Surfactant protein A is a required mediator of keratinocyte growth factor after experimental marrow transplantation

We reported an association between the ability of recombinant human keratinocyte growth factor (rHuKGF) to upregulate the expression of surfactant protein A (SP-A) and to downregulate pulmonary inflammation that occurs after allogeneic bone marrow transplantation (BMT). To establish a causal relationship, rHuKGF (5 mg/kg) was administered subcutaneously for three consecutive days before irradiation to SP-A-sufficient and -deficient [SP-A(+/+) and SP-A(-/-), respectively] mice given inflammation-inducing allogeneic spleen T cells at the time of BMT. In contrast with SP-A(+/+) mice, rHuKGF failed to suppress the high levels of TNF-alpha, IFN-gamma, and nitric oxide contained in bronchoalveolar lavage fluids collected on day 7 after BMT from SP-A(-/-) mice. Early post-BMT weight loss was attenuated by rHuKGF in both SP-A(+/+) and SP-A(-/-) recipients. In the absence of supportive respiratory care, however, SP-A deficiency eventually abolished the ability of rHuKGF to prevent weight loss and to improve survival monitored for 1 mo after allogeneic BMT. In further experiments, the addition of cyclophosphamide (which is known to cause severe injury to the alveolar epithelium in donor T cell-recipient mice) to the conditioning regimen prevented rHuKGF-induced upregulation of SP-A and suppression of lung inflammation in both SP-A(+/+) and SP-A(-/-) mice. We conclude that endogenous baseline SP-A levels and optimal upregulation of SP-A are required for the anti-inflammatory protective effects of KGF after allogeneic transplantation.     

Allogeneic bone marrow transplantation for hepatocellular carcinoma: hepatocyte growth factor suppresses graft-vs.-host disease

Allogeneic bone-marrow transplantation (BMT) can induce a powerful graft-vs.-tumor (GVT) effect not only on hematological malignancies but also on solid tumors. However, graft-vs.-host disease (GVHD) is a major complication of allogeneic BMT. We assessed GVT effect on hepatocellular carcinoma (HCC) and the effects of hepatocyte growth factor (HGF) gene transduction on GVHD in HCC transplanted mice. (C57BL/6 x C3H/HeJ)F(1)(B6C3F1, H-2(bxk)) mice were used as recipients and C3H/HeJ(H-2(k)) mice were used as donors. Hepa1-a (a C57L mouse-derived hepatoma cell, H-2(b)) was subcutaneously injected into the recipient mice. Tumor bearing mice were treated in the following ways: group 1, no treatment; group 2, total body irradiation (TBI); group 3, TBI and BMT; group 4, TBI and BMT with empty vector; group 5, TBI and BMT with HGF gene transduction; group 6, TBI and BMT with administration of FK506, a representative immunosuppressive agent. Acute GVHD was assessed by histological examination of the liver, small intestines, and large intestines. Tumor growth was markedly suppressed in mice that received an allogeneic BMT. Donor-derived CD8(+) T cells had infiltrated into the tumor, and cytotoxic CD8(+) T cells against HCC were present. However, among the four groups that received a BMT, this suppressive effect was weaker in group 6 compared with the other three groups (groups 3, 4, and 5). HGF gene transduction improved GVHD while preserving the GVT effects. Allogeneic BMT markedly suppresses the growth of HCC. Simultaneous HGF gene transfer can suppress GVHD while preserving the GVT effect.     

Interactions of bone marrow cells from young and old mice with syngeneic and allogeneic thymic tissue

Age-related changes manifested in MHC-linked recognition of bone marrow (BM) cells by the thymic stroma were studied in vitro model of thymus-BM chimeras. Fetal thymuses (FT) depleted of self-lymphocytes were colonized with BM cells from syngeneic and allogeneic donor mice. When cells from young (3-month-old) or old (24-month-old) donors syngeneic to the stroma were seeded in a mixture with cells of allogeneic young origins (C57BL/6J-Thy1.2 and ARK/J-Thy1.1 seeded onto C57BL/6J FT), the syngeneic cells showed an age-related developmental advantage. Accordingly, cells from the old syngeneic mice manifested a significantly reduced capacity to compete with allogeneic cells when compared with the young syngeneic cells. When allogeneic BM cells from young or old mice were seeded onto the thymic stroma in a mixture with BM cells from young donors syngeneic to that stroma (BALB/c-Thy1.2 mixed with C57BL/Ka-Thy1.1 seeded onto C57BL/6J or C57BL/Ka FT), the Thy1+ cells which developed were mainly of syngeneic origin. The age of the allogeneic cells had no significant effect on the results. However, when old allogeneic cells were mixed with old syngeneic cells, the developmental advantage of the syngeneic cells was not manifested. When seeding of allogeneic cells was followed 1 day later by seeding of syngeneic cells, the syngeneic advantage was eliminated, suggesting that the MHC-linked competition began during the first 24 hr of contact with the thymic tissue. When BM-derived thmocytes grown in FT explants were transferred onto second FT recipient explants of the same genotype as the first ones, the syngeneic advantage was abolished, suggesting either that the thymic microenvironment was modified as a result of colonization or that it induced a change in the BM cells. In this respect, the young allogeneic BM-derived thymocytes showed a significant advantage when compared with the old cells. Thus, the MHC-linked syngeneic preference in the early development of BM cells is also manifested in aging mice, yet at a level that is significantly reduced compared with that seen in the young mice.     

Critical role of TLR9 in acute graft-versus-host disease

Graft-vs-host disease (GVHD) is a major complication after allogeneic bone marrow transplantation. Different studies have demonstrated that intestinal bacterial breakdown products and loss of gastrointestinal tract integrity, both induced by conditioning regiments, are critical in the pathogenesis of acute GVHD. Using C57BL/6 knockout mice, we evaluated the role of TLR4 and TLR9, which recognize bacterial LPS and DNA, respectively, in the GVHD associated with allogeneic bone marrow transplantation. When myeloablative-irradiated TLR9 knockout (TLR9(-/-)) mice were used as graft recipients, survival and clinical score of acute GVHD were improved as compared with the wild-type recipient mice (18/30 vs 1/31 mice still alive at day 70 in a total of four experiments); while no differences were observed using recipient TLR4 knockout (TLR4(-/-)) mice. The reduced mortality and morbidity in TLR9(-/-) mice related with reduced stimulatory activity of TLR9(-/-) spleen APCs after conditioning and reduced proliferation of allogeneic donor T cells. Experiments using TLR9(+/+) into TLR9(-/-) and TLR9(-/-) into TLR9(+/+) chimeric mice as recipients indicated a critical role for nonhematopoietic TLR9(+/+) cells interacting with bacterial breakdown products released in myeloablated mice. Altogether these data reveal a novel important role of TLR9 in GVHD, a finding that might provide tools to reduce this complication of allogeneic transplantation.     

Studies on the induction of tolerance to alloantigens. II. The generation of serum factor(s) able to transfer alloantigen-specific tolerance for delayed-type hypersensitivity by portal venous inoculation with allogeneic cells

The administration of C3H/He spleen cells into allogeneic BALB/c mice via portal venous (p.v.) route resulted in C3H/He alloantigen-specific tolerance for delayed-type hypersensitivity (DTH) responses. When serum from these tolerant BALB/c mice were transferred into naive syngeneic BALB/c mice, the recipient mice lost the capability of generating DTH responses as induced by s.c. immunization with C3H/He cells. Tolerance was transferred only by serum from BALB/c mice inoculated p.v. with C3H/He cells, but not by serum from C3H/He mice inoculated p.v. with C3H/He cells, or BALB/c mice inoculated i.v. with C3H/He cells. This tolerogenic activity in serum from p.v. inoculated BALB/c mice was C3H/He alloantigen specific, because the transfer of the serum did not interfere with the development of anti-C57BL/6 DTH responses in recipient BALB/c mice. Such a serum factor(s) was inducible as early as 1 wk after the inoculation of C3H/He cells into BALB/c mice and not associated with anti-C3H/He alloantibody activity. Moreover, anti-C3H/He or C57BL/6-specific tolerogenic factor(s) prepared in the respective BALB/c or C3H/He mice was successfully transferred into totally allogeneic recipient mice, indicating no requirement of H-2, as well as non-H-2 restriction for the function of serum tolerogenic factor(s). Thus this study demonstrates that p.v. inoculation of allogeneic cells generates serum factor(s) able to transfer in H-2 and non-H-2-unrestricted manners the in vivo tolerance of the alloreactivity specific for alloantigens used for p.v. inoculation.     

Identification of a single non-H-2 gene regulating graft-versus-host disease response

Non-MHC loci have been shown to play an important role in the development and regulation of graft-vs-host disease (GVHD). In the murine model of GVHD under study, injection of C57BL/6 spleen cells into unirradiated (C57BL/6 x DBA/2)F1 hybrid recipient mice results in an acute form of GVHD characterized by CTL, suppressor cells, and runting. In contrast, injection of DBA/2 spleen cells into the same recipients results in a chronic form of GVHD that is characterized by a lack of CTL and hyperproduction of Ig and autoantibodies. After preliminary studies with the use of congenic mice showed that non-MHC loci were controlling GVHD responses in this model, genetic analysis of GVHD response of BXD recombinant inbred strains and (B10.D2 x DBA/2) X DBA/2 BC mice identified a single locus, Gvh, on chromosome 7 that controls whether acute or chronic GVHD results from injection of parental lymphocytes into unirradiated (C57BL/6 x DBA/2)F1 recipient mice.     

CD4+ T cells generated de novo from donor hemopoietic stem cells mediate the evolution from acute to chronic graft-versus-host disease

Acute and chronic graft-versus-host disease (GVHD) remain the major complications limiting the efficacy of allogeneic hemopoietic stem cell transplantation. Chronic GVHD can evolve from acute GVHD, or in some cases may overlap with acute GVHD, but how acute GVHD evolves to chronic GVHD is unknown. In this study, in a classical CD8+ T cell-dependent mouse model, we found that pathogenic donor CD4+ T cells developed from engrafted hemopoietic stem cells (HSCs) in C57BL/6SJL(B6/SJL, H-2(b)) mice suffering from acute GVHD after receiving donor CD8+ T cells and HSCs from C3H.SW mice (H-2(b)). These CD4+ T cells were activated, infiltrated into GVHD target tissues, and produced high levels of IFN-gamma. These in vivo-generated CD4+ T cells caused lesions characteristic of chronic GVHD when adoptively transferred into secondary allogeneic recipients and also caused GVHD when administered into autologous C3H.SW recipients. The in vivo generation of pathogenic CD4+ T cells from engrafted donor HSCs was thymopoiesis dependent. Keratinocyte growth factor treatment improved the reconstitution of recipient thymic dendritic cells in CD8+ T cell-repleted allogeneic hemopoietic stem cell transplantation and prevented the development of pathogenic donor CD4+ T cells. These results suggest that de novo-generated donor CD4+ T cells, arising during acute graft-versus-host reactions, are key contributors to the evolution from acute to chronic GVHD. Preventing or limiting thymic damage may directly ameliorate chronic GVHD.     

IFN-gamma- and cell-to-cell contact-dependent cytotoxicity of allograft-induced macrophages against syngeneic tumor cells and cell lines: an application of allografting to cancer treatment.

In allogeneic tumor or skin transplantation, the rejection process that destroys the allogeneic cells leaves syngeneic cells intact by discrimination between self and nonself. Here, we examined whether the cells infiltrating into the allografts could be cytotoxic against syngeneic immortal cells in vitro and in vivo. The leukocytes (i.e., macrophages (Mphi; 55-65% of bulk infiltrates), granulocytes (20-25%), and lymphocytes (15-20%)) infiltrating into allografts, but not into autografts, in C57BL/6 mice were cytotoxic against syngeneic tumor cells and cell lines, whereas the cytotoxic activity was hardly induced in allografted, IFN-gamma-/- C57BL/6 mice. Among the leukocytes, Mphi were the major population of cytotoxic cells; and the cytotoxic activity appeared to be cell-to-cell contact dependent. When syngeneic tumor cells were s.c. injected into normal C57BL/6 mice simultaneously with the Mphi-rich population or allogeneic, but not syngeneic, fibroblastic cells, tumor growth was suppressed in a cell number-dependent manner, and tumor cells were rejected either with a Mphi:tumor ratio of about 30 or with an allograft:tumor ratio of approximately 200. In the case of IFN-gamma-/- C57BL/6 mice, however, the s.c. injection of the allograft simultaneously with tumor cells had no effect on the tumor growth. These results suggest that allograft or allograft-induced Mphi may be applicable for use in cancer treatment and that IFN-gamma induction by the allograft may be crucial for the treatment.     

Absence of host tumor necrosis factor receptor 1 attenuates manifestations of idiopathic pneumonia syndrome

The interaction of TNF-alpha with TNF receptor 1 (TNFR1) activates several signal transduction pathways that lead to apoptosis or NF-kappa B-dependent inflammation and immunity. We hypothesized that host TNFR1 expression contributes to noninfectious lung injury and inflammation commonly observed after bone marrow transplantation (BMT), termed idiopathic pneumonia syndrome (IPS). C57BL/6 TNFR1-sufficient (TNFR1(+/+)) and -deficient (TNFR1(-/-)) mice were total body irradiated with or without cyclophosphamide conditioning and were given bone marrow plus IPS-inducing donor spleen T cells from B10.BR wild-type mice. TNFR1(-/-) recipient mice exhibited improved early post-BMT survival associated with decreased permeability edema. In addition, the low lung compliance measured in anesthetized, ventilated TNFR1(+/+) mice on day 7 after BMT was restored to baseline during TNFR1 deficiency. Importantly, bronchoalveolar lavage fluid (BALF) inflammatory cells from TNFR1(-/-) vs. TNFR1(+/+) mice generated less nitric oxide (.NO) and nitrating species and exhibited suppressed programmed cell death as assessed using flow cytometry. However, cellular infiltration and levels of proinflammatory cytokines and chemokines were generally higher in BALF collected on day 7 after BMT from TNFR1(-/-) compared with TNFR1(+/+) recipient mice. Our results support a major role of host TNFR1 in regulation of .NO production and lung dysfunction after allogeneic BMT.     

Simultaneous absence of surfactant proteins A and D increases lung inflammation and injury after allogeneic HSCT in mice

The relative contributions of the hydrophilic surfactant proteins (SP)-A and -D to early inflammatory responses associated with lung dysfunction after experimental allogeneic hematopoietic stem cell transplantation (HSCT) were investigated. We hypothesized that the absence of SP-A and SP-D would exaggerate allogeneic T cell-dependent inflammation and exacerbate lung injury. Wild-type, SP-D-deficient (SP-D(-/-)), and SP-A and -D double knockout (SP-A/D(-/-)) C57BL/6 mice were lethally conditioned with cyclophosphamide and total body irradiation and given allogeneic bone marrow plus donor spleen T cells, simulating clinical HSCT regimens. On day 7, after HSCT, permeability edema progressively increased in SP-D(-/-) and SP-A/D(-/-) mice. Allogeneic T cell-dependent inflammatory responses were also increased in SP-D(-/-) and SP-A/D(-/-) mice, but the altered mediators of inflammation were not identical. Compared with wild-type, bronchoalveolar lavage fluid (BALF) levels of nitrite plus nitrate, GM-CSF, and MCP-1, but not TNF-alpha and IFN-gamma, were higher in SP-D-deficient mice before and after HSCT. In SP-A/D(-/-) mice, day 7 post-HSCT BALF levels of TNF-alpha and IFN-gamma, in addition to nitrite plus nitrate and MCP-1, were higher compared with mice lacking SP-D alone. After HSCT, both SP-A and SP-D exhibited anti-inflammatory lung-protective functions that were not completely redundant in vivo.     

Comparative signature-tagged mutagenesis identifies Pseudomonas factors conferring resistance to the pulmonary collectin SP-A

The pulmonary collectin, surfactant protein A (SP-A), is a broad spectrum opsonin with microbicidal membrane permeabilization properties that plays a role in the innate immune response of the lung. However, the factors that govern SP-A's microbial specificity and the mechanisms by which it mediates membrane permeabilization and opsonization are not fully understood. In an effort to identify bacterial factors that confer susceptibility or resistance to SP-A, we used comparative signature-tagged mutagenesis to screen a library of 1,680 Pseudomonas aeruginosa mutants for evidence of differential pulmonary clearance in SP-A-sufficient (SP-A) and SP-A-deficient (SP-A) mice. Two SP-A-sensitive P. aeruginosa mutants harboring transposon insertions in genes required for salicylate biosynthesis (pch) and phosphoenolpyruvate-protein-phosphotransferase (ptsP) were recovered. The mutants were indistinguishable from the parental wild-type PA01 with regard to opsonization by SP-A, but they exhibited increased susceptibility to SP-A-mediated membrane permeabilization. These results suggest that bacterial gene functions that are required to maintain membrane integrity play crucial roles in resistance of P. aeruginosa to the permeabilizing effects of SP-A.     

A subset of asialo GM1+ cells play a protective role in the occurrence of graft-versus-host disease in mice

In three different murine models of bone marrow (BM) transplantation the capacity of asialo GM1+ cells to suppress graft-vs-host disease (GVHD) was investigated. In a first model, total lymphoid irradiation (TLI)-treated BALB/C mice were given 1 mg of anti-asialo GM1 antibody. This led to the disappearance of functional suppressor cells after TLI. Injections of anti-asialo GM1 into TLI-treated BALB/C mice before infusion of 30 x 10(6) fully allogeneic (C3H) BM cells, led to a significantly decreased survival rate as compared to TLI-treated mice injected with control serum before BM transplantation (survival 29 and 83%, respectively, at 120 days after transplantation, p = 0.0032 log rank). The mortality of the former group was due to GVHD as 1 degree all dying animals showed clinical and histologic signs of GVHD, 2 degrees all animals were chimeric and 3 degrees mice receiving no or syngeneic BALB/C BM had excellent survival rates excluding BM aplasia or increased susceptibility for infections as reason for the mortality of the allogeneic BM recipients. In a second model, asialo GM1+ cells were removed in vitro from the C3H BM inoculum before injection into lethally irradiated (9 Gy) BALB/C recipients. In mice kept in specific pathogen-free conditions, this procedure resulted into a significant mortality (12/12) as compared to mice receiving BM pretreated with control serum (1/12, p = 0.0001 log rank). When kept in conventional housing, GVHD occurred in both groups but much earlier in the group receiving anti-asialo GM1-treated BM (median survival time 6 vs 46 days for the control mice, p = 0.001 log rank). No animal receiving anti-asialo GM1 and treated with syngeneic BM died, thus excluding toxicity, increased susceptibility to infections, or decreased graft take as a cause of mortality. In a last model, asialo GM1 cells were removed from syngeneic BM in a BM transplantation model in which T cell-depleted syngeneic (BALB/C) and non-T cell-depleted allogeneic (C3H) BM was administered to lethally irradiated (9 Gy) BALB/C mice. Also in this model GVHD-related mortality only occurred in the group of mice receiving syngeneic BM from which asialo GM+ cells were depleted before infusion (3/12). Our experiments thus clearly show that asialo GM1+ cells from both recipient (the TLI model) as well as donor origin (the TBI experiments) can suppress the occurrence of GVHD.     

Fas-deficient lpr mice are more susceptible to graft-versus-host disease

The Fas/Fas ligand (FasL) pathway is involved in a variety of regulatory mechanisms that could be important for the development of graft-vs-host disease (GVHD) after bone marrow transplantation (BMT), such as cytolysis of target cells by cytotoxic T cells, regulation of inflammatory responses, peripheral deletion of autoimmune cells, costimulation of T cells, and activation-induced cell death. To further evaluate the role of Fas/FasL in the complex pathophysiology of GVHD, we used Fas-deficient B6.lpr mice as recipients in a MHC-matched minor histocompatibility Ag-mismatched murine model for GVHD after allogeneic BMT (C3H.SW-->B6). We found a significantly higher morbidity and mortality from GVHD compared with control B6 recipients. In contrast, B6.lpr recipients had very little hepatic GVHD, although all other specific GVHD target organs (skin, intestines, and thymus) were more severely affected than in the control B6 recipients. B6.lpr recipients with GVHD demonstrated intact donor lymphoid engraftment and an increase in expansion of donor T cells and monocytes/macrophages compared with control B6 recipients. Serum levels of IFN-gamma and TNF-alpha were higher in B6.lpr recipients than in control B6 recipients, and monocytes/macrophages in B6.lpr recipients appeared more sensitized. B6.lpr recipients had more residual peritoneal macrophages after BMT, and peritoneal macrophages from B6.lpr mice could induce a greater proliferative response from C3H.SW splenocytes. This study demonstrates that the expression of Fas in the recipient is required for GVHD of the liver, but shows unexpected consequences when host tissues lack the expression of Fas for the development of GVHD in other organs and systemic GVHD.     

Mechanism of protection from graft-versus-host disease mortality by IL-2. III. Early reductions in donor T cell subsets and expansion of a CD3+CD4-CD8- cell population.

Reducing the graft-vs-host disease (GVHD)-promoting capacity of allogeneic T cells while maintaining alloengraftment and graft-vs-leukemia effects remains an important but elusive goal in clinical bone marrow transplantation (BMT). We have recently demonstrated that a short course of high dose IL-2 administered at the time of BMT has a powerful protective effect against GVHD mortality in mice. This short course of IL-2 is able to protect mice from both acute and chronic GVHD without sacrificing alloengraftment or graft-vs-leukemia effects of allogeneic T cells. Because the early administration of IL-2 seems to be crucial for this effect, we have studied the early lymphoid repopulation events after lethal irradiation and allogeneic BMT. These studies show that there are consistent delays in splenic repopulation by allogeneic cells after BMT in IL-2-treated animals compared with their untreated cohorts. Even greater percent reductions were seen in donor splenic T cell populations in the first few days after BMT in IL-2-treated animals. Splenic cells with the CD3+CD4-CD8- phenotype were increased in IL-2 treated animals at days 3 and 4 after BMT. This phenotype resembles that of bone marrow-derived cells which have been previously shown to inhibit GVHD, suggesting a possible mechanism for the protective effect of IL-2.     

Recipient nonhematopoietic antigen-presenting cells are sufficient to induce lethal acute graft-versus-host disease

The presentation pathways by which allogeneic peptides induce graft-versus-host disease (GVHD) are unclear. We developed a bone marrow transplant (BMT) system in mice whereby presentation of a processed recipient peptide within major histocompatibility complex (MHC) class II molecules could be spatially and temporally quantified. Whereas donor antigen presenting cells (APCs) could induce lethal acute GVHD via MHC class II, recipient APCs were 100-1,000 times more potent in this regard. After myeloablative irradiation, T cell activation and memory differentiation occurred in lymphoid organs independently of alloantigen. Unexpectedly, professional hematopoietic-derived recipient APCs within lymphoid organs had only a limited capacity to induce GVHD, and dendritic cells were not required. In contrast, nonhematopoietic recipient APCs within target organs induced universal GVHD mortality and promoted marked alloreactive donor T cell expansion within the gastrointestinal tract and inflammatory cytokine generation. These data challenge current paradigms, suggesting that experimental lethal acute GVHD can be induced by nonhematopoietic recipient APCs.     

KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT

We investigated keratinocyte growth factor (KGF) as a pretreatment therapy for idiopathic pneumonia syndrome (IPS) generated as a result of lung damage and allogeneic T cell-dependent inflammatory events occurring in the early peri-bone marrow (BM) transplant (BMT) period. B10.BR (H2(k)) recipient mice were transplanted with C57BL/6 (H2(b)) BM with spleen cells after lethal irradiation with and without cyclophosphamide conditioning with and without subcutaneous KGF pretreatment. KGF-pretreated mice had fewer injured alveolar type II (ATII) cells at the time of BMT and exhibited ATII cell hyperplasia at day 3 post-BMT. The composition of infiltrating cells on day 7 post-BMT was not altered by KGF pretreatment, but the frequencies of cells expressing the T-cell costimulatory molecules B7.1 and B7.2 and mRNA for the cytolysin granzyme B (usually increased in IPS) were decreased by KGF. Sera from KGF-treated mice had increases in the Th2 cytokines interleukin (IL)-4, IL-6, and IL-13 4 days after cessation of KGF administration (i.e., at the time of BMT). These data suggest that KGF hinders IPS by two modes: 1) stimulation of alveolar epithelialization and 2) attenuation of immune-mediated injury as a consequence of failure to upregulate cytolytic molecules and B7 ligand expression and the induction of anti-inflammatory Th2 cytokines in situ.     

Syngeneic or autologous graft-versus-host disease

Graft-versus-host disease (GVHD) until recently was supposed to occur only when immunocompetent T lymphocytes are infused into immunoincompetent allogeneic hosts that possess histocompatibility antigens not possessed by the donor that could act as targets for cell-mediated cytotoxicity. A syndrome clinically and histologically identical to mild allogeneic GVHD occurs infrequently, following syngeneic or autologous bone marrow transplantation (BMT). This syndrome called syngeneic or autologous GVHD can be regularly produced with Cyclosporine (CsA) in animals undergoing syngeneic or autologous BMT. Animals with this syndrome develop T cells that are autocytotoxic to Ia antigen-bearing cells. The presence of an irradiated thymus and CsA administration is necessary to produce this disease. Operationally, this disease results from a disturbance of balance between a normally present autoregulatory cell and an autocytotoxic T cell. The study of mechanisms involved in the generation of this disease will add to our fundamental understanding of the cellular regulation of autocytotoxic T cell-medicated reactions and diseases. Most recently, we have been able to induce this disease in man with the aim of investigating its therapeutic effect in autologous BMT in patients with Ia-bearing tumors.     

Bioimaging for the monitoring of the in vivo distribution of infused mesenchymal stem cells in a mouse model of the graft-versus-host reaction

Cell therapy using MSCs (mesenchymal stem cells) might be effective treatment for refractory GVHD (graft-versus-host disease). However, the fate and distribution of MSCs after transplantation remains unclear. In this study, an animal model was developed to monitor the dynamic distribution of MSCs in mice with GVHD. A GVHD mouse model was established by transplanting C57BL/6 donor bone marrow cells and C57BL/6 EGFP (enhanced green fluorescent protein) splenocytes into lethally irradiated BALB/c nude recipient mice. Donor MSCs were obtained from MHC-identical C57BL/6 RFP (red fluorescent protein) mice and infused into the recipient mice on the same transplantation day. In vivo movement of the donor splenocytes (EGFP) and MSCs (RFP) were evaluated by measuring the biofluorescence (IVIS-Xenogen system). Donor splenocytes and MSCs reached the lungs first, and then the gastrointestinal tract, lymph nodes and skin, in that order; the transit time and localization site of these cells were very similar. In the recipient mouse with GVHD, the number of detectable cells declined with time, as assessed by biofluorescence imaging and confirmed by RT (real-time)-PCR. This bioimaging system might be useful for preclinical testing and the design of therapeutic strategies for monitoring the dynamic distribution of MSCs with GVHD.     

Effects of T cell depletion in radiation bone marrow chimeras. I. Evidence for a donor cell population which increases allogeneic chimerism but which lacks the potential to produce GVHD

The opposing problems of graft-vs-host disease (GVHD) and failure of alloengraftment present major obstacles to the application of bone marrow transplantation (BMT) across complete MHC barriers. The addition of syngeneic T-cell-depleted (TCD) bone marrow (BM) to untreated fully allogeneic marrow inocula in lethally irradiated mice has been previously shown to provide protection from GVHD. We have used this model to study the effects of allogeneic T cells on levels of chimerism in recipients of mixed marrow inocula. The results indicate that T cells in allogeneic BM inocula eliminate both coadministered recipient-strain and radioresistant host hematopoietic elements to produce complete allogeneic chimerism without clinical GVHD. To determine the role of GVH reactivity in this phenomenon, we performed similar studies in an F1 into parent combination, in which the genetic potential for GVHD is lacking. The presence of T cells in F1 marrow inocula led to predominant repopulation with F1 lymphocytes in such chimeras, even when coadministered with TCD-recipient-strain BM. These results imply that the ability of allogeneic BM cells removed by T cell depletion to increase levels of allochimerism may be mediated by a population which is distinct from that which produces GVHD. These results may have implications for clinical BM transplantation.     

Cytotoxic antibodies against tumor cells in the blood of intact C3H mice]

Cytotoxic antibodies against mouse mammary tumour cells, L-cells and hepatoma 22a cells have been found in the serum of C3H/f and C3H/He mice over 8 months of age. Analogues antibodies were found in the serum of young and old BALB/c mice, but not in C57BL/6 mice. The cytotoxic activity of antimammary tumour cell serum has been completely abolished by its depletion by renal tissue of syngeneic and allogeneic animals.