Falk Hiepe

Disturbed Peripheral B Lymphocyte Homeostasis in Systemic Lupus Erythematosus

In patients with active systemic lupus erythematosus (SLE), a marked B lymphocytopenia was identified that affected CD19+/CD27− naive B cells more than CD19+/CD27+ memory B cells, leading to a relative predominance of CD27-expressing peripheral B cells. CD27high/CD38+/CD19dim/surface Iglow/CD20−/CD138+ plasma cells were found at high frequencies in active but not inactive SLE patients. Upon immunosuppressive therapy, CD27high plasma cells and naive CD27− B cells were markedly decreased in the peripheral blood. Mutational analysis of V gene rearrangements of individual B cells confirmed that CD27+ B cells coexpressing IgD were memory B cells preferentially using VH3 family members with multiple somatic mutations. CD27high plasma cells showed a similar degree of somatic hypermutation, but preferentially employed VH4 family members. These results indicate that there are profound abnormalities in the various B cell compartments in SLE that respond differently to immunosuppressive therapy.

Short-lived Plasmablasts and Long-lived Plasma Cells Contribute to Chronic Humoral Autoimmunity in NZB/W Mice

The current view holds that chronic autoimmune diseases are driven by the continuous activation of autoreactive B and T lymphocytes. However, despite the use of potent immunosuppressive drugs designed to interfere with this activation the production of autoantibodies often persists and contributes to progression of the immunopathology. In the present study, we analyzed the life span of (auto)antibody-secreting cells in the spleens of NZB × NZW F1 (NZB/W) mice, a murine model of systemic lupus erythematosus. The number of splenic ASCs increased in mice aged 1–5 mo and became stable thereafter. Less than 60% of the splenic (auto)antibody-secreting cells were short-lived plasmablasts, whereas 40% were nondividing, long-lived plasma cells with a half-life of >6 mo. In NZB/W mice and D42 Ig heavy chain knock-in mice, a fraction of DNA-specific plasma cells were also long-lived. Although antiproliferative immunosuppressive therapy depleted short-lived plasmablasts, long-lived plasma cells survived and continued to produce (auto)antibodies. Thus, long-lived, autoreactive plasma cells are a relevant target for researchers aiming to develop curative therapies for autoimmune diseases.

Antigen-driven clonal proliferation of B cells within the target tissue of an autoimmune disease, the salivary glands of patients with Sjögren's syndrome

Structures resembling germinal centers are seen in the salivary glands of patients with Sjögrens syndrome, but it is not known whether the microenvironment of these cell clusters is sufficient for the induction of a germinal center response. Therefore, we cloned and sequenced rearranged Ig V genes expressed by B cells isolated from sections of labial salivary gland biopsies from two Sjögrens syndrome patients. Rearranged V genes from B cells within one cell cluster were polyclonal and most had few somatic mutations. Two adjacent clusters from another patient each contained one dominant B cell clone expressing hypermutated V genes. None of the rearranged V genes was found in both clusters, suggesting that cells are unable to migrate out into the surrounding tissue and seed new clusters. The ratios of replacement to silent mutations in the framework and complementarity determining regions suggest antigen selection of high-affinity mutants. These results show that an antigen-driven, germinal center-type B cell response is taking place within the salivary glands of Sjögrens syndrome patients. In view of the recent demonstration of a germinal center response within the rheumatoid synovial membrane and the existence of similar structures in the target tissues of other autoimmune diseases, we propose that germinal center- type responses can be induced in the nonlymphoid target tissues of a variety of autoimmune diseases.

Phase I/II trial of autologous stem cell transplantation in systemic sclerosis : procedure related mortality and impact on skin disease

BACKGROUND Systemic sclerosis (SSc, scleroderma) in either its diffuse or limited skin forms has a high mortality when vital organs are affected. No treatment has been shown to influence the outcome or significantly affect the skin score, though many forms of immunosuppression have been tried. Recent developments in haemopoietic stem cell transplantation (HSCT) have allowed the application of profound immunosuppression followed by HSCT, or rescue, to autoimmune diseases such as SSc. METHODS Results for 41 patients included in continuing multicentre open phase I/II studies using HSCT in the treatment of poor prognosis SSc are reported. Thirty seven patients had a predominantly diffuse skin form of the disease and four the limited form, with some clinical overlap. Median age was 41 years with a 5:1 female to male ratio. The skin score was >50% of maximum in 20/33 (61%) patients, with some lung disease attributable to SSc in 28/37 (76%), the forced vital capacity being <70% of the predicted value in 18/36 (50%). Pulmonary hypertension was described in 7/37 (19%) patients and renal disease in 5/37 (14%). The Scl-70 antibody was positive in 18/32 (56%) and the anticentromere antibody in 10% of evaluable patients. Peripheral blood stem cell mobilisation was performed with cyclophosphamide or granulocyte colony stimulating factor, alone or in combination. Thirty eight patients had ex vivo CD34 stem cell selection, with additional T cell depletion in seven. Seven conditioning regimens were used, but six of these used haemoimmunoablative doses of cyclophosphamide +/- anti-thymocyte globulin +/- total body irradiation. The median duration of follow up was 12 months (3–55). RESULTS An improvement in skin score of >25% after transplantation occurred in 20/29 (69%) evaluable patients, and deterioration in 2/29 (7%). Lung function did not change significantly after transplantation. One of five renal cases deteriorated but with no new occurrences of renal disease after HSCT, and the pulmonary hypertension did not progress in the evaluable cases. Disease progression was seen in 7/37 (19%) patients after HSCT with a median period of 67 (range 49–255) days. Eleven (27%) patients had died at census and seven (17%) deaths were considered to be related to the procedure (direct organ toxicity in four, haemorrhage in two, and infection/neutropenic fever in one). The cumulative probability of survival at one year was 73% (95% CI 58 to 88) by Kaplan-Meier analysis. CONCLUSION Despite a higher procedure related mortality rate from HSCT in SSc compared with patients with breast cancer and non-Hodgkins lymphoma, the marked impact on skin score, a surrogate marker of mortality, the trend towards stabilisation of lung involvement, and lack of other treatment alternatives justify further carefully designed studies. If future trials incorporate inclusion and exclusion criteria based on this preliminary experience, the predicted procedure related mortality should be around 10%.

Depletion of autoreactive immunologic memory followed by autologous hematopoietic stem cell transplantation in patients with refractory SLE induces long-term remission through de novo generation of a juvenile and tolerant immune system

Clinical trials have indicated that immunoablation followed by autologous hematopoietic stem cell transplantation (ASCT) has the potential to induce clinical remission in patients with refractory systemic lupus erythematosus (SLE), but the mechanisms have remained unclear. We now report the results of a single-center prospective study of long-term immune reconstitution after ASCT in 7 patients with SLE. The clinical remissions observed in these patients are accompanied by the depletion of autoreactive immunologic memory, reflected by the disappearance of pathogenic anti-double-stranded DNA (dsDNA) antibodies and protective antibodies in serum and a fundamental resetting of the adaptive immune system. The latter comprises recurrence of CD31(+)CD45RA(+)CD4(+) T cells (recent thymic emigrants) with a doubling in absolute numbers compared with age-matched healthy controls at the 3-year follow-up (P = .016), the regeneration of thymic-derived FoxP3(+) regulatory T cells, and normalization of peripheral T-cell receptor (TCR) repertoire usage. Likewise, responders exhibited normalization of the previously disturbed B-cell homeostasis with numeric recovery of the naive B-cell compartment within 1 year after ASCT. These data are the first to demonstrate that both depletion of the autoreactive immunologic memory and a profound resetting of the adaptive immune system are required to reestablish self-tolerance in SLE.

Inflamed kidneys of NZB / W mice are a major site for the homeostasis of plasma cells.

(NZB × NZW)F1 (NZB / W) mice develop a disease similar to human systemic lupus erythematosus (SLE), including autoantibody production, hypergammaglobulinaemia and inflammation of the kidneys. It is known that large numbers of lymphocytes infiltrate the kidneys of these mice. Here, we compare the roles of bone marrow, spleen and inflamed kidneys of NZB / W mice in the activation of B cells and the persistence of antibody‐secreting cells (ASC). ASC are present in the kidneys of NZB / W mice with full‐blown disease, as many as in the spleen and bone marrow. The specificity of the ASC in the inflamed kidneys is not restricted to self‐antigens. After immunization of NZB / W mice with ovalbumin (OVA) the OVA‐specific ASC are found initially in the spleen. Weeks later, OVA‐specific ASC are found in high numbers in the bone marrow and the kidneys of these mice, but no longer in the spleen. As determined by FACS, B cells with a germinal center phenotype (B220+ / PNA+) are found only in very low numbers in the kidneys, but in high numbers in the spleen of NZB / W mice. Germinal centers could not be detected in the kidneys, but in the spleen, and plasma cells appear to be scattered over the tissue. These data suggest that in autoimmune NZB / W mice, plasma cells generated in immune reactions of secondary lymphoid organs, later accumulate and persist in the inflamed kidneys, were they enhance the local concentrations of Ab and immunocomplexes. These experiments identify the inflamed kidneys of NZB / W mice as a site of prime relevance for the homeostasisof plasma cells, irrespective of their specificity.

Autologous stem cell transplantation for systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is responsive to treatment with immunosuppressives and steroids, but often pursues a relapsing or refractory course resulting in increasing incapacity and reduced survival. Autologous stem cell transplantation (ASCT) following immunoablative chemotherapy is a newer therapy for autoimmune disease of potential use in severe SLE. A retrospective registry survey was carried out by the European Blood and Marrow Transplant and European League Against Rheumatism (EBMT/EULAR) registry. Data was collected from 53 patients with SLE treated by ASCT in 23 centres. Disease duration before ASCT was 59 (2-155) months (median, range), 44 (83%) were female, and median age was 29 (9-52) years. At the time of ASCT a median of seven American College of Rheumatology (ACR) diagnostic criteria for SLE were present (range 2-10) and 33 (62%) had nephritis. Peripheral blood stem cells were mobilized with cyclophosphamide and granulocyte colony stimulating factor in 93% of cases. Ex vivo CD34 stem cell selection was performed in 42% of patients. Conditioning regimens employed cyclophosphamide in 84%, anti-thymocyte globulin in 76% and lymphoid irradiation in 22%. The mean duration of follow-up after ASCT was 26 (0-78) months. Remission of disease activity (SLEDAI < 3) was seen in 33/50 (66%; 95%CI 52-80) evaluable patients by six months, of which 10/31 (32%; 95%CI 15-50) subsequentlyrelapsed after six (3-40) months. Relapse was associated with negative anti-double stranded DNA (anti-dsDNA) antibodies before ASCT (P 0.007). There were 12 deaths after 1.5 (0-48) months, of which seven (12%; 95%CI 3-21) were related to the procedure. Mortality was associated with a longer disease course before ASCT (P 0.036). In conclusion, this registry study demonstrates the efficacy of ASCT for remission induction of refractory SLE, although mortality appeared high. The safety of this procedure is likely to be improved by patient selection and choice of conditioning regimen. The return of disease activity in one-third of patients might be reduced by long-term immunosuppressive therapy post-ASCT.

Activated memory B cell subsets correlate with disease activity in systemic lupus erythematosus: delineation by expression of CD27, IgD, and CD95.

OBJECTIVE Analysis of peripheral B cell subsets in patients with systemic lupus erythematosus (SLE) has provided evidence of specific alterations, such as an expansion of CD27++ plasma cells/blasts and transitional B cells. However, memory B cells in lupus have not been thoroughly investigated, and only recently a CD27- memory B cell subset was identified in the peripheral blood of lupus patients. Focusing on CD27- B cells, this study aimed to identify abnormalities in peripheral B cell subsets in patients with SLE. METHODS Three independent cohorts of lupus patients were used to characterize CD27- memory B cells, using multiparameter flow cytometry and single-cell reverse transcription-polymerase chain reaction of heavy-chain transcripts. RESULTS We identified a homogeneous subset of CD27-,IgD-,CD95+ memory B cells with an activated phenotype that was increased in patients with disease flares and that correlated with disease activity and serologic abnormalities. In contrast, the entire subset of CD27-,IgD- B cells was found to be heterogeneous, did not correlate significantly with lupus activity, and was also increased in patients with bacterial infections. CONCLUSION We conclude that CD95 is a useful marker to identify CD27- memory B cells with an activated phenotype, which might serve as a biomarker for lupus activity and as a target of further investigations aiming to elucidate the pathogenic potential of these cells and the mechanisms involved in the generation as well as regulation of this CD27-,IgD-,CD95+ memory B cell subset.

Memory B and memory plasma cells

Summary:  Vaccination provides a powerful means to control infections. It exploits and exemplifies the ability of the immune system to preserve the information that a specific pathogen has been encountered in the past. The cells and molecular mechanisms of immunological memory are still being discussed controversially. Here, we review the current concepts of memory B cells, the signals involved in their maintenance, and their role in enhanced secondary reactions. Memory plasma cells, secreting protective antibodies over lifetime, have been recognized only recently. Their characterization as cells resting in terms of proliferation and migration, and surviving in dedicated stromal niches, in the absence of antigen, has generated new concepts of how memory cells in general are organized by stroma cells, the ‘resting memory’. In autoimmunity and chronic inflammation, memory B cells and memory plasma cells can be essential players, and they require special attention, as they do not respond to most conventional therapies. Their selective targeting will depend on a molecular understanding of their lifestyle.

Belimumab reduces autoantibodies, normalizes low complement levels, and reduces select B cell populations in patients with systemic lupus erythematosus

OBJECTIVE To assess the effects of the B lymphocyte stimulator (BLyS)-specific inhibitor belimumab on immunologic biomarkers, including B cell and T cell populations, and maintenance of antibody titers to prior vaccines in autoantibody-positive systemic lupus erythematosus (SLE) patients. METHODS Pooled data from 2 phase III trials, the Study of Belimumab in Subjects with SLE 52-week (BLISS-52) and 76-week (BLISS-76) trials, comparing belimumab 1 mg/kg or 10 mg/kg versus placebo (plus standard SLE therapy for each group) were analyzed for changes in autoantibody, immunoglobulin, and complement levels. BLISS-76 patients were also analyzed for changes in B cell and T cell populations and effects on prior vaccine-induced antibody levels. RESULTS Belimumab-treated patients experienced significant sustained reductions in IgG and autoantibodies and improvement in C3/C4 levels, resulting in greater positive-to-negative conversion rates for IgG anti-double-stranded DNA (anti-dsDNA), anti-Sm, anticardiolipin, and anti-ribosomal P autoantibodies and normalization of hypergammaglobulinemia and low C3/C4 levels. Belimumab-treated patients experienced significant decreases in the numbers of naive and activated B cells, as well as plasma cells, whereas memory B cells and T cell populations did not decrease. Belimumab did not substantially affect preexisting antipneumococcal or anti-tetanus toxoid antibody levels. Post hoc analysis showed greater reductions in SLE disease activity and the risk of severe flares in patients treated with belimumab 10 mg/kg (P≤0.01) who were anti-dsDNA positive and had low C3/C4 levels at baseline. Normalization of the C3 or anti-dsDNA level by 8 weeks, irrespective of therapy, was predictive of a reduced risk of severe flare over 52 weeks. CONCLUSION Belimumab appears to promote normalization of serologic activity and reduce BLyS-dependent B cell subsets in serologically and clinically active SLE. Greater serologic activity may predict a better treatment response to belimumab.