Renée Bazin

Indirect inhibition of in vivo and in vitro T cell responses by intravenous immunoglobulins due to impaired antigen presentation

Several clinical studies done with intravenous immunoglobulin (IVIg)-treated autoimmune patients as well as several in vitro studies have revealed that IVIg can reduce polyclonal T-cell activation and modify their cytokine secretion pattern. However, their effect on (auto)antigen-specific T-cell responses has never been addressed directly. In the present work, we used an in vivo model of induction of antigen-specific T-cell responses and an in vitro antigen presentation system to study the effects of IVIg on T-cell responses. The results obtained showed that IVIg inhibited both the in vivo and in vitro antigen-specific T-cell responses but that this effect was the indirect consequence of a reduction in the antigen presentation ability of antigen-presenting cells. The inhibitory effect of IVIg was FcgammaRIIb-independent, suggesting that IVIg must interfere with activating FcgammaRs expressed on antigen-presenting cells to reduce their ability to present antigens. Such inhibition of T-cell responses by reducing antigen presentation may therefore contribute to the well-known anti-inflammatory effects of IVIg in autoimmune diseases.

Reversal of immune thrombocytopenia in mice by cross-linking human immunoglobulin G with a high-affinity monoclonal antibody

Intravenous immunoglobulins (IVIgs) are used to treat an increasing number of autoimmune diseases, but their exact mechanism of action remains unknown. This study showed that cross‐linking of human IgG present in IVIg preparations using a mouse monoclonal anti‐human IgG generated complexes that prevented or reversed thrombocytopenia in mice more efficiently than IVIg. Furthermore, biologically active complexes were obtained simply by adding the monoclonal antibody to human serum. These results suggest the possible development of an IVIg‐free substitute through the ex vivo, and possibly in vivo, formation of immune complexes containing autologous IgG of immune thrombocytopenic purpura patients.

Tetramolecular immune complexes are more efficient than IVIg to prevent antibody-dependent in vitro and in vivo phagocytosis of blood cells

Intravenous immunoglobulins (IVIg) have immunomodulatory effects in vivo and are widely used in the treatment of autoimmune diseases, such as idiopathic thrombocytopenic purpura (ITP). The mechanisms by which IVIg can prevent platelet clearance in ITP patients are not fully understood but are known to require the participation of low affinity Fcγ receptors (FcγRs), which interact poorly with monomeric immunoglobulin G (IgG). Given the importance of low affinity FcγRs in the treatment of ITP, we hypothesized that immune complexes (IC) produced in vitro could reproduce the effects of IVIg. Small‐size tetramolecular IC were prepared using mouse monoclonal anti‐human IgG and human Fc fragments. The effects of tetramolecular IC and IVIg on the in vitro and in vivo inhibition of phagocytosis of opsonized blood cells were compared. The results obtained showed that tetramolecular IC were at least six times more efficient than IVIg to prevent phagocytosis of opsonized red blood cells in vitro, and clearance of platelets in the thrombocytopenic mouse model.

Increased proportion of B cell hybridomas secreting monoclonal antibodies of desired specificity in cultures containing macrophage-derived hybridoma growth factor (IL-6)

The addition of macrophage feeder cells or conditioned medium has been shown to increase the yield of murine hybridomas obtained after the fusion of myeloma cells and activated B lymphocytes. It has been shown recently that the conditioned medium contains a growth factor (HGF) active on newly formed hybridomas and that the human HGF is similar to B cell stimulatory factor 2 which can induce the synthesis of antibodies in transformed B cells. We have compared in several fusion experiments the stimulatory effects of HGF both on the yield of hybridomas and on the number of antibody-secreting hybridomas. The results obtained clearly showed that while the stimulatory effect of HGF on the yield of growth-positive wells was variable and sometimes barely detectable, the proportion of growth-positive wells containing monoclonal antibodies was consistently much higher in the HGF-containing cultures. These results suggest that the majority of the antibody-secreting newly formed hybridomas are sensitive to HGF and indicate that HGF is a very useful culture supplement for the generation of a high number of antibody-producing hybridomas even if it may not increase significantly the yield of viable hybridomas.

Brain Bioavailability of Human Intravenous Immunoglobulin and its Transport through the Murine Blood–Brain Barrier:

Intravenous immunoglobulin (IVIg) is currently evaluated in clinical trials for the treatment of various disorders of the central nervous system. To assess its capacity to reach central therapeutic targets, the brain bioavailability of IVIg must be determined. We thus quantified the passage of IVIg through the blood–brain barrier (BBB) of C57Bl/6 mice using complementary quantitative and qualitative methodologies. As determined by enzyme-linked immunosorbent assay, a small proportion of systemically injected IVIg was detected in the brain of mice (0.009±0.001% of injected dose in the cortex) whereas immunostaining revealed localization mainly within microvessels and less frequently in neurons. Pharmacokinetic analyses evidenced a low elimination rate constant (0.0053  per hour) in the cortex, consistent with accumulation within cerebral tissue. In situ cerebral perfusion experiments revealed that a fraction of IVIg crossed the BBB without causing leakage. A dose-dependent decrease of brain uptake was consistent with a saturable blood-to-brain transport mechanism. Finally, brain uptake of IVIg after a subchronic treatment was similar in the 3xTg-AD mouse model of Alzheimer disease compared with nontransgenic controls. In summary, our results provide evidence of BBB passage and bioavailability of IVIg into the brain in the absence of BBB leakage and in sufficient concentration to interact with the therapeutic targets.

Inhibition of B cell-mediated antigen presentation by intravenous immunoglobulins (IVIg).

Previous work from our laboratory revealed that IVIg interacted with intracellular proteins involved in antigen presentation in B cells, suggesting that IVIg might interfere with the process of antigen presentation in these cells. In the present work, we used an in vitro assay with ovalbumin as model antigen and showed that IVIg inhibited both BCR-dependent and BCR-independent antigen presentation. The inhibition could not be explained by a modulation of expression of MHC II molecules expressed on B cells and was shown to occur in an FcgammaRIIb-independent manner, suggesting that the events responsible for the inhibitory effect occur at the intracellular level. This was supported by the observation of a direct correlation between the level of spontaneous internalization of two different proteins (IVIg and HSA) and their inhibitory potential. The inhibition of B cell-mediated antigen presentation reported here may help explain some of the anti-inflammatory effects of IVIg observed in treated patients, such as a decrease in autoantibody production.

Spontaneous internalization of IVIg in activated B cells

Previous work from our laboratory showed that IVIg could directly influence the fate of human B cells by inducing their differentiation. The initial goal of the present study was to identify the cell surface molecules recognized by IVIg on human B cells. Purified resting and CD40-activated human B cells were incubated with IVIg and lysed prior to immunoprecipitation. The immunoprecipitated proteins were identified by mass spectrometry (LC-MS). This analysis revealed that BCR, as well as other cell surface receptors or membrane associated proteins were the main targets of IVIg. Surprisingly, intracellular proteins were also found in the immunoprecipitates, suggesting that IVIg could penetrate inside living cells and interact with intracellular targets. We have further studied this unexpected phenomenon and obtained evidence indicating that a significant amount of IVIg was spontaneously internalized inside living cells. We showed that IVIg internalization could occur in a BCR- and FcgammaR-independent pathway. Furthermore, spontaneous IVIg internalization was also observed in whole blood incubated with therapeutic concentrations of IVIg, even in presence of the high endogenous IgG concentration. These observations first suggest that spontaneous internalization can occur in IVIg-treated patients and also that some of the observed alterations in the physiology of IVIg-treated cells may not be only dependent on extracellular interactions of IVIg with cell surface receptors or soluble plasma proteins but may also involve intracellular interactions.

Picogram doses of lipopolysaccharide exacerbate antibody-mediated thrombocytopenia and reduce the therapeutic efficacy of intravenous immunoglobulin in mice

Exacerbation of antibody‐mediated thrombocytopenia following infection with viruses has recently been demonstrated in a mouse model of the disease. The phenomenon was caused by an increased activation of phagocytes through gamma‐interferon secretion in response to infection. Endotoxins from Gram‐negative bacteria are also known to be potent activators of phagocytic cells. The objective of the present work was to determine whether lipopolysaccharide (LPS) could exacerbate antibody‐mediated thrombocytopenia in vivo and so alter the therapeutic efficacy of intravenous immunoglobulin (IVIg), using a mouse model of thrombocytopenia. Very low doses of LPS (picogram range) and of anti‐platelet antibodies (nanogram range), which did not induce thrombocytopenia individually, could synergize in vivo, resulting in significant decreases in platelet counts. The therapeutic efficacy of IVIg in antibody‐mediated thrombocytopenia was significantly reduced in presence of LPS. These in vivo observations further support a role for bacterial infections in the aetiology of immune thrombocytopenic purpura (ITP) and may contribute to better understand the recognized lack of efficacy of IVIg in a significant proportion of patients with ITP.

Immunoglobulin G dimers and immune complexes are dispensable for the therapeutic efficacy of intravenous immune globulin in murine immune thrombocytopenia

BACKGROUND: Several mechanisms have been proposed to explain the therapeutic effects of intravenous immunoglobulin (IVIG) in immune thrombocytopenia (ITP). Noteworthy, a major role has been attributed to immunoglobulin (Ig)G dimers present in IVIG. It has also been suggested that immune complexes formed between IVIG and the patients proteins after infusion could contribute to the therapeutic effect of IVIG in several autoimmune disorders. We recently observed that in‐house preparations of polyclonal human IgG derived from small pools of plasma and devoid of IgG dimers were as efficient as IVIG in a mouse model of thrombocytopenia. In this work, we revisited the role of IgG dimers in the therapeutic effects of IVIG in ITP.

Prevention of T cell activation by interference of internalized intravenous immunoglobulin (IVIg) with MHC II-dependent native antigen presentation.

Activation of self-reactive CD4(+) T cells plays a central role in the initiation and maintenance of autoimmune diseases. We recently reported that intravenous immunoglobulin (IVIg) inhibits the MHC II-restricted CD4(+) T cell activation induced by the presentation of immune complexes. Because native antigens can also play a role in the induction of several autoimmune diseases, we determined whether IVIg could also affect CD4(+) T cell activation following presentation of native antigens by APCs. Here we report that IVIg significantly reduces the activation of CD4(+) T cells by native ovalbumin. The inhibitory effect is FcγR-independent and occurs following internalization of IVIg inside APCs, where it interferes with the intracellular events leading to MHC II-dependent antigen presentation. The effect of IVIg on native antigen presentation could therefore contribute to dampen the autoimmune reaction by reducing CD4(+) T cell activation and the subsequent inflammatory response induced by these cells.