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1.  Donor Hematopoietic Stem Cells Confer Long-Term Marrow Reconstitution by Self-Renewal Divisions Exceeding to That of Host Cells 
PLoS ONE  2012;7(12):e50693.
Dormant hematopoietic stem cells (HSCs) are activated by microenvironmental cues of the niche in response to the injury of bone marrow (BM). It is not clearly understood how engrafted cells respond to these cues and are involved in marrow regeneration. The purpose of this study was to decipher this cellular response in competitive environment. BM cells of CD45.2 mice were transplanted in sub-lethally irradiated CD45.1 mice. The status of the donor and recipient stem cells (LSK: Lin−Sca-1+c-Kit+) were determined by flowcytometry using CD45 alleles specific antibodies. The presence of long-term engraftable stem cells was confirmed by marrow repopulation assay in secondary hosts, and cell cycle status was determined by staining with Ho33342 and pyronin Y, and BrdU retention assay. The expressions of different hematopoietic growth factor genes in stromal compartment (CD45− cells) were assessed by real-time reverse transcriptase- polymerase chain reaction (RT-PCR). The presence of donor cells initially stimulated the proliferation of host LSK cells compared with control mice without transplantation. This was expected due to pro-mitotic and anti-apoptotic factors secreted by the donor hematopoietic cells. Upon transplantation, a majority of the donor LSK cells entered into cell cycle, and later they maintained cell cycle status similar to that in the normal mouse. Donor-derived LSK cells showed 1000-fold expansion within 15 days of transplantation. Donor-derived cells not only regenerated BM in the primary irradiated host for long-term, they were also found to be significantly involved in marrow regeneration after the second cycle of irradiation. The proliferation of LSK cells was associated with the onset of colossal expression of different hematopoietic growth factor genes in non-hematopoietic cellular compartment. Activation of donor LSK cells was found to be dynamically controlled by BM cellularity. Long-term study showed that a high level of hematopoietic reconstitution could be possible by donor cells in a sub-lethally irradiated host.
doi:10.1371/journal.pone.0050693
PMCID: PMC3515605  PMID: 23227199
2.  Immunologically nonspecific mechanisms of tissue destruction in the rejection of skin grafts 
The Journal of Experimental Medicine  1994;179(5):1645-1652.
When mice are lethally irradiated and reconstituted with allogeneic bone marrow cells, their skin is repopulated over a period of several months with Langerhans cells (LC) of marrow donor origin. Skin from such mice, when transplanted to unirradiated syngeneic recipients, became in many cases the sites of intense inflammatory responses that led to varying degrees of destruction of the transplanted skin and in some instances, to rejection of the entire graft. The frequency and intensity of these responses were influenced by the nature of the immunogenetic disparity between the donors and recipients of the marrow cells. Chimeric skin placed on hybrid mice derived from crosses between the marrow donors and recipients behaved in all respects as syngeneic grafts or autografts. When the recipients of the chimeric skin were presensitized to the antigens of the marrow donor, the responses were especially intense, and resulted in all cases in complete rejection. Thus the immunologically mediated attack on the allogeneic LCs was accompanied by widespread and nonspecific destruction of bystander cells. In all cases, the inflammation and tissue damage were confined sharply to the grafted skin, showing clearly that nonspecific or indirect tissue destruction is entirely consistent with highly selective destruction of grafted tissues. This finding removes a major objection to postulated mechanisms of rejection that involve indirect destruction of grafted tissues.
PMCID: PMC2191467  PMID: 8163942
3.  IL-11 separates graft-versus-leukemia effects from graft-versus-host disease after bone marrow transplantation 
Journal of Clinical Investigation  1999;104(3):317-325.
We recently showed that IL-11 prevents lethal graft-versus-host disease (GVHD) in a murine bone marrow transplantation (BMT) model of GVHD directed against MHC and minor antigens. In this study, we have investigated whether IL-11 can maintain a graft-versus-leukemia (GVL) effect. Lethally irradiated B6D2F1 mice were transplanted with either T cell–depleted (TCD) bone marrow (BM) alone or with BM and splenic T cells from allogeneic B6 donors. Animals also received host-type P815 mastocytoma cells at the time of BMT. Recipients were injected subcutaneously with recombinant human IL-11 or control diluent twice daily, from 2 days before BMT to 7 days after BMT. TCD recipients all died from leukemia by day 23. All control- and IL-11–treated allogeneic animals effectively rejected their leukemia, but IL-11 also reduced GVHD-related mortality. Examination of the cellular mechanisms of GVL and GVHD in this system showed that IL-11 selectively inhibited CD4-mediated GVHD, while retaining both CD4- and CD8-mediated GVL. In addition, IL-11 treatment did not affect cytolytic effector functions of T cells after BMT either in vivo or in vitro. Studies with perforin-deficient donor T cells demonstrated that the GVL effect was perforin dependent. These data demonstrated that IL-11 can significantly reduce CD4-dependent GVHD without impairing cytolytic function or subsequent GVL activity of CD8+ T cells. Brief treatment with IL-11 shortly after BMT may therefore represent a novel strategy for separating GVHD and GVL.
PMCID: PMC408425  PMID: 10430613
4.  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
5.  Reduced GVHD in C3-deficient mice is associated with the decrease of donor Th1/Th17 differentiation 
Graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is mediated by the activation of recipient dendritic cells (DCs) and subsequent proliferation of donor T cells. Recently complement system has been shown to modulate adaptive immunity through the interaction between the complement system and lymphocytes. Complement proteins participate in the activation of DCs, antigen presentation to T cells, and proliferation of T cells. Our studies with a murine model of bone marrow transplantation (BMT) demonstrate that complement system regulates alloimmune responses in GVHD. Mice deficient in the central component of the complement system (C3−/−) had significantly lower GVHD-related mortality and morbidity compared to the wild type (WT) recipient mice. The number of donor-derived T cells including IFNγ+, IL17+ and IL17+IFNγ+ subsets was decreased in secondary lymphoid organs of C3−/− recipients. Furthermore, there was a reduction of recipient CD8α+CD11c+ in lymphoid organs. We conclude C3 regulates Th1/17 differentiation in BMT, and define a novel function of the complement system in GVHD.
doi:10.1016/j.bbmt.2012.05.014
PMCID: PMC3437318  PMID: 22664751
6.  Ultimate erythropoietic repopulating abilities of fetal, young adult, and old adult cells compared using repeated irradiation 
Erythropoietic repopulating abilities of fetal liver cells and young and old adult marrow cells were compared as follows: Equal numbers of cells from a donor of each age were mixed with a constant portion of cells pooled from genetically distinguishable competitors. These mixtures were transplanted into stem cell-depleted recipients, and the proportions of recipient hemoglobin that were donor type measured the relative effectiveness of early erythropoietic precursor cells from the various donors (Fig. 1). At intervals of 3-6 mo, recipients were sublethally irradiated, requiring a new round of competitive repopulation. When B6 mice were used as donors, with WBB6F1 competitors and recipients, the highest levels of stem cell activity were found using old donors (Tables I, III). This was true even with unirradiated, immune-competent W/Wv recipients (Table III). When donors and recipients were WBB6F1 hybrids, with B6 competitors, fetal cells initially gave higher levels of repopulating ability, and they were similar to the adult and old marrow cells after 400 d and after recovery from two sublethal irradiations (Table II). These effects were mostly insignificant and probably reflect small differences in initial stem cell concentrations that are brought out by the sensitivity of the competitive repopulation assay. Clearly, ultimate erythropoietic stem cell proliferative capacities did not decline as a result of the proliferation required between 15 d of fetal life and old age. Repopulating abilities of 12-d fetal liver cells were not detectable. We also showed that the proportions of newly synthesized hemoglobins made by the two types of stem cells in tetraparental mice remained nearly constant when tested at 3-d intervals over 30 d. Minimum numbers of stem cells producing erythrocytes over a single 3-d period were calculated as 62 and 128, but these are too low, since variances were similar in the tetraparental mice and in the F1 hybrid control. This contradicts the hypothesis that erythropoietic stem cells reserve limited proliferative capacities by proliferating one or a few at a time. We suggest that erythropoietic stem cells have essentially unlimited proliferative capacities and are found in approximately equal concentrations in the primary erythropoietic organs after 15 or 16 d of fetal life.
PMCID: PMC2187395  PMID: 6147387
7.  Rapid, deletional peripheral CD8 T cell tolerance induced by allogeneic bone marrow: role of donor class II MHC and B cells1 
Mixed chimerism and donor-specific tolerance are achieved in mice receiving 3 Gy total body irradiation and anti-CD154 monoclonal antibody (mAb) followed by allogeneic bone marrow transplantation (BMT). In this model, recipient CD4 cells are critically important for CD8 tolerance. To evaluate the role of CD4 cells recognizing donor MHC class II directly, we used class II-deficient donor marrow and were not able to achieve chimerism unless recipient CD8 cells were depleted, indicating that directly alloreactive CD4 cells were necessary for CD8 tolerance. To identify the MHC class II+ donor cells promoting this tolerance, we used donor bone marrow (BM) lacking certain cell populations or used positively selected cell populations. Neither donor CD11c+ dendritic cells, B cells, T cells nor donor-derived IL-10 were critical for chimerism induction. Purified donor B cells induced early chimerism and donor-specific cell-mediated lympholysis (CML) tolerance in both strain combinations tested. In contrast, positively selected CD11b+ monocytes/myeloid cells did not induce early chimerism in either strain combination. Donor cell preparations containing B cells were able to induce early deletion of donor-reactive TCR transgenic 2C CD8 T cells, whereas those devoid of B cells had reduced activity. Thus, induction of stable mixed chimerism depends on the expression of MHC class II on the donor marrow, but no requisite donor cell lineage was identified. Donor BM-derived B cells induced early chimerism, donor-specific CML tolerance and deletion of donor-reactive CD8 T cells, whereas CD11b+ cells did not. Thus, BM-derived B cells are potent tolerogenic APCs for alloreactive CD8 cells.
PMCID: PMC2628539  PMID: 18768896
B cells; cytotoxic T cells; transplantation; tolerance; MHC class II
8.  Microglial stability and repopulation in the retina 
Background/aims: Parenchymal central nervous system microglia are repopulated by bone marrow derived monocytes more slowly than any other reticuloendothelial cells. The contribution of bone marrow derived monocytes to the uninflammed retina has not been studied. The present study sought to determine repopulation of retinal microglia in uniflammed retina by bone marrow derived monocytes in bone marrow chimeric rats.
Methods: Chimeric (Y→X) Lewis rats were constructed by transplanting 5×107 male bone marrow cells into lethally irradiated female recipient rats. The chimeras were sacrificed 8, 10, 12, 30, and 52 weeks after bone marrow transplant, and retina, brain, lung, and spleen samples were collected. DNA was extracted and quantified. Y positive infiltrating cells in the collected samples were detected by polymerase chain reaction amplification of a Y chromosome specific 104 bp fragment.
Results: There was a rapid repopulation of haematopoietic tissues in the spleen (at 8 weeks), confirming the establishment of chimerism, and to a lesser extent, of lung (at 30 weeks). This repopulation was absent in the brain parenchyma and retina until 52 weeks after transplantation.
Conclusions: These data indicate that resident microglia in the retina, much like those in the brain, are stable in number in the retinal compartment (up to 1 year), and repopulation by bone marrow derived cells may be delayed for a year.
doi:10.1136/bjo.2004.060293
PMCID: PMC1772746  PMID: 15965175
transplant chimera; bone marrow transplant; sry gene; retina
9.  Combination of Intra-Bone Marrow–Bone Marrow Transplantation and Subcutaneous Donor Splenocyte Injection Diminishes Risk of Graft-Versus-Host Disease and Enhances Survival Rate 
Stem Cells and Development  2010;20(5):759-768.
The combination of allogeneic bone marrow transplantation (allo-BMT) and donor lymphocyte infusion (DLI) is a useful method for establishing donor chimerism and preventing a relapse of leukemia/lymphoma. However, there is a risk of inducing uncontrollable fatal graft-versus-host disease (GVHD). In fact, allo-BMT plus intravenous (IV)-DLI using donor splenocytes induces fatal GVHD in recipient mice. In this study, we examined the effects of the combination of intra-bone marrow (IBM)-BMT and the subcutaneous injection of donor splenocytes (SC-DLI) on the allo-BMT system. Recipient BALB/c mice were conditioned by sublethal irradiation (5 Gy), followed by IBM-BMT plus IV-DLI or SC-DLI in C57BL/6 mice. The IV-DLI group showed better engraftment of donor hemopoietic cells than the control group (without DLI) but showed fatal GVHD. The SC-DLI group, however, showed good reconstitution and mild GVHD. These results suggest that the combination of SC-DLI and IBM-BMT promotes the reconstitution of hemopoiesis and helps reduce the risk of GVHD.
doi:10.1089/scd.2010.0232
PMCID: PMC3128777  PMID: 20954841
10.  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
11.  Recipient B Cells Are Not Required For GVHD Induction 
Recipient antigen presenting cells (APCs) are required for CD8-mediated GVHD and have an important and nonredundant role in CD4-mediated GVHD in mouse MHC-matched allogeneic bone marrow transplantation (alloBMT). However, the precise roles of specific recipient APCs — dendritic cells, macrophages, and B cells — are not well defined. If recipient B cells are important APCs they could be depleted with Rituximab, an anti-CD20 monoclonal antibody. On the other hand, B cells can downregulate T cell responses and consequently B cell depletion could exacerbate GVHD. Patients with B cell lymphomas undergo allogeneic hematopoietic stem cell transplantation (alloSCT) and many are B-cell-deficient due to prior Rituximab. We therefore studied the role of recipient B cells in MHC-matched murine models of CD8- and CD4-mediated GVHD by using recipients genetically deficient in B cells and with antibody-mediated depletion of host B cells. In both CD4-and CD8-dependent models, B cell deficient recipients developed clinical and pathologic GVHD. However, although CD8-mediated GVHD was clinically less severe in hosts genetically deficient in B cells, it was unaffected in anti-CD20-treated recipients. These data indicate that recipient B cells are not important initiators of GVHD and that efforts to prevent GVHD by APC depletion should focus on other APC subsets.
doi:10.1016/j.bbmt.2010.03.015
PMCID: PMC3135976  PMID: 20338255
12.  INDUCTION OF DONOR-SPECIFIC TRANSPLANTATION TOLERANCE TO SKIN AND CARDIAC ALLOGRAFTS USING MIXED CHIMERISM IN (A + B → A) IN RATS 
Cell transplantation  1993;2(4):345-353.
Mixed allogeneic chimerism (A + B → A) was induced in rats by reconstitution of lethally irradiated LEW recipients with a mixture of T-cell depleted (TCD) syngeneic and TCD allogeneic ACI bone marrow. Thirty-seven percent of animals repopulated as stable mixed lymphopoietic chimeras, while the remainder had no detectable allogeneic chimerism. When evaluated for evidence of donor-specific transplantation tolerance, only those recipients with detectable allogeneic lymphoid chimerism exhibited acceptance of donor-specific skin and cardiac allografts. Despite transplantation over a major histocompatibility complex (MHO)- and minor-disparate barrier, animals accepted donor-specific ACI skin and primarily vascularized cardiac allografts permanently, while rejecting third party Brown Norway (BN) grafts. The tolerance induced was also donor-specific in vitro as evidenced by specific hyporeactivity to the allogeneic donor lymphoid elements, yet normal reactivity to MHC-disparate third party rat lymphoid cells. This model for mixed chimerism in the rat will be advantageous to investigate specific transplantation tolerance to primarily vascularized solid organ grafts that can be performed with relative ease in the rat, but not in the mouse, and may provide a method to study the potential existence of organ- or tissue-specific alloantigens in primarily vascularized solid organ allografts.
PMCID: PMC2976585  PMID: 8162277
Bone marrow; Transplantation tolerance; Chimerism; Skin
13.  Effects of Sub-lethal Irradiation on Patterns of Engraftment after Murine Bone Marrow Transplantation 
Attempts to reduce the toxicity of hematopoietic stem cell transplantation have led to the use of a variety of immunosuppressive yet non-myeloablative preparative regimens that often include low dose irradiation. To determine the effects of low dose irradiation on the dynamics of donor cell engraftment after bone marrow transplantation (BMT), we coupled standard end-point flow cytometric analysis with in vivo longitudinal bioluminescence imaging performed throughout the early (<10 days) and late (days 10–90) post transplant period. To exclude the contribution of irradiation on reducing immunological rejection, severely immune deficient mice were chosen as recipients of allogeneic bone marrow. Flow cytometric analysis showed that sub-lethal doses of total body irradiation (TBI) significantly increased long-term (14 weeks) donor chimerism in the bone marrow compared to non-irradiated recipients (p<0.05). Bioluminescence imaging demonstrated that the effect of TBI (p<0.001) on chimerism was seen only after the first 7 days post BMT. Flow cytometry analysis at Day 3 showed no increase in the number of donor cells in irradiated BM, confirming that sub-lethal irradiation does not enhance marrow chimerism early after transplantation. Local irradiation also significantly increased late (but not early) donor chimerism in the irradiated limb. Intra-femoral injection of donor cells provided efficient early chimerism in the injected limb, but long-term systemic donor chimerism was highest with intravenous (IV) administration (p<0.05). Overall, the combination of TBI and IV administration of donor cells provided the highest levels of long-term donor chimerism in the marrow space. These findings suggest that the major effect of sub-lethal irradiation is to enhance long-term donor chimerism by inducing proliferative signals after the initial phase of homing.
doi:10.1016/j.bbmt.2010.12.697
PMCID: PMC3086732  PMID: 21176787
14.  Early immunisation with dendritic cells after allogeneic bone marrow transplantation elicits graft vs tumour reactivity 
British Journal of Cancer  2013;108(5):1092-1099.
Background:
Perspectives of immunotherapy to cancer mediated by bone marrow transplantation (BMT) in conjunction with dendritic cell (DC)-mediated immune sensitisation have yielded modest success so far. In this study, we assessed the impact of DC on graft vs tumour (GvT) reactions triggered by allogeneic BMT.
Methods:
H2Ka mice implanted with congenic subcutaneous Neuro-2a neuroblastoma (NB, H2Ka) tumours were irradiated and grafted with allogeneic H2Kb bone marrow cells (BMC) followed by immunisation with tumour-inexperienced or tumour-pulsed DC.
Results:
Immunisation with tumour-pulsed donor DC after allogeneic BMT suppressed tumour growth through induction of T cell-mediated NB cell lysis. Early post-transplant administration of DC was more effective than delayed immunisation, with similar efficacy of DC inoculated into the tumour and intravenously. In addition, tumour inexperienced DC were equally effective as tumour-pulsed DC in suppression of tumour growth. Immunisation of DC did not impact quantitative immune reconstitution, however, it enhanced T-cell maturation as evident from interferon-γ (IFN-γ) secretion, proliferation in response to mitogenic stimulation and tumour cell lysis in vitro. Dendritic cells potentiate GvT reactivity both through activation of T cells and specific sensitisation against tumour antigens. We found that during pulsing with tumour lysate DC also elaborate a factor that selectively inhibits lymphocyte proliferation, which is however abolished by humoral and DC-mediated lymphocyte activation.
Conclusion:
These data reveal complex involvement of antigen-presenting cells in GvT reactions, suggesting that the limited success in clinical application is not a result of limited efficacy but suboptimal implementation. Although DC can amplify soluble signals from NB lysates that inhibit lymphocyte proliferation, early administration of DC is a dominant factor in suppression of tumour growth.
doi:10.1038/bjc.2013.39
PMCID: PMC3619065  PMID: 23511628
dendritic cells; graft vs tumour reaction; allogeneic bone marrow transplantation; immunisation; cytotoxic T cells
15.  LYMPHOID CELL DEPENDENCE OF EOSINOPHIL RESPONSE TO ANTIGEN 
Quantitative determinations were made of the capacity of isogenic bone marrow, spleen, and thymic cells from primed and/or nonprimed mice to repopulate the hemopoietic tissues and to mount an inflammatory and antibody response to specific antigen (tetanus toxoid) in heavily irradiated and reconstituted recipients. Spleen cells from primed mice but not from normal mice had the capacity to adoptively transfer an anamnestic antitoxin titer in irradiated animals in the absence of transplanted bone marrow cells, and during retarded myeloid regeneration. Spleen cells alone or bone marrow cells alone produced an insignificant and a moderate peritoneal eosinophil response, respectively, to antigen. In the presence of bone marrow cells, normal spleen cells augment the capacity of recipient animals to mount an eosinophil response to antigen. A much greater augmentation occurs in animals reconstituted with splenic or thymic cells from primed animals. The increase in antitoxin titers appears to be independent of the response of eosinophils since: (a) marked accumulation of eosinophils can occur in animals with no measurable humoral antitoxin, and (b) high antitoxin titers can occur in animals which do not have marked eosinophil responses. It is suggested that a thymic-derived or thymic-dependent mononuclear cell population is necessary for optimal eosinophil response to antigen. The neutrophil and mononuclear cell responses to antigen are determined by different mechanisms from those which determine the eosinophil response. These studies together with earlier findings strongly indicate that the eosinophil granulocytes play a role in the immune response to antigen.
PMCID: PMC2139085  PMID: 15776576
16.  Hematopoietic cell specific deletion of Toll-like receptor 4 ameliorates hepatic and adipose tissue insulin resistance in high fat fed mice 
Cell metabolism  2009;10(5):419-429.
Summary
Chronic, low-grade inflammation, particularly in adipose tissue, is an important modulator of obesity-induced insulin resistance and the toll-like receptor 4 (Tlr4) is a key initiator of inflammatory responses in macrophages. We performed bone marrow transplantation (BMT) of Tlr4lps-del or control C57Bl/10J bone marrow cells into irradiated wild type C57Bl6 recipient mice to generate hematopoietic cell specific Tlr4 deletion mutant (BMT-Tlr4-/-) and control (BMT-wt) mice. When mice were fed a high-fat diet (HFD) for 16 weeks, BMT-wt mice developed obesity, hyperinsulinemia, glucose intolerance and insulin resistance. In contrast, BMT-Tlr4-/- mice became obese, but did not develop fasting hyperinsulinemia, and had improved hepatic and skeletal muscle insulin sensitivity during euglycemic clamp studies compared to HFD BMT-wt mice. The HFD BMT-Tlr4-/- mice showed markedly reduced adipose tissue inflammatory markers and macrophage content compared to HFD BMT-wt mice. In summary, our results indicate that Tlr4 signaling in hematopoietic-derived cells is important for the development of hepatic and adipose tissue insulin resistance in obese mice.
doi:10.1016/j.cmet.2009.09.006
PMCID: PMC2790319  PMID: 19883619
17.  Stable mixed chimerism and tolerance using a nonmyeloablative preparative regimen in a large-animal model 
Journal of Clinical Investigation  2000;105(2):173-181.
Bone marrow transplantation (BMT) has considerable potential for the treatment of malignancies, hemoglobinopathies, and autoimmune diseases, as well as the induction of transplantation allograft tolerance. Toxicities associated with standard preparative regimens for bone marrow transplantation, however, make this approach unacceptable for all but the most severe of these clinical situations. Here, we demonstrate that stable mixed hematopoietic cell chimerism and donor-specific tolerance can be established in miniature swine, using a relatively mild, non-myeloablative preparative regimen. We conditioned recipient swine with whole-body and thymic irradiation, and we depleted their T-cells by CD3 immunotoxin-treatment. Infusion of either bone marrow cells or cytokine-mobilized peripheral blood stem cells from leukocyte antigen-matched animals resulted in stable mixed chimerism, as detected by flow cytometry in the peripheral blood, thymus, and bone marrow, without any clinical evidence of graft-versus-host disease (GvHD). Long-term acceptance of donor skin and consistent rejection of third-party skin indicated that the recipients had developed donor-specific tolerance.
PMCID: PMC377429  PMID: 10642595
18.  Enhanced allostimulatory activity of host antigen-presenting cells in old mice intensifies acute graft-versus-host disease 
The Journal of Clinical Investigation  2002;109(9):1249-1256.
Older bone marrow transplantation (BMT) recipients are at heightened risk for acute graft-versus-host disease (GVHD) after allogeneic BMT, but the causes of this association are poorly understood. Using well-characterized murine BMT models we have explored the mechanisms of increased GVHD in older mice. GVHD mortality, morbidity, and pathologic and biochemical indices were all worse in old recipients. Donor T cell responses were significantly increased in old recipients both in vivo and in vitro when stimulated by antigen-presenting cells (APCs) from old mice, which also secreted more TNF-α and IL-12 after LPS stimulation. In a B6 → B6D2F1 model, CD4+ donor T cells but not CD8+ T cells mediated more severe GVHD in old mice. We confirmed the role of aged APCs in GVHD using B6D2F1 BM chimeras created with either old or young BM. Four months after chimera creation, allogeneic BMT from B6 donors caused significantly worse GVHD in old BM chimeras. APCs from these mice also stimulated greater responses from allogeneic cells in vitro. These data demonstrate a hitherto unsuspected mechanism of amplified donor T cell responses by aged allogeneic host APCs that increases acute GVHD in aged recipients in this BMT model.
doi:10.1172/JCI14793
PMCID: PMC150964  PMID: 11994414
19.  The Prolonged Life-Span of Alveolar Macrophages 
To further examine the half-life of alveolar macrophages, chimeric CD 45.2 mice were generated through bone marrow transplantation of donor CD 45.1 cells. Before administration of donor cells, recipient mice were divided into two cohorts: the first cohort received total body irradiation; the second cohort also received irradiation—however, the thorax, head, and upper extremities were shielded with lead. Flow cytometric analysis was then performed on blood, peritoneal, and bronchoalveolar lavage cells over time to quantify engraftment. The data generated for the unshielded cohort of mice revealed a macrophage half-life of 30 days. In the shielded cohort, however, we found that by 8 months there was negligible replacement of recipient alveolar macrophages by donor cells, despite reconstitution of the blood and peritoneum by donor bone marrow. Consistent with these findings, the mean fluorescent intensity of alveolar macrophages remained stable over a 4-week period after in vivo PKH26 dye loading. Together, these data show that previous alveolar macrophage half-life studies were confounded by the fact that they did not account for the toxic effects of irradiation conditioning regimens, and demonstrate that the bone marrow does not significantly contribute to the alveolar macrophage compartment during steady-state conditions.
doi:10.1165/rcmb.2007-0224RC
PMCID: PMC2274942  PMID: 18192503
macrophages; half-life; PKH26
20.  Effect of bone marrow transplantation on enzyme levels and clinical course in the neurologically affected twitcher mouse. 
Journal of Clinical Investigation  1988;81(6):1790-1794.
The effect of allogeneic bone marrow transplantation (BMT) was investigated in the neurologically affected twitcher mouse, a model for human Krabbe's disease. Twitcher mice have a hereditary deficiency of the lysosomal enzyme galactosylceramidase, which causes growth delay, tremor, and paralysis of the hind legs. Death occurs at 30-40 d of age. After BMT galactosylceramidase activity increased to donor levels in hemopoietic organs. In lung, heart, and liver, galactosylceramidase activity rose to levels intermediate between those of twitcher and normal mice. Increased galactosylceramidase activity in liver parenchymal cells indicated uptake of the donor enzyme by recipient cells of nonhemopoietic origin. Enzyme activity also increased in kidney tissue. BMT resulted in a gradual increase in galactosylceramidase activity in the central nervous system to 15% of normal donor levels. A 5-6-fold increase in galactosylceramidase activity was found in the peripheral nervous system. This increase in enzyme activity was accompanied by a partial alleviation of neurological symptoms. In particular, paralysis of the hind legs was prevented by BMT. BMT led to a modest restoration of growth and prolonged survival. In several cases, the mice survived for more than 100 d, but eventually all animals died with severe neurological disease.
PMCID: PMC442626  PMID: 3290253
21.  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.
Purpose
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.
Methods
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.
Results
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.
Conclusions
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
22.  Role of Regulatory T Cells in Transferable Immunological Tolerance to Bone Marrow Donor in Murine Mixed Chimerism Model 
Journal of Korean Medical Science  2013;28(12):1723-1728.
Constructing a bone marrow chimera prior to graft transplantation can induce donor-specific immune tolerance. Mixed chimerism containing hematopoietic cells of both recipient- and donor-origin has advantages attributed from low dose of total body irradiation. In this study, we explored the mechanism of mixed chimerism supplemented with depletion of Natural Killer cells. Mixed chimerism with C57BL/6 bone marrow cells was induced in recipient BALB/c mice which were given 450 cGy of γ-ray irradiation (n = 16). As revealed by reduced proliferation and cytokine production in mixed leukocyte reaction and ELISpot assay (24.6 vs 265.5), the allo-immune response to bone marrow donor was reduced. Furthermore, the induction of transferable immunological tolerance was confirmed by adoptive transfer and subsequent acceptance of C57BL/6 skin graft (n = 4). CD4+FoxP3+ regulatory T cells were increased in the recipient compartment of the mixed chimera (19.2% → 33.8%). This suggests that regulatory T cells may be therapeutically used for the induction of graft-specific tolerance by mixed chimerism.
doi:10.3346/jkms.2013.28.12.1723
PMCID: PMC3857366  PMID: 24339700
Bone Marrow Transplantation; Immune Tolerance; Natural Killer Cells; Regulatory T Cells
23.  Murine Model of Interstitial Cytomegalovirus Pneumonia in Syngeneic Bone Marrow Transplantation: Persistence of Protective Pulmonary CD8-T-Cell Infiltrates after Clearance of Acute Infection 
Journal of Virology  2000;74(16):7496-7507.
Interstitial pneumonia (IP) is a severe organ manifestation of cytomegalovirus (CMV) disease in the immunocompromised host, in particular in recipients of bone marrow transplantation (BMT). Diagnostic criteria for the definition of CMV-IP include clinical evidence of pneumonia together with CMV detected in bronchoalveolar lavage or lung biopsy. We have used the model of syngeneic BMT and simultaneous infection of BALB/c mice with murine CMV for studying the pathogenesis of CMV-IP by controlled longitudinal analysis. A disseminated cytopathic infection of the lungs with fatal outcome was observed only when reconstituting CD8 T cells were depleted. Neither CD8 nor CD4 T cells mediated an immunopathogenesis of acute CMV-IP. By contrast, after efficient hematolymphopoietic reconstitution, viral replication in the lungs was moderate and focal. The histopathological picture was dominated by preferential infiltration of CD8 T cells confining viral replication to inflammatory foci. Notably, after clearance of acute infection, CD62Llo and CD62Lhi subsets of CD44+ memory CD8 T cells were found to persist in lung tissue. One can thus operationally distinguish an early CMV-positive IP (phase 1) and a late CMV-negative IP (phase 2). According to the definition, phase 2 histopathology would not be diagnosed as a CMV-IP and could instead be misinterpreted as a CMV-induced immunopathology. We document here that phase 1 as well as phase 2 pulmonary CD8 T cells are capable of exerting effector functions and are effectual in protecting against productive infection. We propose that antiviral “stand-by” memory-effector T cells persist in the lungs to prevent virus recurrence from latency.
PMCID: PMC112270  PMID: 10906203
24.  MURINE MOBILIZED PERIPHERAL BLOOD STEM CELLS HAVE A LOWER CAPACITY THAN BONE MARROW TO INDUCE MIXED CHIMERISM AND TOLERANCE 
Allogeneic bone marrow transplantation (BMT) under costimulation blockade allows induction of mixed chimerism and tolerance without global T cell depletion. The mildest such protocols without recipient cytoreduction, however, require clinically impracticable bone marrow (BM) doses. The successful use of mobilized peripheral blood stem cells (PBSC) instead of BM in such regimens would provide a substantial advance, allowing transplantation of higher doses of hematopoietic donor cells. We thus transplanted fully allogeneic murine granulocyte colony-stimulating-factor (G-CSF) mobilized PBSC under costimulation blockade (anti-CD40L and CTLA4Ig). Unexpectedly, PBSC did not engraft, even when very high cell doses together with non-myeloablative total body irradiation (TBI) were used. We show that, paradoxically, T cells contained in the donor PBSC triggered rejection of the transplanted donor cells. Rejection of donor bone marrow was also triggered by the co-transplantation of unmanipulated donor T cells isolated from naïve (non-mobilized) donors. Donor-specific transfusion and transient immunosuppression prevented PBSC-triggered rejection and mixed chimerism and tolerance were achieved, but graft-versus-host disease (GVHD) occurred. The combination of in vivo T cell depletion with costimulation blockade prevented rejection and GVHD. Thus, if allogeneic PBSC are transplanted instead of BM, costimulation blockade alone does not induce chimerism and tolerance without unacceptable GVHD-toxicity, and the addition of T cell depletion is required for success.
doi:10.1111/j.1600-6143.2008.02371.x
PMCID: PMC2992943  PMID: 18828766
mixed chimerism; tolerance; costimulation blockade; PBSC
25.  CD4+ T Cell–Depleted Lymphocyte Infusion Impairs Neither the Recovery of Recipient Thymus nor the Development of Transplanted Thymus 
Thymus transplantation, in conjunction with bone marrow transplantation (BMT), has been attracting attention for the treatment of various diseases. Recently, donor lymphocyte infusion (DLI) has been used as a helpful tool for establishing donor chimerism and preventing a relapse of leukemia/lymphoma. However, the effects of DLI on transplanted and recipient thymuses have not been explored. We therefore performed DLI in the intrabone marrow–BMT + thymus transplantation setting. We have found that DLI leads to derangements in both recipient thymuses and transplanted thymuses; by 2 wk after BMT, we saw a decrease in total cell number, a lower percentage of CD4+CD8+ cells, and the obliteration of the thymic corticomedullary junction. Four weeks later, the thymic impairment became more serious. However, when we depleted the CD4+ T cells (CD4−-DLI), the recipient thymic recovery and transplanted thymic development were significantly restored by the treatment. In addition, there were much greater levels of TNF-α and Fas ligand, and a lower percentage of regulatory T cells in the DLI group than in the CD4−-DLI group. These findings indicate that inflammation induced by DLI, especially by CD4+ T cells, plays a crucial role in the thymic impairment.
doi:10.4049/jimmunol.1201605
PMCID: PMC3586976  PMID: 23382561

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