder on the left half of the body and deficit of the visual field appeared in 2001, and paraesthesia on the lower part of the chest appeared in 2003. T2-weighted magnetic resonance imaging (MRI) showed a high-intensity lesion in lower segment of the medulla oblongata and cervical spinal cord and the white matter of the cortex. Steroid, mizoribine for 2 yr and IV-CY eight times were not effective for these manifestations, and a new high-intensity lesion was detected on MRI in 2004. After administration of rituximab, these disorders diminished and the abnormal lesion on MRI disappeared, along with the resolution of skin and mucous lesions.

The above five patients were in remission more than 6 months after the commencement of rituximab therapy. Their oral daily steroid doses have been decreased and immunosuppressants have been discontinued in all patients because of remission of the clinical state.

Adverse effects
No infusion-related serious adverse effects of rituximab were observed. In case 1, the serum IgG level decreased significantly to about 200–300 mg/dl 1 month after treatment, and she was treated with infusion of Ig when her serum IgG level was <300 mg/dl. During this period, she had infection with herpes zoster localized at temporal, which appeared on day 25, but it improved smoothly with Ig therapy and acyclovir. Other patients had no severe infections.

Phenotypic analysis of SLE B cells
Costimulatory molecules such as CD40–CD40L and CD28–CD80/CD86 are known to be prerequisites for the activation of autoreactive T cells, and increases in these molecules are reported in active SLE lymphocytes [12–22]. To examine the effects of rituximab on refractory SLE, we assessed the changes in costimulatory molecules on B cells in our patients before treatment and weekly for 1 month after the initial infusion by using flow cytometry. Rituximab therapy markedly reduced CD40 on B cells. In four cases (cases 2, 3, 4 and 5), the mean expression level of CD40 on B cells was 1668 ± 657 (S.D.) molecules/cell before treatment (day 0). CD19+ cells significantly decreased within the first week after the administration of rituximab (data not shown). CD40 expression was also down-regulated within 1 week in all cases, and remained low for the first month (Fig. 3); on day 28 it was significantly lower than that on day 0 (664 ± 336 molecules/cell). Furthermore, CD80 was also down-regulated with time after treatment and had decreased significantly by day 28 (Fig. 4) (7834 ± 1775 vs 1396 ± 637 molecules/cell).



View larger version (19K):
[in this window]
[in a new window]
 
FIG. 3. Serial changes in expression level of CD40 on CD19+ cell in four patients (cases 2, 3, 4 and 5) analysed by FACScan. Data are the amounts of cell surface antigen measured on single CD19+ cells, calculated using Qifkit.

 


View larger version (21K):
[in this window]
[in a new window]
 
FIG. 4. Serial changes in expression level of CD80 on CD19+ cells in three patients (cases 3, 4 and 5) analysed by FACScan. Data are the amounts of cell surface antigen measured on CD19+ cells, calculated using Qifkit. Histograms of CD80 on CD19+ cells at day 0 and at the 7th month after the administration in case 4 are shown.

 
The expression levels of CD40 and CD80 were followed for 7 months in case 3 and case 4. The down-regulation of CD40 and CD80 among CD19+ cells lasted for 7 months after rituximab therapy in these two patients and a marked change in the histogram of CD80 expression on CD19+ cells was observed in case 4 (Fig. 4 and data not shown). Case 3 was still in clinical remission 1 yr after rituximab therapy, although the B-cell count increased to 6.6%. The percentages of CD40+ and CD80+ cells among her CD19+ cells were 94.6 and 97.2% respectively before treatment, but had decreased to 55.5 and 70.8% respectively 1 yr after treatment (data not shown). These results suggest that rituximab normalizes the expression of surface molecules or autoreactivity of B cells, and may have long-term stabilizing effects on the immune system in SLE.


    Discussion
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 
In the present study, rituximab was administrated to five patients with SLE who had showed progressive disease and had failed to respond to conventional immunosuppressive therapy. At the start of rituximab treatment, patients received low to moderate dose of corticosteroid, corresponding to 15–40 mg of prednisolone. Rituximab treatment resulted in improvement of their clinical condition and had no significant adverse effects. Though the five patients had various manifestations, such as CNS lupus (cases 1–5), peripheral nervous system disorder (cases 4 and 5), lupus nephritis (cases 1 and 4), haemolytic crisis (case 3) and cardiomyopathy (case 4), the clinical symptoms and signs and laboratory data rapidly improved in all five patients, as evidenced by reduction in the SLEDAI score. In particular, case 1 became fully alert on day 5, and a similar effect was noted in case 2 on day 2, indicating that the rapid effect of rituximab (a few days) on life-threatening CNS disorder.

The clinical manifestations appear to improve rapidly in parallel with the deletion of B cells from peripheral blood after administration of rituximab. However, autoantibodies will remain in the serum at least for a few weeks and could be produced from CD20 plasma cells, since rituximab does not work on these cells [24]. Furthermore, rituximab was effective in three patients who did not improve with PE and IA, which are thought to eliminate immune complexes and/or cytokines. These results suggest that some of the clinical manifestations, including the CNS disorder observed in these SLE patients, may be largely elicited by cell-mediated immunity rather than autoantibodies.

Rituximab was originally designed to deplete B cells. In our five SLE patients, the quantity of B cells diminished rapidly after treatment. Interestingly, since the B-cell count returned to normal levels, all patients did not relapse and they continue to be in remission. For instance, case 1 received rituximab in July 2002 and the CD19-positive B-cell count was 2.5% of PBMC before treatment and decreased to 0.4% 1 week after the first administration of rituximab. In August 2003, she remained in remission despite the recovery of CD19- and CD20-positive cells to 5.1 and 5.8% respectively. The anti-dsDNA titre, complement, ESR, and white blood cells were still within the normal range and proteinuria was not detected at that stage. These cases suggest that the clinical effects of rituximab can be explained by mechanisms other than its effect on B-cell count.

Leandro et al. [25] reported that treatment with rituximab caused repopulation of naive B cells in RA and SLE. Our results indicate that rituximab not only reduces the number of B-cell and IgG levels but down-regulates CD40 and CD80 on B cells, indicating that mutual T-cell activation through these costimulatory molecules may be disturbed. It has been reported that the CD40–CD40L and CD28–CD80 pathways are necessary for the activation of autoreactive T cells and for polyclonal B-cell activation. In fact, CD40L–CD40 interaction correlated with the anti-dsDNA level [14, 15] and SLEDAI scores [22] and the percentage of CD80+ cells in the activated B-cell subset in SLE was significantly higher than in controls [19].

Thus, the facts that clinical manifestations improved rapidly in all five patients, remission lasted for 20 months in one patient, and changes in both the quantity and the quality of B cells were observed, suggests that rituximab treatment could improve the disease course in patients with SLE. The long-term effects of rituximab on disease activity may be due to ‘resetting’ of the whole autoimmune system through the down-regulation of B-cell activity and the repopulation of inactive B cell clones. This pilot study, therefore, provides sufficient evidence of the excellent tolerability and high efficacy of anti-CD20 rituximab therapy in refractory SLE that is resistant to conventional treatments, and a formal clinical trial is justified.


    Acknowledgments
 
This work was supported in part by a Research Grant-In-Aid for Scientific Research from the Ministry of Health, Labor and Welfare of Japan, the Ministry of Education, Culture, Sports, Science and Technology of Japan and the University of Occupational and Environmental Health, Japan.

The authors have declared no conflicts of interest.


    References
 Top
 Abstract
 Introduction
 Patients and methods
 Results
 Discussion
 References
 

  1. Reff ME, Carner K, Chambers KS et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood 1994;15;83:435–45.[Abstract/Free Full Text]
  2. Stasi R, Pagano A, Stipa E, Amadori S. Rituximab chimeric anti-CD20 monoclonal antibody treatment for adults with chronic idiopathic thrombocytopenic purpura. Blood 2001;98:952–7.[Abstract/Free Full Text]
  3. Stasi R, Stipa E, Forte V, Meo P, Amadori S. Variable patterns of response to rituximab treatment in adults with chronic idiopathic thrombocytopenic purpura. Blood 2002;99:3872–3.[Free Full Text]
  4. Ouartier P, Brethon B, Philippet P, Landman-Parker J, Deist FL, Fischer A. Treatment of childhood autoimmune haemolytic anemia with rituximab. Lancet 2001;358:1511–3.[CrossRef][ISI][Medline]
  5. Hongeng S, Tardtong P, Worapongpaiboon S, Ungkanont A, Jootar S. Successful treatment of refractory autoimmune haemolytic anaemia in a post-unrelated bone marrow transplant paediatric patient with rituximab. Bone Marrow Transplant 2002;29:871–2.[CrossRef][ISI][Medline]
  6. Perrotta S, Locatelli F, La Manna A, Cennamo L, De Stefano P, Nobili B. Anti-CD20 monoclonal antibody (rituximab) for life-threatening autoimmune haemolytic anaemia in a patient with systemic lupus erythematosus. Br J Haematol 2002;116:465–7.[CrossRef][ISI][Medline]
  7. Berentsen S, Tjonnfjord GE, Brudevold R et al. Favourable response to therapy with the anti-CD20 monoclonal antibody rituximab in primary chronic cold agglutinin disease. Br J Haematol 2001;115:79–83.[CrossRef][ISI][Medline]
  8. Specks U, Fervenza FC, McDonald TJ, Hogan MC. Response of Wegener's granulomatosis to anti-CD20 chimeric monoclonal antibody therapy. Arthritis Rheum 2001;44:2836–40.[CrossRef][ISI][Medline]
  9. Remuzzi G, Chiurchiu C, Abbate M, Brusegan V, Bontempelli M, Ruggenenti P. Rituximab for idiopathic membranous nephropathy. Lancet 2002;360:923–4.[CrossRef][ISI][Medline]
  10. Leandro MJ, Edwards JC, Cambridge G, Ehrenstein MR, Isenberg DA. An open study of B lymphocyte depletion in systemic lupus erythematosus. Arthritis Rheum 2002;46:2673–7.[CrossRef][ISI][Medline]
  11. Saito K, Nawata M, Nakayamada S, Tokunaga M, Tsukada J, Tanaka Y. Successful treatment with anti-CD20 monoclonal antibody (rituximab) of life-threatening refractory systemic lupus erythematosus with renal and central nervous system involvement. Lupus 2003;12:798–800.[CrossRef][ISI][Medline]
  12. Datta SK, Kalled SL. CD40-CD40 ligand interaction in autoimmune disease. Arthritis Rheum 1997;40:1735–45.[ISI][Medline]
  13. Koshy M, Berger D, Crow MK. Increased expression of CD40 ligand on systemic lupus erythematosus lymphocytes. J Clin Invest 1996;98:826–37.[Abstract/Free Full Text]
  14. Desai-Mehta A, Lu L, Ramsey-Goldman R, Datta SK. Hyperexpression of CD40 ligand by B and T cells in human lupus and its role in pathogenic autoantibody production. J Clin Invest 1996;97:2063–73.[Abstract/Free Full Text]
  15. Kaneko Y, Hirose S, Abe M, Yagita H, Okumura K, Shirai T. CD40-mediated stimulation of B1 and B2 cells: implication in autoantibody production in murine lupus. Eur J Immunol 1996;26:3061–5.[ISI][Medline]
  16. Yellin MJ, Thienel U. T cells in the pathogenesis of systemic lupus erythematosus: potential roles of CD154-CD40 interactions and costimulatory molecules. Curr Rheumatol Rep 2000;2:24–31.[Medline]
  17. Grammer AC, Lipsky PE. CD154-CD40 interactions mediate differentiation to plasma cells in healthy individuals and persons with systemic lupus erythematosus. Arthritis Rheum 2002;46:1417–29.[CrossRef][ISI][Medline]
  18. Kovacs B, Thomas DE, Tsokos GC. Elevated in vivo expression of the costimulatory molecule B7-BB1 (CD80) on antigen presenting cells from a patient with SLE. Clin Exp Rheumatol 1996;14:695–7.[ISI][Medline]
  19. Folzenlogen D, Hofer MF, Leung DY, Freed JH, Newell MK. Analysis of CD80 and CD86 expression on peripheral blood B lymphocytes reveals increased expression of CD86 in lupus patients. Clin Immunol Immunopathol 1997;83:199–204.[CrossRef][ISI][Medline]
  20. Bijl M, Horst G, Limburg PC, Kallenberg CG. Expression of costimulatory molecules on peripheral blood lymphocytes of patients with systemic lupus erythematosus. Ann Rheum Dis 2001;60:523–6.[Abstract/Free Full Text]
  21. Nagafuchi H, Shimoyama Y, Kashiwakura J, Takeno M, Sakane T, Suzuki N. Preferential expression of B7.2 (CD86), but not B7.1 (CD80), on B cells induced by CD40/CD40L interaction is essential for anti-DNA autoantibody production in patients with systemic lupus erythematosus. Clin Exp Rheumatol 2003;21:71–7.[ISI][Medline]
  22. Grammer AC, Shinohara S, Vazquez E, Gur H, Illei G, Lipsky PE. Normalization of peripheral B cells following treatment of active SLE patients with humanized anti-CD154 (5c8, BG9588). Arthritis Rheum 2001;44(Suppl. 9):S282.
  23. Bombardier C, Gladman DD, Urowitz MB, Caron D, Chang CH. Derivation of the SLEDAI. A disease activity index for lupus patients. The Committee on Prognosis Studies in SLE. Arthritis Rheum 1992;35:630–40.[ISI][Medline]
  24. Arpin C, Dechanet J, Van Kooten C et al. Generation of memory B cells and plasma cells in vitro. Science 1995;268:720–2.[ISI][Medline]
  25. Leandro MJ, Jo C, Edwards W. B-cell repopulation occurs mainly from naïve B cells in patients with rheumatoid arthritis and systemic lupus erythematosus treated with rituximab. Arthritis Rheum 2003;48:S1160.[CrossRef]
Submitted 27 June 2004; revised version accepted 17 September 2004.