Human Allogeneic Bone Marrow Research Articles

Mesenchymal Stem Cell Therapy in Acute Intracerebral Hemorrhage: A Dose-Escalation Safety and Tolerability Trial

BackgroundWe conducted a preliminary phase I, dose-escalating, safety, and tolerability trial in the population of patients with acute intracerebral hemorrhage (ICH) by using human allogeneic bone marrow–derived mesenchymal stem/stromal cells.MethodsEligibility criteria included nontraumatic supratentorial hematoma less than 60 mL and Glasgow Coma Scale score greater than 5. All patients were monitored in the neurosciences intensive care unit for safety and tolerability of mesenchymal stem/stromal cell infusion and adverse events. We also explored the use of cytokines as biomarkers to assess responsiveness to the cell therapy. We screened 140 patients, enrolling 9 who met eligibility criteria into three dose groups: 0.5 million cells/kg, 1 million cells/kg, and 2 million cells/kg.ResultsIntravenous administration of allogeneic bone marrow–derived mesenchymal stem/stromal cells to treat patients with acute ICH is feasible and safe.ConclusionsFuture larger randomized, placebo-controlled ICH studies are necessary to validate this study and establish the effectiveness of this therapeutic approach in the treatment of patients with ICH.

Allogeneic peripheral blood haematopoietic stem cell transplantation for the treatment of dogs with high-grade B-cell lymphoma.

Autologous peripheral blood haematopoietic stem cell transplantation (HCT) cures 33%-40% of dogs with high-grade B-cell lymphoma. We hypothesized, based on human allogeneic bone marrow transplantation literature, that transplanting dogs using canine donor leukocyte-matched CD34+ cells would lead to fewer relapses and increased cure rates. We retrospectively reviewed medical records of dogs diagnosed with high-grade B-cell lymphoma who received an identical allogeneic HCT. A total of 15 dogs transplanted at four facilities were identified. Five of fifteen dogs relapsed before transplant. The mean number of donor CD34+ cells/kg harvested and infused into recipient dogs was 8.0 × 106 /kg (range: 2.08 × 106 /kg-2.9 × 107 /kg). The median disease-free interval and overall survival of all dogs was 1095 days (range: 9-2920 days) and 1115 days (range: 9-2920 days), respectively. Two of five dogs, not in remission at transplant, died in the hospital. The median disease-free interval and overall survival of the remaining three dogs was 25 days (range: 15-250 days) and 1100 days (range: 66-1902 days), respectively. The median disease-free interval and overall survival of the 10 dogs who had not relapsed was 1235 days (range: 19-2920 days) and 1235 days (range: 19-2920 days), respectively. One dog died soon after discharge of presumed gastric-dilatation-volvulus. Eight of nine remaining dogs lived >4yrs post-alloHCT, leading to a cure rate of 89%. Acute graft versus host disease was seen in three dogs. These results suggest that allogeneic HCT can cure ~50% more dogs than those treated with autologous HCT.

Efficacy of Remestemcel-L Treatment in Steroid Refractory Acute Graft-Versus-Host Disease in Children: An Aggregate Analysis

Introduction: Steroid-refractory acute graft-versus-host dose disease (SR-aGVHD) is a frequent and potentially fatal complication of hematopoietic stem cell transplantation (HSCT). There are no approved therapies specifically indicated for use in children with SR-aGVHD < 12 years old. Treatment with culture-expanded, adult human allogeneic bone marrow (BM)-derived mesenchymal stromal cells (remestemcel-L) has shown excellent safety and efficacy in clinical trials conducted over the past 10 years. Here we describe aggregate results of remestemcel-L treatment of SR-aGVHD in the pediatric population. Methods: 309 pediatric subjects with SR-aGVHD were treated with remestemcel-L across 3 protocols. Data were derived from a phase 3, single arm study as first line after steroid failure with no other second line aGVHD therapies added through day 28 (NCT02336230, n=54), an expanded access program in subjects with aGVHD that had not responded to multiple other treatments (NCT00759018, n=241) and the pediatric subset of a randomized controlled trial (NCT00366145, n=14) in which remestemcel or placebo were given with a concomitant second line aGVHD therapy. Subjects in each protocol received 8 intravenous infusions of remestemcel-L (2 x 106 cells/kg twice weekly for 4 weeks). Those achieving a partial response could receive an additional 4 doses (once weekly for the subsequent 4 weeks). This analysis included 309 subjects who received at least one remestemcel-L dose. Response was assessed 28 days after the first dose. Survival was evaluated through day 100. Results: At baseline, mean age was 8.9 years (range 0.3 to 18.2 years) and aGVHD severity by IBMTR grade was C or D in n=252 (82%). Sources of grafts were bone marrow (BM: 46%), mobilized blood (MB: 20%) and cord blood (CB: 31%). Donor grafts were HLA matched related (9%) or unrelated (44%) or HLA-mismatched (46%). Subjects received a mean (standard deviation) of 10.3 (4.2) remestemcel-L infusions for a mean cumulative dose of 660.8 (484.1) x 106 cells. The primary endpoint, overall response (complete + partial response) at day 28 (OR) was observed in 66% of subjects (204/309) and survival at day 100 (OS) was 68% (211/309). Day 28 OR strongly predicted day 100 survival. Among subjects who were alive at day 28 (N=284), 170 of the 204 (83%) responders survived through day 100. In contrast, 41/80 (51%) of non-responders survived through day 100. Day 28 OR frequencies and day 100 OS rates were consistent across baseline aGVHD grades: Grade B OR 70%, OS 77%; Grade C or D OR 65%, OS 66%. Comparable day 28 response frequencies and day 100 survival rates were observed across type of HSCT: OR was achieved in 65%, 64% and 71% of BM, MB and CB, respectively and 67%, 70% and 70% of BM, MB and CB recipients survived through day 100. Comparable OR and OS were observed in HLA compatibility and donor types with OR in 71%, 66% and 66% and day 100 OS in 64%, 66% and 71% of matched related or unrelated and mismatched, respectively. Remestemcel-L was well tolerated with no infusion-related toxicity or other safety concerns. Most treatment-emergent serious adverse events were attributable to aGVHD or the underlying pre-transplant disease. Conclusions: In this large, diverse sample of pediatric subjects with SR- aGVHD, treatment with remestemcel-L was associated with a high and consistent overall response rate and survival rate through 100 days. Day 28 OR was highly predictive of day 100 survival. Remestemcel-L was well tolerated and effective across transplant types, donor sources, moderate to severe grades of GVHD and prior or concurrent treatment paradigms as defined by the individual protocols in this aggregate analysis. Remestemcel-L is a safe and effective treatment for children with SR- aGVHD addressing an unmet medical need for this highly vulnerable population. Disclosures Kurtzberg: Mesoblast, Inc,: Consultancy, Honoraria. Martin:Abgenomics: Research Funding; Enlivex: Consultancy; Procter and Gamble: Current equity holder in publicly-traded company; Pharmacyclics LLC: Membership on an entity's Board of Directors or advisory committees; Neovii: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfzier: Membership on an entity's Board of Directors or advisory committees; Xenikos: Research Funding; Talaris: Consultancy; Rigel: Consultancy; Mesoblast, Inc.: Consultancy; Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria. Prockop:Atara: Research Funding; Jasper: Research Funding; Mesoblast, Inc,: Consultancy, Honoraria, Research Funding. Burke:Mesoblast, Inc,: Current Employment. Segal:Mesoblast, Inc,: Current Employment.

Effects of bone marrow on the microenvironment of the human pancreatic islet: A Protein Profile Approach

Stem cells are a new therapeutic modality that may support the viability and function of human organs and tissue. Our previous studies have revealed that human allogeneic bone marrow (BM) sustains pancreatic β cell function and survival. This paper examines whether BM creates a microenvironment that supports human pancreatic islets in vitro by evaluating 107 proteins in culture media from BM, islet, and islet/bone marrow (IB) with mass spectrometry. Proteins were considered up- or down-regulated if p-values < 0.05 and fold change was greater than 2 fold I VS. IB. In addition, proteins identified that were uniquely found in islets co-cultured with bone marrow, but not in islets or bone marrow. A 95% protein probability was used as a threshold. Twenty three proteins were upregulated, and sixteen proteins were downregulated. The function of each protein is listed based on the protein database, which include structural proteins (9 upregulated, 4 downregulated); anti-protease and anti-endopeptidase enzymes (8 upregulated); cation binding proteins (6 up-regulated). Six proteins were uniquely identified in islet co-cultured with bone marrow. Three are anti-proteases or anti-endopeptidases, and 1 is a structural protein. These findings suggest that BM, by changing culture media proteins, may be one of mechanisms to maintain human islet function and survival.

Development of a surrogate potency assay to determine the angiogenic activity of Stempeucel\xae, a pooled, ex-vivo expanded, allogeneic human bone marrow mesenchymal stromal cell product

BackgroundMesenchymal stromal cells (MSCs) have emerged as a more beneficial alternative to conventional therapy and may offer a potential cure for unmet medical needs. MSCs are known to possess strong immunomodulatory and anti-inflammatory properties. Moreover, they promote angiogenesis and tissue regeneration through the secretion of trophic factors. For these reasons, the past decade witnessed a sharp increase in the number of clinical trials conducted with stem cells for various vascular diseases requiring angiogenesis. In this study, we evaluated the in vitro angiogenic potency of Stempeucel®, which is an allogeneic pooled human bone marrow-derived mesenchymal stromal cell (phBMMSC) product. We previously established the safety of Stempeucel® in our pre-clinical studies, and clinical trials conducted for critical limb ischaemia and acute myocardial infarction.MethodsBecause the proposed mechanism of action of phBMMSCs is mainly through the secretion of pro-angiogenic cytokines, we developed a surrogate potency assay by screening various batches of large-scale expanded phBMMSCs for the expression of angiogenic factors and cytokines through gene expression and growth factor analyses, followed by in vitro functional assays.ResultsThe well characterized angiogenic vascular endothelial growth factor (VEGF) was selected and quantified in twenty six manufactured batches of phBMMSCs to establish consistency following the United States Food and Drug Administration recommendations. According to recommendations 21 CFR 211.165(e) and 211.194(a)(2), we also established and documented the specificity and reproducibility of the test methods employed through validation. Moreover, we also attempted to elucidate the mechanism of action of the cell population to ensure appropriate biological activity. The functional role of VEGF has been established through in vitro angiogenic assays and a dose-dependent correlation was observed with in vitro functional results.ConclusionsThe data generated from this study suggest the selection of VEGF as a single surrogate marker to test the angiogenic potency of phBMMSCs. Our study reports the quantification of VEGF in twenty six batches of large-scale manufactured phBMMSCs, and a concentration-dependent correlation of secreted VEGF to endothelial cell functions of migration, proliferation and tube formation, in the conditioned medium obtained from nine phBMMSC batches. To our cognizance, this is the first study in which a single angiogenic factor (VEGF) has been qualified as a surrogate potency marker through all three in vitro functional assays to determine the angiogenic potency of the phBMMSC population.

Allogeneic mesenchymal stem cell infusion for the stabilization of focal segmental glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is the most frequent acquired renal condition resulting in end stage kidney disease in children. We describe a cell therapy treatment with human allogeneic bone marrow mesenchymal stem cells (MSC) in a 13-year-old patient developing recurrent FSGS after renal transplantation, which was not responding to conventional therapy.This treatment relied on the following measurements:clinical and laboratory evaluation of renal function, proteome array, biopsy, short tandem repeat assay.Before MSC treatment, the patient needed weekly plasmapheresis to achieve proteinuria-to-creatininuria ratio below 5. After three MSC infusions without adverse events, the patient has a stable renal function and the proteinuria target was reached without plasmapheresis. In addition, some circulating inflammatory factors decreased and their levels were still low after one year.This is the first report of an MSC treatment in an FSGS patient. Even though different factors may have contributed to the clinical results, after MSC infusion a stable reduction in the serum level of several inflammatory factors has been registered and the patient does not need anymore plasmapheresis to keep proteinuria under control.In addition, this encouraging single case let us identify some putative efficacy biomarkers that could be of clinical interest in chronic kidney diseases.

Bone marrow increases human islets insulin positive cells in co-culture: quantification with flow cytometry

We have previously demonstrated that allogeneic human bone marrow (BM) supports human islet function and longevity in vitro. We hypothesize that BM supporting human islets may include to increase β-cell in cultured islets. In this study, we developed a method to quantify insulin-producing β cells from cultured islets by using immunofluorescent staining and flow cytometry analysis to explore this possibility. The results show that human islets cocultured with BM for 39 days contained a significantly higher number of insulin-positive β cells (42.3% ± 4.5%) compared to the islet-only cultures (1.15% ± 0.78%), and increased insulin release levels evaluated by ELISA is consistent with increased β cells in same culture condition. Human islet culture with BM significantly increase β-cells while islet only culture lost β-cells in same culture period supports the possibility of BM increasing β-cells in cultured islets.

Immune Reconstitution: How It Should Work, What's Broken, and Why It Matters

Of the multiple cell lineages undergoing reconstitution in the setting of hematopoietic stem cell transplantation (HSCT), this overview will focus on reconstitution of the T cell arm of the immune system. In clinical HSCT, the autologous setting is free of constraints imposed by reactions to allogeneic antigens, but markers are lacking to distinguish reconstitution from transferred cells versus stem or progenitor cells left residual in the host. In allogeneic transplants, the converse is true – the stem cell origins of the reconstituted populations can be determined, but the biology of such reconstitution is confounded by concomitant allogeneic responses. Because of these constraints, the basic biology of T cell immune reconstitution was first delineated in mouse models in which the availability of congenic strains allowed stem cell origins of reconstituted cell populations to be investigated without the presence of confounding allogenic reactions. Two pathways of T cell regeneration were identified: the thymic-dependent maturation of new T cells from marrow progenitors, and thymic-independent expansion of mature peripheral T cells. 1 These initial studies also determined that no other pathways existed by which substantial peripheral populations of T cells were generated, and also established a means for investigating T cell reconstitution in humans by analyzing the expression of isoforms of the leukocyte common antigen CD45 as markers of naive and memory populations which correlated with the pathway of regeneration. They also demonstrated that homeostatic expansion of mature T cells occurred in lymphopenic hosts. Studies of such expansion have formed central concepts of homeostasis – that T cell levels are maintained by a balance between cytokine-consuming cells and homeostatic cytokines that support the maturation, proliferative expansion and survival of T cells.2 In translating these murine findings to humans, the recovery of CD4+ T cells in pediatric patients who were severely lymphopenic following chemotherapy treatment were initially tracked (Figure 1). There was a strong correlation between the recovery of total CD4+ T cell numbers and the recovery of naive, CD45RA+CD45RO- CD4+ T cells after as few as six months. 3 Furthermore, recovery of naive CD4+ cells was associated with a marked expansion of thymus volume, consistent with a dynamic regulation of the thymus.

Extracorporeal photopheresis in the treatment of graft-vs-host disease.

Graft-vs-host disease (GVHD) remains a major cause of morbidity and mortality after allogeneic stem cell transplantation. GVHD was first described by Barnes and Mole as a fatal disease manifest by skin changes, diarrhea, and runting in irradiated mice given allogeneic spleen cells. Early human allogeneic bone marrow transplant patients were noted to manifest a similar syndrome of dermatitis, hepatitis, and enteritis occurring within the first 100 days after infusion of allogeneic stem cells. The etiology of GVHD is related to alloreactivity between immunocompetent donor lymphocytes and transplantation alloantigens on host tissues. The incidence of acute GVHD (aGVHD) ranges from 27% to 50% after sibling-matched related donors and from 42% to 72% after unrelated donor bone marrow or peripheral blood stem cell transplants. Despite aggressive immunosuppressive therapies, including high-dose corticosteroids, cyclosporine A, antithymocyte globulin, tacrolimus, and mycophenolate mofetil, the mortality from acute GVHD remains high. Chronic GVHD (cGVHD) is a syndrome of immune dysregulation whose onset has been arbitrarily defined as occurring at least 100 days after allogeneic stem cell infusion. Clinical features of cGVHD are distinguished from aGVHD in that they more closely resemble autoimmune disorders. Histopathologic changes include sclerodermatous skin changes from collagen deposition, pulmonary fibrosis, esophageal dysfunction, dry mouth or mucocutaneous ulcerations, cholestasis, and myositis or fasciitis and are thought to be initiated, in part, by autoantibodies to cell surface and intracellular proteins. The incidence of cGVHD ranges from 30% in siblingmatched donors to over 70% in matched unrelated donor transplants. Factors associated with cGVHD include increased donor and recipient age, prior aGVHD, and the use of alloimmune female donors. Conventional therapeutic approaches for cGVHD, including corticosteroids and immunosuppressive agents, have demonstrated limited efficacy in patients with extensive disease. Oral methoxsalen followed by ultraviolet A therapy (PUVA), while effective in alleviating the symptoms of chronic skin GVHD, has had no impact on visceral involvement. Novel agents demonstrating activity include T-cell directed therapies, such as humanized anti-CD25 antibody (dacluzimab) and pentostatin, as well as anti-TNF-a antibody (infliximab) and thalidomide. While aGVHD is understood as a disease of acute alloreactivity, the etiology of cGVHD is controversial and is believed to be either an extension of aGVHD and/ or a result of dysfunctional immune reconstitution with generation of autoantibodies and tissue autoreactive Tcell clones. Acute GVHD is believed to be a Th1-driven disease wherein inflammatory cytokines, such as IL-2 and IFN-c as well as the monokines IL-1 and TNF-a, contribute to tissue damage. In murine models, donor CD4-enriched cells of Th2 phenotype have been shown to prevent aGVHD without affecting engraftment, suggesting that they play a role in downregulating the Th1 response, and, in other studies, administration of a Th1 inhibitor (TAK-603) markedly reduced the mortality associated with aGVHD. Chronic GVHD has been proposed by others to be a Th2-mediated disease, but recent studies demonstrating upregulation of Th1 cytokines IFN-c and IL-12 in peripheral blood mononuclear cells implicate, in part, a Th1-derived mechanism similar to that seen in acute alloreactivity.

Distinct TCRAV and TCRBV repertoire and CDR3 sequence of T lymphocytes clonally expanded in blood and GVHD lesions after human allogeneic bone marrow transplantation.

Acute graft-versus-host disease (GVHD) is a disorder involving the skin, gut and liver that is caused by mismatches of major and/or minor histocompatibility antigens between the HLA-identical donor and recipient. If T lymphocytes infiltrating GVHD lesions recognize antigens expressed in these organs, T cell clones should expand in inflammatory tissues. We previously reported that recipients of allogeneic bone marrow grafts have clonally expanded TCRalphabeta(+) T lymphocytes soon after transplantation, which leads to a skew of TCR repertoires. To establish whether or not the same antigens cause clonal expansion of T lymphocytes in both blood and GVHD tissues, we examined the usage of TCR alpha and beta chain variable regions (TCRAV and TCRBV) and determined the complementarity-determining region 3 (CDR3) of T lymphocytes clonally expanded in circulating blood and GVHD lesions. We found that the repertoires and CDR3 diversity of TCRAV and TCRBV differed between the GVHD lesions and circulating blood, suggesting the selective recruitment of antigen-specific T cells into GVHD tissues. We also found that the usage of TCRAV and TCRBV by the clonally expanded T lymphocytes and their CDR3 sequences differed between the GVHD tissues and blood. These results suggest that the antigen specificity of TCRalphabeta(+) T lymphocytes clonally expanded in blood and GVHD lesions is different.

CD40 ligand-stimulated B cell precursor leukemic cells elicit interferon-gamma production by autologous bone marrow T cells in childhood acute lymphoblastic leukemia.

Childhood B cell precursor acute lymphoblastic leukemia (BCP-ALL) cells, collected from bone marrow (BM) at diagnosis, were cultured, after thawing, on allogeneic human bone marrow stroma (HBMS) for 48 h in the presence of a soluble trimeric CD40 ligand (stCD40L) molecule. HBMS maintained leukemic cells viability in all tested cases (mean viability 85%). Under these culture conditions we noticed upregulation or de novo expression of costimulatory molecules CD40, CD80 (B7-1) and CD86 (B7-2) in 22/22, 15/23 and 21/23 cases, respectively. Upregulation, in terms of fluorescence intensity, was also observed in the expression of MHC I, MHC II, CD54 (ICAM 1) and CD58 (LFA 3) molecules. HBMS alone, although to a lesser extent, was able to induce modulation of these molecules, but not CD80, in a similar proportion of cases. Neither stCD40L nor HBMS induced modulation of CD10 and CD34 molecules. Moreover, in 4/4 tested cases, stCD40L-stimulated ALL cells were able to induce allogeneic T cells proliferation. To evaluate whether leukemia-reactive T cells were detectable in the BM of ALL patients at diagnosis, stCD40L-stimulated ALL cells were co-cultured with autologous T cells (ratio 1:1), isolated from BM at diagnosis, for 4 days and a 24 h ELISPOT assay was applied to detect the presence of interferon-gamma (IFN-gamma)-producing cells. In four of seven cases IFN-gamma-producing cells were detected with frequencies of 1/900, 1/1560, 1/2150 and 1/1575 autologous T cells. These data confirm that stCD40L exposure can activate the antigen-presenting cell (APC) capacity of BCP-ALL cells cultured on HBMS and that ELISPOT assay can be used to measure the frequency of leukemia-reactive autologous T cells in the BM of ALL patients even after short-term culture with stCD40L-stimulated ALL cells.

Highly efficient targeted transduction of undifferentiated human hematopoietic cells by adenoviral vectors displaying fiber knobs of subgroup B.

Human hematopoietic stem cells (HSCs) are poorly transduced by vectors based on adenovirus serotype 5 (Ad5). This is primarily due to the paucity of the coxsackievirus-Ad receptor on these cells. In an attempt to change the tropism of Ad5, we constructed a series of chimeric E1-deleted Ad5 vectors in which the shaft and knob of the capsid fibers were exchanged with those of other Ad serotypes. In all these vectors, the Ad E1 region was replaced by an expression cassette containing the cytomegalovirus immediate-early promoter and the gene for enhanced green fluorescent protein (GFP). Experiments performed in vitro showed an efficient transduction of umbilical cord blood (UCB) monocytes, granulocytes, and their precursors as well as the undifferentiated CD34(+) CD33(-) CD38(-) CD71(-) cells by Ad5 vectors carrying Ad subgroup B-specific fiber chimeras (Ad5FBs). In the latter subpopulation, which comprises less than 1% of the CD34(+) cells and is highly enriched with cells repopulating immunodeficient mice, more than 90% of the cells were GFP(+). Transduction by Ad5FBs of the less primitive fraction within UCB CD34(+) cells was significant lower. Actually, the transduction frequency and GFP level declined gradually with increased expression of the CD33, CD38, and CD71 antigens. Flow cytometric analysis of transduced UCB CD34(+) cells that were cultured for 5 days on an allogeneic human bone marrow stroma layer showed maintenance of the phenotypically defined HSCs at levels similar to those of control cultures. The latter finding indicates that neither the transduction procedure nor the high levels of GFP were toxic for these cells.

Pure red cell aplasia.

Pure red cell aplasia.

The human bone marrow as an immunoregulatory organ.

It was 45 years ago that in a virtual revolution in thinking in immunology there developed the acceptance and the subsequent expansion of two new dogmas: (1) that to eliminate toxins and pathogens as the major mode of defense, individual immune cells were, in their ontogeny of differentiation, internally programmed to react singly and then clonally against the virtually limitless individual stimuli of the outside world (1–3); (2) that before this programming was manifested the immune system would fail to recognize any antigenic stimulus as foreign, thereby not differentiating non-self from self-recognition. This allowed for non-self-antigens, if introduced in this early stage, to be immunologically tolerated on subsequent testing. In the chronology of the evolution of these two dogmas, there were the earlier descriptions of specific immunological paralysis and unresponsiveness to certain defined polysaccharides and haptens demonstrated in adult mice and guinea pigs respectively by Felton (4) and Chase (5). It remained for Medawar and his colleagues (6) in allotransplantation experiments to clearly define “acquired specific immunological tolerance” as an ontogenic concept involving the lack of maturation or differentiation were tolerance to be evoked. As early as 1953, Billingham, Brent, and Medawar demonstrated that allogeneic donor bone marrow-derived cells could confer such a specifically acquired tolerant state to the immune system of the murine recipient before self versus non-self recognition occurred in immune ontogeny, the test being subsequent acceptance of skin allografts from the same donors. The developmental processes of positive versus negative selection only a decade later began to be demonstrated to predominantly involve the thymus gland in maturational events of cells of the recipient immune system in studies first performed in neonatal rodents (7). By experimental manipulation of the immune system predominantly involving immunoablation by whole body x-irradiation, these findings of specific immunological tolerance as a result of the infusion of bone marrow-derived cells could be extended to adult animals as the system regenerated from stem cells in the bone marrow of either that of the donor (8), the recipient (9), or both (10). Nonetheless, except in the setting of clinical bone marrow transplantation using major (lethal) immunoablation followed by the salvage and replacement of the recipient immunohematopoietic system with donor cell lineages and only in the context of little or no donor-recipient MHC disparity, with few exceptions (11–15), to date, specific immunological tolerance in organ transplantation in humans has not been possible.

Essential Role of the Thymus to Reconstitute Naive (CD45RA+) T-Helper Cells After Human Allogeneic Bone Marrow Transplantation

To contribute to the understanding of the role of the thymus in humans in the reconstitution of naive (CD45RA+) T cells after bone marrow transplantation (BMT), we compared T-cell regeneration in a unique situation, namely a thymectomized cancer patient (15 years old), with that of thymus-bearing patients after allogeneic BMT. These cases shared features of transplantation (total body irradiation, HLA-matched donors, and graft-versus-host disease prophylaxis with cyclosporine A) and all had an uncomplicated posttransplantation course. As shown by fluorescence-activated cell sorting analyses, the thymectomized host failed to reconstitute CD45RA+ T-helper cells even 24 months after BMT (11% CD45RA+ of CD4+ cells). In this patient, preferentially CD45RO+ cells contributed to the recovery of CD4+ cells (206 of 261/μL at 6 months and 463 of 558/μL at 24 months after BMT, CD45RA+ of CD4+ cells), whereas CD45RA+ cells remained low (&lt;60/μL). In contrast, nine thymus-bearing hosts (5 children and 4 adults) examined between 6 and 24 months after BMT effectively reconstituted CD4+/CD45RA+ cells according to their normal age-related range (≥28% in adults and ≥50% in children). Five of these were analyzed sequentially at 6 and 9 months after BMT. Within this period, CD45RA+ cells increasingly contributed to the recovery of CD4+ cells (median, +21%), even when total CD4+ cells decreased. With respect to T-cytotoxic/suppressor cells, the thymectomized host retained the capacity to recover CD45RA+ cells (137 of 333/μL at 6 months and 596 of 1,046/μL at 24 months after BMT, CD45RA+ of CD8+ cells), a proportion similar to that seen in thymus-bearing hosts. These findings suggest that a thymus-independent pathway exists to regenerate CD45RA+ T-cytotoxic/suppressor cells, but residual thymus is essential to reconstitute naive (CD45RA+) T-helper cells after BMT in humans.

Essential Role of the Thymus to Reconstitute Naive (CD45RA+) T-Helper Cells After Human Allogeneic Bone Marrow Transplantation

AbstractTo contribute to the understanding of the role of the thymus in humans in the reconstitution of naive (CD45RA+) T cells after bone marrow transplantation (BMT), we compared T-cell regeneration in a unique situation, namely a thymectomized cancer patient (15 years old), with that of thymus-bearing patients after allogeneic BMT. These cases shared features of transplantation (total body irradiation, HLA-matched donors, and graft-versus-host disease prophylaxis with cyclosporine A) and all had an uncomplicated posttransplantation course. As shown by fluorescence-activated cell sorting analyses, the thymectomized host failed to reconstitute CD45RA+ T-helper cells even 24 months after BMT (11% CD45RA+ of CD4+ cells). In this patient, preferentially CD45RO+ cells contributed to the recovery of CD4+ cells (206 of 261/μL at 6 months and 463 of 558/μL at 24 months after BMT, CD45RA+ of CD4+ cells), whereas CD45RA+ cells remained low (<60/μL). In contrast, nine thymus-bearing hosts (5 children and 4 adults) examined between 6 and 24 months after BMT effectively reconstituted CD4+/CD45RA+ cells according to their normal age-related range (≥28% in adults and ≥50% in children). Five of these were analyzed sequentially at 6 and 9 months after BMT. Within this period, CD45RA+ cells increasingly contributed to the recovery of CD4+ cells (median, +21%), even when total CD4+ cells decreased. With respect to T-cytotoxic/suppressor cells, the thymectomized host retained the capacity to recover CD45RA+ cells (137 of 333/μL at 6 months and 596 of 1,046/μL at 24 months after BMT, CD45RA+ of CD8+ cells), a proportion similar to that seen in thymus-bearing hosts. These findings suggest that a thymus-independent pathway exists to regenerate CD45RA+ T-cytotoxic/suppressor cells, but residual thymus is essential to reconstitute naive (CD45RA+) T-helper cells after BMT in humans.

Localization to chromosome 22 of a gene encoding a human minor histocompatibility antigen.

In human allogeneic bone marrow transplantation, graft-vs-host disease and graft rejection can occur even if the patient and donor are genotypically matched by inheritance for HLA. By definition, these allogeneic reactions are due to disparities in minor histocompatibility Ags (minor HAs). Minor HAs are presented to T lymphocytes as peptides bound to HLA molecules, and appear to be encoded by genes throughout the genome. We derived T lymphocyte clones from the PBL of a patient suffering from chronic graft-vs-host disease after bone marrow transplant from his HLA-identical sister. Clones reactive against minor HAs were selected on the basis of reactivity with pretransplant patient cells, and absence of reactivity with donor cells. One clone (MD2) was found to use HLA-B7 as a restricting element. A plasmid vector (pHEBo) containing cDNA encoding the HLA-B7 molecule was transfected into lymphoblastoid cell lines derived from two large families that previously had been saturation mapped for hundreds of polymorphic loci. When clone MD2 was tested against family K1362, it was found to be reactive with three of four grandparents, both parents, and eight of eleven offspring. The same clone was tested with family K1331, with two of three tested grandparents reactive, one of two parents, and nine of eleven offspring. Computer analysis showed that both family segregation patterns linked to an area on the long arm of chromosome 22, localizing the gene encoding this minor HA near the platelet-derived growth factor-beta and IL-2Rbeta genes.

The Severe Combined Immunodeficient-Human Peripheral Blood Stem Cell (SCID-huPBSC) Mouse: A Xenotransplant Model for huPBSC-Initiated Hematopoiesis

Mononuclear cells (MNCs) containing peripheral blood stem cells (PBSCs) were obtained from solid-tumor patients undergoing mobilizing chemotherapy followed by granulocyte colony-stimulating factor for PBSC transplantation-supported dose-intensified anticancer chemotherapy and were transplanted into unconditioned "nonleaky" young severe combined immunodeficient mice. Multilineage engraftment was shown by flow cytometry and immunocytochemistry using monoclonal antibodies to various human cell surface antigens as well as identification of human immunoglobulin in murine sera. Within a dose range of MNCs suitable for transplantation (10 to 36 x 10(6) cells/graft) the number of CD34+ cells injected (optimal at > 0.7 x 10(6)/graft) determined the yield of human cells produced in recipient animals. Engraftment of hu PBSC preparations resulted in prolonged generation of physiologic levels of human cytokines including interleukin-3 (IL-3), IL-6, and granulocyte-macrophage colony-stimulating factor, which were detectable in the murine blood over a period of at least 4 months. In vivo survival of immature human progenitor cells was preserved even 9 months after transplantation. Because human IL-3 is known to stimulate early hematopoiesis, a rat fibroblast cell line was stably transfected with a retroviral vector carrying the human IL-3 gene and cotransplanted subcutaneously as additional source of growth factor. Cotransplants of this cell line producing sustained in vivo levels of circulating human IL-3 for at least 12 weeks significantly accelerated the process of engraftment of huPBSC and spurred the spread of mature human cells to the murine spleen, liver, thymus, and peripheral blood. Cotransplants of allogeneic human bone marrow stromal cells derived from long-term cultures resulted in a comparable--though less prominent--support of engraftment.

Cytokine gene expression after allogeneic bone marrow transplantation.

Cytokines produced by T lymphocytes, monocytes/macrophages, and fibroblasts play a central role in the immune response and in the development of graft-versus-host disease (GVHD). Also, it has been reported that dysregulated production of cytokines maybe the primary mediator of clinical manifestation of acute GVHD. Regarding cytokine gene expression after human allogeneic bone marrow transplantation (allo BMT), we have demonstrated increased IL-1 beta, IL-6, and TNF-alpha mRNA expression in peripheral blood mononuclear cells during the development of acute and chronic GVHD and that the degree of the increase was dependent on the severity of the disease. Furthermore, overexpression of these cytokine mRNAs could be detected before the clinical manifestations of GVHD developed. In contrast, IL-2 mRNA expression was not detected in peripheral blood mononuclear cells in GVHD patients. On the other hand, we have reported that increased mRNA expression and protein product of IL-2 and IFN-gamma were evident in the mixed lymphocyte culture of the cases who developed severe lethal transplantation-related complications. Therefore, the detection of increased IL-2 and IFN-gamma gene expression in MLC appeared to be useful for predicting transplantation-related complications in BMT patients. Furthermore, we found increased IL-2 receptor alpha subunit mRNA expression in the peripheral blood mononuclear cells during GVHD. These findings may indicate the important role of inflammatory cytokines such as IL-1 beta, IL-6 and TNF-alpha in the development of the clinical manifestation of GVHD and also may be indicative of the important role of IL-2 and the IL-2 receptor in allo response perhaps mainly as an autocrine effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytotoxic T-lymphocyte response to cytomegalovirus after human allogeneic bone marrow transplantation: pattern of recovery and correlation with cytomegalovirus infection and disease

The high rate of severe cytomegalovirus (CMV) disease after bone marrow transplantation (BMT) is related to the profound immunodeficiency posttransplant. Because cytotoxic T lymphocytes (CTL) have been implicated in resistance to viral infections, we examined the restoration of the CMV-specific CTL response in 20 patients who received bone marrow from HLA-matched, CMV-seropositive donors. Blood specimens were obtained from patients at 1, 2, and 3 months after BMT and from the donors on a single occasion. Peripheral blood mononuclear cells were cocultured with CMV-infected donor-derived fibroblasts for 2 weeks and then tested for cytotoxicity against CMV-infected and uninfected autologous or HLA-mismatched fibroblasts. Cytolytic activity was detected in all 20 donors, with specificity for autologous CMV- infected targets demonstrable in 17 (median CMV-specific lysis at an effector:target ratio of 15:1 was 32%, range 18% to 83%). Specific lysis was mediated by CD8+, class I-restricted T cells, as shown by inhibition with anti-class I monoclonal antibody and by selective depletion of effector cells. By contrast, CMV-specific CTL were detected in only 10 of 20 patients after BMT (median lysis 29% at 3 months post-BMT). None of these 10 patients developed CMV pneumonia, whereas 6 of the 10 patients with an undetectable CMV-specific CTL response after BMT died with CMV pneumonia. These results demonstrate that restoration of CMV-specific CTL responses may require an extended time period after BMT in some patients, and that such patients are at increased risk of developing severe CMV disease. Approaches to reconstitute CMV immunity in BMT patients by adoptive transfer of CMV- specific CD8+ CTL clones derived from the bone marrow donor are now being pursued.