Marcus Odendahl

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.

Correlation analysis between frequencies of circulating antigen-specific IgG-bearing memory B cells and serum titers of antigen-specific IgG

Recent studies in mice have indicated that the long‐lasting specific antibody responses seen after vaccination are probably due to the existence of long‐lived plasma cells. Therefore, because the maintenance of humoral immunity does not necessarily reflect continuous restimulation of long‐lived memory B cells, the question arises as to what degree antibody immunity, as determined by measuring serum immunoglobulin titers against a particular antigen, and memory B cell immunity, as determined by counting circulating memory B cells with specificity for that same antigen, correlate. Here, using a new assay combining two‐step immunomagnetic enrichment with multiparameter flow cytometry to detect, enumerate and characterize antigen‐specific memory B cells, we show for tetanus toxin C‐fragment in blood of normal tetanus toxoid vaccinized donors, and for wasp venom phospholipase A1B in blood of wasp venom‐allergic donors undergoing an immune therapy with wasp venom, that there is no statistically significant linear correlation between the frequencies of circulating antigen‐specific IgG‐bearing memory B cells and the serum titers of antigen‐specific IgG. This lack of a statistically significant linear correlation is in accordance with the idea that B memory cells and plasma cells represent independently controlled forms of immunological memory.

CD38 low IgG-secreting cells are precursors of various CD38 high-expressing plasma cell populations

Despite the important role immunoglobulin G (IgG)‐secreting plasma cells play in memory immune responses, the differentiation and homeostasis of these cells are not completely understood. Here, we studied the differentiation of human IgG‐secreting cells ex vivo and in vitro, identifying these cells by the cellular affinity matrix technology. Several subpopulations of IgG‐secreting cells were identified among the cells isolated from tonsils and bone marrow, particularly differing in the expression levels of CD9, CD19, and CD38. CD38 low IgG‐secreting cells were present exclusively in the tonsils. A major fraction of these cells appeared to be early plasma cell precursors, as upon activation of B cells in vitro, IgG secretion preceded up‐regulation of CD38, and on tonsillar sections, IgG‐containing, CD38 low cells with a plasmacytoid phenotype were found in follicles, where plasma cell differentiation starts. A unitary phenotype of migratory peripheral blood IgG‐secreting cells suggests that all bone marrow plasma cell populations share a common precursor cell. These data are compatible with a multistep model for plasma cell differentiation and imply that a common CD38 low IgG‐secreting precursor gives rise to a diverse plasma cell compartment.

The role of long-lived plasma cells in autoimmunity

Summary Recent results on the biology of plasma cells have shown that these cells can survive as long as memory B cells. Possibly, such long-lived plasma cells are also involved in the production of autoantibodies. Here, we discuss the potential involvement of long-lived plasma cells in the pathogenesis of autoimmune disease and the consequences it has for the development of effective therapeutic strategies.

Aberrant Activation of B Cells in Patients with Rheumatoid Arthritis

Fluorescence-activated cell sorting (FACS) analysis of peripheral blood mononuclear cells (PBMCs) was used to define the differentiation status of peripheral blood B cells. Staining with CD19, CD20, CD38, and CD27 allowed us to distinguish three different CD19+ B cell subsets: CD20+, CD38–, CD27– naïve B cells; CD20+, CD38–, CD27+ memory cells; and CD20–, CD38+, CD27hi plasma cells (FIG. 1). In the peripheral blood of healthy controls, approximately 60% of B cells have the phenotype of naïve cells, and 40% have that of memory cells. Typically, in such healthy donors, less than 2% of the peripheral B cells are plasma cells (CD19+, CD20–, CD38+, and CD27hi). The analysis of blood samples from patients with rheumatoid arthritis (RA) showed a clear change in the B cell populations with a shift toward cells expressing an activated and differentiated phenotype. A typical example is given in FIGURE 1. The majority of B cells are CD27+. In addition to memory B cells, one sees a substantial population of CD27hi plasma cells.

Immunoglobulin V? light chain gene usage in patients with Sjgren's syndrome

Objective To determine whether patients with Sjogrens syndrome (SS) have abnormalities in Ig Vλ and Jλ gene usage, differences in somatic hypermutation, defects in selection, or indications for perturbations of B cell maturation. Methods Individual peripheral B cells from SS patients were analyzed for their Vλ gene usage by single-cell polymerase chain reaction amplification of genomic DNA and compared with those from normal controls. Results Molecular differences from controls in Vλ–Jλ recombination were identified that were reflected by findings in the nonproductive Vλ repertoire of the patients, including enhanced rearrangement of Vλ10A and Jλ2/3 gene segments. In addition, a number of abnormalities in the productive repertoire were identified, indicating disordered selection. A greater usage of 4 Vλ genes (2A2, 2B2, 2C, and 7A), representing 56% of all productive Vλ rearrangements, was observed, suggesting positive selection of these genes. Overutilization of Jλ2/3 and underutilization of Jλ7 in both nonproductive and productive Vλ rearrangements of SS patients compared with controls suggested decreased receptor editing in SS. The mutational frequency did not differ from that in controls, and positive selection of mutations into the productive V gene repertoire was found, similar to that in controls, although mutational targeting toward RGYW/WRCY motifs, typically found in controls, was not found in SS patients. Conclusion Disturbed regulation of B cell maturation with abnormal selection, defects in editing Ig receptors, and abnormal mutational targeting may contribute to the emergence of autoimmunity in SS.

Zytometrische Analysen bei systemischen Autoimmunerkrankungen

Zusammenfassung Im Rahmen von rheumatisch-entzündlichen Erkrankungen, insbesondere den Kollagenosen sind Autoantikörper von diagnostischer und auch ätiopathogenetischer Relevanz. Über die Zellen, die diese Autoantikörper produzieren, ist sehr wenig bekannt. Zytometrische Analysen gestatten, Zellen des Immunsystems anhand spezifischer Markermoleküle zu identifizieren und so die Veränderungen des Immunsystems bei Autoimmunpatienten zu erfassen. Diese Untersuchungen können neben Einsichten in die Pathogenese auch neue diagnostische und therapeutische Ansätze bieten. Beispielhaft werden im Folgenden jüngere Daten zu veränderten B-Zellpopulationen bei Patienten mit SLE und Sjögren-Syndrom dargestellt.Summary A number of autoantibodies play a significant role in collagen vascular diseases and represent diagnostic markers of some of these entities. Despite increasing knowledge of these serological findings, data are limited about potential disturbances of precursor cells that finally lead to the autoantibody producing plasma cells. Recent evidence of disturbed B cell homeostasis indicates that the peripheral B cell compartments in systemic lupus erythematodes (SLE) and Sjögren’s syndrome are characteristically different to normal. Although the identification of autoreactive B cells in peripheral blood is still subject of ongoing studies, the differences in B cell subsets add to the understanding of the immunopathogenesis of these diseases and may provide new diagnostic clues and therapeutical avenues of these entities.