Isabelle André-Schmutz

Restoration of Human B-cell Differentiation Into NOD-SCID Mice Engrafted With Gene-corrected CD34+ Cells Isolated From Artemis or RAG1-deficient Patients

Severe combined immunodeficiency (SCID) caused by mutation of the recombination-activating gene 1 (RAG1) or Artemis gene lead to the absence of B- and T-cell differentiation. The only curative treatment is allogeneic bone marrow (BM) transplantation, which displays a high survival rate when an HLA compatible donor is available but has a poorer prognosis when the donor is partially compatible. Consequently, gene therapy may be a promising alternative strategy for these diseases. Here, we report that lentiviral gene-corrected BM CD34(+) cells (isolated from Artemis- or RAG1-deficient patients) sustain human B-cell differentiation following injection into non-obese diabetic/SCID (NOD-SCID) mice previously infused with anti-interleukin-2 receptor beta chain monoclonal antibody. In most of the mice BM, engrafted with Artemis-transduced cells, human B-cell differentiation occurred until the mature stage. The B cells were functional as human immunoglobulin M (IgM) was present in the serum. Following injection with RAG1-transduced cells, human engraftment occurred in vivo but B-cell differentiation until the mature stage was less frequent. However, when it occurred, it was always associated with human IgM production. This overall approach represents a useful tool for evaluating gene transfer efficiency in human SCID forms affecting B-cell development (such as Artemis deficiency) and for testing new vectors for improving in vivo RAG1 complementation.

Human T‐Lymphoid Progenitors Generated in a Feeder‐Cell‐Free Delta‐Like‐4 Culture System Promote T‐Cell Reconstitution in NOD/SCID/γc−/− Mice

Slow T‐cell reconstitution is a major clinical concern after transplantation of cord blood (CB)‐derived hematopoietic stem cells. Adoptive transfer of in vitro‐generated T‐cell progenitors has emerged as a promising strategy for promoting de novo thymopoiesis and thus accelerating T‐cell reconstitution. Here, we describe the development of a new culture system based on the immobilized Notch ligand Delta‐like‐4 (DL‐4). Culture of human CD34+ CB cells in this new DL‐4 system enabled the in vitro generation of large amounts of T‐cell progenitor cells that (a) displayed the phenotypic and molecular signatures of early thymic progenitors and (b) had high T lymphopoietic potential. When transferred into NOD/SCID/γc−/− (NSG) mice, DL‐4 primed T‐cell progenitors migrated to the thymus and developed into functional, mature, polyclonal αβ T cells that subsequently left the thymus and accelerated T‐cell reconstitution. T‐cell reconstitution was even faster and more robust when ex vivo‐manipulated and nonmanipulated CB samples were simultaneously injected into NSG mice (i.e., a situation reminiscent of the double CB transplant setting). This work provides further evidence of the ability of in vitro‐generated human T‐cell progenitors to accelerate T‐cell reconstitution and also introduces a feeder‐cell‐free culture technique with the potential for rapid, safe transfer to a clinical setting. STEM CELLS2012;30:1771–1780

RUNX1-dependent RAG1 deposition instigates human TCR-δ locus rearrangement

Within the human TCR-α/δ locus, ordered rearrangements requires RUNX1, which binds to the Dδ2-23RSS and interacts with RAG1 to enhance RAG1 deposition at this site. Absence of this RUNX1 binding site in the homologous murine Dδ1-23RSS offers an explanation for the lack of ordered TCR-δ gene assembly in mice.

The BLNK adaptor protein has a nonredundant role in human B-cell differentiation

BACKGROUNDnExpression of the pre-B-cell receptor (pre-BCR) by pre-BII cells constitutes a crucial checkpoint in B-cell differentiation. Mutations that affect the pre-B-cell receptor result in early B-cell differentiation blockades that lead to primary B-cell immunodeficiencies. BLNK adaptor protein has a key role in the pre-B-cell receptor signaling cascade, as illustrated by the abnormal B-cell development in the 4 patients with BLNK gene defects reported to date. However, the BLNK proteins precise function in human B-cell differentiation has not been completely specified.nnnMETHODSnB-cell development, including IgVH and Vk chain repertoires analysis, was studied in the bone marrow of a new case of BLNK deficiency in vitro and in vivo.nnnRESULTSnHere, we report on a patient with agammaglobulinemia, with a total absence of circulating B cells. We detected a homozygous mutation in BLNK, which leads to the complete abrogation of BLNK protein expression. In the bone marrow, we identified a severe differentiation blockade at the pre-BI- to pre-BII-cell transition. IgVH gene rearrangements and selection of the IgH repertoire were normal, whereas the patients pre-BI cells showed very restricted usage of the IgVκ repertoire. Complementation of bone marrow progenitors from the patient with the BLNK gene and transplantation into NOD/SCID/γcko mice allowed the complete restoration of B-cell differentiation and a normal usage of the IgVκ genes.

CXCR4-related increase of circulating human lymphoid progenitors after allogeneic hematopoietic stem cell transplantation.

Immune recovery after profound lymphopenia is a major challenge in many clinical situations, such as allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recovery depends, in a first step, on hematopoietic lymphoid progenitors production in the bone marrow (BM). In this study, we characterized CD34+Lin−CD10+ lymphoid progenitors in the peripheral blood of allo-HSCT patients. Our data demonstrate a strong recovery of this population 3 months after transplantation. This rebound was abolished in patients who developed acute graft-versus-host disease (aGVHD). A similar recovery profile was found for both CD24+ and CD24− progenitor subpopulations. CD34+lin−CD10+CD24− lymphoid progenitors sorted from allo-HSCT patients preserved their T cell potentiel according to in vitro T-cell differentiation assay and the expression profile of 22 genes involved in T-cell differentiation and homing. CD34+lin−CD10+CD24− cells from patients without aGVHD had reduced CXCR4 gene expression, consistent with an enhanced egress from the BM. CCR7 gene expression was reduced in patients after allo-HSCT, as were its ligands CCL21 and CCL19. This reduction was particularly marked in patients with aGVHD, suggesting a possible impact on thymic homing. Thus, the data presented here identify this population as an important early step in T cell reconstitution in humans and so, an important target when seeking to enhance immune reconstitution.

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

BackgroundCommon variable immunodeficiency (CVID) is characterized by infections and hypogammaglobulinemia. Neutropenia is rare during CVID.MethodsThe French DEFI study enrolled patients with primary hypogammaglobulinemia. Patients with CVID and neutropenia were retrospectively analyzed.ResultsAmong 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 (11xa0years) with an increased percentage of deaths compared to the whole DEFI group (31.3 vs 3.4%, Pxa0<xa00.05). Neutropenia was diagnosed for 10 patients before 22xa0years 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 2.6xa0g/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.ConclusionNeutropenia 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.

682. Correction of CTLs Cytotoxic Function Defect by SIN-lentiviral Mediated Expression of Munc13-4 in Type 3 Familial Hemophagocytic Lymphohistiocytosis

Patients with mutations in UNC13D gene, coding for Munc13-4 protein, suffer from type 3 Familial hemophagocytic lymphohistiocytosis (FHL3), a life-threatening disorder of the immune system. Munc13-4 controls docking of lytic granules before they fused with the plasma membrane in cytotoxic T and NK lymphocytes and its defect results in defective cytotoxic function of these cells. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, is partially successful even when a compatible donor is available because of the important inflammatory background of patients. In this context gene therapy could be a promising therapeutic option especially for those patients without any compatible donor. As Munc13-4s function is to allow proper cytotoxic activity in mature cytotoxic CD8+ T cells we proposed that these laters may constitute target for gene correction. We constructed a self-inactivating HIV-1 derived lentiviral vector encoding human Munc13-4 in two different pseudotypes, the high tropism VSV-G and the measles virus glycoproteins (H/F) envelope which target more efficiently lymphoid cells through the signaling lymphocyte activation molecule (SLAM) as described by Verhoeyen et al. 2011. We demonstrated that both vectors are able to stably transduce FHL3 CD8+ T cells resulting in correction of defective degranulation capacity of these cells. However comparative analysis showed that H/F pseudotyped vector was more efficient than VSV-G vector to transduce FHL3 T cells. Adoptive transfer of the gene-corrected FHL3 T cells in SCID mice bearing autologous B-LCL lymphoma led to significant tumor regression due to an efficient homing into the tumor mass and long persistence of corrected T cells in peripheral blood as compared to non-corrected T cells receiving mice. Our study shows for the first time that a lentiviral mediating gene transfer in T cells could be proposed to treat a hemophagocytic lymphohistiocytosis disorder.

759. Reduction of HLH-like Manifestations in Murine Model of Munc13-4 Deficiency Following Lentiviral Gene Transfer into Hematopoietic Stem and Progenitor Cells

Patients with mutations in UNC13D gene, coding for Munc13-4 protein, suffer from type 3 Familial hemophagocytic lymphohistiocytosis (FHL3), a life-threatening disorder of the immune system which represents 25% of all FHLs. Munc13-4 controls docking of lytic granules before they fused with the plasma membrane in cytotoxic T and NK lymphocytes and its defect results in defective cytotoxic function of these cells. Hematopoietic stem and progenitor cell (HSPC) transplantation, which is the only curative treatment for FHL3 to date, is partially successful even when a compatible donor is available because of the important inflammatory background of patients. In this context gene therapy could be a promising therapeutic option especially for those patients without any compatible donor. In this study, we took advantages from a murine model of FHL3, the Jinx mice, to investigate the feasibility of HSPC gene therapy for this pathology. Jinx mice do not spontaneously develop clinical features of hemophagocytic lymphohistiocytosis (HLH), but do so when infected with lymphocytic choriomeningitis virus (LCMV). We generated and used a self-inactivated lentiviral vector to complement HSC from Unc13d -/- (Jinx) mice and transplanted them back into the irradiated Jinx recipients. This transplantation led to the complete reconstitution of the immune system at levels comparable to that of control mice. The recipients were then challenged with LCMV. While Jinx mice reconstituted with GFP expressing HSPC developed leukopenia, anemia and body weight loss, characteristic of HLH in this murine model, gene corrected Jinx recipients developed only mild or no HLH manifestations. This reduction in HLH manifestation correlated with a significant reduction of virus titer in the liver and serum level of IFN-g and inflammatory cytokines. All these ameliorations might be explained by the restoration of cytotoxic function of CTLs as demonstrated in an in-vitro degranulation assay. Overall, this study provides data supporting the potential of HSC gene therapy in a FHL immune dysregulation such as UNC13D deficiency.

225. Preserving CD4+ T-Cells Phenotype and Function Upon Ex Vivo Lentiviral Transduction

Ex vivo gene modification of T-cell is an appealing strategy for the treatment of both immunodeficiencies and neoplasia. Cell activation remains an essential prerequisite to genetic modification by lentiviral transduction. However, most current protocols for T-cell activation alter significantly the phenotype of transduced cells. This phenotypic change can constitute a drawback when the gene modification aims to correct a gene defect while preserving the T cell-subsets diversity. As we plan to conduct an open label phase I/II gene therapy trial for HIV-1 infected patients presenting with lymphoma where the patients will receive autologous hematopoietic stem cells transplantation with gene modified CD34+ cells and CD4+ T-cells, we investigate the use of the TransAct™ reagent (a CD3/CD28 nanoscale activator reagent from Miltenyi Biotec, Bergisch Gladbach, Germany) for the clinical grade transduction of CD4+ T-cells with the LVsh5/C46 lentiviral vector (Cal-1, Calimmune, Inc. Tucson, USA). LVsh5/C46 is a SIN lentiviral vector that inhibits two crucial steps of CD4+ T cell infection by the HIV virus: (i) attachment of the virus to its target by downregulation of CCR5 via a short hairpin RNA, (ii) fusion of the virus to the target cell through expression of the C46 inhibitor. Compared to previously published T-cells transduction protocols, the use of Miltenyi TransAct™ permits an efficient transduction - evaluated by measurement of vector copy number through quantitative PCR - without major phenotypic modification. Indeed, after activation with the TransAct™ reagent, ex vivo transduced CD4+ T-cells display very few changes in their surface markers with conservation of naive (CCR7+CD62L+CD45RA+), central memory (CCR7+CD62L+CD45RA-) and effector memory (CCR7-CD62L-CD45RA-) subsets in superimposable proportions as initially. Moreover, expression of CD25 remains below 50% of cells suggesting a more “gentle “ activation of the transduced CD4+ T-cells. Phenotypic analysis of transduced cells show no overt modification of cell subsets containing TH1, TH2 and TH17 memory T-cells. Furthermore, transduced T-cells retained the ability to be primed towards the TH1, TH2 and TH17 pathways. Cell sorting of ex vivo modified cells demonstrate a preferential transduction of naive CD31+CCR7+CD62L+CD45RA+ T-cells as compared to other subsets. Besides, we did not find any significant impact of the transduction process on the TCRβ repertoire diversity as evaluated by high-throughput sequencing and analysis of diversity through the Gini-Simpson index or the Shannon index. This optimized protocol could be of valuable use for clinical trials aiming to provide the patient with diversified gene-corrected T-cells.

Alterations of circulating lymphoid committed progenitor cellular metabolism after allogeneic stem cell transplantation in humans.

Lymphoid-committed CD34(+)lin(-)CD10(+)CD24(-) progenitors undergo a rebound at month 3 after allogeneic hematopoietic stem cell transplantation (allo-HSCT) in the absence of acute graft-versus-host disease (aGVHD). Here, we analyzed transcriptional programs of cell-sorted circulating lymphoid-committed progenitors and CD34(+)Lin(-)CD10(-) nonlymphoid progenitors in 11 allo-HSCT patients who had (nxa0=xa05) or had not (nxa0=xa06) developed grade 2 or 3 aGVHD and in 7 age-matched healthy donors. Major upregulated pathways include protein synthesis, energy production, cell cycle regulation, and cytoskeleton organization. Notably, genes from protein biogenesis, translation machinery, and cell cycle (CDK6) were overexpressed in progenitors from patients in the absence of aGVHD compared with healthy donors and patients affected by aGVHD. Expression of many genes from the mitochondrial oxidative phosphorylation metabolic pathway leading to ATP production were more specifically increased in lymphoid-committed progenitors in the absence of aGVHD. This was also the case for genes involved in cell mobilization such as those regulating Rho GTPase activity. In all, we found that circulating lymphoid-committed progenitors undergo profound changes in metabolism, favoring cell proliferation, energy production, and cell mobilization after allo-HSCT in humans. These mechanisms are abolished in the case of aGVHD or its treatment, indicating a persistent cell-intrinsic defect after exit from thexa0bone marrow.