Experimental research on the cellular interactions during hemopoiesis

We present a review of experimental studies performed at the Laboratory of Histogenesis of the Institute of Developmental Biology, Russian Academy of Sciences, on the problem of cell interactions during hemopoiesis. Special attention has been given to original experimental models, such as production of hemopoietic foci on underlayers of fibroblasts encapsulating a foreign body in the peritoneal cavity of rodents (after intraperitoneal transplantation of hemopoietic cells) and repopulation of ectopic hemopoietic territories under the kidney capsule of mice by syngeneic or xenogeneic hemopoietic cells. We describe the competitive interactions of genetically different hemopoietic cells after the transplantation of their mixtures to irradiated mice (multicomponent radiation chimeras). Xenogeneic and multicomponent chimeras have also been obtained in long-term bone marrow culture. We have examined characteristics of hemopoiesis on stromal cell underlayers produced by cells of various origins in vitro and then transplanted into the peritoneal cavity of irradiated mice. We discuss the results obtained and possible mechanisms of these phenomena.     

Mixed xenogeneic chimerism induces donor-specific humoral and cellular immune tolerance for cardiac xenografts

Xenotransplantation has been suggested as a potential solution to the critical shortage of donor organs. However, success has been limited by the vigorous rejection response elicited against solid organs transplanted across species barriers. Mixed xenogeneic bone marrow chimeras resulting from the transplantation of a mixture of host and donor marrow (B10 mouse + F344 rat --> B10 mouse) results in donor-specific cross-species transplantation tolerance for subsequent nonvascularized skin and islet grafts. Furthermore, compared with fully xenogeneic chimeras (rat --> mouse), mixed xenogeneic chimeras exhibit superior immunocompetence for infectious agents in vivo and in vitro, suggesting that the immune system is intact. The ability to establish long-term humoral and cellular tolerance for primarily vascularized xenografts in vivo, in the setting of both recipient and donor Ig and effector cell production, has not previously been characterized. Mixed xenogeneic chimeras exhibit donor-specific humoral tolerance as evident by the absence of anti-donor Ab and Ab-dependent donor-specific cytotoxicity in vitro and intravascular IgM deposition within donor-strain (F344) cardiac xenografts in vivo. F344 cardiac xenografts are accepted (median > or =180 days) without clinical or histologic evidence of rejection, suggesting cellular tolerance. In contrast, MHC-disparate third-party mouse (B10.BR) and rat (ACI or WF) grafts are rejected (median of 23 and 41 days, respectively) in association with extensive mononuclear cell infiltration and vascular deposits of mouse IgM. These results demonstrate that mixed xenogeneic chimerism establishes donor-specific humoral and cellular tolerance and permits the successful transplantation of even primarily vascularized xenografts in the setting of intact Ab production.     

H-2-restricted graft-versus-host reaction: Foreign determinants and restriction elements

Nylon-wool-purified T cells from radiation chimeras cause a lethal graft-versus-host reaction (GVHR) in irradiated, bone-marrow-protected recipients only if the recipient shares a restriction element with the T-cell donor and also expresses antigens foreign to the donor. Class I molecules (H-2K and H-2D) can act as restriction elements, but restriction to class II molecules could not be demonstrated. However, class II molecules as well as H-2K and some non-H-2 determinants could serve as foreign antigens.     

Characteristics of a xenogeneic lymphocyte transfer reaction: its use in the study of graft versus host capability of mouse lymphoid cells from various anatomic sites.

A xenogeneic intradermal lymphocyte transfer test is described in which mouse lymphoid cells from various anatomic compartments were injected intó guinea pigs. Murine peripheral blood lymphocytes and peritoneal exudate (mineral oil induced) cells gave significantly greater responses than did cells from the spleen or non-inflammatory peritoneal exudates. The “flare” reaction, characteristic of the allogeneic lymphocyte transfer test, was absent. Similar experiments conducted with presensitized donor cells produced a marked “flare” reaction, but only with blood and inflammatory exudate lymphocytes. The fact that the flare could be ablated by prior in vitro irradiation of donor cells suggests that the flare reaction is related, at least in part, to the mitosis of these cells in the host. Effects of host manipulations such as prior irradiation and decomplementation suggest that host factors may play a role in the expression of dermal inflammation. It is concluded that this particular xenogeneic transfer response varies with either; a) the number of effector cells in the transferate or; b) the differential ability of a given murine lymphoid population to survive in guinea pig skin or, to varying degrees, a combination of both.     

Transplantable liver production plan

Organ grafts developed in the xenogeneic pig scaffold are expected to resolve most issues of donor safety and ethical concerns about living-donor liver transplantation in Japan. We have been working on so-called “Yamaton” projects to develop transplantable organs using genetically engineered pigs. Our goal is to produce chimeric livers with human parenchyma in such pigs. The Yamaton-Liver project demonstrated the proof of concept by showing that rat–mouse chimeric livers could develop in mice and be successfully transplanted into syngeneic or allogeneic rats. Under conventional immunosuppression, the transplanted livers showed long-term function and protection against rejection. Because chimeric liver grafts have xenogeneic components, additional strategies, such as humanization of pig genes, induction of hematopoietic chimeras in donors, and replacement of pig endothelial cells with human ones, might be required in clinical use. Our projects still need to overcome various hurdles but can bring huge benefits to patients in the future.     

Contrasting feature in the repopulation of host-type T cells in the spleens of F1----P and P----F1 radiation bone marrow chimeras

The regeneration and persistence of host- and donor-derived T cells were examined in the thymus as well as the spleen of mouse radiation bone marrow chimeras of two semiallogeneic combinations (F1----P, P----F1) with different Thy-1 markers on T cells of donor and host origins. An unexpectedly large number of host-type T cells were recovered from the spleens of F1----P chimeras, amounting to as high as 45 and 25% of total T cells at 6 and 14 weeks after bone marrow transplantation (BMT), respectively. To the contrary, the residual host-type T cells in the spleens of P----F1 chimeras disappeared quickly, resulting in less than 0.1% of total T cells at 6 weeks after BMT. It was also revealed that the number of host-type T cells in the spleens of F1----P chimeras decreased in proportion to increase of radiation dose given to the recipients.     

Elimination of porcine hemopoietic cells by macrophages in mice.

The difficulty in achieving donor hemopoietic engraftment across highly disparate xenogeneic species barriers poses a major obstacle to exploring xenograft tolerance induction by mixed chimerism. In this study, we observed that macrophages mediate strong rejection of porcine hemopoietic cells in mice. Depletion of macrophages with medronate-encapsulated liposomes (M-liposomes) markedly improved porcine chimerism, and early chimerism in particular, in sublethally irradiated immunodeficient and lethally irradiated immunocompetent mice. Although porcine chimerism in the peripheral blood and spleen of M-liposome-treated mice rapidly declined after macrophages had recovered and became indistinguishable from controls by wk 5 post-transplant, the levels of chimerism in the marrow of these mice remained higher than those in control recipients at 8 wks after transplant. These results suggest that macrophages that developed in the presence of porcine chimerism were not adapted to the porcine donor and that marrow-resident macrophages did not phagocytose porcine cells. Moreover, M-liposome treatment had no effect on the survival of porcine PBMC injected into the recipient peritoneal cavity, but was essential for the migration and relocation of these cells into other tissues/organs, such as spleen, bone marrow, and peripheral blood. Together, our results suggest that murine reticuloendothelial macrophages, but not those in the bone marrow and peritoneal cavity, play a significant role in the clearance of porcine hemopoietic cells in vivo. Because injection of M-liposomes i.v. mainly depletes splenic macrophages and liver Kupffer cells, the spleen and/or liver are likely the primary sites of porcine cell clearance in vivo.     

Nonspecific suppressor elements in murine allogeneic radiation chimeras.

Spleen cells from long-term mouse allogeneic radiation chimeras were tested for their ability to modulate the graft-versus-host (GVH) or plaque-forming cell (PFC) response of normal lymphocytes transplanted in lethally X-irradiated recipients. In vivo GVH proliferation of normal lymphocytes (syngeneic to donor cells of the chimera) against antigens of host-type in which the chimeric state had been established was reduced by chimera cells. Inhibition varied, some chimeras suppressing GVH more than others and a few not suppressing at all. The suppressive effect was abrogated if the chimera cells were treated with anti-θ; treatment with anti-IgM did not eliminate this activity. When mixtures of normal donor lymphocytes and chimera cells were given to irradiated recipients genetically different from host or donor, reduction of donor cell GVH also occurred. Further, chimera cells reduced the GVH activity of normal host cells in irradiated recipients differing from the host at one H-2 locus and from the donor at minor histocompatibility loci. The modulating effect of spleen cells from chimeras on the PFC response by normal lymphocytes also varied. Six chimeras induced a 25 to 90% suppression, two enhanced the response, and one showed no effect. Where suppression occurred, treatment of chimera cells with anti-θ most often, but not always, restored PFC production. Our results show that the suppressive action of splenic lymphoid cells by chimeras is highly nonspecific and variable in expression. We suggest that tolerance in chimeras may be mediated by nonspecific suppressor elements leading to unresponsiveness to a variety of antigens including SRBC.     

Long-term xenogeneic chimeras. Full differentiation of rat T and B cells in SCID mice

To test whether T and B cell differentiation can proceed across species barriers, rat fetal liver (FL) cells were used to reconstitute SCID mice. Provided that the hosts were conditioned with light irradiation, i.v. injection of FL cells caused near-complete repopulation with rat-derived lymphohematopoietic cells, including myeloid and erythroid cells, Ia+ cells of the macrophage/dendritic cell lineages, and mature T and B cells. In keeping with the known hypersensitivity of SCID cells to irradiation, host hematopoietic cells in the chimeras were almost undetectable, even with hosts exposed to as low as 250 rad. In the case of T cells, the distribution of immature and mature cells in the thymus of rat FL----SCID chimeras closely resembled the normal rat thymus in terms of architecture and expression of CD4, CD8, and alpha beta-TCR molecules. Thymopoiesis was followed by the appearance of large numbers of typical rat CD4+ and CD8+ cells in spleen and lymph nodes. These organs also contained substantial numbers of rat B (mu+) cells. The data thus indicate that the xenogeneic environment of SCID mice is fully capable of sustained de novo differentiation of rat T and B cells.     

Expression of exogenous genes in blastodermal cells of chicken in vitro

Chicken blastodermal cells (BCs) from stage X embryos produce both somatic and germline chimeras when injected into the subgerminal cavity of recipient embryos. Transfection of the donor cells in vitro could lead to the production of chimeras capable of transmitting the transgene to their offspring. The aim of this study was to transfer and express foreign genes under control of the ovalbumin promoter in the BCs. The results showed that luciferase activity in the BCs reached a plateau value with a 2.0:1.0 or 5.0:1.0 liposome-DNA ratio and using 1 microg of DNA. Under this same condition, no difference was found in relative activity between the pGL-control and pOVALUC plasmid. The expression of other exogenous genes (green fluorescent protein and interferon alpha2a) driven by the chicken ovalbumin promoter in cultured chicken blastodermal cells in vitro is possible by this assay. Hatchability of recipient embryos after injection of 1,500 or 800 transfected BCs was compared. The advantage of using a smaller number (800) of injected transfected BCs was that early embryonic mortality was reduced and resulted in higher (P<0.01) hatchability (24.5%) than in the case of 1,500 BCs injected.     

Distinct effects of IL-18 on the engraftment and function of human effector CD8 T cells and regulatory T cells

IL-18 has pleotropic effects on the activation of T cells during antigen presentation. We investigated the effects of human IL-18 on the engraftment and function of human T cell subsets in xenograft mouse models. IL-18 enhanced the engraftment of human CD8(+) effector T cells and promoted the development of xenogeneic graft versus host disease (GVHD). In marked contrast, IL-18 had reciprocal effects on the engraftment of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) in the xenografted mice. Adoptive transfer experiments indicated that IL-18 prevented the suppressive effects of Tregs on the development of xenogeneic GVHD. The IL-18 results were robust as they were observed in two different mouse strains. In addition, the effects of IL-18 were systemic as IL-18 promoted engraftment and persistence of human effector T cells and decreased Tregs in peripheral blood, peritoneal cavity, spleen and liver. In vitro experiments indicated that the expression of the IL-18Ralpha was induced on both CD4 and CD8 effector T cells and Tregs, and that the duration of expression was less sustained on Tregs. These preclinical data suggest that human IL-18 may have use as an adjuvant for immune reconstitution after cytotoxic therapies, and to augment adoptive immunotherapy, donor leukocyte infusions, and vaccine strategies.     

Bone Marrow Transplantation Concurrently Reconstitutes Donor Liver and Immune System across Host Species Barrier in Mice

Liver immunopathologic mechanisms during hepatotropic infection, malignant transformation, and autoimmunity are still unclear. Establishing a chimeric mouse with a reconstituted liver and immune system derived from a single donor across species is critical to study regional donor immune responses in recipient liver. Using a strain of mice deficient in tyrosine catabolic enzyme fumarylacetoacetate hydrolase (fah ^-/-) and bone marrow transplantation (BMT), we reconstituted the donor''s hepatocytes and immune cells across host species barrier. Syngeneic, allogeneic or even xenogeneic rat BMT rescued most recipient fah^-/- mice against liver failure by donor BM-derived FAH⁺ hepatocytes. Importantly, immune system developed normally in chimeras, and the immune cells together with organ architecture were intact and functional. Thus, donor BM can across host species barrier and concurrently reconstitutes MHC-identical response between immune cells and hepatocytes, giving rise to a new simple and convenient small animal model to study donor''s liver immune response in mice.     

Expression of Recipient CD47 on Rat Insulinoma Cell Xenografts Prevents Macrophage-Mediated Rejection through SIRPα Inhibitory Signaling in Mice

We have previously proven that the interspecies incompatibility of CD47 is responsible for in vitro phagocytosis of xenogeneic cells by host macrophages. Utilizing an in vivo model in the present study, we investigated whether genetically engineered expression of mouse CD47 in rat insulinoma cells (INS-1E) could inhibit macrophage-mediated xenograft rejection. INS-1E cells transfected with the pRc/CMV-mouse CD47 vector (mCD47-INS-1E) induced SIRPα-tyrosine phosphorylation in mouse macrophages in vitro, whereas cells transfected with the control vector (cont-INS-1E) did not. When these cells were injected into the peritoneal cavity of streptozotocin-induced diabetic Rag2^−/−γ chain ^−/− mice, which lack T, B, and NK cells, the expression of mouse CD47 on the INS-1E cells markedly reduced the susceptibility of these cells to phagocytosis by macrophages. Moreover, these mice became normoglycemic after receiving mCD47-INS-1E, whereas the mice that received cont-INS-1E failed to achieve normoglycemia. Furthermore, injection of an anti-mouse SIRPα blocking monoclonal antibody into the mouse recipients of mCD47-INS-1E cells prevented achievement of normoglycemia. These results demonstrate that interspecies incompatibility of CD47 significantly contributes to in vivo rejection of xenogeneic cells by macrophages. Thus, genetic induction of the expression of recipient CD47 on xenogeneic donor cells could provide inhibitory signals to recipient macrophages via SIPRα; this constitutes a novel approach for preventing macrophage-mediated xenograft rejection.     

Immunodeficient mice as hosts for hemoparasitic infections

Thiruchandurai Rajan, Julie Moore and Leonard Shultz here review the evolution of technology in murine xeno-lymphohemopoietic chimeras, produced by engraftment with xenogeneic (fetal or adult) progenitor cells or mature lymphohemopoietic tissues into immunodeficient mice, and their use as hosts for hemoprotozoan parasites. Particular attention is paid to the development of chimeras that house xenogeneic peripheral red blood cells (xeno-RBC). These chimeras are potentially invaluable models for hemoprotozoan parasites, such as Babesia and Plasmodium. There are, however, daunting limitations that have to be overcome before these models can become universally acceptable systems for the study of these parasitic agents.     

Production of germ-line chimeras in rainbow trout by blastomere transplantation

We describe a technique for producing germ-line chimeric rainbow trout, Oncorhynchus mykiss, by microinjection of the isolated blastomeres. FITC-labeled donor cells and non-labeled recipient embryos at various developmental stages between the early blastula and early gastrula stages were used for cell transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40-somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient embryos, the combination of midblastula (2.5 dpf) donor cells and early blastula (1.5 dpf) recipient embryos gave the highest chimera formation rate and the best distribution pattern of donor cells. Using this combination, chimeric rainbow trout were produced with donor blastomeres from dominant orange-colored mutant embryos and wild-type recipient embryos. Of the 238 chimeric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor-derived orange body color. To test for germ-line transmission of donor cells, gametes obtained from the matured chimeras were fertilized with gametes from wild-type fish. Of the 19 matured chimeras, 6 (32%) yielded donor-derived orange-colored progeny, in addition to wild-type siblings. The contribution rates of donor cells in the germ-line ranged from 0.3 to 14%. This technique for producing germ-line chimeras should be a powerful tool for cell-mediated gene transfer in rainbow trout. Especially, if body color mutants are used for either donor cells or the host embryos, it will be possible to easily concentrate F1 transgenic embryos derived from transplanted donor cells by body color screening. Mol. Reprod. Dev. 59: 380-389, 2001.     

Immunofluorescent study of the origin of connective tissue cells in xenogenic radiation chimeras under normal conditions and in aseptic inflammation]

The origin of elements of the focus of aseptic inflammation and the normal subcutaneous connective tissue in the xenogenic (mouse-rat) radiation chimaeras was investigated by means of indirect Coons method with antiserum to the rat bone marrow cells. The cells of the imflammation focus (leucocytes, macrophages, fibroblasts or fibroblast-like cells, polynuclear giant cells of foreign bodies), as well as leucocytes, macrophages and some fibroblasts of the normal subcutaneous connective tissue, were shown to take their origin from the transplanted bone marrow cells of the donor.     

The facilitation of enduring engraftment of allogeneic bone marrow and avoidance of secondary disease in mice

A method for transplantation of allogeneic bone marrow has been developed and tested in mice. It consists of a treatment preceding supralethal, total-body irradiation (preconditioning) in which a combination of three drugs acting on neuroendocrine regulation are administered, followed by inoculation of a large number of allogeneic bone marrow cells. A second inoculation of allogeneic marrow from the same immunogenetically different donor is given after irradiation. This system provides a high level of protection to mice against radiation damage and facilitates engraftment of the foreign marrow. A large proportion of the engrafted mice become enduring chimeras, manifesting no secondary disease.     

Vascular endothelial cells in cell-mediated immunity: adoptive transfer with in vitro conditioned cells is genetically restricted at the endothelial cell barrier

Delayed-type hypersensitivity (DTH) is a cell-mediated immune response that can be adoptively transferred in rats when greater than 2 X 10(8) cells from peritoneal exudate, lymph nodes, or spleen are used. We have shown that by using an in vitro conditioning step with antigen, transfer can be subsequently carried out with as few as 2 X 10(7) spleen cells. The magnitude of DTH was reflected in ear swelling after intradermal injection of antigen [tuberculin or keyhole limpet hemocyanin (KLH)] and confirmed histologically. The transfer was antigen specific, requiring the sensitizing antigen in both the in vitro conditioning step and in the ear test challenge. Adoptive transfer with conditioned cells was genetically restricted by alleles of the RT-1 region [major histocompatibility complex (MHC) of the rat]. Brown Norway strain (n haplotype) immune cells would not transfer DTH to Lewis (1 haplotype), ACI (a haplotype), or Buffalo (b haplotype) rats, whereas each strain would transfer DTH to syngeneic recipients. Moreover, this pattern of restriction held for all strains when tested in reciprocal fashion. In additional experiments, F1 to parental bone marrow chimeras were constructed so that bone-marrow-derived cells and non-bone-marrow-derived cells were of different RT-1 haplotypes. When these chimeras were used as recipients, transfer of DTH was only observed when immune donor cells and recipient non-bone-marrow-derived cells were syngeneic. These results point to the critical role of non-bone-marrow-derived cells (endothelial cells) in the DTH reaction.     

IgG- and C3-dependent adhesion of neutrophils in systems with allogeneic and xenogeneic ligands]

Interactions of human and rat neutrophils with allogenic and xenogeneic ligands were investigated. Human blood neutrophils reacted equally with human and rat IgG; IgG-dependent adhesion of rat neutrophils (from aseptic peritoneal exudate or blood) was observed only in allogenic system. C3-dependent reactions of neutrophils were observed both in allogenic and xenogeneic systems. The advantage of allogenic combinations in this case was reliable only for rat neutrophils. The relation of obtained results to structural and functional heterogeneity of receptors and ligands is discussed.     

Production of donor T cells is critical for induction of donor-specific tolerance and maintenance of chimerism

Nonmyeloablative conditioning has significantly reduced the morbidity associated with bone marrow transplantation. The donor hemopoietic cell lineage(s) responsible for the induction and maintenance of tolerance in nonmyeloablatively conditioned recipients is not defined. In the present studies we evaluated which hemopoietic stem cell-derived components are critical to the induction of tolerance in a total body irradiation-based model. Recipient B10 mice were pretreated with mAbs and transplanted with allogeneic B10.BR bone marrow after conditioning with 100-300 cGy total body irradiation. The proportion of recipients engrafting increased in a dose-dependent fashion. All chimeric recipients exhibited multilineage donor cell production. However, induction of tolerance correlated strictly with early production of donor T cells. The chimeras without donor T cells rejected donor skin grafts and demonstrated strong antidonor reactivity in vitro, while possessing high levels of donor chimerism. These animals lost chimerism within 8 mo. Differentiation into T cells was aborted at a prethymic stage in recipients that did not produce donor T cells. Moreover, donor Ag-driven clonal deletion of recipient T cells occurred only in chimeras with donor T cells. These results demonstrate that donor T cell production is critical in the induction of transplantation tolerance and the maintenance of durable chimerism. In addition, donor T cell production directly correlates with the deletion of potentially alloreactive cells.