Geneviève de Saint-Basile and I have devoted a great deal of effort to the genetic and molecular characterization of a form of DICS whose gene is located on the X chromosome and is responsible for a complete defect in the development of T lymphocytes and Natural Killer (NK) lymphocytes. We were able to locate the gene locus on the X chromosome in 1984, and above all to observe a spontaneous partial improvement in this serious pathology linked to the occurrence of a "revertant". This phenomenon is the consequence of a somatic mutation event occurring in a T lymphocyte precursor cell which, by chance, led to the correction of the inherited mutation and thus enabled, by selection, the differentiation of T lymphocytes. This observation made it possible to estimate the number of T lymphocytes that can be generated from a single precursor. It was also a key factor in the reasoning behind the development of gene therapy for this disease (see below): a single cell dividing a large number of times can generate a very large number of T-lymphocytes. We considered that a form of "natural" gene therapy had thus, by chance, partially corrected this child's DICS. This observation illustrates how the detailed analysis of an exceptional situation can contribute to our understanding of physiology (T cell development dynamics) and to the development of a new therapy.

The diversification of the antibody response and its genetic anomalies

B lymphocytes produce antibodies (immunoglobulins) which recognize antigens through their variable regions and activate cellular functions through their constant regions. Variable regions are generated by the process of genetic recombination described above. The constant regions are of several types (M, G, A, E) with different functions. A second process of genetic recombination enables an immunoglobulin to be modified in the course of an immune response in such a way that 1) the constant region changes from M to G, A or E, and 2) the variable region acquires (after selection) a higher affinity for the antigen. These are known as immunoglobulin isotype switching (CII) and somatic mutations respectively. These B-cell-specific events occur in secondary lymphoid organs (lymph nodes, spleen) and enhance the immune capacity of B-cells against infectious agents.

A systematic and in-depth study of patients with IHD characterized by a CII defect, notably led by Anne Durandy, has led to the identification or clarification of the role of molecules essential to this secondary diversification of immune regions: firstly, the CD40L molecule, present on the surface of T lymphocytes required for immunoglobulin production ("T-B" cooperation); secondly, the Activation Induced Deaminase (AID) molecule, which induces DNA modifications, responsible for initiating the recombination process (CII) and somatic mutations of variable regions, Uracil N Glycosylase, which "cleans up" the DNA lesion generated by AID (and enzymes for repairing DNA mismatches). The AID molecule, first identified by T Honjo in Kyoto and whose importance has been revealed by the study of human hereditary pathologies, is often referred to as the "mutator" of immunoglobulin genes.

The cytotoxic function of T lymphocytes and genetic abnormalities

One of the effector functions of T lymphocytes (and NK lymphocytes) is to destroy other cells infected by micro-organisms (notably viruses), as well as tumor cells. These "killer" lymphocytes are an essential part of anti-infectious immunity. We know that it is linked to the excretion of the contents of cytoplasmic granules, known as secretory granules, on contact with the target cell. These proteins (perforin and proteases) cause the death of the target cell by a process known as "apoptosis". With Geneviève de Saint-Basile, we have identified a series of hereditary diseases all characterized by a functional defect in the cytotoxic capacity of T (and NK) lymphocytes. This work is of interest from several angles. They draw attention to the fact that a defect in cytotoxicity actually leads to an exacerbated immune response, as if killer lymphocytes were physiologically necessary for the termination of an immune response. They have led to the identification of a series of molecules whose function is to enable exocytosis of cytotoxic granules according to a structured program: intracellular migration, membrane docking, activation and membrane fusion. Last but not least, this process displays striking molecular analogies with the exocytosis of granules containing neuromediators at synapses between nerve cells, despite differences in time scale and size. Thus, an analogous biological question - the regulated excretion of proteins at cell-cell contact - has been similarly addressed in two distinct cell types.

This work has also led to the understanding that these genetic diseases cause a syndrome known as "lymphohistiocytic activation syndrome", in which the persistent secretion of interferon γ is crucial. These results show how cytotoxic cells contribute to completing an antiviral immune response, and open up an interesting therapeutic avenue by neutralizing the effects of interferon γ.

Genetic abnormalities of the immune system and autoimmunity

Autoimmune diseases are common, and their prevalence increases with age. They bear painful witness to the fact that cells of the adaptive immune system (T and B lymphocytes) are able to recognize components of the self. These autoreactive lymphocytes are not normally pathogenic, in other words, they are not involved in immune responses outside autoimmune diseases. How can they be effectively controlled? The answer to this question of both theoretical ("tolerance" to the self) and medical interest once again comes in part from the study of rare hereditary diseases responsible for a susceptibility to autoimmunity. Our main contribution with Frédéric Rieux-Laucat concerns the identification of hereditary anomalies in the gene encoding a protein called FAS in patients suffering from lymphoproliferative syndrome with autoimmunity. FAS is a membrane receptor found notably on the surface of T and B lymphocytes. Its binding by a ligand: the FAS ligand on the surface of activated T lymphocytes causes chronic death of the activated lymphocytes (which is exactly the situation encountered with self antigen-specific lymphocytes). This molecular communication system, which can act intracellularly ("cis") or intercellularly ("trans"), thus controls the eventual escape of self-reactive T and B lymphocytes.

It has also been shown that somatic ("acquired") mutations in the same gene can favor the emergence of autoreactive lymphocytes and thus autoimmune diseases, since such lymphocytes acquire a selective survival advantage. This observation forms the basis of a hypothesis that could account for common autoimmune diseases: the accumulation of somatic mutations in genes controlling cell death or division within autoreactive T and/or B lymphocytes, would favor their escape and thus the induction of autoimmunity, according to a model close to oncogenesis. This hypothesis is testable (and in the process of being tested), and is likely to lead to the identification of important new molecules in the control of autoimmunity.

Today, the analysis of inherited defects in the immune system, in particular its adaptive component (T and B lymphocytes) through the identification of genetically determined defects, is continuing. Current genome sequencing capabilities, in particular exomes, i.e. the coding parts of genes or the entire genome, greatly facilitate this approach. A number of mutations are currently being studied in relation to defects in the development of T or B lymphocytes and the onset of autoimmune or inflammatory diseases. Some of these diseases correspond to a rare genetic form (monogenic cause) of more frequent pathologies, such as Crohn's disease (inflammatory bowel disease). It is hoped that this work will identify molecular pathways whose more complex abnormalities could explain these pathologies, and thus open up possible therapeutic avenues.

We postulate that the continued study of rare genetic disease models responsible for autoimmune diseases will lead to a better understanding of the functioning of the various physiological control systems of lymphocytes reactive against components of the self. In parallel, the systematic search for somatic genome alterations in self-reactive lymphocytes (see above) will enable us to test the hypothesis of the role of the accumulation of such mutations in the genesis of autoimmunity, and to identify potential therapeutic targets.

Genetic abnormalities of the immune system and therapeutics

Based on our growing understanding of the pathophysiology of IHDs, we have sought to develop therapeutics for these disorders, some of which have subsequently been applied to the treatment of other diseases. This work has focused on three areas: the use of monoclonal antibodies, hematopoietic stem cell allografts and gene therapy: based on the observation that rare patients whose leukocytes lack the leukocyte adhesion protein "b2 integrin" (which results in a very severe predisposition to infections due to a defect in leukocyte adhesion and migration) have a reduced capacity for bone marrow graft rejection, we proposed the injection of an anti-b2 integrin monoclonal antibody as a method of preventing bone marrow graft rejection. This approach proved partially successful.

Epstein Barr virus (EBV) causes uncontrolled proliferation of B lymphocytes in immunocompromised individuals (e.g., genetically or as a result of immunosuppressive therapy after organ allograft transplantation), because the virus selectively infects these cells and induces their cell division. In the absence of an effective immune response, this cell proliferation is lethal. We have shown that injecting such patients with monoclonal antibodies specific for B lymphocytes stops this cell proliferation and, in a large number of cases, cures them. In a way, these results foreshadowed the current success of monoclonal antibodies in the treatment of primary B-cell lymphomas.

In the same way, we were involved in the first applications of the therapeutic use of anti-B-cell monoclonal antibodies to treat an autoimmune disease (autoimmune hemolytic anemia) by destroying B-cells, including those producing the pathological autoantibodies.

Hematopoietic stem cell (HSC) allografts

Growing knowledge of the pathophysiology of many severe hereditary immune system disorders has led to the use of HSC allografts to replace deficient cells with functional ones.

Based on this simple principle, tangible results have been obtained for a number of IIDs, inflammatory and autoimmune diseases (mevalonate kinase and Fox P3 deficiencies) and metabolic diseases (adrenoleukodystrophy).

Gene therapy for hereditary immune deficiencies

Despite these successes, allogeneic HSC transplantation has inherent limitations, due to the risk of fatal complications, which can be too high when donor and recipient do not share sufficient HLA tissue group compatibility. An alternative is gene therapy. The principle is based on the introduction of a normal copy of the mutated gene into the stem cells of the hematopoietic system, so that the daughter cells carry this "therapeutic" gene and are able to express the deficient protein in the patient. Stem cells are modified "ex-vivo" using a viral vector ("retrovirus") whose characteristics enable its genome to be integrated into the cell genome. In this way, the therapeutic gene is replicated during each cell division and, thanks to a system for regulating its expression (transcription), the corresponding protein is produced.

The first application we developed with Marina Cavazzana-Calvo and Salima Hacein-Bey-Abina was in the treatment of X-linked SCID, based on the observation of the selective advantage conferred on revertant T-cell precursors. This approach proved successful, as for the first time it was possible to correct the manifestations of the disease in a stable manner over time. Ten patients in Paris, then 10 in London, were treated, 17 of whom are now living well with a functional immune system, with a median follow-up of over 10 years.

However, this success was tempered by the occurrence of leukemia (5 cases/20) due to aberrant deregulation of oncogenes located close to the retrovirus integration site. Although these leukemias were cured in 4 out of 5 cases, their occurrence led to a halt in the trials, a pause used to understand the mechanism of this oncogenic effect. This has now been identified, and is linked to the ability of a regulatory element in the viral vector to permanently transactivate a gene alongside which the virus has integrated. If this gene is an oncogene, its aberrant expression accounts for the risk of leukemia. In a major international collaboration, this work has led to the development of modified vectors in which the transactivator element has been removed. Such vectors have been shown to be experimentally effective and are currently being tested in new clinical trials for the treatment of several diseases: adrenoleukodystrophy (DICS, Wiskott-Aldrich syndrome). Over the next few years, we'll be able to establish whether their use will enable us to maintain their proven efficacy while reducing the risk of side-effects. Results to date are encouraging, but caution is called for.

An unexpected aspect of this research is the analysis of T-cell population dynamics in humans. In treated patients, all T-lymphocytes carry a "molecular signature" corresponding to the integration of the viral vector's cellular genome at a precise location. This signature can be identified and the number of cells bearing the same signature, i.e. descended from the same progenitor cell, quantified. It is therefore possible to monitor the number of progenitors involved in lymphocyte differentiation, quantitative variations in this diversity and the abundance of each clone. Coupling this information with the study of the clonal diversity of newly-formed T lymphocytes (i.e. the receptor for the antigen) and "memory" T lymphocytes (previously engaged in an immune response), it becomes possible to generate a dynamic picture of the evolution of T lymphocyte populations in vivo, particularly with regard to their longevity, renewal and expansion during an immune response.