Human cystic fibrosis embryonic stem cell lines derived on placental mesenchymal stromal cells.

This study describes the production of two new human embryonic stem cell (hESC) lines affected by cystic fibrosis. These cell lines are heterozygous compounds, each a carrier of the DF508 mutations associated either with E585X or with 3849+10 kb C-->T. The derivation process was performed on irradiated human placental mesenchymal stromal cells and designed to minimize contact with xeno-components. This new source of feeder cells is easy to obtain and devoid of ethical concerns. The cells have a great capacity to proliferate which reduces the need for continuous preparation of new feeder cell lines. In addition, three normal hESC lines were obtained in the same conditions. The five stem cell lines retained hESC-specific features, including an unlimited and undifferentiated proliferation capacity, marker expression and the maintenance of stable karyotype. They also demonstrated pluripotency in vitro, forming cell lineages of the three germ layers, as indicated by immunolocalization of beta-tubulin, alpha-fetoprotein and actin. These new genetic cell lines represent an important in-vitro tool to study the physiological processes underlying this genetic disease, drug screening, and tissue engineering.

A rapid test of alloreactivity based on interleukin-2 mRNA expression might identify liver transplant recipients with donor-specific hyporesponsiveness.

BACKGROUND: Immunosuppression withdrawal is feasible in some liver transplant (OLT) recipients but may lead to severe rejection in others, underlying the need for reliable biomarkers to identify patients with tolerant profile in whose weaning/withdrawal could be safely proposed. We evaluated the value of real-time polymerase chain reaction (PCR)-based measurement of interleukin (IL)-2 mRNA in mixed lymphocyte reaction (MLR) to monitor in vitro anti-donor reactivity in OLT patients. METHODS: MLR were performed in three patients undergoing living donor OLT using a tolerogenic protocol including donor stem cells. IL-2 mRNA production in MLR was measured by PCR at several intervals after OLT. RESULTS: In the early posttransplant period, three patients presented with global immunodeficiency, as indicated by low IL-2 mRNA production against both donor and third-party antigens. In the two patients who has immunosuppression successfully withdrawn, donor-specific hyporesponsiveness was observed thereafter: IL-2 mRNA production against donor cells remained low, while IL-2 mRNA production against a third-party antigen-presenting cells progressively recovered. No such modulation of the anti-donor response was observed in the patient in whom withdrawal led to rapid rejection. CONCLUSION: Measurement of IL-2 mRNA production in MLR might prefer a tool to monitor anti-donor reactivity after OLT for decisions to minimize or withdraw immunosuppression in patients displaying donor-specific hyporesponsiveness.

Transient expansion of peptide-specific lymphocytes producing IFN-gamma after vaccination with dendritic cells pulsed with MAGE peptides in patients with mage-A1/A3-positive tumors.

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.

Blockade of proliferation and tumor necrosis factor-alpha production occurring during mixed lymphocyte reaction by interferon-gamma-specific natural antibodies contained in intravenous immunoglobulins.

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.

Optimal control of interferon-gamma and tumor necrosis factor-alpha by interleukin-10 produced in response to one HLA-DR mismatch during the primary mixed lymphocyte reaction.

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.

Tumor necrosis factor-alpha and interleukin-6 production induced by variations of DR4 polymorphism during the primary mixed lymphocyte reaction.

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.

Interleukin 12 unmasks HLA class I differences during mixed lymphocyte reaction induced interferon gamma production.

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.

Alloactivation induced during mixed-lymphocyte reaction provokes release of tumor necrosis factor alpha and interleukin 6 by macrophages and primes them to lipopolysaccharides.

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.

[New therapeutic approachs in melanoma]. / Approches thérapeutiques innovantes dans le mélanome.

During the last decade, new insights in cellular and molecular biology have opened new avenues in cancer immunotherapy. Two distinct modalities have been developed: adoptive immunotherapy and anti-tumoral vaccination (active immunotherapy). We will first describe the main strategies of adoptive immunotherapy and then elaborate on the protocols of anti-tumoral vaccination against tumor associated antigens (TAA). In that context, we will pay peculiar attention on the pivotal role of dendritic cells (DC) as natural adjuvant.

[The immunology-hematology-transfusion department]. / Le Service d'Immunologie-Hématologie-Transfusion.

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.

Recommended indications for the administration of polyclonal immunoglobulin preparations.

The following recommendations, which aim at standardising and rationalising the clinical indications for administering polyclonal immunoglobulins in Belgium, were drawn up by a working group of the Superior Health Council. To this end, the Superior Health Council organised an expert meeting devoted to"Guidelines for the use of immunoglobulins". The experts discussed the indications for immunoglobulin use, the'ideal'immunoglobulin preparation, its mechanisms of action, the practical issues involved in administering immunoglobulins and their potential side effects. The recommendations formulated by the experts were validated by the Superior Health Council working group with the purpose of harmonising immunoglobulin use in Belgium

Guidelines for the use of fresh frozen plasma.

Recommendations, which aim at standardising and rationalising clinical indications for the transfusion of fresh frozen plasma (FFP) in Belgium, were drawn up by a working group of the Superior Health Council. For this purpose the Superior Health Council organised an expert meeting devoted to "Transfusion Guidelines: Pathogen reduction, products and indications for the transfusion of plasma" in collaboration with the Belgian Haematological Society.The experts discussed the indications for the transfusion of FFP, pathogen reduction for FFP and the practical issues of administering FFP and plasma-derived concentrates. The recommendations formulated by the experts were validated by the working group with the purpose of harmonising FFP transfusion in Belgian hospitals.

Guidelines for the transfusion of platelets.

Recommendations aiming at standardising and rationalising clinical indications for the transfusion of platelets in Belgium were drawn up by a working group of the Superior Health Council. To this end the Superior Health Council organised an expert meeting devoted to "Guidelines for the transfusion of platelets" in collaboration with the Belgian Hematological Society. The experts discussed the indications for platelet transfusions, the ideal platelet concentrate and the optimal platelet transfusion therapy. The recommendations prepared by the experts were validated by the working group with the purpose of harmonising platelet transfusion in Belgian hospitals.

Increased in vitro immunosuppressive action of anti-CMV and anti-HBs intravenous immunoglobulins due to higher amounts of interferon-gamma specific neutralizing antibodies.

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.

Transplantation tolerance and mixed chimerism: at the frontier of clinical application.

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.