menu

Phenotypic Changes of Lymphocytes in Patients with Systemic Lupus Erythematosus Who Are in Longterm Remission After B Cell Depletion Therapy with Rituximab

SHIGERU IWATA, KAZUYOSHI SAITO, MIKIKO TOKUNAGA, KUNIHIRO YAMAOKA, MASAO NAWATA, SONOSUKE YUKAWA, KENTARO HANAMI, SHUNSUKE FUKUYO, IPPEI MIYAGAWA, SATOSHI KUBO, and YOSHIYA TANAKA

Background:

ABSTRACT. Objective. Rituximab has recently emerged as a novel treatment strategy for systemic lupus erythematosus (SLE). We investigated longitudinally the differentiation and phenotypic changes of peripheral B cells and T cells in patients with SLE after rituximab treatment. Methods. Phenotypic changes on B cells and T cells in 10 patients with SLE treated with rituximab were analyzed before, 28 days after, and 2 years after rituximab treatment, and at relapse. Results. Rituximab rapidly depleted naive and memory B cells from the peripheral blood. In the patients with prolonged remission, the memory B cells remained depleted while naive B cells recovered within 3.9 months, and the expression levels of CD40 and CD80 remained downregulated for 2 years. There was also a decrease of memory T cells relative to naive T cells, and the expression of CD40L and inducible costimulator (ICOS) on CD4-positive T cells rapidly decreased and remained downregulated for 2 years. In 1 patient, an increase in the number of memory B cells with upregulation of CD40 and CD80 expression was noted just before relapse. In another patient with relapse, however, recovery of CD4-positive memory T cells with upregulation of ICOS expression was noted, with no change in the number of memory B cells. Conclusion. Our results suggest that the phenotypic changes of peripheral B cells result in inhibition of T cell differentiation and activation mediated by B cells and thereby bring about longterm remission of SLE. Activated memory B cells or ICOS-positive CD4-positive memory T cells reappeared in association with relapse, probably reflecting the heterogeneity of SLE. (First Release Dec 15 2010; J Rheumatol 2011;38:633.41; doi:10.3899/jrheum.100729) Key Indexing Terms: SYSTEMIC LUPUS ERYTHEMATOSUS RITUXIMAB B CELL DEPLETION THERAPY Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease induced by activation of autoreactive T cells and overproduction of autoantibodies by B cells. From the First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, Kitakyushu, Japan. 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. Dr. Tanaka has received consultant fees from Mitsubishi-Tanabe Pharma and Pfizer Inc. and lecture fees from Mitsubishi-Tanabe Pharma, Takeda Pharmaceutical Co. Ltd., Abbott, Eisai Pharma, and Chugai Pharma. S. Iwata, MD; K. Saito, MD, PhD; M. Tokunaga, MD, PhD; K. Yamaoka, MD, PhD; M. Nawata, MD; S. Yukawa, MD; K. Hanami, MD; S. Fukuyo, MD; I. Miyagawa, MD; S. Kubo, MD; Y. Tanaka, MD, PhD, First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health. Address correspondence to Prof. Y. Tanaka, The First Department of Internal Medicine, School of Medicine, University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahata-nishi, Kitakyushu 807-8555, Japan. E-mail: tanaka@med.uoeh-u.ac.jp Accepted for publication October 12, 2010. Rituximab is a human-mouse chimeric monoclonal antibody that targets the CD20 antigen, a B cell-specific antigen, and causes depletion of CD20-expressing pre-B to mature B cells. Rituximab has recently been reported to show a rapid onset of effect and prolonged efficacy in patients with refractory SLE1,2,3,4,5,6,7,8, emerging as a promising new agent for the treatment of this disease. In regard to the mechanism underlying the longterm remission of SLE induced by rituximab, Anolik, et al9 reported that the drug caused peripheral blood depletion of memory B cells10,11 and plasma cells12 that play important roles in the pathogenesis of SLE, as well as a depletion of memory B cells from the secondary lymphoid tissue13. It has been reported that not only activated B cells, but also T cells and dendrocytes are involved in the pathogenesis of SLE in humans14,15,16,17. Treatment with rituximab has been demonstrated to produce an increase in the number of CD4+CD25brightFoxp3+ regulatory T cells in the peripheral blood during the recovery phase from SLE or an increase in the messenger RNA expression of Foxp3 at 1 to 3 months after treatment18,19. We have also shown that rituximab may downregulate the expression of CD40L, a costimulatory molecule expressed on T cells, in patients with SLE7. To our knowledge, however, there are no reported comprehensive studies that have investigated the effects of rituximab on the differentiation of T and B cells or on the expression of costimulatory molecules on these cells. The precise mechanisms underlying the longterm remission of SLE induced by rituximab and the lymphocyte subsets involved in the pathogenesis of SLE remain unknown. We investigated longitudinally the pattern of B cell and T cell differentiation and the changes in the expression levels of costimulatory molecules on these cells in rituximab-treated patients with SLE showing longterm remission and relapse, in order to determine the mechanisms underlying both the longterm remission of SLE induced by rituximab and the lymphocyte subsets involved in the pathogenesis of SLE. MATERIALS AND METHODS Patients. Our cohort study involved 10 patients diagnosed with SLE based on their fulfilling at least 4 of the 11 modified American College of Rheumatology (ACR) criteria for the diagnosis of SLE20. Table 1 shows the background variables of the patients before they started rituximab treatment. The subjects were 9 women and 1 man, with a mean age of 27.4 ± 8.8 years (range 16–41 yrs). The mean duration of illness from SLE diagnosis to administration of rituximab was 96.7 ± 113.3 months (range 3–324 mo). The mean steroid dose prior to the start of rituximab treatment was 35.0 ± 16.8 mg/day. Despite receiving conventional treatments such as pulse steroid therapy, intermittent intravenous cyclophosphamide pulse therapy (IVCY), cyclosporine A, mizoribine, azathioprine, mycophenolate, plasma exchange, and immunoadsorption therapy, all the patients had highly active disease, with a mean SLE Disease Activity Index (SLEDAI) of 16.2 ± 9.6 and a British Isles Lupus Assessment Group (BILAG) activity index of 19.8 ± 8.2 (all patients falling in the BILAG score category) at the start of rituximab treatment. The organ involvement was as follows: lupus nephritis in 7 patients [World Health Organization (WHO) type I in 1 patient and type IV in 6 patients], neuropsychiatric SLE in 5 patients, and thrombotic thrombocytopenic purpura in 1 patient. All patients were treated with rituximab at our facility between 2004 and 2009, and all completed the course of the anti-CD20 antibody treatment protocol formulated for our study. Written informed consent was obtained from each patient in accord with the requirement of the study protocol approved by the ethics committee of our university. Treatment schedule. Rituximab was administered twice, with a 1-week interval between administrations, at a dose of 375 mg/m2 in all patients. Assessment. Laboratory measurements included the serum levels of complements, and the titers of antinuclear antibody, antiribonucleoprotein antibody, anti-SSA antibody, anti-SSB antibody, anti-Sm antibody, and antidsDNA antibody. To assess the activity of SLE, the BILAG index21 and SLEDAI22 were calculated. Disease activity was scored on a 5-category scale by the BILAG index: A (severely active), B (moderately active), C (stable mild disease), D, and E. Responses to rituximab were categorized according to the improvement of the BILAG index, as major clinical response (MCR), partial clinical response (PCR), and no clinical response (NCR). More specifically, MCR was defined as improvement of the BILAG index to C or better at 2 years, PCR as improvement of the BILAG index to B in at least 1 domain at 2 years, and NCR as failure to meet the definition of either MCR or PCR5. Flow cytometry. Analysis of the B cell and T cell phenotypes and expression of the surface molecules on these cells was carried out by flow cyto - metry before, 28 days after, and 2 years after rituximab treatment, as well as at the time of relapse. Mononucleated cells were isolated from the peripheral blood, and were treated with the following antibodies: FITC-labeled mouse IgG1 κ, FITC-conjugated anti-CD40, FITC-conjugated anti-CD80, FITC-conjugated anti-CD69, FITC-conjugated anti-CD45RA, phycoerythrin (PE)-conjugated anti-IgD, PE-conjugated anti-CD45RO, PE-conjugated anti-inducible costimulator (ICOS), PE-Cy5-conjugated anti-CD4, PE-Cy7-conjugated anti-CD19 (Pharmin - gen, San Diego, CA, USA), FITC-conjugated anti-CD40L (Ancell, Bayport, MN, USA), and allophycocyanin-conjugated anti-CD27 (BioLegend, San Diego, CA, USA). They were then incubated 30 minutes at 4˚C, and the cells were washed 3 times with FACS solution and analyzed using the FACSCalibur (Becton-Dickinson, San Jose, CA, USA) and FlowJo software (Digital Biology, Tokyo, Japan). The numbers of CD40 and CD80 molecules expressed per CD19-positive cell were counted using QIFIKIT Beads (Dako Japan, Kyoto, Japan). Statistical analysis. The results were analyzed using SPSS version 16. The statistical significance of differences between the pretreatment and posttreatment values was tested by Wilcoxon’s test. P values › 0.05 were considered statistically significant. RESULTS Changes in levels of B cell and T cell surface antigens following rituximab treatment. We analyzed longitudinally the pattern of B cell and T cell differentiation and the changes in the expression levels of the costimulatory molecules on these cells in the longterm responders to rituximab therapy. Figures 1A and 1B show a representative patient with longterm remission after the treatment (Patient 7). Rituximab treatment resulted in a disappearance of CD19+IgD+CD27– naive B cells, CD19+IgD–CD27– memory B cells, and CD19+IgD–CD27+ class-switched memory cells from the peripheral blood within 28 days after treatment. On the other hand, CD19lowCD27high or IgD–CD38+ plasma cells persisted in the peripheral blood of these patients until Day 28, although these cells also disappeared completely from the peripheral blood by 6 months after rituximab treatment. The naive B cells recovered within 3 to 9 months after treatment, while the memory B cells and plasma cells remained depleted for 2 years. The similar changes of B cell phenotype were observed in all of the 8 patients with longterm remission of SLE (Figure 1C, 1D). The ritux- imab therapy resulted, by 2 years after treatment, in a significant fall in the percentage of CD19+IgD+CD27+ memory B cells (3.0 ± 1.8% to 1.2 ± 0.7%, p › 0.05; 1.0 ± 0.8 cells/μl to 0.9 ± 0.9 cells/μl, p = 0.8203), CD19+IgD–CD27+ class-switched memory B cells (43.5 ± 10.0% to 5.4 ± 3.2%, p › 0.001; 22.8 ± 24.4 cells/μl to 6.7 ± 5.9 cells/μl, p = 0.1187), and CD19+IgD– CD27– memory B cells (34.7 ± 8.8% to 5.9 ± 3.2%, p › 0.001; 14.7 ± 11.0 cells/μl to 5.8 ± 5.9 cells/μl, p = 0.1096); and a significant reduction of the plasma cells and memory B cells/naive B cells ratio (4.80 ± 2.12 to 0.15 ± 0.07, p › 0.001; 4.8 ± 2.1 to 0.1 ± 0.2, p › 0.001). Next, we assessed the effects of rituximab treatment on the expression levels of costimulatory molecules on CD19-positive cells. Figure 2A shows the changes of phenotypes in a representative case and similar trends were noted in all of the 8 patients with SLE who showed longterm remission following rituximab therapy (Figure 2B). At 2 years after the initial infusion, no significant change from the baseline level of the percentage and the number of CD40-expressing cells among the CD19-positive cells was observed (88.8 ± 10.6% to 76.4 ± 16.0%, p = 0.0742; 78.5 ± 69.1 cells/μl to 80.1 ± 54.5 cells/μl, p = 0.5212), even though the number of CD40 molecules per CD19-positive cell had fallen significantly within 28 days after the treatment (from 1957.8 ± 769.6 to 1200.9 ± 120.2 mole - cules/cell, p = 0.0357). In contrast to CD40, a significant reduction from baseline levels remained at 2 years in the percentage and number of CD80-expressing cells among the CD19-positive cells (55.8 ± 27.3% to 10.0 ± 5.4%, p = 0.0008; 46.8 ± 29.0 cells/μl to 8.9 ± 9.3 cells/μl, p = 0.0042) and in the number of CD80 molecules per CD19-positive cell (1657.2 ± 1936.1 to 158.4 ± 88.4 molecules/cell, p = 0.0016), indicating the reduction of memory B cells, since CD80 is expressed only on memory B cells. On the other hand, the percentage of CD4+CD45RObright memory T cells in the peripheral blood was high before the start of rituximab therapy, and a similar trend was observed until Day 28 in a representative case of Patient 4 (Figure 3A), and similar trends were observed among 8 patients treated with rituximab (Figure 3B). At 2 years after initial infusion, a significant reduction was observed in the percentage of memory T cells among the CD4-positive T cells (66.2 ± 16.2% to 45.9 ± 16.2%, p = 0.0124) and a significant increase in the number of naive T cells (64.6 ± 31.6 to 165.8 ± 97.2 cells/μl, p = 0.0087). Further, the expression of CD69, an activation marker expressed on CD4-positive cells, and of the costimulatory molecules CD40L and inducible costimulator decreased rapidly by Day 28 in a representative patient (Figure 4A). Expression of these molecules remained reduced for 2 years in 8 patients treated with rituximab (CD69, 17.9 ± 18.7% to 3.8 ± 5.7%, p = 0.0117; 30.5 ± 34.3 cells/μl to 12.1 ± 17.3 cells/μl, p = 0.032; CD40L, 10.0 ± 6.6% to 2.3 ± 1.0%, p = 0.0008; 23.6 ± 21.9 cells/μl to 6.9 ± 2.7 cells/μl, p = 0.0502; and ICOS, 8.7 ± 5.0% to 2.3 ± 1.7%, p = 0.0063; 21.1 ± 10.6 cells/μl to 8.4 ± 7.3 cells/μl, p = 0.0311; Figure 4B). Changes in expression of lymphocyte surface antigens in patients showing relapse after prolonged remission of SLE. We observed 1 case with B cell-dominant relapse and another with T cell-dominant relapse. The patient with B cell-dominant relapse was a 16-year-old girl (Patient 9) with lupus nephritis (WHO type IV). In this patient, despite intense immunosuppressive therapy, disease activity remained high (SLEDAI 13 and BILAG 23). She achieved remission of SLE after rituximab treatment, with rapid disappearance of the CD19+IgD–CD27– memory B cells and CD19+IgD–CD27+ class-switched memory B cells from the peripheral blood by Day 28, along with rapid reduction in the expression of the costimulatory molecules CD40L and ICOS on the T cells. However, the disease relapsed at 1.5 years after rituximab treatment, and simultaneously the butterfly rash reappeared, the anti-dsDNA antibody titer increased again, and proteinuria recurred. Just before relapse, the percentages of CD19+IgD–CD27– memory B cells and CD19+IgD–CD27+ class-switched memory B cells, as well as the levels of CD40 and CD80 on the CD19-positive cells, increased, without any significant change in the number of T cells (Figure 5A). This patient was treated again with rituximab. The retreatment resulted in the disappearance again of memory B cells from peripheral blood and a decrease in disease activity. The butterfly rash disappeared, the anti-dsDNA antibody test was negative, and urinary protein excretion and occult blood disappeared. The patient with T cell-dominant relapse was a 29-year-old woman (Patient 6). Despite intense immunosuppressive therapy, she continued to have central nervous system (CNS) symptoms and a high disease activity level (SLEDAI 9, BILAG 12). The results of tests for anti-dsDNA antibody and anti-Sm antibody were negative. Treatment with rituximab induced remission of SLE along with rapid disappearance of both naive and memory B cells from the peripheral blood. Two years later, however, the disease relapsed and the patient presented with CNS disease manifestations. While no changes in B cells were seen either before or after the relapse, an increase in the population of memory T cells was noted, along with markedly elevated levels of ICOS on CD4-positive cells (Figure 5B). In this patient, disease activity was found to be worse and to involve predominantly T cell abnormalities. Therefore, she was retreated with IVCY, because this drug is considered to be effective against T cells as well. Systemic symptoms, such as fever, malaise, and lymph node swelling, and also the psychiatric symptoms improved with IVCY treatment. In addition, brain perfusion scintigraphy showed an improvement of blood flow, and the level of ICOS on CD4-positive T cells decreased (data not shown). DISCUSSION Rituximab has recently been demonstrated to be effective in the treatment of SLE1,2,3,4,5,6,7,8 and we undertook our study to determine the mechanisms of the longterm remission of SLE induced by rituximab and the relapse after remission. When patients with highly active SLE were treated with rituximab, rapid depletion of CD19+IgD+CD27– naive B cells, CD19+IgD–CD27– memory B cells, and CD19+IgD–CD27+ memory B cells from the peripheral blood was observed, while CD19lowCD27high or IgD–CD38+ plasma cells persisted in the blood until Day 28. For patients with clinical remission for about 2 years after rituximab treatment, the plasma cells as well as memory B cells remained depleted or in markedly reduced numbers, although the naive B cells recovered. Analysis of the changes in the levels of the costimulatory molecules on the B cells revealed that levels of both CD40 and CD80 remained suppressed until 2 years after rituximab therapy. However, in Patient 9, who showed relapse after prolonged remission, an increase in the percentage of CD19+IgD–CD27– memory B cells and CD19+IgD–CD27+ memory B cells, as well as levels of CD40 and CD80 on these cells, was noted just before the relapse of SLE. Further, in these patients who showed relapse, the anti-dsDNA antibody titers increased, along with development of lupus nephritis as organ involvement, suggesting the correlation between changes in the B cells and the pathophysiology of SLE. Thus, in the patients in whom B cells were successfully depleted by rituximab therapy, the numbers of memory B cells and plasma cells, which express costimulatory molecules, remained suppressed for prolonged periods of time, even though the naive B cells recovered. These findings suggest that reconstitution of the peripheral B cell compartment is crucial for sustaining longterm SLE remission and that recovery of memory B cells expressing costimulatory molecules precedes the SLE relapse. A significant finding was that rituximab used to produce B cell depletion also affected T cell differentiation and activation. In cases with highly active SLE complicated by lupus nephritis or CNS disorders, findings suggestive of T cell subset involvement in the pathophysiology of SLE have been reported, such as reduction in the population of naive T cells and an inverse correlation with the antibody-forming potential23,24,25,26,27. In patients with sustained SLE remission for 2 years after rituximab treatment, however, there were significant increases in the peripheral blood CD4-positive and CD4+CD45RA+ naive T cells. Further, although no changes were seen in the number of CD4+CD45RO+ memory T cells, the expression of CD45RO decreased (CD45RObright to CD45ROintermediate), suggesting the reduced activation potential of the cells. In fact, reduction or disappearance of the expression of CD69 and the costimulatory molecules CD40L and ICOS was noted. As described, activated B cells in patients with SLE showed enhanced expression of MHC class II antigens and costimulatory molecules and an antigen-presenting potential close to that of dendritic cells, suggesting T cell activation. However, the costimulatory molecule-expressing B cells disappeared, thereby reducing the costimulatory molecule-expressing memory T cells, a change that probably contributes to longterm remission in patients with SLE. The case of Patient 6 in this study is interesting because the SLE relapse was associated with predominant T cell abnormalities. With regard to the clinical presentation of this patient, there were marked systemic symptoms such as fever (over 38˚C), polyarthritis, and lymphadenopathy, along with CNS involvement. However, this patient cannot be viewed as a specific or extraordinary case of SLE. The fact that an increase in the memory T cells and an increase in the levels of ICOS on the CD4-positive cells preceded the relapse of SLE, without any changes in the number of B cells, B cell subsets, or surface antigen expression, indicated that T cell activation was predominantly involved in the SLE relapse. Many clinical studies revealed that some patients do not benefit at all from peripheral B cell depletion therapy with rituximab1,2,7,13. When those findings are considered with our findings, it would appear that the existence of T cell-dependent/B cell-independent abnormalities may be involved in the pathogenesis of SLE. This may reflect the heterogeneity in the pathophysiology of SLE. Indeed, the patient with B cell-dominant relapse in our study responded well to retreatment with rituximab, and a favorable outcome of the patient with T cell-dominant relapse was obtained following treatment with IVCY. Thus, a higher efficacy of B cell-targeted therapy may be obtained in patients with B cell-dominant SLE, while T cell-targeted therapy may be needed for patients with T cell-dominant SLE. Our findings support the notion that activated T cells, in addition to activated B cells, may be potentially involved in the pathogenesis of SLE, and that interaction between activated B cells and T cells may worsen the pathophysiology of SLE. Depletion of B cells by rituximab may result in the reconstitution of B cells in the peripheral compartment. That could cause inhibition of T cell activation and differentiation mediated by memory B cells, which in turn might lead to longterm remission of SLE. ACKNOWLEDGMENT The authors thank T. Adachi, N. Sakaguchi, and K. Noda for their excellent technical assistance. REFERENCES 1. Looney RJ, Anolik JH, Campbell D, Felgar RE, Young F, Arend LJ, et al. B cell depletion as a novel treatment for systemic lupus erythematosus: a phase I/II dose-escalation trial of rituximab. Arthritis Rheum 2004;50:2580-9. 2. 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. 3. Rastetter W, Molina A, White CA. Rituximab: expanding role in therapy for lymphomas and autoimmune diseases. Annu Rev Med 2004;55:477-503. 4. Anolik J, Sanz I, Looney RJ. B cell depletion therapy in systemic lupus erythematosus. Curr Rheumatol Rep 2003;5:350-6. 5. Tanaka Y, Yamamoto K, Takeuchi T, Nishimoto N, Miyasaka N, Sumida T, et al. A multicenter phase I/II trial of rituximab for refractory systemic lupus erythematosus. Mod Rheumatol 2007;17:191-7. 6. Tokunaga M, Fujii K, Saito K, Nakayamada S, Tsujimura S, Nawata M, et al. Down-regulation of CD40 and CD80 on B cells in patients with life-threatening systemic lupus erythematosus after successful treatment with rituximab. Rheumatology 2005;44:176-82. 7. Tokunaga M, Saito K, Kawabata D, Imura Y, Fujii T, Nakayamada S, et al. Efficacy of rituximab (anti-CD20) for refractory systemic lupus erythematosus involving the central nervous system. Ann Rheum Dis 2007;66:470-5. 8. Lu TY, Ng KP, Cambridge G, Leandro MJ, Edwards JC, Ehrenstein M, et al. A retrospective seven-year analysis of the use of B cell depletion therapy in systemic lupus erythematosus at University College London Hospital: the first fifty patients. Arthritis Rheum 2009;61:482-7. 9. Anolik JH, Barnard J, Cappione A, Pugh-Bernard AE, Felgar RE, Looney RJ, et al. Rituximab improves peripheral B cell abnormalities in human systemic lupus erythematosus. Arthritis Rheum 2004;50:3580-90. 10. Wei C, Anolik J, Cappione A, Zheng B, Pugh-Bernard A, Brooks J, et al. A new population of cells lacking expression of CD27 represents a notable component of the B cell memory compartment in systemic lupus erythematosus. J Immunol 2007;178:6624-33. 11. Cappione A, Anolik JH, Pugh-Bernard A, Barnard J, Dutcher P, Silverman G, et al. Germinal center exclusion of autoreactive B cells is defective in human systemic lupus erythematosus. J Clin Invest 2005;115:3205-16. 12. Jacobi AM, Odendahl M, Reiter K, Bruns A, Burmester GR, Radbruch A, et al. Correlation between circulating CD27 high plasma cells and disease activity in patients with systemic lupus erythematosus. Arthritis Rheum 2003;48:1332-42. 13. Anolik JH, Barnard J, Owen T, Zheng B, Kemshetti S, Looney RJ, et al. Delayed memory B cell recovery in peripheral blood and lymphoid tissue in systemic lupus erythematosus after B cell depletion therapy. Arthritis Rheum 2007;56:3044-56. 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. 15. Grammar AC, Slota R, Fischer R, Gur H, Girschick H, Yarboro C, et al. Abnormal germinal center reactions in systemic lupus erythematosus demonstrated by blockade of CD154-CD40 interactions. J Clin Invest 2003;112:1506-20. 16. Harris DP, Haynes L, Sayles PC, Duso DK, Eaton SM, Lepak NM, et al. Reciprocal regulation of polarized cytokine production by effector B and T cells. Nat Immunol 2000;1:475-82. 17. Skok J, Poudrier J, Gray D. Dendritic cell-derived IL-12 promotes B cell induction of Th2 differentiation: a feedback regulation of Th1 development. J Immunol 1999;163:4284-91. 18. Vallerskog T, Gunnarsson I, Widhe M, Risselada A, Klareskog L, van Vollenhoven R, et al. Treatment with rituximab affects both the cellular and the humoral arm of the immune system in patients with SLE. Clin Immunol 2007;122:62-74. 19. Sfikakis PP, Souliotis VL, Fragiadaki KG, Moutsopoulos HM, Boletis JN, Theofilopoulos AN. Increased expression of the FoxP3 functional marker of regulatory T cells following B cell depletion with rituximab in patients with lupus nephritis. Clin Immunol 2007;123:66-73. 20. Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, et al. The 1982 revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1982;25:1271-7. 21. Hay EM, Bacon PA, Gordon C, Isenberg DA, Maddison P, Snaith ML, et al. The BILAG index: a reliable and valid instrument for measuring clinical disease activity in systemic lupus erythematosus. Q J Med 1993;86:447-58. 22. Bencivelli W, Vitali C, Isenberg DA, Smolen JS, Snaith ML, Sciuto M, et al. Disease activity in systemic lupus erythematosus: report of the Consensus Study Group of the European Workshop for Rheumatology Research. III. Development of a computerised clinical chart and its application to the comparison of different indices of disease activity. The European Consensus Study Group for Disease Activity in SLE. Clin Exp Rheumatol 1992;10:549-54. 23. Morimoto C, Steinberg AD, Letvin NL, Hagan M, Takeuchi T, Daley J, et al. A defect of immunoregulatory T cell subsets in systemic lupus erythematosus patients demonstrated with anti-2H4 antibody. J Clin Invest 1987;79:762-8. 24. Sato K, Miyasaka N, Yamaoka K, Okuda M, Yata J, Nishioka K. Quantitative defect of CD4+2H4+ cells in systemic lupus erythematosus and Sjögren’s syndrome. Arthritis Rheum 1987;30:1407-11. 25. Raziuddin S, Nur MA, Alwabel AA. Selective loss of the CD4+ inducers of suppressor T cell subsets (2H4+) in active systemic lupus erythematosus. J Rheumatol 1989;16:1315-9. 26. Tanaka S, Matsuyama T, Steinberg AD, Schlossman SF, Morimoto C. Antilymphocyte antibodies against CD4+2H4+ cell populations in patients with systemic lupus erythematosus. Arthritis Rheum 1989;32:398-405. 27. Mimura T, Fernsten P, Jarjour W, Winfield JB. Autoantibodies specific for different isoforms of CD45 in systemic lupus erythematosus. J Exp Med 1990;172:653-6. Iwata, et al: Rituximab for SLE 641