Reduced memory B-cell populations in boys with B-cell dysfunction after bone marrow transplantation for X-linked severe combined immunodeficiency

X-linked severe combined immunodeficiency (XSCID) is a lethal disease resulting in death in infancy. In many instances, haploidentical bone marrow transplantation (BMT) offers reconstitution of T-cell immunity alone, with residual hypogammaglobulinaemia. The exact nature of B-cell dysfunction in these patients is unclear, although differentiation arrest of the B cells is a potential explanation. To ascertain the differentiation status of peripheral blood B lymphocytes from XSCID patients after BMT, the surface expression of CD19, CD10, CD34, CD5, serum immunoglogulin (sIg)M, sIgD, sIgG and CD27 on these B cells was investigated using three-colour flow cytometry. CD27 is a marker of memory B cells. Populations of CD19+IgM-D- B cells, CD19+IgM-only, CD19+IgG+CD27+ and CD19+IgM+ CD27+ B cells were found to be diminished in the XSCID patients after BMT with persistent hypogammaglobulinaemia, compared with both post-BMT patients with B-cell function and age-matched normal controls. This indicated the lack of CD19+IgM-D- B cells, which represent Ig isotype-switched B cells, as well as CD19+IgM-only and CD19+IgG+CD27+ or CD19+IgM+CD27+ memory B-cell populations. Interaction between CD27 and its ligand CD70 has been shown to induce IgG and IgM production by CD27+ B cells. Therefore, the lack of CD27/70 interaction is a probable explanation for the hypogammaglobulinaemia in these patients after BMT.     

Allogenic fetal liver cells have a distinct competitive engraftment advantage over adult bone marrow cells when infused into fetal as compared with adult severe combined immunodeficient recipients

In utero transplantation (IUT) is becoming a viable option for the treatment of various immune and metabolic disorders diagnosed early in gestation. In this study, donor fetal liver cells had a 10-fold competitive engraftment advantage relative to adult bone marrow in allogeneic fetal severe combined immunodeficient (SCID) recipients compared with adult recipients. In contrast, adult bone marrow cells engrafted slightly better than fetal liver cells in allogeneic adult SCID transplant recipients. By using different ratios of fetal and adult cell mixtures, fetal liver cells repopulated 8.2 times better than adult bone marrow cells in fetal recipients, but only 0.8 times as well in adult recipients. Fetal SCID recipients were more permissive to an allogeneic donor graft than adult recipients. These data indicate that the recipient microenvironment may regulate the engraftment efficiency of a given stem cell source and suggest that the use of cord blood should be tested in clinical IUT.     

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.     

In utero bone marrow transplantation of fetal baboons with mismatched adult marrow: initial observations

Recent advances in prenatal diagnoses of sickle cell anemia and thalassemia permit early identification of affected fetuses. However, the only intervention possible to date is abortion of the affected fetuses. Transplantation of normal marrow into fetuses in utero could correct these life-threatening disorders, but to accomplish this techniques must be developed for fetal transplantation in man. Therefore, we have transplanted fetal baboons with mismatched adult baboon bone marrow from donors that differed at the glucose phosphate isomerase locus. Twenty-two fetuses between 60 and 160 days of gestation (term gestation is 182 days) were transplanted intraperitoneally with 10(9) marrow mononuclear cells/kg body weight using an ultrasonic technique. No immunosuppressive or preparative regimen was given prior to or after transplantation, and all fetuses tolerated the procedure well. One month after transplantation fetal blood samples were obtained to assess chimerism. Three chimeras were detected among 10 fetuses transplanted at 80 days' gestation, and no chimeras were detected in fetuses greater than 80 days' gestation at the time of transplantation. All chimeras died in utero during the third trimester of pregnancy: one of an intrauterine infection at 160 days' gestation, one at 135 days' gestation and one at 145 days' gestation. In contrast, the other 19 non-chimeric fetuses survived. These data suggest: (1) in utero fetal bone marrow transplantation is technically feasible in primates; (2) that allogeneic adult bone marrow can engraft and persist for at least 1 month in fetal baboons transplanted at 80 days of gestation; and (3) that delineation of the causes for loss of fetal chimeras should prove valuable in assessing the therapeutic potential for in utero bone marrow transplantation in man.     

Long-term chimerism and B-cell function after bone marrow transplantation in patients with severe combined immunodeficiency with B cells: A single-center study of 22 patients.

We retrospectively analyzed the B-cell function and leukocyte chimerism of 22 patients with severe combined immunodeficiency with B cells (B(+) SCID) who survived more than 2 years after bone marrow transplantation (BMT) to determine the possible consequences of BMT procedures, leukocyte chimerism, and SCID molecular deficit on B-cell function outcome. Circulating T cells were of donor origin in all patients. In recipients of HLA-identical BMT (n = 5), monocytes were of host origin in 5 and B cells were of host origin in 4 and of mixed origin in 1. In recipients of HLA haploidentical T-cell-depleted BMT (n = 17), B cells and monocytes were of host origin in 14 and of donor origin in 3. Engraftment of B cells was found to be associated with normal B-cell function. In contrast, 10 of 18 patients with host B cells still require Ig substitution. Conditioning regimen (ie, 8 mg/kg busulfan and 200 mg/kg cyclophosphamide) was shown neither to promote B-cell and monocyte engraftment nor to affect B-cell function. Eight patients with B cells of host origin had normal B-cell function. Evidence for functional host B cells was further provided in 3 informative cases by Ig allotype determination and by the detection, in 5 studied cases, of host CD27(+) memory B cells as in age-matched controls. These results strongly suggest that, in some transplanted patients, host B cells can cooperate with donor T cells to fully mature in Ig-producing cells.     

Retroviral transduction of IL2RG into CD34(+) cells from X-linked severe combined immunodeficiency patients permits human T- and B-cell development in sheep chimeras

X-linked severe combined immunodeficiency (XSCID) is caused by mutations of the common gamma chain of cytokine receptors, gamma(c). Because bone marrow transplantation (BMT) for XSCID does not provide complete immune reconstitution for many patients and because of the natural selective advantage conferred on lymphoid progenitors by the expression of normal gamma(c), XSCID is a good candidate disease for therapeutic retroviral gene transfer to hematopoietic stem cells. We studied XSCID patients who have persistent defects in B-cell and/or combined B- and T-cell function despite having received T cell-depleted haploidentical BMT. We compared transduction of autologous B-cell lines and granulocyte colony-stimulating factor-mobilized peripheral CD34(+) cells from these patients using an MFGS retrovirus vector containing the gamma(c) gene IL2RG pseudotyped with amphotropic, gibbon ape leukemia virus, or RD114 envelopes. Transduced B-cell lines and peripheral CD34(+) cells demonstrated provirus integration and new cell-surface gamma(c) expression. The chimeric sheep model was exploited to test development of XSCID CD34(+) cells into mature myeloid and lymphoid lineages. Transduced and untransduced XSCID CD34(+) cells injected into developing sheep fetuses gave rise to myeloid cells. However, only transduced gamma progenitors from XSCID patients developed into T and B cells. These results suggest that gene transfer to autologous peripheral CD34(+) cells using MFGS-gc retrovirus may benefit XSCID patients with persistent T- and B-cell deficits despite prior BMT.     

Reconstitution after transplantation with T-lymphocyte-depleted HLA haplotype-mismatched bone marrow for severe combined immunodeficiency.

Severe combined immunodeficiency (SCID) is potentially correctable by bone marrow transplantation if a patient has a suitable histocompatible donor. In the absence of an HLA-matched donor, lethal graft-versus-host disease (GVHD), which is mediated by alloreactive donor T cells, may occur. In an attempt to prevent GVHD in one SCID patient lacking a matched donor, we treated maternal haplomismatched bone marrow with a unique nonmitogenic T-cell-specific monoclonal antibody (anti-T12) and complement to remove mature T cells. Despite the removal of greater than 99% mature T cells, the child developed significant life-threatening GVHD, which was terminated by a 5-day course of intravenous anti-T12. Subsequently, immune reconstitution occurred by 6 wk: the mature circulating T cells proliferated in response to soluble and allo-antigens in vitro and provided help for B-cell immunoglobulin synthesis. The patient was removed from a protective environment and discharged without evidence of further infection. Both HLA and chromosomal analyses showed that the circulating cells in the patient were of maternal origin. More importantly, the maternal T cells were no longer reactive with recipient cells. Mixing experiments indicated that the state of tolerance that resulted in this chimera was not due to active suppression. We conclude that HLA-mismatched transplantation for SCID can be undertaken if mature alloreactive donor T lymphocytes are depleted before and after bone marrow grafting.     

Reconstitution of lymphoid development and function in ZAP-70-deficient mice following gene transfer into bone marrow cells

Mutations in the ZAP-70 protein tyrosine kinase gene result in a severe combined immunodeficiency (SCID) characterized by a selective inability to produce CD8(+) T cells and a signal transduction defect in peripheral CD4(+) cells. Transplantation of genetically modified hematopoietic progenitor cells that express the wild-type ZAP-70 gene may provide significant benefit to some of these infants. The feasibility of stem cell gene correction for human ZAP-70 deficiency was assessed using a ZAP-70 knock-out model. ZAP-70-deficient murine bone marrow progenitor cells were transduced with a retroviral vector expressing the human ZAP-70 gene. Engraftment of these cells in irradiated ZAP-70-deficient animals resulted in the development of mature CD4(+) and CD8(+) T cells. In marked contrast, both populations were absent in ZAP-70(-/-) mice undergoing transplantation with bone marrow progenitor cells transduced with a control vector. Importantly, ZAP-70-reconstituted T cells proliferated in response to T-cell receptor stimulation. Moreover, these ZAP-70-expressing T cells demonstrated a diverse T-cell receptor repertoire as monitored by the relative usage of each T-cell receptor beta chain hypervariable region subfamily. The presence of ZAP-70 in B cells did not affect either lipopolysaccharide- or lipopolysaccharide/interleukin-4-mediated immunoglobulin isotype switching. Altogether, these data indicate that retroviral-mediated gene transfer of the ZAP-70 gene may prove to have a therapeutic benefit for patients with ZAP-70-SCID.     

Development of immunologic functions after bone marrow transplantation in 33 patients with severe combined immunodeficiency

We retrospectively analyzed the development of lymphocytes and of the main immunological functions in 33 patients with severe combined immunodeficiency who survived at least 6 months after bone marrow transplantation (BMT). Eighteen patients received HLA-identical BM and 15 received HLA-nonidentical BM. Development of immune functions occurred faster after HLA-identical BMT as full T- and B-lymphocyte- mediated responses were present at day 186 versus 505, respectively (P = .05). In addition, antibody responses remain completely or partially absent in 8 of 15 patients of the second group. Detection of antibody response after HLA-incompatible BMT correlated with engraftment of donor B cells in informative cases. In patients who received an HLA- nonidentical BMT after chemotherapy (6 of 15), development of immune functions occurred more rapidly and 6 of 6 had B-cell functions, including normal antibody production. Autoimmunity was not uncommon and was found after HLA-incompatible BMT (4 of 15) or after HLA-partially phenotypically identical BMT (2 of 3). Antibodies were in most cases specific for blood cells. Occurrence of autoimmunity correlates with poor B-cell functions and to a lesser extent with defective T-cell responses. This type of study may lead to definition of a more accurate strategy for performing BMT in patients with severe combined immunodeficiency.     

Immunoreconstitution in severe combined immunodeficiency after transplantation of HLA-haploidentical, T-cell-depleted bone marrow

Immunological reconstitution by transplantation of HLA-haploidentical, paternal bone marrow was attempted in four infants with severe combined immunodeficiency who lacked HLA-identical donors. To prevent graft-versus-host disease, T lymphocytes were removed from the grafts by agglutination with soybean agglutinin and rosette formation with sheep red blood cells. None of the patients received conditioning treatment before transplantation. Normal, donor-derived cellular immune functions developed in all four patients within several months of transplantation. Threatening complications of graft-versus-host reactions were not seen. All four patients remain in excellent health 12-15 months after discharge home, with persisting normal T-cell functions.     

HLA-haploidentical bone marrow transplantation for severe combined immunodeficiency using E rosette fractionation and cyclosporine

Eight patients with severe combined immunodeficiency received bone marrow cells from their HLA haplotype-identical fathers after bone marrow T cell depletion by rosetting with neuraminidase-treated sheep red cells. Because the method led to the infusion of a small percentage of T lymphocytes (0.1% to 0.3%), cyclosporin was given by continuous intravenous infusion for two months in order to prevent the occurrence of graft-v-host disease (GVHD). Three patients who did have residual nonfunctional T lymphocytes received busulfan and cyclophosphamide before transplantation. Engraftment was observed in seven patients, and severe GVHD was not seen. Two patients died early after the bone marrow transplantation because of prior infections, and a third died at day 90 from a B cell lymphoproliferative syndrome. The five other patients are doing well. Stable engraftment has been achieved with reconstitution of cell-mediated immunity in 5/5 and humoral immunity in 4/5 patients.     

In vivo gene transfer into rat bone marrow progenitor cells using rSV40 viral vectors

Hematopoietic stem cell (HSC) gene transfer has been attempted almost entirely ex vivo and has been limited by cytokine-induced loss of self-renewal capacity and transplantation-related defects in homing and engraftment. Here, we attempted to circumvent such limitations by injecting vectors directly into the bone marrow (BM) to transduce HSCs in their native environment. Simian virus 40 (SV40)-derived gene delivery vectors were used because they transduce resting CD34+ cells very efficiently. Rats received SV-(Nef-FLAG), carrying FLAG marker epitope--or a control recombinant SV40 (rSV40)--directly into both femoral marrow cavities. Intracellular transgene expression by peripheral blood (PB) or BM cells was detected by cytofluorimetry. An average of 5.3% PB leukocytes expressed FLAG for the entire study--56 weeks. Transgene expression was sustained in multiple cell lineages, including granulocytes (average, 3.3% of leukocytes, 20.4% of granulocytes), CD3+ T lymphocytes (average, 0.53% of leukocytes, 1% of total T cells), and CD45R+ B lymphocytes, indicating gene transfer to long-lived progenitor cells with multilineage capacity. An average of 15% of femoral marrow cells expressed FLAG up to 16.5 months after transduction. Thus, direct intramarrow administration of rSV40s yields efficient gene transfer to rat BM progenitor cells and may be worthy of further investigation.     

In vitro transdifferentiation of adult bone marrow Sca-1+ cKit- cells cocultured with fetal liver cells into hepatic-like cells without fusion

Several research groups have recently reported that certain bone marrow cells (BMCs) differentiate into hepatocytes in vitro as well as in vivo in rodents. However, it has yet to be elucidated what factors effectively trigger and sustain transdifferentiation of BMCs. In the present study, we specifically asked whether the presence of murine fetal liver cells (FLCs) triggered and supported in vitro transdifferentiation of murine BMCs. Fractionated BMCs from green fluorescence protein (GFP)-expressing transgenic mice and FLCs from ROSA26 mice (X-gal(+) FLCs) were cocultured in the presence of hepatocyte growth factor in laminin-coated dishes. We found that Sca-1(+) BMCs gave rise to adherent hepatic-like cells, which expressed albumin as assessed with immunocytochemistry and RNA-polymerase chain reaction (PCR), and alpha-fetoprotein and cytokeratin 19 as examined with RNA-PCR. When GFP(+)Sca-1(+)cKit(-) cells were cocultured with X-gal(+) FLCs, all GFP(+) albumin-producing cells were negative for X-gal, showing that cell fusion was not associated in the observed BMCs' differentiation into hepatic-like cells. Titration analysis revealed that 1 of 5,943 Sca-1(+)cKit(-) cells had the ability to proliferate and differentiate into hepatic-like cells. These data strongly suggest that BMCs differentiate into hepatic-like cells in the presence of FLCs and that the present method may be useful for propagating BMC-derived hepatocytic progenitors and for investigating the nature of those cells.     

The lethal effects of transplantation of Socs1-/- bone marrow cells into irradiated adult syngeneic recipients

Injection of neonatal bone marrow cells from mice lacking the gene encoding suppressor of cytokine signaling 1 (SOCS1) into irradiated syngeneic 129/Sv or C57BL/6 mice led to a decreased survival, more rapidly occurring in 129/Sv than in C57BL/6 mice. Moribund mice did not exhibit the acute or chronic diseases developed by Socs1-/- mice but developed a pathology characteristic of graft-versus-host disease with typical chronic inflammatory lesions in the liver, skin, lungs, and gut. The results indicate that cells derived from the Socs1-/- bone marrow are autoaggressive but did not identify the cell types involved. Failure of the engrafted Socs1-/- marrow cells to reproduce the tissue damage typical of Socs1-/- disease indicates that loss of SOCS1 from target tissues may also be required for the development of the Socs1-/- diseases, such as fatty degeneration of the liver, polymyositis, or corneal inflammation.     

Evaluation of different protocols for gene transfer into non-obese diabetes/severe combined immunodeficiency disease mouse repopulating cells

Purpose Although gene transfer with retroviral vectors has shown distinct clinical success in defined settings, efficient genetic manipulation of hematopoietic progenitor cells remains a challenge. To address this issue we have evaluated different transduction protocols and retroviral constructs in the non-obese diabetes (NOD)/severe combined immunodeficiency disease (SCID) xenograft model. Methods An extended transduction protocol requiring 144 h of in vitro manipulation was compared to a more conventional protocol requiring 96 h only. Result While pretransplantation analysis of cells transduced with a retroviral vector, expressing the enhanced green fluorescent protein (EGFP) marker gene, demonstrated significantly higher overall transduction rates for the extended protocol (33.6 ± 2.3 vs. 22.1 ± 3.8%), EGFP expression in CD34+ cells before transplantation (4.0 ± 0.9 vs. 11.6 ± 2.5%), engraftment of human cells in NOD/SCID bone marrow 4 weeks after transplantation (4.5 ± 1.7 vs. 36.5 ± 9.4%) and EGFP expression in these cells (0 ± 0 vs. 11.3 ± 2.8%) were significantly impaired. When the 96 h protocol was used in combination with the spleen focus forming virus (SFFV)/murine embryonic stem cell (MESV) hybrid vector SFβ11-EGFP, high transduction rates for CD45+ (41.0 ± 5.3%) and CD34+ (38.5 ± 3.7%) cells prior to transplantation, as well as efficient human cell engraftment in NOD/SCID mice 4 weeks after transplantation (32.4 ± 3.5%), was detected. Transgene expression was observed in B-lymphoid (15.9 ± 2.0%), myeloid (36.5 ± 3.5%) and CD34+ cells (10.1 ± 1.5%). Conclusion Our data show that CD34+ cells maintained in cytokines for multiple days may differentiate and loose their capacity to contribute to the haematological reconstitution of NOD/SCID mice. In addition, the SFFV/MESV hybrid vector SFβ11-EGFP allows efficient transduction of and gene expression in haematopoietic progenitor cells.     

Full hematopoietic engraftment after allogeneic bone marrow transplantation without cytoreduction in a child with severe combined immunodeficiency

Bone marrow transplantation (BMT) for severe combined immunodeficiency (SCID) with human leukocyte antigen (HLA)-identical sibling donors but no pretransplantation cytoreduction results in T-lymphocyte engraftment and correction of immune dysfunction but not in full hematopoietic engraftment. A case of a 17-month-old girl with adenosine deaminase (ADA) deficiency SCID in whom full hematopoietic engraftment developed after BMT from her HLA-identical sister is reported. No myeloablative or immunosuppressive therapy or graft-versus-host disease (GVHD) prophylaxis was given. Mild acute and chronic GVHD developed, her B- and T-cell functions became reconstituted, and she is well almost 11 years after BMT. After BMT, repeated studies demonstrated: (1) Loss of a recipient-specific chromosomal marker in peripheral blood leukocytes (PBLs) and bone marrow, (2) conversion of recipient red blood cell antigens to donor type, (3) conversion of recipient T-cell, B-cell, and granulocyte lineages to donor origin by DNA analysis, and (4) increased ADA activity and metabolic correction in red blood cells and PBLs.     

In vitro differentiation of mouse bone marrow mononuclear cells into hepatocyte-like cells.

AIM:: It is imperative to explore some ways to gain the functional hepatocytes for hepatocyte transplantation. Bone marrow stem cells can differentiate into hepatocytes in vivo and in vitro. We select fibroblast growth factor-4 (FGF-4), oncostatin M (OSM), hepatocyte growth factor (HGF) and epithermal growth factor (EGF) as differentiation factors, and design the appropriate directed differentiation medium in order to gain hepatocytes through directed differentiation of bone marrow stem cells. METHODS:: Bone marrow mononuclear cells (BMMCs) were cultured in the directed differentiation media including FGF-4, OSM, HGF and EGF. In the course of cell differentiation, cell morphology was observed, and the expression patterns of some genes of the hepatocyte were validated and confirmed by RT-PCR. The ALB-, and CK18-expressed cells were gone further step to be confirmed by Western blot analysis, immunofluorescence and flow cytometric analysis. Hepatocyte functional activity, including glycogen synthesis and urea production, were confirmed by periodic acid-Shiff (PAS) staining and urea assay. RESULTS:: Some epithelial-like cells or polygonal cells appeared in the directed differentiation medium within 12 days, and the number and sizes of colonies of epithelial-like cells or polygonal cells increased in the course of the cell directed differentiation. AFP, HNF-3ss, ALB and CK18 mRNA expressions first appeared within day 7, and lasted throughout the later directed differentiation. TTR, G-6-P and TAT mRNA expressions could be detected within day 14, and their expressions lasted in the course of the later directed differentiation. ALB and CK18 were confirmed to exist in the differentiated BMMCs by Western blot analysis. ALB was found in the cytoplasm and cell membrane, while CK18 scattered in the cytoplasm by immunofluorescent staining. On day 21,the ratio of ALB-positive cells was 69.45%, and the ratio of CK18-positive cells was 67.36%. The accumulation of glucogen was detected in the cytoplasm of the differentiated cells. The directed differentiated BMMCs produced urea 3 days later, and they produced urea in a time-dependent manner. CONCLUSIONS:: BMMCs could differentiate into hepatocytes or hepatocyte-like cells in the differentiation media including HGF, FGF-4, EGF, and OSM. These hepatocyte-like cells were identified at the gene level and protein level. Furthermore, these hepatocyte-like cells had some hepatocellular synthesis and metabolism functions.     

In vitro functional defects of bone marrow-derived CD34+ progenitors from newly diagnosed mature B-cell malignancies with bone marrow tumor involvement

OBJECTIVE: We hypothesized that the presence of tumor cells in bone marrow (BM) could alter hematopoietic progenitor cell functions. Therefore, we evaluated phenotypic and in vitro functional properties of BM-derived CD34+ progenitors issued from untreated and newly diagnosed patients presenting a mature B-lymphoproliferative disorder (LPD) involving the BM (Inv+). PATIENTS AND METHODS: In vitro proliferation and differentiation capacities of primitive and committed progenitors were evaluated by cobblestone area-forming cell (CAFC) and colony-forming cell (CFC) assays, and ex vivo cell expansion. Migratory capacities of CD34+ cells were explored by chemotaxis assays using a CXCL12alpha gradient. RESULTS: Our results showed that CD34+ cells from Inv+ patients overexpressed CD117 and had a significant decrease of week-3 and -6 CAFC, and CFC frequencies, compared to cells obtained from healthy volunteers and LPD patients without BM involvement (Inv-). In addition, progenitors from Inv+ patients maintained a significantly decreased CFC capacity after ex vivo cell expansion, compared to healthy volunteers. However, the former cells held their migratory capacity in response to CXCL12alpha. CONCLUSION: Functional defects of primitive and committed CD34+ progenitors detected among LPD patients with BM tumor involvement suggest either that tumor cells may induced bystander effects on progenitors or that "unusual" CD34+ cells may exist in the BM that could belong to the proliferating tumor tissue.