Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

[The bone marrow: a reserve of stem cells able to repair various tissues?]. / Moelle osseuse: réservoir de cellules souches réparatrices de différents tissus ?

Hematopoietic stem cells (HSC) have been widely used for autologous and allodeneic transplantation during decades, although little was known about their migration, survival, self-renewal and differentiation process. Their sorting by the CD34(+) marker they express at the cell surface in human has been challenged by the recent discovery of HSC in the CD34(-) compartment that may precede CD34(+) HSC in the differentiation process. Until recently, stem cells present in the bone marrow were thought to be specific for hematopoiesis. Some experiments including clinical trials showing the formation of various tissues, muscle, neural cells and hepatocytes for instance, after transplantation of medullar cells, have challenged this dogma. In fact, the proofs of such a transdifferentiation process by HSC are still missing and the observations may result from the differentiation of other mulipotent stem cells present in the bone marrow, such as mesenchymal stem cells and more primitive multipotent adult progenitor cells (MAPC) and side population (SP) cells.

Concurrent engagement of CD40 and the antigen receptor protects naive and memory human B cells from APO-1/Fas-mediated apoptosis.

Naive and memory B cells were isolated from human tonsils and examined for expression of APO-1/Fas and for their sensitivity to the APO-1-dependent apoptosis. APO-1 was found to be constitutively expressed on memory but not on naive B cells. The susceptibility of both cell types to the APO-1 apoptotic pathway was acquired upon CD40 triggering and was correlated with increased expression of the APO-1 receptor. Both naive and memory B cells were protected from the APO-1-mediated death signal after dual ligation of the Ag receptor adn CD40. Our findings suggest that the APO-1 pathway controls the specificity of B cell responses to T-dependent Ags and that occupancy of the Ag receptor dictates the outcome of APO-1-ligation on B cell survival.

The differentiation of human memory B cells into specific antibody-secreting cells is CD40 independent.

It is generally accepted that memory B cells can be defined by their ability to produce, upon antigenic challenge, somatically mutated antibody molecules characterized by an increased affinity and by the expression of a downstream heavy chain isotype. However, the inability to isolate this particular B cell compartment has precluded the study of memory B lymphocyte physiology in man. We previously reported on the identification of an IgD- B cell subset in human tonsils that we defined as CD38- B cells, whose phenotype is highly reminiscent of that of memory B lymphocytes from the splenic marginal zone of rodents. In the present study, we developed a model of the measles virus (MV)-specific secondary antibody response in vitro to assess the presence of memory B lymphocytes in different B cell subsets isolated from human tonsils and explore the activation requirements of human memory B cells. Our findings show that the memory B cell pool resides in the CD38- B cell subpopulation and that the differentiation of MV-activated memory B cells into antibody-secreting cells can be achieved upon co-stimulation with interleukin (IL)-2 and IL-10, but does not require engagement of CD40. Interestingly, the CD40-mediated signal was found to synergize with Ig-cross-linking agents for the proliferation of memory B cells, but strongly suppressed their capacity to differentiate along the plasmacytoid pathway. Collectively, our results suggest that the CD40 signaling pathway is instrumental for the clonal expansion of the memory B cell pool, but does not operate in the later phase of the response, which allows their maturation into antibody-secreting cells.

Regulation of germinal center B cell differentiation. Role of the human APO-1/Fas (CD95) molecule.

We previously described the existence of a tonsillar IgD- B cell subset with memory B cell features. To test the possibility that these cells could derive from germinal center (GC) B cell precursors, we examined the proliferation, differentiation, and phenotype of GC B cells after culturing with either anti-CD40 Abs or activated T cells, presumably mimicking the signals received by centrocytes in the light zone of GC. We show in this work that GC B cells proliferate and secrete Igs in both activation systems, thus indicating that CD40 ligation is also required for differentiation of GC B cells along the plasmacytoid pathway. T cell-dependent activation of GC B cells induced down-regulation of most GC-related markers (CD10, CD38, and CD77) and up-regulation of CD44 and CD62-L which are both expressed on the putative memory B cells subset. Moreover, T cell-mediated stimulation of GC B cells resulted in the strong induction of CD5 and up-regulation of APO-1/Fas (CD95). In contrast, stimulation performed with immobilized anti-CD40 Abs did not affect expression of CD10 and CD38 and failed to induce CD62-L and CD5, suggesting that the CD40 signaling pathway is necessary but not sufficient for the development of memory B cells. CD95 ligation on GC B cells was found to antagonize the stimulatory effect of immobilized anti-CD40 Abs on their proliferation, survival, and Bcl-2 expression. The possible role of CD95 in the expansion and selection of the Ag-activated B cell clones in GC is discussed.

Phenotypic and functional heterogeneity of the IgD- B cell compartment: identification of two major tonsillar B cell subsets.

Two major B cell subpopulations were identified in the IgD- compartment of tonsils and subsequently isolated. They displayed the following phenotypes: CD10+CD38+CD44- (CD38+ B cells) and CD10-CD38-CD44+ (CD38- B cells). Of the CD38- B cells, 70% also expressed CD24 and CD39, whereas CD77 was specifically distributed on 40% of CD38+ B cells, suggesting an additional level of heterogeneity in the cellular composition of these two B cell types. Whereas the majority of CD38+ B cells were in cycle, most CD38- B cells were quiescent. Conversely, Bcl-2 was expressed in CD38- B cells but was not detected in CD38+ B cells. Of the CD38- B cells, 30% bore the homing receptor Leu-8/Mel-14, whereas CD38+ B cells lacked this marker. Thus, CD38- B cells have both survival capacity and migratory competence. Both subsets expressed surface (s) Igs which were mainly of the IgG class, implying that most of these cells have already undergone isotype switching. CD38- B cells proliferated vigorously and produced large amounts of IgG in response to cytokines, following ligation of slgs or CD40. In contrast, CD38+ B cells were only stimulated for DNA synthesis by a combination of IL-4 and anti-CD40 antibodies, and failed to differentiate into Ig-secreting cells regardless of the stimulus applied. We propose that CD38- B cells represent an extra-follicular mature B cell population which has been positively selected and rescued from apoptosis, whereas the CD38+ B cell subset is composed of germinal centre B cells.