Biomedical subjects
Publications and source records attributed to M Toungouz.
New immunotherapies derived from biotechnology offer fascinating perspectives in different fields of medicine including anti-infectious vaccines, cancer, organ transplantation and autoimmune diseases. In this paper, we illustrate how the Department of Immunology can contribute to the development of these new treatments within a academic hospital such as the Erasme Hospital at the Université Libre de Bruxelles.
Although the persistence of donor-type hematopoietic cells in low numbers (microchimerism) is well established in some transplant recipients, its relevance for graft acceptance is still a matter of debate. On the other hand, clonal deletion of donor-specific alloreactive cells associated with mixed chimerism (macrochimerism) has reliably produced long-term graft tolerance in pre-clinical models. So far, the cytoablative conditioning regimens required to achieve mixed chimerism have hampered the clinical development of such protocols. Here, we discuss recent observations suggesting that the deliberate induction of hematopoietic cell chimerism might become a feasible strategy to achieve transplantation tolerance in clinics.
Assessment of T-cell activation is pivotal for evaluation of cancer immunotherapy. We initiated a clinical trial in patients with MAGE-A1 and/or -A3 tumors using autologous DC pulsed with MAGE peptides aimed at analyzing T-cell-derived, IFN-gamma secretion by cytokine flow cytometry and ELISPOT. We also tested whether further KLH addition could influence this response favorably. Monocyte-derived DC were generated from leukapheresis products. They were pulsed with the relevant MAGE peptide(s) alone in group A (n=10 pts) and additionally with KLH in group B (n=16 pts). A specific but transient increase in the number of peripheral blood T lymphocytes secreting IFN-gamma in response to the vaccine peptide(s) was observed in 6/8 patients of group A and in 6/16 patients of group B. We conclude that anti-tumor vaccination using DC pulsed with MAGE peptides induces a potent but transient anti-MAGE, IFN-gamma secretion that is not influenced by the additional delivery of a nonspecific, T-cell help.
In cancer immunotherapy, the use of dendritic cells (DC) loaded with tumor-associated antigens (TAA) emerged as a promising strategy. We initiated 3 pilot clinical trials with immunological endpoints using TAA loaded autologous DC. These trials showed that this approach was safe and associated with the induction of potent TAA specific IFN-gamma responses, which were transient despite the providing a further help through KLH presentation. Subcutaneous (s.c.) IL-2 administration was associated with long-lasting TAA specific IL-5 production. Clinical responses were observed in about 1/3 of the patients. Further improvements will take advantage of the use of a new type of DC cells (IL-3/IFN-beta DC) and of tumor cell-DC hybrids.
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BACKGROUND AND OBJECTIVES: We previously found that interferon-gamma (IFN-gamma) antibodies in intravenous immunoglobulins (IVIG) can block not only IFN-gamma production and tumor necrosis factor-alpha secretion, but also T-cell proliferation. Since the presence of IFN-gamma antibodies has been attributed to previous viral infection, we hypothesized that the viral status of the plasma donors used for IVIG pools might be a decisive factor in controlling the immunosuppressive capacity of IVIG. MATERIALS AND METHODS: We tested three different pooled, human IVIG preparations for the presence of IFN-gamma antibodies by ELISA. RESULTS: Comparison of the immunomodulatory activity of polyvalent IVIG with that of specific CMV and HBs IVIG showed that the latter-had higher levels of IFN-gamma antibodies and an increased capacity to block mixed lymphocyte reaction and cytokine production. CONCLUSION: We propose that these in vitro assays constitute a basis for the selection of plasma intended for manufacturing IVIG aimed at immunosuppression in the transplant setting.
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The mechanism of action of intravenous immunoglobulins (IVIg) for prevention of graft rejection and graft-versus-host disease (GVHD) is poorly understood. Recently, it has been shown that these preparations contain natural antibodies directed toward interferon (IFN)-gamma. During mixed lymphocyte reaction (MLR), which constitutes an in vitro model of allograft rejection and GVHD, T cell recognition of HLA differences induces IFN-gamma release. This cytokine promotes T cell proliferation and acts as a macrophage-activating factor to provoke tumor necrosis factor-alpha secretion. The aim of the present work is to investigate the influence of IVIg on IFN-gamma production occurring during MLR and its subsequent impact on T cell proliferation and tumor necrosis factor (TNF)-alpha secretion. We tested IVIg preparations for the presence of anti-IFN-gamma and anti-TNF-alpha antibodies. High amounts of anti-IFN-gamma, but not anti-TNF-alpha antibodies, were found. IVIg addition at the initiation of culture resulted in IFN-gamma secretion blockade. Likewise, lymphocyte proliferation and TNF-alpha secretion were inhibited. This inhibition was reversed by the addition of recombinant human IFN-gamma. Furthermore, the inhibitory properties of IVIg were mimicked by an IFN-gamma-specific neutralizing monoclonal antibody. We conclude that the capacity of IVIg to inhibit proliferation and TNF-alpha release during MLR is due to IFN-gamma blockade by natural antibodies. This immunosuppressive mechanism could contribute to the effect of IVIg on prophylaxis of organ graft rejection and GVHD after allogeneic bone marrow transplantation.
Interleukin (IL)-10 is an immunosuppressive cytokine potentially involved in the control of the allogeneic response. Several studies failed to detect it in mixed lymphocyte reaction supernatants. However, experiments using IL-10-specific antibodies, revealing its inhibitory action on interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, provided indirect evidence that endogenous IL-10 was produced. The aim of the present work is to elucidate the role of IL-10 during mixed lymphocyte reaction and to investigate the influence of HLA-DR antigens on its production and on the regulatory loop involving TNF-alpha and IFN-gamma. Using a highly sensitive ELISA, a significant (P < 0.0001) but low IL-10 release could be detected (33.7 +/- 3.6 pg/ml) in response to HLA-DR disparities. However, IL-10 release was not graded as 1 DR mismatch (MM)-induced maximal secretion (32.3 +/- 5.1 pg/ml). This contrasted with TNF-alpha and IFN-gamma productions, which significantly increased in 2 DR MM pairs. Addition to IL-10-specific antibodies resulted in higher enhancement of INF-gamma (235 +/- 38% vs. 122 +/- 39%, P = 0.02) and, to a lesser extent, TNF-alpha (147 +/- 56% vs. 112 +/- 20%, NS) in 1 compared with 2 DR MM pairs. We conclude that the 1 DR MM setting is associated with optimal IL-10 secretion and more efficient inhibition of IFN-gamma and TNF-alpha compared with the 2 DR MM configuration. Although promoting enhanced IFN-gamma and TNF-alpha release, introduction of an additional DR MM does not result in increased IL-10 production. These data indicating that the IL-10 regulatory feedback loop is more effective in 1 DR rather than complete DR incompatibility could have an impact on matching policies for planned transfusion.
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We investigated the genetic control of IFN-gamma release during MLR and its relationship with TNF-alpha and IL-12. Blocking experiments demonstrated the IFN-gamma dependence of TNF-alpha production and the significant contribution of IL-12 to IFN-gamma secretion. We studied informative pairs allowing the evaluation of the relative importance of HLA class I and class II antigens. Maximal IFN-gamma secretion allowing discrimination between fully HLA different and identical subjects required 5 days. In class I different but DRB1 identical pairs, a moderate but discriminant IFN-gamma release was found. Exogenous IL-12 addition after 24 hours of preactivation by MLR resulted in a marked enhancement of IFN-gamma production at day 2. In pairs differing only by class I antigens, the discriminating capacity was significantly increased as compared to values obtained in absence of IL-12 at day 2 (p < 0.004) and at day 5 (p < 0.004). The crucial role of class I antigens on IFN-gamma release was further substantiated by the blocking action of the W6/32 mAb directed against a monomorphic epitope common to all HLA-A, -B, and -C antigens. We conclude that IFN-gamma production during MLR is under the control of class I antigens. Furthermore, exogenous IL-12 strongly amplifies their influence.
In vitro data about the action of pooled immunoglobulins (Ig) on cytokine (CK) production are controversial. The recent finding of natural antibodies against staphylococcal toxins neutralizing superantigen-induced activation prompted us to design an assay determining their ability to modulate staphylococcal enterotoxin B (SEB) induced CK production (IL-6 and TNF-alpha). Presence of anti-SEB antibodies was demonstrated by a dot-blot assay in the three preparations tested. Preincubation of SEB with pooled Ig prior to addition into the test tube containing PBMCs (neutralizing condition) resulted in a strong inhibition of both TNF-alpha and IL-6 release (TNF alpha: 59 +/- 5% inhibition, P < 0.0001; IL-6: 71 +/- 7% inhibition, P < 0.0001, n = 15). Anti-CD3 MoAb-induced CK production was not modified. During our study it was found that experimental conditions were critical to observe this inhibitory effect. Reversing the previous procedure by adding PBMCs into the test tube containing pooled Ig mixed with SEB resulted in a marked induction of TNF-alpha and IL-6 production. The same observation was made when pooled Ig solely was added (coating condition). F(ab')2 fragments of pooled Ig displayed similar inhibitory capacity when added in neutralizing condition, indicating that the mechanism involved was not Fc dependent. The fragments lost the activating properties of intact Ig when incubated in coating condition, showing that Fc receptor activation occurs in this setting. The present work demonstrates that inhibition of SEB-induced CKs release by pooled Ig can be achieved by SEB neutralization, provided that the experimental conditions avoid activation through the Fc receptor. It can be assumed that similar mechanisms take place in some clinical conditions during which pooled Ig are infused.
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Serologically defined MHC class II differences provoke release of TNF-alpha and IL-6 during MLR. In order to assess the influence of micropolymorphism defined at the genomic level, we selected informative donors pairs within DR2 DR4 serologically defined unrelated subjects by combining those differing only by DR4 alleles, as assessed by PCR-SSOP (DRB1*0401 to 07). Two groups of MLR combinations were tested including DRB1-identical (group 1, n = 12) and one DRB1 difference (group 2, n = 16). Pairs of HLA-identical siblings (n = 4) and of unrelated subjects differing by two major DR incompatibilities detected by serology (n = 27) were used as controls. We further investigated whether DP and DQ differences contributed to the observed CK production. Comparison of group 2 with group 1 showed that one DRB1 difference had a marked influence on CK production at day 3 (TNF-alpha: 401.8 +/- 85 pg/ml vs. 128.7 +/- 34.5 pg/ml, P = 0.001; SI = 2.97 +/- 0.23 vs. 1.27 +/- 0.09, P < 0.0001; IL-6: 317.6 +/- 44.8 pg/ml vs. 108 +/- 13 pg/ml, P = 0.003; SI = 2.53 +/- 0.37 vs. 1.11 +/- 0.05, P < 0.0001). However, CK release in group 2 was significantly lower than that observed in subjects with two serologically defined DR differences (TNF-alpha: 515.1 +/- 61.4 pg/ml, P = 0.05; SI = 5.61 +/- 0.48, P < 0.0001; IL-6: 545.9 +/- 75.8 pg/ml, P = 0.03; SI = 4.75 +/- 0.58, P < 0.0004). Addition of LPS after one day of MLR resulted in discriminant production of CK in group 2 as compared with group 1. Neither DP nor DQ differences affected CK production. In conclusion, DR subtypic differences induce significant CK release during primary MLR. This in vitro study demonstrates the immunodominance of the DR system in eliciting strong inflammatory mediators release.
We tested various factors affecting the production of the CKs IL-6 and TNF-alpha during in vitro alloactivation induced by MLR. Different MLR combinations involving familial and unrelated pairs were evaluated. In family studies, MLRs involving pairs of HLA-identical siblings (n = 6) were characterized by IL-6 and TNF-alpha secretion comparable to the one of autologous controls, in marked contrast with HLA-different combinations (n = 6). These displayed a strong and early (day 3) release of both CKs. In combinations of unrelated individuals involving HLA-A, -B, -C-different but -DR, -DQ-identical pairs (n = 3), low CK release was observed. Addition of LPS (1 micrograms/ml) considerably increased production of IL-6 and TNF-alpha. Clear discrimination of MHC class II differences required a 24-hour preculture followed by addition of LPS for 4 hours, a time relationship compatible with a priming phenomenon due to alloactivation. We conclude that MHC class II alloactivation not only provokes IL-6 and TNF-alpha secretion, but also primes macrophages to LPS so that the production of these CKs is markedly increased and occurs much earlier after LPS addition.
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