RAG1/2 knockout pigs with severe combined immunodeficiency.

Pigs share many physiological, biochemical, and anatomical similarities with humans and have emerged as valuable large animal models for biomedical research. Considering the advantages in immune system resemblance, suitable size, and longevity for clinical practical and monitoring purpose, SCID pigs bearing dysfunctional RAG could serve as important experimental tools for regenerative medicine, allograft and xenograft transplantation, and reconstitution experiments related to the immune system. In this study, we report the generation and phenotypic characterization of RAG1 and RAG2 knockout pigs using transcription activator-like effector nucleases. Porcine fetal fibroblasts were genetically engineered using transcription activator-like effector nucleases and then used to provide donor nuclei for somatic cell nuclear transfer. We obtained 27 live cloned piglets; among these piglets, 9 were targeted with biallelic mutations in RAG1, 3 were targeted with biallelic mutations in RAG2, and 10 were targeted with a monoallelic mutation in RAG2. Piglets with biallelic mutations in either RAG1 or RAG2 exhibited hypoplasia of immune organs, failed to perform V(D)J rearrangement, and lost mature B and T cells. These immunodeficient RAG1/2 knockout pigs are promising tools for biomedical and translational research.

FOXN1 mutation abrogates prenatal T-cell development in humans.

BACKGROUND: The transcription factor FOXN1 is implicated in the differentiation of thymic and skin epithelial cells, and alterations in it are responsible for the Nude/SCID phenotype. During a genetic counselling programme offered to couples at risk in a community where a high frequency of mutated FOXN1 had been documented, the identification of a human FOXN1(-/-) fetus gave the unique opportunity to study T cell development in utero. RESULTS: Total blockage of CD4(+) T cell maturation and severe impairment of CD8(+) cells were documented. Evaluation of the variable-domain ß-chain (Vß) families' usage among T lymphocytes revealed that the generation of T cell receptor (TCR) diversity occurred to some extent in the FOXN1(-/-) fetus, although it was impaired compared with the control. A few non-functional CD8(+) cells, mostly bearing TCRγδ in the absence of CD3, were found. DISCUSSION: FOXN1 is crucial for in utero T cell development in humans. The identification of a limited number of CD8(+) cells suggests an extrathymic origin for these cells, implying FOXN1-independent lymphopoiesis.

Induction of transplantation tolerance in humans using fetal cell transplants.

When engrafted with donor stem cells and lymphoid cells, patients develop transplantation tolerance to donor antigens. We analyzed the mechanism of tolerance induction in immunoincompetent recipients whose immunity has been reconstituted by transplantation of mismatched stem cells. Seven infants or human fetuses received fetal liver transplants as a treatment for severe combined immunodeficiency disease. After reconstitution of immunity by lymphocytes developed from donor stem cells, T-cell clones were produced and analyzed. Because donors and recipients were HLA mismatched, it was easy to demonstrate the donor origin of the T-cell clones. These clones were shown to have developed tolerance to histocompatibility antigens of the stem cell donor via a process of clonal deletion (probably as a result of contact with donor-derived macrophages and dendritic cells). They were also tolerant to histocompatibility antigens of the host but through a different mechanism: many clones recognized these antigens but had no detrimental effect on the target cells exhibiting host antigens, either in vitro or in vivo. Clonal anergy was therefore the cause of this tolerance to host determinants, resulting in a lack of graft-versus-host disease and of autoimmunity. The contact between developing T cells of donor origin and host epithelial cells within the host thymus may explain this colonal anergy. It should be noted that all patients had high serum levels of interleukin-10, which might have contributed to the persistent engraftment and tolerance.

[Hematopoietic stem cell transplant in combined immunodeficiency syndromes of childhood: an optimal treatment for primary immunodeficiency]. / Trasplante de células progenitoras hematopoyéticas en pediatría: una alternativa de tratamiento en inmunodeficiencias primarias.

The congenital immunodeficiency disorders in which the defect has been clearly traced to the stem cell can be cured with allogeneic stem-cell transplantation (SCT) from an unaffected donor. Widespread application of this treatment modality has been tempered by the fact that risk-benefit considerations do not always favor a procedure that carries a significant risk for morbidity and mortality. Some malignant disorders of childhood eventually have to be treated by an autologous or allogeneic SCT, however nonmalignant disorders can also be treated with this approach. This article reviews the current status of SCT for nonmalignant inherited immunodeficiency disorders.

B cell function after haploidentical in utero bone marrow transplantation in a patient with severe combined immunodeficiency.

An in utero paternal CD34(+) cell transplant was performed in a T-B+NK+ SCID fetus. We report here the results of the 3-year humoral immune reconstitution study. The methods used were ApoB VNTR typing, flow cytometry, nephelometry, hemagglutination, ELISA, ELISPOT and lymphoproliferative assays. The T cells were of donor origin whereas monocytes, B and NK cells were of host origin. Peripheral B cell counts and IgM levels were normal since birth. IVIG therapy was required at 5 months of age until 2 years old. IgA levels > or =20 mg/dl were detected from month 17 post transplantation. Isohemagglutinins were present since month 8 post transplantation, the highest titers (anti-A:1/128, anti-B:1/32) were obtained at month 33 post-transplantation. After immunization with rHBsAg, circulating anti-HBsAg IgG secreting cells and a 7.8-fold increase in serum anti-HBsAg Ab were detected. We conclude that split chimerism following in utero haploidentical BMT allows complete humoral immune reconstitution in a T-B+NK+ SCID patient.

Bowman-Birk protease inhibitor activates DNA-dependent protein kinase and reduces formation of radiation-induced dicentric chromosomes.

PURPOSE: To test a stimulatory effect of the radioprotector Bowman Birk protease inhibitor (BBI) upon DNA repair processes. MATERIALS AND METHODS: An effect of BBI upon DNA repair was investigated by quantification of radiation-induced dicentric chromosomes. Sensitivity to ionizing radiation was determined by clonogenic survival assay. Quantification of activity of the DNA-dependent kinase was performed by immunoprecipitation and phosphorylation of a TP53-derived peptide. RESULTS: The formation of radiation-induced dicentric chromosomes was reduced significantly after pretreatment of cells with BBI. By using a cell line with an inducible expression of a mutated TP53, it was shown that the BBI-mediated reduction of dicentric chromosome formation depended on the presence of wild-type TP53. To get further insights into the molecular mode of action of BBI, activity of the DNA-dependent protein kinase (DNA-PK) was quantified. BBI treatment resulted in a stimulation of basal (DNA-PK) activity. In SCID mouse fibroblasts deficient in DNA-PK activity, BBI failed to reduce the amount of radiation-induced dicentric chromosomes and the radioprotective effect was absent. Likewise, cells expressing mt.TP53 did not show radioprotection by BBI. CONCLUSIONS: It was observed that BBI exerts its radioprotective effect by a reduction of incorrect DNA repair, resulting in a reduced amount of dicentric chromosomes. This effect on the fidelity of DNA repair is TP53 dependent and correlated with induction of DNA-PK activity.

In-utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDXI).

BACKGROUND: X-linked severe combined immunodeficiency (SCIDXI) is an inherited immune defect which leads to death in infancy from severe infections. The defect is caused by mutations of the IL-2RG gene that encodes for the common gamma chain shared by several cytokine receptors. The disease is characterised by lack of T and NK cells with normal numbers of B cells. SCIDXI can be cured by bone marrow transplantation (BMT) or prevented by abortion after prenatal diagnosis. METHODS: A male fetus was diagnosed as having SCIDXI by molecular, immunophenotypic, and functional analyses. The fetus was injected intraperitoneally under ultrasound guidance with CD34 haematopoietic progenitor cells purified from paternal bone marrow and T-cell depleted by E rosetting. Chimerism analysis was by HLA-DQ alpha typing and gamma-chain staining on cord blood. FINDINGS: A healthy 3.6 kg boy was delivered by caesarean section at 38 weeks of gestation with no clinical or laboratory signs of graft-versus-host disease. Engraftment of donor-derived CD2 cells was found at birth. At 3.5 months of age the infant is well and his T-cell counts and function are normal. INTERPRETATION: In-utero transplantation of haematopoietic progenitor cells allowed immune reconstitution of a fetus with SCIDXI and may be an alternative to elective abortion. Our report should encourage applications of this method to other inherited disorders curable by BMT.

Engrafted maternal T cells in a severe combined immunodeficiency patient express T-cell receptor variable beta segments characterized by a restricted V-D-J junctional diversity.

To better understand the peculiar functional behavior of engrafted maternal T cells in a severe combined immunodeficiency (SCID) patient, we characterized, at the molecular level, the T-cell repertoire of a SCID child with a high number of engrafted, mature, activated lymphocytes. We found that, although these transplacentally acquired T cells express a random set of T-cell receptor variable beta (TCRBV) segments, the TCRBV transcripts are characterized by an extremely restricted V-D-J junctional diversity. Only a few cDNA clones were dominant among the TCRBV4+, TCRBV6+, and TCRBV20+ populations in engrafted cells, whereas the same TCRBV chains expressed by the mother's lymphocytes had the expected junctional hetero-geneity. Highly diverse and polyclonal junctions were also expressed by maternal cells activated in mixed lymphocyte reaction by Epstein-Barr virus (EBV)-transformed B lymphocytes from the patient, indicating that the strong clonal selection that characterizes the engrafted cells repertoire is probably not due to allorecognition. Furthermore, we report that the repertoire of the transplacentally acquired lymphocytes is dynamic over time and is characterized by waves of expression and contraction of selected clones, expressing different TCRBV segments. These results help to explain some of the abnormal functional behaviors of engrafted maternal cells and raise new questions regarding the mechanisms responsible for the restricted clonal diversity.

In utero transplantation of fetal liver stem cells into human fetuses.

Based on our experience in the field of fetal liver transplantation (FLT) that we have developed since 1976, we initiated, in 1988, in utero FLT into human fetuses, taking advantage of the immunologic tolerance in young fetuses. The transplants have involved fetuses suffering from various diseases at 12-28 weeks postfertilization, with 2 of the 6 cases eventually resulting in abortion. With the 4 other fetuses, a favorable outcome was observed. Three children are more than 4 years old, and they are alive and well, with evidence of engraftment, reconstitution of immunity, and partial correction of beta-thalassemia. In the fourth case, the fetus is alive and well and birth is expected soon. In utero transplantation of stem cells is a therapy with remarkable advantages: (a) tolerance induction due to immune immaturity of the host, (b) lack of graft-versus-host disease (GVHD) due to immaturity of the donor, (c) ideal isolation of the fetus in the maternal uterus, and (d) optimal environment for donor fetal cell development in the vicinity of host fetal cells and growth factors.

Abnormal T cell-dependent B-cell responses in SCID mice receiving allogeneic bone marrow in utero. Severe combined immune deficiency.

In allogeneic hematopoietic stem cell transplant recipients, restoration of humoral immunity is delayed and can remain impaired for years. In many severe combined immune deficiency (SCID) patients given haploidentical bone marrow (BM), lesions in humoral immunity are exacerbated by poor engraftment of donor B cells. The nature of these defects is important to understand as they render patients susceptible to infection. Previous work in mice suggested that in utero transplantation (IUT) of allogeneic BM might offer several advantages for the correction of primary immune deficiencies. In SCID mice given fully allogeneic BM in utero, the lymphoid compartment was restored with minimal evidence of graft-versus-host disease (GVHD). The present report examines B-cell reconstitution and function in mice that have received allogeneic IUT. Results are compared with those of adult mice given total body irradiation (TBI) followed by transplantation with allogeneic BM. In addition to enumerating the various B-cell subsets present in BM, spleen, and peritoneal cavity (PC), B-cell competence was assessed by challenging mice with T cell-independent (TI) and T cell-dependent (TD) antigens. The results demonstrated that all B-cell subsets in the BM and periphery were restored in allogeneic IUT and TBI mice, as were antibody responses after TI challenge. Upon immunization with TD antigens, however, IUT and TBI mice exhibited suboptimal responses as measured by the capacity to isotype switch and generate germinal center (GC) B cells. Thus, although allogeneic BM transplantation results in complete recovery of the B-cell compartment, certain elements of the humoral response remain defective.

Trasplante de células progenitoras hematopoyéticas en pediatría: Una alternativa de tratamiento en inmunodeficiencias primarias / Hematopoietic stem cell transplant in combined immunodeficiency syndromes of childhood: An optimal treatment for primary immunodeficiency

The congenital immunodeficiency disorders in which the defect has been clearly traced to the stem cell can be cured with allogeneic stem-cell transplantation (SCT) from an unaffected donor. Widespread application of this treatment modality has been tempered by the fact that risk-benefit considerations do not always favor a procedure that carries a significant risk for morbidity and mortality. Some malignant disorders of childhood eventually have to be treated by an autologous or allogeneic SCT, however nonmalignant disorders can also be treated with this approach. This article reviews the current status of SCT for nonmalignant inherited immunodeficiency disorders.

Tradicionalmente el trasplante de células progenituras hematopoyéticas (TCPH) se ha utilizado en pacientes pediátricos para el tratamiento de padecimientos malignos. Sin embargo, también existen indicaciones y experiencia para padecimientos benignos dentro de los cuales se encuentran los síndromes de inmunodeficiencia combinada primaria. Estos síndromes de la infancia constituyen una serie de padecimientos que aun cuando son infrecuentes en la patología infantil constituyen un grupo de alteraciones que hasta hace más de tres décadas eran irremediablemente fatales. Con el advenimiento del TCPH el pronóstico de estos síndromes ha mejorado sustancialmente, por lo que es importante conocer sus resultados, así como su morbimortalidad asociada.