Johannes Irsch

Isolation and characterization of allergen-binding cells from normal and allergic donors.

BACKGROUND Flow cytometry of the immune system so far has been limited to the analysis of subpopulations according to lineage markers. The cells involved in a particular immune response could not be assayed due to their low frequency. Here we show the potential of antigen-specific high gradient magnetic cell sorting to enrich cells for visualisation in multiparameter cytometry, functional studies and immortalization. OBJECTIVES The aim of this study was the development of an efficient technology for staining and isolation of antigen-binding cells from human peripheral blood. In particular, allergen-specific cells from normal and allergic donors should be analysed and compared to develop a cellular diagnosis of allergy. STUDY DESIGN The rare antigen-specific cells were sorted by high-gradient magnetic cell sorting with MACS. Haptenized phospholipase A2 (PLA2), the major allergen of bee venom, or haptenized ParoI, the major allergenic component of Parietaria officinalis, were used as antigens. The cells from normal and allergic donors, binding to the allergen were characterized phenotypically by immuno-fluorescence. Allergen-specific B-cells were immortalized by EBV transformation. RESULTS AND CONCLUSION Allergen-specific cells can be enriched from blood of both allergic and normal donors to purities of up to 75%, by high gradient magnetic cell sorting. The specificity of labelling with allergen was confirmed by establishing allergen-specific EBV-transformed B-cell lines from the sorted cells. Clear differences exist in the cellular composition of allergen-binding cells from normal compared to allergic donors. In normal donors the allergen-binding cells are B-cells expressing CD19 and CD21. In allergic donors, in addition to allergen-binding B-cells, occurring in about equal absolute numbers as in normal donors, basophilic granulocytes are labeled by allergen. These cells express CD38, CD9 and CD25 on their surface, and stain for IgE.

Class switch recombination was specifically targeted to immunoglobulin (Ig)G4 or IgA in Hodgkin's disease-derived cell lines

In T cell‐dependent immune responses, class switch recombination occurs in germinal centres. There is now evidence that Hodgkin/Reed–Sternberg cells are derived from germinal centre B cells. Cytokines specifically determine the direction of class switching, i.e the isotype of the new antibodies. We performed restriction analyses and polymerase chain reaction on the immunoglobulin heavy chain loci for five Hodgkins disease‐derived B‐cell lines and one Hodgkins disease‐derived T‐cell line in order to analyse class switch recombination. In all the B‐cell lines, class switch recombination had been targeted to Cα4 or Cα1/2. This showed that cell‐line precursors had undergone class switching, probably under the influence of TH2 or TH3 cell‐derived cytokines. Deletions comprising several constant region genes were observed in cell lines L428, L1236, L591 and KMH2. Karyotype analyses of two of these revealed translocational breakpoints within the immunoglobulin heavy chain gene locus. Our data support the view that a chromosomal instability may occur during class switch recombination in Hodgkin/Reed–Sternberg cells causing chromosomal breaks. Thus, as in other germinal centre B cell‐derived lymphomas, the immunoglobulin gene locus may be frequently involved in structural chromosomal aberrations in Hodgkins disease.

The frequency of phospholipase A2 binding of basophilic granulocytes does not decrease during bee-venom-specific immunotherapy

Background: The major allergenic component of bee venom is phospholipase A2 (PLA2).