Mesenchymal stem cells (MSC) have recently been used successfully in humans to control severe graft-versus-host disease. However, the mechanisms involved in their immunomodulatory effects remain a matter of debate. Here, we show that MSC are unable to activate allogeneic T cells even in the presence of T-cell growth factors. We then found that MSC inhibit T-cell proliferation triggered either by allogeneic, mitogenic or antigen-specific stimuli. Interestingly, MSC inhibit T-cell proliferation by inducing apoptosis of activated T cells, but have no effect on resting T cells. Furthermore, we show that this apoptosis could be related to the conversion of tryptophan into kynurenine by indoleamine 2,3-dioxygenase expressed by MSC in the presence of IFNc. Moreover, we show that the inhibitory effect of MSC is neither abrogated nor modified during expansion in culture or after irradiation. Together, these results bring new insight to the mechanisms of immunosuppression induced by MSC and might help to develop their clinical use controlling immune-related adverse effects in humans.

SummarySome lupus anticoagulants (LA) have been shown to be directed against phospholipid-bound prothrombin. While developing an ELISA to detect anti-prothrombin autoantibodies in patient serum or plasma, no or very low signal was observed using human prothrombin immobilized on plain polystyrene plates. In contrast, the same LA-positive samples bound specifically to prothrombin coated on γ-irradiated plates, depending on the radiation dose, in the absence of added calcium and phospholipid. Optimization of the assay required the addition of 0.1% Tween 20 to the buffers. Antibody specificity for immobilized prothrombin was ascertained by competition using liposome-bound prothrombin, since fluid-phase prothrombin competed poorly. Seventy-seven of 139 patients (55.4%) with LA related to a variety of underlying diseases possessed anti-prothrombin antibodies (27 IgG, 35 IgM and 15 both isotypes), either isolated or more often associated with anti-(β2 glycoprotein I (β2GPI) antibodies. These included 67-71% of the patients with systemic lupus erythematosus and related disorders, primary antiphospholipid antibody syndrome or drug-induced LA (autoimmune groups), but only 19-20% of those with infection or malignancy (p <0.001). As previously shown for anti-β2GPI antibodies, IgG2 was the predominant IgG subclass reactive with prothrombin. Thus, autoimmune patients with LA have a high incidence of antibodies to β2GPI and prothrombin, the binding of which could similarly require high antigen density and/or exposure of cryptic epitopes resulting from protein interaction with an irradiated (i. e. more anionic) polystyrene surface.

This work aims to further characterize the newly described leukemic plasmacytoid dendritic cells (LPDC), for which we had previously demonstrated their normal, PDC-like ability to produce IFN- § . In addition, LPDC also express the specific antigens BDCA-2 and BDCA-4. Importantly, they become fully competent antigen-presenting cells (APC) after a short maturation induced by IL-3 + CD40L or virus, exhibiting a characteristic APC phenotype (high expression of CD83 and of the costimulatory molecules CD40, CD80, CD86). Whereas IL-3 + CD40L-activated LPDC prime naive CD4 + T cells towards a Th2 pathway (IL-4-secreting T cells), virus-activated LPDC drive a Th1 profile (IFN-+ -secreting T cells). Moreover, we show in one case that LPDC are able to capture, process and present exogenous antigens, leading to the activation of both CD4 + and CD8 + T cell clones in an antigen-specific manner. This study further characterizes the phenotype and immunological functions of LPDC.

Saponin, a detergent like molecule, can permeabilise cell membranes without destroying them, and thus can be used for the detection of intracellular antigens on intact cells with a flow cytometer. First experiments were reported that demonstrated the detection of intracytoplasmic antigens such as intermediate filaments and CD3 in T acute lymphoblastic leukemia (ALL). Further experiments were also performed to prove that intranuclear structures were equally accessible: dyes such as propidium iodide (PI) and monoclonal antibodies (mAb) such as Ki67 could penetrate the nucleus and lead to the analysis of DNA content and to the discrimination between the different cell cycle phases (G0, G1, S, G2‐M). This rapid and sensitive method retained sufficient integrity of cells being treated to enable differentiation of cell types on the basis of morphology. Furthermore, it did not alter membrane expression of most antigens. Therefore, it was of particular interest for multiparametric analysis, especially for simultaneous study of membrane and intracellular structures.

The capacity of cultured human monocytes to synthesize and to secrete the subcomponents of C1 and C1 inhibitor was examined. Non-stimulated monocytes secreted C1q and C1s from day 5 of culture. C1s reached a plateau immediately at its maximum level, whereas C1q secretion increased progressively until the end of the second week. Between day 12 and day 25, C1q secretion remained nearly constant (1-15 fmol/day per microgram of DNA, depending on the donor), whereas C1s secretion decreased and even in some cases stopped. C1r and C1 inhibitor were not secreted in detectable amounts by these resting cells. Stimulation of monocytes by yeasts, immunoglobulin G-opsonized sheep red blood cells or latex beads did not modify consistently C1q and C1s secretion. Activation by conditioned media from mitogen-, antigen- or allogeneic-stimulated lymphocyte cultures increased C1q production from 2 to 7 times and re-activated C1s secretion. Under the same conditions of activation, C1 inhibitor was secreted (up to 300 fmol/day per microgram of DNA) and C1r became detectable in culture supernatants. Isolated human monocytes are thus able to synthesize the whole C1 subcomponents; C1, if assembled, could be protected from non-immunological activation by locally produced C1 inhibitor. Activated monocytes appear to be a good tool for studying the assembly of C1 subcomponents and the role of C1 inhibitor in this process.