Marina Cavazzana & Isabelle André-Schmutz
Human Lymphohematopoiesis Lab
- Laure Caccavelli
- Aurélie Gabrion
- Cécile Roudaut
- Alessandra Magnani
- Laurent Laganier
- Elisa Magrin
- Caroline Tuchmann-Durand
- Maxime Bouabdelli
- Horiya Amrane
- Brigitte Ternaux
- Chloé Couzin
- Rania Gatri
- Jennifer Masselis
- Quentin Nicolas
- Jennifer Nisoy
- Steicy Sobrino
- Cindy Da Silva
- Alexandrine Garrigue
- Anne Durandy
- Chantal Lagresle-Peyrou
- Emmanuelle Six
- Shabi Soheili
- Sven Kracker
- Corinne de Chappedelaine
- Soëli Charbonnier
- Ranjita Moiranghtem
- Juliette Olivré
- Kuiying Ma
- Marianne Delville
- Julien Zuber
- Leslie Weber
- Florence Bellier
- Amine Bouafia
- Loïc Chentout
- Sabrina Lizot
- Inès Rajbaoui
- Sarah Enouz
- Adeline Denis
- Chloé Mollet
- Fanny Montoya
- Elodie Edmond
The common denominator of our project is the human lymphohaematopoietic system, characterized by cells with differing self-renewal and differentiation capacities as a function of the individual's age and clinical status (i.e. healthy or diseased). In adult mammals, haematopoiesis (i.e. the expansion and differentiation of haematopoietic stem cells into blood cells in the bone marrow) undergoes constant, tightly regulated renewal and undergoes profound changes over the lifespan.
Understanding of the hierarchy of human haematopoiesis and the different steps in T and B cell differentiation in the healthy body and in very particular disease situations constitutes the most fundamental part of our research project. Overall, the knowledge generated by these studies will help us to actively implement new treatment protocols. Haematopoietic stem and progenitor cells (HSPCs) harvested from a healthy or diseased individual and ex vivo gene modifications constitute essential tools for curing most severe, cell-intrinsic, inherited defects of the lymphohaematopoietic system. Nevertheless, several issues still compromise the full success of these types of therapies.
Improvements in this HSPC-based strategy have resulted from progress and discoveries provided by the first part of our project and by other research groups. The most recent findings on the characteristics of human T cells (i.e. their long life, self-renewal capacity, homeostasis and functions) have prompted us and others to consider their in vivo use after ex vivo manipulation - paving the way for less toxic therapeutic approaches.
1. HSPC homeostasis and hematopoietic hierarchy.
The follow-up of gene therapy trials performed in the Biotherapy Department give us the unique opportunity to track progenitor cells and their descendants through deep sequencing analysis of retroviral integration sites (RIS) (a collaboration with F. Bushman (University of Pennsylvania, Philadelphia, USA)). This extensive RIS analysis is conducted in the context of several immunodeficiencies (such as SCID-X1, WAS, and beta-thalassemia). The major advances in integrome knowledge provide unique information on human hematopoietic ontogeny that can be inferred by integration sites tracking in fractionated blood cell populations. In the Wiskott-Aldrich Syndrome (WAS) patients, this clonal tracking highlights a diversity of hematopoietic differentiation programs with different levels of contribution to the myeloid and lymphoid lineages. These new findings provide unique data on human hematopoiesis.
Circos plot showing integration sites sharing between lineages. Sharing of integration site is depicted for one WAS patient 3 years after gene therapy treatment. The proportion of integration sites shared between myeloid (Neutrophils and Monocytes) and lymphoid (B cells, T cells and NK cells) lineages is represented by the ribbons connecting each combination of two lineages. Six Emmanuelle
2. Study of pathological T and B cell differentiation.
We have a unique opportunity to study a cohort of patients presenting intrinsic cellular defects at different stages in the hematopoietic differentiation process. These “natural” models provide us with key information for implementing our knowledge of human hematopoietic development and homeostasis. Our cohort of primary immunodeficiency (PID) patients includes those with combined T and B cell defects. Some PIDs are related to a peripheral defect in the late phase of lymphoid differentiation or maturation, whereas others are related to a central defect with an early block in B and T differentiation. Two examples are presented here:
a. Moesin deficiency: a T-cell and B-cell defect compromising the migration/survival of the two lineages
We have been interested by the similar clinical features of 7 male patients. During childhood, most of them developed severe varicella, pneumopathies and recurrent pulmonary infections. All the patients have a severe peripheral leucopenia, hypogammaglobulinaemia and a poor response to vaccinal Ag. Despite the severe leucopenia, Igs and prophylactic treatment appeared sufficient to prevent severe infections. Among the T lymphocytes, the naïve compartment was particularly low and T cell proliferation in vitro decreased as compared to the controls. Using exome-sequencing analysis, we have identified in all patients the same missense mutation in the moesin gene, a member of the ERM protein family, which links plasma membrane proteins with actin–based cytoskeletons and is implicated in various cellular functions such as survival, adhesion, migration and activation.
b. B-cell defects: immunoglobulin switch recombination deficincies
We have a unique opportunity to study a cohort of primary immunodeficiency (PID) patients including primary antibody deficiencies and combined T and B cell defects. Some PIDs are related to a peripheral defect in the late phase of lymphoid differentiation or maturation, whereas others are related to a central defect with an early block in B and T differentiation. Disturbed PI3K/AKT/mTOR signaling as disease causing mechanism for PIDs is one of our main focus of studies.
3. From bench to bedside: the challenge of translational medicine.
The ideal treatment for a number of PIDs is replacement of the patient’s HSPCs by allogeneic healthy or autologous gene modified ones. However, allogeneic or gene modified autologous hematopoietic stem cell transplantation (HSCT) faces common as well as specific obstacles Common obstacles are related to the inflammatory cytokines present at the time of treatment, the need to obtain “empty niches” without inducing toxicity and the long time period needed to reconstitute the adoptive T-cell compartment. This last obstacle is responsible for 30% of deaths when the patient presents on-going, severe, opportunistic infections and the HSPC donor is only partially HLA-compatible. This obstacle could be solved through an acceleration of T-cell generation by ex vivo generated T-cell progenitors.
a. Acceleration of T cell generation by transplantation of ex vivo generated T-cell progenitors
We have developed a new culture system based on the immobilized Notch ligand DL-4. Culture of human HSPCs in this system enabled the in vitro generation of large amounts of T-cell progenitor cells and accelerated T-cell reconstitution after HSCT in NSG mice. This culture system thus provides a feeder-cell-free culture technique mimicking the thymic niche, with the potential for rapid, safe transfer to a clinical setting. In this context, the project aims at (i) translating the protocol into a clinical trial, (ii) further enriching this artificial thymic niche by identifying thymic factors implicated in the recruitment, growth and commitment of HSPCs, and (iii) understanding how ex vivo generated T-cell precursors migrate to the thymus and participate to the regeneration of the thymic stroma.
CD34+ lymphoid progenitors (green) seed the thymus (pink) and immediately engage in the process of thymopoiesis in specialized thymic niches (as illustrated by CD7 expression (red). André-Schmutz Isabelle.
b. Gene therapy and hereditary disorders
With the Biotherapy Department and the fundamental research laboratory, gene therapy trials constitute a real challenge to not only cure patients with rare genetic diseases but also to better understand the clinical aspects of these disorders. Today, gene therapy is necessary as a means of treatment of extreme power and extraordinary efficiency.
The use of haematopoietic stem cells to correct genetic disorders is the main task in our laboratory and induces high expectations for paediatric patients enrolled into our clinical studies:
- Pre-clinical studies: Fanconi anemia, sickle cell disease, IPEX syndrome or severe combined immunodeficiency, familial hemophagocytic lymphohistiocytosis type 3, HIV.
- Clinical studies: Wiskott-Aldrich syndrome, sickle cell disease, beta-thalassemia, CGD, CD45RA