Gunther E. Dannecker

Anti-histone autoantibodies react specifically with the B cell surface

In an attempt to induce an immune response against Mls-1a antigens by immunizing C57B1/6 mouse (Mls-1b) with purified B cells from DBA/2 mouse (Mls-1a), we generated a panel of monoclonal antibodies from which the 5B9.6 mAb, taken as a representative antibody, was thoroughly investigated. This antibody specifically reacts with B cells from all mouse strains studied including C57Bl/6 mice as shown by FACS analysis of double-antibody labelled spleen cells. Using enzyme immunoassays and immunoprecipitation techniques, 5B9.6 mAb was found to be specific for histones. Amino acid sequence analysis of a peptide derived from a 5B9.6-immunoprecipitated polypeptide from DBA/2 B cells showed a 100% homology with a sequence within H2B histones. Furthermore, 5B9.6 mAb was able to interact with the cell surface of 7OZ/3 cell line, known as a typical pre-B cell line. The presence of histones can be modulated on the surface of 7OZ/3 cells since this antigen was upregulated after exposure of these cells to a cocktail of IL-1 and cAMP. Finally, 5B9.6 mAb was shown to interact with freshly isolated B cells from human peripheral blood.

Expression and regulation of B7 family molecules on macrophages (MΦ) in preterm and term neonatal cord blood and peripheral blood of adults

Macrophage (MΦ) receptors of the B7 family (CD80, CD86) play a crucial role in T cell activation: the lack of costimulation leads to anergy or apoptosis of reactive T cells. MΦ may differentiate into different subsets, the balance of which defines MΦ‐dependent T cell reactions. The aim of this study was to examine neonatal and adult T cell response with respect to the costimulatory MΦ‐potential in order to identify molecular predictors for the neonatal immune defense.

Activation of human T cells by the superantigen staphylococcus enterotoxin B : analysis on a cellular level

Superantigens interact with and activate a sizeable fraction of T cells characterized by expression of specific V beta gene segments of their antigen receptor. The massive activation of T cells in an organism is considered responsible for clinical symptoms associated with superantigen-producing bacteria. Here we studied the in vitro activation of human T cells by the superantigen Staphylococcus Enterotoxin B on a cell by cell basis. Superantigen-reactive T cells were stained with a V beta 12-specific monoclonal antibody and analyzed in a cytofluorograph. Blast formation of SEB-reactive T cells occurs within 12 h and reaches a plateau after 24 h. Double-staining of V beta 12+ T cells with antibodies against different T cell activation or adhesion surface molecules revealed a time-dependent differential upregulation for CD2, CD11 = LFA-1, CD25, CD28, CD69, and HLA-DR. The expression of CD3, CD4 and CD5 was not influenced by the superantigen. The rapid phenotypic changes of superantigen reactive T cells in terms of marker expression and cell size could provide early tools in diagnosing diseases caused by superantigens.

Superantigen-staphylococcal-enterotoxin-A-dependent and antibody-targeted lysis of GD2-positive neuroblastoma cells.

Superantigens such as the staphylococcal enterotoxin A (SEA) are among the most potent T cell activators known. They bind to major histocompatibility complex (MHC) class II molecules and interact with T cells depending on their T cell receptor (TCR) Vβ expression. Superantigens also induce a variety of cytokines and trigger a direct cytotoxic effect against MHC-class-II-positive target cells. In order to extend superantigen-dependent cell-mediated cytotoxicity (SDCC) to MHC-class-II-negative neuroblastoma cells, SEA was linked to the anti-ganglioside GD2 human/mouse chimeric monoclonal antibody (mAb) ch14.18. Ganglioside GD2 is expressed on most tumours of neuroectodermal origin but is expressed to a lesser extent on normal tissues. The linkage of ch 14.18 to SEA was achieved either with a protein-A-SEA fusion protein or by chemical coupling. Both constructs induced T-cell-mediated cytotoxicity towards GD2-positive neuroblastoma cells in an effector-to-target(E∶T)-ratio-and dose-dependent manner in vitro. To reduce the MHC class II affinity of SEA, a point mutation was introduced in the SEA gene (SEAm9) that resulted in 1000-fold less T cell killing of MHC-class-II-expressing cells as compared to native SEA. However, a protein-A-SEAm9 fusion protein mediated cytotoxicity similar to that of protein-A-SEA on ch14.18-coated, MHC-class-II-negative neuroblastoma cells. Taken together, these findings suggest that superantigen-dependent and monoclonal-antibody-targeted lysis may be a potent novel approach for neuroblastoma therapy.

The toxic shock syndrome toxin-1 induces anergy in human T cells in vivo

TSST-1 is a Staphylococcus aureus-derived superantigen which has been implicated in the pathogenesis of toxic shock syndrome. In mice, superantigen-induced proliferation is followed by deletion or anergy of reactive T cells. So far, superantigen-induced T-cell anergy has not been observed in humans. We therefore examined PBMCs derived from a 15-year-old patient suffering from severe toxic shock syndrome. Markedly elevated levels of circulating TSST-1-reactive T cells were found by cytofluorometric analysis. Upon in vitro restimulation with TSST-1, hyporesponsiveness of TSST-1-responsive V beta 2+ T cells was detected, thus confirming results obtained in the murine system.

Effect of Dexamethasone on B7 Regulation and T Cell Activation in Neonates and Adults

The safety of dexamethasone for neonates has been questioned, partly because of its multiple unspecific effects on the immune system. Specific effects of dexamethasone on co-stimulatory and immune suppressive functions of neonatal compared with adult macrophages (MΦ) are not known. We evaluated the effect of dexamethasone on the expression and regulation of MΦ B7 family receptors (B7-1, CD80; B7-2, CD86) and on their ability to co-stimulate T cells. Cord blood macrophages (CBMΦ) and MΦ from healthy adults (PBMΦ) were isolated, and cell surface markers were phenotyped by flow cytometry. In tissue culture, cells were exposed to dexamethasone, interferon-γ (IFN-γ), cAMP, or a T cell mitogen (αCD3) and examined for their capacity to activate or destroy T cells. CBMΦ were less able to up-regulate CD80 and CD86 than PBMΦ (p < 0.05). Dexamethasone inhibited the up-regulation of CD80, CD86, and HLA-DR on PBMΦ and even more so on CBMΦ (p < 0.05 versus PBMΦ for CD80 and CD86). In the presence of dexamethasone, stimulation with αCD3 MAb enhanced cytotoxic functions of PMBMΦ and CBΜΦ with an increase in deleted T cells, a reduced fraction of enlarged T cells, and an inhibition of T cell CD28 up-regulation, which again were more pronounced with CBMΦ (p < 0.05 versus PBMΦ). In conclusion, neonatal MΦ are exquisitely sensitive to the inhibitory effects of dexamethasone on B7 expression. Although perhaps producing the desired therapeutic effect, dexamethasone may do so in newborns at the expense of a near complete paralysis of MΦ-dependent T cell function.

Diminished Response to Interleukin-10 and Reduced Antibody-Dependent Cellular Cytotoxicity of Cord Blood Monocyte-Derived Macrophages

Monocyte-derived macrophage (MΦ) subsets are generated by antagonistic induction pathways. A helper MΦ-type (Mh-MΦ) is induced by interferon gamma (IFN-γ), whereas a cytotoxic MΦ-type (Mc-MΦ), induced by interleukin-10 (IL-10), is a potent mediator of antibody-dependent cellular cytotoxicity (ADCC). Compared with MΦ from healthy adults [peripheral blood monocyte-derived macrophages (PBMΦ)], cord blood MΦ (CBMΦ) were found less capable of generating Mh-MΦ. Here we tested the hypothesis that their generation of Mc-MΦ via IL-10 is also impaired. MΦ surface markers were phenotyped. IL-10 protein and mRNA production were detected after stimulation [αCD3 monoclonal antibody (mAb)]. CBMΦ or PBMΦ were co-cultured with MΦ-depleted mononuclear cells of adults and CD4-targeting antibodies as models for ADCC were added. In cord blood, we found diminished αCD3-induced IL-10 protein and mRNA production (p < 0.05 versus adults). Basal CD16 and HLA-DR expressions on CBMΦ of preterm and full-term neonates were lower (p < 0.05 versus PBMΦ). IL-10 had reduced effects on CD16 up- and HLA-DR down-modulation on CBMΦ (p < 0.05 versus PBMΦ). CD4-directed receptor modulation and deletion were reduced in the presence of CBMΦ (p < 0.05 versus PBMΦ). IL-10 failed to enhance their ADCC capacity, which was in contrast to PBMΦ (p < 0.05). These data suggest that CBMΦ have an impaired cytotoxic capacity via lower sensitivity toward IL-10.

[Updated statement by the German Society for Pediatric and Adolescent Rheumatology (GKJR) on the FDA's report regarding malignancies in anti-TNF-treated patients from Aug. 4, 2009].

TNF inhibitors and other biologicals have greatly expanded the therapeutic options for juvenile idiopathic arthritis (JIA). While the efficacy of etanercept and adalimumab has been proven in randomized controlled clinical trials, their long-term safety remains the subject of ongoing investigations. Reports of leukaemia and tumours in children and adolescents treated with etanercept, infliximab and adalimumab have raised questions about an increased risk for malignancies, with lymphoma accounting for the largest group at 50% of all 48 malignancies reported by the FDA.Consequently, TNF inhibitors should be indicated under careful consideration of individual risk factors, such as increased family occurrence of malignancies, or pre-treatment with carcinogenic substances such as cyclophosphamide. This is particularly true for non-approved substances, and non-approved indications, and for combination therapy of TNF inhibitors with immunosuppressive drugs. On the other hand, however, treatment should not be stopped or started in any patient in whom treatment is necessary due to the current knowledge. Adequate patient information, surveillance and documentation of treatment in the registry of the GKJR is strongly recommended.

Aktualisierte Stellungnahme der GKJR zur Meldung der FDA über Fälle von Malignomen bei Anti-TNF-behandelten Patienten vom 04.08.2009

TNF inhibitors and other biologicals have greatly expanded the therapeutic options for juvenile idiopathic arthritis (JIA). While the efficacy of etanercept and adalimumab has been proven in randomized controlled clinical trials, their long-term safety remains the subject of ongoing investigations. Reports of leukaemia and tumours in children and adolescents treated with etanercept, infliximab and adalimumab have raised questions about an increased risk for malignancies, with lymphoma accounting for the largest group at 50% of all 48 malignancies reported by the FDA.Consequently, TNF inhibitors should be indicated under careful consideration of individual risk factors, such as increased family occurrence of malignancies, or pre-treatment with carcinogenic substances such as cyclophosphamide. This is particularly true for non-approved substances, and non-approved indications, and for combination therapy of TNF inhibitors with immunosuppressive drugs. On the other hand, however, treatment should not be stopped or started in any patient in whom treatment is necessary due to the current knowledge. Adequate patient information, surveillance and documentation of treatment in the registry of the GKJR is strongly recommended.

Differential Expression of T Cell Receptor Variable β Genes on CD4+ and CD8+ T Cells: Influence by Sex Linked Genes?

We examined the expression of seven V alpha or V beta T cell receptor (TCR) segments on human CD4+ and CD8+ T cells. Confirming previously published results, we found a preferential expression of four V segment gene products on CD4+ T cells. One of these markers (V beta 6.7) was constantly expressed on more CD4+ T cells than CD8+ T cells. None of the analyzed blood samples showed a complete deletion of T cells expressing a particular V beta gene segment. In addition, our data provide the first evidence that genes on sex chromosomes may influence the formation of the human T cell repertoire. The ratio of CD4+/CD8+ T cells expressing V beta 12 gene products was always > or = 1 in female donors, whereas approximately 30% male donors exhibited more CD8+V beta 12+ T cells than CD4+V beta 12+ T cells.