Abstract: Laboratory predictors of severe forms of dengue virus (DENV) infection are needed. These clinical forms seem to be associated with high viremia, stressing the importance of immune responses, which could involve dendritic cells (DC). Yet, very few studies have evaluated DC after DENV infection. We assessed peripheral blood DC subset numbers in mild and severe forms of dengue in 44 patients, older than 15Â years old, infected with serotypes DENV-2, 3 or 4. Patients were divided in high, intermediate and no detectable viremia according to results of molecular biology amplification of DENV. Serological status of anti-DENV IgG or IgM determined primary or secondary infections. Plasmacytoid and myeloid DC absolute and relative numbers were reduced in infected patients when compared to an age-matched healthy control group, but no significant differences in DC numbers were observed between mild or severe forms of disease. A severe disease was more frequent in patients infected with DENV-2 serotype and with secondary infection but no significant differences in DC subset numbers were found related to these variables. Viremia levels did not correlate to disease severity yet were associated to lower DC numbers. Plasmacytoid DC numbers were significantly lower in high and intermediate viremia groups compared to non-infected controls, but not in no detectable viremia patients. Myeloid DC numbers were also significantly lower than controls, even in no detectable viremia patients. These results confirm that circulating DC subset numbers are reduced after DENV infection, although this is not a biomarker of severe forms of dengue in adults.
Abstract: In the bone marrow, hematopietic and mesenchymal stem cells form a unique niche in which the oxygen tension is low. Hypoxia may have a role in maintaining stem cell fate, self renewal and multipotency. However, whereas most studies addressed the effect of transient in vitro exposure of MSC to hypoxia, permanent culture under hypoxia should reflect the better physiological conditions.
Abstract: A cross-regulation between two regulatory T cell (T(reg) ) subsets [CD4(+) CD25(+) and invariant natural killer (NK) T - iNK T] has been described to be important for allograft tolerance induction. However, few studies have evaluated these cellular subsets in stable recipients as correlates of favourable clinical outcome after heart transplantation. T(reg) and iNK T cell levels were assayed by flow cytometry in peripheral blood samples from 44 heart transplant recipients at a 2-year interval in 38 patients, and related to clinical outcome. Multi-parameter flow cytometry used CD4/CD25/CD127 labelling to best identify T(reg) , and a standard CD3/CD4/CD8/Vα24/Vβ11 labelling strategy to appreciate the proportions of iNK T cells. Both subtypes of potentially tolerogenic cells were found to be decreased in stable heart transplant recipients, with similar or further decreased levels after 2 years. Interestingly, the patient who presented with several rejection-suggesting incidents over this period displayed a greater than twofold increase of both cell subsets. These results suggest that CD4(+) CD25(+) CD127(low/neg) T(reg) and iNK T cells could be involved in the local control of organ rejection, by modulating immune responses in situ, in clinically stable patients. The measurement of these cell subsets in peripheral blood could be useful for non-invasive monitoring of heart transplant recipients, especially in the growing context of tolerance-induction trials.
Abstract: Adenovirus (ADV) infections are one of the major causes of morbidity and mortality after hematopoietic stem cell transplantation, despite new antiviral treatment strategies. We describe here a complete clinical-grade generation of human anti-ADV cytotoxic T cells to propose an adoptive immunotherapy. Peripheral blood mononuclear cells (PBMC) from 7 healthy donors, known for their good cellular immunity against ADV, were stimulated for 6 hours with a synthetic peptide pool covering the ADV5 Hexon protein interferon-gamma (IFN-gamma) secreting cells were isolated on a clinical device. After immunoselection, a mean number of 1.01 +/- 0.84 x 10(6) total nucleated cells was obtained. The isolated ADV-specific T cells were mainly CD4+ (mean=56% +/- 20.8%, yield=51% +/- 32.4%) but also CD8+ (mean=42% +/- 27%, yield = 56% +/- 39.3%). Isolated T lymphocytes (CTL) were expanded to carry out functional tests. Ability of the expanded CTL to secrete IFN-gamma and to proliferate after restimulation with the ADV peptide pool was confirmed. A high cytotoxicity against autologous target cells loaded with ADV antigens was observed but not against nonloaded target cells. We observed a decrease of 1.27 log of the allogeneic reaction against non HLA identical healthy donor PBMC with CTL compared with the PBMC before selection. Clinical-grade generation of ADV-specific T cells was achieved with a synthetic antigen. This technology has the advantage of being fast, and is sufficiently reactive to be proposed for immunotherapy if antiviral treatment fails.
Abstract: Epigenetic modifications of chromatin may play a role in maintaining viral latency and thus persistence of the human T-lymphotropic virus type 1 (HTLV-1), which is responsible for HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP). A major determinant of disease progression is increased peripheral blood proviral load (PVL), possibly via the accumulation of infected cells in the central nervous system (CNS) creating a damaging inflammatory response. Current therapeutic approaches that focus on reducing either cell proliferation, viral replication, or tissue invasion are still unsatisfactory. Contrasting with these inhibitory strategies, we evaluated the efficacy of a novel approach aimed, paradoxically, at activating viral gene expression to expose virus-positive cells to the host immune response. We used valproate (VPA), a histone deacetylase inhibitor that has been used for decades as a chronic, safe treatment for epileptic disorders. Based on in vitro and in vivo data, we provide evidence that transient activation of the latent viral reservoir causes its collapse, a process that may alleviate the condition of HAM/TSP. This represents the first such approach to treating HAM/TSP, using gene activation therapy to tilt the host-pathogen balance in favor of an existing antiviral response. This trial is registered at http://clinicaltrials.gov/as no. NCT00519181.
Abstract: Apoptotic leukocytes are endowed with immunomodulatory properties that can be used to enhance hematopoietic engraftment and prevent graft-versus-host disease (GvHD). This apoptotic cell-induced tolerogenic effect is mediated by host macrophages and not recipient dendritic cells or donor phagocytes present in the bone marrow graft as evidenced by selective cell depletion and trafficking experiments. Furthermore, apoptotic cell infusion is associated with TGF-beta-dependent donor CD4+CD25+ T-cell expansion. Such cells have a regulatory phenotype (CD62L(high) and intracellular CTLA-4+), express high levels of forkhead-box transcription factor p3 (Foxp3) mRNA and exert ex vivo suppressive activity through a cell-to-cell contact mechanism. In vivo CD25 depletion after apoptotic cell infusion prevents the apoptotic cell-induced beneficial effects on engraftment and GvHD occurrence. This highlights the role of regulatory T cells in the tolerogenic effect of apoptotic cell infusion. This novel association between apoptosis and regulatory T-cell expansion may also contribute to preventing deleterious autoimmune responses during normal turnover.
Abstract: Treatment (from 5 to 25 weeks of age) with a novel blocking monoclonal antibody, mAb I-10, directed against the plasma membrane (pm) form of LAMP-1, protected against development of autoimmune diabetes in the NOD mouse. A shorter course of treatment, i.e. from 5 to 12 weeks of age, significantly reduced the occurrence of insulitis as well as disease onset. Interfering with pm-LAMP-1 required continuous treatment as tolerance was not observed when treatment was stopped, and no higher proportion of cells with a T regulatory phenotype (e.g. CD4(+)CD25(+)) were induced. The mechanism appears to involve modulating a proinflammatory cytokine, as the proportion of pancreatic-infiltrating IFN-gamma-positive cells was significantly reduced in the mAb I-10-treated group. These results demonstrate an unexpected role for pm-LAMP-1 in autoimmune disease progression, and suggest that further investigation should be performed to understand how this molecule modulates IFN-gamma-driven responses.
Abstract: Intravenous infusion of apoptotic donor or third-party leukocytes simultaneously with an allogeneic donor bone marrow (BM) graft favors engraftment across major histocompatibility barriers. While verifying that such apoptotic cell infusion might not also be associated with antibody (Ab)-mediated allo-immune responses, we found, rather strikingly, that apoptotic cell infusion could in fact successfully prevent a humoral allo-immunization against a BM graft in mice. Indeed, among recipients having rejected their BM graft, prior apoptotic cell infusion was associated with a near absence of Ab-mediated allo-responses, while such an immunization was frequently observed in the absence of apoptotic cell infusion. This was also observed when infusing host apoptotic cells, thus showing that the prevention of immunization was linked to the apoptotic state of the cells rather than mediated by residual anti-recipient activity. In vivo anti-transforming growth factor-beta (TGF-beta) treatment resulted in the loss of this apoptotic cell infusion-associated protective effect on humoral allo-responses. Further studies will determine whether apoptotic cell infusion, in addition to hematopoietic graft facilitation might also contribute to preventing deleterious Ab-mediated allo-responses in various transplantation settings.
Abstract: Flow cytometry crossmatch (FCXM) is a more sensitive technique than classical complement-dependent cytotoxicity (CDC) for the detection of donor-directed antibody before renal transplantation. Nevertheless, the role of FCXM in predicting long-term survival of kidney grafts is still unclear. The purpose of our study was to evaluate the impact of a positive T-cell FCXM (T-FCXM) on long-term kidney allografts outcome. Of the 184 consecutive kidney transplantations performed in our center between 1 January1991 and 15 November 1996 a FCXM, performed concurrently to the pre-transplant CDCXM, was available for 170 patients. The CDCXM was negative in all recipients. Among these recipients, 12 (7.1%) had a positive T-FCXM. These patients were not different from patients with a negative T-FCXM for donor and recipient age, sex, frequency of second transplantation, number of human leukocyte antigen matches or mismatches. Frequency of immunized patients was higher in kidney recipients with a positive FCXM (58.3% vs. 24.7%; p=0.02, chi-square test). Survival analysis revealed that kidney graft outcome was better in negative T-FCXM recipients (p=0.03), while patient survival was not statistically different. Our results suggest that a positive pre-transplant T-FCXM despite a negative CDCXM is associated with an impaired long-term graft survival in renal allotransplantation.
Abstract: Allergic contact dermatitis, the clinical manifestation of contact hypersensitivity, is one of the most common disorders of the skin. It is elicited upon multiple cutaneous re-exposure of sensitized individuals to the sensitizing agent. In this study, we demonstrate that using IL-18 binding protein (IL-18BP) to neutralize IL-18 significantly reduced clinical symptoms in a murine model of contact hypersensitivity. Furthermore, IL-18BP alleviated the relapses during established disease, as indicated by significant protection during re-exposure of mice that had previously undergone a contact hypersensitivity response without treatment. Although edema was not influenced, IL-18BP reduced the number of T cells homing to sites of inflammation, resulting in diminished local production of IFN-gamma. Thus, by preventing the accumulation of effector T cells to the target tissue, IL-18BP appears to be a potent protective mediator to counter skin inflammation during contact hypersensitivity. Taken together with the evidence that IL-18 is present in tissue samples of the human disease, our data reinforces IL-18BP as a candidate for this therapeutic indication.
Abstract: The ultimate objective of organ transplantation is to obtain a state of tolerance, i.e. long-term acceptance of the graft without immunosuppressive therapy in order to limit the complications of these treatments (viral infections, tumours, etc.). The various immunological mechanisms allowing a state of tolerance will be described in this review. Among these various experimental strategies, combined bone marrow (or haematopoietic stem cell) transplantation and organ transplantation, made possible by the development of non-myeloablative or less intensive conditioning, appears to be one of the most promising lines of research. This approach leads to colonization of the recipient by donor cells. This state is described as "macro-chimerism" and achieves a real state of central tolerance in relation to an organ derived from the bone marrow donor. We have shown recently that intravenous injection of apoptotic cells in combination with allogeneic bone marrow cells increases the success rate of bone marrow transplantation. In a model of combined bone marrow/solid organ transplantation, these apoptotic cells induce tolerance limited to the donor's bone marrow cell antigens without inducing auto-immunization. We therefore propose a new approach to cell-based therapy (using the immunomodulating properties of apoptotic cells) to promote the success of haematopoietic stem cell transplantation. This approach can be particularly useful in combined haematopoietic stem cell and organ transplantation in order to induce a state of macro-chimerism.
Abstract: Gene-transfer techniques are commonly employed for both in vitro and in vivo studies. However, modifications of the target cell following the introduction of the gene of interest are not often examined. These modifications can alter the immunogenicity and/or the susceptibility of the target cell to apoptosis and may produce unwanted consequences in vivo.
Abstract: To modulate alloreactivity after hematopoietic stem cell (HSC) transplantation, suicide gene-expressing donor T cells can be administered with an allogeneic T cell-depleted HSC graft. Immune competence of such cells is a critical issue. We have examined the impact of our ex vivo gene transfer protocol (12-day culture period including CD3/IL-2 activation, retrovirus-mediated gene transfer, and G418-based selection) on the phenotype and functional properties of gene-modified cells (GMC). GMC were compared with control cells that had been cultured in parallel with GMC, but nontransduced and nonselected, as well as with peripheral blood mononuclear cells (PBMC). Our data show that phenotypical modifications are similar in control cells and GMC, demonstrating that alterations result from the 12-day culture rather than from the transduction and/or selection process itself. Such modifications include a reversal of CD4/CD8 ratio, activated phenotype (increased expression of CD45RO, CD95, and HLA-DR), and acquisition or increased expression of co-stimulatory molecules (CD80, CD86, and CD40). This led to an enhanced allostimulating potential of GMC, as compared with resting T cells, when used as stimulating cells in mixed lymphocyte reactions. Conversely, when using them as responder cells in mixed lymphocyte reactions, GMC exhibited a rapid loss of alloreactivity that resulted both from culture-dependent and from transduction and/or selection-dependent events. In conclusion, the retrovirus-mediated gene transfer can be associated with major phenotypical and functional alterations that could have strong clinical implications (increased immunogenicity, reduced anti-leukemic effect). Thus, future T cell expansion protocols should try to improve not only cell expansion or gene transfer efficiency, but also T cell functions.
Abstract: Cell-based therapies are promising approaches to treat uncontrolled pathologies, such as tumours. Apoptotic tumour cells have recently been proposed as a source of tumour-associated antigens to stimulate an efficient immune response. However, a complex relationship exists between apoptosis and the immune system. In this review, the different factors that may influence immune responses against apoptotic cells are detailed and discussed in the light of recent publications. These factors include the nature of the phagocytes and the receptors involved in apoptotic cell uptake, as well as the environment in which cells are dying. A possible distinction between apoptosis and necrosis by immune system sentinels adds a further level of complexity. The potential use of the immunomodulatory properties associated with apoptosis to favour engraftment and induce tolerance in transplantation is then discussed. In conclusion, this review will suggest appropriate conditions to efficiently and safely use apoptotic cells as a new cell therapy product.
Abstract: Cross-tolerization of T lymphocytes after apoptotic cell uptake by dendritic cells may be involved in self-tolerance maintenance. Furthermore, immunosuppressive properties are attributed to apoptotic cells. This study evaluated the consequences of apoptotic leukocyte administration in a restrictive engraftment model of murine bone marrow (BM) transplantation. Sublethally irradiated recipients received a limited number of allogeneic BM, with or without irradiated apoptotic leukocytes of different origins. No graft-versus-host disease was observed. Whereas only a low proportion of mice receiving BM cells alone engrafted, addition of apoptotic irradiated leukocytes, independently of the origin (donor, recipient, third-party mice, as well as xenogeneic peripheral blood mononuclear cells), significantly enhanced engraftment. Similar results were obtained after infusion of leukocytes rendered apoptotic by UVB irradiation or by anti-Fas monoclonal antibody stimulation, thus confirming the role of apoptotic cells in engraftment facilitation. Overall, these results suggest that apoptotic leukocytes can nonspecifically facilitate allogeneic BM engraftment. Such a simple approach could be of interest in BM transplantation settings involving an important HLA donor/recipient disparity, a T-cell-depleted graft, or reduced conditioning regimen intensity.
Abstract: The use of donor T cells expressing the herpes simplex thymidine kinase (HSV-TK) gene followed by ganciclovir (GCV) treatment could allow for specific modulation of the alloreactivity occurring after bone marrow transplantation. We are presently exploring such an approach in a phase I clinical trial.
Abstract: Flow-PRA is a flow cytometric method for both anti-HLA class I and class II antibody (Ab) detection. We evaluated this technique for Ab screening in patients awaiting kidney transplantation. After having established a rigorous threshold for positivity, a three-dilution difference in sensitivity between Flow-PRA and complement-dependent cytotoxicity (CDC) persisted. The sensitivity of the method was satisfactory since all CDC-positive sera were also found to be positive with the Flow-PRA method. Discrimination between anti-HLA class I and class II Abs was excellent. Furthermore, all sera responsible for a positive flow cytometry crossmatch (FCXM) and a negative CDC-crossmatch (CDCXM) at the time of a putative transplant were found to be positive with Flow-PRA beads. The specificity was excellent for anti-class I Ab detection since no false positive serum was found. On the other hand, the specificity was lower for anti-class II detection, since 8.3% (2/24) false positive results were detected among all the negative sera tested. Overall, our results suggested that Flow-PRA should be of value for anti-HLA Ab screening prior to kidney transplantation.
Abstract: We are presently investigating the therapeutic potential of herpes simplex-thymidine kinase-expressing donor T cells in the setting of a T cell-depleted allogeneic bone marrow transplantation. The generation, expansion, and selection of the gene-modified T cells require a 12-day ex vivo culture period in high-dose interleukin (IL)-2 that could significantly alter their in vivo alloreactivity.
Abstract: The clinical and immunological relevance of a positive B-cell flow-cytometry (B-FCXM) crossmatch in renal transplantation is still controversial.
Abstract: Uniparental isodisomy is defined as the inheritance of two copies of the same parental chromosome and can result in defects when it produces homozygosity for a recessive mutation or in the presence of imprinting. We describe the detection of a chromosome 6 uniparental isodisomy in a 9 year old girl, discovered during a search for an HLA identical sib. HLA typing, erythrocyte phenotyping, and genotypes of microsatellite polymorphisms were compatible with a paternal isodisomy of chromosome 6, with normal biparental origin of the other chromosomes. Paternal cells were not responsive to the patient's cells in mixed lymphocyte cultures. This fortuitous detection of a chromosome 6 isodisomy suggests that cases of chromosome 6 UPD may not be deleterious and may therefore go undetected.
