A novel blocking monoclonal antibody recognizing a distinct epitope of human CD40 molecule.

CD40, a member of the tumor necrosis factor receptor superfamily, is an important costimulatory molecule during the immune response. Here, we report a blocking mouse antihuman CD40 monoclonal antibody, mAb 3G3, of which the specificity was verified by flow cytometry and Western blot. It was shown by competition test that 3G3 bound to a different site (epitope) of CD40 from the reported CD40 mAbs, including clone mAb89, 3B2, and 5C11. It was also found that mAb 3G3 could inhibit homotypic aggregation of Daudi cells induced by the agonistic anti-CD40 mAb 5C11. Furthermore, mAb 3G3 effectively inhibited the proliferation of peripheral blood mononuclear cells in mixed lymphocyte reaction assay. Finally, a sensitive and specific soluble CD40 (sCD40) ELISA kit was established by matching mAb 3G3 with 5C11, and it was found that the levels of sCD40 in sera from patients with immune disorders such as hyperthyroidism, chronic nephritis, and rheumatoid arthritis were obviously higher than those from normal individuals. Thus, this blocking anti-CD40 mAb provides a novel tool for the study of CD40.

Section 1C: Assessment of the functional activity and IgG Fc receptor utilisation of 64 IgG Rh monoclonal antibodies. Coordinator's report.

Sixty-four IgG Rh monoclonal antibodies (Mabs) submitted to the Fourth International Workshop on Monoclonal Antibodies Against Human Red Blood Cells and Related Antigens were characterised and tested in quantitative functional assays at five laboratories. The biological assays measured the ability of anti-D to mediate phagocytosis or extracellular lysis of RBC by IgG Fc receptor (Fc gamma R)-bearing effector cells. Interactions of RBC pre-sensitised with anti-D (EA-IgG) with monocytes in chemiluminescence (CL) assays were found proportional to the amount of IgG anti-D on the RBC. Using antibodies to inhibit Fc gamma RI, Fc gamma RII or Fc gamma RIII, the only receptor utilised in the monocyte CL and ADCC assays for interactions with EA-IgG1 was found to be Fc gamma RI. In these assays, enhanced interactions were promoted by EA-IgG3 and additional Fc gamma receptors may have contributed. IgG2 anti-D was not reactive in these assays and EA-IgG4 promoted weak reactions through Fc gamma RI. A macrophage ADCC assay showed that haemolysis of EA-IgG3 was greater than that of EA-IgG1, mediated mainly through Fc gamma RIII. In ADCC assays using lymphocytes (NK cells) as effector cells and papainised RBC target cells, only a minority of IgG1 anti-D Mabs were shown to be able to mediate haemolysis in the presence of monomeric IgG (AB serum or IVIg). These interactions were mediated solely through Fc gamma RIII. Haemolysis via Fc gamma RIII may depend on the presence of certain sugars on the oligosaccharide moiety of IgG. Most Mabs (IgG1, IgG2, IgG3 and IgG4) elicited intermediate, low or no haemolysis in these assays. Blocking studies indicated that low activity IgG1 and IgG4 anti-D utilised only Fc gamma RI. Other IgG1 and IgG3 Mabs appeared to promote haemolysis through Fc gamma RI and Fc gamma RIII while IgG2 was inhibited by Mabs to both Fc gamma RII and Fc gamma RIII, suggesting a variety of Fc gamma R are utilised for anti-D of low haemolytic activity. Excellent agreement between the results of the lymphocyte ADCC assays and antibody quantitation was observed between the participating laboratories.

Human cytomegalovirus infection of bone marrow myofibroblasts enhances myeloid progenitor adhesion and elicits viral transmission.

Human cytomegalovirus (CMV) infection of bone marrow transplant recipients can cause pancytopenia, as well as life-threatening interstitial pneumonia. CMV replicates actively in bone marrow stromal cells, whereas it remains latent in hematopoietic progenitors. Our aim was to study the influence of CMV infection on adherence of CD34(+) cells to the myofibroblastic component of human bone marrow and examine transmission of virus from myofibroblasts to CD34(+) cells. We show that smooth actin, but not fibronectin, organization is markedly modified by CMV infection of bone marrow stromal myofibroblasts. Nonetheless, CMV infection led to increased adherence of the CD34(+) progenitor cell line, KG1a, relative to adherence to uninfected myofibroblasts from the same donors. Adherence of CD34(+) cells to infected bone marrow myofibroblasts resulted in transfer of virions and viral proteins through close cell-to-cell contacts. This phenomenon may play a role in the pathophysiology of CMV bone marrow infection and in eventual virus dissemination.

Remote tumor cells in the case of breast cancers: significance of their presence for prognosis [corrected].

Through two clinical studies, tumor cells were searched for in the bone biopsies and cytapherisis of patients suffering from inflammatory tumors and who had undergone intensive therapy and autografts (Pegase 2 program). In these studies we used immunocytochemical test with two monoclonal antibodies. The results have shown the presence of tumor cells in 14% of the patients (respectively 18%), with no correlation to the appearance of metastases after 4 years in the first study. Nevertheless, the presence of these tumor cells seems to be an important factor in the number of relapses. It seems important to develop research into tumor contamination especially in the selection of grafts over the next few years.

Obtaining a functional recombinant anti-rhesus (D) antibody using the baculovirus-insect cell expression system.

The cloning and production of a human anti-rhesus (Rh) D monoclonal antibody (mAb) using the baculovirus-insect cell expression system is described. This monoclonal recombinant antibody R.D7C2 derived from a human parental IgM lambda immunoglobulin was obtained after immortalization of lymphocytes by Epstein-Barr virus (EBV). The human heavy (VH) and light (VL) variable regions were cloned from the parental cell line and genetically fused to the human constant IgG1 heavy (H) and light (L) chain genes (gamma 1 and lambda, respectively). A recombinant baculovirus was constructed that directs the co-expression of genes encoding both genetically fused heavy and light chains under the control of two late and strong baculovirus promoters. After infecting the Spodoptera frugiperda (Sf9) insect cell line with this baculovector, a complete IgG1 mAb was secreted in the culture medium indicating that each immunoglobulin chain was correctly processed and assembled with a functional glycosylation into a tetrameric form. In vitro analysis showed that the functional properties of R.D7C2 using agglutination tests were efficient for the specific recognition of Rh-D-positive red blood cells (RBC). In addition, R.D7C2 showed effector functions of the gamma 1 heavy chain resulting in the lysis of Rh+ papain RBC by an antibody-directed cellular cytotoxicity mechanism. These results demonstrate that R.D7C2 can be produced in the baculovirus-insect cell expression system as a source for potential therapeutic application in the treatment of the haemolytic disease of the newborn.

Serum interleukin-10 in acquired immunodeficiency syndrome lymphoma patients. Seroco-Hemoco Study Group.

Interleukin-10 (IL-10) has multiple effects on lymphoid development, particularly as a stimulant of activated B-cell proliferation and differentiation. It is thought that IL-10 might play a role in the development of B lymphoid malignancies based on the observation that lymphomatous tissues from HIV+ patients contain numerous cells containing IL-10 mRNA as well as IL-10 protein. The aim of this study using an Elisa test was to analyze IL-10 in the serum of 18 HIV+ patients with non Hodgkin's B lymphoma (NHL) and compared the presence of this cytokine in the serum of 18 HIV+ patients without NHL. In this comparative study we also considered the different parameters such as the mode of contamination, sex, age and number of CD4 cells. 44% of the patients with HIV-related NHL had significant levels of IL-10 (> or = 12 pg/ml) in their serum, in comparison to the patients without NHL who did not show detectable serum IL-10.