Immunological reactions to neural grafts in the central nervous system

Immunological rejection is a lasting, although highly variable, threat to allo- and xenogeneic neural tissue grafted to the CNS of rodents, monkeys and man. One major determinant for rejection of intracerebral CNS grafts appears to be induction of major histocompatibility complex (MHC) antigens on the donor CNS cells. We have previously examined the cellular immune response against neural mouse xenografts undergoing rejection in the adult rat brain. In this study we focus on the astro- and microglial reactions within and around the graft, and the potential of individual host rat and donor mouse brain cells to express MHC antigens. Previous light microscopical observations of expression of rat MHC antigen class I by endothelial cells, microglial cells, and invading leukocytes were extended to the ultrastructural level and found to include a few astrocytes. Rat and mouse MHC antigen class II was only detected on leukocytes and activated microglial cells. The findings imply that within grafts of brain or spinal cord tissue donor astrocytes, microglial cells and endothelial cells can be induced to act as target cells for class I specific host T cytotoxic cells, while only (graft and host) microglial cells can be induced to express MHC antigen class II and present antigen to sensitized (and possibly also resting) host T helper cells.     

Methylprednisolone prevents rejection of intrastriatal grafts of xenogeneic embryonic neural tissue in adult rats

We studied the effects of high-dose methylprednisolone on the survival of intrastriatal neural xenografts and the host responses against them. Dissociated mesencephalic tissue from inbred mouse (CBA-strain) embryos was transplanted to the intact striatum of adult Sprague-Dawley rats. The rats received either daily injections of methylprednisolone (30 mg/kg), or cyclosporin A (10 mg/kg), or no immunosuppressive treatment. Two or six weeks after transplantation, there was good survival of xenografts in both the methylprednisolone- and cyclosporin A-treated rats. In contrast, the xenografts in untreated control rats were all rejected by six weeks. There was no marked difference in the degree of expression of MHC class I and II antigens and the accumulation of activated astrocytes and microglial cells/macrophages between the three groups. However, both methylprednisolone and cyclosporin A reduced infiltration of T lymphocytes to the transplantation sites. The expression of pro-inflammatory cytokines (interferon-λ, tumour necrosis factor-a, interleukin-6) in and around the grafts was lower in the methylprednisolone- and cyclosporin A-treated groups than in untreated control rats. Although high-dose methylprednisolone caused significant body weight loss, we conclude that this treatment can prevent rejection of intrastriatal grafts of xenogeneic embryonic neural tissue in the adult.     

BN rats do not reject F344 brain allografts even after systemic sensitization

Embryonic brain tissue allografts under many circumstances survive transplantation into the brain. It is generally believed that such grafts will not survive if the host animal is systemically sensitized, by skin grafting or other means, to major histocompatibility complex (MHC) antigens of the donor animal. We have found that F344 brain grafts survive in BN hosts even when the host is systemically sensitized to F344 tissue. Embryonic cerebral neocortex from F344 donors was transplanted into BN host rats (n = 95). Subsequently, the host rats were systemically sensitized with donor skin (n = 25), brain tissue (n = 41), or spleen cells (n = 6) and compared with a control group of rats consisting of allografts with no sensitization or sham procedures (n = 23). Rejection of the transplants in BN rat hosts was not provoked by any of the sensitization methods tested. Minor immunological responses that did not result in rejection were, however, present in many host animals. We did not observe infiltration of W3/13+ T cells and OX8+ cytotoxic lymphocytes in any of the groups. Nevertheless, substantial infiltrations of OX6+ antigen-presenting cells and W3/25+ helper T cells were present. There was also an extensive enhancement of MHC class I immunoreactivity in parts of the grafted tissue developing within the third ventricle, but not for the same type of graft in the lateral ventricle. This increase of MHC class I expression was not accompanied by infiltration of cytotoxic T cells. Our findings thus suggest that neural graft rejection depends on general genetic susceptibility to immune reactions, particularly experimental allergic encephalomyelitis and not only on disparity between donor and host antigens encoded by the MHC. Moreover, enhancement of MHC class I and class II expression within transplanted tissue does not predict graft rejection.     

An in vivo and in vitro analysis of systemic immune function in mice with histologic evidence of neural transplant rejection

Histologic and immunocytochemical analyses of fetal neocortical tissue transplanted to the lateral ventricle of inbred adult mice indicate that this tissue survives transplantation well if the donor and host are isogeneic. The major histocompatibility complex (MHC) of the mouse is known as the H-2 locus. H-2-incompatible neural transplants (allografts), unlike their H-2-identical counterpart (isografts), are characterized by the presence of T cells comprising both major T-cell subsets and macrophages, and by a marked increase in the expression of both class I and class II (Ia) MHC antigens. These findings suggest a recognition of H-2 alloantigens by the host's immune system followed by an appropriate effector response. We report here our attempts to demonstrate systemic host sensitization to alloantigens in mice bearing H-2-incompatible intraventricular neural transplants. We measured the time to rejection of orthotopic skin grafts subsequent to neural transplantation, splenocyte proliferative responses to alloantigens in mixed lymphocyte cultures (MLC), and class I-restricted antigen-specific cytolytic T lymphocyte (CTL) activity. No significant differences were found in any of these tests of host systemic sensitization between mice with allogeneic neural transplants and those with isogeneic transplants or control animals. We conclude that intraventricular neural transplants, while recognized and affected by cells of the host's immune system, do not elicit a detectable systemic sensitization to class I H-2 alloantigens. Rejection of neural transplants may depend on sensitization to class II H-2 alloantigens, to so-called minor histocompatibility antigens, or some combination thereof.     

Patterns of immune rejection of mouse neocortex transplanted into neonatal rat brain, and effects of host immunosuppression

We studied the histological and immunological characteristics of graft rejection in the rodent central nervous system (CNS) using embryonic mouse neocortex transplanted into the CNS of neonatal rats. Grafts from animals aged 8-145 days (n = 210) were examined using standard histological techniques for demonstrating cell morphology and fiber projections. Immunohistochemical techniques were used to identify graft projections into the host CNS. The incidence of graft rejection was 18% for animals between 18 and 30 days of age, but increased abruptly to 73% for animals older than 30 days. No graft rejection was seen in animals younger than 18 days. In a smaller group of xenograft recipient rats sacrificed at specific time points before and after one month of age, detailed immunohistochemical studies were performed to correlate the histological appearance of the graft with the level of major histocompatibility complex (MHC) class I and II immunoreactivity, and microglial, astrocytic and lymphocytic staining within the graft and host brain. Evidence of mild rejection as manifested by the appearance of scattered lymphocytes within the graft coincided with the development of Class I and II immunoreactivity within the graft and at the graft-host interface, which was demonstrated in some animals as early as 24 days. At 29 days of age, rejecting grafts showed diffuse MHC expression within the graft and at the graft-host interface; in contrast, unrejected grafts failed to show MHC immunoreactivity. Thirty-four day-old grafts often showed severe rejection with perivascular lymphocytic cuffing within the graft and in host parenchyma remote from the graft associated with increased MHC immunoreactivity within the host brain. In grafts older than 34 days there was frequently a violent rejection reaction with disruption of the cytoarchitecture of the graft and surrounding host tissues, and widespread MHC antigen expression. Immunosuppression with cyclosporin A was effective in avoiding rejection. The high incidence of rejection with neocortical xenografts is in striking contrast to the much lower incidence seen with retinal xenografts. This suggests that there are immunological features unique to neocortex which incite host recognition and rejection.     

Immunohistochemical analysis on the rejection of xenogeneic brain grafts

The central nervous system (CNS) of mammals has long been thought of as an immunologically privileged site. However, this concept is now changing because the rejection of histo-incompatible neural grafts has been frequently observed in the CNS. In neural transplantation used as therapy for some human neurodegenerative diseases, it is important to determine which factors are related to brain graft rejection. In this study, we examined immunological reactions in brains that had received isogeneic (rat to rat) and xenogeneic (mouse to rat) neural transplants. In the immunohistochemical analysis, antibodies against T cell receptor αβ (R73), macrophage and microglia (0X42), MHC class II antigens (0X6), CD4 (W3/25), CD8 (0X8), NK cell (3.2.3), B cell (RLN-9D3), T cell receptor (TCR) Vβ8.2 (R78), TCR Vβ8.5 (B73) and TCR Vβl0 (G101) were used. At the early stage of both isogeneic and xenogeneic transplantation, a nonspecific inflammatory reaction characterized by macrophage infiltration was observed along the needle track which was produced by the grafting procedure. From the day 7 stage onwards, the non-specific inflammatory reaction was replaced by the specific immune reactions of T cell infiltration, neovascularization and necrosis of xenogeneic grafts. Marked T cell infiltration was detected in the lesions, whereas NK and B cells were not. Quantitative analysis of T cell subsets revealed that both CD4+ and CD8+ T cells were found in the xenogeneic transplants. Microglia became activated and strongly expressed MHC class II antigens at the time of graft rejection. Isogeneic transplants, in contrast, showed no histological characteristics of rejection, and numerous dopaminergic neurons with several neurites were observed in the grafts. Based on these findings, we concluded that T cells are the principal effectors in the rejection of xenogeneic neural grafts, and that activated microglia may have some role in presenting antigens to the infiltrating T cells during the rejection process.     

Human fetal neural precursor cells can up-regulate MHC class I and class II expression and elicit CD4 and CD8 T cell proliferation

The use of allogeneic fetal neural precursor cells (NPCs) as a cell replacement therapy in neurodegenerative disorders holds great promise. However, previous studies concerning the possibility of alloimmune rejection of the transplanted cells have been inconclusive. Here, we used flow cytometry to quantify the expression of major histocompatibility complex (MHC) molecules by human NPCs, obtained from the cortex or ventral mesencephalon of fetuses with gestational ages between 7 and 11 weeks. MHC class I was undetectable on the surface of freshly isolated primary fetal tissue from either location, but increased over time in proliferating NPC cultures; after 7days in vitro, MHC class I was detectable on most cells. Following differentiation, MHC class I expression persisted on non-neuronal cells. MHC class II levels remained low at all time points but were inducible by pro-inflammatory cytokines, whereas the co-stimulatory molecules, CD80 and CD86, remained undetectable. Nonetheless, CD4+ and CD8+ T cells proliferated when peripheral blood mononuclear cells (PBMCs) were cultured with allogeneic NPCs. Weaker responses were obtained when NPCs were co-cultured with purified allogeneic responder T cells, suggesting that indirect allorecognition contributed significantly to PBMC responses. In conclusion, differentiating human NPCs are immunogenic in vitro, suggesting that they may trigger immune rejection unless transplant recipients are immunosuppressed.     

Leukocyte infiltration and glial reactions in xenografts of mouse brain tissue undergoing rejection in the adult rat brain. A light and electron microscopical immunocytochemical study

Neural mouse xenografts undergoing rejection in the adult recipient rat brain were characterized with regard to infiltrating host leukocytes and reactions of graft and host astro- and microglial cells. Rejection occurred within 35 days with infiltration of the grafts by in particular macrophages and T-cells as well as blood-brain barrier (BBB) leakage for IgG. In the surrounding host brain microglial cells showed increased histochemical staining for nucleoside diphosphatase (NDPase) and increased immunocytochemical expression of complement receptor type 3 (CR3), while astroglial cells displayed an increased immunoreactivity for glial fibrillary acidic protein (GFAP). Light microscopic findings of rat major histocompatibility complex (MHC) antigen class I on microglial cells, endothelial cells and leukocytes were confirmed at the ultrastructural level and extended to include a few astrocytes. Rat and mouse MHC antigen class II was only detected on leukocytes and activated microglia. We suggest that host macrophages and activated host and xenograft microglial cells act in situ as immunostimulatory cells on T-helper cells, and that increased levels of donor MHC antigen class I may further enhance the killer activity exerted by host T-cytotoxic cells.     

Time-dependent expression of donor- and host-specific major histocompatibility complex class I and II antigens in allogeneic dopamine-rich macro- and micrografts: comparison of two different grafting protocols

Neural transplantation, as a therapeutic approach to Parkinson’s disease, still requires allogeneic graft material and raises questions of immunosuppression and graft rejection. The present study investigated the time course of major histocompatibility complex (MHC) expression and astrocytic response in allogeneic dopaminergic grafts, comparing two different grafting protocols. Adult 6-hydroxydopamine-lesioned Lewis 1.W rats received intrastriatal cell suspension grafts from the ventral mesencephalon of DA rat fetuses, either as single 1-μl macrograft via metal cannula or as four micrografts of 250 nl/deposit via a glass capillary. No immunosuppression was administered. Immunohistochemistry was performed at 1, 3, 6, and 12 weeks after grafting, using antibodies against donor- and host-specific MHC class I and II antigen, glial fibrillary acidic protein (GFAP) and tyrosine hydroxylase (TH). Most animals showed good allograft survival up to 12 weeks after transplantation with no signs of rejection. Reinnervation of the lesioned striatum by TH-positive neurites was observed from 3–6 weeks on. Expression of donor-specific MHC class I was comparably low in both allogeneic grafting groups, while host MHC class I and II reaction as well as astrocytic response tended to be higher in the macrografted animals. Donor MHC class II was not observed at any time point. It is concluded that intraparenchymal allografts of fetal mesencephalic cell suspensions can survive well in the rat Parkinson model without immunosuppression for at least 12 weeks, and that the expression of moderate amounts of donor-specific MHC class I antigen does not suffice to initiate a rejection process. In addition, the microtransplantation approach may reduce the level of trauma and subsequent MHC and GFAP expression and may, thereby, minimize the risk of graft rejection. Received: 14 May 1997 / Revised, accepted: 17 July 1997     

Long-term survival of mouse corpus callosum grafts in neonatal rat recipients, and the effect of host sensitization.

Previous studies have suggested that the incidence of spontaneous rejection among immunogenetically mismatched neural transplants in neonatal recipients varies significantly depending on the cellular composition of the graft material. For example, neuron-rich grafts of embryonic mouse retina generally survive for extended periods without showing signs of rejection after implantation into neonatal rats, whereas cortical xenografts, which contain abundant glial and endothelial cells as well as neurons, typically undergo rejection 4-6 weeks after implantation. To determine whether the presence of donor glia is responsible for this high incidence of spontaneous rejection, we examined the fate of a non-neuronal graft material composed predominantly of xenogeneic glial cells (post-natal day 3, PD3, CD-1 mouse corpus callosum) implanted into the mesencephalon of PD1 Sprague-Dawley rats. The distribution and survival of donor astrocytes were assessed using a monoclonal antibody specific for a mouse astrocyte surface antigen, M2. Thirteen of 16 animals sacrificed within 2 months of implantation had detectable transplants. In these animals, M2-positive cells frequently migrated well away from body of the graft, clustering in large numbers in several characteristic regions of the host brain. Unlike cortical grafts of similar age, the vast majority (93%) of callosal transplants showed no histological signs of rejection or major histocompatibility complex antigen expression in and around the transplant-derived cells. As previously noted in the neonatal retinal transplant paradigm, however, well-integrated 1-month-old corpus callosum grafts could be induced to reject by appropriate sensitization of the host immune system, implying that the host was not immunologically tolerant to the foreign neural graft. With longer survival times in unsensitized hosts, a progressively smaller percentage of animals had detectable donor astrocytes (5 of 10 animals at 3 months postimplantation and 4 of 16 animals at 4 months); in those 9 animals with surviving grafts, only small numbers of M2-positive cells were seen within the graft bed and surrounding host brain. However, only 2 of the 26 "long-term" animals showed evidence of graft rejection. These results indicate that mouse astrocytes show characteristic patterns of migration into the host brain when implanted into neonatal rats; however, these xenogeneic cells have a limited duration of survival. The infrequency with which even subtle signs of spontaneous rejection were detected in animals that had received corpus callosum xenografts suggests that an immune-mediated process is unlikely to be responsible for the time-dependent elimination of the donor astrocytes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Immunological reactions following intracerebroventricular transplantation of adrenal medulla

Immunological reactions after intracerebroventricular syn-, allo- and xenogenic transplantation of adrenal medulla were investigated histologically. In xenografts only, T cell infiltration and graft rejection were observed. Syngrafts and allografts were not rejected and were not infiltrated by T cells, although expression of MHC class II antigen was observed at all survival times. Major histocompatibility complex (MHC) class I immunoreactivity was strongly expressed in adrenal cortex syngrafts, which could play a role in the rejection of grafts containing mixed cell populations. The survival of chromaffin cells in allografts was decreased as compared to syngrafts, and there were fewer allograft animals with large numbers of surviving chromaffin cells. There was some increased cellularity (microglia and macrophages) in allografts even though no T cell infiltration was found. Therefore, it appears that this limited survival of intracerebral adrenal medulla allografts is not due to T cell-mediated graft rejection.     

Modulation of MHC class II antigen expression in human myoblasts after treatment with IFN-gamma

Some investigators have proposed myoblast transfer as a potential therapy for the treatment of Duchenne muscular dystrophy. Little is known about the immunobiology of myoblast transplantation. Transplantation rejection is mediated to a large extent by CD8+ T cells, which recognize alloantigens encoded by class I HLA genes, and by CD4+ T cells, which recognize alloantigens encoded by class II HLA genes. Gamma interferon (IFN-gamma) is a potent inducer of HLA class II molecules as well as beta 2-microglobulin, which is co-expressed with HLA class I. IFN-gamma may be a critical cytokine involved in graft rejection. We purified human myoblasts by flow cytometry and incubated them in vitro for varying time periods with recombinant human IFN-gamma. The inducibility of HLA-DR and -DP molecules raises a note of caution concerning possible rejection phenomenon which might occur following myoblast transplantation.     

The blood-brain barrier protects foreign antigens in the brain from immune attack

To examine the role of the blood-brain barrier (BBB) in maintaining immune privilege in the brain, the BBB in the region of stably integrated mouse neural grafts implanted in neonatal rat brains was transiently disrupted by intracarotid infusion of hypertonic mannitol. This led to graft rejection and to prominent expression of major histocompatibility complex (MHC) antigens on cells adjacent to the graft. Grafts in control animals receiving an intracarotid infusion of isotonic saline showed only rare MHC expression and no increased incidence of rejection. Opening the barrier in the absence of a graft caused neither MHC expression nor cellular infiltration within the brain, suggesting that the effects of the hypertonic infusion were not produced by an indirect injury-mediated effect on the host brain. We conclude that the integrity of the blood-brain barrier is an important factor in the relative immune privilege of nonsyngeneic neural grafts.     

Engraftment of freshly isolated or cultured human umbilical cord blood cells and the effect of cyclosporin A on the outcome

Human umbilical cord blood (HUCB) is a potentially valuable resource for cell therapy. The present study investigated the short-term survival of intrastriatal grafts of either freshly isolated or cultured HUCB cells and the effect of the immunosuppressive agent cyclosporin A (CSA) in host rat brains. The group injected with either freshly isolated or cultured HUCB cells was subdivided into CSA or saline controls. Freshly isolated and cultured HUCB cells displayed surface markers CD33, CD44, CD45, CD51/61 and CD90/Thy-1. The hematopoietic progenitor marker CD34 was expressed only in freshly isolated cells. The majority of injected HUCB cells were localized within a 500-mum radius from the injection site in the striatum; however, a subpopulation migrated along the corpus callosum. There was no significant statistical difference in the cell count between freshly isolated and cultured HUCB cells with or without CSA. Some grafted HUCB cells expressed either a neural or microglial marker. There was weak up-regulation of major histocompatibility complex (MHC) class I antigen in rats either with or without CSA. However, there were considerably fewer positive cells labeled with an MHC class II antigen in CSA groups. These results suggest that neither freshly isolated nor cultured HUCB cells induce acute rejection after intrastriatal transplantation up to 14 days. CSA suppressed up-regulation of MHC class II antigen in the host brain.     

Dual-label immunocytochemistry of the active multiple sclerosis lesion: major histocompatibility complex and activation antigens

Fresh-frozen autopsy material containing active inflammatory lesions from 9 different patients with multiple sclerosis (MS) was analyzed by immunocytochemistry using a panel of monoclonal antibodies, and a dual-label immunocytochemical method was developed which permitted the simultaneous detection of two different surface markers on a single cell. We now report the following. (1) The predominant T-cell phenotype within MS lesions is CD2,3,8. This phenotype marks the suppressor-cytotoxic subset. (2) These cells do not express the natural killer cell marker NKH-1, which is present on a subset of CD8-positive cells in peripheral blood. (3) The infiltrating cell expresses class I (HLA A, B, C), but not class II (DR and DQ), major histocompatibility complex (MHC) molecules. (4) Other T-cell surface molecules, including the activation antigens interleukin-2 receptor, Ta1, and T11-3, as well as the marker 2H4, are largely not expressed. (5) Endothelial cells express both class I and class II MHC molecules and the 4B4 molecule in both MS and control tissue. (6) Astrocytes within the vicinity of MS lesions are predominantly class II MHC-negative. These results demonstrate that the T-cell infiltrate present in MS tissue on autopsy has a restricted phenotypic profile, but they also raise the possibility that, within this population, few activated effector cells are present.     

Vacuolar myositis with expression of both MHC class I and class II antigens on skeletal muscle fibers.

We describe here a 10-year-old patient with high levels of serum IgE and inflammatory myopathy whose muscle fibers exhibit excessive autophagy. Previous studies have demonstrated surface expression of class I MHC antigens on muscle fibers from patients with inflammatory myopathy. The muscle fibers of this patients showed marked expression of both class I and class II MHC antigens. The reaction products were demonstrated not only on sarcolemma but also in and around some vacuoles. Both CD4-positive and CD8-positive T-lymphocytes were noted in inflammatory exudates surrounding these fibers but B-lymphocytes were rare. We hypothesize that myocyte expression of both class I and class II antigens may play a role in the pathogenesis of this new type of inflammatory myopathy.     

Treatment of rat hemiparkinson model with xenogeneic neural transplantation: Tolerance induction by anti-T-cell antibodies

To obtain basic knowledge for the application of xenogeneic neural transplantation to patients with Parkinson's disease, the rejection process of xenogeneic neural grafts in rats was examined and a therapy to control it was developed. Tissues including the ventral mesencephalon were taken from mouse embryos and transplanted into the right lateral ventricle of mature male rats. Transplanted xenografts were usually rejected by day 15. To prevent the graft rejection, host rats were treated with anti-T-cell receptor αβ (anti-TCRαβ) or anti-CD2 monoclonal antibody (mAb) or by a combination of the two. Anti-TCRαβ (1 mg/kg) and anti-CD2 (7 mg/kg) mAb were administered for 3 consecutive days (day -2, -1, and 0 of transplantation). Although the administration of mAb against either CD2 or TCRαβ did not induce tolerance, the combination therapy with anti-CD2 and anti-TCR αβ mAb produced graft survival for more than 100 days. The tolerance induced by this combined antibody therapy is antigen specific because rats with long-term surviving neural xenograft accepted a second neural graft from the same donor strain C3H/He mouse, but not from a third-party strain BALB/c mouse, without additional treatment. In addition, T cells isolated from these rats did not respond to cultured C3H/He brain cells, but did respond vigorously to BALB/c brain cells in mixed lymphocyte reaction. More importantly, the finding that xenograft transplantation with the proper treatment reduced the rotation rate of 6-OHDA-lesioned rats confirmed that surviving grafts functioned properly. The results of the present study suggest that xenogeneic neural transplantation in combination with T-cell-targeted immunotherapy is an effective approach for treatment of Parkinson's disease. J. Neurosci. Res. 48:385–396, 1997. © 1997 Wiley-Liss, Inc.     

Cellular events associated with peripherally induced rejection of mature neural xenografts placed into neonatal rat brains

Various circumstances have brought about a dispute concerning the immunologically priviledged status of the central nervous system (CNS). Using a transplantation paradigm, we have examined the cellular events associated with an experimentally induced focal assault on the CNS by the immune system. Chunks of embryonic mouse cortex were transplanted into neonatal rat brains and allowed to survive for 4 weeks. The adult rats then received a skin graft of donor origin to induce rejection of the transplanted tissue. Animals were sacrificed at various time points and examined histologically and immunocytochemically. Under these circumstances, the transplant is rejected via a first-set rejection response, and astrocytes of donor origin appear to be the primary target of the host immune system. Expression of class I and class II major histocompatibility antigens is noted to correlate with lymphocytic invasion of the transplant.     

Expression of Ia antigen on vascular endothelial cells in mouse cerebral tissue grafted into the third ventricle of rat brain

The mechanisms of the immunological rejection after xenogeneic neural transplantation were investigated with special reference to the expression of class II major histocompatibility complex (MHC) antigen (Ia antigen) on the grafted tissue. Tissue from a newborn mouse cerebral cortex was transplanted into the third ventricle of a 4-week-old rat brain. Infiltration of cytotoxic T-cells into the grafted tissue was investigated immunohistochemically by using a monoclonal antibody (OX-8). The infiltration began 8 days after transplantation and continued until about 4 weeks when the tissue was completely rejected. The expression of Ia antigen was also investigated immunohistochemically. The Ia antigen was first detected in the grafted tissue at 6 days after transplantation. The Ia antigen was considered to be expressed on the vascular endothelial cells judging from the staining patterns and the location of India ink which was perfused from the host's left cardiac ventricle. The perfusion experiments with India ink also revealed that blood was supplied to the grafted tissue from 5 days after transplantation. These results suggest that the expression of Ia antigen on the vascular endothelial cells renders the grafted tissues competent to initiate and participate in the immune reaction. The results also raise a possibility that the expression of Ia antigen is triggered by blood supplied from the host brain. In addition, the results indicate that the Ia-positive blood vessels do not originate in the host brain but are intrinsic to the grafted tissue.     

Expression of Major Histocompatibility Complex Antigens and Induction of Human T-Lymphocyte Proliferation by Astrocytes and Macrophages from Porcine Fetal Brain

Porcine fetal brain cells are of potential use as donor cells for transplantation therapies of neurodegenerative diseases in humans. Our aim was to determine the immunestimulatory properties of astrocytes and macrophages from porcine fetal brain in vitro. By flow cytometry, freshly isolated porcine fetal brain cells were nonautofluorescent, while primary cultures of these cells, prepared to favor growth of astrocytes and macrophages/microglia, consisted of both an autofluorescent and a nonautofluorescent cell population. The cultured autofluorescent cells had qualities typical of macrophages: CD18 (beta(2) integrin subunit) expression, high granularity, and phagocytic activity. The cultured nonautofluorescent cells stained positive for the astrocyte marker glial fibrillary acidic protein and CD56 (NCAM isoform). While freshly isolated porcine fetal brain cells expressed very low levels of major histocompatibility complex (MHC) class I and no MHC class II antigens, primary culture of these cells resulted in upregulation of MHC class I antigens on astrocytes and macrophages and MHC class II antigens on a subpopulation of the macrophages. Single-cell suspensions prepared from the primary cultures were flow sorted into astrocyte and macrophage populations on the basis of cell granularity and autofluorescence or on the basis of CD56 expression. Pure suspensions (>98%) of astrocytes induced a low proliferative response in human T lymphocytes, as determined by [(3)H]thymidine incorporation after 4 days of coculture. A suspension of 91% macrophages was a strong inducer of human T-cell proliferation, even stronger than allogeneic mononuclear blood cells. For neural xenotransplantation, our findings suggest that depletion of macrophages from the donor-cell suspensions may enhance graft survival by reducing cell-mediated rejection.