Syngeneic Bone Research Articles

Abstract 2191: Alteration of anti-tumoral immunity in the pre-metastatic bone microenvironment via autonomic nerve system dysfunction

Abstract Bone metastasis is a major cause of morbidity and mortality for breast cancer patients. Bone is a unique metastatic microenvironment because of complex interactions among numerous distinct cell types such as osteo-blasts, -clasts, -cytes and marrow immune cells in physiological and pathological conditions. Imbalanced bone homeostasis by diverse factors such as the sympathetic nerve system (SNS) activation, potentially contribute to the progression of bone metastasis, yet precise mechanisms remain unclear. We investigated the effects of beta 2 adrenergic receptor (β2AR) activation in osteoblasts induced by prolonged SNS stimulation on anti-tumoral immunity in bone metastasis. We treated female Balb/C with chronic immobilization stress (CIS; 2 h. daily) and found that CIS increased syngeneic intra-tibial 4T1 bone metastasis growth as well as the number of CD11b+Gr1+ myeloid-derived suppressor cells (MDSC) counterbalancing anti-tumoral cytotoxic T cells in bone microenvironment. A β2AR-specific inhibitor (ICI-118551) or MDSC depletion using anti-Gr1 antibodies reversed CIS-induced bone metastatic tumor growth. More importantly, 2-week CIS pre-treatment increased EDU+ MDSC proliferation in the bone marrow, contributing to subsequent bone metastatic tumor growth and micro-metastatic seeding in intra-tibial and intra-cardiac tumor injection models, respectively. When CIS was administered after the establishment of bone metastatic tumors, the pro-tumorigenic effects were marginal. In osteoblast-specific β2AR knockout mice (Adrb2flox/flox; murine 2.3kb Col1-CreC57BL6 mice), CIS did not increase MDSC proliferation compared with littermate floxed controls, suggesting that β2AR in osteoblasts upregulates MDSC in bone microenvironment. RNA-sequencing transcriptome analysis of saline- or clenbuterol (a selective β2AR agonist)-treated murine osteoblasts showed that interleukin-6 (IL6) was the most significantly increased cytokine. Indeed, CIS or clenbuterol treatment increased IL-6 expression in vivo (immunohistochemistry) and in vitro (cultured osteoblasts), respectively. MDSC isolated from the bone marrow of CIS-treated mice had significantly increased STAT3 phosphorylation by flow cytometry and the expression of immuno-suppressive functional molecules such as arginase 1 (Arg1) and indolamine 2,3-dioxygenase 1 (Ido1) compared to those from control mice. Our data collectively demonstrated that SNS-dependent β2AR in osteoblasts expands and activates MDSC in the pre-metastatic bone microenvironment via the IL6-STAT3-Arg1/IDO1 pathway, creating a fertile soil for bone metastasis progression. Our data provided the first direct evidence for anti-tumoral immunity and pro-tumorigenic effects of autonomous nerve system dysfunction specific to the metastatic bone microenvironment. Citation Format: Eun Jung Lee, Da Hyeon Yun, Seungpil Jung, Serk In Park. Alteration of anti-tumoral immunity in the pre-metastatic bone microenvironment via autonomic nerve system dysfunction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2191.

MO066GASDERMIND MUTATION IS PROTECTIVE AGAINST RENAL ISCHEMIA REPERFUSION INJURY

Abstract Background and Aims Ischemia reperfusion injury (IRI) is an important contributor to acute kidney injury (AKI) and manifests as delayed graft function following kidney transplantation. Limiting the damage of IRI has implications on graft outcomes and has driven further exploration of the underlying pathophysiology. We hypothesize that pyroptosis, a pro-inflammatory form of cell death, has an important role in IRI and AKI. The pyroptosis pathway converges to the cleavage and release of N-terminal of the Gasdermin-D protein, leading to pore formation in the cell membrane and cell death. We examined the effects of Gasdermin-D mutation on inflammation in acute kidney injury. Method Male C57BL/6 mice were exposed to ethyl-N-nitrosourea mutagenesis, leading to a loss-of-function, single nucleotide polymorphism (isoleucine to asparagine mutation, I105N) in the Gasdermin-D gene. Age- and gender-matched littermate control wild-type, heterozygous and homozygous Gasdermin-DI105N mice were subjected to bilateral renal IRI (36°C, 22mins) and sacrificed 24-hours post-reperfusion for analysis of renal function, histology and biomolecular phenotyping. To delineate if the GasderminD mutation in renal parenchymal or hematopoietic cells were key drivers of IRI, we generated chimeric mice with whole body irradiation and infusion of syngeneic donor bone marrow. Following 8 weeks of engraftment, bilateral renal IRI was performed with analysis at 24 h reperfusion. Results Homozygote and heterozygote Gasdermin-DI105N mice were protected from renal IRI in a gene dose-dependent manner when compared to wild-type, with lower mean serum creatinine (15.7, 48.1 and 85.5µmol/L respectively, p<0.001), less histological tubular injury and cell death (1.8, 3.6 and 5.1 TUNEL+ cells/hpf, p<0.01) and significantly decreased expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, RANTES). Homozygote GasderminDI105N chimeric mice (reconstituted with wild-type donor bone marrow) were more susceptible to IRI, and serum creatinine was similar to that of wild-type chimeric control mice, indicating that hematopoietic cells rather than parenchymal cells, are likely predominant drivers of injury. Similarly, adoptive transfer of CpG-activated CD11c+ dendritic cells into homozygous Gasdermin-DI105N mice augmented renal injury compared to GpC-treated cells. Conclusion GasderminDI105N mice were protected from IRI and demonstrates the importance of the pyroptosis pathway on acute kidney injury. Manipulation of GasderminD is potentially an attractive target to mitigate inflammation and cellular death following injury.

A Comparative Evaluation of Commercially Available Cell-Based Allografts in a Rat Spinal Fusion Model.

To evaluate the comparative abilities of commercially available, viable, cellular bone allografts to promote posterolateral spinal fusion. Human allografts containing live cells were implanted in the athymic rat model of posterolateral spine fusion. Three commercially available allogeneic cellular bone matrices (Trinity Evolution, Trinity ELITE and Osteocel Plus) were compared with syngeneic iliac crest bone as the control. All spines underwent radiographs, manual palpation, and micro-computed tomography (CT) analysis after excision at 6 weeks. Histological sections of randomly selected spines were subjected to semiquantitative histopathological scoring for bone formation. By manual palpation, posterolateral fusion was detected in 40% (6/15) of spines implanted with syngeneic bone, whereas spines implanted with Trinity Evolution and Trinity ELITE allografts yielded 71% (10/14) and 77% (10/13) fusion, respectively. Only 7% (1/14) of spines implanted with Osteocel Plus allografts were judged fused by manual palpation (statistically significantly less than ELITE, P < .0007, and Evolution, P < .0013). The mineralized cancellous bone component of the allografts confounded radiographic analysis, but Trinity Evolution (0.452 ± 0.064) and Trinity ELITE (0.536 ± 0.109) allografts produced statistically significantly higher bone fusion mass volumes measured by quantitative micro-CT than did syngeneic bone (0.292 ± 0.109, P < .0001 for ELITE and P < .003 for Evolution) and Osteocel Plus (0.258 ± 0.103, P < .0001). Semiquantitative histopathological scores supported these findings because the total bone and bone marrow scores reflected significantly better new bone and marrow formation in the Trinity groups than in the Osteocel Plus group. The Trinity Evolution and Trinity ELITE cellular bone allografts were more effective at creating posterolateral fusion than either the Osteocel Plus allografts or syngeneic bone in this animal model. The superior fusion rate of Trinity cellular bone allografts may lead to better clinical outcome of spinal fusion surgeries.

Abstract 3936: Efficacy of single agent radium-223 in the syngeneic MBT-2 bladder cancer bone growth model in mice

Abstract Radium-223 dichloride (Ra-223, Xofigo®) is a targeted alpha therapy which prolongs the survival of castration-resistant prostate cancer (CRPC) patients with symptomatic bone metastases. As a calcium-mimetic, Ra-223 selectively binds to hydroxyapatite and targets areas of high bone turnover such as bone metastases. Here, we report the effects of Ra-223 on development and progression of osteolytic bone lesions and on survival in the syngeneic intratibial MBT-2 murine bladder cancer model in immunocompetent mice. Female 5-7-week-old C3H/HeNHsd mice were injected intratibially with 5x105 MBT-2 cells on day 0. The mice were stratified based on body weight (n=40 per group) on day 5 and Ra-223 (300 kBq/kg, i.v.) or vehicle control was administered on days 5, 33, and 61. The tumor-induced osteolytic lesion area was imaged using radiography on days 10, 18, 25, 76, and at sacrifice. The mice were sacrificed when they met the pre-defined sacrifice criteria (&amp;gt; 20% body weight loss, palpable tumor outgrowth from the bone or general deterioration of health status). The study was terminated on day 158. In this study, the first mice met the sacrifice criteria on study day 24 and the survival curves plateaued at 52% after 84 days in the control group and at 68% after 112 days in the Ra-223 treatment group. Survival on day 40 was significantly improved in the Ra-223 treatment group compared to the vehicle control (p&amp;lt; 0.001), but the effect was not statistically significant at the end of the study. Based on X-ray imaging 25 days after cancer cell inoculation, Ra-223 treatment also significantly decreased the bone lesion area compared to the vehicle treatment (p=0.0041). After initially sacrificing the mice with extensive bone lesions, tumor growth in bone was stabilized around day 25. The main reason for sacrifice at a later stage was breathing difficulties due to lung metastases, and in the Ra-223-treated mice only one mouse was sacrificed due to bone metastases. Ra-223 treatment was well-tolerated; no body weight loss was observed. In summary, radium-223 dichloride (Xofigo®) demonstrates moderate single agent in vivo efficacy in the intratibial MBT-2 murine bladder cancer model of osteolytic bone metastasis. Retaining a functional immune system in syngeneic mouse models is particularly relevant for the characterization of potential immune-stimulatory effects of Ra-223. The promising results obtained in this model warrant further investigation of Ra-223 in combination with immuno-oncological treatments like checkpoint inhibitors. Citation Format: Tiina E. Kähkönen, Mari I. Suominen, Jenni H. Mäki-Jouppila, Birgitta Sjöholm, Ilmari Ahonen, Dominik Mumberg, Karl Ziegelbauer, Jussi M. Halleen, Sanna-Maria Käkönen, Arne Scholz. Efficacy of single agent radium-223 in the syngeneic MBT-2 bladder cancer bone growth model in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3936.

Influence of the induced membrane filled with syngeneic bone and regenerative cells on bone healing in a critical size defect model of the rat’s femur

IntroductionThe induced membrane technique for the treatment of large bone defects consists of a 2-stage procedure. In the first stage, a polymethylmethacrylate (PMMA) cement spacer is inserted into the bony defect of a rat’s femur and over a period of 2–4 weeks a membrane forms that encapsulates the defect/spacer. In a second operation the membrane is opened, the PMMA spacer is removed and the resulting cavity is filled with autologous bone.Since little effort has been made to replace the need for autologous bone this study was performed to elucidate the influence of different stem cells and the membrane itself on bone healing in a critical size femur defect model in rats.Especially the question should be addressed whether the use of stem cells seeded on a β-TCP scaffold is equivalent to syngeneic bone as defect filling in combination with the induced membrane technique. Materials and MethodsA total of 96 male Sprague-Dawley (SD) rats received a 10 mm critical size defect of the femur, which was stabilized by a plate osteosynthesis and filled with PMMA cement. In a second step the spacer was extracted and the defects were filled with syngeneic bone, β-TCP with MSC + EPC or BM-MNC. In order to elucidate the influence of the induced membrane on bone defect healing the induced membrane was removed in half of the operated femurs. The defect area was analysed 8 weeks later for bone formation (osteocalcin staining), bone mineral density (BMD) and bone strength (3-point bending test). ResultsNew bone formation, bone mineral density and bone stiffness increased significantly, if the membrane was kept. The transplantation of biologically active material (syngeneic bone, stem cells on b-TCP) into the bone defect mostly led to a further increase of bone healing. Syngeneic bone had the greatest impact on bone healing however defects treated with stem cells were oftentimes comparable. ConclusionFor the first time we demonstrated the effect of the induced membrane itself and different stem cells on critical size defect healing. This could be a promising approach to reduce the need for autologous bone transplantation with its’ limited availability and donor site morbidity.

Abstract P5-05-06: Development of advanced pre-clinical in vivo models of metastatic breast cancer

Abstract Backgrounds: The fact that we continue to lose 40,000 women with breast cancer every year in the US despite the recent advance in basic research clearly demonstrate disconnect in translation of basic research findings to clinic. This is largely due to lack of appropriate animal model that mimic clinical conditions for preclinical studies that result in high failure rate of clinical trials. To date, we had established many syngeneic mouse models, which are not free from limitations; 1) few clinically relevant animal models with bone metastasis have been established, 2) syngeneic mouse model cannot address human cancer genomics and tumor heterogeneity. Patient-Derived Xenograft (PDX) model has emerged as pre-clinical model to address these issues, however, it suffers low tumor take rate of around 20-40%, and lack metastatic model. Here, we describe development of orthotopic implantation, and bone and liver metastatic breast cancer mouse models to overcome these limitations. Methods: 1) 4T1.2-luc3 cells that has metastatic potential to the bone were orthotropically inoculated as a syngeneic mouse model, imaged with IVIS and MRI. 2) Patient tumor tissues of 1mm(3) were implanted surgically into dorsal subcutaneous space (SQ), or orthotropically into mammary fat pat #2 and #4 (MFP). Results: 1) We established a syngeneic breast cancer bone metastasis model. Primary tumors were surgically resected days after 4T1.2-luc3 cells were orthotopically implanted under direct vision. Removal of primary tumor allowed bioluminescent visualization and quantification of bone metastasis by IVIS. We found that MRI was effective in evaluating bone metastasis and bone related events in these mice. MRI allows differentiation of bone metastasis from metastasis to the surrounding organs with bone destruction image, whereas conventional bioluminescence imaging shows only existence of cancer cells. 2) The overall tumor take rate of the tumor in PDX model was 46.0% (74/161 implantation site). Take rate from triple-negative breast cancer tumors was 56.1% (74/132), on the other hand, that from ER positive tumors was 0% (0/39). Tumor take rate was significantly better in MFP implantation than SQ (39.5%, 30/76 vs 51.2%, 44/85, p&amp;lt;0.01). Tumor weight were significantly heavier in MFP compared to SQ (0.072g vs 0.328g, p&amp;lt;0.00001). With more passage, the difference in tumor weight between SQ and MFP was significantly increased(p&amp;lt;0.0001). Finally, we developed a PDX breast cancer liver metastasis model by surgically implanting tissue fragment into liver using direct vision technique. We found MRI to be very useful as a living imaging modality to evaluate cancer progression in the deeply located metastatic sites of PDX models. Conclusions: We have established orthotropic syngeneic breast cancer bone metastasis model as well as improved breast cancer PDX model with synchronous liver metastasis utilizing MRI. Our novel models could be powerful tools for preclinical studies. Citation Format: Okano M, Kawaguchi T, Okano I, Katsuta E, Takabe K. Development of advanced pre-clinical in vivo models of metastatic breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P5-05-06.

MHC Class I Expression by Donor Hematopoietic Stem Cells Is Required to Prevent NK Cell Attack in Allogeneic, but Not Syngeneic Recipient Mice

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs.

Feasibility of establishment of rat model of bone cancer pain by using Walker 256 cells cultured in vitro or in vivo

To investigate the possibility of establishing rat model of bone cancer pain using cancer cells cultured in vitro or by ascites passaging and verify the reliability of this method. Syngeneic SD rat carcinoma cells of the line Walker 256 were cultured in vitro and inoculated into the peritoneal cavity of SD rats respectively. Two kinds of Walker 256 cell suspension were made. Thirty-two SD rats were randomly divided into 4 equal groups: Group N (undergoing injection of Hank's solution into cavitas medullaris of left tibia), Group K (undergoing injection of heat-killed Walker 256 cells), Group V (injected with Walker 256 cells cultured in vitro), and Group A (injected with Walker 256 cells passaged in ascites). After 6, 12, and 18 days, the rats underwent roentgenography. Radio nuclide emission computed tomography (ECT) was conducted 12 days later. And MRI was conducted 15 days later. Thermal withdrawal latency (TWL) and pressure withdrawal threshold (PWT) were measured. Von Frey threshold and weight bearing of left hind limb were examined. On the 18th day after the establishment of model the rats were killed with their left tibia taken out to undergo microscopy. The rats of Groups A and V began to display decrease of left hint limb activity and roentgenography showed minute defect of bone trabecula in the proximal epiphysis by day 6. By day 12 roentgenography showed multiple defect of bone trabecula, ECT showed reactive bone formation. The rats of Groups A and V displayed signs of weight loss by day 14, and by day 18 roentgenography showed full thickness bicortical bone loss and formation of soft tissue tumor. Histological examination 18 days later revealed that the bones inoculated with live cells showed infiltration of bone marrow spaces by malignant tumor. The PWT values gradually decreased since day 6 to day 18, and the PWT values in this period of Groups A and V were all significantly lower than those of Groups K and N (all P < 0.01). The von Frey values gradually decreased since day 6 to day 18, and the von Frey values in this period of Groups A and V were all significantly lower than those of Groups K and N (all P < 0.01). The weight bearing value of left hind limb gradually decreased and the weight bearing difference between the 2 hind limbs gradually increased since day 6 to day 18, and the values of weight bearing difference between the 2 hind limbs of Groups A and V were significantly higher than those of Groups K and N (all P < 0.01). Sixteen rats underwent subcutaneous injection of morphine, and the PWT values increased 20, 30, and 40 min later in a dose-dependent manner (P < 0.01). Naloxone injected 1 h later antagonized the analgesic effect of morphine. A SD rat model of bone cancer pain has been successfully established by using syngeneic rat bone carcinoma cells cultured in vitro or in vivo, and the latter being more convenient.

Effects of ex vivo activated immune cells on syngeneic and semi-allogeneic bone marrow transplantation in mice

We have previously shown that a novel immunotherapy using ex vivo activated immune cells is capable of promoting survival and hematopoietic recovery in mice after combined chemotherapy and radiotherapy. In this study, we investigated whether the immunotherapy with ex vivo activated immune cells had the same beneficial effects after syngeneic and semiallogeneic bone marrow transplantation (BMT) in BALB/c mice subjected to a lethal dose of total body irradiation (TBI). Immune cells were cultured in vitro with a combination of cytokines and a calcium ionophore for 2 days and subsequently injected to mice daily for 4 days starting 1 day after BMT. The immunotherapy enhanced survival and multilineage peripheral blood recovery in BMT mice with limited numbers of transplanted bone marrow cells when a low dose of ex vivo activated immune cells were used. However, the beneficial effects were completely lost when a higher dose of the same therapeutic immune cells were tested, and instead the immunotherapy significantly exacerbated complications associated with the lethal radiation and BMT. This detrimental effect appeared to be the result of strong in vivo nonspecific immune responses induced by either activated therapeutic immune cells or interaction between therapeutic immune cells and MHC-mismatched bone marrow cells transplanted or both. Our data suggest that the immunotherapy with appropriately selected dosages may be beneficial to BMT but vigorous in vivo immune responses soon after BMT may exacerbate post-transplant complications.

Transplantation of Bioencapsulated Bone Marrow Stem Cells Improve Hepatic Regeneration and Survival of 90% Hepatectomized Rats: A Preliminary Report

We transplanted bioencapsulated bone marrow stem cells intraperitoneally into 90% hepatectomized rats and found that this increases both the rates of hepatic regeneration and survival of the animals. Bone marrow cells isolated from Wistar rats were bioencapsulated using alginate-polylysine-alginate method. These bioencapsulated bone marrow cells were transplanted intraperitoneally into 90% hepatectomized syngeneic wistar rats. Control groups included 90% hepatectomized group receiving intraperitoneal injection of either empty microcapsules or free bone marrow cells. Unlike the control groups, transplantation of bioencapsulated bone marrow cells improved the survival of 90% hepatectomized rats, with an efficacy similar to that of bioencapsulated hepatocytes or free hepatocytes. These results suggest that syngeneic bioencapsulated bone marrow stem cells can increase the survival rates of 90% hepatectomized rats. We also discuss the potential for a new alternative to hepatocyte transplantation for cellular therapy of acute liver failure. In particular, bone marrow stem cells can be obtained from the same patient with no immunorejection, whereas in hepatocyte transplant, immunosuppressant will be needed to prevent immunorejection of the donor hepatocytes.

Linomide administration following bone marrow transplantation in mice.

The effect of linomide, an immunomodulatory drug, on natural killer (NK) cells and T cell-dependent immune responses following syngeneic or allogeneic bone marrow transplantation (BMT) was investigated in BALB/c mice inoculated with B-cell leukemia (BCL1). Linomide given in the drinking water had no impact on graft survival or graft versus leukemia (GVL) effects. Although linomide regulates anti-self reactivity in mice with experimental and spontaneous autoimmune disorders, the anti-tumor effects induced by allogeneic donor lymphocytes were not affected. This indicates that different mechanisms regulate anti-self and anti-leukemia effects. Alternatively, linomide might affect the homing of self-reactive lymphocytes to specific target organs in autoimmune disorders, although the homing process may not be relevant to the control of leukemia by alloreactive lymphocytes.

Synthetic peptide thymodepressin promoted take of transplanted hemopoietic precursor cells in the bone marrow of irradiated mice.

We studied the possibility of using a new synthetic heme-inhibiting peptide thymodepressin for improving the efficiency of transplantations of syngeneic and allogeneic bone marrow. Thymodepressin was injected to recipients 3 times (48, 24, and 2 h) before irradiation and transplantation of bone marrow suspension. The yield of 9- and 12-day colonies increased, the number of proliferating CFU-S-12 and pre-CFU-S in recipient bone marrow increased in comparison with the control. In case of an allogeneic donor-recipient combination, the number of 9-day splenic colonies in thymodepressin-treated animals increased 5-fold compared to the control. We hypothesized that thymodepressin therapy and irradiation form an adaptive response of the recipient hemopoietic microenvironment in the bone marrow, which provides conditions for optimal functioning of donor hemopoietic precursors.

Disease of the liver following bone marrow transplantation in children: incidence, clinical course and outcome in a long-term perspective.

Sixty-four consecutive cases of allogeneic (n = 16), autologous (n = 47) or syngeneic (n = 1) bone marrow transplantation (BMT) in children with haematological or lymphoid malignancy, aplasia or metabolic disease were reviewed to assess the incidence, clinical presentation and outcome of liver disease. Median follow-up time was 5 y (1.0-10). No liver diagnosis was established at the pre-transplant check-up. During the first 100 d post-transplant, 81% of the patients had impaired liver function as documented by various biochemical parameters. Three of 64 patients (5%) met diagnostic criteria for veno-occlusive disease. Four (25%) of the 16 receiving allografts were diagnosed as having acute graft vs host disease (GVHD) with liver involvement (grades II-III). No patient died of liver disease. During the late post-transplant follow-up, one patient developed HCV hepatitis after packed erythrocyte transfusion. Four patients were diagnosed as having chronic GVHD with liver involvement; three of them also had an episode of CMV hepatitis. At their latest follow-up, the patients with chronic GVHD had aminotransferase values 1.5-3 times the normal, whereas all other long-term survivors had normal or near-normal liver function tests. We conclude that the incidence of serious liver disease was low in this paediatric population of bone marrow recipients.

IN VIVO REACTIVITY OF T CELL CLONES ISOLATED FROM MICE WITH SYNGENEIC GRAFT-VERSUS-HOST DISEASE

Syngeneic graft-versus-host disease (SGVHD) has been shown to occur in murine syngeneic radiation bone marrow chimeras following a short course of cyclosporine. To analyze the effector mechanisms present in diseased animals, four T cell clones (1D5, 1D8, 1C10, 2D8) were isolated from the spleens of C3H/HeN mice late in the disease course by cloning on irradiated syngeneic spleen cells. These clones were CD4+, alpha beta TCR+ and responded to I-Kk in vitro. In addition to I-Ek reactivity, three of the clones exhibited crossreactivity with the superantigen MIs 1a (mtv 7). Clones 1D5 and 1C10 were found to express TCR V beta chains (V beta 4 and V beta 8.1, respectively), which are normally present in the T cell repertoire of C3H/HeN mice. All SGVHD clones were found to be autoreactive in that they responded to syngeneic stimulator cells in the absence of xenogeneic serum proteins. To test in vivo activity, the 1D5 SGVHD clone was injected into the hind footpad of mice where it was shown to induce footpad swelling in a cell dose-dependent, I-Ek-specific manner in sublethally irradiated, but not normal mice. Histological analysis indicated that the clone induced dermal and subcutaneous edema that correlated directly with injection of 1D5 and not the control clone. Preliminary experiments suggested that the other three autoreactive clones behaved in a similar manner. These data are consistent with the involvement of a self-class II-specific CD4+ T cell in murine SGVHD.

Induction of immunological tolerance in full major and multiminor histocompatibility-disparate mice using a mixed bone marrow transplantation model.

Bone marrow transplantation (BMT) has been used to induce and maintain immunological tolerance. Such tolerance could facilitate tissue and organ transplantation between the donor and the recipient without need for continuous immune suppression. A protocol employing transplantation of a mixture of T-cell depleted (TCD) syngeneic plus TCD-allogeneic bone marrow cells has been successfully used for induction of transplantation tolerance between mice that differ at components of the major histocompatibility barrier (MHC), or for crossing the xenogenic barrier. We examined the production of specific immunological tolerance using mixed syngeneic plus allogeneic TCD-BMT to cross the entire major plus multiminor histocompatibility barriers. The transplanted mice were repopulated in a stable manner with a mixture of both donor and recipient phenotypes. On histological examination, the mice were reconstituted with hemopoietic and immunocompetent lymphocytes as assayed by their responses to the thymus-dependent cellular antigen, sheep red blood cells (SRBC) in an in vivo plaque-forming cell assay. The transplanted mice were found to be stable chimeras and expressed long lasting tolerance of both donor and recipient cells and yet they were fully reactive to third party cells in mixed lymphocyte culture. These results provide evidence that "supportive" or accessory cells in the syngeneic marrow facilitate maturation of donor marrow cells into fully functioning immunocytes in an allogeneic environment crossing the MHC barrier, which represents the greatest known challenge to allogeneic marrow transplantation in mice. The MHC-mismatched mixed allogeneic transplantation method may improve organ engraftment in human recipients of BMT from a partially mismatched donor or from a cadaver donor, and may significantly improve graft acceptance from a fully matched sibling donor.

The mechanism of recurrence of mouse myeloid leukaemia after total body irradiation and bone marrow transplantation.

RFM mice were X-irradiated (9.5 Gy) 3, 4, 5, 6 or 7 days after inoculation of a transplantable strain-specific myeloid leukaemia (ML) and were reconstituted or not with syngeneic or allogeneic bone marrow cells. Recurrent leukaemia was observed in mice with either type of the bone marrow transplant, indicating that ML cells survived the dose of 9.5 Gy of X-rays. ML cells exposed in vitro to high doses of X-rays (20, 30, 40, 50, 60 Gy) and injected into lethally irradiated syngeneic recipients were still capable of forming leukaemic colonies on the spleens. Higher doses (70, 80, 90 and 100 Gy) abolished the colony formation completely. Irradiated ML cells were also capable of causing leukaemia (hepatosplenomegaly) if inoculated into lethally irradiated CBA mice reconstituted with bone marrow cells of CBA or RFM mice. That was attributed to the release of a leukaemogenic factor from the ML cells, capable of transforming transplanted normal cells.

CD4+ T CELLS, BUT NOT CD8+ T CELLS, MEDIATE THE BREAKING OF TOLERANCE IN MIXED ALLOGENEIC CHIMERAS (B10 + B10.BR ± B10)

Reconstitution of mouse recipients with a mixture of syngeneic plus allogeneic bone marrow (A+B-->A) results in stable mixed lymphohematopoietic chimerism and donor-specific transplantation tolerance. Previously, it was reported that administration of large numbers of unmanipulated host-type splenocytes to neonatal or adult radiation bone marrow chimeras resulted in a loss of chimerism and donor-specific transplantation tolerance. To characterize the phenotype(s) of cells that were responsible for this loss of chimerism, we performed depletion of various subsets of unmanipulated B10 splenocytes prior to infusion into mixed allogeneic chimeras (B10 + B10.BR-->B10). Recipients were followed serially to identify changes in the level of donor chimerism and by in vitro functional assays of tolerance. We report here that CD4+ T cells, but not CD8+ T cells, were sufficient to mediate the loss of donor chimerism. In all recipients in which allogeneic chimerism became undetectable, there was a simultaneous loss of donor-specific transplantation tolerance.

Induction of specific T-cell responsiveness to allogeneic bone.

The capacity of fresh murine allogeneic bone to induce a specific immune response in vitro was studied. T-cells stimulated by allogeneic bone in vitro were collected and were characterized for state of activation, cell-surface phenotype, and antigen specificity. The stimulating antigens were determined by genetic mapping with use of recombinant inbred strains of mice and by blocking of mixed lymphocyte cultures with use of neutralizing antibodies. Purified T-cells were cultured alone or with allogeneic or syngeneic bone. In some experiments, the bone marrow was removed before in vitro culture. Responding cells were recovered after a secondary exposure to the stimulating bone. Primed cells were used immediately or cell-lines were developed. The data demonstrated that (1) allogeneic bone activated T-cells and induced their proliferation; (2) bone-induced proliferation of T-cells was specific for antigens that map to the major histocompatibility complex of the bone donor; (3) within the major histocompatibility complex, the antigens responsible for proliferation of T-cells were apparently class-I and class-II determinants; (4) removal of bone-marrow cells had no effect on the ability of that bone to stimulate alloreactivity; and (5) all of the alloreactive T-cells had the cell-surface phenotype Thy-+ CD8+ CD4-.

Revascularisation of bone grafts in rats.

Revascularisation of syngeneic and allogeneic intramuscular bone grafts have been studied using radioactive microspheres to measure the ingrowth of blood vessels. New bone formation and resorption were measured by 85strontium uptake and by graft weight reduction. Revascularisation, and mineralisation rate were significantly higher in syngeneic grafts than in allogeneic grafts at two, three and six weeks after implantation. The syngeneic grafts lost weight faster indicating that the allogeneic grafts resorbed more slowly. The ingrowth of new vessels is impaired in allogeneic bone, and this probably inhibits the rate of bone formation and resorption of the grafts.

Prolonged nitrous oxide exposure inhibits settlement of transplanted hemopoietic stem cells in murine spleen.

In order to clarify the mechanism of hemopoietic depression induced by nitrous oxide inhalation, effects of prolonged nitrous oxide exposure on the settlement of transplanted bone marrow cells were investigated. Mice were continuously exposed to mixed gas containing 50% nitrous oxide, 21% oxygen and 29% nitrogen for 7 days and then they were irradiated with 850 rads. By the irradiation, endogenous pluripotent hemopoietic stem cells (CFU-S) almost disappeared in the mice. Normal syngenic murine bone marrow cells were injected intravenously and the numbers of CFU-S, which settled in the bone marrow and spleen 2 hr after injection, were measured. There was no difference of the numbers of CFU-S settled in the bone marrow between nitrous oxide and control gas exposed mice. In contrast, the numbers of CFU-S in the spleen of nitrous oxide exposed mice were approximately 60% of the control. These results and our previous data suggest that hemopoietic inhibitory effects of nitrous oxide in mice are due to a damage of splenic hemopoietic microenvironment, that supports the settlement of hemopoietic stem cells.