Assessing the growth, yield, and biochemical composition of greenhouse cherry tomatoes with special emphasis on the progressive growth report.

The growth of plants hinges on a complex interplay of biochemical and physiological activities across various growth stages. These intricate processes dynamically adapt to different environmental conditions, shaping both plant development and productivity. This study explores the impact of greenhouse climate on the growth, yield, and biochemistry of winter-grown cherry tomatoes 'Cheramy F1'. A randomized complete block design (RCBD) under split plot arrangements (3 Rows) with three replications (3 plants from each row) was adopted. The data were collected on various dates during the period extending from December to March of two consecutive growing seasons in 2022 and 2023, and presented as averages. An analysis of variance was applied to statistically analyze the collected data at a confidence level of p < 0.05. The climatic conditions in the greenhouse were calculated as temperature ranging from a minimum of 10.5 °C to the maximum of 41.3 °C by an average of 21.2 °C during the vegetative stage and from 8.2 °C to 32.3 °C by an average of 20.9 °C during the fruit-bearing stage, with an average CO2 concentration fluctuated within the range of 385.61 ppm to 510.30 ppm and an average light intensity of 94.62 to 240.45 W/m². This study assessed various growth parameters such as plant height, leaf growth, stem diameter, leaf spacing, leaf count, leaf area, and inflorescence count per plant, and suggested the optimum range of greenhouse conditions for each stage. The key results of this study revealed the Progressive Growth Report (PGR), predicting daily potential growth rates of plants: plant height, 2.86 to 3.81 cm/day; growth rate of mature older leaf: 0.003988 m2/day; middle younger leaf: 0.008733 m2/day; top nascent leaf: 0.010722 m2/day; three to five leaves per week; and one inflorescence per week. In our accidental observation, we noticed unusual plant growth and yield responses because of the various growing postures and positions that the plants adopted in the greenhouse. An exceedingly significant difference among the inflorescences was found in view of their growth, productivity and biochemical composition. A non-significant interaction was found between the fruit keeping quality (shelf days), fruit height, fruit diameter, and inflorescence number. The present study results highlight the possible responses of greenhouse-grown cherry tomatoes to different ranges of temperature, light intensity, and CO2 concentrations, offering valuable insights for optimizing greenhouse cherry tomatoes cultivation.

Efficacy and safety of anti-CD45-saporin as conditioning agent for RAG deficiency.

BACKGROUND: Mutations in the recombinase-activating genes cause severe immunodeficiency, with a spectrum of phenotypes ranging from severe combined immunodeficiency to immune dysregulation. Hematopoietic stem cell transplantation is the only curative option, but a high risk of graft failure and poor immune reconstitution have been observed in the absence of myeloablation. OBJECTIVES: Our aim was to improve multilineage engraftment; we tested nongenotoxic conditioning with anti-CD45 mAbs conjugated with saporin CD45 (CD45-SAP). METHODS: Rag1-KO and Rag1-F971L mice, which represent models of severe combined immune deficiency and combined immune deficiency with immune dysregulation, respectively, were conditioned with CD45-SAP, CD45-SAP plus 2 Gy of total body irradiation (TBI), 2 Gy of TBI, 8 Gy of TBI, or no conditioning and treated by using transplantation with lineage-negative bone marrow cells from wild-type mice. Flow cytometry and immunohistochemistry were used to assess engraftment and immune reconstitution. Antibody responses to 2,4,6-trinitrophenyl-conjugated keyhole limpet hemocyanin were measured by ELISA, and presence of autoantibody was detected by microarray. RESULTS: Conditioning with CD45-SAP enabled high levels of multilineage engraftment in both Rag1 mutant models, allowed overcoming of B- and T-cell differentiation blocks and thymic epithelial cell defects, and induced robust cellular and humoral immunity in the periphery. CONCLUSIONS: Conditioning with CD45-SAP allows multilineage engraftment and robust immune reconstitution in mice with either null or hypomorphic Rag mutations while preserving thymic epithelial cell homeostasis.

Expanded circulating hematopoietic stem/progenitor cells as novel cell source for the treatment of TCIRG1 osteopetrosis.

Allogeneic hematopoietic stem cell transplantation is the treatment of choice for autosomal recessive osteopetrosis caused by defects in the TCIRG1 gene. Despite recent progress in conditioning, a relevant number of patients are not eligible for allogeneic stem cell transplantation because of the severity of the disease and significant transplant-related morbidity. We exploited peripheral CD34+ cells, known to circulate at high frequency in the peripheral blood of TCIRG1-deficient patients, as a novel cell source for autologous transplantation of gene corrected cells. Detailed phenotypical analysis showed that circulating CD34+ cells have a cellular composition that resembles bone marrow, supporting their use in gene therapy protocols. Transcriptomic profile revealed enrichment in genes expressed by hematopoietic stem and progenitor cells (HSPCs). To overcome the limit of bone marrow harvest/ HSPC mobilization and serial blood drawings in TCIRG1 patients, we applied UM171-based ex-vivo expansion of HSPCs coupled with lentiviral gene transfer. Circulating CD34+ cells from TCIRG1-defective patients were transduced with a clinically-optimized lentiviral vector (LV) expressing TCIRG1 under the control of phosphoglycerate promoter and expanded ex vivo. Expanded cells maintained long-term engraftment capacity and multi-lineage repopulating potential when transplanted in vivo both in primary and secondary NSG recipients. Moreover, when CD34+ cells were differentiated in vitro, genetically corrected osteoclasts resorbed the bone efficiently. Overall, we provide evidence that expansion of circulating HSPCs coupled to gene therapy can overcome the limit of stem cell harvest in osteopetrotic patients, thus opening the way to future gene-based treatment of skeletal diseases caused by bone marrow fibrosis.

Efficacy of lentivirus-mediated gene therapy in an Omenn syndrome recombination-activating gene 2 mouse model is not hindered by inflammation and immune dysregulation.

BACKGROUND: Omenn syndrome (OS) is a rare severe combined immunodeficiency associated with autoimmunity and caused by defects in lymphoid-specific V(D)J recombination. Most patients carry hypomorphic mutations in recombination-activating gene (RAG) 1 or 2. Hematopoietic stem cell transplantation is the standard treatment; however, gene therapy (GT) might represent a valid alternative, especially for patients lacking a matched donor. OBJECTIVE: We sought to determine the efficacy of lentiviral vector (LV)-mediated GT in the murine model of OS (Rag2R229Q/R229Q) in correcting immunodeficiency and autoimmunity. METHODS: Lineage-negative cells from mice with OS were transduced with an LV encoding the human RAG2 gene and injected into irradiated recipients with OS. Control mice underwent transplantation with wild-type or OS-untransduced lineage-negative cells. Immunophenotyping, T-dependent and T-independent antigen challenge, immune spectratyping, autoantibody detection, and detailed tissue immunohistochemical analyses were performed. RESULTS: LV-mediated GT allowed immunologic reconstitution, although it was suboptimal compared with that seen in wild-type bone marrow (BM)-transplanted OS mice in peripheral blood and hematopoietic organs, such as the BM, thymus, and spleen. We observed in vivo variability in the efficacy of GT correlating with the levels of transduction achieved. Immunoglobulin levels and T-cell repertoire normalized, and gene-corrected mice responded properly to challenges in vivo. Autoimmune manifestations, such as skin infiltration and autoantibodies, dramatically improved in GT mice with a vector copy number/genome higher than 1 in the BM and 2 in the thymus. CONCLUSIONS: Our data show that LV-mediated GT for patients with OS significantly ameliorates the immunodeficiency, even in an inflammatory environment.

Autonomous role of Wiskott-Aldrich syndrome platelet deficiency in inducing autoimmunity and inflammation.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked immunodeficiency characterized by eczema, infections, and susceptibility to autoimmunity and malignancies. Thrombocytopenia is a constant finding, but its pathogenesis remains elusive. OBJECTIVE: To dissect the basis of the WAS platelet defect, we used a novel conditional mouse model (CoWas) lacking Wiskott-Aldrich syndrome protein (WASp) only in the megakaryocytic lineage in the presence of a normal immunologic environment, and in parallel we analyzed samples obtained from patients with WAS. METHODS: Phenotypic and functional characterization of megakaryocytes and platelets in mutant CoWas mice and patients with WAS with and without autoantibodies was performed. Platelet antigen expression was examined through a protein expression profile and cluster proteomic interaction network. Platelet immunogenicity was tested by using ELISAs and B-cell and platelet cocultures. RESULTS: CoWas mice showed increased megakaryocyte numbers and normal thrombopoiesis in vitro, but WASp-deficient platelets had short lifespan and high expression of activation markers. Proteomic analysis identified signatures compatible with defects in cytoskeletal reorganization and metabolism yet surprisingly increased antigen-processing capabilities. In addition, WASp-deficient platelets expressed high levels of surface and soluble CD40 ligand and were capable of inducing B-cell activation in vitro. WASp-deficient platelets were highly immunostimulatory in mice and triggered the generation of antibodies specific for WASp-deficient platelets, even in the context of a normal immune system. Patients with WAS also showed platelet hyperactivation and increased plasma soluble CD40 ligand levels correlating with the presence of autoantibodies. CONCLUSION: Overall, these findings suggest that intrinsic defects in WASp-deficient platelets decrease their lifespan and dysregulate immune responses, corroborating the role of platelets as modulators of inflammation and immunity.

Wiskott-Aldrich syndrome protein deficiency in natural killer and dendritic cells affects antitumor immunity.

Wiskott-Aldrich syndrome (WAS) is a primary immunodeficiency caused by reduced or absent expression of the WAS protein (WASP). WAS patients are affected by microthrombocytopenia, recurrent infections, eczema, autoimmune diseases, and malignancies. Although immune deficiency has been proposed to play a role in tumor pathogenesis, there is little evidence on the correlation between immune cell defects and tumor susceptibility. Taking advantage of a tumor-prone model, we show that the lack of WASP induces early tumor onset because of defective immune surveillance. Consistently, the B16 melanoma model shows that tumor growth and the number of lung metastases are increased in the absence of WASP. We then investigated the in vivo contribution of Was(-/-) NK cells and DCs in controlling B16 melanoma development. We found fewer B16 metastases developed in the lungs of Was(-/-) mice that had received WT NK cells as compared with mice bearing Was(-/-) NK cells. Furthermore, we demonstrated that Was(-/-) DCs were less efficient in inducing NK-cell activation in vitro and in vivo. In summary, for the first time, we demonstrate in in vivo models that WASP deficiency affects resistance to tumor and causes impairment in the antitumor capacity of NK cells and DCs.

Preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vector for the treatment of Wiskott-Aldrich syndrome.

Gene therapy with ex vivo-transduced hematopoietic stem/progenitor cells may represent a valid therapeutic option for monogenic immunohematological disorders such as Wiskott-Aldrich syndrome (WAS), a primary immunodeficiency associated with thrombocytopenia. We evaluated the preclinical safety and efficacy of human CD34(+) cells transduced with lentiviral vectors (LV) encoding WAS protein (WASp). We first set up and validated a transduction protocol for CD34(+) cells derived from bone marrow (BM) or mobilized peripheral blood (MPB) using a clinical grade, highly purified LV. Robust transduction of progenitor cells was obtained in normal donors and WAS patients' cells, without evidence of toxicity. To study biodistribution of human cells and exclude vector release in vivo, LV-transduced CD34(+) cells were transplanted in immunodeficient mice, showing a normal engraftment and differentiation ability towards transduced lymphoid and myeloid cells in hematopoietic tissues. Vector mobilization to host cells and transmission to germline cells of the LV were excluded by different molecular assays. Analysis of vector integrations showed polyclonal integration patterns in vitro and in human engrafted cells in vivo. In summary, this work establishes the preclinical safety and efficacy of human CD34(+) cells gene therapy for the treatment of WAS.

Exonic knockout and knockin gene editing in hematopoietic stem and progenitor cells rescues RAG1 immunodeficiency.

Recombination activating genes (RAGs) are tightly regulated during lymphoid differentiation, and their mutations cause a spectrum of severe immunological disorders. Hematopoietic stem and progenitor cell (HSPC) transplantation is the treatment of choice but is limited by donor availability and toxicity. To overcome these issues, we developed gene editing strategies targeting a corrective sequence into the human RAG1 gene by homology-directed repair (HDR) and validated them by tailored two-dimensional, three-dimensional, and in vivo xenotransplant platforms to assess rescue of expression and function. Whereas integration into intron 1 of RAG1 achieved suboptimal correction, in-frame insertion into exon 2 drove physiologic human RAG1 expression and activity, allowing disruption of the dominant-negative effects of unrepaired hypomorphic alleles. Enhanced HDR-mediated gene editing enabled the correction of human RAG1 in HSPCs from patients with hypomorphic RAG1 mutations to overcome T and B cell differentiation blocks. Gene correction efficiency exceeded the minimal proportion of functional HSPCs required to rescue immunodeficiency in Rag1-/- mice, supporting the clinical translation of HSPC gene editing for the treatment of RAG1 deficiency.

Employment success of social assistance recipients: A provincial analysis by industry.

Poverty continues to burden millions of Canadians each year, and social assistance (SA) is one program that provides last-resort financial assistance, conditional upon looking for and accepting work. Using tax panel data of SA recipients from across seven Canadian regions between 2000 and 2018, we model the probabilities of employment success (ES) across industry of employment, SA benefit amounts, unionization, and individual-level characteristics. We adopt an economic stance to explain reliance upon SA, examining the broader macroeconomic indicators of ES, and to demonstrate the factors associated with exiting SA. We find that many SA recipients do not present evidence of recent employment, indicating a disconnect between stated SA program aims and their outcomes. We provide evidence for increased SA benefits and unionization as significant predictors of ES of SA recipients.

La pauvreté continue de peser sur des millions de Canadiens chaque année, et l'aide sociale (AS) est un programme qui fournit une aide financière de dernier recours, à condition de chercher et d'accepter un emploi. À l'aide de données fiscales de panel sur les bénéficiaires de l'aide sociale de sept régions canadiennes entre 2000 et 2018, nous modélisons les probabilités de réussite professionnelle en fonction du secteur d'emploi, du montant des prestations d'aide sociale, de la syndicalisation et des caractéristiques individuelles. Nous adoptons une position économique pour expliquer le recours à l'AS, en examinant les indicateurs macroéconomiques plus larges de la réussite professionnelle, et pour démontrer les facteurs associés à la sortie de l'AS. Nous constatons que de nombreux bénéficiaires de l'AS ne présentent pas de preuve d'emploi récent, ce qui indique un décalage entre les objectifs déclarés du programme d'AS et leurs résultats. Nous fournissons des preuves que l'augmentation des prestations d'AS et la syndicalisation sont des prédicteurs importants de la réussite de l'emploi des bénéficiaires de l'AS.

Partial correction of immunodeficiency by lentiviral vector gene therapy in mouse models carrying Rag1 hypomorphic mutations.

Introduction: Recombination activating genes (RAG) 1 and 2 defects are the most frequent form of severe combined immunodeficiency (SCID). Patients with residual RAG activity have a spectrum of clinical manifestations ranging from Omenn syndrome to delayed-onset combined immunodeficiency, often associated with granulomas and/or autoimmunity (CID-G/AI). Lentiviral vector (LV) gene therapy (GT) has been proposed as an alternative treatment to the standard hematopoietic stem cell transplant and a clinical trial for RAG1 SCID patients recently started. However, GT in patients with hypomorphic RAG mutations poses additional risks, because of the residual endogenous RAG1 expression and the general state of immune dysregulation and associated inflammation. Methods: In this study, we assessed the efficacy of GT in 2 hypomorphic Rag1 murine models (Rag1F971L/F971L and Rag1R972Q/R972Q), exploiting the same LV used in the clinical trial encoding RAG1 under control of the MND promoter. Results and discussion: Starting 6 weeks after transplant, GT-treated mice showed a decrease in proportion of myeloid cells and a concomitant increase of B, T and total white blood cells. However, counts remained lower than in mice transplanted with WT Lin- cells. At euthanasia, we observed a general redistribution of immune subsets in tissues, with the appearance of mature recirculating B cells in the bone marrow. In the thymus, we demonstrated correction of the block at double negative stage, with a modest improvement in the cortical/medullary ratio. Analysis of antigenspecific IgM and IgG serum levels after in vivo challenge showed an amelioration of antibody responses, suggesting that the partial immune correction could confer a clinical benefit. Notably, no overt signs of autoimmunity were detected, with B-cell activating factor decreasing to normal levels and autoantibodies remaining stable after GT. On the other hand, thymic enlargement was frequently observed, although not due to vector integration and insertional mutagenesis. In conclusion, our work shows that GT could partially alleviate the combined immunodeficiency of hypomorphic RAG1 patients and that extensive efficacy and safety studies with alternative models are required before commencing RAG gene therapy in thesehighly complex patients.

Lentiviral-mediated gene therapy leads to improvement of B-cell functionality in a murine model of Wiskott-Aldrich syndrome.

BACKGROUND: Wiskott-Aldrich syndrome (WAS) is an X-linked primary immunodeficiency characterized by thrombocytopenia, eczema, infections, autoimmunity, and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical donors is curative, but it is not available to all patients. We have developed a gene therapy (GT) approach for WAS by using a lentiviral vector encoding for human WAS promoter/cDNA (w1.6W) and demonstrated its preclinical efficacy and safety. OBJECTIVE: To evaluate B-cell reconstitution and correction of B-cell phenotype in GT-treated mice. METHODS: We transplanted Was(-/-) mice sublethally irradiated (700 rads) with lineage marker-depleted bone marrow wild-type cells, Was(-/-) cells untransduced or transduced with the w1.6W lentiviral vector and analyzed B-cell reconstitution in bone marrow, spleen, and peritoneum. RESULTS: Here we show that WAS protein(+) B cells were present in central and peripheral B-cell compartments from GT-treated mice and displayed the strongest selective advantage in the splenic marginal zone and peritoneal B1 cell subsets. After GT, splenic architecture was improved and B-cell functions were restored, as demonstrated by the improved antibody response to pneumococcal antigens and the reduction of serum IgG autoantibodies. CONCLUSION: WAS GT leads to improvement of B-cell functions, even in the presence of a mixed chimerism, further validating the clinical application of the w1.6W lentiviral vector.

Thymic Epithelial Cell Alterations and Defective Thymopoiesis Lead to Central and Peripheral Tolerance Perturbation in MHCII Deficiency.

Major Histocompatibility Complex (MHC) class II (MHCII) deficiency (MHCII-D), also known as Bare Lymphocyte Syndrome (BLS), is a rare combined immunodeficiency due to mutations in genes regulating expression of MHCII molecules. MHCII deficiency results in impaired cellular and humoral immune responses, leading to severe infections and autoimmunity. Abnormal cross-talk with developing T cells due to the absence of MHCII expression likely leads to defects in thymic epithelial cells (TEC). However, the contribution of TEC alterations to the pathogenesis of this primary immunodeficiency has not been well characterized to date, in particular in regard to immune dysregulation. To this aim, we have performed an in-depth cellular and molecular characterization of TEC in this disease. We observed an overall perturbation of thymic structure and function in both MHCII-/- mice and patients. Transcriptomic and proteomic profiling of murine TEC revealed several alterations. In particular, we demonstrated that impairment of lymphostromal cross-talk in the thymus of MHCII-/- mice affects mTEC maturation and promiscuous gene expression and causes defects of central tolerance. Furthermore, we observed peripheral tolerance impairment, likely due to defective Treg cell generation and/or function and B cell tolerance breakdown. Overall, our findings reveal disease-specific TEC defects resulting in perturbation of central tolerance and limiting the potential benefits of hematopoietic stem cell transplantation in MHCII deficiency.

Activation of the aryl hydrocarbon receptor promotes allograft-specific tolerance through direct and dendritic cell-mediated effects on regulatory T cells.

VAF347 is a low-molecular-weight compound, which activates the aryl hydrocarbon receptor (AhR). Herein, we report that oral administration of a water-soluble derivative of VAF347 (VAG539) promotes long-term graft acceptance and active tolerance in Balb/c mice that receive a transplant of MHC-mismatched pancreatic islet allografts. In vivo VAG539 treatment results in increased frequency of splenic CD4(+) T cells expressing CD25 and Foxp3, markers associated with regulatory T (Tr) cells, and in vitro VAF347 treatment of splenic CD4(+) T cells improved CD4(+)CD25(+)Foxp3(+) T-cell survival. Interestingly, transfer of CD11c(+) dendritic cells (DCs), but not of CD4(+) T or CD19(+) B cells, from VAG539-treated long-term tolerant hosts into mice that recently underwent transplantation resulted in donor (C57Bl/6)-specific graft acceptance and in a significantly higher frequency of splenic CD4(+)CD25(+)Foxp3(+) Tr cells. Furthermore, the transfer of CD4(+)CD25(+) T cells from these mice into mice that recently underwent transplantation promoted graft acceptance. Similarly, cell therapy with in vitro VAF347-treated bone marrow-derived mature DCs prevented islet graft rejection, and reduced OVA-specific T-cell responses in OVA-immunized mice. Collectively, our data indicate that AhR activation induces islet allograft-specific tolerance through direct as well as DC-mediated effects on Tr-cell survival and function.

Evidence for long-term efficacy and safety of gene therapy for Wiskott-Aldrich syndrome in preclinical models.

Wiskott-Aldrich Syndrome (WAS) is a life-threatening X-linked disease characterized by immunodeficiency, thrombocytopenia, autoimmunity, and malignancies. Gene therapy could represent a therapeutic option for patients lacking a suitable bone marrow (BM) donor. In this study, we analyzed the long-term outcome of WAS gene therapy mediated by a clinically compatible lentiviral vector (LV) in a large cohort of was(null) mice. We demonstrated stable and full donor engraftment and Wiskott-Aldrich Syndrome protein (WASP) expression in various hematopoietic lineages, up to 12 months after gene therapy. Importantly, we observed a selective advantage for T and B lymphocytes expressing transgenic WASP. T-cell receptor (TCR)-driven T-cell activation, as well as B-cell's ability to migrate in response to CXCL13, was fully restored. Safety was evaluated throughout the long-term follow-up of primary and secondary recipients of WAS gene therapy. WAS gene therapy did not affect the lifespan of treated animals. Both hematopoietic and nonhematopoietic tumors arose, but we excluded the association with gene therapy in all cases. Demonstration of long-term efficacy and safety of WAS gene therapy mediated by a clinically applicable LV is a key step toward the implementation of a gene therapy clinical trial for WAS.

Gene Modification and Three-Dimensional Scaffolds as Novel Tools to Allow the Use of Postnatal Thymic Epithelial Cells for Thymus Regeneration Approaches.

Defective functionality of thymic epithelial cells (TECs), due to genetic mutations or injuring causes, results in altered T-cell development, leading to immunodeficiency or autoimmunity. These defects cannot be corrected by hematopoietic stem cell transplantation (HSCT), and thymus transplantation has not yet been demonstrated to be fully curative. Here, we provide proof of principle of a novel approach toward thymic regeneration, involving the generation of thymic organoids obtained by seeding gene-modified postnatal murine TECs into three-dimensional (3D) collagen type I scaffolds mimicking the thymic ultrastructure. To this end, freshly isolated TECs were transduced with a lentiviral vector system, allowing for doxycycline-induced Oct4 expression. Transient Oct4 expression promoted TECs expansion without drastically changing the cell lineage identity of adult TECs, which retain the expression of important molecules for thymus functionality such as Foxn1, Dll4, Dll1, and AIRE. Oct4-expressing TECs (iOCT4 TEC) were able to grow into 3D collagen type I scaffolds both in vitro and in vivo, demonstrating that the collagen structure reproduced a 3D environment similar to the thymic extracellular matrix, perfectly recognized by TECs. In vivo results showed that thymic organoids transplanted subcutaneously in athymic nude mice were vascularized but failed to support thymopoiesis because of their limited in vivo persistence. These findings provide evidence that gene modification, in combination with the usage of 3D biomimetic scaffolds, may represent a novel approach allowing the use of postnatal TECs for thymic regeneration. Stem Cells Translational Medicine 2019;8:1107-1122.

Rapamycin and interleukin-10 treatment induces T regulatory type 1 cells that mediate antigen-specific transplantation tolerance.

Islet transplantation is a cure for type 1 diabetes, but its potential is limited by the need for constant immunosuppression. One solution to this problem is the induction of transplantation tolerance mediated by T regulatory cells. T regulatory type 1 (Tr1) cells are characterized by their production of high levels of interleukin (IL)-10, which is crucial for their differentiation and suppressive function. We investigated the effects of IL-10 administered in combination with rapamycin on the induction of Tr1 cells that could mediate a state of tolerance in diabetic mice after pancreatic islet transplantation. The efficacy of this treatment was compared with IL-10 alone and standard immunosuppression. Stable long-term tolerance that was not reversible by alloantigen rechallenge was achieved only in mice treated with rapamycin plus IL-10. Tr1 cells that produced high levels of IL-10 and suppressed T-cell proliferation were isolated from splenocytes of rapamycin plus IL-10-treated mice after treatment withdrawal. In rapamycin plus IL-10-treated mice, endogenous IL-10 mediated an active state of tolerance, as was observed when the blockade of IL-10 activity rapidly induced graft rejection >100 days after transplantation. CD4(+) T-cells from rapamycin plus IL-10-treated mice transferred antigen-specific tolerance in mice that received new transplants. Thus rapamycin plus IL-10 not only prevented allograft rejection but also induced Tr1 cells that mediated stable antigen-specific, long-term tolerance in vivo.

Induction of tolerance in type 1 diabetes via both CD4+CD25+ T regulatory cells and T regulatory type 1 cells.

Success in developing novel therapies to recommence self-tolerance in autoimmunity depends on the induction of T regulatory (Tr) cells. Here, we report that rapamycin combined with interleukin (IL)-10 efficiently blocks type 1 diabetes development and induces long-term immunotolerance in the absence of chronic immunosuppression in nonobese diabetic (NOD) mice. Rapamycin mediates accumulation in the pancreas of suppressive CD4(+)CD25(+)FoxP3(+) Tr cells, which prevent diabetes. IL-10 induces Tr type 1 (Tr1) cells, which reside in the spleen and prevent migration of diabetogenic T-cells to the draining lymph nodes. These two Tr cell subsets act in concert to control diabetogenic T-cells that are still present in long-term tolerant mice. Rapamycin plus IL-10 treatment, promoting distinct subsets of Tr cells, may constitute a novel and potent tolerance-inducing protocol for immune-mediated diseases.

Relevance of hyperglycemia on the timing of functional loss of allogeneic islet transplants: implication for mouse model.

BACKGROUND: The role of recipient hyperglycemia on timing of allograft survival is unknown. In this study, we investigated if and how variation in recipient glycemia affects the ability to achieve and maintain normoglycemia after transplant of C57BL/6 islets into diabetic BALB/c mice. METHODS AND RESULTS: 85 diabetic BALB/c mice with non-fasting glycaemia ranging between 275 and 600 mg/dL were transplanted with 400 C57BL/6 islets. The time of rejection inversely correlated with the pre-transplant blood glucose concentration (P=0.004). All the 13 mice with normoglycemia beyond 50 days had pretransplant glycemia <450 mg/dL and the presence of autologous beta cell function was demonstrated in 8 (>100 days function) by the persistence of normoglycemia after allograft removal. The presence of immunosuppression (rapamycin plus FK506 plus anti-IL-2Ra chain mAbs, n=31; rapamycin plus IL-10; n=29) removed the influence of pretransplant hyperglycemia but after treatment withdrawn the timing and the probability of graft loss correlate with the pretransplant hyperglycemia. Pretransplant glycemia was inversely correlated with HOMA-B and serum insulin showing that a significant residual beta cell mass was present in mice with glycemia <450 mg/dL. CONCLUSION: This study demonstrates that the timing of functional loss of islets allotransplantation depends on the degree of recipient hyperglycemia. This potential bias should be kept in count in experimental results and a threshold that excludes moderate diabetes should be used in defining recipient's eligibility.

The role of tissue adaptation and graft size in immune tolerance.

Understanding how immune tolerance is induced and maintained is critical for our approach to immune-related diseases. Ecoimmunity is a new theory that views the immune system-tissue interaction as a co-adapting predator-prey system. Ecoimmunity suggests that tissues adapt to the selective immune pressure during ontogeny and throughout life. Therefore, immune tolerance towards 'self' represents a symmetric balance between the propensity of the immune system to prey on 'self' cells, and the tissue's specific capacity to undergo phenotypic adaptations in order to avoid destructive immune interaction. According to this theory, we hypothesized that tissues of adult immune-deficient mice, which are not exposed to selective immune pressure, will not withstand immune activity and will therefore display higher susceptibility to graft rejection. To test this prediction, C57Bl/6 wild type female mice were rendered diabetic by streptozotocin and transplanted with syngeneic pancreatic islets isolated from either immune-deficient C57Bl/6 SCID or wild type females. Remarkably, recipients of islet grafts from immune-deficient syngeneic donors displayed significantly impaired glucose homeostasis compared to mice transplanted with islets of wild type donors (p<0.001, two way repeated measures ANOVA). The severity of this impairment was correlated with islet graft size, suggesting a capacity of transplanted islets to gradually acquire a tolerogenic phenotype. These findings support the view of graft survival that is based on 'natural selection' of tissue cells. In addition, we describe a new experimental system for molecular characterization of self-tolerance.

Good Laboratory Practice Preclinical Safety Studies for GSK2696273 (MLV Vector-Based Ex Vivo Gene Therapy for Adenosine Deaminase Deficiency Severe Combined Immunodeficiency) in NSG Mice.

GSK2696273 (autologous CD34+ cells transduced with retroviral vector that encodes for the human adenosine deaminase [ADA] enzyme) is a gamma-retroviral ex vivo gene therapy of bone marrow-derived CD34+ cells for the treatment of adenosine deaminase deficiency severe combined immunodeficiency (ADA-SCID). ADA-SCID is a severe monogenic disease characterized by immunologic and nonimmunologic symptoms. Bone-marrow transplant from a matched related donor is the treatment of choice, but it is available for only a small proportion of patients. Ex vivo gene therapy of patient bone-marrow CD34+ cells is an alternative treatment. In order to prepare for a marketing authorization application in the European Union, preclinical safety studies in mice were requested by the European Medicines Agency (EMA). A pilot study and a main biodistribution study were performed according to Good Laboratory Practice (GLP) at the San Raffaele Telethon Institute for Gene Therapy test facility. In the main study, human umbilical cord blood (UCB)-derived CD34+ cells were transduced with gamma-retroviral vector used in the production of GSK2696273. Groups of 10 male and 10 female NOD-SCID gamma (NSG) mice were injected intravenously with a single dose of transduced- or mock-transduced UCB CD34+ cells, and they were observed for 4 months. Engraftment and multilineage differentiation of blood cells was observed in the majority of animals in both groups. There was no significant difference in the level of chimerism between the two groups. In the gene therapy group, vector was detectable in lymphohemopoietic and nonlymphohemopoietic tissues, consistent with the presence of gene-modified human hematopoietic donor cells. Given the absence of relevant safety concerns in the data, the nonclinical studies and the clinical experience with GSK2696273 supported a successful application for market authorization in the European Union for the treatment of ADA-SCID patients, for whom no suitable human leukocyte antigen-matched related donor is available.