Abstract

Objective. Accumulating evidence suggests that B-cell depletion therapy by rituximab may be effective for autoimmune disorders. However, an optimal dose of rituximab and a mechanism of its action remain to be established. We performed a dose-escalation study for treatment of Japanese patients with autoimmune diseases including eight with SLE and one with Evans’ syndrome.

Methods. Rituximab was infused intravenously, weekly 4 times in a dose-escalating fashion at three different doses of 100, 250 or 375 mg/m2 to three patients each. Immunological parameters were monitored at certain points until 12 months after the treatment.

Results. Rituximab was well tolerated and safe in these patients. Seven out of eight SLE patients and one with Evans’ syndrome clinically responded completely or partially to the treatment. Four patients achieved long-term remission (18–30 months) without any additional treatment. In these patients, a significant decrease in circulating B cells continued for 6 months after the treatment. The mean fluorescence intensities of CD19, CD21, CD40 and BR3 on the residual B cells as well as the percentage of CD69+ CD4+ T cells decreased significantly. Serum TNF-α levels decreased significantly on day 2. The Th1/Th2 balance of CD4+ T cells gradually shifted towards a Th1 type by 6 months.

Conclusion. In addition to B-cell depletion, modification of B-cell and T-cell phenotypes as well as cytokine profiles may be involved in the action of rituximab.

Introduction

Accumulating evidence supports that B lymphocytes are actively involved in the pathogenesis of SLE [1]. Studies from lupus model mice indicate that dysregulated B cells contribute to the pathogenesis of the disease not only by an autoimmune mechanism mediated by autoantibodies but also by antibody-independent mechanisms, such as a direct interaction with other cell types [2, 3]. Intrinsic defects have also been shown in B cells obtained from SLE patients. Increased expression of CD154 [4], CD86 [5] and IL-10 [6] as well as enhanced mutational activity of Vk gene rearrangement [7] have been implicated for the hyperreactivity of B cells from SLE patients. B-cell receptor (BCR) signalling defects have also been reported in SLE patients. Calcium mobilization and tyrosine phosphorylation are up-regulated upon activation of BCR on B cells from SLE patients [8]. In addition, we and others have demonstrated molecular defects closely related to BCR signalling events: Lyn [9], HS1 [10, 11] and SHIP [12].

In recent years, a B-cell targeted therapy with the use of an anti-CD20 antibody, rituximab, has been introduced in the treatment of autoimmune diseases such as RA and SLE [13, 14]. Rituximab is a mouse/human chimeric IgG1 anti-CD20 monoclonal antibody, which was first approved for treatment of non-Hodgkin's B-cell lymphoma [15], and is very effective for depleting both normal and malignant CD20+ B cells in vivo. In patients with RA, a B-cell targeted therapy by rituximab has been confirmed to be effective in recent controlled clinical trials [16, 17]. Several uncontrolled, open-labelled studies as well as numerous case reports of SLE and other less-common autoimmune diseases have been performed to assess a therapeutic role of rituximab [17, 18]. However, there still remain many issues including a mechanism of action, an optimal dose of and long-term efficacy of the drugs. In the present study, we evaluate a dose-escalation of rituximab for treatment of autoimmune diseases (eight patients with SLE and one with Evans’ syndrome). We found that substantial decrease in the expression of CD19, CD21 and BR3 on B cells, a shift towards a Th1 type of CD4+ T cells, and reduction in the serum levels of TNF-α may be involved in the mechanisms of action for rituximab.

Patients and methods

Study design

This study was an uncontrolled, open-labelled, pilot study for dose-escalation of rituximab to evaluate its safety and clinical efficacy in the treatment of patients with autoimmune diseases refractory to conventional corticosteroids and immunosuppressive therapies. This clinical research protocol was approved by the Institutional Review Board of Kyushu University Hospital.

Rituximab was administered intravenously in a dose-escalating fashion at a low dose of 100 mg/m2 (Level 1 for first three patients), an intermediate dose of 250 mg/m2 (Level 2 for next three patients) and a high dose of 375 mg/m2 (Level 3 for final three patients) weekly on days 1, 8, 15 and 22. Patients were allowed to receive corticosteroids or immunosuppressive agents that did not exceed the doses at study entry, although concomitant medication could be tapered according to clinical needs at the discretion of the investigating physicians (Y.T., T.H., H.T.) at any time during the trial period.

Patients

A total of nine patients participated in the study. Diagnosis was SLE in eight patients and Evans’ syndrome in one. All of the patients were refractory to at least one previous conventional therapy for their diseases, >16 yrs old, and gave written informed consent prior to their participation. All of the patients with SLE fulfilled the ACR 1997 revised criteria for SLE [19, 20].

Characteristics of the nine patients enrolled in this study are shown in Table 1. The median age was 40 yrs (range 23–57) and the median duration of the disease was 9 yrs (range 0.4–16). Clinical manifestations at study entry were as follows. Three had definite antibody-associated cytopenias (Patients 1 and 8 with Coombs test-positive autoimmune haemolytic anaemia and Patient 9 with autoimmune thrombocytopenia). Three patients (Patients 2, 6 and 7) had neurological manifestations. Six (Patients 3, 4, 5, 6, 8 and 9) were carrying proteinuria and renal biopsy was performed in four of them; three of them (Patients 3, 5 and 6) were histologically diagnosed as WHO Class IV and the remaining one (Patient 4) as WHO Class V. These four patients developed chronic renal dysfunction. At the study entry, the mean dosage of prednisolone-adjusted corticosteroid was 29.6 ± 13.5 mg/day in nine patients, and four of them were treated with immunosuppressive agents.

Table 1.

Characteristics of the patients at study entry

PatientDiagnosisAge/sexDisease duration (yrs)Disease manifestations at study entryPrevious therapyTherapy at study entry
Evans’ syndrome 57/F 16 Haemolytic anaemia CS (oral, pulse) AZA, CSA, CY PSL 30 mg 
SLE 23/F 0.4 Transverse myelitis CS (oral, pulse) IV-CY, PE PSL 40 mg IV-CY 
SLE 26/F Nephritis (class IV), leucopenia, anaemia CS (oral) MMF, CSA PSL 15 mg, CSA 175 mg 
SLE 44/F Nephritis (class V), rash CS (oral) AZA, CSA PSL 12.5 mg, CSA 75 mg 
SLE 40/M 11 Nephritis (class IV), lymphopenia, anaemia CS (oral, pulse) MZ, IV-CY mPSL 16 mg 
SLE APS 39/F CNS lupus (psychosis), thrombosis, nephritis (class IV), lymphopenia CS (oral, pulse) IV-CY, CSA, PE PSL 19 mg, CSA 175 mg 
SLE 33/F Fever, arthralgia, discoid rash, polyneuropathy, lymphopenia CS (oral, iv) PSL 40 mg 
SLE AIHA 46/F Haemolytic anaemia, proteinuria, lymphopenia CS (oral, pulse) AZA, IV-CY, PE PSL 40 mg 
SLE 49/F Thrombocytopenia, haemolytic anaemia, lymphopenia, proteinuria, arthralgia CS (oral, pulse), AZA, CSA, CY PSL 50 mg 
PatientDiagnosisAge/sexDisease duration (yrs)Disease manifestations at study entryPrevious therapyTherapy at study entry
Evans’ syndrome 57/F 16 Haemolytic anaemia CS (oral, pulse) AZA, CSA, CY PSL 30 mg 
SLE 23/F 0.4 Transverse myelitis CS (oral, pulse) IV-CY, PE PSL 40 mg IV-CY 
SLE 26/F Nephritis (class IV), leucopenia, anaemia CS (oral) MMF, CSA PSL 15 mg, CSA 175 mg 
SLE 44/F Nephritis (class V), rash CS (oral) AZA, CSA PSL 12.5 mg, CSA 75 mg 
SLE 40/M 11 Nephritis (class IV), lymphopenia, anaemia CS (oral, pulse) MZ, IV-CY mPSL 16 mg 
SLE APS 39/F CNS lupus (psychosis), thrombosis, nephritis (class IV), lymphopenia CS (oral, pulse) IV-CY, CSA, PE PSL 19 mg, CSA 175 mg 
SLE 33/F Fever, arthralgia, discoid rash, polyneuropathy, lymphopenia CS (oral, iv) PSL 40 mg 
SLE AIHA 46/F Haemolytic anaemia, proteinuria, lymphopenia CS (oral, pulse) AZA, IV-CY, PE PSL 40 mg 
SLE 49/F Thrombocytopenia, haemolytic anaemia, lymphopenia, proteinuria, arthralgia CS (oral, pulse), AZA, CSA, CY PSL 50 mg 

AIHA: autoimmune haemolytic anaemia; F: female; M: male; nephritis (class IV): World Health Organization class IV glomerulonephritis; nephritis (class V): World Health Organization class V glomerulonephritis; CS: corticosteroids; CY: cyclophosphamide; IV-CY: intravenous cyclophosphamide; PE: plasmapheresis; MMF: mycophenolate mofetil; MZ: mizoribine; PSL: prednisolone; mPSL: methylprednisolone; CSA: cyclosporine A.

Table 1.

Characteristics of the patients at study entry

PatientDiagnosisAge/sexDisease duration (yrs)Disease manifestations at study entryPrevious therapyTherapy at study entry
Evans’ syndrome 57/F 16 Haemolytic anaemia CS (oral, pulse) AZA, CSA, CY PSL 30 mg 
SLE 23/F 0.4 Transverse myelitis CS (oral, pulse) IV-CY, PE PSL 40 mg IV-CY 
SLE 26/F Nephritis (class IV), leucopenia, anaemia CS (oral) MMF, CSA PSL 15 mg, CSA 175 mg 
SLE 44/F Nephritis (class V), rash CS (oral) AZA, CSA PSL 12.5 mg, CSA 75 mg 
SLE 40/M 11 Nephritis (class IV), lymphopenia, anaemia CS (oral, pulse) MZ, IV-CY mPSL 16 mg 
SLE APS 39/F CNS lupus (psychosis), thrombosis, nephritis (class IV), lymphopenia CS (oral, pulse) IV-CY, CSA, PE PSL 19 mg, CSA 175 mg 
SLE 33/F Fever, arthralgia, discoid rash, polyneuropathy, lymphopenia CS (oral, iv) PSL 40 mg 
SLE AIHA 46/F Haemolytic anaemia, proteinuria, lymphopenia CS (oral, pulse) AZA, IV-CY, PE PSL 40 mg 
SLE 49/F Thrombocytopenia, haemolytic anaemia, lymphopenia, proteinuria, arthralgia CS (oral, pulse), AZA, CSA, CY PSL 50 mg 
PatientDiagnosisAge/sexDisease duration (yrs)Disease manifestations at study entryPrevious therapyTherapy at study entry
Evans’ syndrome 57/F 16 Haemolytic anaemia CS (oral, pulse) AZA, CSA, CY PSL 30 mg 
SLE 23/F 0.4 Transverse myelitis CS (oral, pulse) IV-CY, PE PSL 40 mg IV-CY 
SLE 26/F Nephritis (class IV), leucopenia, anaemia CS (oral) MMF, CSA PSL 15 mg, CSA 175 mg 
SLE 44/F Nephritis (class V), rash CS (oral) AZA, CSA PSL 12.5 mg, CSA 75 mg 
SLE 40/M 11 Nephritis (class IV), lymphopenia, anaemia CS (oral, pulse) MZ, IV-CY mPSL 16 mg 
SLE APS 39/F CNS lupus (psychosis), thrombosis, nephritis (class IV), lymphopenia CS (oral, pulse) IV-CY, CSA, PE PSL 19 mg, CSA 175 mg 
SLE 33/F Fever, arthralgia, discoid rash, polyneuropathy, lymphopenia CS (oral, iv) PSL 40 mg 
SLE AIHA 46/F Haemolytic anaemia, proteinuria, lymphopenia CS (oral, pulse) AZA, IV-CY, PE PSL 40 mg 
SLE 49/F Thrombocytopenia, haemolytic anaemia, lymphopenia, proteinuria, arthralgia CS (oral, pulse), AZA, CSA, CY PSL 50 mg 

AIHA: autoimmune haemolytic anaemia; F: female; M: male; nephritis (class IV): World Health Organization class IV glomerulonephritis; nephritis (class V): World Health Organization class V glomerulonephritis; CS: corticosteroids; CY: cyclophosphamide; IV-CY: intravenous cyclophosphamide; PE: plasmapheresis; MMF: mycophenolate mofetil; MZ: mizoribine; PSL: prednisolone; mPSL: methylprednisolone; CSA: cyclosporine A.

The exclusion criteria included serious organ dysfunctions that were considered to be independent of their primary autoimmune diseases, history of hypersensitivity to drugs, uncontrollable active infection, human immunodeficiency virus positivity, hepatitis B or C virus positivity, pre-existing known malignancy, pregnancy and unwillingness to comply with the protocol.

Assessments of clinical data

Adverse events were monitored until the time of flare of the disease in each of the patients. Clinical and laboratory assessments were performed prior to and every 1 or 2 week for the first month after the initial infusion, and monthly thereafter. In the case of SLE, clinical assessments were performed based on SLEDAI for the general disease activity of SLE.

Flowcytometric analysis

Flowcytometric analyses were performed as previously described [21]. Peripheral blood mononuclear cells (PBMCs) were stained with combinations of FITC, phycoerythrin (PE) or APC-conjugated monoclonal antibodies against the molecules of interest.

Measurement of serum cytokine levels

Serum levels of cytokines [IL-6, soluble IL-2 receptor (sIL-2R), TNF-α, TGF-β1] were measured prior to the rituximab treatment, on days 2 and 8, and at 1, 3 and 6 months after the treatment by ELISA kits (R&D Systems, Minneapolis, MN, USA; Fujirebio, Tokyo, Japan and Diagnostic Products Corporation, Los Angeles, CA, USA).

Th1/Th2 balance of CD4+ T cells

The percentages of IFN-γ+ and IL-4+ CD4+ T cells (%IFN-γ and%IL-4) were calculated from flowcytometric intracellular cytokine analyses and the Th1/Th2 balance was evaluated as the ratio of IFN-γ+ cells (%) to IL-4 + cells (%). Intracellular cytokine detection from CD4+ T cells was performed as described previously [22].

RNA extraction and real-time PCR

Total RNA was extracted from CD4+ T cells with RNAqueous-4PCR Kit (Ambion, Austin, TX, USA) according to the manufacturer's instructions. The cDNA was synthesized using RETRO script First Strand Synthesis Kit for RT-PCR (Ambion) according to the manufacturer's instructions; expression of the genes encoding GATA3 (Hs00231122_m1), T-bet (Hs00203434_m1), IFN-γ (Hs00174143_m1), IL-4 (Hs00174122_m1), FoxP3(Hs00203958_m1) or fos (Hs00170630_m1) was quantified with Taqman Gene Expression Assay kits (Applied Biosystems, Brunchburg, NJ, USA) using ABI Prism 7700 sequence detection system (Applied Biosystems). All gene-expression results are expressed as arbitrary units relative to expression of the gene encoding glyceraldehyde phosphate dehydrogenase (GAPDH).

Statistical analysis

Since the clinical variables might be non-normally distributed, Wilcoxon signed-rank tests (paired differences) were employed for within-group comparisons and Mann–Whitney tests (unpaired differences) for between-group comparisons. Data were presented as means ± s.d. All of the calculations were performed using STATA Version 8.2 (Stata Corporation, College Station, TX, USA) software.

Results

B-cell depletion

B-cell depletion (defined as peripheral CD19+ B lymphocyte count of <5 cells/μl or >90% depletion) was achieved in eight of nine patients (89%). In the low-dose group (Level 1), B-cell depletion was observed in two out of three patients, and in the intermediate (Level 2) and high (Level 3) dose groups, B-cell depletion was achieved in all six patients. Overall, the average number of B cells was significantly reduced, and reached the nadir in 3 months after the treatment (Table 2). The significant decrease in B cells continued for at least 6 months. In contrast, the number of total lymphocytes as well as those of T-cell subsets and NK cells was not affected in our patients. When the percent decrease in B cells was compared between the groups treated with low (four infusions of 100 mg/m2) and high (four infusions of 375 mg/m2) doses, there was no significant difference except on day 2. The percent decrease in B cells on day 2 in the low-dose group was 59.0 ± 24.1%, while that in the high-dose group was 25.4 ± 16.8% (P < 0.05).

Table 2.

Immunological analysis of lymphocytes after rituximab treatmenta

BaselineDay 21 week1 month3 months6 months12 months
Lymphocyte subsets (count/μl)        
    Total lymphocytes 940 ± 1029 915 ± 859 977 ± 917 941 ± 584 1215 ± 994 971 ± 558 1008 ± 589 
    B cells (CD19+) 75.5 ± 64.5 27.6 ± 25.0 18.7 ± 18.3* 4.6 ± 4.3** 4.1 ± 7.0** 11.6 ± 16.3** 16.3 ± 12.0 
    T cells (CD3+) 744 ± 949 754 ± 812 846 ± 876 823 ± 596 1053 ± 971 821 ± 541 979 ± 553 
    CD4+ 340 ± 549 302 ± 456 363 ± 511 338 ± 325 389 ± 532 267 ± 272 327 ± 362 
    CD8+ 375 ± 362 398 ± 343 420 ± 374 433 ± 257 571 ± 408 471 ± 305 501 ± 70.9 
    CD4/CD8 ratio 0.71 ± 0.42 0.71 ± 0.56 0.78 ± 0.59 0.72 ± 0.38 0.62 ± 0.37 0.61 ± 0.44 0.63 ± 0.63 
    CD45RA+CD4+ 107 ± 125 83 ± 88 107 ± 113 104 ± 93 132 ± 175 124 ± 135 ND 
    CD45RO+CD4+ 216 ± 385 103 ± 88 177 ± 253 175 ± 235 183 ± 246 150 ± 197 ND 
    CD25+CD4+ 45 ± 39 54 ± 53 50 ± 49 66 ± 61 71 ± 82 43 ± 32 ND 
    NK cells (CD3-56+) 74 ± 72 91 ± 130 93 ± 95 100 ± 158 105 ± 120 94 ± 90 94 ± 98 
MFI on B cells        
    CD19 429 ± 199 90 ± 25** 96 ± 28** 145 ± 87** 184 ± 120** 204 ± 111* 271 ± 67 
    CD21 185 ± 100 45 ± 24** 45 ± 23** 49 ± 45* 60 ± 86* 68 ± 55** 198 ± 118 
    CD40 32.5 ± 15.2 30.7 ± 16.6 19.8 ± 8.6 23.0 ± 13.0 17.6 ± 9.9* 19.5 ± 10.3 ND 
    BR3 47.1 ± 36.9 18.4 ± 9.3 18.5 ± 2.9 12.7 ± 6.1* 9.3 ± 4.5** 12.1 ± 9.9* ND 
    CD69 5.9 ± 4.4 5.4 ± 2.9 5.1 ± 1.8 4.6 ± 1.0 5.5 ± 2.8 6.3 ± 5.7 ND 
    HLA-DR 312 ± 159 217 ± 132 191 ± 56 175 ± 169 184 ± 102 224 ± 58 ND 
    CD80 7.3 ± 2.3 7.5 ± 3.5 7.9 ± 3.9 9.3 ± 6.4 8.4 ± 1.6 16.2 ± 12.5 ND 
    CD86 10.7 ± 7.9 19.5 ± 13.1 23.4 ± 19.1 19.1 ± 10.3 36.1 ± 6.8** 25.9 ± 8.2 ND 
Positive T cells/subsets (%)        
    CD69+/CD4+ 9.9 ± 6.8 6.1 ± 5.6 6.8 ± 5.5 7.4 ± 6.3 2.9 ± 2.5* 4.7 ± 1.9* 5.2 ± 5.2 
    CD154+/CD4+ 2.0 ± 1.6 1.3 ± 1.8 1.1 ± 1.2 0.9 ± 1.1 0.7 ± 0.7 0.7 ± 0.8 0.29 ± 0.35 
    HLA-DR+/CD4+ 32.1 ± 15.5 31.6 ± 15.4 36.5 ± 24.4 36.4 ± 22.0 35.2 ± 20.3 29.2 ± 13.2 ND 
    CD45RA+/CD4+ 32.4 ± 20.3 27.3 ± 17.8 27.6 ± 15.2 26.3 ± 11.3 31.9 ± 16.9 32.2 ± 14.1 ND 
    CD45RO+/CD4+ 56.0 ± 19.6 46.0 ± 23.5 51.8 ± 21.6 49.2 ± 24.0 45.3 ± 16.7 48.8 ± 17.4 ND 
    CD28+/CD4+ 67.8 ± 20.9 70.2 ± 22.2 67.1 ± 27.1 68.0 ± 23.4 67.9 ± 22.0 63.5 ± 23.7 ND 
    CD25+/CD4+ 25.5 ± 17.0 27.5 ± 16.5 23.2 ± 16.2 21.9 ± 14.7 24.1 ± 19.1 21.0 ± 13.2 13.3 ± 14.2 
    CD69+/CD8+ 16.1 ± 9.0 14.2 ± 12.7 13.9 ± 8.1 13.2 ± 9.5 12.0 ± 10.4 13.1 ± 12.7 ND 
    CD154+/CD8+ 2.4 ± 1.8 2.0 ± 1.7 3.0 ± 3.2 2.3 ± 2.3 1.7 ± 1.8 2.5 ± 3.7 0.80 ± 1.0 
    HLA-DR+/CD8+ 57.4 ± 23.1 58.1 ± 21.8 64.1 ± 23.9 61.8 ± 31.8 66.4 ± 22.3 65.0 ± 19.6 69.3 ± 4.65 
    CD28+/CD8+ 30.6 ± 21.3 31.7 ± 21.1 26.5 ± 19.1 34.3 ± 25.3 27.1 ± 18.8 21.4 ± 11.4 ND 
    CD25+/CD8+ 3.0 ± 1.4 3.4 ± 2.2 4.2 ± 2.6 4.5 ± 5.0 1.9 ± 1.4 1.4 ± 0.5 1.7 ± 0.96 
BaselineDay 21 week1 month3 months6 months12 months
Lymphocyte subsets (count/μl)        
    Total lymphocytes 940 ± 1029 915 ± 859 977 ± 917 941 ± 584 1215 ± 994 971 ± 558 1008 ± 589 
    B cells (CD19+) 75.5 ± 64.5 27.6 ± 25.0 18.7 ± 18.3* 4.6 ± 4.3** 4.1 ± 7.0** 11.6 ± 16.3** 16.3 ± 12.0 
    T cells (CD3+) 744 ± 949 754 ± 812 846 ± 876 823 ± 596 1053 ± 971 821 ± 541 979 ± 553 
    CD4+ 340 ± 549 302 ± 456 363 ± 511 338 ± 325 389 ± 532 267 ± 272 327 ± 362 
    CD8+ 375 ± 362 398 ± 343 420 ± 374 433 ± 257 571 ± 408 471 ± 305 501 ± 70.9 
    CD4/CD8 ratio 0.71 ± 0.42 0.71 ± 0.56 0.78 ± 0.59 0.72 ± 0.38 0.62 ± 0.37 0.61 ± 0.44 0.63 ± 0.63 
    CD45RA+CD4+ 107 ± 125 83 ± 88 107 ± 113 104 ± 93 132 ± 175 124 ± 135 ND 
    CD45RO+CD4+ 216 ± 385 103 ± 88 177 ± 253 175 ± 235 183 ± 246 150 ± 197 ND 
    CD25+CD4+ 45 ± 39 54 ± 53 50 ± 49 66 ± 61 71 ± 82 43 ± 32 ND 
    NK cells (CD3-56+) 74 ± 72 91 ± 130 93 ± 95 100 ± 158 105 ± 120 94 ± 90 94 ± 98 
MFI on B cells        
    CD19 429 ± 199 90 ± 25** 96 ± 28** 145 ± 87** 184 ± 120** 204 ± 111* 271 ± 67 
    CD21 185 ± 100 45 ± 24** 45 ± 23** 49 ± 45* 60 ± 86* 68 ± 55** 198 ± 118 
    CD40 32.5 ± 15.2 30.7 ± 16.6 19.8 ± 8.6 23.0 ± 13.0 17.6 ± 9.9* 19.5 ± 10.3 ND 
    BR3 47.1 ± 36.9 18.4 ± 9.3 18.5 ± 2.9 12.7 ± 6.1* 9.3 ± 4.5** 12.1 ± 9.9* ND 
    CD69 5.9 ± 4.4 5.4 ± 2.9 5.1 ± 1.8 4.6 ± 1.0 5.5 ± 2.8 6.3 ± 5.7 ND 
    HLA-DR 312 ± 159 217 ± 132 191 ± 56 175 ± 169 184 ± 102 224 ± 58 ND 
    CD80 7.3 ± 2.3 7.5 ± 3.5 7.9 ± 3.9 9.3 ± 6.4 8.4 ± 1.6 16.2 ± 12.5 ND 
    CD86 10.7 ± 7.9 19.5 ± 13.1 23.4 ± 19.1 19.1 ± 10.3 36.1 ± 6.8** 25.9 ± 8.2 ND 
Positive T cells/subsets (%)        
    CD69+/CD4+ 9.9 ± 6.8 6.1 ± 5.6 6.8 ± 5.5 7.4 ± 6.3 2.9 ± 2.5* 4.7 ± 1.9* 5.2 ± 5.2 
    CD154+/CD4+ 2.0 ± 1.6 1.3 ± 1.8 1.1 ± 1.2 0.9 ± 1.1 0.7 ± 0.7 0.7 ± 0.8 0.29 ± 0.35 
    HLA-DR+/CD4+ 32.1 ± 15.5 31.6 ± 15.4 36.5 ± 24.4 36.4 ± 22.0 35.2 ± 20.3 29.2 ± 13.2 ND 
    CD45RA+/CD4+ 32.4 ± 20.3 27.3 ± 17.8 27.6 ± 15.2 26.3 ± 11.3 31.9 ± 16.9 32.2 ± 14.1 ND 
    CD45RO+/CD4+ 56.0 ± 19.6 46.0 ± 23.5 51.8 ± 21.6 49.2 ± 24.0 45.3 ± 16.7 48.8 ± 17.4 ND 
    CD28+/CD4+ 67.8 ± 20.9 70.2 ± 22.2 67.1 ± 27.1 68.0 ± 23.4 67.9 ± 22.0 63.5 ± 23.7 ND 
    CD25+/CD4+ 25.5 ± 17.0 27.5 ± 16.5 23.2 ± 16.2 21.9 ± 14.7 24.1 ± 19.1 21.0 ± 13.2 13.3 ± 14.2 
    CD69+/CD8+ 16.1 ± 9.0 14.2 ± 12.7 13.9 ± 8.1 13.2 ± 9.5 12.0 ± 10.4 13.1 ± 12.7 ND 
    CD154+/CD8+ 2.4 ± 1.8 2.0 ± 1.7 3.0 ± 3.2 2.3 ± 2.3 1.7 ± 1.8 2.5 ± 3.7 0.80 ± 1.0 
    HLA-DR+/CD8+ 57.4 ± 23.1 58.1 ± 21.8 64.1 ± 23.9 61.8 ± 31.8 66.4 ± 22.3 65.0 ± 19.6 69.3 ± 4.65 
    CD28+/CD8+ 30.6 ± 21.3 31.7 ± 21.1 26.5 ± 19.1 34.3 ± 25.3 27.1 ± 18.8 21.4 ± 11.4 ND 
    CD25+/CD8+ 3.0 ± 1.4 3.4 ± 2.2 4.2 ± 2.6 4.5 ± 5.0 1.9 ± 1.4 1.4 ± 0.5 1.7 ± 0.96 

aData are summarized from effective eight cases (Patients 1–3, 5–9). ND: not determined. *P < 0.05. **P < 0.01 vs baseline by Wilcoxon signed-rank test. The values are ± s.d.

Table 2.

Immunological analysis of lymphocytes after rituximab treatmenta

BaselineDay 21 week1 month3 months6 months12 months
Lymphocyte subsets (count/μl)        
    Total lymphocytes 940 ± 1029 915 ± 859 977 ± 917 941 ± 584 1215 ± 994 971 ± 558 1008 ± 589 
    B cells (CD19+) 75.5 ± 64.5 27.6 ± 25.0 18.7 ± 18.3* 4.6 ± 4.3** 4.1 ± 7.0** 11.6 ± 16.3** 16.3 ± 12.0 
    T cells (CD3+) 744 ± 949 754 ± 812 846 ± 876 823 ± 596 1053 ± 971 821 ± 541 979 ± 553 
    CD4+ 340 ± 549 302 ± 456 363 ± 511 338 ± 325 389 ± 532 267 ± 272 327 ± 362 
    CD8+ 375 ± 362 398 ± 343 420 ± 374 433 ± 257 571 ± 408 471 ± 305 501 ± 70.9 
    CD4/CD8 ratio 0.71 ± 0.42 0.71 ± 0.56 0.78 ± 0.59 0.72 ± 0.38 0.62 ± 0.37 0.61 ± 0.44 0.63 ± 0.63 
    CD45RA+CD4+ 107 ± 125 83 ± 88 107 ± 113 104 ± 93 132 ± 175 124 ± 135 ND 
    CD45RO+CD4+ 216 ± 385 103 ± 88 177 ± 253 175 ± 235 183 ± 246 150 ± 197 ND 
    CD25+CD4+ 45 ± 39 54 ± 53 50 ± 49 66 ± 61 71 ± 82 43 ± 32 ND 
    NK cells (CD3-56+) 74 ± 72 91 ± 130 93 ± 95 100 ± 158 105 ± 120 94 ± 90 94 ± 98 
MFI on B cells        
    CD19 429 ± 199 90 ± 25** 96 ± 28** 145 ± 87** 184 ± 120** 204 ± 111* 271 ± 67 
    CD21 185 ± 100 45 ± 24** 45 ± 23** 49 ± 45* 60 ± 86* 68 ± 55** 198 ± 118 
    CD40 32.5 ± 15.2 30.7 ± 16.6 19.8 ± 8.6 23.0 ± 13.0 17.6 ± 9.9* 19.5 ± 10.3 ND 
    BR3 47.1 ± 36.9 18.4 ± 9.3 18.5 ± 2.9 12.7 ± 6.1* 9.3 ± 4.5** 12.1 ± 9.9* ND 
    CD69 5.9 ± 4.4 5.4 ± 2.9 5.1 ± 1.8 4.6 ± 1.0 5.5 ± 2.8 6.3 ± 5.7 ND 
    HLA-DR 312 ± 159 217 ± 132 191 ± 56 175 ± 169 184 ± 102 224 ± 58 ND 
    CD80 7.3 ± 2.3 7.5 ± 3.5 7.9 ± 3.9 9.3 ± 6.4 8.4 ± 1.6 16.2 ± 12.5 ND 
    CD86 10.7 ± 7.9 19.5 ± 13.1 23.4 ± 19.1 19.1 ± 10.3 36.1 ± 6.8** 25.9 ± 8.2 ND 
Positive T cells/subsets (%)        
    CD69+/CD4+ 9.9 ± 6.8 6.1 ± 5.6 6.8 ± 5.5 7.4 ± 6.3 2.9 ± 2.5* 4.7 ± 1.9* 5.2 ± 5.2 
    CD154+/CD4+ 2.0 ± 1.6 1.3 ± 1.8 1.1 ± 1.2 0.9 ± 1.1 0.7 ± 0.7 0.7 ± 0.8 0.29 ± 0.35 
    HLA-DR+/CD4+ 32.1 ± 15.5 31.6 ± 15.4 36.5 ± 24.4 36.4 ± 22.0 35.2 ± 20.3 29.2 ± 13.2 ND 
    CD45RA+/CD4+ 32.4 ± 20.3 27.3 ± 17.8 27.6 ± 15.2 26.3 ± 11.3 31.9 ± 16.9 32.2 ± 14.1 ND 
    CD45RO+/CD4+ 56.0 ± 19.6 46.0 ± 23.5 51.8 ± 21.6 49.2 ± 24.0 45.3 ± 16.7 48.8 ± 17.4 ND 
    CD28+/CD4+ 67.8 ± 20.9 70.2 ± 22.2 67.1 ± 27.1 68.0 ± 23.4 67.9 ± 22.0 63.5 ± 23.7 ND 
    CD25+/CD4+ 25.5 ± 17.0 27.5 ± 16.5 23.2 ± 16.2 21.9 ± 14.7 24.1 ± 19.1 21.0 ± 13.2 13.3 ± 14.2 
    CD69+/CD8+ 16.1 ± 9.0 14.2 ± 12.7 13.9 ± 8.1 13.2 ± 9.5 12.0 ± 10.4 13.1 ± 12.7 ND 
    CD154+/CD8+ 2.4 ± 1.8 2.0 ± 1.7 3.0 ± 3.2 2.3 ± 2.3 1.7 ± 1.8 2.5 ± 3.7 0.80 ± 1.0 
    HLA-DR+/CD8+ 57.4 ± 23.1 58.1 ± 21.8 64.1 ± 23.9 61.8 ± 31.8 66.4 ± 22.3 65.0 ± 19.6 69.3 ± 4.65 
    CD28+/CD8+ 30.6 ± 21.3 31.7 ± 21.1 26.5 ± 19.1 34.3 ± 25.3 27.1 ± 18.8 21.4 ± 11.4 ND 
    CD25+/CD8+ 3.0 ± 1.4 3.4 ± 2.2 4.2 ± 2.6 4.5 ± 5.0 1.9 ± 1.4 1.4 ± 0.5 1.7 ± 0.96 
BaselineDay 21 week1 month3 months6 months12 months
Lymphocyte subsets (count/μl)        
    Total lymphocytes 940 ± 1029 915 ± 859 977 ± 917 941 ± 584 1215 ± 994 971 ± 558 1008 ± 589 
    B cells (CD19+) 75.5 ± 64.5 27.6 ± 25.0 18.7 ± 18.3* 4.6 ± 4.3** 4.1 ± 7.0** 11.6 ± 16.3** 16.3 ± 12.0 
    T cells (CD3+) 744 ± 949 754 ± 812 846 ± 876 823 ± 596 1053 ± 971 821 ± 541 979 ± 553 
    CD4+ 340 ± 549 302 ± 456 363 ± 511 338 ± 325 389 ± 532 267 ± 272 327 ± 362 
    CD8+ 375 ± 362 398 ± 343 420 ± 374 433 ± 257 571 ± 408 471 ± 305 501 ± 70.9 
    CD4/CD8 ratio 0.71 ± 0.42 0.71 ± 0.56 0.78 ± 0.59 0.72 ± 0.38 0.62 ± 0.37 0.61 ± 0.44 0.63 ± 0.63 
    CD45RA+CD4+ 107 ± 125 83 ± 88 107 ± 113 104 ± 93 132 ± 175 124 ± 135 ND 
    CD45RO+CD4+ 216 ± 385 103 ± 88 177 ± 253 175 ± 235 183 ± 246 150 ± 197 ND 
    CD25+CD4+ 45 ± 39 54 ± 53 50 ± 49 66 ± 61 71 ± 82 43 ± 32 ND 
    NK cells (CD3-56+) 74 ± 72 91 ± 130 93 ± 95 100 ± 158 105 ± 120 94 ± 90 94 ± 98 
MFI on B cells        
    CD19 429 ± 199 90 ± 25** 96 ± 28** 145 ± 87** 184 ± 120** 204 ± 111* 271 ± 67 
    CD21 185 ± 100 45 ± 24** 45 ± 23** 49 ± 45* 60 ± 86* 68 ± 55** 198 ± 118 
    CD40 32.5 ± 15.2 30.7 ± 16.6 19.8 ± 8.6 23.0 ± 13.0 17.6 ± 9.9* 19.5 ± 10.3 ND 
    BR3 47.1 ± 36.9 18.4 ± 9.3 18.5 ± 2.9 12.7 ± 6.1* 9.3 ± 4.5** 12.1 ± 9.9* ND 
    CD69 5.9 ± 4.4 5.4 ± 2.9 5.1 ± 1.8 4.6 ± 1.0 5.5 ± 2.8 6.3 ± 5.7 ND 
    HLA-DR 312 ± 159 217 ± 132 191 ± 56 175 ± 169 184 ± 102 224 ± 58 ND 
    CD80 7.3 ± 2.3 7.5 ± 3.5 7.9 ± 3.9 9.3 ± 6.4 8.4 ± 1.6 16.2 ± 12.5 ND 
    CD86 10.7 ± 7.9 19.5 ± 13.1 23.4 ± 19.1 19.1 ± 10.3 36.1 ± 6.8** 25.9 ± 8.2 ND 
Positive T cells/subsets (%)        
    CD69+/CD4+ 9.9 ± 6.8 6.1 ± 5.6 6.8 ± 5.5 7.4 ± 6.3 2.9 ± 2.5* 4.7 ± 1.9* 5.2 ± 5.2 
    CD154+/CD4+ 2.0 ± 1.6 1.3 ± 1.8 1.1 ± 1.2 0.9 ± 1.1 0.7 ± 0.7 0.7 ± 0.8 0.29 ± 0.35 
    HLA-DR+/CD4+ 32.1 ± 15.5 31.6 ± 15.4 36.5 ± 24.4 36.4 ± 22.0 35.2 ± 20.3 29.2 ± 13.2 ND 
    CD45RA+/CD4+ 32.4 ± 20.3 27.3 ± 17.8 27.6 ± 15.2 26.3 ± 11.3 31.9 ± 16.9 32.2 ± 14.1 ND 
    CD45RO+/CD4+ 56.0 ± 19.6 46.0 ± 23.5 51.8 ± 21.6 49.2 ± 24.0 45.3 ± 16.7 48.8 ± 17.4 ND 
    CD28+/CD4+ 67.8 ± 20.9 70.2 ± 22.2 67.1 ± 27.1 68.0 ± 23.4 67.9 ± 22.0 63.5 ± 23.7 ND 
    CD25+/CD4+ 25.5 ± 17.0 27.5 ± 16.5 23.2 ± 16.2 21.9 ± 14.7 24.1 ± 19.1 21.0 ± 13.2 13.3 ± 14.2 
    CD69+/CD8+ 16.1 ± 9.0 14.2 ± 12.7 13.9 ± 8.1 13.2 ± 9.5 12.0 ± 10.4 13.1 ± 12.7 ND 
    CD154+/CD8+ 2.4 ± 1.8 2.0 ± 1.7 3.0 ± 3.2 2.3 ± 2.3 1.7 ± 1.8 2.5 ± 3.7 0.80 ± 1.0 
    HLA-DR+/CD8+ 57.4 ± 23.1 58.1 ± 21.8 64.1 ± 23.9 61.8 ± 31.8 66.4 ± 22.3 65.0 ± 19.6 69.3 ± 4.65 
    CD28+/CD8+ 30.6 ± 21.3 31.7 ± 21.1 26.5 ± 19.1 34.3 ± 25.3 27.1 ± 18.8 21.4 ± 11.4 ND 
    CD25+/CD8+ 3.0 ± 1.4 3.4 ± 2.2 4.2 ± 2.6 4.5 ± 5.0 1.9 ± 1.4 1.4 ± 0.5 1.7 ± 0.96 

aData are summarized from effective eight cases (Patients 1–3, 5–9). ND: not determined. *P < 0.05. **P < 0.01 vs baseline by Wilcoxon signed-rank test. The values are ± s.d.

Adverse events

Chest discomfort as a mild infusion reaction was observed but relieved without any treatment in one patient (Patient 9). Other patients did not have any infusion reactions.

Five patients experienced bacterial infection including nasopharyngitis (Patients 1, 3 and 4), bacterial bronchitis (Patient 1), phlegmone (Patient 5) and bacterial pneumonia (Patient 6). Patient 8 had cutaneous candidiasis. Patient 5 died of renal insufficiency 14 months after rituximab treatment, because of the disease progression.

Clinical outcome

Eight of nine patients clinically responded to the treatment (Table 3). One patient (Patient 3), who flared 4 months after rituximab treatment, failed to achieve B-cell depletion. According to the clinical manifestations, three out of three patients with haematological disorders, three out of three with neurological disorders, five out of six with renal disorders responded at least partially to the treatment. Patient 4 (WHO Class V lupus nephritis) did not show any response to rituximab treatment. Especially, in all of the patients carrying haematological disorders (Patients 1, 8 and 9), the effect of rituximab continued for 30, 20 and 18 months, respectively, and the dose of corticosteroids could be tapered without any additional immunosuppressive therapy. In eight patients with SLE, the SLEDAI was determined before and after the treatment. The average SLEDAI score was 17.6 ± 10.2 at baseline, which was significantly improved to 7.3 ± 2.4 after rituximab treatment (P = 0.028). Along with the improvement of the SLEDAI score, the dose of corticosteroids could be reduced from 27.4 ± 13.3 mg/day at baseline to 13.9 ± 5.0 mg/day after the treatment with rituximab, which was statistically significant (P = 0.018).

Table 3.

Clinical outcome

Changes in disease parametersChanges in CS dose (mg/day)
PatientTotal dose of RTX (mg/m2)B-cell depletion/ clinical efficacyParametersBaseline/ post-RTXSLEDAI baseline/ post RTXAnti-dsDNA (IU/ml) baseline/ post RTXHypo-complement/ normalizationCICs (C1q) (μ g/ml) Baseline/ post RTXCSBaseline/ post RTXFollow up/time to flare (months)
400 Yes/yes Hb (g/dl) 6.1/13.1    NA/NA PSL 30/15 30/no 
400 Yes/yes paraplegia Improved 8/8 Neg/neg No/- Neg/neg PSL 40/1 29/no 
400 No/yes Upr (g/day) 0.8/0.2 17/4 304/360 Yes/no 8.2/3.7 PSL 15/15 6/4 
1000 Yes/no Upr (g/day) 2.5/2.6 10/10 Neg/neg No/- Neg/neg PSL 12.5/12.5 12/0 
1000 Yes/yes Upr/Ucr 4.4/0.8 12/9 Neg/neg No/- 2.6/neg mPSL 16/14 9/9 
1000 Yes/yes Psychosis Improved 40/10 48/neg Yes/no 2.1/neg PSL 19/18 8/8 
   Upr/Ucr 8.4/2.8        
1500 Yes/yes Poly- neuropathy Improved 15/7 Neg/neg No/- Neg/neg mPSL 24/18 6/3 
   Arthralgia No change        
1500 Yes/yes Hb (g/dl) 8.9/14.3 16/4 31/neg Yes/yes 5.6/neg PSL 40/20 20/no 
   Upr/Ucr 0.8/0.1        
1500 Yes/yes Plt (×104/μl) 1.2/25.1 23/6 >400/neg Yes/yes 15.0/neg PSL 50/9 18/no 
   Hb (g/dl) 6.7/14.0        
   Upr (g/day) 4.1/0.6        
Changes in disease parametersChanges in CS dose (mg/day)
PatientTotal dose of RTX (mg/m2)B-cell depletion/ clinical efficacyParametersBaseline/ post-RTXSLEDAI baseline/ post RTXAnti-dsDNA (IU/ml) baseline/ post RTXHypo-complement/ normalizationCICs (C1q) (μ g/ml) Baseline/ post RTXCSBaseline/ post RTXFollow up/time to flare (months)
400 Yes/yes Hb (g/dl) 6.1/13.1    NA/NA PSL 30/15 30/no 
400 Yes/yes paraplegia Improved 8/8 Neg/neg No/- Neg/neg PSL 40/1 29/no 
400 No/yes Upr (g/day) 0.8/0.2 17/4 304/360 Yes/no 8.2/3.7 PSL 15/15 6/4 
1000 Yes/no Upr (g/day) 2.5/2.6 10/10 Neg/neg No/- Neg/neg PSL 12.5/12.5 12/0 
1000 Yes/yes Upr/Ucr 4.4/0.8 12/9 Neg/neg No/- 2.6/neg mPSL 16/14 9/9 
1000 Yes/yes Psychosis Improved 40/10 48/neg Yes/no 2.1/neg PSL 19/18 8/8 
   Upr/Ucr 8.4/2.8        
1500 Yes/yes Poly- neuropathy Improved 15/7 Neg/neg No/- Neg/neg mPSL 24/18 6/3 
   Arthralgia No change        
1500 Yes/yes Hb (g/dl) 8.9/14.3 16/4 31/neg Yes/yes 5.6/neg PSL 40/20 20/no 
   Upr/Ucr 0.8/0.1        
1500 Yes/yes Plt (×104/μl) 1.2/25.1 23/6 >400/neg Yes/yes 15.0/neg PSL 50/9 18/no 
   Hb (g/dl) 6.7/14.0        
   Upr (g/day) 4.1/0.6        

Hb: haemoglobin; Upr: urinary protein; Upr/Ucr: urinary protein/urinary creatinine ratio; Plt: platelet count; Neg: negative; NA: not assessed; RTX: rituximab. Patient 5 died of renal insufficiency 14 months after rituximab treatment.

Table 3.

Clinical outcome

Changes in disease parametersChanges in CS dose (mg/day)
PatientTotal dose of RTX (mg/m2)B-cell depletion/ clinical efficacyParametersBaseline/ post-RTXSLEDAI baseline/ post RTXAnti-dsDNA (IU/ml) baseline/ post RTXHypo-complement/ normalizationCICs (C1q) (μ g/ml) Baseline/ post RTXCSBaseline/ post RTXFollow up/time to flare (months)
400 Yes/yes Hb (g/dl) 6.1/13.1    NA/NA PSL 30/15 30/no 
400 Yes/yes paraplegia Improved 8/8 Neg/neg No/- Neg/neg PSL 40/1 29/no 
400 No/yes Upr (g/day) 0.8/0.2 17/4 304/360 Yes/no 8.2/3.7 PSL 15/15 6/4 
1000 Yes/no Upr (g/day) 2.5/2.6 10/10 Neg/neg No/- Neg/neg PSL 12.5/12.5 12/0 
1000 Yes/yes Upr/Ucr 4.4/0.8 12/9 Neg/neg No/- 2.6/neg mPSL 16/14 9/9 
1000 Yes/yes Psychosis Improved 40/10 48/neg Yes/no 2.1/neg PSL 19/18 8/8 
   Upr/Ucr 8.4/2.8        
1500 Yes/yes Poly- neuropathy Improved 15/7 Neg/neg No/- Neg/neg mPSL 24/18 6/3 
   Arthralgia No change        
1500 Yes/yes Hb (g/dl) 8.9/14.3 16/4 31/neg Yes/yes 5.6/neg PSL 40/20 20/no 
   Upr/Ucr 0.8/0.1        
1500 Yes/yes Plt (×104/μl) 1.2/25.1 23/6 >400/neg Yes/yes 15.0/neg PSL 50/9 18/no 
   Hb (g/dl) 6.7/14.0        
   Upr (g/day) 4.1/0.6        
Changes in disease parametersChanges in CS dose (mg/day)
PatientTotal dose of RTX (mg/m2)B-cell depletion/ clinical efficacyParametersBaseline/ post-RTXSLEDAI baseline/ post RTXAnti-dsDNA (IU/ml) baseline/ post RTXHypo-complement/ normalizationCICs (C1q) (μ g/ml) Baseline/ post RTXCSBaseline/ post RTXFollow up/time to flare (months)
400 Yes/yes Hb (g/dl) 6.1/13.1    NA/NA PSL 30/15 30/no 
400 Yes/yes paraplegia Improved 8/8 Neg/neg No/- Neg/neg PSL 40/1 29/no 
400 No/yes Upr (g/day) 0.8/0.2 17/4 304/360 Yes/no 8.2/3.7 PSL 15/15 6/4 
1000 Yes/no Upr (g/day) 2.5/2.6 10/10 Neg/neg No/- Neg/neg PSL 12.5/12.5 12/0 
1000 Yes/yes Upr/Ucr 4.4/0.8 12/9 Neg/neg No/- 2.6/neg mPSL 16/14 9/9 
1000 Yes/yes Psychosis Improved 40/10 48/neg Yes/no 2.1/neg PSL 19/18 8/8 
   Upr/Ucr 8.4/2.8        
1500 Yes/yes Poly- neuropathy Improved 15/7 Neg/neg No/- Neg/neg mPSL 24/18 6/3 
   Arthralgia No change        
1500 Yes/yes Hb (g/dl) 8.9/14.3 16/4 31/neg Yes/yes 5.6/neg PSL 40/20 20/no 
   Upr/Ucr 0.8/0.1        
1500 Yes/yes Plt (×104/μl) 1.2/25.1 23/6 >400/neg Yes/yes 15.0/neg PSL 50/9 18/no 
   Hb (g/dl) 6.7/14.0        
   Upr (g/day) 4.1/0.6        

Hb: haemoglobin; Upr: urinary protein; Upr/Ucr: urinary protein/urinary creatinine ratio; Plt: platelet count; Neg: negative; NA: not assessed; RTX: rituximab. Patient 5 died of renal insufficiency 14 months after rituximab treatment.

Four of eight SLE patients were positive for anti-dsDNA antibody and also had low complement titres. Anti-dsDNA antibody became negative in three of these patients, two of whom showed normalized complement titres. Patient 3 did not show either improvement of anti-dsDNA titre or complement titre, which was eventually followed by the deterioration of proteinuria and the SLEDAI score 4 months after rituximab treatment. The titres of ANA and anti-Sm antibodies were analysed before and after treatment of rituximab in SLE patients. Patients 2, 3 and 4 were all carrying marginal titres of ANA (×40–×80), which showed no significant change after rituximab treatment. In contrast, Patients 6, 8 and 9, who were carrying significantly increased ANA titres, ×160, ×320 and >×2560, respectively, presented apparent decrease to levels <×40 after rituximab treatment. In the case of anti-Sm antibodies, the titres (cut-off index; COI) were estimated by ELISA. Patients 3, 4 and 6 showed titres of 10, 38 and 46, respectively, before treatment, which were decreased to 7, 22 and 20.7. The other patients were negative for anti-Sm antibodies both before and after rituximab treatment.

Serum immunoglobulin levels gradually decreased in 6 months after the treatment. The levels of IgG, IgA and IgM at baseline were 982 ± 431, 233 ± 119 and 91 ± 43 mg/dl, respectively, while they dropped down to 716 ± 71, 172 ± 66 and 60 ± 30 mg/dl, respectively, at 6 months. Only IgM levels at 3 (P = 0.012) and 6 (P = 0.012) months after the treatment were significantly low.

Surface molecules of B cells

The expression of surface molecules on the residual B cells was analysed by the mean fluorescence intensity (MFI). As shown in Table 2, the MFIs of CD19 and CD21 were decreased as early as day 2, which continued until 6 months after rituximab treatment. The expression of BR3 was decreased at 1, 3 and 6 months after the treatment. The expression of CD40 was gradually decreased, and this decrease became statistically significant at 3 months after the treatment. In contrast, the expression of CD86 was significantly increased at 3 months after the treatment. The expression levels of CD69 and CD80 were not affected during the period. The expression level of CD19 on the residual B cells was shown in Fig. 1. The subtypes of the B cells were classified according to the expression level of CD27. CD27− naïve B cells and CD27+ memory B cells showed decreased expression of CD19 after rituximab treatment, while CD27 highly positive plasmablast cells did not alter the expression level of CD19.

Fig. 1.

Changes in the expression levels of CD19 on B cells after treatment with 375 mg/m2 of rituximab. Expression levels of CD19 and CD27 on B cells were estimated by FACS analysis after rituximab treatment in Patient 8 (baseline, day 2, 1 week, 1, 3 and 6 months). CD27− naïve B cells (R2) and CD27+ memory B cells (R3) showed decreased expression of CD19 after rituximab treatment, while CD27 high plasmablast cells (R4) did not alter the expression levels of CD19.

Surface molecules of T cells

The percentage of CD69+ CD4+ T cells was significantly decreased at 3 and 6 months after rituximab treatment. Although there was a trend, no significant decrease was observed in CD154 (CD40L) after the treatment (Table 2). These changes in surface molecules of T cells were preceded by an immediate decrease in B cells which was observed 2 days after rituximab treatment. The percentage of other T-cell subsets carrying such molecules as HLA-DR, CD45RA and CD45RO did not show any change during the course of 6 month analysis.

Th1/Th2 balance

FACS analysis of intracellular cytokines showed that the ratio of IFN-γ to IL-4-expressing cells among CD4+ T cells was increased at 3 and 6 months after the treatment, indicating that the Th1/Th2 balance was skewed towards Th1 (Fig. 2A).

Fig. 2.

(A) Gradual shift towards Th1 in Th1/Th2 balance after rituximab treatment. The percentage of IFN-γ+ and IL-4+ CD4+ T cells (%IFNγ and %IL-4) was counted by FACS and the Th1/Th2 balance was evaluated by the ratio of %IFNγ–%IL-4 (%IFNγ/%IL-4). (B) Real-time RT-PCR analysis for T-bet and GATA-3. The gene expression results are expressed as arbitrary units relative to expression of the gene encoding GAPDH. The ratio of T-bet to GATA-3 expression is also shown (lower right).

At the same time, the expression of transcription factors essential for Th1 induction (T-bet) and Th2 induction (GATA-3) was estimated in CD3+ T cells by using real-time RT-PCR analysis (Fig. 2B). The mRNA level of T-bet was significantly increased 3 (P = 0.012) and 6 (P = 0.036) months after the treatment compared with that of GAPDH. In contrast, GATA-3 expression was not affected after the treatment. Therefore, the ratio of T-bet/GATA-3 was significantly increased 3 months after the treatment of rituximab (P = 0.036), which indicated a shift towards Th1 at the level of transcription and was consistent with the result obtained by FACS analysis of intracellular cytokines at the protein level. The message levels of IL-2, IFN-γ, fos and Foxp3 in CD3+ T cells were estimated at 3 months by using real-time RT-PCR analysis as well. Only fos was significantly suppressed 3 months after rituximab treatment (P = 0.012) (data not shown).

Serum cytokine level

Serum levels of IL-6, TNF-α, sIL-2 receptor and TGF-β1 were determined until 6 months after the treatment. Overall, there was no apparent change during the course of observation, however, the level of TNF-α was significantly decreased on day 2 (P = 0.028) (Table 4). The tendency of decreased TNF-α was observed at 3 months (P = 0.058) and 6 months (P = 0.086).

Table 4.

Serum cytokine levels after rituximab treament

BaselineDay 21 week1 month3 months6 months
IL-6 (pg/ml) 3.3 ± 2.6 3.6 ± 2.8 3.9 ± 3.8 5.3 ± 4.8 3.7 ± 2.5 3.4 ± 3.8 
TNF (pg/ml) 45.0 ± 28 34.8 ± 18.4* 37.9 ± 24.1 40.7 ± 26.0 30.9 ± 15.1 25.0 ± 15.3 
sIL-2R (U/ml) 742 ± 427 795 ± 537 757 ± 483 895 ± 491 829 ± 681 588 ± 518 
TGF-β1 (ng/ml) 11.0 ± 13.1 8.1 ± 7.1 7.1 ± 6.8 5.4 ± 2.6 5.0 ± 3.7 10.5 ± 13.8 
BaselineDay 21 week1 month3 months6 months
IL-6 (pg/ml) 3.3 ± 2.6 3.6 ± 2.8 3.9 ± 3.8 5.3 ± 4.8 3.7 ± 2.5 3.4 ± 3.8 
TNF (pg/ml) 45.0 ± 28 34.8 ± 18.4* 37.9 ± 24.1 40.7 ± 26.0 30.9 ± 15.1 25.0 ± 15.3 
sIL-2R (U/ml) 742 ± 427 795 ± 537 757 ± 483 895 ± 491 829 ± 681 588 ± 518 
TGF-β1 (ng/ml) 11.0 ± 13.1 8.1 ± 7.1 7.1 ± 6.8 5.4 ± 2.6 5.0 ± 3.7 10.5 ± 13.8 

The normal ranges are <4.0 pg/ml for IL-6, <4.8 pg/ml for TNF, 188–570 U/ml for sIL-2R and 0.89–1.80 ng/ml for TGFβ1. *P < 0.05 by Wilcoxon signed-rank test. The values are ± s.d.

Table 4.

Serum cytokine levels after rituximab treament

BaselineDay 21 week1 month3 months6 months
IL-6 (pg/ml) 3.3 ± 2.6 3.6 ± 2.8 3.9 ± 3.8 5.3 ± 4.8 3.7 ± 2.5 3.4 ± 3.8 
TNF (pg/ml) 45.0 ± 28 34.8 ± 18.4* 37.9 ± 24.1 40.7 ± 26.0 30.9 ± 15.1 25.0 ± 15.3 
sIL-2R (U/ml) 742 ± 427 795 ± 537 757 ± 483 895 ± 491 829 ± 681 588 ± 518 
TGF-β1 (ng/ml) 11.0 ± 13.1 8.1 ± 7.1 7.1 ± 6.8 5.4 ± 2.6 5.0 ± 3.7 10.5 ± 13.8 
BaselineDay 21 week1 month3 months6 months
IL-6 (pg/ml) 3.3 ± 2.6 3.6 ± 2.8 3.9 ± 3.8 5.3 ± 4.8 3.7 ± 2.5 3.4 ± 3.8 
TNF (pg/ml) 45.0 ± 28 34.8 ± 18.4* 37.9 ± 24.1 40.7 ± 26.0 30.9 ± 15.1 25.0 ± 15.3 
sIL-2R (U/ml) 742 ± 427 795 ± 537 757 ± 483 895 ± 491 829 ± 681 588 ± 518 
TGF-β1 (ng/ml) 11.0 ± 13.1 8.1 ± 7.1 7.1 ± 6.8 5.4 ± 2.6 5.0 ± 3.7 10.5 ± 13.8 

The normal ranges are <4.0 pg/ml for IL-6, <4.8 pg/ml for TNF, 188–570 U/ml for sIL-2R and 0.89–1.80 ng/ml for TGFβ1. *P < 0.05 by Wilcoxon signed-rank test. The values are ± s.d.

Discussion

Clinical and immunological response to rituximab treatment was evaluated in Japanese patients with refractory autoimmune diseases. The dose of rituximab was increased stepwise from Level 1 (a total of 400 mg/m2) for the first three patients, to the Level 2 (a total of 1000 mg/m2) for the next three patients and to the Level 3 (a total of 1500 mg/m2) for the final three patients. The treatment with rituximab at these doses gave rise to remission in eight of nine patients with refractory autoimmune diseases. Four weekly administration of rituximab at a dose of 375 mg/m2 was safe and well tolerated.

In addition, here we report a number of novel findings on the modulation of immunological parameters after treatment with rituximab. First, we showed for the first time that the expression levels of CD19, CD21 and BR3 on the residual B cells were significantly decreased shortly after the treatment of rituximab. These effects were continuing for at least 6 months after rituximab treatment even when the recovery of B cells was achieved (Table 2, Fig. 1). CD19 is associated with CD21, CD81 and Leu-13, and serves as a positive regulator of BCR signalling events [23]. Increased CD19 function is involved in the pathogenesis of human systemic sclerosis [24]. In addition, possible association of a CD19 gene polymorphism with SLE has been demonstrated in Japanese [25]. BR3 is the receptor for B-cell activating factor belonging to the tumor necrosis factor (TNF) family, which is an important cytokine for B-cell activation and is supposed to be associated with SLE [26]. In addition, down-regulation of CD40 on residual B cells was shown in the present study, which is consistent with a previous report [27]. Taken together, reduction of these surface molecules is likely to cause qualitative changes in residual B-cell function. The expression of CD86, a co-stimulatory molecule for T cells, was unexpectedly increased on B cells 3 months after rituximab treatment (Table 2), which was not explained by the decreased expression of CD40 on B cells, an inducer of CD86. A number of other CD86 inducers, such as Toll-like receptors and IL-5 [28], might have overcome the effect of CD40 decrease and eventually caused increased CD86 expression on B cells.

Second, shift of Th1/Th2 balance towards Th1 in CD4+ T cells and concomitant increase in T-bet/GATA-3 message were for the first time shown in the present study (Fig. 2A, 2B). Th cell polarization after B-cell depletion is consistent with the finding that B cells play an important role for Th2 polarization of CD4+ T cells [29]. As Th1/Th2 balance in SLE is controversial [22, 30], it is not clear whether this gradual shift towards Th1 after rituximab treatment is involved in the improvement of the disease activities or not. Although there was no significant decrease in CD154, CD69 on CD4+ T cells, an activation surface marker, was suppressed after rituximab treatment (Table 2), as previously reported [31].

Third, serum TNF-α levels were significantly decreased after rituximab treatment on day 2 (Table 4). The tendency of decreased TNF-α was observed at 3 months (P = 0.058) and 6 months (P = 0.086). Because stimulated B cells can produce various cytokines including TNF-α [32], B-cell depletion induced by rituximab might have contributed to the reduction in the levels of serum TNF-α, although the exact mechanism for this reduction is unclear at this point. TNF-α has been increasingly considered to play a role in the pathogenesis of SLE [33, 34]. However, considering that only partial reduction in serum TNF-α was achieved in our patients, the involvement of TNF-α in the clinical improvement after rituximab treatment should be small.

Fourth, serial analyses for T-cell surface molecules in the present study clearly revealed that drastic B-cell depletion precedes the reduction in the expression of activation marker of T cells (Table 2). This time lag might imply that reduction in B cell number directly or indirectly may cause improvement of T-cell phenotypes.

Finally, the degree of B-cell depletion was not different until 6 months between a low-dose group (4 weekly infusions of 100 mg/m2) and a high-dose group (4 weekly infusions of 375 mg/m2) except for day 2. The finding that the degree of B-cell depletion is not correlated with escalating doses has also been reported [35], though the available evidence is limited in number of patients treated in the present study. It is still unknown whether the clinical efficacy of rituximab is dose dependent, while many of studies have applied 4 weekly administrations of 375 mg/m2, which is a dosage originally used for malignant lymphoma [36]. Since the total number and the nature of B-cells to be depleted are different between malignant lymphoma and autoimmune diseases, an optimal dose of rituximab in autoimmune diseases will need further investigation [18]. In fact, considering that B-cell depletion is an important factor to achieve clinical response [17, 18], it would be of interest to further investigate an optimal dose of rituximab in autoimmune diseases. In our study, long-term remission over 18 months was achieved in both a low-dose group (Patients 1, 2) and a high-dose group (Patients 8, 9).

Three of the four patients achieving long-term remission were complicated by haematological disorders: one with Evans’ syndrome and two with SLE carrying autoimmune haemolytic anaemia or autoimmune thrombocytopenic purpura. Although the number of reported patients with autoimmune haematological disorders and SLE or Evans’ syndrome is small [37–40], rituximab appears to provide overall favourable results for autoimmune cytopenia.

In conclusion, we performed a dose-escalation study for rituximab for treatment of Japanese patients with SLE. Our observation indicates that rituximab is safe and well tolerated at escalating doses from 400 to 1500 mg/m2. Although confirmatory phase III study is needed, our results suggest that rituximab may be effective for autoimmune diseases refractory to conventional treatment. Immunological analysis shows that in addition to B-cell depletion, qualitative changes in B cells and T cells as well as modulation of cytokine expression are actively involved in the improvement of SLE.

graphic

Acknowledgements

Funding: This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (T.H.).

Disclosure statement: The authors have declared no conflicts of interest.

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