Éric Aubin

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.

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.

A plant-derived quadrivalent virus like particle influenza vaccine induces cross-reactive antibody and T cell response in healthy adults

Recent issues regarding efficacy of influenza vaccines have re-emphasized the need of new approaches to face this major public health issue. In a phase 1-2 clinical trial, healthy adults received one intramuscular dose of a seasonal influenza plant-based quadrivalent virus-like particle (QVLP) vaccine or placebo. The hemagglutination inhibition (HI) titers met all the European licensure criteria for the type A influenza strains at the 3μg/strain dose and for all four strains at the higher dosages 21days after immunization. High HI titers were maintained for most of the strains 6months after vaccination. QVLP vaccine induced a substantial and sustained increase of hemagglutinin-specific polyfunctional CD4 T cells, mainly transitional memory and TEMRA effector IFN-γ(+) CD4 T cells. A T cells cross-reactive response was also observed against A/Hong-Kong/1/1968 H3N2 and B/Massachusetts/2/2012. Plant-based QVLP offers an attractive alternative manufacturing method for producing effective and HA-strain matching seasonal influenza vaccines.

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.

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.

Absence of cytokine modulation following therapeutic infusion of intravenous immunoglobulin or anti‐red blood cell antibodies in a mouse model of immune thrombocytopenic purpura

Human intravenous immunoglobulin (IVIg) and anti‐D immunoglobulin preparations are used in the treatment of immune thrombocytopenic purpura (ITP). One mechanism proposed to explain their therapeutic effects in ITP patients is the induction of expression of anti‐inflammatory cytokines, such as interleukin (IL)‐10 or IL‐1ra, leading to a reduction of phagocytic activity of the reticuloendothelial system. However, increased expression of pro‐inflammatory cytokines was also noted following treatment of ITP patients, raising doubt on the actual contribution of anti‐inflammatory cytokines in the therapeutic effects of IVIg and anti‐D immunoglobulins. The present study evaluated the in vivo modulation of expression of a large array of inflammatory cytokines using a mouse model of thrombocytopenia. IVIg was not found to modulate cytokine expression although it efficiently prevented thrombocytopenia. In contrast, protective (M1/69) and non‐protective (TER‐119) anti‐mouse red blood cell (RBC) antibodies (mimicking anti‐D treatment) both increased the expression of CXCL‐1 and CXCL‐5. Thus, there was no relationship between inflammatory cytokine expression and prevention of thrombocytopenia by IVIg or anti‐mouse RBC in the ITP mouse model. These results suggest that the increase in cytokine expression observed in ITP patients following IVIg or anti‐D infusion is not required for their therapeutic effects but may rather represent a side‐effect of the treatment.

Reduced frequency of blood donors with false‐positive HIV‐1 and ‐2 antibody EIA reactivity after elution of low‐affinity nonspecific natural antibodies

BACKGROUND : The detection by EIA of antibodies (Abs) specific to HIV antigens in the serum of blood donors is important for transfusion safety. A small but significant number of donor sera (0.1‐0.3%) yield false‐positive results in EIA, and these donors must be permanently deferred from the blood donor list, causing operational and public relations problems.

Increased secretion of hyperimmune antibodies following lipopolysaccharide stimulation of CD40-activated human B cells in vitro

Human B cells can be cultured ex vivo for a few weeks, following stimulation of the CD40 cell surface molecule in the presence of recombinant cytokines such as interleukin‐4 (IL‐4). However, attempts to produce polyclonal antigen‐specific human antibodies by in vitro culture of human B cells obtained from immunized donors have not been successful. It has been shown in mice that lipopolysaccharide (LPS) is a potent mitogen for B cells and plays an important role in the generation of antigen‐specific antibody responses. Although it has long been believed that LPS has no direct effect on human B cells, recent data indicating that IL‐4‐activated human B cells are induced to express Toll‐like receptor‐4, the main LPS receptor, prompted us to study the effects of LPS on the proliferation and antibody secretion of human B cells. Our results showed that LPS caused a reduction in the expansion of CD40‐activated human B cells, accompanied by an increase in antigen‐specific antibody secretion. This result suggested that some, but not all, B cells were able to differentiate into antibody‐secreting cells in response to LPS. This increased differentiation could be explained by the observation that LPS‐stimulated human B cells were induced to secrete higher amounts of IL‐6, a pleiotropic cytokine well‐known for its B‐cell differentiation activity. In vivo, the effect of LPS on cytokine secretion by B cells may not only enhance B‐cell differentiation but also help to sustain a local ongoing immune response to invading Gram‐negative bacteria, until all pathogens have been cleared from the organism.

Immunomodulatory effects of therapeutic preparations of human albumin

Background and Objectives  Albumin is the most abundant protein in plasma and is considered to be immunologically inert. However, we recently observed that therapeutic human albumin preparations, used as protein control in studies involving high doses of IVIg, modulated the MHC II‐restricted activation of antigen‐specific T cells. In the present work, we characterized this effect in more details.