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Keywords:

  • Chimerism;
  • T cells;
  • Tolerance;
  • Transplantation

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Stable mixed chimerism has been considered the most robust tolerance strategy. However, rejection of solid donor tissues by chimeras has been observed, a state termed split tolerance. Since new non-myeloablative mixed chimerism approaches are being actively pursued, we sought to determine whether they lead to full tolerance or split tolerance and to define the mechanisms involved. Fully mismatched mixed chimeras generated by induction with various lymphocyte-depleting antibodies along with either low-dose irradiation or busulfan and temporary sirolimus, maintained stable mixed chimerism but nevertheless rejected donor skin grafts. Generation of stable mixed chimerism using antibody targeting CD40L, but not depleting antibodies to CD4 and CD8, could prevent split tolerance when skin grafts were given together with donor bone marrow. Minor antigen matching abrogated the ability of effector T cells to reject donor skin grafts. A CFSE killing assay indicated that chimeras were both directly and indirectly tolerant of donor hematopoietic cell antigens, suggesting that minor mismatches triggered a tissue-specific response. Thus, split tolerance due to tissue-restricted polymorphic antigens prevents full tolerance in a number of non-myeloablative mixed chimerism protocols and a ‘tolerizing’ agent is required to overcome split tolerance. A model of the requirements for split tolerance is presented.

Abbreviations:
ALS:

anti-lymphocyte serum

B6:

C57BL/6

BMT:

bone marrow transplantation

BUS:

busulfan

SRL:

sirolimus

TBI:

total body irradiation

Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Much of the effort to develop donor-specific transplantation tolerance has been focused on inducing peripheral tolerance through costimulation blockade. While initially promising, with more extensive tests of this approach the success has been somewhat limited, particularly when translated to larger animal models and to the clinic 1. In hindsight this may not be surprising given that tolerance naturally occurs primarily in the thymus and only secondarily in the periphery 2. In contrast, the approach of generating hematopoietic chimerism via bone marrow transplantation (BMT) takes advantage of the thymic central tolerance mechanisms, and is considered the most robust method of inducing donor-specific tolerance 36. The chimerism approach is limited clinically by the harsh recipient conditioning needed to establish chimerism and the possibility of graft-vs.-host disease 7.

More recently less toxic strategies have been developed that establish mixed allogeneic chimerism, where substantial levels of donor and recipient hematopoietic cells co-exist in the recipient, and have raised hope that robust transplantation tolerance will soon be routine clinically 6. Mixed chimerism has been considered to induce tolerance to all other donor tissues. If true, mixed chimerism could be a solution for both solid organ transplantation and cellular transplants, such as allogeneic islets used to treat type-1 diabetes. However, studies in full chimeras, where virtually all hematopoietic cells in the recipient are of donor origin, indicated that chimerism did not always allow acceptance of donor skin grafts; the recipients could show split tolerance 810. Rejection of donor skin grafts in full chimeras suggested that in fact the chimeras were ‘autoimmune’, as donor T cells rejected the donor skin 11. Given the presence of a substantial portion of host T cells in mixed chimeras, it cannot be assumed that split tolerance in mixed chimeras will necessarily occur by the same mechanisms as in full chimeras.

Despite the recognition of split tolerance in the early studies of experimental 7 and natural 12 mixed chimerism, it has received little attention since then. While split tolerance has been observed in some mixed chimerism strategies 7, 13, many current studies are specifically designed to avoid the issue by using donor and recipient combinations matched for minor histocompatibility antigens 1418. The prevention of split tolerance by minor antigen matching could result from a number of mechanisms, including reduced indirect reactivity to the donor or elimination of allelic tissue-specific antigens. Although split tolerance can be eliminated or reduced by minor antigen matching 7, this artificial approach cannot be applied practically to clinical transplantation. We therefore embarked on studies aimed at testing whether split tolerance is a common outcome in current non-myeloablative mixed chimerism strategies, assessed the mechanisms that can generate split tolerance, and examined the ability of split tolerance to be overcome with different antibody treatments.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Stable chimeras via non-myeloablative conditioning reject donor skin grafts

Previously we found that minimal conditioning with total body irradiation (TBI) at 1–3 Gy, followed by a 4-wk treatment with sirolimus (SRL), could induce stable mixed chimerism across a strongly mismatched strain combination if the recipients were depleted of lymphocytes before BMT 19. These chimeras accepted islet transplants from the same donor 19. To test whether they were tolerant to another donor tissue, we generated stable mixed chimeras using a similar low-dose TBI, SRL and anti-lymphocyte serum (ALS) protocol and transplanted donor skin grafts 14 wk post-BMT. Although the donor skin grafts were accepted well initially, eventually they were all rejected chronically in spite of maintenance of donor hematopoietic chimerism (Fig. 1A), indicating a state of split tolerance. Donor skin grafts gradually became thin, lost hair and shrank. Furthermore, the rejection rate did not correlate with chimerism level, since median survival times in the three groups were 86 days (3 Gy), 43 days (2 Gy) and 111 days (1 Gy). Our findings are unlike the apparent tolerance observed in mixed chimeras generated by myeloablative conditioning using the same donor and recipient combination and given skin grafts many weeks post-BMT 20.

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Figure 1. Rejection of donor skin grafts in stable chimeras. B6 mice were given BALB/c bone marrow cells on day 0, and all received SRL treatment beginning from day 0 (terminated on day 14 or 28). In addition, they received either TBI (1–3 Gy) or BUS on day –1. Recipients’ lymphocytes were depleted by injection of ALS (A), or by anti-CD4 ± anti-CD8 mAb (B). Chimerism data (top panels) are presented as percentage of lymphocytes that are donor in origin (mean ± SD), and represent those mice that achieved chimerism (100% of mice, n=10, treated with anti-CD4 ± anti-CD8 or ALS/3 Gy, n=4; 80% of mice, n=10, treated with ALS/<3 Gy). Donor BALB/c skin grafts were transplanted 14 wk (A) or 17–23 wk (B) after BMT.

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In order to test whether split tolerance was limited to protocols including ALS and/or low-dose TBI during the conditioning, we transplanted donor skin grafts to mixed chimeras which were conditioned by modified SRL-based protocols. We depleted the recipient's CD4 or CD4 and CD8 T cells, an approach pioneered by Waldmann and colleagues 21, 22, and used an irradiation-free conditioning therapy with busulfan (BUS). We found that all the SRL-treated mice conditioned with BUS/anti-CD4 (five of five) and BUS/anti-CD4/anti-CD8 (five of five) developed high levels of chimerism (Fig. 1B). Again, nearly all the chimeras tested (nine of ten) in these two groups rejected donor skin grafts while the level of chimerism instead increased over time (Fig. 1B). Thus, in all of the protocols tested, donor chimerism either increased or was maintained at peak levels for the additional 10–20 wk during which chimerism was monitored following rejection of donor skin.

Direct and indirect tolerance to hematopoietic cells but not polymorphic tissue antigens

Mixed chimerism has been shown in many studies to generate deletional tolerance in directly reactive anti-donor T cells. For example, we showed previously that mixed chimerism generated using the ALS/3 Gy/SRL protocol employed herein resulted in a loss of Vβ5+ T cells with direct anti-donor specificity 19. In contrast, little is known about the state of T cells responding indirectly to donor antigens in these chimeras. Lack of full indirect tolerance of donor bone marrow cell antigens could potentially explain split tolerance in the mixed chimeras. Skin grafts are known to be sensitive to indirect rejection 23, 24, and a differential sensitivity between skin and hematopoietic cells to indirect rejection mechanisms could therefore result in the appearance of split tolerance. Another potential explanation for split tolerance is the presence of tissue-specific antigens in skin that are absent in bone marrow cells 2527.

To begin to examine whether mixed chimeras may be split-tolerant due to indirect rejection of donor skin grafts but not donor bone marrow, we examined the state of reactivity to donor antigens in recipient MHC. We transplanted to our BALB/c to C57BL/6 (B6) chimeras skin from BALB.B donors that possess the minor-H antigens of BALB/c but the MHC of the recipient, H-2b. Stable chimeras generated with anti-CD4/anti-CD8/3 Gy/SRL, which had already rejected BALB/c donor skin grafts, rejected BALB.B but not control syngeneic skin grafts placed 34 wk post-BMT (Fig. 2A). This indicated that the mixed chimeras were not tolerant of all of the BALB/c minor-H antigens in the context of recipient MHC, despite maintaining stable chimerism levels (∼70% donor cells when assessed ⩾7 wk after rejection of BALB.B skin).

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Figure 2. Indirect reactivity to donor skin but not hematopoietic cells in mixed chimeras. Chimeras generated with anti-CD4/anti-CD8/3 Gy/SRL rejected donor BALB/c skin grafts and were transplanted with BALB.B (n=5) and syngeneic B6 skin grafts (n=3) 34 wk post-BMT. (A) Skin graft survival. (B) In vivo killing assay; chimeras generated with anti-CD4/anti-CD8/3 Gy/SRL or anti-CD4/3 Gy/SRL that had rejected donor BALB/c skin grafts were injected with labeled donor (CFSEhi) vs. syngeneic control (CFSElo) targets at a 1:1 ratio. Donor targets were BALB/c, BALB.B or CB6F1. Syngeneic B6 targets were used as a negative control. Chimeras and positive-control non-chimeric B6 mice were immunized prior to the assay with spleen cells corresponding to the type of donor target assayed (BALB/c, BALB.B or CB6F1). Naive B6 mice were negative controls. Targets were recovered from the recipients’ spleen 16–20 h later and data representative of at least three separate tests are shown. (C) CB6F1 plus BALB/c bone marrow into B6 chimeras (n=6) were generated with ALS/2 Gy, and 15 wk post-BMT challenged with BALB/c skin grafts (first graft) and then a second BALB/c skin graft 12 wk later. Donor chimerism at 30 wk post-BMT in these mice ranged from 4–8% for BALB/c cells and 58–70% for CB6F1 cells.

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However, responses to BALB/c minors in recipient MHC could indicate a lack of tolerance to either indirectly presented antigens of donor hematopoietic cells or minor antigens present in donor skin but not donor hematopoietic cells. To test whether the chimeras may retain some indirect reactivity to antigens in donor hematopoietic cells, we applied an in vivo cytotoxicity assay 28. We immunized the chimeras with donor spleen cells, and tested whether they could reject fluorescently labeled donor (CFSEhi) vs. syngeneic (B6 H-2b, CFSElo) control targets injected at a 1:1 ratio and recovered in the spleen ∼20 h later. Killing of donor cells would lead to a low donor-to-recipient target ratio in recovered cells. Donor targets were BALB/c (H-2d; to test direct reactivity), BALB.B (H-2b; to test indirect reactivity to donor minor-H antigens) or CB6F1 (H-2d/b; to test total direct and indirect reactivity to donor, including reactivity to peptides of donor MHC presented in recipient MHC).

Fig. 2B shows that BALB/c, BALB.B and F1 targets were not killed in these chimeric recipients, unlike the non-chimeric controls. Even when the length of the killing assay was extended to 7 days, we did not observe significant specific killing of donor target cells by the chimeras (Supporting Information Table 1). However, recovery of targets at late time points was quite variable and additional experiments suggested that the variability was due to priming and CTL activity of the targets themselves (data not shown).

These data suggested that, even though deletion of alloreactive T cells may be incomplete 19, the chimeras appear tolerant of donor hematopoietic cells, directly and indirectly. To further test the possibility that mixed chimeras may have effective direct but not indirect tolerance to donor antigens, we created BALB/c plus CB6F1 into B6 mixed chimeras. In this way the population of recipient T cells in mixed chimeras that normally recognizes donor BALB/c antigens only via indirect presentation on recipient MHC, would also recognize these antigens presented directly 29 on the F1 donor cells. Even though BALB/c hematopoietic cell antigens could be presented directly in the context of both donor and recipient MHC, these chimeras also rejected BALB/c donor skin (Fig. 2C). Therefore, direct presentation to the ‘indirect’ pathway-responding anti-donor T cells did not overcome split tolerance. Taken together the data indicate that mixed chimerism does lead to indirect tolerance of donor hematopoietic cell antigens.

To test whether the rejection of donor skin in stable chimeras is caused by allelic antigens present in the skin grafts, we simultaneously challenged chimeras with skin grafts from both donor BALB/c and an MHC-congenic B6.C-H-2d donor, which lacks BALB/c minor-H antigens. Fig. 3A shows that four of four chimeras transplanted with the two grafts 23 wk post-BMT rejected the BALB/c donor grafts while none rejected the congenic skin grafts (p=0.007). The congenic grafts appeared healthy macroscopically and had no evidence of infiltration (Fig. 3B). Thus, effector T cells in chimeras that are in the midst of rejecting BALB/c skin fail to reject B6.C-H-2dskin. Taken together with the evidence that the mixed chimeras are indirectly tolerant of donor hematopoietic cell antigens, these results indicate that rejection of donor skin is triggered by allelic tissue-specific antigens present in donor skin but not in donor bone marrow.

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Figure 3. Effector cells that reject donor skin grafts do not reject MHC-congenic skin. B6 recipients (n=4) given 3 Gy on day –1, BALB/c BMT on day 0, and MR1 on days 0, 2, 4 and 6. All chimeras received BALB/c and B6.C-H-2d skin grafts given at 23 wk post-BMT (donor cells in the chimeras ranged from 56–77% at 23 wk). One of these chimeras also had a prior BALB/c skin graft on day 0 (rejected at day 140). (A) Skin graft survival. (B) Representative macroscopic (top) and histologic (bottom; hematoxylin and eosin, 100× magnification) appearance of B6.C-H-2d skin grafts at 186 days post-skin grafting.

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Antibody targeting CD40L, but not T depleting antibodies, prevent split tolerance

If split tolerance in non-myeloablative chimeras was due to tissue-specific antigens, as our data suggested, this would predict that the presence of the tissue-specific antigens during the ‘tolerizing’ protocol should prevent split tolerance, leading to acceptance of donor skin grafts. Indeed, it has been observed that donor skin grafts are frequently accepted when placed early after BMT, during treatment with anti-CD40L, but not when given much later 13. However, it is not clear whether the ability to prevent split tolerance is dependent on a non-myeloablative chimerism approach that targets CD40L. We therefore tested for split tolerance in stable mixed chimeras generated using a low-dose TBI-based approach that included treatment with either anti-CD40L (MR1) 30 or anti-CD4 plus anti-CD8 and SRL [without SRL in this group the stability of chimerism was reduced (not shown)].

In two separate experiments using the MR1 protocol, we induced stable mixed chimerism in 75% of recipients. Six of 15 recipients were transplanted with donor skin grafts and bone marrow cells on the same day, another nine were given a donor skin graft later, i.e. 14–24 wk post BMT. The chimerism levels of these two groups were similar (Fig. 4A). All nine chimeras that were transplanted with donor skin grafts later on rejected the grafts. In contrast, four of six recipients that had skin grafts and bone marrow transplanted simultaneously (early group) accepted the grafts indefinitely (p=0.002 compared to late group). Furthermore, only the four chimeras that accepted the first graft in the ‘early’ group accepted a second donor skin graft indefinitely (Fig. 4A, lower panel); they rejected third party grafts within 12 days (data not shown).

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Figure 4. Induction protocol and timing of skin transplantation determine full tolerance vs. split tolerance in mixed chimeras. B6 mice were given 3 Gy on day –1, BALB/c BMT on day 0 and antibody to either CD40L (A) or CD4 and CD8, and SRL (B). Top panels: Chimerism results for the 15 of 20 anti-CD40L recipients and the ten of ten anti-CD4/anti-CD8 recipients that achieved stable chimerism. Middle panels: Donor skin grafts were given at the time of BMT (early; anti-CD40L n=6; anti-CD4/anti-CD8 n=5) or more than 14 wk post-BMT (late; anti-CD40L n=9; anti-CD4/anti-CD8 n=5). Lower panels: For anti-CD40L recipients, second donor grafts were transplanted 14 or 24 wk post-BMT in four chimeras that accepted the first graft (from ‘early’ group) and five chimeras that rejected the first skin graft (from ‘early’ and ‘late’ groups). Ticks on survival curves represent loss of a recipient with skin graft still intact. For anti-CD4/anti-CD8 recipients (n=5) that had rejected their first donor graft, a second donor and third party graft was given 17 wk post-BMT.

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We then replaced MR1 treatment with depleting antibodies specific to CD4 and CD8 and found that a nearly identical level of stable mixed chimerism was achieved (Fig. 3B). Furthermore, unlike with the MR1 protocol, 100% of recipients treated with anti-CD4 plus anti-CD8 achieved stable mixed chimerism. Nevertheless, split tolerance was not overcome by giving the skin graft early in these recipients; skin grafts were rejected when placed at the time of BMT or later than 17 wk post-BMT. In recipients given their first donor skin graft at the time of BMT, second donor and third party skin grafts given 17 wk post-BMT were also rejected (Fig. 4B, lower panel), with the donor graft being rejected in a rapid second set fashion. Together the data indicate that the ability to overcome split tolerance is neither related to the capacity of the induction protocol to achieve stable mixed chimerism nor to a particular level of chimerism. Instead, preventing split tolerance depends on the choice of targeting antibody induction protocol.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Acceptance of donor bone marrow but not donor skin grafts could result from a number of mechanisms. One possibility is the presence of tissue-specific antigens in skin that are absent in bone marrow cells 2527, 31. Tissue-specific antigens as the explanation for split tolerance has been questioned 32. Another possibility is that the chimeras may not be tolerant of indirectly presented antigens from donor hematopoietic cells, with differential sensitivity between skin and hematopoietic cells to indirect rejection resulting in split tolerance. Skin grafts are known to be sensitive to indirect rejection 23, 24; in contrast, we found that hematopoietic cells do indeed demonstrate some resistance to this rejection pathway (Chan et al., manuscript in preparation).

However, data from a number of different experimental approaches herein were consistent with a role for tissue-specific antigens as the explanation for split tolerance in mixed chimeras generated by non-myeloablative regimens. Firstly, while the BALB/c to B6 mixed chimeras rejected skin grafts presenting donor antigens in MHC of the recipient type (BALB.B skin grafts), suggesting a lack of tolerance in T cells with indirect anti-donor specificity, the same T cells did not kill BALB.B hematopoietic cell targets. Thus, chimeras appeared indirectly tolerant of donor hematopoietic cells but not skin. Furthermore, to fully exclude the possibility that split tolerance is a result of effective direct but not indirect tolerance, we created chimeras where donor antigens could be presented on recipient type MHC directly on donor bone marrow (BALB/c plus CB6F1 into B6). Even under these conditions, split tolerance was the outcome.

Secondly, split tolerance could be prevented by the presence of the donor skin graft early after BMT, during treatment with tolerance-promoting antibody to CD40L. This effect is similar to the ability of skin epidermal cell injection to prevent split tolerance in lethal irradiation chimeras 25, and suggests that the bone marrow cells do not possess the antigens needed to induce tolerance to the skin. Lastly, skin grafts possessing donor MHC, but lacking donor minor-H antigens, were not rejected despite rejection of simultaneously placed donor skin grafts. Thus, the effector cells rejecting donor skin appeared not to have the appropriate target antigen on MHC-congenic donor skin.

An important aspect of our data that would not have been predicted based on older studies in lethal irradiation chimeras 25, is that overcoming split tolerance in non-myeloablative mixed chimeras requires not only the early presence of the donor skin and its antigens, but also a specific tolerance-promoting treatment (in this case using MR1). In contrast to the ability of antibody targeting the CD40 costimulatory pathway, simple depletion of T cells facilitates tolerance of donor bone marrow but is not sufficient to prevent split tolerance in our model. Since treatment with MR1 has been associated with a tolerance that involves regulation and not simply deletion of donor-reactive T cells 33, it could be that a regulatory mechanism is required for tolerance to the tissue-restricted antigens in skin but not for tolerance to the more widely distributed antigens on donor bone marrow cells.

If this scenario is correct, then non-depleting antibodies to CD4 and CD8, which can also trigger a regulatory cell response 3436, are likely to be more effective at preventing split tolerance than their T cell-depleting counterparts. In this regard, an interesting recent study found that non-depleting anti-CD4 and anti-CD8 synergized with anti-CD40L in generating mixed chimerism and skin graft acceptance. Tolerance did not appear to involve a regulatory mechanism as assessed by a test of linked suppression using an F1 skin graft expressing additional allogeneic MHC 37. However, only a small amount of linked suppression is likely to be required to prevent a response to tissue-restricted skin antigens, and this level of suppression may be more readily detected using grafts expressing only additional minor antigens.

Given the frequency of split tolerance we found in non-myeloablative mixed chimeras, the question becomes why numerous other studies in mixed chimeras do not find split tolerance. One could surmise that researchers in the field have a pre-determined bias (full tolerance is the preferred outcome clinically), as evidenced by the numerous studies that chose to use clinically irrelevant MHC-congenic donors (minor antigen-matched) that are likely to give a false impression of full tolerance (skin acceptance) 16, 3844, including in protocols very similar to our own 45. No explanation is given in such studies for the unusual choice of donor/recipient combination.

However, since there are also many studies using fully mismatched combinations that did not find split tolerance even with skin grafts given late after BMT 20, 21, 4649, other factors must also be involved. Rejection of skin grafts in chimeras can be quite a protracted process, and in many studies skin grafts are not followed sufficiently long 50. Other factors that may play a role in the different outcomes include cryopreservation of donor skin 20, the type and size of skin graft used, the timing of skin transplantation relative to tolerizing antibody treatment (residual antibody may induce tolerance), the relative strength of reactivity in different strain combinations, and the absence of second donor skin graft challenge. These issues may also be relevant to the apparent absence of split tolerance in some neonatal tolerance models 51.

One might suggest that split tolerance is simply an academic curiosity occurring with skin transplants, and having little clinical relevance. While this might be a clinically hopeful point of view, no substantial theoretical basis has been suggested for the expectation that tolerance to one type of donor tissue (e.g. hematopoietic cells) would lead to full tolerance of other donor tissues. What currently remains more difficult to explain is the absence of split tolerance under certain conditions, such as in islet transplants to mixed chimeras using non-autoimmune strain combinations 19. We have shown that, as with lethal irradiation chimeras, split tolerance in non-myeloablative mixed chimeras has at least two requirements: (1) the presence of a tissue-specific antigen, and (2) polymorphism in the antigens targeted.

To our knowledge the requirement for polymorphism has not been explained. We suggest that indirect presentation of non-polymorphic tissue-specific antigens on donor bone marrow-derived APC leads to tolerance of these antigens, hence a requirement for polymorphism to generate split tolerance (Fig. 5). In addition to skin, split tolerance in chimeric canine recipients of heart transplants has also been reported, suggesting that polymorphisms in tissue-specific antigens may not be an exclusive property of the skin 52. Most recently, clinical trials involving combined kidney transplantation and non-myeloablative BMT were conducted in human patients 53. Surprisingly, long-term acceptance of donor kidney was not associated with maintenance of donor hematopoietic cells, which, as the authors suggested, may reflect the differential expression of donor antigens that are in fact tissue-specific. While the split tolerance observed in their studies operates in the reverse manner as compared to what we have described here, it nevertheless warrants the same caution over the clinical potential of hematopoietic chimerism in terms of generating full donor-specific transplantation tolerance.

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Figure 5. A model for split tolerance requirements. Indirect tolerance of tissue-specific antigens makes polymorphism a requirement for split tolerance. BALB/c to B6 mixed bone marrow chimeras possess both donor and host DC. Left side: Indirect presentation of non-polymorphic tissue-specific antigens (e.g. made in skin) leads to tolerance of these antigens presented in the context of both donor and recipient MHC. Tolerogenic indirect presentation may occur in the tissue, as donor and host DC migrate into the tissue, or in draining lymph nodes. B6.C-H-2d skin (not shown) grafted to these chimeras is accepted. Right side: T cells are neither directly nor indirectly tolerant of a polymorphic tissue-specific antigen present in a BALB/c skin graft; the graft is rejected.

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Therefore, one possible explanation for lack of islet rejection might be a lack of polymorphisms in the islet-specific antigens in mice. Interestingly, however, pancreatic isoantigens were previously detected in rabbits 54. Hence, split tolerance is potentially relevant to chimerism strategies in islet 55 or pancreas transplantation that is currently used to treat type-1 diabetes. Supporting this possibility, we recently found that mixed chimerism generated in pre-diabetic NOD mice does lead to split tolerance, with donor islets being rapidly rejected (Chan et al., manuscript in preparation). Given the genetic diversity of the human population, allelic tissue-specific antigens in islets or other tissues may be expected to provide targets for rejection in chimeric recipients. In addition, because polymorphisms probably play a critical role in many cases of split tolerance, one can anticipate that split tolerance will be particularly frequent in chimerism approaches to xenotransplantation 18, 5658.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

Mice

Adult B6 (H-2b), BALB/c (H-2d), CBA/J (H-2k), CB6F1 (H-2d/b), BALB.B (H-2b) and B6.C-H2d (H-2d) mice were purchased from Jackson Laboratory (Bar Harbor, ME). All care and handling of animals were carried out in accordance with the guidelines established by the Canadian Council on Animal Care.

Bone marrow transplantation and treatment protocols

BALB/c bone marrow cells were prepared by flushing the femur and tibia with PBS followed by passage of cell suspensions through a nylon mesh. B6 mice were given low-dose TBI, (1–3 Gy as indicated) on day –1 using a 137Cs irradiator (Gammacell 40; Atomic Energy of Canada, Ottawa, Canada) at an exposure rate of approximately 0.56 Gy/min. In the irradiation-free conditioning groups, B6 recipients were intraperitoneally (i.p.) injected with 20 mg/kg BUS (Busulfex®; kindly provided by Orphan Medical Inc., Minneapolis, MN) on day –1. Forty million unmodified BALB/c bone marrow cells (0.5 mL in PBS) were injected intravenously into B6 recipient mice on day 0.

SRL (Rapamune; Wyeth Canada, Montreal, Canada) was purchased from University of Alberta Hospital Pharmacy, diluted to 0.2 mg/mL with normal saline, and injected i.p. at 3 mg/kg immediately after BMT, and then daily for a total of 28 days (14 days in BUS-conditioned group). Where indicated, recipient lymphocytes were depleted before BMT by rabbit anti-mouse lymphocyte serum (ALS; Accurate Chemical & Scientific, Westbury, NY) at a dose of 0.3 mL i.p. on day –5 and –2 or by anti-CD4 mAb (GK1.5) and/or anti-CD8 mAb (2.43) at a dose 0.25 mg i.p. on day –3 to –1. In the costimulation blockade-based treatment groups, anti-CD40L (MR1; Bio Express, West Lebanon, NH) was injected on days 0, 2, 4 and 6 at a dose of 0.5 mg i.p.

Flow cytometry

Recipient blood or spleen samples were stained with both donor- and recipient-specific mAb (PE-conjugated anti-H-2Dd, 34-2-12, and biotinylated anti-H-2Db, KH95, for BALB/c to B6 chimeras; BD Pharmingen, San Diego, CA) for 15 min at 4°C and then washed. Biotinylated mAb was detected with APC-conjugated streptavidin. The percentage of lymphocyte-gated donor cells in recipients was defined as donor cells divided by the total number of recipient plus donor cells. Data were collected with a FACSCalibur® (Becton Dickson, Sunnyvale, CA) and analyzed using CellQuest™.

Skin transplantation

Full-thickness tail skin of ∼1 cm2 was transplanted onto the lateral thoracic wall of the recipients. The grafts were secured with sutures and covered with a band aid for a minimum of 7 days. The day at which 100% of donor tissue was lost was defined as the day of rejection. Zinc formalin-fixed, paraffin-embedded skin graft tissue sections were stained with hematoxylin and eosin.

In vivo cytotoxic assay

To assess whether the chimeras were tolerant to donor antigens from hematopoietic cells, we used spleen cells labeled with CFSE (Molecular Probes, Eugene, OR) as targets. The chimeras were immunized with five million donor spleen cells, corresponding to the type of donor target assayed (see below), 2 and/or 1 wk before intravenous injection of labeled donor (CFSEhi, 5 μM) vs. syngeneic control (CFSElo, 0.5 μM) targets at a 1:1 ratio (20–30 million cells of each). Donor targets were BALB/c (H-2d; to test direct reactivity), BALB.B (H-2b; to test indirect reactivity to donor minor-H antigens in recipient MHC) or CB6F1 (H-2d/b; to test total direct and indirect reactivity to the donor). Syngeneic B6 targets were used as a negative control. Targets were recovered from the recipients’ spleen 16–20 h later and analyzed by flow cytometry. Killing of donor cells leads to a low donor-to-syngeneic target ratio in recovered cells.

Statistical analysis

The log-rank test was used for survival curves; p<0.05 was considered statistically significant. Statistical analyses were done using Prism (GraphPad, San Diego, CA).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

We thank Dawne Colwell for artwork. This study was supported by grants from the National Institutes of Health (DK066512-01) to A.M.J.S. and C.C.A, and from the Canadian Institutes of Health Research and the Alberta Heritage Foundation for Medical Research (AHFMR) to C.C.A. A.M.J.S. and C.C.A. are supported by scholar awards from the AHFMR. W.F.N.C. is supported by doctoral research studentships from the Muttart Diabetes Research & Training Centre and the Canadian Diabetes Association.

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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Results
  5. Discussion
  6. Materials and methods
  7. Acknowledgements
  8. Supporting Information

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