Theimmuneresponseto intracerebralneuralgrafts D. J. Sloan, M. J. Wood and H. M. Charlton Neural transplantation offers a potential therapeutic For example, Faustman et al. 11, reported that prior approach to a variety of neurological disorders, most treatment of pancreatic islets of Langerhans with notably those of a degenerative nature. However, the antibody to deplete dendritic cells led to prolonged degree of immunological privilege (i. e. isolation from an graft survival in allogeneic hosts. Reconstitution of the immune response) in the brain, which is not absolute, recipients of long-standing islet grafts with donor may be a significant impediment to the survival of strain dendritic cells resulted in the rejection of the histoincompatible grafts. The nature of this privilege, islet grafts. However, this result was dependent upon together with the specific immune events leading to the strains of mice or rats used 12. It should be added neural graft rejection, are discussed. As a consequence that donor leucocytes other than dendritic cells might of this immune-mediated rejection, immunosuppression also be capable of initiating rejection, but present in some form might be necessary to guarantee long-term evidence favours dendritic leucocytes as the primary graft survival. Various strategies are being explored to immunostimulating cell type. The above mechanisms lead to sensitization of the suppress the immune response to neural grafts, not only for future use in clinical therapies, but also to bring host, causing the activation and proliferation of intracerebral allo- and xenotransplantation to the suitably specific precursor lymphocytes within the host lymphoid organs. Together they represent the attention of the general neurobiologist. afferent arc of the immune response, since they The concept that the CNS represents an immunologi- involve the passage of cells or antigens from the graft cally privileged site has evolved since the early days of to the host lymphoid organs and the activation of tissue transplantation to the brain1. The fact that this lymphocytes, which takes place prior to the onset of privilege is not absolute has been pointed out in graft destruction. The efferent arc comprises the activated lymphoseveral reports z-6, and it is now possible to examine this concept of privilege with new immunological and cytes and antibodies that are released from the lymph histological techniques, and from the standpoint of an nodes or spleen into the blood circulation. It is obvious improved knowledge of the cellular immune response that there must be a route of entry for these to organ and skin grafts. A resum6 of the main lymphocytes into the graft and, indeed, there is events thought to be involved in graft rejection is evidence that lymphoid cells actively adhere to and cross the endothelium of blood vessels adjacent to and shown diagrammatically in Box 1. Why should intracerebral neural grafts survive within grafts 13. Finally, specific cytotoxic T cells and helper T cells, better than peripherally placed grafts, despite similar histocompatibility differences between the donors and which represent an important arm of the rejection hosts? In order to answer this question it is important process, depend upon the expression in donor tissue to consider, in the first instance, why the peripherally of foreign major histocompatibility complex (MHC) class I and class II molecules, respectively, for placed grafts are rapidly rejected. Medawar first proposed that transplantation efficient T-cell binding and function. Most peripheral immunity was the result of a systemic and not a local tissues express MHC class I molecules, but the reaction7. This was borne out in the elegant exper- expression of MHC class II molecules is restricted to iments of Barker and Billingham8, in which skin certain leucocyte populations, though their expression allografts (between strains of the same species) were is inducible in some non-leucocytic cells 14. By maintained for prolonged periods on pedicles of host interacting with MHC class II molecules on antigenskin that received a blood and nerve supply, but were presenting cells (APCs), within grafts, activated isolated from the host lymphatics and, therefore, the helper T cells can augment rejection by the production regional lymph nodes. However, Strober and of cytokines. Gowans 9 showed that extracorporeal kidney transplants were rejected even though these had only Immunity in the CNS vascular connections to the host, thus suggesting that In considering the above processes, how does the sensitization was also possible via the blood circu- CNS differ from the majority of peripheral sites? lation, probably to the spleen. Therefore, on the basis Lymph drainage from the brain to the peripheral of these experiments it can be argued that cells or lymph nodes is not well defined; nevertheless, antigens must be able to reach the lymphoid organs of experiments have shown that macromolecules dethe host via lymphatic or vascular connections in order posited within the brain can reach the deep cervical lymph nodes 15'16. for rejection to occur. With respect to the experiments of Strober and With regard to classical APCs, the dendritic leucoGowans9, Larsen et al. lO have recently shown that cytes that reside in almost all tissues of the body are donor leucocytes, in particular the specialized absent from the brain. However, the presence of cells immunostimulating cells called dendritic cells, appear with many APC surface markers is found within the in the spleen over a period of days following transplan- choroid plexus, but their possible role in the host tation of cardiac allografts in mice. These cells are immune response remains to be determined. found in most tissues and evidence that they are The blood-brain barrier exhibited by cerebral primarily responsible for stimulating graft rejection capillaries, when intact, might prevent surveillance of comes from depletion and reconstitution experiments. the immune system by cells, although it has been TINS, VoL 14, No. 8, 1991
© 1991, ElsevierSciencePublishersLtd, (UK) 0166- 2236191/$02.00
D. J. Sloan,M. J. Woodand H. M. £harlton areat the Deptof Human Anatomy, University of Oxford, South ParksRoad, Oxford OXl 3QX, UK.
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Box 1. Immune events in allograft rejection Foreign antigen is presented to host helper T-cell precursors by specialized antigen-presenting cells (APCs) (classically, dendritic leucocytes). Antigen dentritic leucocyte in processed form, and in association with the major histocompatibility complex (MHC) class II molecule - a heterodimer consisting of o~ and [3 chains - is presented to helper T-cell precursors. The helper T cells selected are those whose T-cell receptors (TCRs) bind favourably to the MHC(+ peptide I ) ~ antigen complex. The TCR is a polymorphic heterodimer comprising o~ and 1~ chains, each prTcie~°r X ~ TCR consisting of both variable and constant domains, and unique to a particular T-cell precursor. The interaction between the TCR and the MHCantigen complex is stabilized by several cell surface accessory molecules, among which, in the TCR-CD4 and MHC class II-peptide interaction case of helper T cells, is the CD4 glycoprotein; this c=constant domain interaction culminates in T-cell activation. v=variable domain The transcription rates of many cytokine genes IL-2~~ / are up-regulated in the activated cell and as a consequence numerous cytokines are secreted, of activated helper which interleukin-2 (IL-2) is of central importance in the generation and amplification of the immune response. Its actions are autocrine, resulting in the proliferation of helper T cells themselves, and paracrine, mobilizing various other effector-cell APO~ populations. The TCR of cytotoxic T-cell precursors, an important effector-cell group, interacts with processed antigen in association with an MHC class I molecule, a heterodimer consisting of an o~chain with one constant and two variable domains and the invariant I~2 microglobulin. In this case the TCR-MHC interaction is stabilized by a CD8 accessory molecule, although this binding alone is ( L-4, L-6, IFN-y) insufficient for cytotoxic T-cell stimulation and IL2 is also required for full activation and prolifera ation. ~l ulll~,l aLivl i The interplay between a number of cytokines TCR-CD8 and MHC class I-peptide interaction produced dictates the character of the immune c=constant domain response and results in the generation and acv=variable domain tivation of particular effector-cell populations: macrophages; lymphokine-activated killer cells recruitment of (LAK cells) and natural killer cells (NK cells), for effector cells which IL-2 and interferon-y (IFN-y) are particularly important; and B cells, which act via the production of cytotoxic antibodies and are dependent on IL-4 and IL-6. LAK cell Effector cells are recruited to the graft site APC where their actions may result in graft rejection. This sequestration is brought about by the proNK cell duction of local chemotactic factors (notably IL-I and tumour necrosis factor), which increases the B cell adhesive properties of neighbouring endothelial macrophage surfaces. These factors are produced as a consequence of increased expression of intercellular and endothelial adhesion molecules, thereby facilitating the subsequent migration of effector cells into the grafted tissue. Their continued cytokine production results in up-regulation of local MHC plasma cell cytotoxic antibody expression and perpetuation of the inflammatory cascade.
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demonstrated that it does not represent a barrier to activated lymphocytestT. The breakdown of the bartier during the grafting procedure could also provide a route for donor cells or tissue antigens to enter the host lymphoid tissue via the blood circulation to the spleen. 342
The transplantation antigens of the MHC, which play a key role in the generation and propagation of immune responses, are not expressed at detectable levels in healthy CNS tissue, apart from endothelial cells. However, in both adult and fetal rats, most cells of the CNS do have the capacity to express TINS, VoL 14, No. 8, 1991
certain of these antigens under pathological condifions. Astrocytes, microglia and capillary endothelial cells have all been shown to express MHC class II molecules and to produce some cytokines on stimulation with intefferon-y (IFN-y) in vitro 18-21. Perhaps the most likely cell type to be involved in augmenting rejection within the graft by expressing foreign MHC class II molecules and co-stimulatory cytokine signals to host helper T cells is the bone marrow-derived microglial cell, which probably represents the resident tissue macrophage within the CNS 22-24. Whether or not these cells can initiate rejection awaits investigation. Finally, the concept that neural tissue per se is nonimmunogenic has been conclusively denied by the fact that such tissue transplanted beneath the host kidney capsule is rejected as rapidly as an orthotopic skin graft2s. Is the brain an 'immunologically privileged' site? The use of specific monoclonal antibodies against a variety of lymphoid cell markers and against MHC antigens has made it possible to follow host-graft interactions. There is now overwhelming evidence that, while some protection is offered to foreign tissue grafts, delayed rejection can take place in the brain, particularly if there is wide genetic disparity between donor and host. Mason et al. 25 reported the survival of neural grafts to the third cerebral ventricle in rats when the donor strain differed from the recipient at the MHC loci alone, or multiple non-MHC loci alone. When the genetic disparity was increased to include both the MHC and multiple non-MHC loci, survival was more variable and leucocytic infiltration containing T cells, together with widespread MHC antigen expression on the donor tissue, was evident in the majority of the grafts. This suggests that differences in non-MHC or minor transplantation antigens can have a significant effect on the survival of neural grafts if they occur in addition to disparities between the MHC loci of host and graft. Even in the grafts with only MHC or only non-MHC differences, small foci of leucocytic infiltration and MHC expression were observed in some histological sections, suggesting that rejection was occurring at localized 'hot-spots' within the grafts, and also that matching the donor and host tissue at the MHC loci alone might not be sufficient to abrogate rejection in the long term. We have extended the observations of Mason by showing that a similar situation exists for grafts to the lateral ventricles in rats 26 (Fig. 1A, B), also demonstrated in this site in fully histoincompatible mice by Nicholas et al. 27 In the above examples the site of transplantation was within the ventricular system of the brain. As long ago as 1923, Murphy and Sturm28 demonstrated that transplanted tissue impinging on the lateral cerebral ventricles was rejected, while grafts within the brain parenchyma survived for longer periods. This has been confirmed in our own studies where a greater proportion of fully mismatched grafts (i.e. MHC and multiple non-MHC mismatches) survived when grafted to the striatum rather than to the ventricles26. In these grafts, MHC class I expression was minimal, apart from patches of donor blood TINS, Vol. 14, No. 8, 1991
vessels, and host lymphocytes were largely absent (Fig. 1C,D). In another recent report it was shown that loci of host leucocytes were present in such grafts and that these were associated with a local increase in donor MHC expression, indicating that a slow, but ongoing, immune response was taking place 29. In addition, Lawrence et al. 29 have also claimed that cells of dendritic leucocyte morphology were seen in their electron microscope study of allografts to the brain in rats. They also state in their report that clusters of lymphocytes were in contact with these ceils, in much the same fashion as those observed in the lymph nodes and spleen. In this case the numbers of host dendritic leucocytes and of precursor lymphocytes with relevant specificities entering the graft from the blood circulation would probably be low, perhaps leading to protracted rejection in situ. There is one further factor that can influence the immune response to allografts. Among highly inbred rat strains, combinations of donors and recipients exist where the recipient can be classified as a high or low responder to the donor antigens3°'31. Mason et al. 25 demonstrated that neural grafts transplanted to high-responder recipients were rejected, whereas those transplanted to low-responder hosts resulted in long-term survival. As it is difficult to envisage how we can determine the 'responder status' of human recipients, it would be prudent to be cautious and assume that, in the absence of immunosuppressive therapy, grafts of allogeneic tissue are likely to be rejected in the long term. Despite the evidence from experiments in animals in favour of this cautious approach, some controversy still exists and the need for immunosuppressive therapy has not been accepted by everybody involved in clinical neural transplantation trials 32. Thus far we can conclude that the CNS does indeed represent an immunologically privileged site, but that this privilege is far from absolute. Even within groups there is a disparity of response in which some allografts show little or no evidence of rejection, while others may be undergoing extensive lymphocytic infiltration. There can be no guarantee from operation to operation that tissue damage (and therefore local inflammatory responses) will be similar, and this might play a central part in determining the outcome of grafting into the brain. If sensitization is very slow, then the ectopic expression of MHC antigens induced by cytokines may subside before any significant response is mounted. Thus, any immune effector cells that were generated would be unable to bind to their targets. Likewise, the rate of vascularizafion of grafts and the time that the blood-brain barrier remains open will not be the same from animal to animal33; this might have profound effects on the generation of an immune response and the ability of a graft to survive. In animals that have already been sensitized to donor antigens prior to neural transplantation, grafts within the CNS are rejected rapidly 7. If, at the time of grafting to the brain, donor neural tissue is also placed beneath the kidney capsule, then rapid rejection of both grafts ensues 25'26. Even established, surviving neural allo- and xenografts (between species) can be rejected rapidly by subsequent host sensitization to donor antigens, which is readily accomplished by orthotopic skin grafting26'34. These experiments 343
Fig. t. (A) Coronal section of rat brain containing a graft of postnatal day I neonatal cortex tissue in the lateral cerebral ventricle 30 days after transplantation (arrow). The recipient rat was of the highresponder strain, and the donor tissue was mismatched at the major histocompatibility complex (MHC) (class I and class II loci) and multiple non-MHC immunogenetic loci. A monoclonal antibody specific for the donor MHC class I molecules only revealed high levels of surface expression of this antigen throughout the grafted tissue. (B) This photomicrograph shows the adjacent histological section to (,4), stained with an antibody to all the donor and host rat MHC class II molecules. The graft (arrow) was filled with class II-positive cells, the majority of which were probably host macrophages. The presence of infiltrating leucocytes was confirmed in a serial histological section stained with a monoclonal antibody to the rat leucocyte common antigen (found on all rat leucocytes). (C) Coronal section of rat brain containing a graft of cortex tissue in the caudate putamen 30 days after transplantation (arrow). Again the recipient was of the high-responder strain and the donortissue was mismatched at MHC (class I and class II loci) and multiple non-MHC immunogenetic loci. Using the same antibody described above in (A), donor MHC class I molecules on the graft were visualized. Most of the graft shows only low expression of class I antigen, except for the blood vessels (bv), which constitutively express it in the normal rat brain, and small foci of high expression contained within the grafts (open arrow). (D) The adjacent histological section to (C) was stained for all rat MHC class II molecules. Localized foci of MHC class II-positive cells were seen throughout the graft (open arrow). Generally, infiltration with class II-positive cells was significantly lower than that found in the intraventricular allografts. Scale bars are 150 pm.
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indicate that there is no a priori deficiency in the efferent arc from the lymphoid tissue to neural grafts, but rather that the afferent arc might be compromised in some way. In the case of xenografts where antigenic differences might be even more disparate, we and others 25'35 have found that these are rapidly rejected, usually within 20 days. For these xenografts, destruction might be the result of a rapid generation of effector cells, or a pre-existing state of immunity in the recipients at the time of grafting. In the former case, it has already been mentioned that macromolecules injected into the brain can reach the deep cervical lymph nodes rapidly 15'16, and there is evidence from studies in vitro that xenogeneic antigens might be presented by host APCs to host T cells 36. With respect to a pre-existing state of immunity, some species have naturally occurring 'preformed' antibodies and therefore B-ceU specificities, which can cross-react or bind to the antigens of other species 37. Also, in the rat, abnormally high titres of circulating antibody have been shown to result from the deposition of xenogeneic antigens into the brain or subarachnoid space38,39. This provides further evidence that xenogeneic antigens do reach the deep cervical lymph nodes 39 to generate an immune response that is driven by T cells. Such antibodies are probably important in the rejection of vascularized xenografts, where their main target is thought to be the foreign vascular endothelial cells 4°. Nevertheless they could also cause considerable damage in brain grafts, as bound antibody can activate the complement cascade, and lead to nonspecific, antibody-dependent cellmediated cytotoxicity by macrophages. There can be no doubt that neural tissue is immunogenic and that the failure of neural allo- and xenografts to survive is due to immunological processes. For example, rat neural xenografts in immunodeficient (nude) mice survive without signs of rejection 25, and grafting mouse tissue to neonatal rats, at the time when immunocompetence is just developing, results in permanent survival in many cases 34. In this TINS, VoL 14, No. 8, 1991
Fig. 2. Coronal section of rat brain containing a graft of postnatal day 1 cortex tissue in the lateral cerebral ventricle 30 days after transplantation (G). Again the recipient was of the high-responder strain and the donor tissue was mismatched at the major histocompatibility complex (MHC) (class I and class II loci), and multiple non-MHC immunogenetic loci. The recipient was given an intraperitoneal injection of an anti-interleukin-2 (IL-2) receptor monoclonal antibody, each day from the time of transplantation, for ten days. (A) This section was stained for donor MHC class I antigen as before (Fig, 1A and C), and it is clear that this short-term immunosuppressive treatment prevented the induction of significant amounts of this antigen on the donor tissue. (B) The adjacent section was stained to reveal all rat MHC class II antigen expression by use of the same monoclonal antibody as before (Fig. 1B, D). The graft did not contain any class II-positive cells, confirming that it was not undergoing rejection. Note that positive cells were present within the host choroid plexus (ch.p). These cells are present in the ungrafted adult rat choroid plexus and may represent a population of resident macrophages or dendritic leucocytes, but this awaits further investigation, Scale bar is 150 i~m. treatment is necessary for neural aUograft survival has yet to be determined. In Box 1, it can be seen that both helper and cytotoxic T cells depend upon cell surface molecules closely associated with their individual T-cell receptors for activation. For helper T cells the associated molecule is CD4 and for cytotoxic T cells, it is CD8. Immunosuppression Injections of anfi-CD4 antibodies in mice may result in If neuroanatomical and behavioural studies in ani- the induction of tolerance to allogeneic tissue and mals are used to justify the use of neural grafts in long-term survival of, for example, heart grafts 45. humans, then our present knowledge suggests that Nicholas et aL 46 have shown that by using an antisome form of immunosuppression should be included CD4 monoclonal antibody to deplete helper T cells, in the therapeutic regimen. the rejection of intraventricular neural allografts in Use of the drug Cyclosporin-A has revolutionized mice can be prevented, and we have used anti-CD4 the field of organ transplantation in humans and has antibodies to prevent rejection in the hypogonadal been successful in maintaining long-term survival of mouse model system. In these mice there is a xenografts in the rat model of Parkinson's disease, deletion associated with the gene encoding the provided the daily treatment is continued throughout hypothalamic ~onadotrophic hormone releasing horthe timecourse of the experiment 4a. By this means it mone (GnRH) . As a consequence pituitary gonadohas been possible to demonstrate that human substan- trophic hormone content is depleted and there is a tia nigra can indeed supply a dopaminergic input to the failure of postnatal gonadal development in both rat host striatum, and it has also been possible to sexes. Transplantation of late fetal/early neonatal determine the optimum age of such grafted fetal mouse neural tissue containing GnRH cell bodies neurones 44. Continuous injection of a nephrotoxic within the third cerebral ventricle reverses the hypodrug in older patients may have undesirable side gonadism 48. By transplanting GnRH-rich tissue from effects, although whether or not such long-term the medial pre-optic area of rats into hypogonadal second example, it is interesting that later disruption of the blood-brain barrier results in graft rejection41, and that activation of host glial cells by lesioning host fibre tracts can also lead to graft rejection 42. This raises the possibility that host APCs could be involved in initiation of neural graft rejection in situ.
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mice treated with anti-CD4 antibody for the first ten days post-operatively, the extreme hypogonadism observed prior to grafting was initially reversed InternationalSpinal (Wood, K. J. and Charlton, H. M., unpublished Research Trust, the observations). However, we have since found that by Belt Trust, the MRC 60 days there is evidence that these grafts undergo and the Wellcome rejection. At the moment permutations and combiTrust. nations of dose and timing are under way to test the model further. Helper T cells, when activated, secrete the lymphokine intefleukin-2 (IL-2), which induces the expression of high affinity IL-2 receptors upon both the helper T-cell and cytotoxic T-cell populations; the end result is that both populations expand enormously in numbers. Blocking the CD4 molecule obviously affects the initial activation process but another logical site for influencing T-cell amplification would be the IL-2 receptor itself. A monoclonal antibody to the rat IL-2 receptor has been raised, the administration of which results in prolonged survival of renal allografts 49. In our own studies, daffy intraperitoneal injections of this antibody for ten days following transplantation in high-responder rats has significantly improved the condition of fully mismatched allograffs in the lateral ventricles (Fig. 2). Furthermore, similar treatment has resulted in long-term survival of human xenografts in the rat model of Parkinson's disease, with apparently permanent reversal of the motor deficiencies in those animals~°. Although solid tissue fragments and cell suspensions of neural tissue are immunogenic as a whole, it could be possible that individual cell types within the graft are themselves immunogenic to a greater or lesser degree. Indeed, evidence is accumulating that CNS neurones may be refractory to the induction of MHC antigens and therefore might not be capable of initiating an immune response, nor of presenting a target to cytolytic T cells. Bartlett et al. 51 claim to have abrogated neural allograft rejection completely by pre-selecting a subpopulation of embryonic neuroepithelial cells for grafting by the use of immunobead separation on the basis of MHC expression. The neuronal precursor cells, which did not express MHC class I in vitro when treated with IFN-y, were successfully maintained in the brain parenchyma in a mouse model where whole neural grafts were normally rejected. For the future, this finding offers the possibility for grafting enriched neuronal cell populations across allogeneic, and perhaps even xenogeneic, histocompatibility barriers, without the need to immunosuppress the recipients. For extensive reviews of this subject the reader is referred to Refs 52-54, which may also be of interest.
Acknowledgements We would like to thank the
Selected references 1 Barker, C. F. and Billingham, R. E. (1977) Adv. Immunol. 25, 1-54 2 Scheinberg, L. C., Edelman, F. L. and Levy, W. A. (1964) Arch. NeuroL 1 1 , 2 4 8 - 2 6 4 3 Lance, E. M. (1967) Transplantation 5(6), 1471-1483 4 Raju, S. and Grogan, B. (1969) Transplant. Proc. 9, 1187-1191 5 Geyer, S. J. and Gill, T. J. (1979) Am. J. PathoL 94, 569-584 6 Freed, W. J. (1983) BioL Psychiatry 18, 1205-1267 7 Medawar, P. B. (1948) Br. J. Exp. PathoL 29, 58-69 8 Barker, C. F. and Billingham, R. E. (1968) J. Exp. /Wed. 28, 197-221 9 Strober, S. and Gowans, J. L. (1965) J. Exp. Med. 122, 347-352 10 Larsen, C. P., Morris, P. J. and Austyn, J. M. (1990) J. Exp. 346
/Vled. 171,307-314 11 Faustman, D. et aL (1984) Proc. Natl Acad. Sci. USA 81, 3864-3868 12 Mason, D. W. and Morris, P. J. (1986) Annu. Rev. Immunol. 4, 119-145 13 Mantovani, A. and Dejana, E. (1989) ImmunoL Today 10, 370-375 14 Milton, A. D. and Fabre, J. W. (1985) J. Exp. /Wed. 161, 98-112 15 Bradbury, M. W. B. (1990)in Pathophysiology of the BloodBrain Barrier (Johansson, B. B., Owman, C. and Widner, H., eds), pp. 403-412, Elsevier 16 Bradbury, M. W. B. and Cole, D. F. (1980) J. Physiol. 299, 353-365 17 Naparstek, Y., Cohen, I. R., Fuks, Z. and Vlodavsky, I. (1984) Nature 310, 241-244 18 Wong, G. H. W., Bartlett, P. F., Clark-Lewis, I., Battye, F. and Schrader, J. W. (1984) Nature 310, 688-691 19 Fierz, W., Endler, B., Reske, K., Wekerle, H. and Fontana, A. (1985) J. ImmunoL 134, 3785-3793 20 Calder, V. L., Wolswijk, G. and Noble, M. (1988) Eur. J. ImmunoL 18, 1195-1201 21 Male, D. K., Pryce, G. and Hughes, C. C. W. (1987) Immunology 60, 453-459 22 Perry, V. H. and Gordon, S. (1987) J. Exp. 4,1ed. 166, 1138-1143 23 Poltorak, M. and Freed, W. J. (1989) Exp. Neurol. 103, 222-233 24 Vass, K. and Lassman, H. (1990) Am. J. PathoL 137,789-800 25 Mason, D. W. et aL (1986) Neuroscience 19, 685-694 26 Sloan, D. J., Baker, B. J., Puclavec, M. and Charlton, H. M. (1990) Pro8. Brain Res. 82, 141-152 27 Nicholas, M. K., Antel, J. P., Stefansson, K. and Arnason, B. G, W. (1987) J. ImmunoL 139, 2275-2283 28 Murphy, J. B. and Sturm, E. (1923) J. Exp. IVled. 38, 183-197 29 Lawrence, J. M,, Morris, R. J., Wilson, D. J. and Raisman, G. (1990) Neuroscience 37, 431-462 30 Butcher, G. and Howard, J. (1982) Transplantation 34, 161-166 31 Zimmermann, F., Davies, H., Knoll, P., Gokel, J. and Schmidt, T. (1984) Transplantation 37, 406-410 32 Hitchcock, E. R., Clough, C., Hughes, R. and Kenny, B. (1988) Lancet i, 1274 33 Broadwell, R. D., Charlton, H. M., Balin, B. J. and Salcman, M. (1987) J. Comp. NeuroL 260, 47-62 34 Lund, R. D., Rao, K., Kunz, H. W. and Gill, T. J. (1988) Transplantation 46, 216-233 35 Finsen, B. R., Pedersen, E. B., Sorensen, T., Hokland, M. and Zimmer, J. (1990) Prog. Brain Res. 82, 111-128 36 Lindahl, K. F. and Bach, F. H. (1976) J. Exp. /vled. 144, 305-318 37 Auchincloss, H. (1988) Transplantation 46, 1-20 38 Santos, T. Q. and Valdimarsson, H. (1982)J. Neuroimmunol. 2,215-222 39 Widner, H., M611er, G. and Johansson, B. B. (1988) 5cand. J. ImmunoL 28, 563-571 40 Platt, J. L. etaL (1990) Immunol. Today 11,450-456 41 Pollack, I. F., Lund, R. D. and Rao, K. (1990) Prog. Brain Res. 82, 129-140 42 Rao, K. and Lund, R. D. (1989) Brain Res. 488, 332-335 43 Brundin, P., Widner, H., Nilsson, O. G., Strecker, R. E. and Bj6rklund, A. (1989) Exp. Brain Res. 75, 195-207 44 Brundin, P, et aL (1988) Dev. Brain Res. 39, 233-243 45 Madsen, J. C., Wood, K. J. and Morris, P. J. (1987) Transplant. Proc. 19, 3991-3997 46 Nicholas, M. K., Chenelle, A. G., Brown, M. M., Stefanson, K. and Arnason, B. G. W. (1990) Prog. Brain Res. 82, 161-167 47 Mason, A. J. et aL (1986) Science 234, 1366-1371 48 Krieger, D. T. et aL (1982) Nature 298, 299-303 49 Tellides, G., Dallman, M. J. and Morris, P. J. (1989) Transplant. Proc, 21,997-998 50 Honey, C. R., Clarke, D. J., Dallman, M. J. and Charlton, H. M. (1991) Neuroreport 1,247-249 51 Bartlett, P. F. etaL (1990) Prog. Brain Res. 82, 153-160 52 Nicholas, M. K. and Arnason, B. G. W., eds (1989) in Neural Regeneration and Transplantation (Seil, F. J., ed.), pp. 239-284, Alan R. Liss 53 Brent, L. (1990) in Pathophysiology of the Blood-Brain Barrier (Johansson, B. B., Owman, C. and Widner, H., eds), pp. 383-402, Elsevier 54 Widner, H. and Brundin, P. (1988) Brain Res. Rev. 13, 287-324 TINS, VoL 14, No. 8, 1991
© 1991, ElsevierSciencePublishersLtd, (UK) 0166- 2236191/$02.00
D. J. Sloan,M. J. Woodand H. M. £harlton areat the Deptof Human Anatomy, University of Oxford, South ParksRoad, Oxford OXl 3QX, UK.
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Box 1. Immune events in allograft rejection Foreign antigen is presented to host helper T-cell precursors by specialized antigen-presenting cells (APCs) (classically, dendritic leucocytes). Antigen dentritic leucocyte in processed form, and in association with the major histocompatibility complex (MHC) class II molecule - a heterodimer consisting of o~ and [3 chains - is presented to helper T-cell precursors. The helper T cells selected are those whose T-cell receptors (TCRs) bind favourably to the MHC(+ peptide I ) ~ antigen complex. The TCR is a polymorphic heterodimer comprising o~ and 1~ chains, each prTcie~°r X ~ TCR consisting of both variable and constant domains, and unique to a particular T-cell precursor. The interaction between the TCR and the MHCantigen complex is stabilized by several cell surface accessory molecules, among which, in the TCR-CD4 and MHC class II-peptide interaction case of helper T cells, is the CD4 glycoprotein; this c=constant domain interaction culminates in T-cell activation. v=variable domain The transcription rates of many cytokine genes IL-2~~ / are up-regulated in the activated cell and as a consequence numerous cytokines are secreted, of activated helper which interleukin-2 (IL-2) is of central importance in the generation and amplification of the immune response. Its actions are autocrine, resulting in the proliferation of helper T cells themselves, and paracrine, mobilizing various other effector-cell APO~ populations. The TCR of cytotoxic T-cell precursors, an important effector-cell group, interacts with processed antigen in association with an MHC class I molecule, a heterodimer consisting of an o~chain with one constant and two variable domains and the invariant I~2 microglobulin. In this case the TCR-MHC interaction is stabilized by a CD8 accessory molecule, although this binding alone is ( L-4, L-6, IFN-y) insufficient for cytotoxic T-cell stimulation and IL2 is also required for full activation and prolifera ation. ~l ulll~,l aLivl i The interplay between a number of cytokines TCR-CD8 and MHC class I-peptide interaction produced dictates the character of the immune c=constant domain response and results in the generation and acv=variable domain tivation of particular effector-cell populations: macrophages; lymphokine-activated killer cells recruitment of (LAK cells) and natural killer cells (NK cells), for effector cells which IL-2 and interferon-y (IFN-y) are particularly important; and B cells, which act via the production of cytotoxic antibodies and are dependent on IL-4 and IL-6. LAK cell Effector cells are recruited to the graft site APC where their actions may result in graft rejection. This sequestration is brought about by the proNK cell duction of local chemotactic factors (notably IL-I and tumour necrosis factor), which increases the B cell adhesive properties of neighbouring endothelial macrophage surfaces. These factors are produced as a consequence of increased expression of intercellular and endothelial adhesion molecules, thereby facilitating the subsequent migration of effector cells into the grafted tissue. Their continued cytokine production results in up-regulation of local MHC plasma cell cytotoxic antibody expression and perpetuation of the inflammatory cascade.
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demonstrated that it does not represent a barrier to activated lymphocytestT. The breakdown of the bartier during the grafting procedure could also provide a route for donor cells or tissue antigens to enter the host lymphoid tissue via the blood circulation to the spleen. 342
The transplantation antigens of the MHC, which play a key role in the generation and propagation of immune responses, are not expressed at detectable levels in healthy CNS tissue, apart from endothelial cells. However, in both adult and fetal rats, most cells of the CNS do have the capacity to express TINS, VoL 14, No. 8, 1991
certain of these antigens under pathological condifions. Astrocytes, microglia and capillary endothelial cells have all been shown to express MHC class II molecules and to produce some cytokines on stimulation with intefferon-y (IFN-y) in vitro 18-21. Perhaps the most likely cell type to be involved in augmenting rejection within the graft by expressing foreign MHC class II molecules and co-stimulatory cytokine signals to host helper T cells is the bone marrow-derived microglial cell, which probably represents the resident tissue macrophage within the CNS 22-24. Whether or not these cells can initiate rejection awaits investigation. Finally, the concept that neural tissue per se is nonimmunogenic has been conclusively denied by the fact that such tissue transplanted beneath the host kidney capsule is rejected as rapidly as an orthotopic skin graft2s. Is the brain an 'immunologically privileged' site? The use of specific monoclonal antibodies against a variety of lymphoid cell markers and against MHC antigens has made it possible to follow host-graft interactions. There is now overwhelming evidence that, while some protection is offered to foreign tissue grafts, delayed rejection can take place in the brain, particularly if there is wide genetic disparity between donor and host. Mason et al. 25 reported the survival of neural grafts to the third cerebral ventricle in rats when the donor strain differed from the recipient at the MHC loci alone, or multiple non-MHC loci alone. When the genetic disparity was increased to include both the MHC and multiple non-MHC loci, survival was more variable and leucocytic infiltration containing T cells, together with widespread MHC antigen expression on the donor tissue, was evident in the majority of the grafts. This suggests that differences in non-MHC or minor transplantation antigens can have a significant effect on the survival of neural grafts if they occur in addition to disparities between the MHC loci of host and graft. Even in the grafts with only MHC or only non-MHC differences, small foci of leucocytic infiltration and MHC expression were observed in some histological sections, suggesting that rejection was occurring at localized 'hot-spots' within the grafts, and also that matching the donor and host tissue at the MHC loci alone might not be sufficient to abrogate rejection in the long term. We have extended the observations of Mason by showing that a similar situation exists for grafts to the lateral ventricles in rats 26 (Fig. 1A, B), also demonstrated in this site in fully histoincompatible mice by Nicholas et al. 27 In the above examples the site of transplantation was within the ventricular system of the brain. As long ago as 1923, Murphy and Sturm28 demonstrated that transplanted tissue impinging on the lateral cerebral ventricles was rejected, while grafts within the brain parenchyma survived for longer periods. This has been confirmed in our own studies where a greater proportion of fully mismatched grafts (i.e. MHC and multiple non-MHC mismatches) survived when grafted to the striatum rather than to the ventricles26. In these grafts, MHC class I expression was minimal, apart from patches of donor blood TINS, Vol. 14, No. 8, 1991
vessels, and host lymphocytes were largely absent (Fig. 1C,D). In another recent report it was shown that loci of host leucocytes were present in such grafts and that these were associated with a local increase in donor MHC expression, indicating that a slow, but ongoing, immune response was taking place 29. In addition, Lawrence et al. 29 have also claimed that cells of dendritic leucocyte morphology were seen in their electron microscope study of allografts to the brain in rats. They also state in their report that clusters of lymphocytes were in contact with these ceils, in much the same fashion as those observed in the lymph nodes and spleen. In this case the numbers of host dendritic leucocytes and of precursor lymphocytes with relevant specificities entering the graft from the blood circulation would probably be low, perhaps leading to protracted rejection in situ. There is one further factor that can influence the immune response to allografts. Among highly inbred rat strains, combinations of donors and recipients exist where the recipient can be classified as a high or low responder to the donor antigens3°'31. Mason et al. 25 demonstrated that neural grafts transplanted to high-responder recipients were rejected, whereas those transplanted to low-responder hosts resulted in long-term survival. As it is difficult to envisage how we can determine the 'responder status' of human recipients, it would be prudent to be cautious and assume that, in the absence of immunosuppressive therapy, grafts of allogeneic tissue are likely to be rejected in the long term. Despite the evidence from experiments in animals in favour of this cautious approach, some controversy still exists and the need for immunosuppressive therapy has not been accepted by everybody involved in clinical neural transplantation trials 32. Thus far we can conclude that the CNS does indeed represent an immunologically privileged site, but that this privilege is far from absolute. Even within groups there is a disparity of response in which some allografts show little or no evidence of rejection, while others may be undergoing extensive lymphocytic infiltration. There can be no guarantee from operation to operation that tissue damage (and therefore local inflammatory responses) will be similar, and this might play a central part in determining the outcome of grafting into the brain. If sensitization is very slow, then the ectopic expression of MHC antigens induced by cytokines may subside before any significant response is mounted. Thus, any immune effector cells that were generated would be unable to bind to their targets. Likewise, the rate of vascularizafion of grafts and the time that the blood-brain barrier remains open will not be the same from animal to animal33; this might have profound effects on the generation of an immune response and the ability of a graft to survive. In animals that have already been sensitized to donor antigens prior to neural transplantation, grafts within the CNS are rejected rapidly 7. If, at the time of grafting to the brain, donor neural tissue is also placed beneath the kidney capsule, then rapid rejection of both grafts ensues 25'26. Even established, surviving neural allo- and xenografts (between species) can be rejected rapidly by subsequent host sensitization to donor antigens, which is readily accomplished by orthotopic skin grafting26'34. These experiments 343
Fig. t. (A) Coronal section of rat brain containing a graft of postnatal day I neonatal cortex tissue in the lateral cerebral ventricle 30 days after transplantation (arrow). The recipient rat was of the highresponder strain, and the donor tissue was mismatched at the major histocompatibility complex (MHC) (class I and class II loci) and multiple non-MHC immunogenetic loci. A monoclonal antibody specific for the donor MHC class I molecules only revealed high levels of surface expression of this antigen throughout the grafted tissue. (B) This photomicrograph shows the adjacent histological section to (,4), stained with an antibody to all the donor and host rat MHC class II molecules. The graft (arrow) was filled with class II-positive cells, the majority of which were probably host macrophages. The presence of infiltrating leucocytes was confirmed in a serial histological section stained with a monoclonal antibody to the rat leucocyte common antigen (found on all rat leucocytes). (C) Coronal section of rat brain containing a graft of cortex tissue in the caudate putamen 30 days after transplantation (arrow). Again the recipient was of the high-responder strain and the donortissue was mismatched at MHC (class I and class II loci) and multiple non-MHC immunogenetic loci. Using the same antibody described above in (A), donor MHC class I molecules on the graft were visualized. Most of the graft shows only low expression of class I antigen, except for the blood vessels (bv), which constitutively express it in the normal rat brain, and small foci of high expression contained within the grafts (open arrow). (D) The adjacent histological section to (C) was stained for all rat MHC class II molecules. Localized foci of MHC class II-positive cells were seen throughout the graft (open arrow). Generally, infiltration with class II-positive cells was significantly lower than that found in the intraventricular allografts. Scale bars are 150 pm.
344
indicate that there is no a priori deficiency in the efferent arc from the lymphoid tissue to neural grafts, but rather that the afferent arc might be compromised in some way. In the case of xenografts where antigenic differences might be even more disparate, we and others 25'35 have found that these are rapidly rejected, usually within 20 days. For these xenografts, destruction might be the result of a rapid generation of effector cells, or a pre-existing state of immunity in the recipients at the time of grafting. In the former case, it has already been mentioned that macromolecules injected into the brain can reach the deep cervical lymph nodes rapidly 15'16, and there is evidence from studies in vitro that xenogeneic antigens might be presented by host APCs to host T cells 36. With respect to a pre-existing state of immunity, some species have naturally occurring 'preformed' antibodies and therefore B-ceU specificities, which can cross-react or bind to the antigens of other species 37. Also, in the rat, abnormally high titres of circulating antibody have been shown to result from the deposition of xenogeneic antigens into the brain or subarachnoid space38,39. This provides further evidence that xenogeneic antigens do reach the deep cervical lymph nodes 39 to generate an immune response that is driven by T cells. Such antibodies are probably important in the rejection of vascularized xenografts, where their main target is thought to be the foreign vascular endothelial cells 4°. Nevertheless they could also cause considerable damage in brain grafts, as bound antibody can activate the complement cascade, and lead to nonspecific, antibody-dependent cellmediated cytotoxicity by macrophages. There can be no doubt that neural tissue is immunogenic and that the failure of neural allo- and xenografts to survive is due to immunological processes. For example, rat neural xenografts in immunodeficient (nude) mice survive without signs of rejection 25, and grafting mouse tissue to neonatal rats, at the time when immunocompetence is just developing, results in permanent survival in many cases 34. In this TINS, VoL 14, No. 8, 1991
Fig. 2. Coronal section of rat brain containing a graft of postnatal day 1 cortex tissue in the lateral cerebral ventricle 30 days after transplantation (G). Again the recipient was of the high-responder strain and the donor tissue was mismatched at the major histocompatibility complex (MHC) (class I and class II loci), and multiple non-MHC immunogenetic loci. The recipient was given an intraperitoneal injection of an anti-interleukin-2 (IL-2) receptor monoclonal antibody, each day from the time of transplantation, for ten days. (A) This section was stained for donor MHC class I antigen as before (Fig, 1A and C), and it is clear that this short-term immunosuppressive treatment prevented the induction of significant amounts of this antigen on the donor tissue. (B) The adjacent section was stained to reveal all rat MHC class II antigen expression by use of the same monoclonal antibody as before (Fig. 1B, D). The graft did not contain any class II-positive cells, confirming that it was not undergoing rejection. Note that positive cells were present within the host choroid plexus (ch.p). These cells are present in the ungrafted adult rat choroid plexus and may represent a population of resident macrophages or dendritic leucocytes, but this awaits further investigation, Scale bar is 150 i~m. treatment is necessary for neural aUograft survival has yet to be determined. In Box 1, it can be seen that both helper and cytotoxic T cells depend upon cell surface molecules closely associated with their individual T-cell receptors for activation. For helper T cells the associated molecule is CD4 and for cytotoxic T cells, it is CD8. Immunosuppression Injections of anfi-CD4 antibodies in mice may result in If neuroanatomical and behavioural studies in ani- the induction of tolerance to allogeneic tissue and mals are used to justify the use of neural grafts in long-term survival of, for example, heart grafts 45. humans, then our present knowledge suggests that Nicholas et aL 46 have shown that by using an antisome form of immunosuppression should be included CD4 monoclonal antibody to deplete helper T cells, in the therapeutic regimen. the rejection of intraventricular neural allografts in Use of the drug Cyclosporin-A has revolutionized mice can be prevented, and we have used anti-CD4 the field of organ transplantation in humans and has antibodies to prevent rejection in the hypogonadal been successful in maintaining long-term survival of mouse model system. In these mice there is a xenografts in the rat model of Parkinson's disease, deletion associated with the gene encoding the provided the daily treatment is continued throughout hypothalamic ~onadotrophic hormone releasing horthe timecourse of the experiment 4a. By this means it mone (GnRH) . As a consequence pituitary gonadohas been possible to demonstrate that human substan- trophic hormone content is depleted and there is a tia nigra can indeed supply a dopaminergic input to the failure of postnatal gonadal development in both rat host striatum, and it has also been possible to sexes. Transplantation of late fetal/early neonatal determine the optimum age of such grafted fetal mouse neural tissue containing GnRH cell bodies neurones 44. Continuous injection of a nephrotoxic within the third cerebral ventricle reverses the hypodrug in older patients may have undesirable side gonadism 48. By transplanting GnRH-rich tissue from effects, although whether or not such long-term the medial pre-optic area of rats into hypogonadal second example, it is interesting that later disruption of the blood-brain barrier results in graft rejection41, and that activation of host glial cells by lesioning host fibre tracts can also lead to graft rejection 42. This raises the possibility that host APCs could be involved in initiation of neural graft rejection in situ.
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mice treated with anti-CD4 antibody for the first ten days post-operatively, the extreme hypogonadism observed prior to grafting was initially reversed InternationalSpinal (Wood, K. J. and Charlton, H. M., unpublished Research Trust, the observations). However, we have since found that by Belt Trust, the MRC 60 days there is evidence that these grafts undergo and the Wellcome rejection. At the moment permutations and combiTrust. nations of dose and timing are under way to test the model further. Helper T cells, when activated, secrete the lymphokine intefleukin-2 (IL-2), which induces the expression of high affinity IL-2 receptors upon both the helper T-cell and cytotoxic T-cell populations; the end result is that both populations expand enormously in numbers. Blocking the CD4 molecule obviously affects the initial activation process but another logical site for influencing T-cell amplification would be the IL-2 receptor itself. A monoclonal antibody to the rat IL-2 receptor has been raised, the administration of which results in prolonged survival of renal allografts 49. In our own studies, daffy intraperitoneal injections of this antibody for ten days following transplantation in high-responder rats has significantly improved the condition of fully mismatched allograffs in the lateral ventricles (Fig. 2). Furthermore, similar treatment has resulted in long-term survival of human xenografts in the rat model of Parkinson's disease, with apparently permanent reversal of the motor deficiencies in those animals~°. Although solid tissue fragments and cell suspensions of neural tissue are immunogenic as a whole, it could be possible that individual cell types within the graft are themselves immunogenic to a greater or lesser degree. Indeed, evidence is accumulating that CNS neurones may be refractory to the induction of MHC antigens and therefore might not be capable of initiating an immune response, nor of presenting a target to cytolytic T cells. Bartlett et al. 51 claim to have abrogated neural allograft rejection completely by pre-selecting a subpopulation of embryonic neuroepithelial cells for grafting by the use of immunobead separation on the basis of MHC expression. The neuronal precursor cells, which did not express MHC class I in vitro when treated with IFN-y, were successfully maintained in the brain parenchyma in a mouse model where whole neural grafts were normally rejected. For the future, this finding offers the possibility for grafting enriched neuronal cell populations across allogeneic, and perhaps even xenogeneic, histocompatibility barriers, without the need to immunosuppress the recipients. For extensive reviews of this subject the reader is referred to Refs 52-54, which may also be of interest.
Acknowledgements We would like to thank the
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