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1.  Prolonged impairment of very late activating antigen-mediated T cell proliferation via the CD3 pathway after T cell-depleted allogeneic bone marrow transplantation. 
Journal of Clinical Investigation  1994;94(2):481-488.
One of the major obstacles in allogeneic bone marrow transplantation (allo-BMT) is prolonged T cell dysfunction resulting in a variety of infectious complications in the months to years after hematologic engraftment. We previously showed that immobilized extracellular matrix (ECM) proteins such as fibronectin (FN), the CS-1 domain of FN, or collagen (CO) acted synergistically with immobilized anti-CD3 to induce T cell proliferation. In addition, the comitogenic effect of ECMs could be mimicked by immobilized mAb reactive with a common beta 1 chain (CD29) of very late activating (VLA) antigens which include ECM receptors. Since the interaction of T cells with ECMs appears to play an important role in the process of T cell reconstitution following allo-BMT, we examined the expression of VLA antigens (alpha 1-alpha 6, beta 1) and their functional roles in CD3-mediated T cell proliferation at various times after T cell depleted allo-BMT. VLA beta 1 as well as VLA alpha 4, alpha 5, and alpha 6 expression was lower than normal controls during the first 3 mo after allo-BMT and auto-BMT, whereas these expressions returned to normal levels by 4 mo after allo-BMT and auto-BMT. Although alpha 1 and alpha 2 were not expressed on lymphocytes from normal controls, these antigens were expressed on lymphocytes at the detectable levels (5-15%) from patients after allo-BMT and auto-BMT. Both CD29 and CD3 were expressed at normal levels on lymphocytes from patients > 3 mo after allo-BMT, whereas T cell interaction with ECM through VLA proteins or crosslinking of VLA beta 1 expressed by T cells with anti-CD29 mAb results in poor induction of CD3-mediated T cell proliferation for a prolonged period (> 1 yr) after allo-BMT. In contrast, T cell proliferation induced by crosslinking of anti-CD2 or anti-CD26 with anti-CD3 was almost fully recovered by 1 yr post-allo-BMT. After autologous BMT, impaired VLA-mediated T cell proliferation via the CD3 pathway after auto-BMT returned to normal levels within 1 yr despite no significant difference in CD3 and CD29 expression following either allo- or auto-BMT. The adhesion of T cells from post-allo-BMT patients to FN-coated plate was normal or increased compared to that of normal controls. Moreover, the induction of the tyrosine phosphorylation of pp105 protein by the ligation of VLA molecules was not impaired in allo-BMT patients. These results suggest that there are some other defects in the process of VLA-mediated signal transduction in such patients. Our results imply that disturbance of VLA function could explain, at least in part, the persistent immunoincompetent state after allo-BMT and may be involved in susceptibility to opportunistic infections after allo-BMT.
PMCID: PMC295109  PMID: 7518837
2.  Novel role for surfactant protein A in gastrointestinal graft-versus-host disease 
Graft-versus-host-disease (GVHD) is a severe and frequent complication of allogeneic bone marrow transplantation (BMT) that involves the gastrointestinal tract and lungs. The pathobiology of GVHD is complex and involves immune cell recognition of host antigens as foreign. We hypothesize a central role for the collectin surfactant protein A (SP-A) in regulating the development of GVHD after allogeneic BMT.
C57BL/6 (H2b; WT) and SP-A deficient mice on C57BL/6 background (H2b; SP-A−/−) mice underwent allogeneic (Allo) or syngeneic (Syn) BMT with cells from either C3HeB/FeJ (H2k; SP-A−/−alloBMT or WTalloBMT) or C57Bl/6 (H2b; SP-A−/−synBMT or WTsynBMT) mice. 5 weeks post BMT, mice were necropsied and lung and gastrointestinal (GI) tissue were analyzed.
SP-A−/−alloBMT or WTalloBMT had no significant differences in lung pathology however, SP-A−/−alloBMT mice developed marked features of GI GVHD including decreased body weight, increased tissue inflammation and lymphocytic infiltration. SP-A−/−alloBMT mice also had increased colon expression of IL-1β, IL-6, TNF-α, and IFN-γ and as well as increased Th17 cells, and diminished regulatory T (Treg) cells.
Our results demonstrate the first evidence of a critical role for SP-A in modulating GI GVHD. In these studies, we demonstrate that mice deficient in SP-A that have undergone an alloBMT have a greater incidence of GI GVHD that is associated with increased Th17 cells and decreased Tregs. The results of these studies demonstrate that SP-A protects against the development of GI GVHD and establishes a role for SP-A in regulating the immune response in the GI tract.
PMCID: PMC3373011  PMID: 22508928
Graft-versus-host disease; Surfactant Protein A; Th17; Regulatory T cells
3.  The critical early proinflammatory events associated with idiopathic pneumonia syndrome in irradiated murine allogeneic recipients are due to donor T cell infusion and potentiated by cyclophosphamide. 
Journal of Clinical Investigation  1997;100(5):1015-1027.
We have hypothesized that lung damage occurring in the peri-bone marrow transplant (BMT) period is critical for the subsequent generation of idiopathic pneumonia syndrome (IPS), a major complication following human BMT. The proinflammatory events induced by a common pre-BMT conditioning regimen, cyclophosphamide (Cytoxan(R)) (Cy) and total body irradiation, were analyzed in a murine BMT model. Electron microscopy indicated that Cy exacerbated irradiation-induced epithelial cell injury as early as day 3 after BMT. Allogenicity was an important contributing factor to lung injury as measured by lung wet and dry weights and decreased specific lung compliance. The most significant pulmonary dysfunction was seen in mice receiving both allogeneic T cells and Cy conditioning. IPS was associated with an influx of T cells, macrophages, and neutrophils early post-BMT. Hydroxyproline levels were not increased, indicating that the injury was not fibrotic early post-BMT. As early as 2 h after chemoradiation, host macrophages increased in number in the lung parenchyma. Continued increases in macrophages occurred if splenic T cells were administered with the donor graft. The expression of costimulatory B7 molecules correlated with macrophage numbers. Frequencies of cells expressing mRNA for the inflammatory proteins TNF-alpha, IL-1beta, and TGFbeta were increased. Cy accelerated the upregulation of TGFbeta and increase in host macrophages. The exacerbation of macrophage activation and severity of IPS was dependent on allogeneic T cells, implicating immune-mediated mechanisms as critical to the outcome of IPS. This demonstration of early injury after BMT indicates the need for very early therapeutic intervention before lung damage becomes profound and irreversible.
PMCID: PMC508276  PMID: 9276718
4.  Serum amyloid A protein concentration in bone marrow transplantation for beta thalassaemia. 
Journal of Clinical Pathology  1992;45(4):348-351.
AIMS: To investigate whether serum amyloid A protein (SAA) and C-reactive protein (CRP) concentrations could be used in the management of beta thalassaemic patients undergoing bone marrow transplantation (BMT). METHODS: Serum SAA and CRP concentrations were determined in paired samples from 66 patients with beta thalassaemia before and after BMT. Serum SAA concentrations were determined by an enzyme linked immunoassay (EIA); serum CRP concentrations were determined by a nephelometric assay. RESULTS: Serum SAA concentrations before transplantation were significantly higher in the group that subsequently rejected the transplant than the group without complications. SAA concentrations increased after BMT in acute graft versus host disease (GvHD) and rejection. No significant increase in SAA or CRP was found in chronic GvHD. Increases in serum in SAA and CRP concentrations were not related to concomitant infection episodes. CONCLUSIONS: The different acute phase response in acute GvHD and rejection compared with chronic GvHD suggests that different immunopathogenic mechanisms are responsible.
PMCID: PMC495278  PMID: 1577974
5.  Tumor necrosis factor- alpha production to lipopolysaccharide stimulation by donor cells predicts the severity of experimental acute graft-versus-host disease. 
Journal of Clinical Investigation  1998;102(10):1882-1891.
Donor T cell responses to host alloantigen are known predictors for graft-versus-host disease (GVHD); however, the effect of donor responsiveness to an inflammatory stimulus such as lipopolysaccharide (LPS) on GVHD severity has not been investigated. To examine this, we used mouse strains that differ in their sensitivity to LPS as donors in an experimental bone marrow transplant (BMT) system. Lethally irradiated (C3FeB6)F1 hosts received BMT from either LPS-sensitive (LPS-s) C3Heb/Fej, or LPS-resistant (LPS-r) C3H/ Hej donors. Mice receiving LPS-r BMT developed significantly less GVHD as measured by mortality and clinical score compared with recipients of LPS-s BMT, a finding that was associated with significant decreases in intestinal histopathology and serum LPS and TNF-alpha levels. When donor T cell responses to host antigens were measured, no differences in proliferation, serum IFN-gamma levels, splenic T cell expansion, or CTL activity were observed after LPS-r or LPS-s BMT. Systemic neutralization of TNF-alpha from day -2 to +6 resulted in decreased intestinal pathology, and serum LPS levels and increased survival after BMT compared with control mice receiving Ig. We conclude that donor resistance to endotoxin reduces the development of acute GVHD by attenuating early intestinal damage mediated by TNFalpha. These data suggest that the responsiveness of donor accessory cells to LPS may be an important risk factor for acute GVHD severity independent of T cell responses to host antigens.
PMCID: PMC509139  PMID: 9819375
6.  Neonatal bone marrow transplantation for severe combined immunodeficiency 
AIMS—To evaluate outcome following neonatal bone marrow transplantation (BMT) for severe combined immunodeficiency (SCID) when there is a family history of a previously affected sibling, and to compare results with those published for in utero BMT.
METHODS—A retrospective review of cases referred and transplanted between 1987 and 1999, focusing on infectious and graft versus host disease (GvHD) complications after BMT, and T and B lymphocyte function. Thirteen patients received 18 stem cell transplants: four whole marrow, one cord blood, 10 parental T cell depleted (TCD) haplo-identical, and three TCD unrelated donor BMT. Nine were conditioned with busulphan and cyclophosphamide.
RESULTS—All are alive and well (six months to 11.5 years after BMT). Six had grade I-II acute GvHD and two chronic GvHD (now resolved). Three had a top up BMT for poor T cell function, one had a third BMT for graft failure and chronic GvHD, and one had a third BMT for graft failure. Twelve have good in vitro proliferation to T cell mitogens, and all have normal serum IgA levels. Three receive intravenous immunoglobulin; for one of these, it is less than one year since BMT. Nine are above the 2nd centile, and 10 of 12 old enough to be assessed have normal neurodevelopment.
CONCLUSION—These results are better than those published for in utero BMT for SCID. Early postnatal BMT should be the preferred option in neonatal SCID.

PMCID: PMC1721317  PMID: 11517204
7.  Thiol/Redox Metabolomic Profiling Implicates GSH Dysregulation in Early Experimental Graft versus Host Disease (GVHD) 
PLoS ONE  2014;9(2):e88868.
Graft-versus-host disease (GVHD) is a common complication of allogeneic bone marrow transplantation (BMT). Upregulation of inflammatory cytokines precedes the clinical presentation of GVHD and predicts its severity. In this report, thiol/redox metabolomics was used to identify metabolic perturbations associated with early preclinical (Day+4) and clinical (Day+10) stages of GVHD by comparing effects in Syngeneic (Syn; major histocompatibility complex- identical) and allogeneic transplant recipients (Allo BMT) in experimental models. While most metabolic changes were similar in both groups, plasma glutathione (GSH) was significantly decreased, and GSH disulfide (GSSG) was increased after allogeneic compared to syngeneic recipient and non-transplant controls. The early oxidation of the plasma GSH/GSSG redox couple was also observed irrespective of radiation conditioning treatment and was accompanied by significant rise in hepatic protein oxidative damage and ROS generation. Despite a significant rise in oxidative stress, compensatory increase in hepatic GSH synthesis was absent following Allo BMT. Early shifts in hepatic oxidative stress and plasma GSH loss preceded a statistically significant rise in TNF-α. To identify metabolomic biomarkers of hepatic GVHD injury, plasma metabolite concentrations analyzed at Day+10 were correlated with hepatic organ injury. GSSG (oxidized GSH) and β-alanine, were positively correlated, and plasma GSH cysteinylglycine, and branched chain amino acids were inversely correlated with hepatic injury. Although changes in plasma concentrations of cysteine, cystathionine (GSH precursors) and cysteinylglycine (a GSH catabolite) were not significant by univariate analysis, principal component analysis (PCA) indicated that accumulation of these metabolites after Allo BMT contributed significantly to early GVHD in contrast to Syn BMT. In conclusion, thiol/redox metabolomic profiling implicates that early dysregulation of host hepatic GSH metabolism and oxidative stress in sub-clinical GVHD before elevated TNF-α levels is associated with GVHD pathogenesis. Future studies will probe the mechanisms for these changes and examine the potential of antioxidant intervention strategies to modulate GVHD.
PMCID: PMC3928313  PMID: 24558439
8.  Differential roles of IL-1 and TNF-α on graft-versus-host disease and graft versus leukemia 
Journal of Clinical Investigation  1999;104(4):459-467.
We demonstrate an increase in graft-versus-host disease (GVHD) after experimental bone marrow transplant (BMT) when cyclophosphamide (Cy) is added to an otherwise well-tolerated dose (900 cGy) of total body irradiation (TBI). Donor T cell expansion on day +13 was increased after conditioning with Cy/TBI compared with Cy or TBI alone, although cytotoxic T lymphocyte (CTL) function was not altered. Histological analysis of the gastrointestinal tract demonstrated synergistic damage by Cy/TBI and allogeneic donor cells, which permitted increased translocation of LPS into the systemic circulation. TNF-α and IL-1 production in response to LPS was increased in BMT recipients after Cy/TBI conditioning. Neutralization of IL-1 significantly reduced serum LPS levels and GVHD mortality, but it did not affect donor CTL activity. By contrast, neutralization of TNF-α did not prevent GVHD mortality but did impair CTL activity after BMT. When P815 leukemia cells were added to the bone marrow inoculum, allogeneic BMT recipients given the TNF-α inhibitor relapsed at a significantly faster rate than those given the IL-1 inhibitor. To confirm that the role of TNF-α in graft versus leukemia (GVL) was due to effects on donor T cells, cohorts of animals were transplanted with T cells from either wild-type mice or p55 TNF-α receptor–deficient mice. Recipients of TNF-α p55 receptor–deficient T cells demonstrated a significant impairment in donor CTL activity after BMT and an increased rate of leukemic relapse compared with recipients of wild-type T cells. These data highlight the importance of conditioning in GVHD pathophysiology, and demonstrate that TNF-α is critical to GVL mediated by donor T cells, whereas IL-1 is not.
J. Clin. Invest. 104:459-467 (1999).
PMCID: PMC408528  PMID: 10449438
9.  Antigenaemia during acute graft versus host disease. 
Journal of Clinical Pathology  1995;48(2):105-109.
AIMS--Animal studies have shown that antigens present within the gut play an important role in the development of acute graft versus host disease (GvHD) following allogeneic bone marrow transplantation (BMT). In previous studies, inert sugars have been found to penetrate the small bowel mucosa after conditioning therapy for BMT; endotoxaemia can also occur during acute GvHD. Data on absorption of antigenic proteins across the gut following BMT in humans have not been presented as yet. METHODS--Six patients undergoing allogeneic BMT were studied to determine whether enteric ovalbumin absorption increased or endotoxaemia developed during acute GvHD. RESULTS--Three patients had minimal antigenaemia and no detectable endotoxaemia before receiving conditioning therapy. At the onset of acute GvHD, however, much higher ovalbumin concentrations were detected in those patients with severe antigenaemia. Serum concentrations of specific antiovalbumin IgG and IgA, or antiendotoxin IgM or IgG had no bearing on detectable IgG or IgM ovalbumin or endotoxin concentrations. In five of six patients, small bowel permeability increased, as tested by the lactulose/mannitol sugar absorption test, but detectable ovalbumin absorption increased in only three of these and only two developed endotoxaemia. CONCLUSIONS--Antigens present within the gut can cross the mucosal epithelium during acute GvHD, probably resulting in an enhanced immune response.
PMCID: PMC502370  PMID: 7745106
10.  Pulmonary, Gonadal, and Central Nervous System Status after Bone Marrow Transplantation for Sickle Cell Disease 
We conducted a prospective, multicenter investigation of human-leukocyte antigen (HLA) identical sibling bone marrow transplantation (BMT) in children with severe sickle cell disease (SCD) between 1991 and 2000. To determine if children were protected from complications of SCD after successful BMT, we extended our initial study of BMT for SCD to conduct assessments of the central nervous system (CNS) and of pulmonary function 2 or more years after transplantation. In addition, the impact on gonadal function was studied. After BMT, patients with stroke who had stable engraftment of donor cells experienced no subsequent stroke events after BMT, and brain magnetic resonance imaging (MRI) exams demonstrated stable or improved appearance. However, 2 patients with graft rejection had a second stroke after BMT. After transplantation, most patients also had unchanged or improved pulmonary function. Among the 11 patients who had restrictive lung changes at baseline, 5 were improved and 6 had persistent restrictive disease after BMT. Of the 2 patients who had obstructive changes at baseline, 1 improved and 1 had worsened obstructive disease after BMT. There was, however, significant gonadal toxicity after BMT, particularly among female recipients. In summary, individuals who had stable donor engraftment did not experience sickle-related complications after BMT, and were protected from progressive CNS and pulmonary disease.
PMCID: PMC2919571  PMID: 19822218
Sickle cell anemia; Bone marrow transplantation; Long-term follow-up
11.  Comparable Outcomes for Hematologic Malignancies after HLA-Haploidentical Transplantation with Posttransplantation Cyclophosphamide and HLA-Matched Transplantation 
Advances in Hematology  2015;2015:431923.
The implementation of high-dose posttransplantation cyclophosphamide (PTCy) has made HLA-haploidentical (haplo) blood or marrow transplantation (BMT) a cost effective and safe alternative donor transplantation technique, resulting in its increasing utilization over the last decade. We review the available retrospective comparisons of haplo BMT with PTCy and HLA-matched BMT in adults with hematologic malignancies. The examined studies demonstrate no difference between haplo BMT with PTCy and HLA-matched BMT with regard to acute graft-versus-host disease (aGVHD), nonrelapse mortality, and overall survival. Chronic GVHD occurred less frequently after haplo BMT with PTCy compared with HLA-matched BMT utilizing standard GVHD prophylaxis. In addition, patients with a high risk of relapse by the disease risk index had a suggestion of improved progression-free and overall survival after haplo BMT with PTCy when compared with a historical cohort of HLA-matched BMT in one analysis. Furthermore, in Hodgkin lymphoma relapse and progression-free survival were improved in the haplo BMT with PTCy compared with the HLA-matched BMT cohort. These findings support the use of this transplantation platform when HLA-matched related donors (MRDs) are unavailable and suggest that clinical scenarios exist in which haplo BMT may be preferred to HLA-matched BMT, which warrant further investigation.
PMCID: PMC4680052  PMID: 26713094
12.  Procalcitonin Does Not Discriminate Infection from Inflammation after Allogeneic Bone Marrow Transplantation 
Procalcitonin (PCT) is an early marker of bacterial infection but little is known about its value in neutropenic allogeneic bone marrow transplant (BMT) recipients. We collected plasma from 12 recipients of T-cell-depleted HLA-matched related BMT recipients who had been treated preemptively with meropenem from the day after BMT for at least 15 days. PCT and C-reactive protein (CRP) concentrations were determined on BMT days 1, 5, 8, 12, and 15, and their relationship to inflammatory events (IE), including mucositis, microbiologically and clinically defined infections, acute graft-versus-host disease (GVDH), and unexplained fever, was then determined. The PCT concentrations were all low and never exceeded 4 μg/liter, unlike CRP concentrations, which spanned the full range up to 350 mg/liter. All patients had mucositis, and there was no significant difference between PCT concentrations associated with mucositis alone and those associated with an additional IE on BMT days 1 to 12. However, on BMT day 15, the mean concentrations of PCT were 0.37 ± 0.05 μg/liter for the 10 patients that had an additional IE, compared with 0.11 ± 0.03 μg/liter for the 2 patients with mucositis only (P = 0.012), and GVHD rather than infection was involved in six cases. PCT was also not a sensitive marker of gram-positive bacteremia or pulmonary aspergillosis. Thus, PCT is of little value in discriminating infections from other inflammatory complications that occur following allogeneic BMT.
PMCID: PMC95980  PMID: 11063493
13.  Growth and growth hormone secretion after bone marrow transplantation. 
Archives of Disease in Childhood  1993;68(4):458-463.
This study analyses the growth and the growth hormone secretion of children given various conditioning protocols before bone marrow transplantation (BMT). Twenty nine children (14 boys, 15 girls) given BMT were classified according to their conditioning protocol: total body irradiation (TBI) given as a single exposure of 10 Grays (Gy, group I, 11 cases), or 8 Gy (group II, four cases), 12 Gy given as six fractionated doses (Group III, seven cases), or chemotherapy alone (group IV, seven cases). The arginine-insulin stimulated growth hormone peak, 2-7.5 years after BMT, was > 10 micrograms/l in all patients except four from group I (6.9-8.9 micrograms/l). A second growth hormone secretion evaluation was performed in 10 group I patients because of persistent low growth velocity despite a normal growth hormone peak. There were no significant changes in the mean (SEM) stimulated growth hormone peak (18.4 (2.2) v 20.1 (3.6) micrograms/l) at 3 (0.3) to 5.2 (0.6) years after BMT. The sleep growth hormone peaks and concentrations (n = 6) were normal. The mean cumulative height changes (SD) during the three years after BMT were: -1.4 (0.2) in group I, -0.1 (0.4) in group II, -0.4 (0.2) in group III, and 1.5 (0.5) in group IV; this was significant in groups I and IV. The final heights of two monozygotic twins (BMT donor and recipient) had differed by 17.5 cm, despite them both having normal growth hormone peaks and puberty. Eight patients, treated for congenital immune deficiency syndrome, were growth retarded at the time of BMT. Of these, only those conditioned by chemotherapy alone had significant catch up growth (2(0.6)SD) while those conditioned by a single Gy exposure did not (0(0.4)SD). It is concluded that the total radiation dose is critical for growth evolution, as is the fractionation schedule. For the TBI doses and the interval since BMT studied, there was no correlation between growth hormone peak and the height loss. The rapidity of decreased growth velocity after TBI and the comparison between the monozygotic twins suggest that radiation induced skeletal lesions are partly responsible for the decreased growth.
PMCID: PMC1029264  PMID: 8503666
14.  Recipient Myeloid-derived Immunomodulatory Cells Induce PD-1 Ligand-Dependent Donor CD4+Foxp3+ Treg Proliferation and Donor-Recipient Immune Tolerance After Murine Non-myeloablative Bone Marrow Transplantation§ 
Journal of immunology (Baltimore, Md. : 1950)  2013;191(11):10.4049/jimmunol.1302191.
We have previously shown that non-myeloablative total lymphoid irradiation/rabbit anti-thymocyte serum (TLI/ATS) conditioning facilitates potent donor-recipient immune tolerance following bone marrow transplantation (BMT) across major histocompatibility complex (MHC) barriers via recipient invariant natural killer T cell (iNKT cell)-derived IL-4-dependent expansion of donor Foxp3+ naturally occurring Treg (nTreg). Here we report a more specific mechanism. Wild-type (WT) BALB/c (H-2d) hosts were administered TLI/ATS and BMT from WT or STAT6−/− C57BL/6 (H-2b) donors. Donor nTreg following STAT6−/− BMT demonstrated no loss of proliferation in vivo, indicating that an IL-4 responsive population in the recipient rather than the donor drives donor nTreg proliferation. In GVHD target organs, three recipient CD11b+ cell subsets (Gr-1highCD11cneg; Gr-1intCD11cneg; and Gr-1lowCD11c+) were enriched early after TLI/ATS + BMT versus TBI/ATS + BMT. Gr-1lowCD11c+ cells induced potent H-2Kb+CD4+Foxp3+ nTreg proliferation in vitro in 72-hr MLR. Gr-1lowCD11c+ cells were significantly reduced in STAT6−/− and iNKT cell-deficient Jα18−/− BALB/c recipients after TLI/ATS + BMT. Depletion of CD11b+ cells resulted in severe acute GVHD, and adoptive transfer of WT Gr-1lowCD11c+ cells to Jα18−/− BALB/c recipients of TLI/ATS + BMT restored day 6 donor Foxp3+ nTreg proliferation and protection from CD8 effector T cell-mediated GVHD. Blockade of PD-L1 or PD-L2, but not CD40, TGF-β, Arginase 1, or iNOS inhibited nTreg proliferation in co-cultures of recipient-derived Gr-1lowCD11c+ cells with donor nTreg. Through iNKT-dependent Th2 polarization, myeloid-derived immunomodulatory DCs are expanded after non-myeloablative TLI/ATS conditioning and allogeneic BMT, induce PD-1 ligand dependent donor nTreg proliferation, and maintain potent graft-versus-host immune tolerance.
PMCID: PMC3863612  PMID: 24190658
Graft-versus-host disease; transplantation; tolerance; T cells; monocytes
15.  Strategic nonmyeloablative conditioning: CD154:CD40 co-stimulatory blockade at primary BMT promotes engraftment for secondary BMT after engraftment failure1 
There is an increased risk of failure of engraftment following nonmyeloablative conditioning. Sensitization resulting from failed bone marrow transplantation (BMT) remains a major challenge for secondary BMT. Approaches to allow successful retransplantation would have significant benefits for BMT candidates living with chronic diseases. We used a mouse model to investigate the effect of preparative regimens at primary BMT on outcome for secondary BMT. We found that conditioning with TBI or recipient T-cell lymphodepletion at primary BMT did not promote successful secondary BMT. In striking contrast, successful secondary BMT could be achieved in mice conditioned with anti-CD154 co-stimulatory molecule blockade at first BMT. Blockade of CD154 alone or combined with T-cell depletion inhibits generation of the humoral immune response after primary BMT as evidenced by abrogation of production of anti-donor Abs. The humoral barrier is dominant in sensitization resulting from failed BMT, as almost all CFSE-labeled donor cells were killed at 0.5 and 3 hr in sensitized recipients in in vivo cytotoxicity assay, reflecting antibody-mediated cytotoxicity. CD154:CD40 co-stimulatory blockade used at primary BMT promotes allogeneic engraftment in secondary BMT after engraftment failure at first BMT. The prevention of generation of anti-donor Abs at primary BMT is critical for successful secondary BMT.
PMCID: PMC2767528  PMID: 18941252
16.  Hemopoietic colony growth-promoting activities in the plasma of bone marrow transplant recipients. 
Journal of Clinical Investigation  1988;82(1):255-261.
Plasma samples were obtained from 34 bone marrow transplant (BMT) recipients before and after administration of the preparative regimen and tested for their ability to promote and/or support growth of hemopoietic colonies. The ability of plasma samples to promote colony formation on their own was tested on normal nonadherent target cells without addition of exogenous growth factors. The growth-supporting activity was examined in the presence of medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM) and/or erythropoietin (EPO). A series of kinetic changes was routinely observed. Pretransplant samples rarely gave rise to colonies without addition of exogenous growth factors. Plasma samples obtained after completion of the preparative regimen demonstrated increments of growth-promoting activities for megakaryocyte and granulocyte-macrophage progenitors (CFU-Meg and CFU-GM), respectively, that peaked between 7 and 21 d after transplantation. By day 30, activity levels of some patients had returned to pretransplant values, whereas in other patients, activities remained elevated. Persisting activity levels were associated with delayed engraftment. In contrast, activities for progenitors committed to erythropoiesis (BFU-E) and pluripotent precursors (CFU-GEMM) were only rarely observed. The activities were independent of febrile episodes. Their growth-promoting influence on CFU-GM could be neutralized completely by anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) antibodies. These data suggest that at least some of the observed activities in post-BMT plasma are related to GM-CSF. The growth-supporting activities of pretransplant plasma samples are lower than normal plasma when tested on CFU-Meg and CFU-GM. The growth-supporting activities improved transiently within the first month after BMT. A decline during the second and third month was followed by a gradual return to activity levels that were comparable to normal plasma. The effects of these plasma samples on BFU-E and CFU-GEMM were assessed with PHA-LCM and EPO. Similar to CFU-Meg- and CFU-GM-supporting capabilities, they improved transiently after BMT with a return of normal support function after 5-6 mo. The observed endogenous production of growth-promoting and growth-supporting activities for hemopoietic progenitors may serve as a background to design clinical trials for the timely administration of recombinant hemopoietic growth factors to BMT recipients.
PMCID: PMC303502  PMID: 3292585
17.  Effects of Mixed Chimerism and Immune Modulation on GVHD, Disease Recurrence and Survival after HLA-identical Marrow Transplantation for Hematologic Malignancies 
The success of allogeneic bone marrow transplantation (allo-BMT) is affected by underlying disease relapse. Although mixed chimerism (MC) is not necessarily a poor prognostic factor, several groups have suggested that MC is associated with an increased risk of disease relapse. There is evidence that patients with MC benefit from additional immunotherapy if the treatment is started in minimal residual disease status (mixed chimerism status), not in frank hematological relapse. The purposes of this study are to evaluate 1) the risk for relapse or graft rejection in correlation to persistent MC status after allo-BMT, and 2) the possibility of preventing relapse by immune modulation treatments (withdrawal or rapid taper-off of post-transplant immuno-suppression, additional interferon treatment, or the administration of donor lymphocytes) in hematologic malignancies.
Patients and Methods
Of 337 allogeneic donor-recipient pairs between March 1996 and August 1998, 12 patients who showed persistent or progressive MC and who received immune modulation treatments were evaluated. Twelve patients, median age 31 years (range 9 to 39 years), received an allo-BMT for: acute myelogenous leukemia (AML, n = 5), chronic myelogenous leukemia (CML, n = 4), acute lymphocytic leukemia (ALL, n = 3). Serial polymerase chain reaction (PCR) analysis of YNZ 22-, 33.6-minisatellites or Y chromosome-specific PCR analysis at short term intervals (pre-and post-transplant 1, 3, 6, 9, … months) was performed. Once MC was detected, immune modulation treatments on the basis of increasing MC in an early phase of recurrence of underlying disease were started.
Nine of 12 patients converted to complete chimerism (CC) (AML 5/5, CML 3/4, ALL 1/3). Four of 9 CC patients developed graft-versus-host disease (GVHD) grade ≤2 during immune modulation. All were treated successfully with steroids. Three patients who were not converted to CC showed relapse of underlying diseases or graft failure.
The results demonstrate that, in patients with hematologic malignancies after allo-BMT, persistent MC is associated with relapse of underlying diseases or graft failure. Furthermore, when patients receive early immune modulation treatment, MC can be changed to complete donor pattern chimerism and ultimately prevent relapse.
PMCID: PMC4531771  PMID: 11242811
mixed chimerism; complete chimerism; allogeneic bone marrow transplantation; graft-versus-host disease
18.  Tumor Control in a Model of Bone Marrow Transplantation and Acute Liver-Infiltrating B-Cell Lymphoma: an Unpredicted Novel Function of Cytomegalovirus 
Journal of Virology  2002;76(6):2857-2870.
Tumor relapse and cytomegalovirus (CMV) infection are major concerns in the therapy of hematopoietic malignancies by bone marrow transplantation (BMT). Little attention so far has been given to a possible pathogenetic interplay between CMV and lymphomas. CMV inhibits stem cell engraftment and hematopoietic reconstitution. Thus, by causing maintenance of bone marrow aplasia and immunodeficiency, CMV could promote tumor relapse. Alternatively, CMV could aid tumor remission. One might think of cytopathogenic infection of tumor cells, induction of apoptosis or inhibitory cytokines, interference with tumor cell extravasation or tumor vascularization, or bystander stimulation of an antitumoral immune response. To approach these questions, the established model of experimental BMT and murine CMV infection was extended by the introduction of liver-infiltrating, highly tumorigenic variant clone E12E of BALB/c-derived B-cell lymphoma A20. We document a remarkable retardation of lymphoma progression. First-guess explanations were ruled out: (i) lymphoma cells were not infected; (ii) lymphoma cells located next to infected hepatocytes did not express executioner caspase 3 but were viable and proliferated; (iii) an inhibitory effect of virus on the formation of tumor nodules in the liver became apparent by day 7 after BMT, long before the reconstitution of immune cells; and (iv) recombinant tumor necrosis factor alpha (TNF-α) did not substitute for virus; accordingly anti-TNF-α did not prevent the inhibition. Notably, while the antitumoral effect required replicative virus, prevention of cytopathogenic infection of the liver by antiviral CD8 T cells did not abolish lymphoma control. These findings are paradigmatic for a novel virus-associated antitumoral mechanism distinct from oncolysis.
PMCID: PMC135996  PMID: 11861853
19.  The skeletal site-differential changes in bone mineral density following bone marrow transplantation: 3-year prospective study. 
Journal of Korean Medical Science  2002;17(6):749-754.
Loss of bone mass is usually detected after bone marrow transplantation (BMT) during the early post-transplant period. However, little is known about the long-term effects of BMT on bone metabolism. We have prospectively investigated 11 patients undergoing BMT. Bone mineral density (BMD) was measured before BMT, and 1, 2, and 3 yr after BMT. Serum markers of bone turnover were serially measured before BMT and 1, 2, 3, 4, and 12 weeks, 6 months, and 1 yr after BMT. The mean change in the lumbar spine (L2-4) BMD, calculated as the percent change from the baseline to the level at 1, 2, and 3 yr was -4.7% (NS), -1.1% (NS), and +6.4% (p<0.05), respectively. The mean change in the total proximal femur BMD from the baseline to the level at 1, 2, and 3 yr was -8.5% (p<0.01), -8.7% (p<0.05) and -5.6% (p<0.05), respectively. In summary, there was little decline in lumbar BMD at 1 yr following BMT and gradual recovery until 3 yr. In contrast, femoral BMD decreased much more than the lumbar area at 1 yr and did not recover until 3 yr. The mechanism of skeletal site-selective differences in the changes of BMD needs to be elucidated.
PMCID: PMC3054966  PMID: 12482996
20.  Specific immunosuppression by mixed chimerism with bone marrow transplantation after Staphylococcal Enterotoxin B pretreatment could prolong corneal allograft survival in mice 
Molecular Vision  2012;18:974-982.
We assessed the combined use of Staphylococcal Enterotoxin B (SEB) superantigen pre-treatment along with allogeneic bone marrow transplant (BMT) to induce immune suppression condition and inhibit corneal keratoplasty rejection in mice.
BALB/C (H-2d) mice were both BMT and corneal allografts donors and C57BL/6(H-2b) mice were recipients. Prior to BMT, recipients received single injections of either SEB, cyclophosphamide (CYP), or normal saline (NS). Allogenic corneal penetrating keratoplasty was performed 7 days after BMT. Bone marrow chimerisms in recipients (donor major histocompatibility complex-II H2-d) were determined on Days 14, 28, and 56 post-BMT. Recipient immune response was assessed by mixed lymphocyte reactions (MLR) using splenocytes from C57BL/6 mice as responders in co-culture with stimulator cells from C57BL/6 (isogeneic), BALB/C (allogeneic), or CBA/1(third party) mice. Cluster of differentiation 4 receptors positive (CD4+) and CD8+T cells in recipient mice were evaluated. Corneal graft survival was assessed using Kaplan–Meier survival curves.
SEB pre-treatment induced higher levels of hematopoietic chimerism on Days 14, 28 and 56 post-BMT than did CYP or NS pre-treatment. Mean corneal allograft survival was significantly prolonged with group SEB-BMT (20.3±7.6 days) compared to group CYP-BMT (13.0±4.0 days) and NS-BMT (9.0±2.2 days). SEB-BMT mice splenocytes had diminished MLR responses compared to CYP-BMT or NS-BMT mice. CD4+ and CD8+ T cells in peripheral blood and spleens were significantly reduced in group SEB-BMT mice.
BMT after SEB pre-treatment could promote mixed chimerism, which inhibited allogeneic cornea transplant rejection. This should possibly relate to CD4+ and CD8+ T cell deletion and acquiring donor-specific immunosuppression.
PMCID: PMC3339037  PMID: 22550390
21.  Less Graft-Versus-Host Disease after Rabbit Antithymocyte Globulin Conditioning in Unrelated Bone Marrow Transplantation for Leukemia and Myelodysplasia: Comparison with Matched Related Bone Marrow Transplantation 
PLoS ONE  2014;9(9):e107155.
One of the major drawbacks for unrelated donor (UD) bone marrow transplantation (BMT) is graft-versus-host disease (GVHD). Despite results from randomized trials, antithymocyte globulin (ATG) is not routinely included for GVHD prophylaxis in UD BMT by many centers. One of ways to demonstrate the usefulness of rabbit ATG in UD BMT is to evaluate how its results approximate to those observed in matched related (MRD) BMT. Therefore, we compared the outcomes between UD BMT with rabbit ATG (Thymoglobulin) for GVHD prophylaxis (n = 25) and MRD BMT (n = 91) for leukemia and myelodysplasia. All but one patient received a myeloablative conditioning regimen. Grades II–IV acute GVHD were similar (39.5% vs. 36%, p = 0.83); however, MRD BMT recipients developed more moderate-severe chronic GVHD (36.5% vs. 8.6%, p = 0.01) and GVHD-related deaths (32.5% vs. 5.6%, p = 0.04). UD BMT independently protected against chronic GVHD (hazard ratio 0.23, p = 0.04). The 6-month transplant-related mortality, 1-year relapse incidence, and 5-year survival rates were similar between patients with non-advanced disease in the MRD and UD BMT groups, 13.8% vs. 16.6% (p = 0.50), 20.8% vs. 16.6% (p = 0.37), and 57% vs. 50% (p = 0.67), respectively. Stable full donor chimerism was equally achieved (71.3% vs. 71.4%, p = 1). Incorporation of rabbit ATG in UD BMT promotes less GVHD, without jeopardizing chimerism evolution, and may attain similar survival outcomes as MRD BMT for leukemia and myelodysplasia especially in patients without advanced disease.
PMCID: PMC4154845  PMID: 25188326
22.  Bone Marrow Transplantation Improves Autoinflammation and Inflammatory Bone Loss in SH3BP2 Knock-In Cherubism Mice 
Bone  2014;71:201-209.
Cherubism (OMIM#118400) is a genetic disorder in children characterized by excessive jawbone destruction with proliferation of fibro-osseous lesions containing a large number of osteoclasts. Mutations in the SH3-domain binding protein 2 (SH3BP2) are responsible for cherubism. Analysis of the knock-in (KI) mouse model of cherubism showed that homozygous cherubism mice (Sh3bp2KI/KI) spontaneously develop systemic autoinflammation and inflammatory bone loss and that cherubism is a TNF-α-dependent hematopoietic disorder. In this study, we investigated whether bone marrow transplantation (BMT) is effective for the treatment of inflammation and bone loss in Sh3bp2KI/KI mice. Bone marrow (BM) cells from wild-type (Sh3bp2+/+) mice were transplanted to 6-week-old Sh3bp2KI/KI mice with developing inflammation and to 10-week-old Sh3bp2KI/KI mice with established inflammation. Six-week-old Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells exhibited improved body weight loss, facial swelling, and survival rate. Inflammatory lesions in the liver and lung as well as bone loss in calvaria and mandibula were ameliorated at 10 weeks after BMT compared to Sh3bp2KI/KI mice transplanted with Sh3bp2KI/KI BM cells. Elevation of serum TNF-α levels was not detected after BMT. BMT was effective for up to 20 weeks in 6-week-old Sh3bp2KI/KI mice transplanted with Sh3bp2+/+ BM cells. BMT also ameliorated the inflammation and bone loss in 10-week-old Sh3bp2KI/KI mice. Thus our study demonstrates that BMT improves the inflammation and bone loss in cherubism mice. BMT may be effective for the treatment of cherubism patients.
PMCID: PMC4274253  PMID: 25445458
Cherubism; SH3BP2; Autoinflammation; Bone loss; Bone marrow transplantation
23.  Immune Reconstitution Kinetics following Intentionally Induced Mixed Chimerism by Nonmyeloablative Transplantation 
PLoS ONE  2015;10(5):e0126318.
Establishing mixed chimerism is a promising approach for inducing donor-specific transplant tolerance. The establishment and maintenance of mixed chimerism may enable long-term engraftment of organ transplants while minimizing the use of immunosuppressants. Several protocols for inducing mixed chimerism have been reported; however, the exact mechanism underlying the development of immune tolerance remains to be elucidated. Therefore, understanding the kinetics of engraftment during early post-transplant period may provide insight into establishing long-term mixed chimerism and permanent transplant tolerance. In this study, we intentionally induced allogeneic mixed chimerism using a nonmyeloablative regimen by host natural killer (NK) cell depletion and T cell-depleted bone marrow (BM) grafts in a major histocompatibility complex (MHC)-mismatched murine model and analyzed the kinetics of donor (C57BL/6) and recipient (BALB/c) engraftment in the weeks following transplantation. Donor BM cells were well engrafted and stabilized without graft-versus-host disease (GVHD) as early as one week post-bone marrow transplantation (BMT). Donor-derived thymic T cells were reconstituted four weeks after BMT; however, the emergence of newly developed T cells was more obvious at the periphery as early as two weeks after BMT. Also, the emergence and changes in ratio of recipient- and donor-derived NKT cells and antigen presenting cells (APCs) including dendritic cells (DCs) and B cells were noted after BMT. Here, we report a longitudinal analysis of the development of donor- and recipient-originated hematopoietic cells in various lymphatic tissues of intentionally induced mixed chimerism mouse model during early post-transplant period. Through the understanding of immune reconstitution at early time points after nonmyeloablative BMT, we suggest guidelines on intentionally inducing durable mixed chimerism.
PMCID: PMC4427480  PMID: 25961559
24.  Distinct genotypic distributions of cytomegalovirus (CMV) envelope glycoprotein in bone marrow and renal transplant recipients with CMV disease. 
A prospective study of the spectrum of glycoprotein B (gB) and glycoprotein H (gH) genotypes of cytomegalovirus (CMV) was conducted with five categories of patients: viremic bone marrow-transplant (BMT) recipients who developed CMV disease after BMT (n = 22), viremic BMT recipients without CMV disease (n = 11), viremic renal-transplant recipients who developed CMV disease after transplantation (n = 14), viremic renal-transplant recipients without CMV disease (n = 13), and premature babies with asymptomatic congenital CMV infections (n = 13). Genotypic stability was observed because the gB and gH genotypes of multiple isolates obtained from a single patient were identical. The distribution of gH genotypes in patients of all groups studied were similar. However, there was a unique distribution of the gB genotype in the first category of patients, i.e., BMT recipients with CMV disease, which was distinct from those of all other categories (P < 0.05). CMV isolates from 54% of BMT recipients with CMV disease exhibited gB type 2, while isolates from 46, 50, 69, and 77% of the BMT recipients without CMV disease, renal-transplant recipients with and those without CMV disease, and premature babies with congenital CMV infection, respectively, were of gB type 1. An analysis of the clinical characteristics of BMT recipients with CMV disease indicated that all underwent either an allogeneic or matched, unrelated donor transplant, and half had severe acute graft-versus-host disease (grades 2 to 4). The statistically significant genotypic difference between CMV isolates from BMT recipients with and without CMV disease was not observed between isolates from renal-transplant recipients with and without CMV disease. We speculate that differences in pathogenesis in different patient groups might account for these observations. These findings would also facilitate decision making about the choice of recombinant CMV glycoprotein vaccine required to immunize transplant donors and the subsequent adoptive transfer of immunity to BMT recipients. When the source of CMV DNA required for genotyping was investigated among renal-transplant recipients, direct use of peripheral blood leukocytes was 95% effective compared to the effectiveness of cells obtained from conventional culture of peripheral blood specimens.
PMCID: PMC170584  PMID: 9302197
25.  Interleukin-18 Regulates Acute Graft-Versus-Host Disease by Enhancing Fas-mediated Donor T Cell Apoptosis 
The Journal of Experimental Medicine  2001;194(10):1433-1440.
Interleukin (IL)-18 is a recently discovered cytokine that modulates both T helper type 1 (Th1) and Th2 responses. IL-18 is elevated during acute graft-versus-host disease (GVHD). We investigated the role of IL-18 in this disorder using a well characterized murine bone marrow transplantation (BMT) model (B6 → B6D2F1). Surprisingly, blockade of IL-18 accelerated acute GVHD-related mortality. In contrast, administration of IL-18 reduced serum tumor necrosis factor (TNF)-α and lipopolysaccharide (LPS) levels, decreased intestinal histopathology, and resulted in significantly improved survival (75 vs. 15%, P < 0.001). Administration of IL-18 attenuated early donor T cell expansion and was associated with increased Fas expression and greater apoptosis of donor T cells. The administration of IL-18 no longer protected BMT recipients from GVHD when Fas deficient (lpr) mice were used as donors. IL-18 also lost its ability to protect against acute GVHD when interferon (IFN)-γ knockout mice were used as donors. Together, these results demonstrate that IL-18 regulates acute GVHD by inducing enhanced Fas-mediated apoptosis of donor T cells early after BMT, and donor IFN-γ is critical for this protective effect.
PMCID: PMC2193680  PMID: 11714750
bone marrow transplantation; Th1/Th2 cytokines; IFN-γ; LPS; TNF-α

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