The Thymic Way to Transplantation Tolerance1

Giuseppe Remuzzi,2 Norberto Perico, Charles B. Carpenter, and Mohamed H. Sayegh

the thymus and the possible mechanism(s) of ac- G. Remuzzi, N. Perico, Mario Negri Institute for Phar- quired thymic tolerance are reviewed. macological Research and Division of Nephrology, Key Words: Thymus. self-tolerance, acquired tolerance, pan- Ospedali Riuniti di Bergamo, Bergamo, Italy creatic islets. kidney, heart, liver, small bowel, skin, MHC C.B, Carpenter. M,H. Sayegh, Laboratory of Immuno- allopeptides. transplantation genetics and Transplantation, Renal Division, Depart- ment of Medicine, Brigham and Women’s Hospital, T he major goal in transplantation research is the Harvard Medical School, Boston, MA development of strategies that lead to specific immunologic unresponsiveness or tolerance to the (J, Am, Soc. Nephrol. 1995; 5:1639-1646) allograft. The thymus plays the major role in development of self-tolerance (1 ,2), and initial work by ABSTRACT Waksman and colleagues more than 20 yr ago (3,4) indicated that the thymus may play a role in the Within the pastthree decades, extensive research has induction of acquired tolerance to exogenous anti- been carried out with the aim to prevent graft rejec- gens. Recently, there has been a renewed interest in tion by minimizing the side effects related to the use of studying the role of the thymus in acquired tolerance, immunosuppressants. The major goal in transplanta- and several investigators have shown that the intra- tion research remains the development of strategies thymic injection of antigen induces a state of specific that would allow one to achieve a state of donor- systemic unresponsiveness in experimental autoim- specific unresponsiveness in order to promote a con- mune (5-7) as well as transplantation models (see dition of true tolerance without the need of immuno- below). The purpose of this article is to provide the suppressants. Recent evidence has been provided reader with an overview of the role of the thymus in that this Is a pursuing goal, at least in experimental acquired tolerance with special emphasis on transplantation tolerance, review the mechanisms of ac- animals. The thymus plays the major role in the devel- quired thymic tolerance, and discuss the potential opment of self-tolerance, and initial work in the late applications of acquired thymic tolerance in humans. 96Os indicated that the thymus also plays a critical role in the induction of acquired tolerance to exoge- THE ROLE OF THE THYMUS IN “SELF”-TOLERANCE nous antigens. Recently, the interest in acquired thy- Studies of the crystal structure and function of the mic tolerance has been renewed by the observation major histocompatibility complex (MHC) molecules that, in the rat, the thymus is an immunologically and the sequencing of T cell receptors (TCR) have privileged site in which isolated pancreatic islets can yielded important information on the mechanisms of T be engrafted and survive indefinitely. Moreover, intra- cell recognition of antigen. Foreign antigens are rec- thymic injection of the islets induced donor-specific ognized after they are processed and presented as unresponsiveness, which allowed survival of a second peptides in the binding groove of cell surface mole- donor-strain islet cell allograft transplanted into an cules encoded by the MHC genes to the cs//3 TCR extrathymic site. These findings on cellular allografts (8-12). The TCR on each T cell are identical and specific for a given self-MHC plus antigenic peptide have been extended to vascularized organ al- complex (9,10,13). TCR are selected to react with lografts. Studies have documented that, in rodents, foreign (nonseif-) antigens: those that recognize autol- the intrathymic injection of donor cells induces a state ogous (self-) antigens are anergized or eliminated. of tolerance and prolongs the survival of allografts, Failure of these mechanisms may result in autoim- including kidney, heart, liver, and small bowel. Unre- mune diseases. Self- /nonself-discrimination is estab- sponsiveness to organ graft is donor but not tissue lished, early in life (14), and is dependent on a func- specific, and evidence is presented here that the tionally intact thymus, as suggested by early thymus has a central role in such a phenomenon. experiments on thymus transplanted mice (15). TCR, The nature of the alloantigen(s) being recognized in unlike the antigen receptors of B cells, are not se- creted after antigen stimulation but remain on the 1 Received August 5, 1994. Accepted October 19, 1994. surface of T lymphocytes in two heterodimeric forms 2correspondence to Dr. G. Remuzzi. Mario Negri Institute for Pharmacological (ie., a and 13 or y and proteins) (10). Throughout life, Research, Via Gavazzeni 11, 24125 Bergamo, Italy. antigen receptors of the immune system are generated 1046-6673/0509-1 639$03.00/0 Journal of the American society of Nephrology by the random rearrangement of DNA segments en- copyright © 1995 by the American society of Nephrology coding the variable parts of the a and f3 chains of TCR,

Journal of the American Society of Nephrology 1639 Transplantation Tolerance

with specificity for self- and nonseif-antigens (16). promote cell deletion (18,24). Whatever the mecha- This implies that the capacity to distinguish self from nisms are, only 1 to 5% of the total thymocyte popu- nonself is not genetically determined but is acquired lation completes maturation; the remaining 95 to 99% during the process ofT cell development. Although the of T cells are deleted in the thymus (25) (Figure 1). random rearrangement of the gene gives rise, by dif- Apoptosis is a form of cell death more subtle than ferent combinations of the variable segments of the necrosis, which requires new gene expression (26,27). two chains, to a different T cell repertoire that has As cells undergo apoptosis, the surface membrane more than iO possible specificities (1 7), mature T becomes ruffled and produces blebs (apoptotic bod- cells have in fact much fewer TCR combinations, les), which are usually phagocytosed by other cells. because of a number of selection events occurring in Coincident with these changes, nuclear DNA is di- the thymus (18). gested by an endogenous nuclease that cleaves chro- Precursor T lymphocytes reach the thymus early in matin between individual nucleosomes (27). Genes fetal life, although there is a continuous input of and mechanisms that control programmed cell death prothymocytes throughout life (1 9). Immature T cells are the subject of intense investigation. One such gene reside in the cortex and lack the TCR, CD4, and CD8 has been identified, bcl-2, and functions as a repres- molecules. These cells undergo population growth, sor of programmed cell death in many, but not all, rearrangement of TCR genes, and surface expression types of cells (28). In the thymus, bcl-2 is present in of CD4 and CD8 molecules (“double-positive” thymo- the mature surviving thymocytes of the medulla, but cytes) (14, 18-22). TCRCD4CD8 T cells interact is not expressed in most cortical thymocytes, the with thymic epithellal cells or bone marrow-derived majority of which die by apoptosis (28). Thus, bcl-2 macrophages/dendritic antigen-presenting cells appears to be involved in the salvation of maturing T (APC) that express self-MHC molecules and undergo cells, as shown by data that the introduction of bct-2 selection processes that ultimately shape the T cell into the normally vulnerable cortical thymocytes pro- repertoire toward self-MHC restriction and self-tolerance. Self-tolerance is mediated by “negative selection” by the deletion of autoreactive T cell clones (14). Precursor Surviving TCR T cells move into the medulla, increase the density ofTCR expression to that of peripheral T cells, and before leaving the thymus, lose one of Thymus the costimulatory accessory molecules, thus becom- if Double-positive CD4 ing “single-positive” (CD4CD8 T cells or CD4CD8 thymocyte T cells) T cells. At the same time, “positively selected” medullary thymocytes acquire MHC Class Il-restricted helper (CD4) or MHC Class I-restricted cy- CD8 totoxic (CD8) functions and migrate to the periphery. The way these two processes occur is still debated, and two working hypothesis have been proposed. von Boehmer and Kisielow (23) provided a fundamental contribution to the current understanding of thymic selection, using transgenic SCID mice bred to express N.gotlv. SIctIon Posittv#{149}51#{149}ction one type of TCR only, specific for the male (H-Y) antigen presented by Class I MHC molecules. Their 11’ APOPTOS1S data suggest that double-positive thymocytes form a MATURATiON CD4 heterogeneous population of potentially “useful,” “useless,” and “harmful” cells. They elaborated the theory that cells with a TCR complementary to self- MHC are selected for further maturation, but not all * MHC-restrlcted T cells reach full maturity. Those with To prlphry a TCR complementary fitting MHC molecule plus self- Figure 1. T cell development In the thymus. Bone marrow- antigen are eliminated by apoptosis (programmed cell derived T cell precursors migrate to the thymus, where TCR death of “harmful” T cells in the thymus). The remain- genes are randomly expressed in parallel to the concomi- ing T lymphocytes, MHC restricted but not autoreac- tant expression of the CD4 and CD8 molecules on their tive, down-regulate one of the two costimulatory mol- surface (“double-positive” thymocytes). At this stage, cells ecules (CD4 or CD8) and colonize the peripheral with TCR complementary to self-MHC and also to self-antigen lymphoid compartments as mature T cells. As a pos- are destroyed by a process of programmed cell death sible alternative, the “affinity-avidity” hypothesis of T (apoptosis); cells that recognize self-MHC molecule but not cell development implies a low-affinity interaction be- the self-antigen are rescued from death and mature to CD4 tween the TCR and the thymic ligand as the factor or CD8 cells, which leave the thymus and colonize the underlying positive selection and survival of the thy- peripheral lymphold tissues. thymic APC, bone marrow- mocyte, whereas a higher-affinity Interaction would derived APC; thymic EC, thymic epithelial cell; Ag, antigen.

1640 Volume 5’ Number 9#{149}1995 Remuzzi et al

tected them from a wide variety of apoptotic stimuli, In the 1960s and 1970s, Waksman and colleagues including radiation and anti-CD3 treatment (29). published several articles (3,4) that established the More recent studies in somatic bcl-2 knockout mice critical importance of the thymus In the process of show that lymphocytes developed normally until acquired tolerance to foreign antigens. They showed about 2 wk postnatally, when they die prematurely, that injection into the thymus ofadult rats ofa soluble indicating a shortened lifespan (30). Moreover, also in antigen induced a state of systemic unresponsiveness these animals, immature thymocytes disappeared that was specific to the injected antigen (3). Moreover, with time, suggesting that the lymphoid precursor Shimonkevitz and Bevan (40) injected into the thymus cells were depleted (30). Thus, clonal deletion still of irradiated, bone marrow-reconstituted mice alloge- occurred, arguing that certain cell deaths are inde- neic CD4/CD8 double-negative thymocytes from pendent of bcl-2 (3 1). There are genes the expression adult donor mice and found a marked decrease in of which increases in apoptotic cells, although their cytotoxic T lymphocyte precursors to donor antigen. role in the process has yet to be determined. They The induction of antigen-specific systemic unrespon- include Fas, a gene with a product that is a mem- siveness to allografts by the intrathymic injection of brane-spanning protein homologous to tumor necro- donor cells was first reported by Posselt et at. (41). sis factor and nerve growth factor receptors (32). In They used the model ofchemically induced diabetes in mice bearing the lprmutation, a defect in the Fas gene the WF rat to show that allogeneic pancreatic islets, has been reported that was related to lymphoprolif- when injected into the thymus accompanied by a eration (33). Also, RP-2 and RP-8 are two of a family of single injection of antilymphocyte serum (ALS) ip, genes the messages of which remarkably increase survive indefinitely and induce specific unresponsive- after the induction ofapoptosis in thymocytes (34,35). ness to subsequent islet allografts transplanted extra- The thymic microenvironment plays a major role in thymically. We then performed experiments aimed at autoreactive T cell elimination through the generation exploring the role of the thymus in acquired tolerance of soluble factors that contribute to establish whether to solid organ allografts (42-45). First, we trans- a given clone will be suppressed. The observation that planted isolated glomeruli from the kidney of Brown- maturing thymocytes, in the earliest stage of T cell Norway rats into the thymus of MHC-incompatible development, but not later on, have very high cell Lewis rats (42). Ten days later, the contralateral kid- surface expression of receptors for thromboxane A. ney from the same donor was orthotopically trans- (TxA2) (36), a potent aggregating and vasoconstrictor planted in the Lewis recipient. All Lewis rats received agent (37), suggested that TxA., elaborated by thymic cyclosporine (40 mg/kg po) for 2 days before intrathy- stromal cells might act as the key signal for cellular mic injection and were given dexamethasone (2.5 events related to T cell development. This possibility is mg/kg sc) on the day of injection. Renal allografts supported by recent findings that, in vitro, a TxA,, survived indefinitely with preserved function for more agonist, but not an inactive metabolite, induced than 2 yr. Antigen specificity was demonstrated by concentration-dependent DNA fragmentation in injecting glomeruli from third party Sprague Dawley CD4CD8 thymocytes (36), an event inhibited by a strain rats and transplanting Brown-Norway renal specific ‘2 receptor antagonist. allografts (43). Such kidneys were promptly rejected, similar to control animals injected intrathymically ACQUIRED THYMIC TOLERANCE IN with medium alone. These initial experiments with the intrathymic in- TRANSPLANTATION jection of donor cells to achieve systemic tolerance Back in 1953, Billingham et at. (38) induced specific were all performed by injecting into the thymus cells tolerance to skin allografts by the systemic injection of from the same tissue (islets) or organ (glomeruli) that donor-strain hematopoietic cells into neonatal mice. was subsequently used for extrathymic transplanta- Despite the multiple mechanisms potentially involved, tion. Because, in transplantation, the principal tar- unresponsiveness may have been the result of intra- gets of the immune response to allografts are the MHC thymic inactivation and/or the deletion of T cells that antigens, we then tested the ability of donor cells would have reacted with donor antigens. Evidence in expressing donor MHC Class I and Class II antigens to support of this possibility derives from recent data induce tolerance to renal allografts. The intrathymic that mice inoculated at birth with bone marrow donor inoculation of donor peripheral blood mononuclear cells selectively deleted donor-reactive T cells in the cells into the thymus induced indefinite survival of the thymus (39). At variance with findings with hemato- subsequent renal allografts, as isolated glomeruli did, poietic cells in neonate mice, liver, testicle, and kidney indicating that acquired thymic tolerance is not tissue homogenates did not produce tolerance to skin al- specific (43). These observations were followed by a lograft (38), which was interpreted as evidence that series of publications demonstrating the effects of cells of nonhematopoietic origin do not reach the intrathymic injections of donor cells to induce sys- thymus. An alternative explanation for these data temic and antigen-specific unresponsiveness to car- could be that hematopoietic and nonhematopoletic diac (46-69), liver (70), small bowel (60,7 1), and skin cells both circulate through the thymus, but only the allografts (72-74) in the rat and mouse models. Donor former retains the capacity of inducing tolerance. cells included splenocytes (47,48,51-53,56-58,60,

Journal of the American Society of Nephrology 1641 Transplantation Tolerance

62,63,73-76), bone marrow cells (50,54,68,76), thy- Oluwole et aL (89) indicate that the intrathymic injec- mocytes (64), liver APC (Kupfer cells) (70,77), and tion of soluble MHC induces systemic unresponsive- recipient’s myocytes (49,59,66) transfected with donor ness to islet allografts. Interestingly, they showed that MHC (69). when a “high dose” of antigen is injected, there was no What is the nature of the alloantigen being recog- need to administer ALS systemically, whereas the nized in the thymus? Two independent investigators administration ofALS was required with “lower doses” have recently shown that the intrathymic injection of of antigen. These data are consistent with our obser- whole viable donor cells is not necessary and that the vations with MHC allopeptides, where we show that intrathymic injection of soluble MHC molecules ob- 100 g is an “optimal” dose, because the intrathymic tamed from mononuclear cells by KC1 extraction was injection of 1 or 1 0 g of peptides had no effect on sufficient to induce systemic unresponsiveness in the renal allograft survival when we do not use concomi- rat cardiac (78,79) as well as islet (75) allograft mod- tant systemic immunosuppression (90). els. Furthermore, we have recently shown (80) that the The mechanisms of intrathymic tolerance to al- intrathymic injection of synthetic polymorphic Class II lografts remain unclear, although functional T cell MHC 25-mer allopeptides representing the full length inactivation/anergy, clonal deletion, and microchi- sequence of RT1 .B”13 and RT1 .DUP (DQ and DR, or I-A merism (presence of donor cells in the recipient) have and I-E like, respectively) (81) was sufficient to induce been suggested by different authors. Odorico et aL specific systemic unresponsiveness to a subsequent (50) showed that the intrathymic injection of T cell- MHC-incompatible renal allograft. More recently (82), depleted allogeneic bone marrow cells, containing we have demonstrated that the intrathymic injection dendritic cells, B cells, and macrophages, which ex- of the immunogenic MHC peptides was tolerogenic, press both Class I and Class II MHC molecules, ef- whereas the intrathymic injection of the nonimmuno- fected the prolongation of vascularized cardiac al- genic MHC peptides was not. Although the mecha- lograft survival in the rat and demonstrated the nisms of the T cell recognition of nominal antigens, presence of donor bone marrow cells in the recipient’s such as viral proteins for example, are fairly well thymus for as long as 3 wk after their intrathymic established, the mechanisms of the T cell recognition injection. In a similar model, Goss et aL (47), however, of alloantigen remain unclear. New evidence confirms showed that microchimerism may not be necessary, the occurrence of at least two distinct, but not neces- because the intrathymic injection of irradiated alloge- sarily mutually exclusive, pathways of allorecognition neic splenocytes was sufficient to induce unrespon- (83,84). In the so-called “direct” pathway, T cells siveness. The results of studies with soluble MHC and recognize intact allo-MHC plus peptide complex on the MHC allopeptides also confirm that microchimerism, surface of donor or stimulator cells. In the so-called although it may be present in recipients rendered “indirect” pathway, T cells recognize processed alloan- tolerant by the intrathymic injection of donor cells, Is tigens as allopeptides presented by self-APC. Recent not necessary for the induction of tolerance. data from several investigators indicate that self-re- In the initial studies by Posselt et aL, it was demon- stricted T cell recognition of processed alloantigens strated that the precursor frequency of donor-reactive occurs during allograft rejection (83,84), and there is cytotoxic T lymphocytes was reduced in recipients of evidence that this pathway may play an important role intrathymic islets (4 1 ,9 1). In our model (80,82), the in allograft rejection (85). Experiments with intrathy- intrathymic injection of Class II MHC allopeptides mic peptides establish that the indirect recognition of effected antigen-specific peripheral T cell hyporespon- MHC allopeptides operates within the thymus and siveness, as determined by significant and sustained that this pathway of allorecognition is important in reduction of the mixed lymphocyte response in vitro as the induction of systemic tolerance. well as significant depression of delayed-type hyper- The uniqueness of the thymus as a privileged site for sensitivity responses in vivo (92). These data could the induction of systemic tolerance has been proved indicate either functional inactivation/anergy of allo- by findings that injecting donor cells iv (44,64, reactive T cell clones or actual deletion. Markmann 70, 73,86) into the peritoneum (43,73,87), into the and coworkers (93) explored the possibility that the mediastinum (88), or into another lymphoid organ tolerant state generated by the intrathymic inocula- such as the spleen (47,80) does not lead to the induction of donor cells was the result of the deletion of tion of tolerance. Furthermore, in several models, It donor-reactive T lymphocytes during their maturation appears that early thymectomy (up to 2 to 4 wk) into the thymus. They used cells from DBA 2/J mice posttransplant abrogates the unresponsive state bearing on their surface the minor lymphocyte-stim- (57,61,78,80,82), indicating that a primed thymus is ulating MLS superantigen, which is recognized by T required at least for the induction phase of tolerance. lymphocytes identified by antibodies specific for the The minimal duration of time between the intrathy- V136 segment of their T cell receptors. By injecting mic injection of donor antigen and subsequent trans- DBA2/J spleen cells into the thymus of MLSadefl plant and the requirement for the administration of cient BALB/c mice, they found substantial deletion of additional transient immunosuppression accompany- Vf36 thymocytes. Data from our group in the renal ing the intrathymic injection vary in the different allograft model (82) showed that the systemic admin- experimental models studied. Recently, data from istration of recombinant interleukin-2 daily for 5 days

1642 Volume 5’ Number 9 ‘1995 Remuzzi et al

starting on the day of transplantation abrogated the tolerogenic effect of intrathymic MHC allopeptides. By contrast, the administration of recombinant lnterleu- kin-2 at 4 to 6 wk after transplantation failed to abrogate the tolerant state. We propose that there are two phases of acquired thymic tolerance. The induction phase appears to be mediated by a state of T cell anergy, whereas the maintenance phase is mediated Intrathymic Injection by the clonal deletion of the previously anergized T Donor cells cells, most likely through the process of apoptosis or Soluble MHC Activated T cells MHC Peptides Thymic regulatory cells circulate through programmed cell death (90). go to periphery thymus How does the intrathymic injection of antigen result in peripheral T cell anergy and/or deletion? Recent ANERGY / DELETION (?Apoptosis) observations by Agus et at. indicate that activated but not resting peripheral T cells circulate to the thymus and stay for a quite prolonged period of time (94). In SPECIFIC TOLERANCE addition, Tanaka et at. (95) studied thymocyte traffick- Figure 2. Mechanisms of acquired thymic tolerance. The Ing by using ‘‘ ‘In-labeled cells and demonstrated that intrathymic injection of donor cells expressing MHC mole- these cells circulate to peripheral lymph nodes and cules, soluble MHC, or MHC allopeptides before the trans- back to the thymus during accelerated cardiac al- plantation of tissue or organ allograffs leads to the induction lograft rejection. It is possible, therefore, that, after of specific systemic tolerance. The unresponsive state is the transplantation of tissues or organs into recipients mediated by specific peripheral T cell anergy/deletion. tolerized by the intrathymic injection of alloantigen, Thymic regulatory cells leave the thymus to the periphery, or alloactivated peripheral T cells (activated by the spe- alloactivated peripheral T cells circulate through the thymus. cific alloantigens of the graft) may circulate through The interaction between thymic APC and alloactivated pe- the thymus and get anergized and/or ultimately de- ripheral T cells results in anergy and, ultimately, the deletion leted. Alternatively, thymic APC (such as dendritic of specific alloreactive T cells clones, most likely through the cells for example) might migrate to the peripheral process of apoptosis. The requirement of concomitant or lymphoid organs and to the graft and anergize and/or transient immunosuppression and the optimal timing of transplantation after the intrathymic injection vary in the delete specifically alloactivated T cells (Figure 2). Re- different experimental models studied. cent evidence has become available that donor-spe- cfflc unresponsiveness in rats injected into the thymus with Class II MHC allopeptides was completely slon were viable up to 4 and 8 wk posttransplant, as abolished when animals were given daily a TxA2 re- compared with animals receiving concomitant immu- ceptor blocker, either starting at the time of intrathy- nosuppresslon. Studies on the mechanisms of ac- mic injection or 15 days after renal transplantation quired thymic tolerance are essential In order to define (90). These findings would suggest that MHC-peptide the cellular Interactions in the thymus that ultimately recognition by alloactivated T cells circulating through lead to the induction of the unresponsive state . This is the thymus presented by thymic APC leads to a series important, because the human thymus is known to of events that Involve the TXA2/TXA2 receptor, thus involute In adult life, but it may continue to host the triggering Intracellular events that increase cytoplas- necessary cells that will recognize injected alloantigen mic calcium, resulting in T cell anergy and ultimately and induce systemic unresponsiveness . Furthermore, deletion by apoptosis. because of the involution of the thymus In adult life, the thymic procedure might be applicable in children and young adults. Another important question that PERSPECTIVES FOR HUMAN STUDIES needs to be answered is, what is the minimum waiting Although it has been relatively easy to achieve long- time between the intrathymic injection and transplan- term graft acceptance in experimental small animal tation? Should more than 24 h be necessary, this models, the induction and maintenance of transplan- might limit the applicability of the procedure to living tation tolerance in large animals (pigs or primates) donors. On the other hand, defining specific immuno- and in humans has proved to be more difficult to logic parameters, such as the mixed lymphocyte re- achieve. An obvious question, therefore, Is what Is the sponse or T cell precursor frequency assays, which are clinical applicability of acquired thymlc tolerance In down-regulated after the intrathymic injection of an- humans? Studies in large animals are needed to tigen, might allow better immunologic monitoring to confirm the feasibility of the intrathymic approach for determine the optimal timing of transplantation. Even tolerance induction. One recent report by Watt et a!. more Important, there is need to define the effect of (96) in the pig model indicated that the adult pig “conventional” immunosuppressive drugs on the un- thymus supports islet transplants. Interestingly, the responsive state induced by the Intrathymic Injection investigators found that islets transplanted in the two of antigen, because at this stage, clinical trials In pigs that received no concomitant immunosuppres- humans need to be designed with some form of con-

Journal of the American Society of Nephrology 1643 Transplantation Tolerance

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1644 Volume 5’ Number 9’ 1995 Remuzzi et al

vival in the rat. Immunol Cell Biol 1991 ;69: 185-189. 66. Seo K, Imal Y, Imamura 5, et at.: Induction of neonatal 47. Goss JA, Nakafusa Y, Flye MW: Intrathymic injection of immunologic tolerance to heart transplantation by intra- donor alloantigens induces donor-specific vascularized thymic myocardial cell inoculation in rats. J Heart Lung allograft tolerance without immunosuppression. Ann Transplant 1993; 12:5230-5235. Surg 1992;216:409-416. 67. Oluwole SF, Chowdhury NC, Jin MX: The relative con- 48. Oluwole SF, Chowdhury N, Fawwaz R, Hardy MA: Induc- tribution of intrathymic inoculation of donor leukocyte tion of tolerance to cardiac allogralts by pretreatment subpopulation In the induction of specific tolerance. Cell with intrathymic UVB donor spleen cells. Transplant Immunol 1994; 153:163-170. Proc 1992;24:2904-2905. 68. Matsuura T, Hara Y, Imanishi M, Kanda H, Kurita T: 49. Sea K, Imai Y, Imamura S. et at.: Inhibition of acute Prolonged survival of rat cardiac allografts induced by rejection of heart graft in rats without immunosuppres- intrathymic injection ofdonor bone marrow cells. Trans- sants after intrathymic myocardlal cell inoculation in the plant Proc 1993:25:3283-3285. neonatal period. Kyobu-Geka 1992;45: 1067-1070. 69. Knechtle SJ, Wang J, Jiao 5, Giessler EK, Sumimoto 50. Odorico J, Barker C, Posselt A, Naji A: Induction of RS, Wolff J: Induction of specific tolerance by intrathy- donor-specific tolerance to rat cardiac allografts by In- mic injection of recipient muscle cells transfected with trathymic inoculation of bone marrow. Surgery 1992; donor class I major histocompatibility complex. Trans- 112:370. plantation 1994:57:990-996.

5 1 . Kline GM, Shen Z, Mohiuddin M, Ruggiero V, Rostami 70. Campos L, Aifrey EA, Posselt AM, Odorico JM, Barker S, DiSesa VJ: Successfull experimental heart transplan- CF, Naji A: Prolonged survival of rat orthotopic liver tation without immunosuppressive drugs. J Heart Lung allograft after intrathymic inoculation of donor-strain Transplant 1993; 12:388-393. cells. Transplantation 1993:55:866-870. 52. Oluwole SF, Chowdhury NC, Fawwaz RA: Induction of 7 1 . Goss JA, Nakafusa Y, Flye MW: Prolongation of small donor-specific unresponsiveness to rat cardiac allografts bowel allografts after intrathymic injection of donor al- by intrathymic injection of UV-B Irradiated donor speen loantigen and ALS. J Surg Res 1993:54:494-498. cells. Transplantation 1993:55:1389-1395. 72. Goss JA, Nakafusa Y, Uchiyama K, Flye MW: Induction 53. Nakafusa Y, Goss JA, Mohanakumar T, Flye MW: Induc- of extended survival of rat skin xenograft in mice by tion of donor-specific tolerance to cardiac but not skin or pretreatment with intrathymic xenoantigen and antilym- renal allografts by intrathymic injection of splenocyte phocyte serum. Transplantation 1992:54:1101-1103. alloantlgen. Transplantation 1993;55:877-882. 73. Ohzato H, Monaco AP: Induction of specific unrespon- 54. Odorico JS, O’Connor T, Campos L, Barker CF, Posselt siveness (tolerance) to skin allografts by intrathymic AM, Naji A: Examination of the mechanisms responsible donor-specific splenocyte injection in antilymphocyte for tolerance induction after Intrathymic inoculation of serum-treated mice. Transplantation 1992:54:1090- allogeneic bone marrow. Ann Surg 1993;218:525-533. 1095. 55. Kampinga J, Kiatter F, Bartels H, et at.: Allotolerance 74. Ohzato H, Monaco AP: Tolerance induction of skin Induced by intrathymic application of alloantigen: Intro- allografts following intrathymic injection with donor- duction of a new and clinically relevant procedure allow- specific splenocytes in major histocompatibility complex ing heart graft survival in high-responder rats. Trans- class I, class I+MLS, and class 1+11 disparities. Trans- plant Proc 1993;25:2850-2851. plant Proc 1993:25:297-298. 56. Mohiuddin M, Kline G, Shen Z, Ruggiero V, Rostami S, 75. Qian T, Schachner R, Brendel M, Kong 5, Alejandro R: DiSesa VJ: Experiments in cardiac xenotransplantatlon. Induction of donor specific tolerance to rat islet allografts Response to intrathymic xenogenelc cells and intrave- by Intrathymic inoculation of solubilized spleen cell nous cobra venom factor. J Thorac Cardiovasc Surg membrane antigens. Diabetes 1993:42:1544-1550. 1993; 106:632-635. 76. James T, Jin MX, Chowdhury NC, Oluwole SF: Toler- 57. Nakafusa Y, Goss JA, Flye MW: Prevention by thymec- ance induction to rat islet allografts by intrathymic tomy of tolerance induced by intrathymic injection of inoculation of donor spleen cells. Transplantation 1993: donor splenocytes. Surgery 1 993; 114:183-190. 56:1148-1152. 58. Goss JA, Nakafusa Y, Yu 5, Flye MW: Intrathymic 77. Di Sllvio M, Murase N, Rio HL, et al.: Intrathymic injection of donor alloantigens induces specific tolerance transplantation of hepatocytes is facilitated by the addi- to cardiac allografts. Transplantation 1993;56: 166-173. tion ofpancreatic islets. Transplant Proc 1992:24:2987. 59. Sea K, Imamura 5, Ohta Y, Nishikawa T, Imai Y: Longer 78. Oluwole SF, Chowdhury NC, Fawwaz R, James T, Hardy transplanted heart allograft survival in rats without MA: Induction of specIfic unresponsiveness to rat car- immunosuppressants after intrathymic myocardial cell diac allografts by pretreatment with intrathymic donor inoculation in the neonatal period. Heart Vessels 1993; major histocompatibility complex class I antigens. 8:67-70. Transplant Proc 1993:25:299-300. 60. Chowdhuiy NC, Fawwaz RA, Oluwole SF: Induction of 79. Oluwole SF, Chowdhury NC, Jin M, Hardy MA: Induc- donor-specific tolerance to rat cardiac and small bowel tion of transplantation tolerance in rat cardiac allografts allografts by intrathymic inoculation of donor T-cells. J by intrathymic inoculation of allogeneic soluble pep- Surg Res 1993;54:368-374. tides. Transplantation 1993:56:1523-1528.

6 1 . Oluwole SF, Chowdhury NC, Fawwaz HA: Induction of 80. Sayegh MH, Perico N, Imberti 0, Hancock WW, Carpen- donor-specific unresponsiveness to rat cardiac allografts ter CB, Remuzzi G: Thymic recognition of class II major by pretreatment with intrathymic donor MHC class I histocompatibility complex allopeptides induces donor- antigens. Transplantation 1993;55: 1396-1402. specific unresponsiveness to renal allografts. Transplan- 62. Odorico JS, Posselt AM, Naji A, Markmann JF, Barker tation 1993:56:461-465.

CF: Promotion of rat cardiac allograft survival by intra- 8 1 . Sayegh MH, Khoury SJ, Hancock WW, Weiner HL, thymic inoculation of donor splenocytes. Transplanta- Carpenter CB: Induction of immunity and oral tolerance tion 1993:55:1104-1107. with polymorphic class II MHC allopeptides in the rat. 63. Goss JA, Nakafusa Y, Flye MW: MHC class II presenting Proc Natl Acad Sci USA 1992:89:7762-7766. cells are necessary for the induction of intrathymic 82. Sayegh MH, Perico N, Gallon L, et al.: Mechanisms of tolerance. Ann Surg 1993:217:492-501. acquired thymic unresponsiveness to renal allografts. 64. Odonco JS, Barker CF, Markmann JF, Posselt AM, Naji Transplantation 1994:58:125-132. A: Prolonged survival of rat cardiac allografts after intra- 83. Shoskes DA, Wood KJ: Indirect presentation of MHC thymic inoculation of donor thymocytes. Transplant antigens in transplantation. Immunol Today 1994:15: Proc 1993:25:295-296. 32-38. 65. Goss JA, Nakafusa Y, Yu 5, Flye MW: Reduction In 84. Sayegh MH, Watschinger B, Carpenter CB: Mechanisms donor-specific cytotoxic T lymphocytes and prolonged of T cell recognition of alloantigen. Role of peptides. cardiac allograft survival following intrathymic donor Transplantation 1994:57:1295-1302. splenocytes injection. Transplant Proc 1993;25:286- 85. Auchincloss HJ, Lee R, Shea 5, Markowitz JS, Grusby 288. MJ, Glimcher LII: The role of “indirect” recognition in

Journal of the American Society of Nephrology 1645 Transplantation Tolerance

initiating rejection of skin grafts from major histocom- ulation of T-cell immunity by intrathymic cellular trans- patibility complex class Il-deficient mice. Proc Nail Acad plantation. Transplant Rev 1993;7:200-2 13. Sci USA 1993:90:3373-3377. 92. Hancock WW, Khoury SJ, Carpenter CB, Sayegh MH: 86. Markmann JF, Bassiri H, Desal NM, Kim JI, Odorico JS, Differential effects of oral versus intrathymic adminis- Barker CF: Donor antigen-specific T-lymphocyte dde- tration of polymorphic major histocompatibility complex tion after intrathymic inoculation. Transplant Proc class II peptides on mononuclear and endothelial cell 1993:25:305-306. activation and cytokine expression during a delayed- 87. Remuzzi G, Perico N: Induction of unresponsiveness via type hypersensitivity response. Am J Pathol 1994; 144: intrathymic Inoculation. Lancet 1991 ;338:450. 1149-1158. 93. Markmann JF, Odonco JS, Bassirl H, Desai N, Kim JI, 88. Krokos NV, Brons 1GM, Srlwatanawongsa V, et a!.: Barker CF: Deletion of donor-reactive T lymphocytes in Intrathymic injection of donor antigen-presenting cells adult mice after intrathymic inoculation with lymphoid prolongs heart graft survival. Transplant Proc 1993:25: cells. Transplantation 1993:55:871-877. 303-304. 94. Agus DB, Surh CD, Sprent J: Reentry of T cells to the 89. Oluwole SF, Jin M-X, Chowdhury NC, Ohajekwe OA: adult thymus Is restricted to activated T cells. J Exp Med Effectiveness of intrathymic inoculation of soluble anti- 1991:173:1039-1046. gens in the induction of specific unresponsiveness to rat 95. Tanaka K, Tilney NL, Kupic-Weglinski JW: Maturing islet allografts without transient recipient immunosup- thymocytes in accelerated rejection of cardiac allografts pression. Transplantation 1994:58:1077-1084. in presensitized rats. Transplantation 1992:54:515- 90. Remuzzl G, Noris M, Benigni A, Imbertl 0, Sayegh MH, 519. Perico N: Thromboxane A2 receptor blocking abrogates 96. Watt PC, Mullen Y, Nomura Y, et a!.: Successful engraft- donor-specific unresponsiveness to renal allografts trig- ment of autologous and allogeneic islets into the porcine gered by thymic recognition of histocompatibility al- thymus. J Surg Res 1994:56:367-371. lopeptides. J Exp Med 1994:180:1967-1972. 97. Sayegh MH: Applicability of acquired thymic unrespon- 91. Posselt AM, Campos L, Mayo GL, et a!.: Selective mod- siveness in humans. J Nephrol 1994;7:3-4.

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1646 Volume 5’ Number 9 ‘ 1995