Role of graft interleukin-10 expression in the tolerogenicity of neonatal skin allografts.

BACKGROUND: Allografts of skin from neonatal donors survive longer than those from adult donors and can induce tolerance in mice that are treated with short-term immunosuppression. Neonatal (< or =24 hr old) epidermal cells (EPC) secrete high levels of interleukin-(IL) 10 and include abundant class II- immature Langerhans cells (LC). In this study, the role of IL-10 in the tolerogenicity of neonatal skin grafts was examined. METHODS: After a preliminary experiment established that tolerogenesis by neonatal grafts could be supported by monoclonal antilymphocyte antibodies, B10.A(5R) recipients were immunosuppressed with anti-CD4 plus anti-CD8 (days 0, +2) and adult C57B1/6 bone marrow cells (day +7). Recipients were grafted with adult or neonatal C57B1/6 skin from wild-type or IL-10 deficient ("knockout" donors). EPC from wild-type and knockout neonatal skin were compared by flow cytometry, before and after 48 hr culture, to adult cells in terms of class II and costimulatory molecule expression. RESULTS: Grafts from knockout neonates survived longer than those from adult donors (median survival, MST=81 vs. 61 days), but not as long as those from wild-type neonates (MST=100 days; P<0.05). As with normal neonatal EPC, neonatal knockout EPC expressed little class II antigen. Both types of neonatal EPC acquired class II in culture, and up-regulated CD80 and CD86 in an adult pattern, but failed to up-regulate class II antigen to the high level seen among cultured adult cells. CONCLUSIONS: The tolerogenicity of neonatal skin grafts derives in part from natural expression of IL-10 by the graft. Another possible contribution to tolerogenicity may be the inability of neonatal antigen presenting cells to up-regulate class II fully. Low expression of class II by neonatal cells is not attributable to epidermal IL-10 secretion.

Requirement for early donor cell chimerism during prolonged survival of murine skin allografts.

BACKGROUND: Posttransplantation infusion of donor bone marrow cells (BMC) can prolong allograft survival in antilymphocyte antibody- (ALA) treated recipients. This study examined the hypothesis that chimerism of donor BMC origin contributes to allograft unresponsiveness. METHODS: Survival of day 0 skin grafts from C3H (H2k) donors was prolonged on ALA-treated B6AF1 mice by day +7 infusion of BMC from C3H or C3H-H2o2 (H2K(d)I(d)D(k)) mice. To test for functional chimerism, depletional anti-H2Kd antibody was injected at intervals after C3H-H2o2 BMC infusion. To confirm the persistence of active cells in the recipients, cells harvested from bone marrow of ALA- and C3H BMC-treated primary recipients were transferred to secondary ALA-treated recipients. Other recipients were infused on day +21 with additional donor BMC or light-density BMC that had been cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) to promote the differentiation of dendritic cells (DC). RESULTS: Injection of depletional antibody targeting infused BMC interrupted skin graft survival, whether the injection was made on day +7, 2 hr after BMC injection, or as late as day +28. Prolonged graft survival was transferred to secondary recipients with cells recovered from primary recipient marrow as long as 4 weeks after initial donor BMC treatment. Graft survival prolonged by day +7 BMC was further enhanced by infusion on day +21 of light density cultured BMC, although a second dose of unfractionated BMC was inactive. Repeated injection of cultured BMC at 2-week intervals after day +21 prolonged graft survival even further. CONCLUSIONS: These data directly demonstrate that at least short-term donor BMC-derived microchimerism is required for prolonged allograft survival. The data further suggest that the active chimeric cells persist in recipient marrow. Finally, the beneficial effect of late infusion of cultured cells containing partially differentiated DC suggest that DC may be the active chimeric cells.

Late adjunctive therapy with single doses of rapamycin in skin-allografted mice treated with antilymphocyte serum and donor bone marrow cells.

Temporary donor-specific unresponsiveness induced by treatment of skin allografted mice with antilymphocyte serum (ALS) and donor bone marrow cells (BMC) can be converted to long-term graft survival and tolerance by adding adjunctive immunosuppressive agents, including rapamycin (Rapa). Rapa was given in a suboptimal dosing schedule to probe several aspects of its use in the promotion of tolerization. Single doses were given at 2 weeks post-transplantation to recipients prepared with ALS and donor BMC. Graft survival was markedly prolonged in a dose-dependent fashion by day +14, Rapa doses ranging from 0.75 to 6.0 mg/kg. Indefinite (> 300 day) graft survival was observed in 26% of recipients given the highest Rapa dose. Short-term treatment with cyclosporin A (CsA) or deoxyspergualin (DSG) was ineffective when injected at this time. Rapa augmented the tolerizing effect of grafting with skin from newborn mice but had no significant additive or synergistic effects with the short course of CsA or with DSG given on days +1 to +3, even though the latter prolonged graft survival when added to the ALS/BMC protocol. Single doses of Rapa on day +1 also prolonged graft survival, but without any of the grafts surviving indefinitely. Later dosing on day +28 resulted in > 70% of grafts surviving > 300 days. Challenge grafting of these mice after day +300 resulted in delayed rejection of donor strain, but not third-party skin grafts. Rapa was very effective when given as widely spaced doses on days +14 and +49. Also, grafts showing the earliest signs of rejection could be rescued with a single Rapa dose in recipients treated with ALS and BMC but not ALS alone. Transfer of prolonged graft survival with spleen cells from ALS plus BMC treated recipients was not adversely affected by Rapa given to the suppressor-like spleen cell donors approximately 1 week before cell harvest. We conclude that the use of Rapa as an adjunctive agent in allograft recipients treated with ALS plus donor BMC is very flexible in terms of timing of administration, and that the drug can be effectively given as widely spaced doses or as a rescue agent after ALS/BMC treatment. Additionally, an active immunoregulatory mechanism induced by ALS/BMC treatment appears to be spared by Rapa.

Effect of monoclonal anti-CD4 and anti-CD8 on skin allograft survival in mice treated with donor bone marrow cells.

Allograft unresponsiveness can be induced by donor bone marrow cells (BMC) in antilymphocyte serum (ALS)-treated recipients. The effect of administering monoclonal anti-CD4 and -CD8 at several points has been examined in a mouse skin allograft model of this protocol. Brief peritransplant administration of anti-CD4 and -CD8 was used to replace ALS. Anti-CD4 treatment prolonged graft survival only slightly and conditioned recipients poorly for the effect of posttransplantation donor BMC infusion. Anti-CD8 was ineffective in both capacities. A mixture of anti-CD4 and anti-CD8 was at least as effective as ALS in prolonging graft survival and in promoting the beneficial effects of donor BMC. Like the monoclonal antibodies, ALS also depleted splenic and lymph node CD4+ and CD8+ cells. Injection of ALS, but not the monoclonal antibodies, altered the CD4/CD8 phenotype of thymocytes, although persistent binding of both types of antibody to thymocytes was demonstrated. Abrogation of the positive effect of BMC by reconstitution of normal spleen cells on day +3 after ALS treatment confirmed that cell depletion is a requirement of this system. Monoclonal antibodies were also given to ALS/BMC-treated recipients after their grafts had become established. Anti-CD8 injection at either 2 or 4 weeks after transplantation further prolonged graft survival. In contrast, anti-CD4 injection at 2 or 6 weeks after grafting precipitated rejection, which suggests that continued allograft survival following ALS and donor BMC treatment is due to the activity of a CD4+ cell population.

Association between epidermal interleukin-10 secretion and the ability of cotransplanted skin from neonatal donors to prolong adult allograft survival.

Cotransplantation of skin from neonatal donors prolongs the survival of adult skin allografts on rabbit anti-mouse lymphocyte serum-treated and donor bone marrow cell-treated mice relative to controls (the cotransplant effect). In B6AF1 (H2a/b) recipients, cotransplants of skin from C3HeB/FeJ (C3H; H2k) neonates up to 6-7 days old prolonged the survival of adult C3H skin grafts. Skin from 9- to 10-day-old neonates was inactive. The magnitude of the cotransplant effect declined with increasing cotransplant age. Although few class II+ cells are present in skin from < 24-hr-old mice, the numbers of these cells increase rapidly after birth. On days 3-4, when the cotransplant effect is strong, their numbers in neonatal skin are greater than in adult skin. Development of class II expression continues when neonatal skin is grafted, but with an apparent 2-day lag. Because class II+ cell numbers decline in grafted adult skin, we speculate that this apparent developmental lag may be due to Langerhans cells migrating from the graft as they mature. Epidermal cells from litters of neonates were cultured overnight and supernatants were tested for interleukin (IL)-10. All 11 samples from 0- to 4-day-old neonates and 4 of 9 samples from 4- to 7-day-old neonates were positive. IL-10 was found in 1 of 9 samples from donors 8-16 days old and 1 of 7 samples from individual adult mice. Thus, there is a temporal association between the ability of neonatal skin to produce a cotransplant effect and its ability to secrete IL-10.

Effect of rapamycin on renal allograft survival in canine recipients treated with antilymphocyte serum, donor bone marrow, and cyclosporine.

Rapamycin (Rapa) monotherapy can promote renal allograft survival in dogs, but it is very toxic. To attempt to augment the effectiveness of Rapa and reduce its toxicity in a tolerance induction protocol, canine renal allograft recipients were treated briefly with antilymphocyte serum (ALS), donor bone marrow cells (BMC), and a limited course of cyclosporine (CsA). Rapa had little effect when CsA-treated recipients were given ALS on days -5 to -1 and BMC on day +1. When combined with CsA given days +13 to +42, ALS on days -5 to +7, and BMC on day +10, Rapa at 0.3 mg/kg on day +8 plus alternate days +15 to +39 significantly increased overall survival and was compatible with long-term survival after immunosuppression (6 grafts, 1 graft > 212 days, 1 graft > 470 days). Rapa appeared to prevent early rejections that can occur during treatment with these ALS/BMC/CsA protocols. Little toxicity of Rapa was observed with any treatment.

Tolerogenic behavior of skin allografts from neonatal mice.

Neonatal skin allografts can be tolerogenic when transplanted to appropriately immunosuppressed hosts. Single grafts of neonatal skin survive longer than adult skin grafts when recipients are treated with antilymphocyte serum (ALS) and donor bone marrow cells (BMC). Neonatal skin grafts can also prolong the survival of adult grafts of the same donor strain simultaneously cotransplanted with the neonatal grafts. To probe the mechanisms involved in this cotransplantation phenomenon, we delayed placement of the neonatal cotransplants relative to grafting with adult skin. Neonatal allografts placed either 7-9 days or 14 days after grafting with adult skin significantly prolonged adult graft survival in mice treated with ALS and BMC. However, day 0-placed neonatal cotransplants must remain on the recipient for > 2 weeks to prolong adult graft survival. Removal of cotransplants from ALS- and BMC-treated recipients after 7 or 14 days abrogated the cotransplantation effect. If left in place until day 21, neonatal cotransplants could significantly prolong adult graft survival, but did not induce the long-term graft survival observed in approximately 50% of the recipients whose cotransplants were not removed. Cotransplant removal after 1 year did not affect subsequent adult graft survival. Additionally, cotransplants were removed from recipients either on day 14 or from long-term graft-bearing mice and retransplanted to other ALS/BMC-treated recipients. These retransplanted grafts were unable to prolong survival of adult grafts on the new recipients. After transplant, but not before transplant, cyclophosphamide treatment of recipients prevented expression of the cotransplant effect in ALS-treated mice. However, recipient splenectomy > or = 1 week before grafting did not interfere with the effect. These results reflect on the contributions of the donor tissue, and the recipients' response, to the tolerogenic signals that permit a neonatal cotransplant to prolong adult graft survival.

Effect of asialofetuin on prolongation of skin allograft survival by donor bone marrow cells.

Survival of skin allografts made to antilymphocyte serum (ALS)-treated recipients is prolonged by posttransplant intravenous injection of donor strain bone marrow cells (BMC). If asialofetuin (ASF) is coinjected with the BMC, the prolonged graft survival is augmented (e.g., median survival time increased from 43 days to 72 days by injection of ASF). We have confirmed that, like peanut agglutinin-binding stem cells, the active BMC are at risk for hepatic sequestration after injection, possibly via hepatic asialoglycoprotein receptors. A fraction enriched for these active cells binds to liver sections in vitro and localizes to liver in vivo. This binding and localization can be partly inhibited by ASF. Although injected cells could also be found in the spleen, the beneficial effect of ASF could be demonstrated in previously splenectomized mice. Also, splenectomy 2 hr after BMC injection (without ASF) had little effect on the BMC-induced prolonged graft survival, while transfer of cells from the removed spleens to secondary ALS-treated recipients did not transfer such prolongation. In contrast, transfer of BMC from primary, donor marrow-injected recipients did transfer graft-prolonging activity, especially if both primary and secondary recipients were ASF injected. Our results suggest that recipient marrow, but not spleen, is the site of short-term localization of graft survival-prolonging BMC. Augmentation of allograft survival with ASF in ALS-treated recipients appears to result from the diversion of BMC away from a microenvironment not conducive to the expression of their graft-prolonging activity.

Abrogation of secondary skin allograft rejection by veto-like cells in donor bone marrow.

To investigate whether a veto cell phenomenon is involved in the ability of donor strain bone marrow cells (BMC) to inactivate second-set skin graft rejection induced by the adoptive transfer of sensitized spleen cells (SSC), mice were grafted with ear skin allografts and injected on the following day with recipient strain SSC or SSC plus donor strain BMC. When skin and BMC donors from an F1 strain (B6AF1) were combined with a parental strain (C57BL/6) as recipient and SSC donors, the BMC were fully active in abrogating second-set rejection even though the SSC were naturally tolerant of the BMC. Thus, immunological recognition of the SSC by the BMC is not necessary for this abrogation. The suppressive activity of the BMC is specific in a manner similar to that reported for veto cells (i.e., they suppress an anti-self response). If BMC from various strains of mice were used to alter the rejection of C3H grafts by B6AF1 recipients, only BMC expressing the MHC antigens to which the SSC had been sensitized significantly suppressed the activity of SSC. Sharing of additional antigens between the graft and BMC (e.g., MHC antigens not recognized by the SSC or minor antigens) was neither necessary nor sufficient. Mitomycin C-treated BMC were unable to inhibit SSC-induced accelerated rejection. Overall, the characteristics of the SSC-suppressive activity of BMC are consistent with a veto cell nature of the BMC.

Allograft and xenograft unresponsiveness induced by transplantation of neonatal skin.

Class I MHC-disparate skin allografts from neonatal donors survive longer than adult grafts in mice treated with antilymphocyte serum (ALS; days -1, +2) or ALS plus donor bone marrow cells (BMC; day +7 relative to grafting on day 0) or ALS, BMC, and a 2-week course of post-transplant CsA. We have now investigated whether this phenomenon extends to more strongly histoincompatible allografts (H2-KI and H2-KID mismatched) made to recipients treated with ALS, ALS plus BMC, or ALS, BMC, and CsA. Survival of neonatal grafts was longer than that of adult grafts in all experimental groups (P < 0.05). In ALS/BMC/CsA-treated mice, for example, median survival of neonatal C3H grafts was 46 days, with adult grafts surviving 22 days on C57BL/6 recipients (full MHC disparity). Survival of neonatal hamster skin grafted to B6AF1 mice was not augmented beyond that for adult skin using a similar immunosuppressive protocol. But if alternate day ALS and CsA injections were begun on day -5, and ALS and CsA continued as in the allograft models, a significant xenogeneic neonatal survival advantage was demonstrated. (Donor BMC had no graft-prolonging effect in the xenograft model.) Donor BMC significantly prolonged full MHC-mismatched grafts from newborn, but not adult donors in ALS-treated recipients. Also, addition of CsA to ALS/BMC treatment prolonged the survival of neonatal, but not adult grafts mismatched at KI or KID. These results indicate that the survival advantage of neonatal grafts in immunosuppressed recipients extends to strong allogeneic incompatibilities and even to a xenograft model.

Prolonged adult skin allograft survival as a result of cotransplantation with neonatal tissue. The requirement for antigen sharing between graft and cotransplant.

The survival of an adult skin allograft can be prolonged by a cotransplant of neonatal, but not adult, skin on the same recipient. We demonstrated this phenomenon using a C3HeB/FeJ (H-2k; C3H) adult graft and neonatal cotransplant donors. The median survival time (MST) for adult graft survival on B6AF1 (H-2a/b) recipients was 59 days on recipients treated with antilymphocyte serum and donor bone marrow cells. With adult or neonatal cotransplants, the MSTs for adult graft survival were 55 and > 100 days, respectively. Our current experiments explore the specificity of this phenomenon by substituting neonatal cotransplants of several allogeneic and partially allogeneic strains. Cotransplants that do not share the antigens presented by the adult graft to the recipient as foreign do not produce any prolongation of adult graft survival. Thus, cotransplants of adult or neonatal C57BL/6J (H-2b) or A/J (H-2a) strain skins had no significant effect on adult C3H graft survival. In contrast to these results, neonatal (but not adult) cotransplants that share presented antigens produce a significant cotransplant effect. The presence, on a recipient, of a neonatal cotransplant of CBA/J (H-2k) resulted in significant prolongation of adult skin grafts (MST > 150 days; P < 0.05). As well, on a different recipient strain (CAF1; H-2a/d), neonatal C3H-H-2o2/SfSn (H-2o2) skin cotransplants, sharing only background antigens and H-2Dk with the adult graft donor, caused a significant prolongation of adult graft survival relative to that seen on recipients bearing only a single adult graft (MSTs = 53 and 31 days; P < 0.05). Our results suggest that this graft-prolonging effect of neonatal cotransplantation requires that the cotransplant shares antigens with the adult graft that are presented as foreign to the recipient.

Prolongation of impure murine islet allografts with antilymphocyte serum and donor-specific bone marrow.

The inclusion of contaminating nonislet tissue in allografts of purified islets of Langerhans has been shown to decrease allograft survival times in unimmunosuppressed mice. Our current work examines the effectiveness of antilymphocyte serum and donor bone marrow cell infusion on the survival of very impure islet allografts. Treatment of B6AF1 mice with peritransplant antilymphocyte serum plus posttransplant infusion of donor (C3H/HeJ) bone marrow was very effective at prolonging contaminated allograft survival. ALS plus BMC was also effective for even more immunogenic allograft situations; mice that received contralateral transplants of adult and neonatal tissue, and mice that received dual transplants of adult islets. Long-term graft-bearing mice that originally received ALS and BMC were challenged with C3H/HeJ skin grafts. These mice exhibited several different types of response. Four of thirteen appeared sensitized to donor antigen, four rejected the skin grafts in approximate first-set fashion, and five showed limited prolongation, with skin grafts surviving 20-48 days. Long-term graft-bearing mice were also tested in in vitro proliferative assays. As responders in mixed lymphocyte reactions, splenocytes (SPC) from only one of eight appeared to show specific unresponsiveness to donor cells. Four of eight had a normal proliferative response and stimulation index to stimulation with C3H/HeJ SPC. SPC from three did not produce a normal stimulation index, but had an abnormally high (3x normal) background proliferation. SPC from seven of eight of these mice were able to suppress the proliferative response of naive B6AF1 SPC to C3H/HeJ cells. Our conclusions are that while ALS plus BMC are very effective in prolonging islet allograft survival, this treatment does not appear to be uniformly inducing a state of immunologic unresponsiveness in these mice. It appears that multiple mechanisms are acting to maintain islet allografts in this system, either independently or sequentially.