ALS-treated Recipients Research Articles

Marked prolongation of rat skin xenografts induced by intrathymic injection of xenogeneic splenocytes and a short course of rapamycin in antilymphocyte serum-treated mice.

The effect of intrathymic (IT) injection of donor splenocytes and a short course of rapamycin (Rapa) treatment on rat to mouse skin xenograft survival was investigated. ACI rat skin xenografts were transplanted to (C57BL/6 x A)F1 mice treated with rabbit anti-mouse lymphocyte serum (ALS) on days -1, +2, and +4 relative to skin grafting on day 0. Fifty million donor-type splenocytes were injected intrathymically on day 7 after transplantation. Rapa was given intraperitoneally every other day from day 0 to day 12 at a dose of 3.0 mg/kg. Prolonged skin xenograft survival was observed in ALS- and Rapa-treated recipients (no IT injection) with a median survival time of 47 days. However, skin graft survival was markedly more prolonged in the group treated with ALS, Rapa, and IT injection of donor splenocytes did not have a beneficial effect on skin xenograft survival in ALS-treated recipients. An increased presence of donor-type cells was observed in the thymus of the ALS- and Rapa-treated recipients for 7 days after IT injection of donor splenocytes. In conclusion, a short course of Rapa markedly augments rat skin xenograft survival in ALS-treated mice injected intrathymically with donor-type splenocytes.

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.

Induction of transplantation tolerance to rat cardiac allografts by intrathymic inoculation of allogeneic soluble peptides.

Since intrathymic (i.t.) injection of UV-B-irradiated spleen cells (SC) or purified resting allogeneic T cells, but not resting B cells, dendritic cells, or macrophages induces specific tolerance in transiently immunosuppressed recipients, we hypothesized that presentation of donor MHC peptide Ag by the host thymic APCs may convey a tolerogenic signal to the recipient. This study examined if i.t. inoculation of allogeneic soluble Ag obtained from 3 M KCl extracts of purified resting T cells can induce specific tolerance to cardiac allografts in transiently immunomodulated recipients. We have now shown that i.t. inoculation of donor soluble Ag on day -7 combined with 1 ml ALS on days -7 and 0 leads to indefinite WF cardiac allograft survival (> 200 days) in Lewis recipients. This finding was reproducible in sublethally irradiated (200 rads TBI) ACI recipients of i.t. Lewis soluble Ag. In contrast, ACI cardiac allografts were promptly rejected in ALS-treated Lewis recipients of i.t. WF soluble Ag, confirming the donor specificity of such immunologic manipulation. Extrathymic inoculation of WF soluble Ag via the intravenous route in controls failed to prevent normal graft rejection in ALS-treated recipients. The long-term unresponsive recipients specifically and permanently accepted donor-type, second-set cardiac allografts. The observation that thymectomy performed 7 days after i.t. Ag injection led to graft rejection strongly suggests that the early phase of induction of donor-specific tolerance is dependent on the presence of donor alloantigens in the host thymus. This approach may have important clinical therapeutic potential in the induction of transplantation tolerance.

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.

THE EFFECT OF ANTILYMPHOCYTE SERUM, FRACTIONATED DONOR BONE MARROW, AND CYCLOSPORINE ON RENAL ALLOGRAFT SURVIVAL IN MONGREL DOGS

The effect of combined treatment with antilymphocyte serum, fractionated donor bone marrow, and a limited course of cyclosporine on renal allograft survival in mongrel dogs was examined. Recipients were treated with ALS from day -5 to day +7, relative to transplantation on day 0 with an MLR-mismatched donor. Fractionated donor bone marrow cells (BMFr3) obtained by density gradient separation were infused 3-7 days after ALS treatment. CsA treatments were begun either 3-7 days after ALS plus BMFr3 infusion or 3-7 days after treatments with ALS alone. Extended allograft survival was achieved at all CsA doses in BMFr3-infused, ALS-treated recipients. Allografts were sustained throughout the CsA treatment period without rejection in the majority of recipients (6 of 8) receiving ALS plus BMFr3 and CsA at 20 Mg/kg/day for 60 days. By contrast, few grafts were sustained through 30 days of treatment with CsA at 3.2 (1 of 12) or 10 mg/kg/day (2 of 9) in ALS plus BMFr3-treated recipients. Cyclosporine treatment was ineffective at all doses in augmenting allograft prolongation in ALS-treated recipients that did not receive BMFr3. Nearly all (6 of 7) long-term survivors (greater than 180 days) were BMFr3-treated. Peripheral blood lymphocytes or bone marrow cells of these long-term survivors proliferated normally in response to Con A and PWM, and were MLR responsive to third-party cells but did not have reduced MLR responsiveness to donor alloantigen in all cases. These long-term survivors promptly rejected third-party renal allografts without adverse effects on the original transplant's function. These results show that long-term renal allograft survival with specific unresponsiveness to the donor can result from the combined treatment with ALS plus donor BMFr3 and a limited course of CsA.

Graft-versus-host reactivity of human lymphoid cells from established cell lines

Cells from three out of five established human lymphoid cell lines gave reactions when injected under the kidney capsule of rats pretreated with cyclophosphamide. These reactions were characterized by mononuclear cell infiltration and destruction of the kidney cortex. No reactions were obtained when the cells were incubated with anti-human lymphocyte serum or were treated with mitomycin. Reactions could not be elicited in irradiated or ALS-treated recipients. The reactions obtained were indistinguishable from the graft-versus-host reactions given by fresh human buffy coat leukocytes.

Immunosuppressive and graft-rejecting anti--bodies in heterologous anti-lymphocytic serum.

Skin allografts survived longer on ALS-treated, complement-deficient (C5 negative) recipients than on ALS-treated, complement-competent (C5 positive) recipients. Administration of C5-positive serum to C5-negative, ALS-treated recipients resulted in reduced graft survival. A percentage of grafts from ALS-treated, C5-positive donors was rejected when transferred to untreated syngeneic recipients; this was not observed when C5-negative, syngeneic animals served as ALS-treated donors and untreated recipients. It was concluded that ALS has graft-rejecting properties which are promoted by late acting complement components. Unlike ALS-mediated graft rejection, ALS-mediated immunosuppression appeared to be independent of the late acting complement components. The effect of ALS on the humoral response to sheep erythrocytes was examined in complement-deficient and complement-competent mice. Immune-suppression was determined by ALS treatment of C5-competent and C5-deficient mice and also by transfer of in vitro ALS-treated spleen cells from C5-negative and C5-positive donors to cyclophosphamide-treated recipients. The ability of ALS to depress the humoral response to sheep cells and to decrease immunological competence of spleen cells was the same in the presence as in the absence of C5.