Neutropenia in Patients with Common Variable Immunodeficiency: a Rare Event Associated with Severe Outcome.

BACKGROUND: Common variable immunodeficiency (CVID) is characterized by infections and hypogammaglobulinemia. Neutropenia is rare during CVID. METHODS: The French DEFI study enrolled patients with primary hypogammaglobulinemia. Patients with CVID and neutropenia were retrospectively analyzed. RESULTS: Among 473 patients with CVID, 16 patients displayed neutropenia (lowest count [0-1400]*106/L). Sex ratio (M/F) was 10/6. Five patients died during the follow-up (11 years) with an increased percentage of deaths compared to the whole DEFI group (31.3 vs 3.4%, P < 0.05). Neutropenia was diagnosed for 10 patients before 22 years old. The most frequent symptoms, except infections, were autoimmune cytopenia, i.e., thrombopenia or anemia (11/16). Ten patients were affected with lymphoproliferative diseases. Two patients were in the infection only group and the others belonged to one or several other CVID groups. The median level of IgG was 2.6 g/L [0.35-4.4]. Most patients presented increased numbers of CD21low CD38low B cell, as already described in CVID autoimmune cytopenia group. Neutropenia was considered autoimmune in 11 cases. NGS for 52 genes of interest was performed on 8 patients. No deleterious mutations were found in LRBA, CTLA4, and PIK3. More than one potentially damaging variant in other genes associated with CVID were present in most patients arguing for a multigene process. CONCLUSION: Neutropenia is generally associated with another cytopenia and presumably of autoimmune origin during CVID. In the DEFI study, neutropenia is coupled with more severe clinical outcomes. It appears as an "alarm bell" considering patients' presentation and the high rate of deaths. Whole exome sequencing diagnosis should improve management.

[Gene therapy of severe combined immunodeficiency disease: proof of principle of efficiency and safety issues. Gene therapy, primary immunodeficiencies, retrovirus, lentivirus, genome]. / Thérapie génique des déficits immunitaires sévères: preuve de principe d'efficacité et problèmes soulevés. Thérapie génique, déficits immunitaires, rétrovirus, lentivirus, génome.

Inherited defects in T lymphocyte development (severe combined immunodeficiencies-SCID) are considered the best available models to assess the effectiveness of gene therapy. Retroviral vectors have been successfully used ex vivo to transduce hematopoietic precursors from patients with two forms of SCID, namely X-linked SCID and ADA deficiency. Fifteen out of 16 patients with XL-SCID and 4 out of 4 patients with ADA deficiency have benefited from gene therapy. Correction of the immunodeficiency has been maintained now for 6 years in the first patients to be treated. These results provide clear-cut proof of the principle of gene therapy in relevant clinical models. However, three patients with XL-SCID have developed a severe complication after gene therapy, consisting of leukemia-like clonal lymphocyte proliferation. One of the three patients has died from this complication. It is due to provirus insertion within or close to proto-oncogenes, and to the enhancer activity of the viral LTR on targeted proto-oncogenes. Vector modifications, based mostly on inactivating the enhancer activity of the LTR, should preserve efficacy while improving safety.

Medical perspectives of adults and embryonic stem cells.

In the last 30 years, allogeneic bone marrow transplantation has become the treatment of choice for many hematologic malignancies or inherited disorders and a number of changes have been registered in terms of long-term survival rate of transplanted patients as well as of available sources of hematopoietic stem cell (HSC). In parallel to the publication of better results in HSC transplantation, several recent discoveries have opened a scientific and ethical debate on the therapeutical potential of stem cells isolated from adult or embryonic tissues. One of the major discoveries in this field is the capacity of bone marrow-derived stem cells to treat a genetic liver disease in a mouse model, thus justifying the concept of transdifferentiation of adult stem cell and raising hopes on its possible therapeutical applications. We have tried here to summarise the advances in this field and to discuss the limits of these biological data.

Gene therapy for severe combined immunodeficiency.

Studies of severe combined immunodeficiency (SCID), a group of rare monogenic disorders, have provided key findings about the physiology of immune system development. The common characteristic of these diseases is the occurrence of a block in T cell differentiation, always associated with a direct or indirect impairment of B cell immunity. The resulting combined immunodeficiency is responsible for the clinical severity of SCID, which, without treatment, leads to death within the first year of life. Eleven distinct SCID phenotypes have been identified to date. Mutations of ten genes have been found to cause SCID. Identifying the pathophysiological basis of most SCID conditions has led to the possibility of molecular therapy as an alternative to allogeneic hematopoietic stem cell transplantation. This review discusses recent developments in SCID identification and treatment.

Clonal evidence for the transduction of CD34+ cells with lymphomyeloid differentiation potential and self-renewal capacity in the SCID-X1 gene therapy trial.

Immune function has been restored in 9 of 10 children with X-linked severe combined immunodeficiency by gamma c gene transfer in CD34+ cells. The distribution of both T-cell receptor (TCR) V beta family usage and TCR V beta complementarity-determining region 3 (CDR3) length revealed a broadly diversified T-cell repertoire. Retroviral integration site analysis in T cells demonstrated a high number of distinct insertion sites, indicating polyclonality of genetically corrected cell clones, in all patients. Detection of gamma c transgene expression on patients' mature myeloid cells has prompted us to investigate the nature of the most immature transduced hematopoietic precursor cells. Insertion sites shared by T and B lymphocytes as well as highly purified granulocytes and monocytes demonstrate the correction of common multipotent progenitor cells. Moreover, our data show that differentiated leukocytes share the same exact insertion sites with CD34+ cells that we obtained 8 months later and that were able to generate long-term culture-initiating cells (LTC-ICs). This finding demonstrates the initial transduction of very primitive multipotent progenitor cells with self-renewal capacity. These results provide a first evidence in the setting of a clinical trial that CD34+ cells maintain both lymphomyeloid potential as well as self-renewal capacity after ex vivo manipulation.

Disruption of the lineage restriction of TCR beta gene rearrangements.

Despite a common lymphoid recombinase, assembly of Ig genes is restricted to B cells, whereas TCR loci rearrange in T cells. Transcriptional promoters and enhancers are critical for the regulation of the recombination process. However, the specific function of such elements in conferring the lineage-restriction of V(D)J recombination remains poorly understood. To gain further insights into the mechanism restricting TCR beta-chain rearrangements to T cells, we generated mice in which an 11 kb region--containing the beta-chain constant region 2 and the TCR beta enhancer (E beta)--was replaced with the B cell specific Ig heavy-chain enhancer (E mu). Unlike the simple E mu to E beta replacement, this mutation allowed significant levels of D beta to J beta as well as V beta to DJ beta rearrangements in both T and B cells. Although the lineage restriction was disrupted, TCR beta allelic exclusion was still efficient in mutated T cells. Together these results demonstrate that changes in the activity of regulatory elements located at the TCR beta constant regions are sufficient to redirect the recombination pattern of TCR beta variable gene segments.

Transgenic expression of the p16(INK4a) cyclin-dependent kinase inhibitor leads to enhanced apoptosis and differentiation arrest of CD4-CD8- immature thymocytes.

In the thymus, T cell development proceeds by successive steps of differentiation, expansion, and selection. Control of thymocyte proliferation is critical to insure the full function of the immune system and to prevent T cells from transformation. Deletion of the cell cycle inhibitor p16(INK4a) is frequently observed in human T cell neoplasias and, in mice, gene targeted inactivation of the Ink4a locus enhances thymocyte expansion and predisposes mutant animal to tumorigenesis. Here, we investigate the mechanism by which p16(Ink4a) controls thymocyte development by analyzing transgenic mice expressing the human p16(INK4a) into the T cell lineage. We show that forced expression of p16(INK4a) in thymocytes blocked T cell differentiation at the early CD4-CD8-CD3-CD25+ stage without significantly affecting the development of gammadelta T cells. Pre-TCR function was mimicked by the induction of CD3 signaling in thymocytes of recombinase activating gene (RAG)-2-deficient mice (RAG-2(-/-)). Upon anti-CD3epsilon treatment in vivo, p16(INK4a)-expressing RAG-2(-/-) thymocytes were not rescued from apoptosis, nor could they differentiate. Our data demonstrate that expression of p16(INK4a) prevents the pre-TCR-mediated expansion and/or survival of differentiating thymocytes.