Critical variables affecting clinical-grade production of the self-inactivating gamma-retroviral vector for the treatment of X-linked severe combined immunodeficiency.

Patients with X-linked severe combined immunodeficiency (SCID-X1) were successfully cured following gene therapy with a gamma-retroviral vector (gRV) expressing the common gamma chain of the interleukin-2 receptor (IL2RG). However, 5 of 20 patients developed leukemia from activation of cellular proto-oncogenes by viral enhancers in the long-terminal repeats (LTR) of the integrated vector. These events prompted the design of a gRV vector with self-inactivating (SIN) LTRs to enhance vector safety. Herein we report on the production of a clinical-grade SIN IL2RG gRV pseudotyped with the Gibbon Ape Leukemia Virus envelope for a new gene therapy trial for SCID-X1, and highlight variables that were found to be critical for transfection-based large-scale SIN gRV production. Successful clinical production required careful selection of culture medium without pre-added glutamine, reduced exposure of packaging cells to cell-dissociation enzyme, and presence of cations in wash buffer. The clinical vector was high titer; transduced 68-70% normal human CD34(+) cells, as determined by colony-forming unit assays and by xenotransplantation in immunodeficient NOD.CB17-Prkdc(scid)/J (nonobese diabetic/severe combined immunodeficiency (NOD/SCID)) and NOD.Cg-Prkdc(scid) Il2rg(tm1Wjl)/SzJ (NOD/SCID gamma (NSG))) mice; and resulted in the production of T cells in vitro from human SCID-X1 CD34(+) cells. The vector was certified and released for the treatment of SCID-X1 in a multi-center international phase I/II trial.

A human severe combined immunodeficiency (SCID) condition with increased sensitivity to ionizing radiations and impaired V(D)J rearrangements defines a new DNA recombination/repair deficiency.

The products of recombination activating gene (RAG)1 and RAG2 initiate the lymphoid-specific phase of the V(D)J recombination by creating a DNA double-strand break (dsb), leaving hairpin-sealed coding ends. The next step uses the general DNA repair machinery of the cells to resolve this dsb. Several genes involved in both V(D)J recombination and DNA repair have been identified through the analysis of in vitro mutants (Chinese hamster ovary cells) and in vivo situations of murine and equine severe combined immunodeficiency (scid). These studies lead to the description of the Ku-DNA-dependent protein kinase complex and the XRCC4 factor. A human SCID condition is characterized by an absence of B and T lymphocytes. One subset of these patients also demonstrates an increased sensitivity to the ionizing radiation of their fibroblasts and bone marrow precursor cells. This phenotype is accompanied by a profound defect in V(D)J recombination with a lack of coding joint formation, whereas signal joints are normal. Functional and genetic analyses distinguish these patients from the other recombination/repair mutants, and thus define a new group of mutants whose affected gene(s) is involved in sensitivity to ionizing radiation and V(D)J recombination.

Intensive and prolonged treatment of focal and segmental glomerulosclerosis recurrence in adult kidney transplant recipients: a pilot study.

No treatment has consistently induced long-term remission of proteinuria in adult patients with focal segmental glomerulosclerosis (FSGS) recurrence after kidney transplantation. We undertook an open-label, nonrandomized pilot trial of intensive and prolonged treatment of FSGS recurrence. Over an 18-month period, 10 adult kidney transplant recipients with FSGS recurrence received concomitantly high-dose steroids, intravenous cyclosporine for 14 days followed by oral cyclosporine therapy, and an intensive and prolonged course of plasma exchanges (PE). We compared this treatment with those of a control group of 19 patients with a FSGS recurrence transplanted between 1997 and 2005. Complete, rapid (mean 23 +/- 7 days) and sustained remission was obtained in 9/10 patients (90%) as opposed to 27% in the control group. At month 3 and month 12, proteinuria was 0.16 g/day (range 0.05-0.3 g/day) and 0.19 g/day (range 0.05-1 g/day) respectively. Only one patient remained in partial remission at month 12 but he had already lost two previous grafts due to FSGS recurrence. PEs were stopped at month 9 in all patients except for the patient with a partial remission who remains PE-dependent. This small pilot study provides very encouraging results demonstrating that this treatment rapidly achieves complete and sustained remission in a high proportion of patients.

Gene therapy of human severe combined immunodeficiency (SCID)-X1 disease.

Severe combined immunodeficiency-X1 (SCID-X1) is an X-linked inherited disorder characterized by an early block in T and natural killer (NK) lymphocyte differentiation. This block is caused by mutations of the gene encoding the gammac cytokine receptor subunit of interleukin-2, -4, -7, -9, and -15 receptors, which participates in the delivery of growth, survival, and differentiation signals to early lymphoid progenitors. After preclinical studies, a gene therapy trial for SCID-X1 was initiated, based on the use of complementary DNA containing a defective gammac Moloney retrovirus-derived vector and ex vivo infection of CD34+ cells. After a 10-month follow-up period, gammac transgene-expressing T and NK cells were detected in two patients. T, B, and NK cell counts and function, including antigen-specific responses, were comparable to those of age-matched controls. Thus, gene therapy was able to provide full correction of disease phenotype and, hence, clinical benefit.

Increased radiosensitivity of granulocyte macrophage colony-forming units and skin fibroblasts in human autosomal recessive severe combined immunodeficiency.

We studied the radiosensitivity of granulocyte macrophage colony-forming units (GM-CFU) in patients with a severe combined immunodeficiency (SCID). Three patients lacking both mature T and B cells showed a twofold higher GM-CFU radiosensitivity calculated as the DO value (dose required to reduce survival to 37%), and an identical observation was made with fibroblasts from one of these patients. A patient with an SCID with hypereosinophilia, i.e., Omenn's syndrome characterized by extremely restricted T cell heterogeneity and a lack of B cells, also showed abnormal GM-CFU radiosensitivity. In contrast, GM-CFU from a patient lacking only T cells (X-linked form of SCID) showed normal GM-CFU radiosensitivity. These data further support the similarity between human T(-) B(-) SCID and the murine acid mutation characterized by a defect in T cell receptor and immunoglobulin gene rearrangement, and by an abnormal double-strand DNA break repair function. In addition, they strongly suggest that the Omenn's immunodeficiency syndrome may be a leaky T(-)B(-) SCID phenotype as previously indicated by the coexistence of the two phenotypes in siblings.

Outcomes of hematopoietic stem cell transplantation for Hurler's syndrome in Europe: a risk factor analysis for graft failure.

Hurler's syndrome (HS), the most severe form of mucopolysaccharidosis type-I, causes progressive deterioration of the central nervous system and death in childhood. Allogeneic stem cell transplantation (SCT) before the age of 2 years halts disease progression. Graft failure limits the success of SCT. We analyzed data on HS patients transplanted in Europe to identify the risk factors for graft failure. We compared outcomes in 146 HS patients transplanted with various conditioning regimens and grafts. Patients were transplanted between 1994 and 2004 and registered to the European Blood and Marrow Transplantation database. Risk factor analysis was performed using logistic regression. 'Survival' and 'alive and engrafted'-rate after first SCT was 85 and 56%, respectively. In multivariable analysis, T-cell depletion (odds ratio (OR) 0.18; 95% confidence interval (CI) 0.04-0.71; P=0.02) and reduced-intensity conditioning (OR 0.08; 95% CI 0.02-0.39; P=0.002) were the risk factors for graft failure. Busulfan targeting protected against graft failure (OR 5.76; 95% CI 1.20-27.54; P=0.028). No difference was noted between cell sources used (bone marrow, peripheral blood stem cells or cord blood (CB)); however, significantly more patients who received CB transplants had full-donor chimerism (OR 9.31; 95% CI 1.06-82.03; P=0.044). These outcomes may impact the safety/efficacy of SCT for 'inborn-errors of metabolism' at large. CB increased the likelihood of sustained engraftment associated with normal enzyme levels and could therefore be considered as a preferential cell source in SCT for 'inborn errors of metabolism'.

The VAD chemotherapy regimen plus a G-CSF dose of 10 microg/kg is as effective and less toxic than high-dose cyclophosphamide plus a G-CSF dose of 5 microg/kg for progenitor cell mobilization: results from a monocentric study of 82 patients.

A study was conducted to compare the efficiency and toxicity of two peripheral blood stem cell (PBSC) mobilization procedures for newly diagnosed patients with multiple myeloma. Patients from group 1 (n=51) were treated by high-dose cyclophosphamide (HD-CY) plus G-CSF (5 microg/kg/day), and the second group (n=31) by VAD regimen plus G-CSF administration (10 microg/kg/day). Successful mobilization, defined by a minimal count of 2.5 x 10(6) CD34(+) cells/kg collected, was achieved in 96 and 90% of patients in groups 1 and 2, respectively (P=0.15). The mean peripheral blood CD34(+) cells concentration and the mean CD34(+) cells/kg collected were higher in group 2 than in the group 1 (P=0.05). The mean number of leukaphereses necessary to collect a count of 2.5 x 10(6) CD34(+) cells/kg was reduced in group 2 compared to group 1. Adverse events, blood products consumption and time spent in the hospital were significantly greater after HD-CY. In conclusion, VAD plus a G-CSF dose of 10 microg/kg administration seems preferential to HD-CY plus a G-CSF dose of 5 microg/kg for PBSC collection because of equivalent or better efficiency in stem cell mobilization, strong favorable toxicity profile and reduced cost.

Allogeneic and autologous transplantation for haematological diseases, solid tumours and immune disorders: definitions and current practice in Europe.

The Accreditation Subcommittee of the EBMT regularly publishes special reports on current practice of haemopoietic stem cell transplantation for haematological diseases, solid tumours and immune disorders in Europe. Major changes have occurred since the first report was published in 1996. Haemopoietic stem cell transplantation today includes grafting with allogeneic and autologous stem cells derived from bone marrow, peripheral blood and cord blood. With reduced intensity conditioning regimens in allogeneic transplantation, the age limit has increased, permitting the inclusion of older patients. New indications have emerged such as autoimmune disorders and AL amyloidosis for autologous, and solid tumours for allogeneic transplants. The introduction of alternative therapies has challenged well-established indications such as imatinib for chronic myeloid leukaemia. An updated report with revised tables and operating definitions is presented here.

Optimization of retroviral gene transfer protocol to maintain the lymphoid potential of progenitor cells.

We have attempted to improve retrovirus-mediated gene transfer efficacy into hematopoietic progenitor cells (HPCs) without causing them to lose their lymphoid potential. Highly purified CD34(+) cells on CH-296 fibronectin fragments have been transduced with three different cytokine combinations. Murine CD2 was used as a marker gene. Transgene expression was assayed by FACS analysis shortly after transduction of CD34(+) cells and after long-term culture (LTC) extended by differentiation of various lymphoid lineages: NK cells, B cells, and dendritic cells. Compared with the historical cytokine mix, i.e., SCF (stem cell factor) + IL-3 (interleukin 3) + IL-6, the combination SCF + FL (Flt-3 ligand) + M-GDF (megakaryocyte growth and differentiation factor) + IL-3 significantly improved the total number of viable cells and CD34(+) cells after transduction and the long term-cultured progenitors after 6 weeks. In addition, the combination of SCF + FL + M-GDF + IL-3 maintained more efficiently the lymphoid potential of the progeny of transduced long term-cultured CD34(+) cells, as attested by the significantly higher number of CD56(+), CD19(+), and CD1a(+) cells recovered when FL and M-GDF were added to SCF + IL-3. Thus, even though additional improvements may still be needed in transduction of HPCs, these conditions were adopted for a clinical trial of gene therapy for X-linked severe combined immunodeficiency.

Prevention of bone marrow and cardiac graft rejection in an H-2 haplotype disparate mouse combination by an anti-LFA-1 antibody.

The aim of the present study was to compare the preventive effect of a nondepleting monoclonal antibody specific for the LFA-1 alpha chain (CD11a) on the rejection of bone marrow, vascularized cardiac, and nonvascularized skin grafts in the same haplotype-disparate mouse strain combination. A 7-day treatment with a total dose of 0.5 mg of anti-LFA-1 antibody (H-129) had no effect on the rejection of BDF1(H-2b/d) skin grafts by CDF1 (H-2k/d)-treated mice. In contrast, the same treatment regimen significantly prolonged the survival of BDF1 cardiac allografts in CDF1 mice: 7 out of 10 mice had a functional graft after 70 days, whereas all control mice had rejected their graft by 11 days. Nevertheless, cardiac allografts were ultimately rejected. In contrast, infusion of anti-LFA-1 antibody was able to promote definitive engraftment of T-depleted BDF1 marrow in 9 gray-irradiated CDF1 recipients: in surviving mice, engraftment increased from 10% in controls to 78% in antibody-treated recipients. In mice that tolerated their cardiac graft for more than 70 days, there was a slight delay in the rejection of donor skin graft but no in vitro evidence of tolerance. In contrast, mice with successful marrow engraftment did not reject donor skin graft and failed to mount proliferative and cytotoxic responses against donor alloantigens, whatever the percentage of engrafted donor leukocytes. These results indicate that a nondepleting anti-LFA-1 antibody can efficiently protect against rejection of MHC-incompatible heart and bone marrow. The protective effect of anti-LFA-1 antibody was associated with the induction of T cell tolerance toward donor antigens after bone marrow transplantation.

Specific elimination of alloreactive T cells by an anti-interleukin-2 receptor B chain-specific immunotoxin.

Graft-versus-host disease and graft rejection remain the two principal causes of morbidity and mortality after major-histocompatibility-complex-mismatched bone marrow transplantation. Human and animal models suggest that both CD4+ and CD8+ T cell subsets present in the donor inoculum are responsible for their initiation. Since the human mixed lymphocyte culture (MLC) and the HLA-restricted cytotoxicity may reflect cellular interactions occurring during GVHD and graft rejection, inhibitions of these responses may represent useful approaches for screening functional T cell depletion in experimental bone marrow transplantation studies. For this purpose, we have tested the possibility of removing the host-specific allogeneic T cells present in the marrow. After a two-day MLC, the specifically activated host alloreactive blood or bone marrow T cells were incubated with the ricin A-chain toxin conjugated with the antibody 33B3.1 directed against the human receptor of interleukin 2 (33B3.1-IT). A complete inhibition of a primary MLC and of cytotoxic activities was observed as well as a disappearance of IL-2R(+) (p55) T cells. This method had limited consequence upon the alloreactivity of blood or marrow T cells toward a third unrelated party. The limiting-dilution analysis of residual alloantigen-reactive T lymphocytes has shown that this depletion results in a twentyfold to fiftyfold reduction of antihost reactivity. The procedure was also shown not to inhibit the growth of marrow precursors for granulocytes and macrophages.

Bone marrow transplantation from genetically HLA-nonidentical donors in children with fatal inherited disorders excluding severe combined immunodeficiencies: use of two monoclonal antibodies to prevent graft rejection.

OBJECTIVE: For children with life-threatening inborn errors of metabolism without a matched related bone marrow donor, transplantation from an HLA genetically nonidentical donor is the only therapeutic option. To reduce the high risk of graft rejection in this setting without increasing the conditioning regimen, a protocol based on the infusion of an antiadhesion antibody directed against the CD11a (leukocyte function-associated antigen 1 [LFA-1]) molecule was performed by the European Bone Marrow Transplantation-European Society for Immunodeficiency group with promising results. To optimize engraftment, and thereby survival, further, the additional blockade of a second important leukocyte adhesion and signalization pathway mediated by the CD2 and LFA-3 interaction was attempted in a multicenter protocol conducted by the European Bone Marrow Transplantation-European Society for Immunodeficiency group. Results of this study (ie, engraftment and survival) were compared with a historical control group that received the anti-LFA-1 antibody alone. Factors that may have affected engraftment and survival were also considered in this study. METHODS: Forty-four children with inborn errors, including inherited immunodeficiencies (excluding severe combined immunodeficiencies), Chédiak-Higashi syndrome, familial hemophagocytic lymphohistiocytosis, and malignant osteopetrosis, received bone marrow from HLA-nonidentical related donors or from HLA-identical unrelated donors at 13 European centers between August 1990 and June 1993. Bone marrow was depleted of T cells by use of either erythrocyte (E) rosetting or monoclonal antibodies (MoAbs) to prevent graft-versus-host disease. The conditioning regimen consisted of busulfan and cyclophosphamide for all patients plus etoposide for patients with osteopetrosis, familial hemophagocytic lymphohistiocytosis, and Chédiak-Higashi syndrome. Infusions of MoAbs specific for the CD11a and the CD2 molecules were started 4 and 3 days, respectively, before and continued through the first 10 and 11 days, respectively, after bone marrow transplantation (a total of 14 injections). RESULTS: The overall sustained engraftment rate was 69.8%, with full chimerism in 80.6% of patients and no late graft rejection with the use of two MoAbs versus 65.7% and 58.1%, respectively, in the control group, in which only one MoAb was infused. The overall actuarial survival rate with a functional graft was 40.9%, with a mean follow-up of 39.3 months with two MoAbs versus 37.8% with one. The engraftment rate was significantly influenced by the T-cell depletion method, with better results for recipients of E rosette- depleted marrow (78.6% vs 20% for Campath 1-M plus complement-depleted marrows). Graft-versus-host disease and the kinetics of immune reconstitution were similar in both groups. CONCLUSIONS: The overall engraftment rate and overall survival rate with engraftment in patients treated with anti-LFA-1 and anti-CD2 were similar to those in patients treated with anti-LFA-1 antibody alone. However, although the number of patients is too small to draw definitive conclusions, results from the combined use of the two MoAbs indicates a trend toward better engraftment and survival after infusion of E rosette-depleted marrow. Further improvement in survival would demand additional strategies to hasten immunologic recovery.

Does haploidentical transplantation in children with primary immunodeficiencies have the potential to exploit donor NK cell alloreactivity?

Donor potential to exert NK cell alloreactivity has been shown to confer survival advantage in haploidentical hematopoietic cell transplantation for hematological malignancies. We investigated killer immunoglobulin receptor (KIR) ligand incompatibility in 40 children receiving haploidentical transplantation for primary immunodeficiencies. The conditioning regimen consisted of busulfan and cyclophosphamide. T-cell depletion of the graft used complement-dependent lysis or CD34+ selection. Two patients died in the first month. The remaining 38 patients were divided into those with (n=13) and those without (n=25) donor potential to exert NK cell alloreactivity. Engraftment was similar in the two groups (61.5 and 64%, respectively). The incidence of grade II-IV acute graft-versus-host disease (GVHD) tended to be lower in the group with donor potential to exert NK cell alloreactivity, but the difference was not significant. In conclusion, in this series of patients with primary immunodeficiencies, donor potential to exert NK cell alloreactivity was not associated with significant advantages in engraftment and prevention of acute GVHD.

Treatment of juvenile chronic myelomonocytic leukemia by allogeneic bone marrow transplantation.

Twelve of 15 patients with juvenile chronic myelomonocytic leukemia (JCMML) referred to our unit underwent allogeneic bone marrow transplantation (BMT) between 1982 and 1992. BMT was not performed in the remaining three cases because of poor overall condition in two and disease progression in one. Six patients received marrow from HLA-identical siblings after a chemotherapy conditioning regimen in five cases. BMT failed in one case. Long-term remission was achieved in three patients and two others are in remission 6 and 11 months after BMT. Remission was associated with autologous recovery in one patient and minimal mixed chimerism in another. In one patient, a first BMT procedure resulted in autologous recovery and relapse. A second transplant, with chemotherapy conditioning including TBI, was successful. BMT with marrow from a matched unrelated donor was also successful. IN contrast, BMT with marrow from mismatched related donors (five patients) failed because of graft failure and/or relapse. This single-center series indicates that HLA-identical BMT is an appropriate treatment for JCMML. However, on the basis of these results it cannot be ascertained whether chemotherapy or splenectomy are necessary prior to BMT. The best chemotherapy conditioning regimen remains to be defined, as regimens consisting exclusively of chemotherapy resulted either in long-term remission or in autologous recovery with relapse.

Impact of HLA matching on outcome of hematopoietic stem cell transplantation in children with inherited diseases: a single-center comparative analysis of genoidentical, haploidentical or unrelated donors.

SUMMARY: Hematological inherited diseases can be cured by hematopoietic stem cell transplantation (HSCT) from an human leukocyte antigen (HLA)-identical sibling donor (MSD), but the outcome of unrelated donors (URD) or haploidentical donors (HMD) has been a cause of concern. In all, 94 children affected with inherited diseases underwent HSCT at a single center using MSD (group A, n=31), URD (group B, n=23) or HMD (group C, n=40). There was no difference in the rate of engraftment or in the incidence of grades III-IV acute graft-versus-host disease (GVHD) between the groups. Survival rate was 80.6% in group A, 62.5% in group B and 47.5% in group C (P=0.023). In group B, survival rate was 73.7% in the subgroup with zero or one class I mismatch, and 25% in the subgroup with two or more class I mismatches (P=0.04). In group C, survival rate was 83.3% in the 9/10-identical subgroup, 64.3% in the seven or 8/10 subgroup, and 25% in the five or 6/10 subgroup (P=0.0007). Thus, engraftment, incidence of GVHD and survival are similar in recipients of grafts from MSD, URD with 0-1 class I-mismatch, or HMD with at least 7/10 HLA matches. The low success of HSCT using more disparate donors suggests reserving them for patients with very poor prognosis.

Comparison of lenograstim vs filgrastim administration following chemotherapy for peripheral blood stem cell (PBSC) collection: a retrospective study of 126 patients.

Mobilization techniques for peripheral blood stem cell (PBSC) collection include the administration of chemotherapy followed by hematopoietic growth factors or growth factors alone. Two forms of recombinant human granulocyte colony-stimulating factor (rhG-CSF) are available for PBSC mobilization: lenograstim and filgrastim which are the glycosylated and non-glycosylated forms respectively. In order to determine the influence of the two forms of G-CSF following chemotherapy on PBSC collection, we conducted a retrospective study in 126 patients with various hematological malignancies: 65 and 61 for the lenograstim and filgrastim groups respectively. No significant differences between the two groups were observed in terms of sex, age and diagnosis. Prior therapies and PBSC mobilization regimen were also equivalent. No significant difference was observed between the groups for the median CD34+ cells harvested. The number of leukapheresis necessary to obtain a minimal number of 3 x 10(6) CD34+ cells/kg was equivalent for the two groups. The proportion of patients affected by a failure in PBSC collection was similar in the two groups. Our data suggest that lenograstim and filgrastim are equivalent for PBSC mobilization after chemotherapy.

In vivo infusion of anti-LFA-1 and anti-CD2 antibodies prevents graft failure after HLA partially incompatible bone marrow transplantation in children with high risk acute lymphoblastic leukaemia.

Bone marrow transplantation (BMT) from matched sibling donors is the therapy of choice for children with high-risk acute lymphoblastic leukaemia in children. It is however not available to more than two-thirds of patients who lack a matched donor. Here, we review the outcome of 28 patients with high-risk ALL who were transplanted in France with alternative marrow sources such as HLA-phenoidentical unrelated volunteers and HLA-partially incompatible relatives. For these patients, we tested the possibility to prevent T-depleted marrow graft rejection by infusing in vivo two monoclonal antibodies directed against adhesion receptors i.e., LFA-1 and CD2. Two previous multicenter trials in children transplanted with partially incompatible bone marrow for inborn errors of metabolism showed their efficacy in this setting. Twenty eight patients were enrolled in this study and followed for a median of 4.4 years. Bone marrow engraftment occurred in 81% of the evaluable patients. Post-transplantation leukaemic relapse was the most frequent cause of death in this group of patients, and occurred in 39% of patients. The second most frequent complication was infectious disease, while an EBV-induced B-lymphocyte proliferative disorder occurred in four patients. In conclusion, T-cell-depletion combined with infusion of anti-LFA-1 and anti-CD2 antibodies is efficient in preventing graft failure and GVHD in this group of children with high-risk leukaemia undergoing partially incompatible BMT. The overall DFS is not improved in contrast to what has been previously observed in patients with immunodeficiencies transplanted with a similar rejection prophylaxis. Other approaches are therefore needed aiming either at preserving donor T-cell mediated immunity or accelerating immune reconstitution.

[The bone marrow: a reserve of stem cells able to repair various tissues?]. / Moelle osseuse: réservoir de cellules souches réparatrices de différents tissus ?

Hematopoietic stem cells (HSC) have been widely used for autologous and allodeneic transplantation during decades, although little was known about their migration, survival, self-renewal and differentiation process. Their sorting by the CD34(+) marker they express at the cell surface in human has been challenged by the recent discovery of HSC in the CD34(-) compartment that may precede CD34(+) HSC in the differentiation process. Until recently, stem cells present in the bone marrow were thought to be specific for hematopoiesis. Some experiments including clinical trials showing the formation of various tissues, muscle, neural cells and hepatocytes for instance, after transplantation of medullar cells, have challenged this dogma. In fact, the proofs of such a transdifferentiation process by HSC are still missing and the observations may result from the differentiation of other mulipotent stem cells present in the bone marrow, such as mesenchymal stem cells and more primitive multipotent adult progenitor cells (MAPC) and side population (SP) cells.

[Residual lymphocytes after specific depletion. Functional study]. / Lymphocytes résiduels après déplétion spécifique. Etude fonctionnelle.

We previously demonstrated that, following a 2-day stimulation of T cells by MHC incompatible cells, a ricin A-chain conjugated to a monoclonal anti-IL2 receptor p55 subunit can kill 1.5 log of the activated alloreactive T cells while non specific killing of alloreactive T cells of a third partner does not exceed 0.5 log. This methodology is of potential use for selective alloreactive T cell depletion in MHC incompatible bone marrow transplantation in order to prevent both graft versus host disease and graft rejection. This study shows that this T cell depletion method does not alter T cell reactivity to microorganism antigens encountered in infection following BMT. It was found that T cell proliferation to cytomegalovirus and to candida antigens is not affected as shown in proliferative assays and by limiting dilution analysis for the latter antigen.