Jean-Jacques Sotto

In Vivo and In Vitro Modulation of HLA-DM and HLA-DO Is Induced by B Lymphocyte Activation

Ag presentation via HLA class II molecules in B lymphocytes depends on the coordinated action of HLA-DM, the catalyst of class II-peptide loading, and HLA-DO, a pH-dependent modulator of DM, the expression of which is almost completely restricted to B lymphocytes. The relative expression levels of both class II modulators are critical for the composition of the HLA class II peptide repertoire. The data in this work demonstrate that DO and DM expression are both dependent on the cellular activation status in primary human B lymphocytes. In vivo low-density activated primary human B lymphocytes show a prominent reduction in DO and DM expression when compared with high-density resting primary B lymphocytes. In vitro, reduction of DO and DM expression can be induced by B lymphocyte activation via the B cell receptor or by use of the phorbol ester, PMA. Specific inhibition of protein kinase C resulted in a significant reduction of HLA-DO and is potentially due to protein degradation in lysosomal compartments as the phenomenon is reversed by chloroquine. Thus, the expression of the dedicated HLA class II chaperone DM and its pH-dependent modulator DO is regulated and tightly controlled by the activation status of the B lymphocyte.

Impairment of death-inducing signalling complex formation in CD95-resistant human primary lymphoma B cells

Multiple mechanisms exist by which tumour cells can escape CD95‐mediated apoptosis. Previous studies by our laboratory have shown that primary B cells from non‐Hodgkins Lymphoma (B‐NHL) were resistant to CD95‐induced cell death. In the current study, we have analysed the mechanisms underlying CD95 resistance in primary human lymphoma B cells. We report that FADD (FAS‐associated death domain protein) and caspase‐8 were constitutively expressed in lymphoma B cells and that the CD95 pathway was blocked upstream to caspase‐8 activation. However, caspase‐8 was processed and functional after treatment with staurosporine (STS). We found that the expression levels of FLICE (FADD‐like interleukin‐1 beta‐converting enzyme)‐Inhibitory Protein (c‐FLIP) and Bcl‐2‐related proteins were heterogeneous in B‐NHL cells and were not related to CD95 resistance. Finally, we report the absence of a CD95‐induced signalling complex [death‐inducing signalling complex (DISC)] in lymphoma B cells, with no FADD and caspase‐8 recruitment to CD95 receptor. In contrast, DISC formation was observed in CD95‐resistant non‐tumoural (NT) B cells. Therefore, we propose that the absence of DISC formation in primary lymphoma B cells may contribute to protect these cells from CD95‐induced apoptosis.

Detection of Suppressor T Lymphocytes and Estimation of Their Frequency in Limiting Dilution Assays by Generalized Linear Regression Modeling

The estimate of the frequency of suppressor T lymphocytes in unfractionated cell populations remains challenging, mainly because these regulatory cells do not display specific immunophenotypic markers. In this paper, we describe a novel theoretical approach for quantifying the frequency of suppressor cells. This method is based on limiting dilution data modeling, and allows the simultaneous estimation of the frequencies of both proliferating and suppressor cells. We used previously published biological data, characterizing the inhibiting activity of suppressor T cell clones. Starting from these data, we propose a mathematical model describing the interaction between suppressor and proliferating T cells, and applied to a Poisson process. Limiting dilution data corresponding to this non-single-hit, suppressor two-target Poisson model were artificially generated, then modeled according to a generalized linear regression procedure. Deviation from the single-hit Poisson model was revealed by a statistical slope test, and a stepwise analysis of the regression appeared to be an efficient method that strongly argued in favor of the presence of suppressor cells. By using the frequency of proliferating T cells calculated in the first step of the regression, we demonstrated the possibility to provide a reasonable estimate of the frequency of suppressor T cells. Based on these findings, a practical decision-making procedure is given to perform standard analyses of limiting dilution data.

Development of autologous cytotoxic CD4+ T clones in a human model of B-cell non-Hodgkin follicular lymphoma.

Immunotherapy for cancer aims to generate cytotoxic cells that are capable of eradicating tumour cells. It has been well demonstrated that helper, non‐cytotoxic CD4+ T cells are important for the induction and maintenance of anti‐tumour immunity exerted by cytotoxic CD8+ T cells. In contrast, the existence of direct anti‐tumour, effector cytotoxic CD4+ T cells remains elusive, mainly due to the paucity of reliable experimental data, especially in human B‐cell non‐Hodgkin lymphomas. This study developed an appropriate, autologous follicular B‐cell non‐Hodgkin follicular lymphoma model, including the in vitro establishment of a malignant, human leucocyte antigen class I (HLA‐I) deficient B‐cell line, and the generation of three autologous anti‐tumour cytotoxic CD4+ T‐cell clones originating from the peripheral blood of the same patient. These three clones were considered as tumour specific, because they were capable of killing the malignant, HLA‐I‐deficient B‐cell line through a classical HLA‐II restricted perforin‐mediated pathway, but did not lyse the Epstein–Barr virus‐infected autologous normal B lymphocytes. All three CD4+clones were T‐cell receptor Vβ17‐Dβ1‐Jβ1·2 and exhibited an identical complementarity‐determining region 3, suggesting the immunodominance of a single peptide antigen presented by tumour cells. Such lymphoma models would provide a useful tool for in vivo expansion and the adoptive transfer of selected CD4+ cytotoxic cells in immunotherapeutic strategies.

Réflexion sur la prise en charge des patients atteints d’hémopathies malignes nécessitant des transfusions itératives

hma.2012.0700 Auteur(s) : Dominique Bordessoule, Dominique Jaulmes, Michele Levy-Soussan, Eric Fiat, Robert Zittoun, Joel Ceccaldi, Jean-Jacques Sotto et les autres membres de la commission d’ethique de la SFH a a Christian Bastard, Chantal Bauchetet, Philippe Casassus, Morgane Cheminant, Philippe Colombat, Diane Damotte, Jean-Pierre Jouet, Sandra Malak, Genevieve Margueritte, Sarah Morin, Henri Rochant, Alice Tellier et Marc Zandecki. Position du probleme De nombreux patients atteints [...]