J.L. Teillaud

Infusion of Fcγ fragments for treatment of children with acute immune thrombocytopenic purpura

Abstract Summary Treatment of acute immune thrombocytopenic purpura (ITP) with intravenous immunoglobulin (IVIG) induces partial or complete responses, shown by transient or persistent increases in platelet count. The clinical benefit could be due to blockade of the Fcγ receptor (FcγR); platelets sensitised by IgG could not be cleared by cells of the reticuloendothelial system if FcγR on these cells was blocked with IVIG. To find out whether this putative mechanism is correct, we treated twelve children who had acute ITP with intravenous infusions of Fcγ fragments. Eleven children showed rapid increases in platelet counts to above the critical value of 50 × 10 9 /L, thereby avoiding major haemorrhagic risk. The response was stable in six patients and transient in five. No adverse reactions were observed. In responders who had detectable platelet-associated IgG before treatment (> 1500 IgG per platelet), platelet IgG fell substantially with treatment. Serum soluble CD16 (sCD16 or sFcγRIII) concentrations, measured in five children, showed transient or stable increases that correlated with the rise in platelet count. No sCD16 was detected in the Fcγ preparation used. We conclude that the infusion of Fcγ fragments is an efficient treatment of acute ITP in children. The efficacy of Fcγ fragments strengthens the hypothesis that FcγR blockade is the main mechanism of action of IVIG in ITP, although other immunoregulatory mechanisms triggered by the presence of increased sCD16 concentrations in serum could be involved in the clinical benefit observed.

Ion channels and b cell mitogenesis

Given the presence of ionic channels at the membrane of lymphocytes, we have analyzed the effect of various channels blockers on B lymphocytes activation. TEA and 4-AP, two K+ channels blockers, quinine, a blocker of Ca2(+)-activated K+ channels, nickel and verapamil, two Ca2+ channels blockers, all inhibited LPS-induced B cell proliferation. However, these drugs neither inhibited the induction of Ia and Fc gamma RII expression nor cell enlargement and early RNA synthesis, indicating that the entry of B lymphocytes into G1 phase was not affected. In contrast, both late RNA synthesis and the induction of the TfR, which occur while the cell progress through G1, were inhibited by these blockers. These data show that TEA, quinine and verapamil block B lymphocyte activation during the G1 phase, probably between G1A and G1B. To question whether these effects were due to the block of voltage-activated K+ channels, we compared the ability of TEA, quinine, verapamil, 4-AP and nickel to block proliferation and K+ channels. A striking correlation was found for all the drugs but less for 4-AP. Moreover, TMA, a TEA analog unable to block K+ currents, did not affect B cell proliferation. Taken together, our data suggests that functional voltage-gated K+ channels are required at a precise stage of the G1 phase of the B cell cycle.

Immunoglobulin G-binding factors (IgG-BF) inhibit IgG secretion by, as well as proliferation of, hybridoma B cells

Mouse immunoglobulin G-binding factors (IgG-BF) produced either by activated T cells (ATC) or by hybridoma T cells (T2D4) directly inhibit the in vitro IgG secretion by hybridoma B cells. This inhibition affects IgG1, IgG2a and IgG2b and can be detected as early as after 2 h incubation of the cells with IgG-BF eluted from non-equilibrium pH gradient electrophoresis gels. Moreover, IgG-BF also exert a strong growth-inhibitory effect on hybridoma B cells without any detectable immediate cytotoxicity. These results provide an experimental basis to analyze the molecular and biological effects induced by IgG-BF on B cells.

Immunoglobulin-binding factors

It has been known for more than a decade that lymphocytes [1] and also granulocytes [2] and macrophages [3] release molecules capable of binding immunoglobulin (Ig) isotypes. First IgG-binding factors (IgG-BF) [1], then IgE-BF [4], IgA-BF [5] and more recently IgD-BF [6], were reported. Soon after the discovery of IBF, three of their major properties were characterized: (i) they are produced by cells, mostly T lymphocytes, bearing Fc receptors (FcR) for the same Ig isotype; thus, IgG-BF are produced by Fcq/R positive cells, lgE-BF, IgA-BF and IgD-BF are released by FceR, FcaR and Fc6Rpositive cells, respectively; (ii) their production increases upon interaction of Ig with FcR, IgG inducing IgG-BF release [7], IgE and IgA inducing the production of IgE-BF and IgA-BF, respectively [5, 8]; when lymphocytes are incubated in serum-free buffer, the appearance of IBF in the supernatant parallels the shedding of membrane FcR [9]; (iii) IBF have been found to regulate Ig production in vitro, in a strictly isotype-specific manner for IgE-BF and IgA-BF, and in a broader way for IgG-BF (which regulates IgG and lgM responses). Moreover, some IBF have been reported to exert dual effects: IgE-BF produced by T cells either potentiated or suppressed IgE production, depending on their glycosylation [10]. Both suppressive and potentiating IgA-BF [5, 11] and suppressive IgG-BF [7, 12] have also been described. Taken together, these obervations led, soon after the description of IBF, to the proposal that these molecules were in fact shed FcR [13] (the term solu-

Intracellular expression and functional properties of an anti-p21Ras scFv derived from a rat hybridoma containing specific λ and irrelevant κ light chains

Abstract A rat single-chain Fv (Y238 scFv) was derived from the Y13–238 monoclonal antibody, a non-neutralizing anti-Ras antibody. The Y13–238 hybridoma expresses two functional light chains. N-terminus microsequencing of these chains showed the presence of the Y3 Ag1.2.3 Vk chain derived from the rat fusion partner and of a rat Vl chain. Primers designed for rat Vl amplification allowed the cloning of a functional scFv that could bind p21Ras. The kinetics of interaction of purified Y238 scFv with the p21Ras protein was evaluated by BIAcore® with a NTA sensor chip and gave an apparent affinity constant in the nanomolar range (KD=4.58±0.63 nM). Immunoprecipitation experiments of Y238 scFv expressed in Xenopus laevis oocytes confirmed the specificity of the scFv for the Ras protein. Y238 scFv could be intracellularly expressed in oocytes and in mammaliam cells without adverse effect on the Ras signalling cascade. This scFv was therefore used as control in experiments where another anti-Ras scFv (Y259 scFv, derived from the neutralizing anti-Ras mAb Y13–259) blocked the Ras pathway in vitro and led to tumor regression in a nude mouse model [Cochet, O., Kenigsberg, M., Delumeau, I., Virone-Oddos, A., Multon, M.C., Fridman, W.H., Schweighoffer, F., Teillaud, J.L., Tocque, B., 1998. Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression. Cancer Res. 58, 1170–1176.]. Finally, BIAcore® analyses indicated that the epitopes recognized by Y238 and Y259 scFvs are not overlapping and allowed a more precise definition of the Y13–238 epitope.

Increased levels of soluble low-affinity Feγ receptors (IgG-binding factors) in the sera of tumour-bearing mice

Soluble forms of low affinity Feγ receptors (FcyR), also called IgG‐binding factors (IgG‐BF), have been shown to play a regulatory role in immune responses. By using an immunodot assay with the anti‐mouse FcyR MoAb, 2.4G2, the levels of IgG‐BF have been measured in the sera of mice bearing syngeneic tumours of lymphoid or non‐lymphoid origin or in mice injected with high doses of murine IgG. These sera contained large amounts of IgG‐BF as compared with controls. In the case of mice bearing IgG2a‐ or IgG2b‐secreting hybridomas or lymphomas, serum IgG‐BF increased progressively with tumour size and serum monoclonal IgG concentration, reaching 4–12 times the normal levels. A less than three‐fold increase was found in mice bearing an IgG1‐secreting hybridoma or tumours which do not secrete IgG (IgA‐secreting hybridoma, non‐immunoglobulin‐secreting lymphoid tumours or melanoma) or in mice injected with 9 mg of monoclonal IgG2a. The enhancement of serum IgG‐BF levels was independent of the expression of FcγR by the tumour cells, suggesting that the majority of IgG‐BF secreted in response to tumours was produced by the host rather than by the tumour. The increased production of IgG‐BF may participate in the control of tumour growth and in the modulation of the host immune responses in tumour‐bearing animals.

Software for the quantitative evaluation of in vitro monoclonal antibody production from ELISA data

Software has been developed which permits the quantitation of monoclonal antibodies secreted by B cell hybridomas. This program does not require the user to enter a large number of complex parameters and can be easily used without any previous computer experience. It fits all the experimental and standard curves by determining overlapping linear domains using the linear least-squares method. The program is based on logarithmic interpolations for determining Ig concentrations comparing experimental samples to Ig concentrations in standards. It provides a complete print-out of the data with editing options and is written in BASIC EDEX 4.0 Commodore computer language. It permits the accurate quantification of minute amounts of monoclonal antibodies and can be used to detect the inhibitory or enhancing effects of lymphokines or cytokines on Ig secretion by hybridoma B cells.

Prognostic value of cytokine and soluble Fcγ receptor assays in oncology

Abstract The clinical course of a cancer is influenced by the interaction of tumour cells with the patients immune system. It is thus conceivable that immunological parameters may be used as markers of prognostic or predictive value. We report here that increased serum levels of IL-6 is a signal of poor prognosis and predicts unresponsiveness to immunotherapy in patients with metastatic melanoma. In cervical cancer, IL-6 produced by infiltrating macophages is a marker of invasive cancer. In patients with multiple myeloma, the plasmatic levels of soluble Fcγ receptors are markers of the disease, sCD16 being drastically decreased and sCD32 being slightly increased.

Vav and SLP-76 recruitment by cross-linking of FcγRIIa1 in promyelocytic HL-60 cells

The signaling events induced upon cross-linking of the human FcgammaRIIa1 (CD32) which contains an immune receptor tyrosine-based activation motif (ITAM) in its intracellular region, were investigated in the promyelocytic HL-60 cells. It is shown here that the FcgammaRIIa1 engagement recruits the Ras pathway in these cells, as evidenced by the tyrosine-phosphorylation of the Shc adaptator protein and of the extracellular signal-regulated kinase 2 (ERK2). However, p95vav, a molecule able to interact with Rac-1 and to regulate cytoskeletal reorganization, was also found to be phosphorylated. In addition, co-immunoprecipitation experiments demonstrated that Vav is associated with SLP-76 upon FcgammaRIIa1 activation. A strong phosphorylation of p120cbl was also observed. The phosphorylation of molecules such as p95vav, SLP-76 and p120cbl suggests that FcgammaRIIa1 triggering also activates signaling pathways other than the Ras pathway.

A sensitive method for testing the effect of immunoglobulin binding factor on Ig secretion by hybridoma B cells

A microtiter plate assay to detect the effect of immunoglobulin binding factor (IBF) on Ig secretion by hybridoma B cells is described. This technique permits the analysis of Ig secretion by only 200-400 hybridoma B cells using a PFC assay and an ELISA, and therefore increases the IBF/cell number ratio. This increase allows the detection of a strong IBF-mediated inhibitory effect on Ig secretion by hybridoma B cells, which is otherwise difficult to obtain reproducibly. The technique is simple, does not require any transferring step and is convenient, since it permits large numbers of samples to be tested. It can be extended to test IBF for all isotypes or other lymphokines that affect Ig secretion.