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Wiskott-Aldrich syndrome (WAS) is a severe X-linked immunodeficiency characterized by recurrent infections, thrombocytopenia, eczema and increased risk of autoimmune disorders and lymphomas. Hematopoietic stem cell (HSC) transplantation from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Transplantation of genetically corrected autologous HSC could represent an alternative treatment, potentially applicable to all patients. In a murine model of WAS (WAS|[minus]|/|[minus]|), we recently demonstrated correction of the T cell defect 4 months after lentiviral vector-mediated gene therapy [Dupr|[eacute]|, Marangoni, et al. Hum Gene Ther. 2006, 17]. The aim of the present study was to investigate the long-term efficacy and safety of our gene therapy approach in WAS|[minus]|/|[minus]| mice.

The initial interaction with host tissues of systemically administered viral vectors is a critical event for stable gene transfer. It is now well established that injection of adenoviral vectors leads to an inflammatory response, mediated by elevations in IL-6 and TNF|[alpha]|. This response triggers the adaptive immune response, and leads to anti-vector and transgene immunity.

Highly purified CD34++CD38−Lin− hematopoietic progenitors isolated from human fetal liver were infected with the murine retroviral vector, MFG nls-LacZ, which encodes a modified version of the Escherichia coli β-galactosidase gene. Progenitors that were cocultured with the packaging cell line could reconstitute human bone marrow or thymus implanted in SCID-hu mice. Expression of the β-galactosidase gene was observed in primitive and committed clonogenic progenitors, mature myeloid, B-lineage cells, and T-lineage cells for up to 4 months after injection into SCID-hu mice. Furthermore, hematopoietic reconstitution by genetically modified progenitor cells could be achieved by the injection of the cells generated from as few as 500 CD34++CD38−Lin− cells, suggesting efficient retroviral gene transfer into fetal liver progenitors.

Induction and maintenance of peripheral tolerance are important mechanisms to maintain the balance of the immune system. In addition to the deletion of T cells and their failure to respond in certain circumstances, active suppression mediated by T cells or T-cell factors has been proposed as a mechanism for maintaining peripheral tolerance. However, the inability to isolate and clone regulatory T cells involved in antigen-specific inhibition of immune responses has made it difficult to understand the mechanisms underlying such active suppression. Here we show that chronic activation of both human and murine CD4+ T cells in the presence of interleukin (IL)-10 gives rise to CD4+ T-cell clones with low proliferative capacity, producing high levels of IL-10, low levels of IL-2 and no IL-4. These antigen-specific T-cell clones suppress the proliferation of CD4+ T cells in response to antigen, and prevent colitis induced in SCID mice by pathogenic CD4+CD45RB(high) splenic T cells. Thus IL-10 drives the generation of a CD4+ T-cell subset, designated T regulatory cells 1 (Tr1), which suppresses antigen-specific immune responses and actively downregulates a pathological immune response in vivo.

Intensity of pretransplant conditioning has been closely correlated with regimen related toxicity in patients receiving allogeneic bone marrow transplantation (BMT). In this review, we summarize evidence for a direct link between inflammatory reactions induced by irradiation and cytotoxic treatment and occurrence of acute graft-versus-host disease (GvHD) as well as endothelial complications: In our studies, de novo release of TNFalpha during conditioning was associated with an increased risk of severe GvHD and mortality following BMT, whereas increased spontaneous production of IL-10, an endogenous TNF-antagonist, prior to conditioning protected from these complications. Immunogenetic differences in cytokine regulation and costimulation by endotoxin proved to be important cofactors determining the extent of inflammatory cytokine release in individual patients. Pathophysiological relevance of these findings seems to be confirmed by experimental as well as first clinical trials using TNF-antibodies and related antagonists during pretransplant conditioning. Preclinical experiments suggest additional, cytokine independent inflammatory reactions induced by irradiation such as expression of ICAM-1 and endothelial cell apoptosis. Although the exact impact of these findings on pathophysiology of BMT related complications needs further clarification by future studies, conditioning related inflammation as a first crucial step in induction of GvHD and complications has to be considered when designing new protocols for preparation of patients for allogeneic BMT.

Adenosine deaminase (ADA) deficiency is a severe combined immunodeficiency (SCID) characterized by systemic toxicity and profoundly impaired lymphoid cell development and functions due to the accumulation of purine metabolites. ADA-deficient (ADA KO) mice die within 3 weeks of age due to metabolic toxicity and can be rescued by enzyme replacement therapy with PEG-ADA. The aim of our study was to investigate the efficacy of gene therapy (GT) in comparison to bone marrow transplantation (BMT) in the ADA KO mouse model. A SIN HIV-1-based lentiviral vector encoding human ADA cDNA under the control of PGK promoter was constructed. ADA KO mice were maintained short-term on PEG-ADA and then transplanted, after non-myeloablative conditioning either with WT BM cells or with ADA KO BM cells transduced ex vivo. We first showed that BMT with donor WT cells rescued mice from lethality, restored T-cell and B-cell counts in the peripheral blood and led to normal proliferation after T or B cells specific stimulation. Similarly, GT with transduced BM cells rescued ADA KO mice from lethality and restored their growth to normal levels. Quantitative PCR for donor cells (Y chromosome in sex-mismatched transplant) and vector-positive cells showed the presence of ADA transduced cells in the peripheral circulation of transplanted ADA KO mice (30|[ndash]|40% 5 months post-GT). Two months after GT, ADA activity in red blood cells reached normal levels and dAXP toxic metabolites decreased to undetectable levels. GT resulted in normal lymphoid differentiation in primary and secondary lymphoid organs, as well as restoration of T-cell and B-cell in vitro proliferative responses after stimulation with anti-CD3/anti-CD28 and anti-IgM antibody, respectively. Finally, serum levels of IgM and IgG were comparable between GT and BMT mice. These studies demonstrate that lentiviral-mediated ADA gene transfer associated with non-myeloablative conditioning corrects both the metabolic and the immunological defects in the ADA KO animal model. We are currently evaluating the long-term safety related to the use of hematopoietic stem/progenitor cells transduced with lentiviral vectors.

Wiskott-Aldrich syndrome (WAS) is a X-linked primary immunodeficiency with a median survival of 14 years of age due to infections, severe hemorrhage, and lymphomas. Transplantation of hematopoietic stem cells (HSC) from HLA-identical sibling donors is a resolutive treatment, but is available for a minority of patients. Transplantation of genetically corrected autologous HSC could represent an alternative treatment applicable to all patients. We investigated the efficacy and safety of WAS gene transfer with HIV-based lentiviral vectors using untransformed T cells from WAS patients and HSC.

Stable gene replacement by in vivo administration of lentiviral vectors (LV) has important therapeutic applications. However, successful gene therapy is often limited by the immune response to the transgene products, which leads to the clearance of transduced cells. Induction of tolerance to a transgene is highly desirable for successful gene therapy trials. We developed a model in which immunocompetent B6 mice were injected with LV expressing GFP under the CMV promoter (LV-GFP). Peak of GFP expression was detected two weeks after injection and a rapid decrease thereafter. Infiltration of CD8+ T cells was observed in the liver two weeks after LV-GFP injection. GFP-specific IFN-g production by splenic CD8+ T cells derived from LV-GFP injected mice was detected by ELISPOT assay starting from the first week until fifteen weeks post injection. Anti-GFP Abs were also present two weeks after injection and gradually decreased over time. Overall these data indicate that immunocompetent mice injected with LV-GFP develop an Ag-specific CD8+ T cell response that leads to clearance of the transgene expressing cells. CD4+CD25+ T regulatory (Tr) cells play a key role in peripheral tolerance and they have been extensively characterized in preclinical models of T cell mediated diseases. In the present model we investigated whether Tr cells could prevent GFP clearance in vivo. B6 mice were co-injected with LV-GFP and Tr cells isolated from syngeneic wt mice (wt Tr). Clearance of GFP expressing cells and anti-GFP immune response was unmodified in LV-GFP+ wt Tr cells co-injected mice, suggesting that injection of wt Tr cells and consequent increase in the Tr cells pool did not protect from the immune response to the transgene. To investigate whether CD4+CD25+ Tr cells need to be GFP specific in order to modulate the anti-GFP immune response, B6 mice were co-injected with LV-GFP and GFP expressing cells obtained from splenocytes of GFP-Tg mice (GFP Tg cells). Interestingly, transfer of GFP Tg cells was able to down-modulate expansion of splenic GFP-specific CD8+ T cells. Since the GFP Tg cells comprise of both CD4+CD25+ Tr (13,5%) and GFP Tg antigen presenting cells (GFP Tg APC) we next investigated the respective role of these two cell populations. We first co-injected mice with LV-GFP and highly purified GFP Tg Tr (99,1%). As with the administration of wt Tr cells, the transfer of highly purified GFP Tg Tr cells did not modulate the immune response to GFP in vivo. On the contrary, the co-administration of LV GFP and highly purified GFP Tg APC significantly modulated both the cellular and humoral anti-GFP immune responses. These results demonstrate that APC isolated from GFP tolerant mice can reduce the immune response to GFP when transferred in vivo. We hypothesize that after LV-GFP injection the transgene expressing GFP Tg APC activate the endogenous CD4+CD25+ Tr in the absence of danger signals leading to down regulation of the anti-GFP response. Transgene presentation by APC is therefore crucial to trigger an immune-regulatory response mediated by CD4+CD25+ Tr cells.

T regulatory cells (Tregs) play a pivotal role in promoting and maintaining tolerance. Several subsets of Tregs have been identified but, to date, the best characterized are the CD4+FOXP3+ Tregs (FOXP3+Tregs), thymic-derived or induced in the periphery, and the CD4+ IL-10-producing T regulatory type 1 (Tr1) cells. In the past decade much effort has been dedicated to develop methods for the in vitro induction and expansion of FOXP3+Tregs and of Tr1 cells for Treg-based cell therapy to promote and restore tolerance in T-cell mediated diseases, and for expanding antigen (Ag)-specific Tregs in vivo. FOXP3+Tregs constitutively express high levels of CD25 and of the transcription factor FOXP3. FOXP3+Tregs are distinguished from activated CD4+ T cells by the low expression of CD127, and by the DNA demethylation of a specific region of the FOXP3 gene called Treg-specific demethylated region (TSDR). FOXP3+Tregs suppress effector T-cell responses through cell-to-cell contact-dependent mechanisms and suppression requires activation via TCR and is IL-2 dependent. In vitro protocols to expand FOXP3+Tregs for adoptive transfer in vivo have been established. We demonstrated that rapamycin permits the in vitro expansion of FOXP3+Tregs while impairing the proliferation of non-Tregs. Moreover, rapamycin-expanded FOXP3+Tregs maintain their regulatory phenotype in a proinflammatory environment and Th17 cells do not expand in the presence of rapamycin. Despite the progress in FOXP3+Tregs expansion protocols, adoptive transfer of FOXP3+Tregs in humans remains a difficult experimental procedure due to the ability to expand a sufficient number of Ag-specific FOXP3+Tregs in vitro. To propagate a homogenous population of FOXP3+Tregs we developed a lentiviral vector (LV)-based strategy to ectopically express FOXP3 in CD4+ T cells. This method results in the development of suppressive cells that are super-imposable to FOXP3+Tregs. Conversion of effector T cells into FOXP3+Tregs upon LV-mediated gene transfer of wild-type FOXP3 was also obtained in CD4+T cells from immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) patients. We also developed a LV platform, which selectively targets expression of the transgene in hepatocytes, to induce tolerance to self or exogenous Ags. Using this approach we showed that systemic administration of LV encoding for the gene of interest leads to the induction of Ag-specific FOXP3+ Tregs, which mediate tolerance even in pre-immunized hosts. Tr1 cells are identified by their cytokine profile (IL-10+TGF-b+IL-4-IL-17-). Tr1 cells express transiently FOXP3 upon activation; but FOXP3 expression never reaches the high levels characteristic of FOXP3+Tregs. Tr1 cell differentiation and function is independent of FOXP3 since suppressive Tr1 cells can be isolated or generated from peripheral blood of IPEX patients. Tr1 cells were first discovered in peripheral blood of patients who developed tolerance after HLA-mismatched fetal liver hematopoietic stem cell transplant (HSCT). Since their discovery, Tr1 cells have proven to be important in mediating tolerance in several immune-mediated diseases. The immuno-regulatory mechanisms of Tr1 cells have been studied over the years thanks to the possibility to generate these cells in vitro. Tr1 cells suppress T-cell responses via the secretion of IL-10 and TGF-β and by the specific killing of myeloid APC via Granzyme B and perforin. Tr1 cells can be induced in vitro in an Ag-specific manner in the presence of IL-10 or of DC-10. Proof-of-principle clinical trials in allogeneic HSCT demonstrated the safety of Treg-based cell therapy with these polarized Tr1 cells. We are currently planning a phase I/II trial using in vitro polarized Tr1 cells with DC-10 in patients after kidney transplantation. An alternative strategy for the induction of high numbers of human Tr1 cells is the LV-mediated gene transfer of human IL-10 into conventional CD4+ T cells. Stable ectopic expression of IL-10 leads to the differentiation of homogeneous populations of Tr1-like cells displaying potent suppressive functions both in vitro and in vivo. A major hurdle, which limited the studies and the clinical use of Tr1 cells, was the lack of specific biomarkers. By gene expression profiling of human Tr1 cell clones we identified two surface markers (CD49b and LAG-3), which are stably and selectively co-expressed on murine and human Tr1 cells induced in vitro or in vivo. The co-expression of CD49b and LAG-3 enables the isolation of highly suppressive Tr1 cells from in vitro IL-10-polarized Tr1 cells and allows tracking of Tr1 cells in peripheral blood of patients who developed tolerance after allogeneic HSCT. The identification of CD49b and LAG-3 as Tr1-specific biomarkers will facilitate the study of Tr1 cells in vivo in healthy and pathological conditions and the use of Tr1 cells for forthcoming therapeutic interventions. In conclusion, Tregs play a key role in maintaining immunological homeostasis in the periphery. Several open questions regarding FOXP3+ Tregs or Tr1 cell-based therapy in humans remain: how long do Tregs survive after transfer? Is their phenotype stable in pathological conditions and inflammatory environments? Is their mechanism of suppression in vivo Ag-specific? Carefully designed and standardized future clinical protocols reflecting a concerted action among different investigators will help to address these questions and to advance the field. Disclosures: No relevant conflicts of interest to declare.

Severe combined immunodeficiency is a congenital syndrome due to various genetic abnormalities that cause susceptibility to infection, failure to thrive, lymphoid hypoplasia, very low levels of T lymphocytes, and hypogammaglobulinemia.1,2 Untreated, the disorder is usually fatal within the first year of life. We report the successful treatment of a fetus with the X-linked variant of severe combined immunodeficiency by the in utero transplantation of paternal bone marrow that was enriched with hematopoietic cell progenitors. Case Report The patient, 11 months old at this writing, is the second son of a 28-year-old woman known to carry a mutation found in . . .

There have been numerous reports of decreased acute and chronic graft-versus-host disease in patients receiving HLA-matched or HLA-disparate umbilical cord transplants. It has been proposed that this may be due to the unique properties of the neonatal immune system, which permit the development of tolerance to alloantigens. This review discusses experimental evidence contrasting the immune functions of cells derived from cord blood and those from peripheral blood.

Despite significant progress in the past 20 years, induction of tolerance to host and donor HLA antigens after allogeneic transplantation, remains a rare event. Graft rejection and graft-versus-host disease (GVHD) are still the major obstacles to reach long-term engraftment and disease-free survival in patients transplanted with allogeneic hematopoietic stem cells, or solid organs.1,2 Allograft rejection and GVHD are mainly due to activation of alloreactive T cells of host and donor origin, respectively. The recognition of alloantigens presented by either allogeneic, or autologous antigen-presenting cells (APC) results in activation of these allogeneic T cells.3 This initial immune response leads to a cascade of events, including up-regulation of adhesion molecules, activation of macrophages, migration of T cells and recruitment of other effector. cells, which cause tissue damage and the appearance of clinical manifestations of graft rejection and GVHD.4,5 A large body of evidence supports the notion that cytokines play a critical role in these events. In particular, IL-2 and IFN-γ produced by the alloreactive T cells are involved in the initial amplification of the allogeneic responses, whereas IL-1 and TNF-α, produced by the APC, significantly contribute to the strong inflammatory responses which are characteristic for these diseases.5–7Therefore, modulation of cytokine production may represent a valuable target for immunosuppression.

The identification of a battery of cytokines which influence the growth, differentiation and functions of hematopoietic cells has resulted in the use of these cytokines for the treatment of human diseases. The number of diseases for which cytokine therapy will be indicated will surely increase in the near future with our increasing knowledge of how cytokines can influence the growth and regulation of hematopoietic tissues. Rodent, canine, feline and nonhuman primate models are currently used for studying experimental cytokine therapies. These model systems have in the past provided credible evidence justifying the clinical trials of a number of cytokines. However, all of these small and large animal models cannot be used to address specific questions of how human hematopoietic cells would respond to a particular treatment. The effects of cytokines on hematopoiesis in nonhuman primates have been shown to closely resemble those observed on humans due to the similarities of our two species.1 However, studies utilizing nonhuman primates are restricted by several concerns including the cost, availability of animals and ethical considerations. For these reasons, C.B-17 scid/scid (SCID) mice implanted with human hematopoietic tissues (SCID-hu) are a potentially powerful preclinical model for studying the effects of cytokines on human hematopoiesis in vivo.

The effects of the recently identified FLK-2/FLT-3 ligand (FL) on the growth of purified human fetal liver progenitors were investigated under serum-deprived culture conditions. FL alone was found to stimulate modest proliferation in shortterm cultures of CD34(++) CD38(+) lineage (Lin)(-) light-density fetal liver (LDFL) cells and the more primitive CD34(++) CD38(-)Lin(-) LDFL cells. However, the low levels of growth induced by FL were insufficient for colony formation in clonal cultures. Synergism between FL and either granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3) or KIT ligand (KL) was observed in promoting the growth of high-proliferative potential (HPP) colony-forming cells (CFC) and/or low-proliferative potential (LPP)-CFC in cultures of CD34(++) CD38(+) Lin(-) and CD34(++) CD38(-) Lin(-) LDFL-cells. FL, alone or in combination with other cytokines, was not found to affect the growth of CD34(+) Lin(-) LDFL cells, the most mature subpopulation of fetal liver progenitors investigated. The growth of the most primitive subset of progenitors studied, CD34(++) CD38(-) Lin(-) LDFL cells, required the interactions of at least two cytokines, because only very low levels of growth were observed in response to either FL, GM-CSF, IL-3 or KL alone. However, the results of delayed cytokine-addition experiments suggested that individually these cytokines did promote the survival of this early population of progenitors. Although two-factor combinations of FL, KL, and GM-CSF were observed to promote the growth of early progenitors in a synergistic manner, neither of these factors was found to make fetal liver progenitors more responsive to suboptimal concentrations of a second cytokine. Only myeloid cells were recovered from liquid cultures of CD34(++) CD38(-) Lin(-) LDFL cells grown in the presence of combinations of FL, KL, and GM-CSF. These results indicate that FL is part of a network of growth factors that regulate the growth and survival of early hematopoietic progenitors. (C) 1995 by The American Society of Hematology.

Thymic development proceeds in stages whereby immature T-cell precursors lacking T-cell receptor (TCR) and CD4 and CD8 expression (double negative: DN), progress through a TCRlo CD4+CD8+ (double positive: DP) stage before differentiating into mature TCRhi CD4+CD8− or CD4−CD8+ (single positive: SP) thymocytes.1 DP thymocytes are subjected to positive and negative selection.2 Positive selection results in the development of a peripheral T-cell population capable of responding to antigen presented in the context of self major histocompatibility complex (MHC). Negative selection eliminates T cells with potentially harmful self-reactivity. This process is defined as clonal deletion. DP thymocytes which are not clonally deleted progress to a mature SP stage.

The phenotypic similarities between natural killer (NK) and T cells have led to the hypothesis that these distinctive lymphocyte subsets may be developmentally related and thus may share a common progenitor (Lanier, L. L., H. Spits, and J. H. Phillips. 1992. Immunol. Today, 13:392; Rodewald, H.-R., P. Moingeon, J. L. Lurich, C. Dosiou, P. Lopez, and E. L. Reinherz. 1992. Cell, 69:139). In this report, we have investigated the potential of human CD34(+) triple negative thymocytes ([TN] CD3(-), CD4(-), CD8(-)) to generate both T cells and NK cells in murine fetal thymic organ cultures (mFTOC) and in vitro clonogenic assays. CD34(+) TN thymocytes, the majority of which express prominent cytoplasmic CD3 epsilon (cytoCD3 epsilon) protein, can be divided into high (CD34(Bright)) and low (CD34(Dim)) surface expressing populations. CD34(Bright) TN thymocytes were capable of differentiating into T and NK cells when transferred into mFTOC, and demonstrated high NK cell clonogenic capabilities when cultured in interleukin (IL)-2, IL-7, and stem cell factor (SCF). Likewise, CD34(Bright) TN thymocyte clones after 5 d in culture were capable of generating NK and T cells when transferred into mFTOC but demonstrated clonogenic NK cell differentiation capabilities when maintained in culture with IL-2. CD34(Dim) TN thymocytes, however, possessed only T cell differentiation capabilities in mFTOC but were not expandable in clonogenic conditions containing IL-2, IL-7, and SCF. No significant differentiation of other cell lineage was detected in either mFTOC or in clonogenic assays from CD34(+) TN thymocytes. These results represent the first definitive evidence of a common T/NK cell progenitor in the human fetal thymus and delineate the point in thymocyte differentiation where T and NK cells diverge.

THE FLT3/FLK2 receptor tyrosine kinase is closely related to two receptors, c-Kit and c-Fms, which function with their respective ligands, Kit ligand and macrophage colony-stimulating factor to control differentiation of haematopoietic and non-haematopoietic cells1–5. FLT3/FLK2 is thought to be present on haematopoietic stem cells and found in brain, placenta and testis3–5. We have purified to homogeneity and partially sequenced a soluble form of the FLT3/FLK2 ligand produced by mouse thymic stromal cells. We isolated several mouse and human complementary DNAs that encode polypeptides with identical N termini and different C termini. Some variants contain hydrophobic transmembrane segments, suggesting that processing may be required to release soluble lig-and. The purified ligand enhances the response of mouse stem cells and a primitive human progenitor cell population to other growth factors such as interleukins IL-3 and IL-6 and to granulocyte-macrophage colony-stimulating factor, and also stimulates fetal thymocytes.

In this review we describe how studies on the cytokine-stimulated growth of murine bone marrow (BM) progenitors have lead to the observations that large increases in progenitor numbers can be achieved in short-term cytokine-stimulated liquid cultures. Transplantation of these ex vivo expanded murine BM cells was shown to decrease the number of BM cells required to confer radioprotection and to increase the recovery rate of both myeloid and erythroid peripheral blood cells. The ex vivo expansion of murine BM cells does not, however, markedly diminish stem cells capable of long-term hematopoietic reconstitution. Investiga- tions on the expansion of human BM, peripheral blood, umbilical cord blood and fetal hematopoietic progenitors have demonstrated that clinically useful increases in progenitor numbers from these tissues are possible. Thus, ex vivo progenitor expansion may soon be of use in transplantation protocols to accelerate hematopoietic reconstitution and in gene therapy protocols if hematopoietic stem cells can be maintained during ex vivo culture.

Transplantation of allogeneic hematopoietic stem cells in man can cure a variety of diseases including primary immunodeficiencies, metabolic diseases, hematological disorders and malignancies [see for review 1]. Graft versus host disease (GVHD), however, continues to be the major clinical problem, with a mortality rate as high as 50% [2]. Although the precise effector mechanisms of the GVHD are not clear, it is well established that three prerequisite are necessary for the development of the disease: a histocompatibility antigen difference between the donor and recipient, a source of donor immunocompetent cells, and the inability of the host to reject the graft. There are, however, cases in which transplantation of HLA mismatched cells in a non immunocompetent recipient is not followed by GVHD but, on the contrary, it results in full tolerance between host and donor cells. One of such an example are children with Severe Combined Immunodeficiency (SCID) successfully transplanted with HLA mismatched fetal liver stem cells. In these patients no signs of GVHD have been observed despite the HLA incompatibility between the donor and the host cells and the in vitro isolation of donor derived T cells with strong anti-host reactivity [3,4]. The activity of these T cells, which are not deleted from the repertoire, should be suppressed in vivo in order to maintain the homeostasis.

Transplantation of normal hemopoietic stem cells from the fetal liver can cure a number of diseases in experimental animals as well as in humans. Because of immune immaturity of the human fetus during the first trimester of gestation, fetal liver cells of 8–12 weeks post-fertilization are devoid of any T lymphocyte ; therefore they do not induce graft-versus-host disease (GvHD) after transplantation into an allogeneic host, despite full mismatch [1]. Prevention of rejection of these transplanted stem cells can be ensured by immunodeficiency disease of the host, by immunosuppressive treatment or by immune immaturity of the host when the transplant is performed into a fetal patient [2]. The stem cells from the fetal donor progressively differentiate into T lymphocytes within the environment of host antigens, and give rise to mature T cells exerting their functional activities and restoring immune defenses to the patient [3]. In most cases, the fetal donor and the patient host are fully mismatched at the class I and the class II loci of the major histocompatibility complex (MHC). T lymphocytes deriving from the donor stem cells are therefore confronted with HLA-different host cells (monocytes-macrophages, B lymphocytes, NK cells, target cells of various kinds, etc) after they have matured in this HLA-different host environment. This situation provides us with a unique model to study recognition of “self” and “allo” by helper and cytotoxic T cells, as well as the acquisition of tolerance during T cell ontogeny. Positive and negative selection processes are further demonstrated to be separate phenomena, likely to be induced by distinct cell categories.

In this article, we report that the human fetal thymus contains CD34(bright) cells (

Regulatory T cells (Tregs) are a specialized subset of T cells able to control immune responses and promote and maintain immune tolerance. Deficiency or defective function of Tregs has been correlated with autoimmunity, whereas their presence has been associated with tolerance. CD4+ Tregs have been classified into two major subsets according to their origin: the natural occurring Tregs, which develop in the thymus and are present in mice and healthy human individuals since birth, and the inducible Tregs that are generated in the periphery under different tolerogenic conditions. Among the inducible Tregs, the T regulatory type 1 (Tr1) cells represent one of the most extensively characterized subset. Tr1 cells develop in the periphery upon chronic antigen stimulation in the presence of IL-10 produced by tolerogenic antigen-presenting cells. Tr1 cells are characterized by the capacity to produce high levels of IL-10 in the absence of IL-4 and can suppress undesired immune responses mainly through cytokine-mediated mechanisms. However, we recently demonstrated that immunomodulatory activities of Tr1 cells reside not only in their ability to secrete suppressive cytokines but also in their property of cell-to-cell contact-dependent killing of target myeloid cells mediated by granzyme B and perforin. Tr1 cells are distinct from natural occurring Tregs since they are independent from FOXP3 expression for both their function and generation, as demonstrated by Tr1 cell identification in IPEX patients. The presence of Tr1 cells in vivo is associated with tolerance, whereas defects in Tr1 cells lead to autoimmune diseases or to chronic inflammation. In humans, we showed that both exogenous IL-10 or IL-10-derived from tolerogenic dendritic cells (DC-10) can be used to generate alloantigen-specific Tr1 cells in vitro suitable for cell therapy. Moreover, a homogeneous population of IL-10-producing T cells with Tr1-like phenotype and functions can be generated by transducing human CD4+ T cells with a lentiviral vector encoding for human IL-10. A cellular therapy protocol for the ex vivo use of IL-10 to induce alloantigen-specific unresponsiveness in donor-derived T cells for adoptive transfer in patients transplanted with haploidentical hematopoietic stem cells is being applied for patients with high-risk hematopoietic malignancies. In our clinical trial, donor T cells anergized in vitro in the presence of IL-10 are infused post-transplantation into the host with the ultimate goal of providing immune reconstitution with donor T cells that are anergic towards host antigens and contain precursors of host-specific Tr1 cells. This cellular therapy has proven to be safe and to provide immunoreconstitution associated, in most of the patients, with only moderate graft-versus-host disease (GVHD) at a cell dose that, if transferred untreated, would have caused severe acute GVHD. A similar approach could be also foreseen in the clinical setting of HLA-matched unrelated donor to prevent acute and chronic GVHD. Ad hoc experimental settings have been established using dendritic cells as antigen-presenting cells to induce anergy in the context of HLA-matched donors. These recently developed methods allowing the ex vivo induction of Tregs for in vivo infusion represent the first step toward an enlarged use of these cells as cellular therapy not only to inhibit GVHD after allogeneic hematopoietic stem cell transplantation, but also to other transplantation settings, or to re-establish tolerance in autoimmune or allergic diseases.

Human stem cells from the fetal liver can be transplanted to immunodeficient patients and reconstitute their immunity by giving rise to immunocompetent T lymphocytes of donor origin. Despite full HLA mismatch between donor and host, the helper T cells and the cytotoxic T cells which develop in these chimeric patients are totally functional. They recognize the antigenic peptides presented in the context of the foreign HLA molecules of the recipient, indicating that donor stem cells have been positively selected in the host environment, probably the thymic epithelial cells. By contrast, negative selection appears to be imposed upon T cells by donor hemopoietic cells, probably macrophages or dendritic cells, migrating from the transplant to the host thymus. Clonal deletion is then responsible for tolerance to donor HLA antigens, while clonal anergy explains tolerance to host HLA antigens.

We have studied the peripheral T cell repertoire of two patients with severe combined immunodeficiency who were successfully treated with human histocompatibility leukocyte antigen (HLA)-mismatched fetal liver stem cell transplantation. The patients presented a split chimerism. T cells were of donor origin, whereas the B cells/monocytes were of the host phenotype. Interestingly, the natural killer (NK) cells in one patient were donor derived and in the other patient of host origin. The NK cells were functional but did not have antihost or donor reactivity. Despite the HLA mismatch between donor and host cells, complete tolerance was achieved in vivo, and a specific unresponsiveness of peripheral blood mononuclear cells from both patients toward the host cells was demonstrated in vitro. Nevertheless, we could isolate T cell receptor (TCR)alpha beta, CD4+ or CD8+, T cell clones specifically reacting with HLA class I and II molecules of the host. The CD4+ host-reactive T cell clones from both patients produced interleukins 2 and 5, interferon-gamma, granulocyte/macrophage colony-stimulating factor but are specifically defective in interleukin 4 production. The frequencies of CD8+ host-reactive T cells were high, and were in the same range as those observed for CD8+ alloreactive T cells. In contrast, no donor-reactive CD8+ T cells or host or donor-reactive TCR gamma delta + T cells were detected. These data indicate that, after fetal stem cell transplantation, donor-reactive, but not host-reactive cells, are deleted from the T cell repertoire. Therefore, a peripheral mechanism of suppression or clonal anergy, rather than clonal deletion, is involved in maintaining in vivo tolerance toward the host.

The effects of interleukin (IL)-2, -3, -4, -5, -6, and -7 and granulocyte-macrophage colony-stimulating factor (GM-CSF) on histamine release from human basophils were evaluated. IL-3 was the only cytokine with histamine-releasing activity. This activity was observed predominantly on basophils from allergic patients (mean release +/- SEM, 33.9 +/- 9.5%; n = 12), whereas basophils from normal subjects responded less frequently to stimulation with IL-3 (mean release +/- SEM, 2.8 +/- 1.0%; n = 22). The effect of IL-3 was time and temperature dependent, since release was optimal after incubation for 120 min at 37 degrees C. When cell-bound IgE were eluted at acid pH, basophils became unresponsive to IL-3; however, IL-3-induced histamine release correlated with anti-IgE-induced histamine release in allergics, but not in normals. IL-3, IL-5, IL-6, and GM-CSF enhanced significantly anti-IgE- and FMLP-induced histamine release. In contrast, IL-2, IL-4, and IL-7 were devoid of any significant histamine-releasing or -potentiating activity. These results indicate that IL-3 can induce and IL-3, -5, and -6 and GM-CSF can enhance histamine release from human basophils, suggesting a possible role of these cytokines in the expression of allergic reactions.

Adenosine deaminase (ADA)-SCID is characterized by increased purine metabolites and severe combined immunodeficiency. Autoimmune manifestations have been observed in milder forms or after enzyme replacement therapy (ERT), and recently in patients following gene therapy (GT). nTregs have a full enzymatic machinery to generate and sustain high concentrations of extracellular adenosine and may be involved in the pathogenesis of autoimmunity. We analyzed the frequency of CD4+CD25+FOXP3+CD127- in the peripheral blood, the methylation profile of the FoxP3 gene and the suppressive function of nTreg in ADA-SCID patients following ERT, GT or bone marrow transplant (BMT) as compared to controls. We found that the proportion and function of nTregs after GT mirrored that of controls, while nTreg from the ERT group showed a reduced frequency and impaired function. We also investigated the maturation state of B cells in the peripheral blood of ADA-SCID patients. After ERT and early after GT, patients displayed an increased proportion of transitional B cells (CD24highCD38high) and of cells with an incomplete BCR (CD21-CD35-) as compared to BMT and healthy controls. At later follow up patients treated with GT showed a tendency to normalize the phenotype in parallel to an increased frequency of gene corrected cells. Collectively, these data provide a first indication of predisposition to autoimmunity in ADA-SCID and the role of different treatments in the maintenance of peripheral tolerance.

Wiskott-Aldrich Syndrome (WAS) is an X-linked immunodeficiency caused by mutations of the gene encoding for WASp, a protein involved in cytoskeletal reorganization of hematopoietic cells. WAS is characterized by microthrombocytopenia, eczema, infections, tumors and high rate of autoimmunity. WASp negative T cells have been extensively studied, while the role of WASp in B cells has been less characterized. Analysis of serum from adult was-/- mice showed increased level of Igs as compared to wt mice, in particular in IgG2a subclass. This result was further confirmed by ELISpot assays performed on sorted Marginal Zone (MZ) and Follicular (FO) B cells from wt and was-/- mice. The presence of autoreactive B cells was supported by the high titers of autoantibodies against dsDNA and tissue Ags in was-/- mice. In in vitro proliferation assays, was-/- B cells are hyper-proliferating to BCR-dependent (IgM) and Toll Like Receptors (TLRs)-dependent stimuli (LPS, CpG) in comparison to wt B cells, although no difference in the expression of TLRs was found. Preliminary results on Ca2+ flux upon CpG stimulus revealed increased Ca2+ mobilization in was-/- MZ B cell as compared to wt MZ B cells. Our data suggest that WASp absence affects B cell function and further studies are ongoing to better characterize BCR and TLR signaling pathways and intracellular trafficking in order to understand the contribution of B cells in autoimmune manifestations found in WAS patients.

The Wiskott-Aldrich Syndrome protein (WASp) is a regulator of actin remodeling in hematopoietic cells. In T cells, WASp is required for a long-lived Immunological Synapse (IS) and TCR-mediated cytokine production. We recently published that WASp is fundamental in the biology of iNKT cells, a peculiar T cell subset activated by glycosphingolipid antigens presented by APC. We showed that murine was-/- iNKT cells were defective in IL-4 and IFN-γ production upon in vivo activation. It remains an open issue whether this functional impairment is caused by a cell autonomous functional defect of was-/- iNKT cells or by an impaired crosstalk between iNKT cells and APC. To address this issue we analyzed cytokine production upon in vitro activation of wt and was-/- iNKT cells by wt and was-/- DC. The data obtained indicate the defective IL-4 production due to a was-/- iNKT cell autonomous defect and the lower IFN-γ production caused by an inefficient crosstalk between was-/- iNKT cells and was-/- DC. However was-/- iNKT cells produced sustained levels of cytokines upon in vitro stimulation with TPA and Ionomycin, suggesting a TCR-mediated functional impairment of these cells. Supporting this hypothesis was-/- iNKT cells presented a decreased TCR avidity. Altogether these results demonstrate the relevance of WASp in TCR-mediated effector function of iNKT cells. Ongoing studies are aimed at evaluating the ability to properly form the IS by was-/- iNKT cell and APC.

Abstract 45 Background: Adoptive transfer of regulatory T cells is a potentially attractive alternative to conventional immunosuppressive therapy in allogeneic hematopoietic stem cell transplantation (HSCT) (Roncarolo MG., Nat Rev Immunol 2007). Among CD4+ T cells, the subset known as Type 1 regulatory T (Tr1) cells are induced in an antigen specific manner by interleukin-10 (IL-10) and suppress via production of high levels of IL-10 (Groux H., Nature 1997). The aim of this phase I/II study was to establish the safety and efficacy of a new cellular therapy with Tr1 cells in a non-randomized study. Patients and Methods: A cellular therapy protocol for the adoptive transfer of IL-10 induced alloantigen specific donor-derived Tr1 cells in patients transplanted with CD34+ selected cells from haploidentical donor, has been applied to patients with high risk hematopoietic malignancies (www.risetfp6.org). Donor T cells, anergized ex vivo toward host alloantigens, presented by monocytes (original protocol) or tolerogenic dendritic cells (modified protocol) as host antigen presenting cells, in the presence of IL-10, are infused post-transplant into the host (IL-10 DLI). The infusion is made in the absence of immunosuppression for graft-versus-host-disease (GVHD) prophylaxis, with the ultimate goal to provide immune reconstitution without severe GVHD. The infused donor T cells, at the dose of 105 CD3+ cells/kg or 3 × 105 CD3+ cells/kg, are anergic towards host-HLA antigens and contain precursors of host-specific Tr1 cells but, at the same time, comprise memory T cells able to respond to nominal and viral antigens. Results: Eighteen patients have been enrolled, sixteen received CD34+ selected cells from haploidentical donor after myeloablative conditioning. Twelve patients have been treated with IL-10 anergized cell therapy at day +30 post transplant, at the dose of 105 CD3+ cells/kg with the exception of two patients who received 3 × 105 CD3+ cells/kg. No severe immediate reactions post infusion were registered. Five patients died from infections by day +30 after Tregs cell infusion and two patients dropped out for graft rejection. Five patients achieved immune reconstitution at a median of 30,5 days (range 15–46 days) after IL-10 DLI, followed by progressive normalization of TCR repertoire, memory/naïve phenotype and T cell functions in vitro and in vivo. Acute GVHD grade III was observed in one patient who received 3 × 105 CD3+ cells/kg; GVHD grade II was observed in 4 patients who received 105 CD3+ cells/kg and were successfully immune reconstituted. The median follow-up of the IL-10 DLI treated patients is 980 days (range 291–1624); 4 patients are alive and disease free and they do not require immunosuppressive treatment. Conclusions: Cellular therapy with IL-10 anergized donor T cells has proven to be safe and feasible, and to sustain immune reconstitution associated with a reduced severity of GVHD and no occurrence of relapse. This trial represents the first step towards an extended use of Tr1 cells as adjuvant treatment in allogeneic HSCT. Disclosures: No relevant conflicts of interest to declare.

Molecular Therapy (2009); 17: 1073–1082. doi:10.1038/mt.2009.31 Following the publication of this article, the authors noted that Dr. Liabeuf's affiliation is incorrect. Her correct affiliation is Genethon, Evry, France.

Hematopoietic Stem Cell Transplantation (HSCT) from haploidentical family donors is a promising therapeutic option for nearly all patients suffering from high-risk leukemia. Until now, its application has been limited by the prolonged immunodeficiency that patients suffer as a consequence of graft T cell depletion, used to prevent severe Graft versus Host Disease (GvHD). When efficient strategies to control GvHD are applied, adoptive immunotherapy with donor T cells grants a significant advantage for immune reconstitution. However, direct evidence for the role of haploidentical donor T cells in controlling leukemia relapse is still missing. Here we report on the in vivo selection of de novo mutant variants of acute myeloid leukemia (AML), accounting for relapse after haploidentical HSCT and adoptive transfer of donor T cells. These novel variants of AML were observed in 5 out of 17 (29%) patients suffering from disease relapse in a series of 43 patients transplanted at the San Raffaele Hospital in Milan from 2002 to 2008. All patients received a myeloablative conditioning regimen and high doses of haploidentical donor stem cells (median 10.2×106 CD34+ cells/kg, range 4.6–15.5). Donor T lymphocytes were infused as part of the graft (n=21, median 438×106 CD3+ cell/kg, range 179–796) or as post-transplant add-backs (n=22, median 111×105 CD3+ cell/kg, range 1–900). Human Leukocyte Antigen (HLA) genomic typing was routinely used for post-transplant donor-recipient chimerism assessment. The five patients with de novo mutant variants of the original leukemia came to our attention because patient-specific HLA alleles could not be detected in bone marrow samples harvested at disease relapse, nor in subsequently sorted AML blasts. A Loss of Heterozygosity (LOH) study was performed on purified blasts from these patients, and demonstrated that patient-specific HLA alleles were lost due to extensive events of homologous recombination, encompassing a region of chromosome 6 comprising the entire HLA locus. We show that donor T cells capable of recognizing the original, HLA-heterozygous, leukemia were efficiently transferred from the haploidentical donor to the patient, granting an in vivo cytotoxic, cytokine and proliferative anti-tumor response by specific recognition of the mismatched HLA molecules. However, consistent with genomic loss of the patientspecific HLA locus in disease recurrence, the same alloreactive T cells were unable to recognize the mutant variant of the leukemia, harvested at the time of relapse. This observation strongly suggests that the genomic rearrangements we identified granted the disease an in vivo selective advantage in escaping from an established donor T cell response. Taken together, our data show that adoptive transfer of alloreactive donor T cells in haploidentical HSCT is efficient in providing a patient-specific antileukemic effect, and that the loss of this effect is an important mechanism underlying the outgrowth of relapsing disease. The frequency we documented for this phenomenon calls for routine assessment of the leukemia HLA genotype in the post-transplant follow-up and for careful consideration in the choice of a putative second haploidentical donor in case of leukemia relapse. Ultimately, our data provide the first direct evidence for the role of donor T cell alloreactivity in controlling minimal residual disease after haploidentical HSCT, favoring the use of donor T cell-based immunotherapeutic strategies to exploit alloreactivity for the cure of high-risk leukemia.

In the developed world, the survival and quality of life of patients with beta thalassemia (Bthal) has dramatically improved with optimization of blood transfusions and iron chelation. By contrast, in countries with limited resources most affected children die before the age of 20 because of the unavailability of safe blood products, expensive iron chelating drugs and inadequate management of co-morbidities. For these patients allogeneic stem cell transplantation (SCT) from matched donors offers a cure with low morbidity and mortality. Between June 2005 and May 2008, 47 consecutive Bthal patients underwent SCT from an HLA identical sibling in our center, among these, 3 patients underwent 2 SCTs. Median age was 8 years (2–15), country of origin: Lebanon (9), Iraq (19), Palestine (3), Syria (16). One pt was classified as Lucarelli class I, 24 as class II and 22 as class III. Most patients had severe iron overload evidenced by irregular iron chelation (83%), median ferritin 2973 (956–14280), median liver iron concentration 22 mg Fe/g (6.9–95.7). Most patients had liver toxicity due to iron overload and hepatitis evidenced by median ALT 71 (12–545), AST 59 (18–371), liver fibrosis Ishak 3 (0–5), HepC pos 16/47 (34%). Patients had inadequate transfusional management evidence by a median pre-transfusion Hb of 8 g/dL and anti HLA antibodies in 81% pts. Class I-II patients were conditioned with a regimen based on iv busulphan (iv Bu, Busilvex®, dosage according to weight, adjusted from the 5th dose to a target AUC 1200 umol/min) and cyclophosphamide (Cy) 200mg/kg (n19) with the addition of thiotepa (TT 10mg/kg) if

5 . Importantly, we have shown that ex vivo culture of T Reg cells isolated from type 1 diabetic patients in the presence of rapamycin leads to expanded cells with in vitro regulatory activity that is identical to those of normal donors. These data suggest that, for cells from type 1 diabetic subjects, specific cell-culture condi - tions can selectively expand functional T Reg cells over contaminating effector CD4 + CD25 + T cells 8 . As an alternative to the ex vivo expan - sion of T Reg cells we also propose in our Review the use of adoptive immuno - therapy with inducible regulatory T cells, such as T R 1 cells, which are generated in vitro in the presence of the antigen and interleukin - 10. This approach overrides the need for isolating and expanding T Reg cells from patients with ongoing acute and chronic disease 1

Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by eczema, recurrent infections, severe hemorrhages and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Therapy based on the transplant of genetically correct autologous stem cells could represent a valid alternative approach. We investigated the efficacy and the safety of WAS gene transfer using HIV-based lentiviral vector encoding for WAS cDNA under the control of an autologous promoter (1.6 kb). T cells obtained from WAS patients showed normal level of WAS expression after lentiviral transduction. Transduced T cells showed a correction in TCR-driven proliferation and IL-2 production. Furthermore, a selective growth advantage of transduced T cells was observed in long-term in vitro cultures. Studies in T cell clones generated from transduced WAS CD4+ T cells revealed that 1–2 vector copies were necessary and sufficient to correct T cells function. CD34+ cells, isolated from mobilized peripheral blood and bone marrow of healthy donors, were transduced using WASP or GFP-encoding lentiviral vectors. Cells were cultured in the presence of different cytokines to investigate if WAS gene transfer could have any effect on short and long-term differentiation (CFU-C, LTC-IC and B/NK assays). Transduction resulted in a comparable number of CFU-C and LTC-IC colonies and normal B and NK cells differentiation with respect to untransduced cells. Furthermore, transduction of CD34+ cells isolated from the bone marrow of a WAS patient was performed under optimized culture conditions. Lentiviral gene transfer led to restoration of WASP expression in differentiated cells with copy number ranging from 1 to 5 copies per cell. In conclusion, our data demonstrate that the WAS promoter/cDNA-containing lentiviral vector can efficiently transduce and restore WASP expression in CD34+ cells and T cells from WAS patients. Experiments in the Rag2−/−/γchain- murine model are ongoing to test the efficacy and safety of the WASP transduced CD34+ cells. Together, our studies provide a preclinical basis for the implementation of a gene therapy trial for WAS patients.

Severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency is a fatal congenital disorder of the immune system associated with systemic toxicity due to accumulation of purine metabolites. We previously showed that retroviral-mediated ADA gene transfer into autologous hematopoietic stem/progenitor cells (HSC) allowed restoration of immune and metabolic functions. We have now enrolled eight ADA-SCID children (age: 7–67 months) in our phase I/II gene therapy trial in which HSC are combined with low intensity conditioning with busulfan (total dose 4 mg/Kg i.v.). Previous treatment included haploidentical bone marrow transplant (n=3) or long-term (>1 year) enzyme replacement therapy (PEG-ADA) (n=4) associated with insufficient immune reconstitution or severe autoimmunity. In the latter case, PEG-ADA was discontinued to favour the growth advantage for gene corrected cells. The patients received a median dose of 8.8x106/Kg bone marrow CD34+ cells (range 0.9–10.8), containing on average 26.2±9.6% transduced CFU-C. Five patients experienced ANC 1 year after gene therapy, we observed a progressive increase in lymphocyte counts which was sustained over time (median at 1.5 years 1.6x109/L), polyclonal thymopoiesis and normalization of T-cell functions in vitro. Serum Ig levels improved and evidence of antigen-specific antibodies was obtained, leading to IVIG discontinuation in five patients. All the children are currently healthy and thriving, and none of them showed severe infections. Sustained ADA activity in lymphocytes and RBC resulted in a dramatic reduction of RBC purine toxic metabolites (dAXP

Adenosine deaminase (ADA) is a key enzyme of the purine salvage pathway, metabolizing adenosine (Ado) and deoxyadenosine (dAdo). ADA deficiency results in increased levels of Ado and dAdo in plasma and cells, leading to severe combined immunodeficiency (SCID). However, the role of intracellular accumulation of dAdo and/or an aberrant extracellular signaling, mediated by G-coupled adenosine receptors, in the pathogenesis of the disease remains to be elucidated. Retroviral-mediated gene transfer of ADA into hematopoietic stem/progenitor cells has been recently shown to be an effective treatment for ADA-SCID children, thus providing a unique model to investigate the influence of purine metabolism in the survival and functions of T lymphocytes. Using untransformed bulk or CD4+ T-cell lines established from ADA-SCID patients before and after gene therapy, we investigated the biochemical pathways responsible for the pathogenesis of the disease and the ability of gene therapy to restore normal metabolic and immunological functions. We found that the expression of functional ADA in gene corrected T cells resulted in the restoration of SAHH activity and in the normalization of apoptosis induced by exposure to Ado/dAdo in defective cells. Interestingly, while a nucleoside transporter inhibitor could prevent Ado-induced cell toxicity, dAdo exerted its cytotoxic effect via the accumulation of intracellular cAMP, mediated by the engagement of Gs-protein coupled adenosine receptors. Functional studies revealed that both proliferative responses and Th1/Th2 cytokine production were impaired in ADA-deficient cells, but were restored in T cells generated after gene therapy. Such impairment was consequent to a defective TCR signalling, as indicated by the intrinsically reduced activation of p44/p42 MAPK pathway after anti-CD3/anti-CD28 mAb stimulation. Furthermore, a reduced transcription of cytokine genes was observed in ADA−/− CD4+ T cells, which was associated to a defective activation of early transcription factor CREB and possibly to an altered nuclear recruitment of NF-kB, as predicted by the decreased phosphorylation of IkBa. Remarkably, in ADA-deficient T cells, but not in gene corrected or healthy donor cells, exposure to non-toxic concentrations of dAdo resulted in a strong inhibition of p44/p42 MAPK activation and complete abrogation of TCR/CD28-driven proliferation and cytokine production. The effects of dAdo were reverted in the presence of a cAMP/PKA inhibitor, highlighting a key role for PKA hyperactivation and involvement of A2 receptors in causing T-cell dysfunctions. Collectively, these findings provide a more clear understanding of the physiopathology of ADA-SCID and confirm the efficacy of ADA-gene transfer in restoring normal metabolic pathways and immunological functions.

Wiskott-Aldrich syndrome (WAS) is a life-threatening X-linked primary immunodeficiency characterized by infections, hemorrhages, autoimmune disorders, and lymphomas. Transplantation of genetically corrected autologous hematopoietic stem cells (HSCs) could represent an alternative treatment to allogeneic HSC transplantation, the latter being often associated with severe complications. We used WAS(-/-) mice to test the efficacy of a gene therapy approach based on nonlethal irradiation followed by transplantation of WAS(-/-) HSCs transduced with lentiviral vectors encoding the WAS protein (WASP) from either the ubiquitous PGK promoter or the tissue-specific WAS promoter. The procedure resulted in significant levels of engraftment of WASP-expressing T cells, B cells, platelets, and myeloid cells. T cells harbored one or two vector copies and displayed partial to full correction of T cell receptor-driven interleukin- 2 production and proliferation. In addition, polymerization of F-actin and localization of WASP at the site of the immunological synapse were restored. The treatment was well tolerated and no pathology was detected by systematic blood analysis and autopsy. The efficacy of WAS gene transfer into HSCs, using the WAS promoter-containing lentiviral vector, combined with nonlethal irradiation provides a strong rationale for the development of gene therapy for WAS patients. Wiskott-Aldrich syndrome (WAS) is a life-threatening X-linked primary immunodeficiency characterized by infections, hemorrhages, autoimmune disorders, and lymphomas. Transplantation of genetically corrected autologous hematopoietic stem cells (HSCs) could represent an alternative treatment to allogeneic HSC transplantation, the latter being often associated with severe complications. We used WAS-/- mice to test the efficacy of a gene therapy approach based on nonlethal irradiation followed by transplantation of WAS-/- HSCs transduced with lentiviral vectors encoding the WAS protein (WASP) from either the ubiquitous PGK promoter or the tissue- specific WAS promoter. The procedure resulted in significant levels of engraftment of WASP-expressing T cells, B cells, platelets, and myeloid cells. T cells harbored one or two vector copies and displayed partial to full correction of T cell receptor-driven interleukin-2 production and proliferation. In addition, polymerization of F-actin and localization of WASP at the site of the immunological synapse were restored. The treatment was well tolerated and no pathology was detected by systematic blood analysis and autopsy. The efficacy of WAS gene transfer into HSCs, using the WAS promoter-containing lentiviral vector, combined with nonlethal irradiation provides a strong rationale for the development of gene therapy for WAS patients

Top of pageAbstract Gene therapy has been proposed as a potential treatment for Wiskott-Aldrich syndrome (WAS), a severe primary immune deficiency characterized by multiple hematopoietic cellular defects. To develop a safe and effective gene transfer system for this application, we designed SIN lentiviral vectors using 5' flanking sequences of the WAS gene as internal promoters to express a human WAS cDNA. This was shown to restrict the expression of the transgene to hematopoeitic cells, as is naturally the case for WAS. Further work was needed to validate this gene transfer system in multiple lineages of patients cells and to assess its performance in comparison with other lentiviral vectors utilizing strong internal promoters. Results showed that either short (0.5 kb) or long (1.6 kb) WAS promoters induced transgene expression in patients T, B and dendritic cells. Both of these promoter sequences behaved equivalently to each other but appeared to be transcriptionally weaker than the other promoters tested (PGK, EF1a, SFFV). Yet, they could correct the proliferation and IL-2 production defects in WAS T cells and the cytoskeletal anomalies in WAS DC, to the same level than achieved with stronger gene expression systems. They also induced gene expression in patient CD34+ cells without apparent hematopoietic toxicity. Thus, several robust SIN lentiviral expression systems exist to express the human WAS transgene. None of those tested here led to transgene over-expression or hematopoietic toxicity and all achieved functional correction in several lineages of patients cells. Having the therapeutic transgene under control of an endogenous WAS promoter sequence appears to be an effective and safe strategy. This preferentially targets the therapeutic transgene to the hematopoietic system, and may allow its physiological regulation in various lineages of cells. The lower strength of the WAS promoters may also reduce the risk of trans-activating neighbouring genes upon integration of the cassette. These results support further development of such lentiviral vectors for clinical application in WAS.

The WAS gene is mutated in Wiskott-Aldrich syndrome (WAS) and in X-linked thrombopenia (XLT). These diseases associate platelet defects with variable immune dysfunction, as a result of abnormal signalling and impaired cytoskeletal regulation in hematopoeitic cells. Conceivably, severe forms of WAS could be treated by gene therapy because retroviral or lentiviral-mediated WAS gene transfer restores protein expression and function in several WAS protein-deficient models. The purpose of the present study was to improve the safety and efficacy of such lentiviral vectors. Endogenous WAS promoter elements were used to restrict transgene expression to the target cell population and to provide the possibility of regulated expression in these cells. Sequences 0.5 or 1.6 kb upstream of transcription start, were operational in the transfer vector and restricted transgene expression to hematopoietic cells. Vectors utilizing either one of the WAS promoter sequences or the ubiquitously-active PGK-1, SFFV or EF1-a promoters, were compared. Equivalent levels of WAS protein were induced in lymphocytes and dendritic cells (DC), although slightly inferior mRNA levels were obtained in B cells using WAS promoters. At the functional level, all vectors restored a similar degree of proliferation and IL-2 production in T cells and equivalent numbers of podosome cytoskeletal structures in DC. The 0.5 kb or 1.6 kb-long WAS promoter sequences functioned similarly in WAS B lymphocytes or in a model of WASP-deficient T cells generated by RNA interference. No toxicity was induced by over-expression of these vectors in CD34+ cells. Altogether, the data show that lentiviral vectors with WAS promoters function efficiently in several lineages of patient cells and support further development of a hematopoietic-restricted approach for the gene therapy of WAS.

Lentiviral vectors (LVs) are an attractive candidate for gene therapy of monogenic diseases such as the hemophilias. LV administration can achieve stable expression of a transgene. Unfortunately, studies in immunocompetent mice indicate that vector delivery of a neo-antigen can result in a transgene-specific immune response. We have previously shown, however, that driving transgene expression from the hepatocyte-specific albumin promoter reduces the probability of triggering an immune response most likely by restricting expression from antigen presenting cells (APCs).