To be or not to be accepted

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Editorial Manager(tm) for Seminars in Immunopathology Manuscript Draft Manuscript Number: Title: To be or not to be accepted: the role of immunogenicity of neural stem cells following transplantation into the brain in animal and human studies Article Type: Review Article (invited) Keywords: Neural progenitor cells; neural transplantation; graft-host interaction; host defense system; immuntolerance; neurodegenerative diseases Corresponding Author: Guido Nikkhah, MD, PhD Corresponding Author's Institution: First Author: Philipp Capetian Order of Authors: Philipp Capetian;Máté Döbrössy;Christian Winkler;Marco Prinz;Guido Nikkhah, MD, PhD Abstract: Grafting of neural stem cells into the mammalian central nervous system (CNS) has been performed for some decades now, both in basic research and clinical applications for neurological disorders such as Parkinson's and Huntington's disease, stroke and spinal cord injuries. Albeit the "proof of principle" status that neural grafts can reinstate functional deficits and rebuild damaged neuronal circuitries many critical scientific questions are still open. Among them are the manifold immunological aspects that are encountered during the graft-host interaction in vivo. For example, the experience with allografted cells in absence of immunosuppressant drugs has raised serious doubts about an immunological privileged site within the CNS as compared to other engraftment sites in the body. This review discusses recent experimental and clinical findings demonstrating that neural stem cells have unique characteristics that help them modulate the host immunological defense, but, under some conditions, may still trigger a rejection process. Implications of these findings on neural grafting and potential new therapeutic applications are discussed.

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To be or not to be accepted: the role of immunogenicity of neural stem cells following transplantation into the brain in animal and human studies

Philipp Capetian1,2, Máté Döbrössy1, Christian Winkler2, Marco Prinz3, Guido Nikkhah1

1)

Laboratory of Molecular Neurosurgery, Department of Stereotactic and Functional

Neurosurgery, Neurocenter, University Medical Center Freiburg, Breisacher Str. 64, D-79106 Freiburg, Germany. 2)

Department of Neurology, Neurocenter, University Medical Center Freiburg, Breisacher Str.

64, D-79106, Freiburg, Germany 3)

Department of Neuropathology, Neurocenter, University Medical Center Freiburg,

Breisacher Str. 64, D-79106 Freiburg, Germany . Corresponding author: Guido Nikkhah, MD, PhD Laboratory of Molecular Neurosurgery, Department of Stereotactic and Functional Neurosurgery, Neurocenter, University Medical Center Freiburg, Breisacher Str. 64, D-79106 Freiburg, Germany. tel: +49-761-270-50630 fax: +49-761-270-50100 e-mail: [email protected]

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Abstract Grafting of neural stem cells into the mammalian central nervous system (CNS) has been performed for some decades now, both in basic research and clinical applications for neurological disorders such as Parkinson’s and Huntington’s disease, stroke and spinal cord injuries. Albeit the “proof of principle” status that neural grafts can reinstate functional deficits and rebuild damaged neuronal circuitries many critical scientific questions are still open. Among them are the manifold immunological aspects that are encountered during the grafthost interaction in vivo. For example, the experience with allografted cells in absence of immunosuppressant drugs has raised serious doubts about an immunological privileged site within the CNS as compared to other engraftment sites in the body. This review discusses recent experimental and clinical findings demonstrating that neural stem cells have unique characteristics that help them modulate the host immunological defense, but, under some conditions, may still trigger a rejection process. Implications of these findings on neural grafting and potential new therapeutic applications are discussed.

Keywords Neural progenitor cells; neural transplantation; graft-host interaction; host defense system; immuntolerance; neurodegenerative diseases

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Introduction As the adult central nervous system (CNS) has only minimal intrinsic regenerative capabilities, damage to it can result in irreversible morphological and/or functional losses. Neuroscientists have strived since many years to overcome these intrinsic regenerative limitations by exogenous restorative attempts, such as the transplantation of neural stem cells. Early in the course of this research it has been discovered that only embryonic or fetal cells are able to survive and induce a functional recovery following the traumatic transplantation procedure (for an extensive review see 1). Based on promising preclinical data, the feasibility and effectiveness of neurotransplantation has been evaluated in numerous clinical trials for two decades. However, it’s development is still in its infancy and has to be regarded as an experimental therapy for neurological diseases like Parkinson's disease (PD)2 or Huntington's disease (HD)3. From an immunological perspective it is interesting that allografting fetal CNS tissue into the adult brain does not share the same problems observed with solid organ transplantation and immunosuppression is, if at all, only temporarily necessary4. The main reason for this phenomenon was thought to be the immunopriviledge of the mammalian CNS, leaving transplanted tissue mainly unaffected by the host defence 5. This, however, has proven not to be entirely correct and the report on a grafted HD patient with signs of overt rejection 14 months

after

grafting

and

alloimmunisation

occurring

in

patients

even

under

immunosuppression6 demonstrated that there still is quite a lack of knowledge on the complex immunological graft-host interactions following intracerebral neural stem cell transplantation. In this review we want to discuss recent findings on the possible causes for the excellent survival of fetal CNS allografts but also reasons for their potential rejection. As we try to depict, neural stem cells (NSC) themselves can actively circumvent and modify the host response and these capabilities can even be utilized for novel therapeutic approaches aiming at inflammatory CNS diseases. For a matter of simplification, the diversity of neural progenitor cells that have been employed in restorative attempts in animal and human

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studies will be summarized in the term neural stem cells (NSC) for this review, mainly referring to the vast body of evidence that has been derived from studies with fetal neural tissue and cell suspension grafts.

Neural stem cells possess a low immunogenic profile

In the beginnings of the neurotransplantation era it has been assumed that the good intracerebral survival of transplanted NSC was foremost a consequence of the brain as an immunopriviledged site: The mammalian CNS was considered to be a "no-go" area for defense cells where allogeneic tissue or cells could be engrafted and survive unaffected by the host's immunity 5 7. Since then increasing evidence has accumulated that has challenged this hypothesis mainly because of the two following observations: Fetal NSC do survive in a non-immunopriviledged site

8

and can also be rejected inside the

CNS under certain circumstances 9. Hori et al. published an elegant study that demonstrates a low immunogenic nature of NSC 8. NSC grown as neurospheres in culture derived from newborn C57BL/6 mice forebrains were placed in the kidney capsule as a nonimmunopriviledged site of BALB/c recipient mice. Although these two strains are immunologically incompatible, NSC survived for 28 days without rejection. This was a result of lacking sensitization of the host against the graft as mice receiving the NSC showed no delayed hypersensitisation against splenocytes from the donor strain. It was not a suppression of host immunity as the grafts were readily rejected when the recipient animal was either pre- or post-sensitized with donor splenocytes. In contrast, cerebellar tissue from these animals, consisting of mature neurons was rejected in most cases. NSC showed no expression of MHC I or II, in contrast to the cerebellar tissue. Expression of both surface molecules could be induced by stimulation with IFN-γ. This demonstrated that NSC themselves are not attacked by defense mechanisms since they do not sensitize the host (low immunogenicity) but can however be rejected if the host is or becomes sensitized (normal antigenicity).

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Is the absence of MHC I and II expression the sole reason for the low immunogenicity of NSC? Most likely not: In contrast to murine cells, prolonged expansion of human neural precursors in culture leads to increased MHC I and II expression, however not of the costimulatory CD40, CD80 and CD86 molecules. Peripheral lymphocytes were unresponsive when co-cultured with human NSC 10. Therefore, tolerance against NSC is therefore probably not soley due to missing MHC expression but also determined by the absence of costimulatory signals. Conversely, the CNS does not protect grafted neural transplants against host defence. Rat striatal allografts were rejected when sensitizing skin allografts were placed either simultaneously with, two or 6 weeks after the neural grafts 9. This shows that the relative immunopriviledge of the CNS is at no time point secure enough to prevent rejection of the graft when sensitization of the host occurs.

Is rejection of neural grafts an important clinical issue or is the discussion about graft immunogenicity a merely academic one?

In the past there have been concerns that the loss or lack of clinical improvement after neural grafting could be due to immunological rejection processes that compromise neural graft survival and function. However, autopsy findings of patients at long-term follow-up posttransplantation showed little evidence of immunological reactions

11 12 13 14 15

..There is only

one documented case where possibly neural graft rejection took place 6. Fourteen months after grafting of whole ganglionic eminence tissue intrastriatally, one female HD patient experienced weight loss and deterioration of the choreatic movements on her left body side. MRI and PET-scans were indicative for an encephalitic process and, as an infection could be ruled out, a rejection process seemed most likely. Notably, a humoral response against the transplanted fetal material with occurrence of donor allele-specific anti-HLA-I antibodies had occurred after transplantation. Acute anti-inflammatory treatment and reinstatement of the original immunosuppressive regimen rapidly led to regression of MRI signs of encephalitis.

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The general clinical status remained altered for 4 months and recovered thereafter. Anti HLA-I-antibodies were no longer present during follow-up. Allo-immunisation, however, is a frequent event (5 out of 12 patients), even under ongoing immunosuppression as this study could demonstrate. Except the one patient reported, none of the others showed overt signs of immunological rejection. Although well documented, this case cannot quite explain the reason for the sudden rejection. The development of graft-specific antibodies cannot serve as an explanation since obviously half of the patients develop them without overt signs of rejection. All patients received adequate immunosuppression for about one year and one patient had developed anti-HLA-I antibodies under ongoing immunosuppression half a year after grafting. As it seems, development of anti-HLA-I antibodies can take place early after grafting. Our group published one case of a HD patient who deceased six months after grafting16. As this is the earliest posthuman examination of a clinical case grafted intracerebrally with allogenic fetal tissue it allows for important insights on the potential mechanisms and the time window during which allo-immunisation may occur. While studies evaluating allografted human HD brains with a longer time delay after transplantation showed only minimal presence of reactive cells of the host defence15, we observed a different immopathological picture: Intraparenchymal

presence

of

host

immune

cells

(as

demonstrated

e.g.

by

macrophage/microglia marker C68) was only slightly elevated above normal (figure 1 a, b). However, in all deposits located in both striata, pronounced cuffing of monunucelar cells around vessels could be found (figure 1 c, d, e). The cells inside these cuffs proved to be grafted neuronal progenitors as shown by the expression of doublecortin (DCX) (figure 1 c, d, e), CD4+ T cells (figure 1 d) and CD8+ T cells (figure 1 e). It could be demonstrated by immune-electron-microscopy that this “homing” of cells occurred in the so called VirchowRobin space, a space between the glia limitans, (a basal membrane formed by astrocyte processes) and the basal membrane of the endothelial cells (figure 2 a, b). This means that intracerebrally grafted cells reside at least temporally outside the blood brain barrier. Interestingly, this seems to be characteristic of the early post-grafting period as studies on

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patients deceased at longer post-grafting periods did not report similar findings. This raises interesting questions about the origin and the immunological significance of these perivascular cuffs. In fact, they may arise from two different scenarios: Either neural precursor cells reside inside this niche and attract host defense cells, or defense cells form cuffs around these vessels and this leads to a homing of grafted cells. As we will demonstrate, there is published data supporting both hypotheses. Oligodendroglial precursor cells, for example, migrate along the Virchow-Robin space when grafted intraspinally17. On the other hand, the apoptosis marker activated caspase 3 (figure 1 c, d, e) was absent inside neural cells grafted into the striatum. So it can be concluded, that the presence of host defense cells alone is not sufficient to induce more vigorous immunological rejections processes leading to apoptosis. In contrast, if we assume that DCX+ positive cells enter the Virchow-Robin spaces first this would mean that the host defense is not unaware of the grafted cells even when not attacking. It could also be imagined that a “rendezvous” between host defense and graft cells could lead to antibody production. Obviously the production of anti-HLA-I antibodies by the host is quite common while overt acute rejection is extremely rare. Therefore, one would expect a rare occurrence inside either the grafted cells or the host defense leading to the transplants being attacked, probably even completely independent from antibody production. There is experimental data showing that murine NSC can become immuno-competent when infected with the Japanese encephalitis virus

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. NSC express in these cases immuno-

stimulatory molecules such as MHC class I, CD40, CD80 and CD86. Furthermore, they start secreting IL-2 and activate T cells which might suggest that an inflammatory environment such as a subclinical viral encephalitis may trigger an immunological host response leading to rejection. This is certainly an interesting hypothesis that awaits further experimental validation. Transplantation of allogenic NSC into patients with PD and HD was, at least, partially successful which has been attributed also to the low immunity and good survival of NSC in humans. Based on these results attempts had been undertaken to use porcine xenografts as

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cell donors to circumvent the problems associated with human fetal tissue (low accessibility and ethical issues). As studies of xeno-transplanting of fetal porcine tissue in rodents were encouraging, clinical transplantation trials in PD patients were initiated

19

. However, the

outcome was very disappointing and resulted in low numbers of surviving and differentiating NSC inside the xenografts. This may not primarily be related to epitope presentation but to galactosyl-epitopes expressed by non-primate mammals. Humans and primates have circulating anti-Gal antibodies leading to xenograft rejection

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.

Influence of the transplantation procedure

Fetal NSC are implanted in most cases by stereotactic techniques into deep-seated target areas of the brain and have to survive, differentiate and functionally integrate afterwards. There are at least three factors to be taken into account that can influence the extent of the host reaction and, therefore, graft survival: First, the extent of the direct tissue trauma during the implantation phase, secondly, the tissue preparation, and, finally, the site of implantation (target area). The implantation of the NSC requires intraparenchymal injection of the cell suspension. This leads to a direct operative trauma and a breakage of the blood-brain-barrier (BBB) for at least 2 weeks, as demonstrated by Evan’s blue injection in rats who received fetal murine cells21. However, cessation of immunosuppression, as used e.g. in the xenograft situation, at later time points after reconstitution of the BBB still leads to rejection. This shows that an intact BBB per se does not protect against neural graft rejection,, at least in the xenografting situation. A larger implantation trauma, as inflicted by using a metal cannula in contrast to a thinner glass capillary enhances MHC class I and II expression in host cells, perivascular cuffing of lymphocytes and gliosis when transplanting fetal ventral mecencephalic tissue in a rat to rat allograft situation22. This reaction is temporal, however, and expression of host MHC class I and II is pronounced when allografts instead of syngenic grafts are used. However, under

8

these experimental conditions no signs of a more vigorous immunological rejection could be found, even without immunosuppression. The extent of the preparation of the cell suspension plays another important role: Solid grafts possess a pre-existing donor-derived vasculature, which may connect with host vessels

23

and thus, through the endothelial cells, expose foreign MHC molecules to the host defence cells

24

. In contrast, cell-suspension grafts are predominantly vascularized by host vessels

25

and thus lack this source of alloantigen expression. Furthermore, the site of implantation can have a considerable effect on the host immune reaction. For example, peri- or intraventricular graft placement can lead to increased rates of immune response in terms of MHC class I expression inside the grafts and lymphocyte invasion26. There are conflicting results concerning the extent of immunological response and accompanying graft destruction, but graft survival has been demonstrated at this site 27 26. The underlying reasons for the increased immunological host-derived response seen with peri- and intraventricular NSC grafts have been attributed to the drainage of the subarachnoid space to cervical lymph nodes

28

, the lack of a BBB in the circumventricular

organs, choroid plexus and subarachnoid space, as well as to the presence of MHC class IIpositive macrophages in the choroid plexus and subarachnoid space

29

. However "leakiness"

of the BBB cannot be the major cause as NSC grafts can survive outside the BBB without immunogenic reaction and destruction 8 (see also discussion above). Most clinical transplantations in human patients up to now have been performed by using the so-called “macrotransplantation” of small tissue pieces. In contrast to the animal situation

25

,

we could demonstrate that allografts in HD patients do not contain endothelial cells of graftderived origin by FISH against X and Y Chromosomes even when employing macrotransplantation methods (figure 3)

16

. 5 to 6 months after grafting all endothelial cells

have obviously been replaced by a host-derived vasculature inside the grafts.

Immunomodulatory effects of neural stem cells

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A study exploring the ability of transplanted neurospheres-derived cells to ameliorate symptoms of

a murine model of Multiple Sclerosis (MS), the so called experimental

autoimmune encephalomyelitis (EAE), was performed some years ago 30. Both intravenously as intraventricularly administered cells led to an accelerated recovery of symptoms and better final outcome after induction of EAE. This effect could not be reproduced with dead cells, fibroblast or bone marrow-derived stem cells and it was concluded that it represents a genuine effect of NSC. As the authors could observe transplant-derived cells ensheathing host axons as well as differentiating themselves into mature neurons, they concluded that the effect of this treatment was achieved by cell replacement. However, an additional immunomodulatory effect of transplanted neurospheres was also considered. When the authors of this experiment studied the fate of the grafted cells over several courses of this relapsing-remitting disease, they could find that transplanted cells when being injected intravenously survived in an undifferentiated state in perivascular areas while the numbers of surviving engrafted cells intraparenchymaly were negligible31. These perivascular niches were also populated by lymphocytes. NSC in these niches expressed factors of angiogenesis, neurogenesis and growth factors. Furthermore it could be demonstrated that NSC enter the CNS actively by a very late antigen (VLA)-4 – VCAM-1 interaction when administered intravenously. A specific pro-apoptotic effect of NSC on inflammatory Th1-cells could be demonstrated in this study in vitro as well as in vivo. A more systemic effect of NSC on the host auto-immunity could be demonstrated in a later study 32: NSC were found in lymph nodes in close interaction with cells of those organs. The activation of myeloid dendritic cells was hampered by NSC and the expansion of antigen specific encephalitogenic T cells was restrained. The cause of these actions was found to be a novel highly neural precursor specific bone morphogenic protein (BMP)-4 dependent mechanism, as the effects could be reverted with the BMP-antagonist noggin. Therefore, the peripheral immune response may also modulate the immune reaction in the CNS. Both, the disease attenuating effect of transplanted neural precursors as well as the formation of perivascular niches could be demonstrated in a later study also in a non-human

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primate model of MS33. NSC derived from human embryonic stem cells do show a similar effect34. These elegant studies introduce an entirely new aspect on the restorative capacity of NSC mediated by the immune system: These cells not only own stealth characteristics leaving them widely ignored and unaffected by the host defense system under normal circumstances, they quite effectively modulate the host’s immune system by unique mechanisms. Keeping these findings in mind another interesting explanation of the afore-mentioned NSClymphocyte-rendezvous can be envisioned. Immature NSC share a low immunogenic profile, whereas more matured neurons obviously don’t, as grafting neonate cerebellar tissue leads to graft rejection when grafted into a non-immunopriviledged site8. At 6 months post-grafting we could find in our necropsy study cells expressing the more mature neuronal marker NeuN and the striatal projection neuron marker DARPP32 (figure 4)18. Therefore, if we assume that the graft as such is attracting host defense cells, their cuffing around vessels could attract neural precursor cells from the graft, actively entering this space and modulating or even suppressing the inflammatory reaction. In this context it is important to note that at least in the rodent model immunosuppression is not necessary for graft allograft survival22. This leaves us with the question on how does the neural graft attract the host defense? As we already discussed the mechanical operative trauma is certainly one major stimulating factor for the host immune system to respond. During this phase the mechanical alteration of the surrounding tissue attracts macrophages (fig. 1 b) that potentially ingest dead donor cell material and present it to host defense cells. As already mentioned, perivascular cuffing was not reported by autopsy studies on grafted patients deceased after a longer timespan following the implantation procedure. Possibly, more mature grafts do no longer attract the host defense to that extent.

Conclusion Transplantation of NSC into the brain is from an immunological viewpoint a true challenge

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both for the host as well as for the donor cells. Will they be accepted or not and what are the mechanisms of graft-host interaction? In contrast to the long held opinion that the immunopriviledge of the CNS is the main reason for the good survival of fetal NSC grafts, we have discussed here more recent studies that present interesting and intriguing novel insights into a complex and multimodal scenario of stimulatory and inhibitory signals traversing from the grafted NSC to the host immune system and backwards. By low or absent expression of immuno-stimulatory or co-stimulatory molecules, NSC avoid the detection by the host defense even when grafted outside the CNS. However, anti-HLAantibodies have been observed in grafted HD patients but their immunological relevance for a potential graft rejection has to be further elucidated 35. Although a vigorous graft rejection is a rare event in clinical and experimental neuro-allografts, it can be seen and the reasons for this are poorly understood. Of course, the concomitant immunosuppressant regime may play a pivotal role but a broader consensus on this issue has not been achieved so far 36. Further knowledge about these issues is clearly warranted in order to increase the safety and efficacy of neural transplantation in patients. Procedural factors of grafting have an impact on graft survival and host response towards the graft. Minimizing trauma and using cell suspensions for grafting seem to improve neuronal survival and lower the inflammatory response, as shown for example recently in animal models PD 37. Cell replacement was thought for a long time to be the mode of action in neural grafting (see also

38

for a recent review). However, more recent results have underlined the

immunomodulatory qualities of NSC on the outcome of the transplantation. Exploring those cellular and molecular mechanisms further seems a promising scientific endeavor as in many traumatic and neurodegenerative disorders of the CNS inflammation plays a crucial role and triggers cerebral damage and encompasses many different cell types. Immunomodulatory effects of grafted NSC may contribute beneficially to halt or modify the damage of the host brain and/or contribute to their own survival and functional integration into the host neural circuitry. In our own opinion the scientific evidence has become strong enough to accept that

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the immunogenicity of NSC in the neural transplantation paradigm is both for bad and good.

Acknowledgements This research was supported by grants from the European Commission under the 7th Framework Programme –HEALTH – Collaborative Project Transeuro (Contract n°242003), the German Parkinson Foundation (dPV) and the Weber-Petri-Foundation. The authors declare that they have no conflict of interest.

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Figure 1: Striatal grafts in a HD patient’s brain deceased five, respectively six months after grafting of fetal ganglionic eminence derived tissue: The graft is predominantly composed of DCX+ neuronal progenitors. CD45+ lymphoid cells (a) and CD28+ macrophages (b) are present. A vast influx is found inside perivascular cuffs in the so called “Virchow-Robin” spaces between the glia limitans composed of the astrocytic processes and the endothelial basal membrane (c). DCX+ progenitors are also present in these spaces. Both CD4+ T-helper- (d) and CD8+ T-killer-cells (e) can be found inside the perivascular cuffs, the presence of cleaved caspase 3 (CCP3) positive apoptotic nuclei is however very low.

Figure 2: Immuno-electron-micrograph of perivascular spaces in the graft region: Lymphoid cells (a, asterisks) and DCX+ cells labelled by the black reaction product (b, asterisks) can be found between the glia limitans (a, b arrows) and the endothelial basal membrane (b, arrowheads).

Figure 3: Fluorescence in situ hybridization (FISH) with probes directed against human X-chromosome (red) and Y-chromosome. Inside striatal grafts derived from a female fetus (XX), all endothelial cells (arrow) surrounding vessels (asterisk, the serum has a green autofluorescence) originate from the male host (XY).

Figure 4: Mature neurons coexpressing the striatal projection neuron marker DARPP32 and neuronal nuclear antigen (NeuN) with surrounding immature neuronal progenitors expressing DCX inside the graft area.

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