Defective apoptosis caused by mutations of the Fas gene can lead to an autoimmune lymphoproliferative syndrome (ALPS). The main autoimmune manifestations are haematological: hemolytic anemia, thrombocytopenia and neutropenia. We described 3 patients with ALPS presenting as a lymphoproliferative syndrome associated with a Coomb's negative autoimmune hemolytic anemia and dyserythropoiesis predominating on the more mature erythroblasts. Fas apoptosis deficiency was evidenced in the 3 patients by the demonstration of an increased number of CD4(-)CD8(-)TCR alpha beta(+) T cells, a decreased apoptotic response of activated T lymphocytes to anti-Apo 1-3 monoclonal antibody and the presence of a heterozygous mutation of the Fas receptor gene. (c) 2006 Elsevier SAS. Tous droits reserves

Defective apoptosis caused by mutations of the Fas gene can lead to an autoimmune lymphoproliferative syndrome (ALPS). The main autoimmune manifestations are haematological: hemolytic anemia, thrombocytopenia and neutropenia. We described 3 patients with ALPS presenting as a lymphoproliferative syndrome associated with a Coomb's negative autoimmune hemolytic anemia and dyserythropoiesis predominating on the more mature erythroblasts. Fas apoptosis deficiency was evidenced in the 3 patients by the demonstration of an increased number of CD4(-)CD8(-)TCRalphabeta(+) T cells, a decreased apoptotic response of activated T lymphocytes to anti-Apo 1-3 monoclonal antibody and the presence of a heterozygous mutation of the Fas receptor gene.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the treatment of choice for many hematological malignancies or inherited disorders. In the partially incompatible HLA setting, ex vivo T-cell depletion of the graft and post-transplantation immunosuppression effectively prevent development of graft-versus-host-disease (GVHD), but lead, in turn, to a delay in immune reconstitution and a concordant increase in the rate of incidence of opportunistic infections and disease relapse. In order to selectively deplete T cells responsible for GVHD, we have designed an ex vivo procedure to eliminate CD25 expressing alloactivated donor T cells. In a first phase I/II study based on the use of an anti-CD25 immunotoxin (IT) 15 pediatric patients with acquired or congenital hematopoietic disorders who had received HSCT were infused with 1 to 8x105 allodepleted T cells/kg. No cases of severe acute (>grade II) GVHD occurred. aGVHD of grade I or II, which developed in four patients, correlated with anti-host residual proliferation above 1% in a mixed lymphocyte reaction. Evidence for early T cell expansion was shown in 3 patients with ongoing viral infections. In an attempt to improve allo-depletion and infuse larger numbers of cells, we have developed a new method based on immuno-magnetical depletion of CD25 expressing cells. In pre-clinical tests, this method lead to a more specific and efficient allo-depletion than that obtained with IT. This new procedure is currently used in a phase I/II clinical trial that will include 25 pediatric patients with inherited disorders of the immune system. 3x105 to 5x106 allo-depleted donor T cells/kg will be infused, with three to five patients per dose. Allodepleted donor T cells are cryopreserved until the obtention of quality control results Inclusion, i.e. infusion of allodepleted T cells is performed only if:1) engraftment is proven, 2) absence of GVHD 3)residual antithymoglobulin <0.2microg/ml,4) allodepletion inhibition more than 99% of residual proliferation against host cells in a mixed lymphocyte reaction. Included patients will be stratified in two groups depending on the presence or absence of a viral infection with clinical symptoms. Toxicity is evaluated by occurrence of aGVHD above grade II. Efficacy is measured by CD4+CD3+ T cell counts in the blood two months post HSCT. A real time bayesian statistical approach will allow to test both criteria as primary objectives and to increase or decrease the dose according to the results obtained. Two patients have been so far included in the “infected” group and infused with 3x105 allodepleted T cells/kg. One patient developed grade II cutaneus GVHD. For both, efficacy was reached, since they presented more than 200 CD4+CD3+ T cells /mm3 as early as 60 days post HSCT. These preliminary results are encouraging but need to be further confirmed.

Two children involved in the successful retroviral gene therapy clinical trial of SCID-X1 (2 year survival: 100%, FFTF 60%) developed a malignant T-cell lymphoproliferation 34 months after treatment. In both cases, vector integration into the vicinity of exon 1 of the protooncogene LMO2 occured (Hacein-Bey-Abina, 2003). The potential for unwanted side effects from retroviral insertional mutagenesis is now a focus of biosafety considerations in gene transfer using integrating vector systems. Several groups could show that gene coding areas, in particular transcription start sites, are preferred insertion loci (Schroder, 2002; Wu, 2003). While these studies mapped a great number of integration sites with statistical significance, they were limited to in vitro gene transfer into nonhematopoietic human cell lines. As T cells do not grow in these cases unless they express the gamma c chain, we reasoned that there may be selection pressure on the retrovirus integration site distribution. Using a highly sensitive and specific linear amplification mediated PCR (LAM-PCR), we analyzed DNA samples from 8 patients enrolled in the X-SCID gene therapy trial. We could perform successive insertion site analysis and subsequently identify more than 400 integration sites after transplantation. In all patient samples examined, a stable polyclonal profile demonstrated polyclonal hematopoietic repopulation. To distinguish the influence of required transgene expression on retrovirus integration site distribution, we analyzed insertion sites in patient CD34+ cells prior to autologous transplantation, shortly after ex vivo transduction. More than 100 integration sites present prior to transplantation could be identified for comparative pre-/post-tranplantation analysis. No obvious difference in insertion site distribution could be detected between pre- and post-transplant samples form a first analysis. However, our data indicate that X-SCID correction, which depends on transgene expression, favors chromosomal locations associated with active transciption. We conclude that the distribution of retrovirus integration sites can be assessed by random sampling in a clinical trial setting and may differ significantly from previously published assumptions, especially in settings of selective advantage related to transgene expression.

Naturally occurring genetic disorders of the immune system provide many models for the study of its development and function. In a way, their analysis complements the information provided by the generation of genetic defects in mice created using homologous recombination techniques. In this review, the recent findings made in three areas are focused upon deficiencies in T cell differentiation and in T lymphocyte activation, and on the control process of peripheral immune response.

Patients undergoing bone marrow transplantation are susceptible to many different bacterial, fungal and viral infections. Among the viral pathogens, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and adenovirus cause the greatest morbidity and mortality and have been the most common infectious causes of death following the grafting of allogeneic marrow. This great susceptibility to viral infections is due to the immunodeficiency in cellular and humoral immune responses lasting for months to years. Contributing factors are high-dose chemo/radiotherapy, graft-versus-host disease (GVHD) prophylaxis/treatment, GVHD itself, the degree of HLA disparity between donor and recipient and the underlying disease. Defects of T cell helper and cytotoxic functions contribute to the great incidence of viral infections. We described here the kinetic of immunological reconstitution and the role of T cell immunodeficiency. At day 30 to 40 after BMT, a minority of patients had recovery of virus-specific CD8+ T-cell response. Between day 40 and day 90 recovery of deficient CD8+ and CD4+ T cell responses occured in the majority of the recipients of HLA identical BMT but only in the minority of the recipients of HLA partially incompatible BMT. New approaches should therefore be envisaged either to preserve donor T-cell-mediate immunity or the accelerate immune reconstitution. Add-back of unmanipulated T-cells, or virus-specific T cells could improve antimicrobial defenses after BMT.

Ligands binding to the CD4 molecule can inhibit TCR-mediated T cell activation. We have previously reported that transcription factors regulating the expression of the IL-2 gene, NF-AT, NF-kappaB, and AP-1, are targets of this inhibitory effect in an in vitro model using peripheral human CD4+ T cells activated by a CD3 mAb. Two T cell activation pathways involved in the regulation of these transcription factors, calcium flux and the p21ras pathway, were investigated as potential targets. Binding of HIV envelope glycoprotein gp160/gp120 or a CD4 mAb to the CD4+ T cells, prior to TCR/CD3 activation, inhibited the intracellular calcium elevation. This event strongly suggested an inhibition of PLCgamma1 activity. Tyrosine phosphorylation of PLCgamma1, induced by CD3 activation, was not affected, but its association with tyrosine-phosphorylated proteins, including a 62-kDa protein, was disrupted. This PLCgamma1-associated p62 was found to be immunoreactive to p62-Sam68 Abs. The activation-induced phosphorylation of two p21ras effectors, Raf-1 and Erk2, was inhibited by the CD4 ligands, indirectly pointing to inhibition of the p21ras activation pathway. In addition, we demonstrate that TCR activation of normal CD4+ T cells induced the formation of p120GAP and PLCgamma1-containing complexes. These complexes also contain other unidentified proteins. CD4 ligand binding induced a defective formation of these transduction complexes. This may result in inefficient signaling, partially accounting for the inhibitory effects of the CD4 ligands on both p21ras and calcium-activation pathways.

Familial hemophagocytic lymphohistiocytosis (FHL) is a rare genetic disorder associated with the onset early in life of overwhelming activation of T lymphocytes and macrophages invariably leading to death. Allogeneic bone marrow transplantation (BMT) from an HLA-identical related donor is the treatment of choice in patients with this disease. However, fewer than 20% of patients have a disease-free HLA-identical sibling. BMT from HLA-nonidentical related donors has previously met with poor results, with graft rejection a major obstacle in all cases. We describe BMTs from HLA-nonidentical related donors (n = 13) and from a matched unrelated donor (n = 1) performed in two centers in 14 consecutive cases of FHL. Remission of disease was achieved before BMT in 10 patients. Marrow was T-cell–depleted to minimize graft-versus-host disease (GVHD). Antiadhesion antibodies specific for the α chain of the leukocyte function–associated antigen-1 (LFA-1, CD11a) and the CD2 molecules were infused pre-BMT and post-BMT to help prevent graft rejection, in addition to a conditioning regimen of busulfan (BU), cyclophosphamide (CP), and etoposide (VP16) or antithymocyte globulin (ATG). Sustained engraftment was obtained in 11 of 17 transplants (3 patients had 2 transplants) and disease-free survival in 9 patients with a follow-up period of 8 to 69 months (mean, 33). Acute GVHD greater than stage I was not observed, and 1 patient had mild cutaneous chronic GVHD that resolved. Toxicity due to the BMT procedure was low. Results obtained using this protocol are promising in terms of engraftment and event-free survival within the limitations of the small sample. We conclude that an immunologic approach in terms of drugs used to obtain disease remission and a conditioning regimen that includes antiadhesion molecules in T-cell–depleted BMT from HLA genetically nonidentical donors is an alternative treatment that warrants further study in FHL patients who lack a suitable HLA genetically identical donor.

We report a detailed comparison of B cell defects in two patients, one XLA and one non-XLA. Both had severe agammaglobulinemia with a total absence of CD19+ cells in the periphery. In the non-XLA case, CD19 expression was also highly impaired in the bone marrow, resulting in the absence of both B and preB compartments. Early proB cells were present since CD34+CD10+ and some CD19+CD10+ mostly CD34+ were identified, although diminished. By contrast, in the XLA patient the CD34+CD19+ proB cells were increased whereas the CD34-CD19+ preB cell population was low. Semi-quantitative RT-PCR analysis performed on mononuclear bone marrow cells from the non-XLA patient indicated that lambda-like, VpreB, Rag-1, Rag-2 and TdT transcripts expressed during proB cell stages were found at normal levels whereas E2A, CD10, Syk, Pax-5, CD19, Ig alpha, Ig beta, VH-C mu and V kappa-C kappa transcripts characteristic of later stages were severely depressed. By contrast in the XLA patient most of these transcripts were observed in normal amounts. The phenotype of the non-XLA patient resembles that of Pax-5 or Ig beta knock-out mice, but since the coding sequence of both cDNAs were shown to be normal, the blockage might rather result from an altered regulation of one of these genes or from defect of other genes. All these data indicate that the non-XLA patient suffers from a new genetic defect that results in an arrest of differentiation within the proB cell compartment, before the onset of Ig gene rearrangements. From all agammaglobulinemias reported so far, including XLA cases and those resulting from C mu gene defects, the non-XLA patient exhibits the earliest blockage in the B cell differentiation pathway.

Mutation of the gamma c chain common to interleukin-2 (IL-2), IL-4, IL- 7, IL-9, and IL-15 receptors has been shown to be responsible for the X chromosome-linked severe combined immune deficiency (SCIDX1). Human SCIDX1 patients are characterized by an absence of T and natural killer cell differentiation. We report the case of a SCIDX1 patient who first had few detectable peripheral T cells, then developed, after haploidentical T-depleted bone marrow transplantation (BMT), up to 2,000/microL autologous T cells. These T cells have persisted over 8 years after BMT and were able to proliferate in the presence of mitogens and of some antigens, although to a lesser extent than control T cells. A stop mutation was identified which predicts that the major part of the cytoplasmic tail of gamma c is truncated. This mutation does not affect high-affinity IL-2 binding, but it partly decreases IL- 2 endocytosis and prevents the downmodulation of the IL-2-receptor beta chain and the tyrosine phosphorylation of Jak 3 protein in response to IL-2. This report raises questions concerning the role of the gamma c chain in IL-2 receptor endocytosis and in T-cell development and differentiation.

Natural killer (NK) cells are characterized by their ability to mediate spontaneous cytotoxicity against susceptible tumor cells and infected cells. They differentiate from hematopoietic progenitor cells. Patients with X-linked severe combined immunodeficiency (SCID X1) carry mutations in the gamma c cytokine receptor gene that result in lack of both T and NK cells. To assess the role of interleukin-2 (IL-2), IL-7, and IL-15 cytokines, which share gamma c receptor subunit, in NK cell differentiation, we have studied NK cell differentiation from cord blood CD34 (+) cells in the presence of either stem cell factor (SCF), IL-2, and IL-7 or SCF and IL-15. The former cytokine combination efficiently induced CD34 (+) CD7 (+) cord blood cells to proliferate and mature into NK cells, while the latter was also able to induce NK cell differentiation from more immature CD34 (+) CD7 (-) cord blood cells. NK cells expressed CD56 and efficiently killed K562 target cells. These results show that IL-15 could play an important role in the maturation of NK cell from cord blood progenitors. Following retroviral-mediated gene transfer of gamma c into SCID X1 bone marrow progenitors, it was possible to reproduce a similar pattern of NK cell differentiation in two SCID-X1 patients with SCF + IL-2 + IL-7 and more efficiently in one of them with SCF + IL-15. These results strongly suggest that the gamma c chain transduces major signal(s) involved in NK cell differentiation from hematopoietic progenitor cells and that IL-15 interaction with gamma c is involved in this process at an earlier step than IL-2/IL-7 interactions of gamma c are. It also shows that gene transfer into hematopoietic progenitor cells could potentially restore NK cell differentiation in SCID X1 patients.