Christian Kneitz

Effective B cell depletion with rituximab in the treatment of autoimmune diseases.

In a pilot study four patients with systemic lupus erythematosus (SLE) and autoimmune thrombocytopenia (ITP) were treated with rituximab, a B cell depicting chimeric human/mouse anti-CD20 antibody. Treatment could be performed without serious side effects and resulted in a depletion of B cells from the peripheral blood for at least 4 months. Examination of one patient three months after treatment revealed a complete depletion of B cells in the bone marrow and in the spleen as well. The time point when peripheral B cells returned into the normal range varied between 8 months and over one year and could be observed also in the spleen. The follow up over more than 12 months revealed no significant treatment-associated side effects. Total immunoglobulin and specific antibody levels did not change except for one SLE-patient receiving additional immunosuppressive treatment including cyclophosphamide because of progressive disease. Clinical effectiveness cannot be judged by the small number of patients. However, one SLE patient with refractory severe thrombocytopenia had a very Favourable response with stable platelet numbers over 100.000/microl now for more than 6 months and disappearance of anti-DNA antibodies. The treatment failure in another SLE patient could be due to the persistence of CD20-negative plasmablasts in peripheral blood which are not targeted by anti-CD20 treatment. Further studies are needed to assess the clinical benefit of B cell depletion in the treatment of autoimmune diseases.

Regeneration of the immunoglobulin heavy-chain repertoire after transient B-cell depletion with an anti-CD20 antibody

B-cell depletive therapies have beneficial effects in patients suffering from rheumatoid arthritis. Nevertheless, the role of B cells in the pathogenesis of the disease is not clear. In particular, it is not known how the regeneration of the B-cell repertoire takes place. Two patients with active rheumatoid arthritis were treated with rituximab, and the rearranged immunoglobulin heavy-chain genes (Ig-VH) were analysed to follow the B-cell regeneration. Patient A was treated with two courses of rituximab, and B-cell regeneration was followed over 27 months by analysing more than 680 Ig-VH sequences. Peripheral B-cell depletion lasted 7 months and 10 months, respectively, and each time was accompanied by a clinical improvement. Patient B received one treatment course. B-cell depletion lasted 5 months and was accompanied by a good clinical response. B cells regenerated well in both patients, and the repopulated B-cell repertoire was characterised by a polyclonal and diverse use of Ig-VH genes, as expected in adult individuals. During the early phase of B-cell regeneration we observed the expansion and recirculation of a highly mutated B-cell population. These cells expressed very different Ig-VH genes. They were class-switched and could be detected for a short period only. Patient A was followed long term, whereby some characteristic changes in the VH2 family as well as in specific mini-genes like VH3–23, VH 4–34 or VH 1–69 were observed. In addition, rituximab therapy resulted in the loss of clonal B cells for the whole period.Our data show that therapeutic transient B-cell depletion by anti-CD20 antibodies results in the regeneration of a diverse and polyclonal heavy-chain repertoire. During the early phase of B-cell regeneration, highly mutated B cells recirculate for a short time period in both the patients analysed. The longitudinal observation of a single patient up to 27 months shows subtle intraindividual changes, which may indicate repertoire modulation.

Sun-induced life-threatening lupus nephritis.

Abstract:  Photosensitivity is a widely known characteristic of both cutaneous and systemic lupus erythematosus (LE). However, sun‐induced organ involvement is rarely reported in LE. We describe a 34‐year‐old woman who had been in complete remission of systemic LE for more than 8 years without treatment. After sunbathing, she developed acute sunburn followed by cutaneous LE in sun‐exposed areas. Six weeks later, a lupus nephritis was diagnosed and high serum levels of anti‐double‐stranded (ds) DNA and anti‐Ro/SSA antibodies were detected. Treatment with systemic cyclophosphamide and prednisolone led to septicemia requiring assisted ventilation for more than 2 weeks and repeated hemodialysis. Clinical remission was achieved 3 months later by the use of prednisolone and mycophenolate mofetile. Meanwhile, mild proteinuria persisted, anti‐dsDNA antibodies normalized. We hypothesize that the sunburn‐induced keratinocyte necrosis/apoptosis exposed intracellular antigens as trigger for the generation of autoantibodies that finally mediated immune‐complex nephritis. The patient highlights the impact of UV light not only on skin but also on internal organ involvement in LE.

Frequency of regulatory T cells is not affected by transient B cell depletion using anti-CD20 antibodies in rheumatoid arthritis.

Objectives Transient B cell depletion with the monoclonal anti-CD20 antibody rituximab has shown favourable clinical responses in patients with rheumatoid arthritis (RA). Recently a characteristic regeneration pattern of B cell subpopulations has been reported. However, little is known about the impact of B-cell depletion on peripheral T cells in particular regulatory T cells. Materials and Methodology 17 patients with RA having failed anti-TNF were treated with rituximab. Four colour staining was performed using CD19, CD3, CD4, CD8, CD16, CD56, CD25, HLA-DR, HLA-G and intracellular Foxp3 at five time points spanning up to 12 months after rituximab. In addition, quantification of the soluble form of the HLA class I molecule HLA-G by ELISA has been performed. Results Peripheral B cell depletion lasted 6 to 9 months. The absolute number of CD3+, CD4+ and CD8+ lymphocytes showed no significant changes up to 1 year after B-cell depletion compared to before therapy. Only the relative frequency for CD3 and CD4 showed a significant increase (p < 0.05). In particular, CD4+CD25++ and Foxp3 positive regulatory T cells remained constant. The percentage of HLA-G positive cells in the CD4+ or CD8+ population did not change significantly either. The amount of sHLA-G remained without significant changes. Conclusion Absolute T cell counts showed no significant changes after rituximab compared to the time point before therapy.In particular, the frequency of regulatory T cells with a CD4+CD25++ phenotype as well as positive Foxp3 expression were numerically stable. Additionally, HLA-G positive regulatory T cells and soluble levels of HLA-G showed no significant changes.

Pax‐5 is a key regulator of the B cell‐restricted expression of the CD23a isoform

Human CD23 (the low affinity IgE receptor) is a B cell differentiation marker involved in inflammatory responses. Two isoforms (CD23a and CD23b) are known, which differ only in their cytoplasmic domain. Whereas CD23b expression is specifically induced by IL‐4 on B cells and cells of the myeloid lineage, CD23a expression is restricted to B cells. Each isoform is regulated by its own promoter. Pax‐5 is a B‐cell‐restricted transcription factor with an essential role in early and late B cell development. Analyses of the CD23a promoter revealed a Pax‐5‐binding site, which can compete a high affinity Pax‐5‐binding site or directly bind Pax‐5 protein in electrophoretic mobility shift assays. Introducing mutations into this site abrogates the binding. Expression of Pax‐5 in 293 cellsresulted in a seven‐ to tenfold activation of a CD23a core promoter construct. Most importantly, ectopic expression of Pax‐5 in the monocytic cell line U‐937, which regularly expresses only the CD23b isoform, led to CD23a expression after stimulation with IL‐4 and PMA. Our results suggest that Pax‐5 is a key regulator of the B‐cell‐restricted expression of the CD23a isoform.

Early phenotypic and functional alterations in lymphocytes from simian immunodeficiency virus infected macaques

Phenotypic and functional changes in lymphocytes from rhesus monkeys (Macaca mulatta) were investigated during the first 6 months after infection with SIV mac 32H. Animals preimmunized with keyhole limpet hemocyanin (KLH) were sacrificed 1, 3, 6, 12, and 24 weeks post infection. Subset composition and function of lymphocytes from blood, spleen, lymph node and thymus were analysed. In addition to a rapid decline in CD4/CD8 ratios, a massive reduction in CD29+ CD4+ cells was seen in the periphery. Although depletion of this subset was observed throughout the course of this experiment, the loss of proliferative T cell responses was most pronounced very early after infection and partially recovered after Month 3. Polyclonal cytotoxic responses were only slightly affected. In the thymus, a gradual, but moderate loss of CD4+CD8+ immature thymocytes, and a relative increase in both CD4+ and CD8+ mature subsets was observed. Infectious virus was readily recovered from homogenates of lymph node and spleen, but not of thymus tissue. Interestingly, however, virus was detected in thymocytes from all infected animals by cocultivation with a simian immunodeficiency virus (SIV) susceptible cell line.

The CD23b promoter is a target for NF-AT transcription factors in B-CLL cells.

CD23 is atypically highly expressed in various chronic diseases, including B-CLL, lupus erythematodes and rheumatoid arthritis. Its expression can be further enhanced by interleukin 4 (IL-4). We have shown before that in B-CLL cells nuclear factor(s) of activated T cells (NF-ATs) show permanent nuclear localization and therefore constitutive transcriptional activity. Here we identify CD23b promoter as a novel target for NF-AT factors in B-CLL cells. The CD23b promoter contains two NF-AT binding sites to which NF-ATp and NF-ATc factors bind with high affinity. Mutations introduced into these sites abolished NF-AT binding and impaired the promoter activity, as did cyclosporin A (CsA), an inhibitor of nuclear transport of NF-ATs. Furthermore, we show that IL-4-induced transcription factor STAT6 cooperates with NF-ATs in the induction of the CD23b promoter activity. These results show that the CD23b promoter is a target for NF-AT factors and suggest that the cooperation between NF-AT and STAT factors might be one of the molecular mechanisms responsible for high-level expression of CD23 on the surface of B-CLL cells.

Evaluation of a novel case-based training program (d3web.Train) in hematology

The new media such as the internet and digital imaging offer new opportunities in medical education. In addition to conventional lectures, we developed a case-based simulation training program of 17 hematology cases using the novel training system d3web.Train. We evaluated the assessment of this internet course by medical students, as well as their results in the hematology exam. From a group of 150 students, 47 worked through at least one case and solved 435 cases in total; in average, these students solved 9.5 cases. Eighteen different students filled in a questionnaire about the training system and 68 questionnaires about individual cases. The main results were the students found the cases very helpful (1.5±0.6 on a scale from 1=very helpful to 5=not at all), the training system very good (1.4±0.5 on a scale from 1 to 6), and want to work with it further (1.2±0.4 on a scale from 1 to 5). During the final examination, those 16 students who answered that they had solved more than 5 from the 17 cases scored significantly better (two-sided t test, p<0.01) in the hematological part of the exam than those 34 students solving 0 to 5 cases. To our knowledge, this is the first student evaluation of a case-based training program in general hematology. The d3web.Train system offers a new and great tool for creating a training program in a reasonable amount of time, because it is able to process available patient records.

Regulation of CD23 isoforms on B-chronic lymphocytic leukemia

CD23 is constitutively and atypically expressed on malignant B-cells in patients with chronic lymphocytic leukemia. It exists in two isoforms that differ only in a short amino acid sequence at the N-terminus. The CD23a isoform exhibits an endocytosis signal, that renders it more efficient in antigen uptake than CD23b. Therefore, we analyzed the regulation of CD23 isoforms and tested the ability to stimulate T-cell clones by targeting antigen to CD23 on CLL B-cells. Investigation of several stimulators to promote CD23a expression on CLL versus normal B-cells confirmed a different CD23 regulation in B-CLL. We did not find any evidence for a differential regulation of the two CD23 isoforms in B-CLL. However, CD23a is always predominantly expressed with a constant ratio of CD23a:CD23b. We show that antigen targeted to CD23 on CLL B-cells is very efficiently presented. Therefore, CD23 is likely to provide a suitable target for receptor-mediated antigen presentation in B-CLL which can be used to activate a T-cell response.

Psoriatic arthritis: therapeutic principles

From the dermatologic point of view, psoriatic arthritis (PsA) was seen for a long time as a rare complication of psoriasis. Recent studies, however, reveal a high prevalence of PsA among patients with psoriasis, and the impact of PsA due to chronic inflammation of peripheral joints, axial joints, and periarticular structures leading to radiologic progression, functional impairment, and reduction in quality of life is well recognized. Substantial improvement in understanding immunopathology of PsA has led to a variety of new therapeutic options. This article reviews the current therapeutic principles for PsA.