The immunological challenges of cell transplantation for the treatment of Parkinson's disease.

Dopaminergic cell transplantation is an experimental therapy for Parkinson's disease (PD). It has many potential theoretical advantages over current treatment strategies such as providing continuous local dopaminergic replenishment, eliminating motor fluctuations and medication-induced dyskinesias, slowing down disease progression or even reversing disease pathology in the host. Recent studies also show that dopaminergic cell transplants provide long-term neuromodulation in the basal ganglia that simulates the combined effects of oral dopaminergic therapy and surgical therapies like deep brain stimulation, the contemporary therapeutic approach to advanced PD. However, dopaminergic cell transplantation in PD as not been optimized and current experimental techniques have many drawbacks. In published experiments to date of attempted dopaminergic grafting in PD, the major challenges are unacceptable graft-induced dyskinesias or failure of such grafts to exceed the benefits afforded by sham surgery. A deleterious host immune response to the transplant has been implicated as a major putative cause for these adverse outcomes. This article focuses on recent advances in understanding the immunology of the transplantation in PD and possible methods to overcome adverse events such that we could translate cell replacement strategies into viable clinical treatments in the future.

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.

A patient with Huntington's disease and long-surviving fetal neural transplants that developed mass lesions.

Transplantation of human fetal neural tissue into adult neostriatum is an experimental therapy for Huntington's disease (HD). Here we describe a patient with HD who received ten intrastriatal human fetal neural transplants and, at one site, an autologous sural nerve co-graft. Although initially clinically stable, she developed worsening asymmetric upper motor neuron symptoms in addition to progression of HD, and ultimately died 121 months post transplantation. Eight neural transplants, up to 2.9 cm, and three ependymal cysts, up to 2.0 cm, were identified. The autologous sural nerve co-graft was found adjacent to the largest mass lesion, which, along with the ependymal cyst, exhibited pronounced mass effect on the internal capsules bilaterally. Grafts were composed of neurons and glia embedded in disorganized neuropil; robust Y chromosome labeling was present in a subset of grafts and cysts. The graft-host border was discrete, and there was no evidence of graft rejection or HD pathologic changes within donor neurons. This report, for the first time, highlights the potential for graft overgrowth in a patient receiving fetal neural transplantation.

Neuroinflammation in the generation of post-transplantation dyskinesia in Parkinson's disease.

The observation that neural grafts can induce dyskinesias has severely hindered the development of a transplantation therapy for Parkinson's disease (PD). We addressed the hypothesis that inflammatory responses within and around an intrastriatal graft containing dopamine neurons can trigger dyskinetic behaviors. We subjected rats to unilateral nigrostriatal lesions with 6-hydroxydopamine (6-OHDA) and treated them with L-DOPA for 21 days in order to induce abnormal involuntary movements (AIMs). Subsequently, we grafted the rats with allogeneic embryonic ventral mesencephalic tissue in the dopamine-denervated striatum. In agreement with earlier studies, the grafted rats developed dyskinesia-like AIMs in response to amphetamine. We then used two experimental approaches to induce an inflammatory response and examined if the amphetamine-induced AIMs worsened or if spontaneous AIMs developed. In one experiment, we challenged the neural graft hosts immunologically with an orthotopic skin allograft of the same genetic origin as the intracerebral neural allograft. In another experiment, we infused the pro-inflammatory cytokine interleukin 2 (IL-2) adjacent to the intrastriatal grafts using osmotic minipumps. The skin allograft induced rapid rejection of the mesencephalic allografts, leading to disappearance of the amphetamine-induced AIMs. Contrary to our hypothesis, the rejection process itself did not elicit AIMs. Likewise, the IL-2 infusion did not induce spontaneous AIMs, nor did it alter L-DOPA-induced AIMs. The IL-2 infusions did, however, elicit the predicted marked striatal inflammation, as evidenced by the presence of activated microglia and IL2Ralpha-positive cells. These results indicate that an inflammatory response in and around grafted dopaminergic neurons is not sufficient to evoke dyskinetic behaviors in experimental models of PD.

Local effects of dexamethasone on immune reaction in neural transplantation.

This study was performed to see whether local injection of dexamethasone may protect the neural grafts from immunological rejection and increase the successive rate of graft. Rats with unilateral 6-hydroxydopamine lesions of the mesostriatal dopamine pathway received fetal ventral mesencephalic (FVM) cells and dexamethasone in two regions of the striatum and showed significant (P<0.001) reduction in rotational asymmetry as compared to the non-immunosuppressed group. A significantly greater number of total TH-ir cells (P<0.001) and fewer number of total GFAP -ir cells (P<0.001) and inflammatory cells were observed in the striatum of animals in immunosuppressed group than those in non-immunosuppressed group. This results indicated that local injection of dexamethasone could not only reduce the immune rejection and increase the survival grafted cell but also avoid the side effects brought by long systemic administer of immunosuppressant.

Olfactory ensheathing glia graft in combination with FK506 administration promote repair after spinal cord injury.

The aim of this study was to determine whether a combination of olfactory ensheathing cell (OEC) graft with the administration of FK506, two experimental approaches that have been previously reported to exert protective/regenerative effects after spinal cord injury, promotes synergic restorative effects after complete or partial spinal cord injuries. In partial spinal cord injury, combination of an OEC graft and FK506 reduced functional deficits evaluated by the BBB score, motor-evoked potentials (MEPs) and H reflex tests, diminished cavitation, astrogliosis and increased sparing/regeneration of raphespinal fibers compared to untreated and single-treatment groups of rats. After complete spinal cord transection, the combined treatment significantly improved functional outcomes, promoted axonal regeneration caudal to the lesion, and diminished astrogliosis compared only to non-transplanted animals. Slightly, but non-significant, better functional and histological results were found in OEC-grafted animals treated with FK506 than in those given saline after spinal cord transection. Nevertheless, the combined treatment increased the percentage of rats that recovered MEPs and promoted a significant reduction in astrogliosis. In conclusion, this study demonstrates that OEC grafts combined with FK506 promote additive repair of spinal cord injuries to those exerted by single treatments, the effect being more remarkable when the spinal cord is partially lesioned.

[Experimental research on immunological rejection in neural stem cells allograft].

AIM: To investigate the occurrence of immunological rejection in brain transplantation of neural stem cells (NSCs) in the rat of Parkinson's disease model. METHODS: NSCs derived from the brain of E14.5d SD rat were cultured in vitro. Single cell suspensions were grafted into the striatum of the rat model of Parkinson's disease. Surviving animals were sacrificed at 10, 21, 35 and 60 d after transplantation, and the brain tissue was stained with HE and immunocytochemically to detect the expression of tyrosine hydroxylase (TH), CD4, CD8 and major histocompatibility complex class II antigens (MHC-II). RESULTS: The expression of elicited CD4, CD8 and MHC-II was detected within and around the allografts in the graft groups at 10 and 21 d after grafting and was subsiding at 35 d. At 60 d only very occasional immunopositive cells were present. The number of TH positive neurons was low at 10, 21 d, but increased at 35 d and 60 d. There was no significant difference between negative and positive control groups at different time points. The rotation behavior of PD was decreased 35 d after transplantation. CONCLUSION: Intracerebral transplantation of NSCs did not cause remarkable immunological rejection.

Survival of rat or mouse ventral mesencephalon neurons after cotransplantation with rat sertoli cells in the mouse striatum.

Transplanting cells across species (xenotransplantation) for the treatment of Parkinson's disease has been considered an option to alleviate ethical concerns and shortage of tissues. However, using this approach leads to decreased cell survival; the xenografted cells are often rejected. Sertoli cells (SCs) are testis-derived cells that provide immunological protection to developing germ cells and can enhance survival of both allografted and xenografted cells. It is not clear whether these cells will maintain their immunosuppressive support of cografted cells if they are transplanted across species. In this study, we investigated the immune modulatory capacity of SCs and the feasibility of xenografting these cells alone or with allografted and xenografted neural tissue. Transplanting xenografts of rat SCs into the mouse striatum with either rat or mouse ventral mesencephalon prevented astrocytic infiltration of the graft site, although all transplants showed activated microglia within the core of the graft. Surviving tyrosine hydroxylase-positive neurons were observed in all conditions, but the size of the grafts was small at best. SCs were found at 1 and 2 weeks posttransplant. However, few SCs were found at 2 months posttransplant. Further investigation is under way to characterize the immune capabilities of SCs in a xenogeneic environment.

Beta1 integrin as a xenoantigen in fetal porcine mesencephalic cells transplanted into the rat brain.

Xenografts of fetal porcine mesencephalic cells implanted into the rat striatum are generally rejected within several weeks. The fetal donor mesencephalon predominantly consists of neurons, but also contains microglial and endothelial cells, which are more immunogenic. In the present work, we investigated the occurrence of donor endothelial cells in grafts of porcine mesencephalic cells implanted into the rat striatum. Pig endothelial cells were monitored by immunochemical methods, using a monoclonal antibody (mAb) that recognizes a peptidic epitope of the porcine beta1 integrin, and isolectin IB4, for the staining of the Galalpha1,3Gal epitope. The analysis also involved the detection of the pig hyaluronate receptor CD44, and the cell adhesion molecule CD31. The anti-beta1 integrin mAb revealed endothelial-like cells in grafts of porcine mesencephalic cells as soon as 1 week after implantation. A similar staining pattern was obtained with the IB4 lectin. Unlike aortic endothelial cells, these pig brain-derived endothelial-like cells were not recognized by the anti-CD44 antibody. They also failed to express the CD31 adhesion molecule, a fact which suggests that they remained poorly mature, even in grafts maintained during 45 days in immunosuppressed rats. Interestingly, a strong expression of beta1 integrin immunoreactivity was noticed in a large proportion (80%) of the cells freshly dissociated from the fetal pig mesencephalic tissue. The immunoreactivity decreased progressively after transplantation of the cells into the rat brain. This observation suggests that dissociated neuroblasts are capable of a temporary expression of beta1 integrin. This molecule is known to participate in the process of cell sorting and migration in the developing brain. Hence, its expression could be the hallmark of a rescue mechanism triggered by the disruption of the cell/matrix interactions during the dissociation of the fetal mesencephalon. This disruption might account for part of the dramatic cell death process that occurs during the manipulation of the donor tissue.

Peripheral nerve allografts stored in green tea polyphenol solution.

BACKGROUND: We previously demonstrated the successful 1-month storage of peripheral nerve segments in a green tea polyphenol extract. We investigated whether this method could reduce the donor-host immune reaction associated with peripheral nerve allotransplantation. METHODS: Sciatic nerve segments (20 mm long) were harvested from Dark Agouti (DA) rats, stored in polyphenol solution (1 mg/mL) for 1 month, and transplanted into recipient major histocompatibility complex-mismatched Lewis rats to bridge 15-mm-long sciatic nerve gaps (polyphenol-treated allograft group). The controls were an isograft group (nerve segments harvested from Lewis rats were immediately transplanted into Lewis rats), a polyphenol-treated isograft group (nerve segments harvested from Lewis rats were treated by polyphenol in the same method and transplanted into Lewis rats), and a fresh allograft group (nerve segments harvested from DA rats were transplanted into Lewis rats without storage). To investigate the origins of the cells in the transplanted nerves, sciatic nerve segments harvested from the male DA rat donors were transplanted into female Lewis rat recipients; genomic DNA was extracted from each nerve segment and amplified by polymerase chain reaction using primers specific for the rat sex-determining region of the Y-chromosome (Sry). RESULTS: Nerve regeneration in the polyphenol-treated allograft group was similar to that in the isografted group. Sry-specific bands were detected in all samples in the sex-mismatched polyphenol-treated allograft specimens despite their major histocompatibility complex incompatibility. CONCLUSIONS: Storage in green tea polyphenol solution can reduce both ischemic damage to nerve tissue and donor-host immune reactions after allotransplantation.

Compartmentalization of TCR repertoire alteration during rejection of an intrabrain xenograft.

Xenograft rejections of embryonic pig neural cells implanted into the adult rat striatum occurs within 3-4 weeks, following a dramatic T cell infiltration. Little is known about the cross-talk between the brain and peripheral lymphoid tissues which results in this recruitment and lymphocyte homing. To better characterize the dynamics of the T cell response against xenogeneic neural cells implanted into the brain parenchyma, we used both qualitative and quantitative methods to follow the alterations of the CDR3 length distribution (CDR3-LD) of the TCR (T cell receptor) beta chain in the transplanted striatum and compared this response to that observed in the deep cervical lymph nodes, spleen, and blood. Data showed that the T cell repertoire diversity was highly altered in the recipient brain during xenograft rejection. Comparison of the alterations of the CDR3-LD between several animals revealed a single public alteration in the Vbeta20 family, and many private alterations of the CDR3-LD which differed from one infiltrated brain to another. Alterations of the T cell repertoire were also observed in lymphocytes homed into the deep cervical lymph nodes. However, they differed from the alterations detected in the infiltrated brains. Conversely, no significant alteration of the CDR3-LD was detected in the spleen or in the blood. These data suggest that the deep cervical lymph nodes play an active role in the process of xenograft recognition or/and rejection. However, they also indicate that the fate of T cells homed in the brain and deep cervical lymph nodes differs.

[Experimental research on immunological rejection of brain tissue allograft].

AIM: To investigate the occurrence of rejection in brain tissue transplantation. METHODS: Ventral mesencephalon(VM) single cell suspensions from newborn rats were allografted into the striata of Parkinson's disease model recipient rats. Surviving animals were sacrificed at 2, 4 and 6 wk after transplantation, and the brain tissues were stained with HE or immunocytochemically to inspect the expression of tyrosine hydroxylase (TH) and major histocompatibility complex class II antigens (MHC II). RESULTS: High MHC II expression was observed within and around the allografts compared with control at 2 wk after grafting. MHC II expression then decreased at 4 wk and at 6 wk only few immunopositive cells remained. The number of TH positive neurons was low at 2 wk, but increased at 4 and 6 wk after transplantation. CONCLUSION: The brain is not an "immunologically privileged site" and graft rejection exists in brain tissue transplantation. This rejection maybe induced by MHC II. Therefore it is necessary to use immunosuppressants in brain transplantation.

Intracerebral cytokine profiles in adult rats grafted with neural tissue of different immunological disparity.

To understand graft rejection in cell based therapies for brain repair we have quantified IL-1beta, IL-2, IL-4, IL-10, IL-12p40, IFN-gamma and TNF-alpha mRNA levels using real-time PCR, at days 4, 14, and 42 post-transplantation, in rats engrafted with syngeneic, allogeneic, concordant and discordant xenogeneic neural tissues. In addition, in the discordant xenografts immunohistochemistry and in situ hybridization were applied to detect local expression of IFN-gamma, TNF-alpha, IL-10 and TGF-beta. Allografts remained non-rejected but expressed IL-1beta, TNF-alpha and IL-4 transcripts but not IL-12p40 and IFN-gamma. Xenografts demonstrated distinct cytokine profiles that differed from syngeneic and allogeneic grafts. Non-rejected discordant xenografts contained higher levels of TNF-alpha transcripts and lower levels of IL-2 transcripts than the rejected ones at day 42. Discordant xenografts displayed a stronger and earlier expression of IL-1beta and TNF-alpha, followed by T-helper 1 and T-helper 2 associated cytokine expression. The number of cells expressing mRNA encoding TNF-alpha and TGF-beta was significantly increased over time in the discordant group. In conclusion, the immunological disparity of the implanted tissue explains survival rates and is associated with different cytokine profiles. In allografts, a chronic inflammatory reaction was detected and in xenogeneic grafts a delayed hypersensitivity like reaction may be involved in rejection.

Activated porcine embryonic brain endothelial cells induce a proliferative human T-lymphocyte response.

Transplantation of allogeneic embryonic neural tissue is a potential treatment for patients with Parkinson's and Huntington's diseases. The supply of human donor tissue is limited, and alternatives such as the use of animal (e.g., porcine) donor tissue are currently being evaluated. Before porcine grafts can be used clinically, strategies to prevent neural xenograft rejection must be developed. Knowledge on how human T lymphocytes recognize porcine embryonic neural tissue would facilitate the development of such strategies. To investigate the ability of porcine embryonic brain microvascular endothelial cells (PBMEC) to stimulate human T-cell proliferation, PBMEC were immuno-magnetically isolated and cocultured with purified human CD4 or CD8 single-positive T cells. PBMEC had a cobblestone-like growth pattern and expressed the endothelial cell markers CD31 and CD106. PBMEC stimulated with the supernatant of phytohemagglutinin-activated porcine peripheral blood mononuclear cells or porcine IFN-gamma, but not nonstimulated PBMEC, induced proliferation of both CD8 and CD4 T cells as assessed by [3H]thymidine incorporation. Flow cytometric analyses showed that the degree of CD8 and CD4 T cell proliferation correlated with the expression levels of class I and II major histocompatibility complex (MHC) antigens, respectively. PBMEC expressed a CTLA-4/Fc-reactive molecule, most likely CD86, suggesting that these cells are able to deliver a costimulatory signal to the T cells. Human TNF-alpha, but not human IFN-gamma, induced class I, but not class II, MHC expression on PBMEC. Within a neural graft or the regional lymph nodes, PBMEC might stimulate human T cells via the direct pathway, and should therefore be removed from the donor tissue prior to transplantation.

Simultaneous inhibition of B7 and LFA-1 signaling prevents rejection of discordant neural xenografts in mice lacking CD40L.

Transplantation of embryonic human neural tissue can restore dopamine neurotransmission and improve neurological function in patients with Parkinson's disease. Logistical and ethical factors limit the availability of human embryonic allogeneic tissue. Embryonic xenogeneic neural tissue from porcine donors is an alternative form of donor tissue, but effective immunomodulatory techniques are warranted for neural xenotransplantation to become clinically feasible. We transplanted embryonic porcine ventral mesencephalic tissue into the brains of adult untreated C57BL/6 mice, untreated CD40L-/-mice and CD40L-/-mice that received injections of anti-LFA-1, CTLA41g or both compounds. Double-treated CD40L-/-mice had large grafts with high numbers of dopaminergic neurons 4 wk after transplantation. The grafts were completely devoid of lymphocytes, macrophages and activated microglia. Untreated C57BL/6 mice had rejected their grafts. Untreated CD40L-/-mice and CD40L-/-mice treated with monotherapy of anti-LFA-1 or CTLA41g had smaller grafts and more microglial and lymphocytic infiltration than double-treated CD40L-/-mice. We conclude that immunomodulation with concomitant inhibition of LFA-1 and B7 signaling in the perioperative period in CD40L-/-mice prevented the rejection of discordant neural xenografts. The treatment most likely reduced antigen presenting capacity and interfered with the costimulatory signaling needed for T cell activation to occur.

hNT neurons express an immunosuppressive protein that blocks T-lymphocyte proliferation and interleukin-2 production.

Ntera2/D1 cells had an A1 B8 Bw6 Cw7 DR3 DR52 major histocompatibility complex (MHC) genotype. Its neuronal derivative, hNT neurons, expressed A1 B8 Bw6 MHC class I molecules, but did not activate, and its hNT supernatant suppressed allogeneic mixed lymphocyte cultures (MLC) >98% (p<0.01), phytohemagglutinin (PHA)-activated T-cell proliferation >87% (p<0.01), even 48 h after stimulation, suppressed phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced T-cell proliferation >99% (p<0.001), and reduced interleukin-2 (IL-2) production (p<0.01), while maintaining T cells in a quiescent G(0)/G(1) state without lowering their viability. This immunosuppressive activity was attributed to a 40-100-kDa anionic hNT protein with an isoelectric point of 4.8.

Porcine neural xenografts in rats and mice: donor tissue development and characteristics of rejection.

Embryonic ventral mesencephalic tissue from the pig is a potential alternative donor tissue for neural transplantation to Parkinson's disease patients. For stable graft survival, the host immune response has to be prevented. This study was performed in order to analyze the mechanisms and dynamics of neural xenograft rejection, as well as neurobiological properties of the donor tissue. Adult normal mice and rats, and cyclosporin A-treated rats, received intrastriatal transplants of dissociated embryonic ventral mesencephalic pig tissue that was 27 or 29 embryonic days of age (E27 and E29). The animals were perfused at 2, 4, 6, and 12 weeks after grafting and the brains were processed for immunohistochemistry of dopaminergic (tyrosine hydroxylase positive) neurons, CD4(+) and CD8(+) lymphocytes, natural killer cells, macrophages, microglia, and astrocytes. Thirty-five rats received daily injections of BrdU for 5 consecutive days at different time points after transplantation and were perfused at 6 weeks. These animals were analyzed for proliferation of cells in the donor tissue, both in healthy and in rejecting grafts. No tyrosine hydroxylase-positive cells proliferated after grafting. Our results demonstrated that E27 was superior to E29 donor tissue for neurobiological reasons. Cyclosporin A immunosuppression was protective only during the first weeks and failed to protect the grafts in a long-term perspective. Grafts in mice were invariably rejected between 2 and 4 weeks after transplantation, while occasional grafts in untreated rats survived up to 12 weeks without signs of an ongoing rejection process. CD8(+) lymphocytes and microglia cells are most likely important effector cells in the late, cyclosporin A-resistant rejection process.

Comparison of reactive processes in the rat brain elicited by xenotransplantation of nervous tissues of chicken or pulmonate snail.

It is known that a histocompatibility system is not developed to the same extent in lower invertebrates as in vertebrate animals. We assumed that the xenografts from the newborn invertebrate nervous system would not exert destructive effects on the brain of the vertebrate recipient even without immunosuppressive therapy. In search of brain xenografts (XG) capable to survive in the brain of a recipient without intensive immunosuppression, we transplanted ganglia of terrestrial snails into the rat brain. We compared effects of transplantation of the XG taken from anterior brain of the 18-day embryo chicken (XGC) and from ganglia of a newborn terrestrial pulmonate snail (Helix aspersa L., XGSn). Part of the XGSn were stained by vital fluorescent dyes Bisbenzimid or Fast Blue before grafting. The XGSn were implanted into the neocortex parenchyma in each hemisphere. Rat brains with the XGC were examined 5 days after, and brains with the XGSn - 5 and 28 days after the transplantation. Nonstained sections with the XGSn labeled with fluorescent dyes prior to transplantation were investigated in fluorescent microscope and stained later with tionin and cresyl-violet. Quantitative videoimage analysis of lymphocyte aggregations, reactive gliosis, morphology of the XG areas, and implantation trace was performed. It was found that the XGSn transplantation did not elicit in the rat brain an intensive immunological conflict 5 and 28 days after transplantation. In contrast, the XGC rapidly elicited a strong immune response resulting in massive obliterations in the rat brain and were rejected in 5 days. Labeled snail glia and vessels were observed in the stained XGSn 28 days after transplantation by fluorescence imaging. Putative snail vessels grew into the rat brain from the place of snail tissue transplantation serving the humoral integration of the XG and the host brain. Migration of molluscan glial cells was observed in the brain of recipients.