First-set Grafts Research Articles

Cellular immunity in allograft rejection: Role of lymphocyte subpopulations and T-cell subsets in rat cardiac allograft rejection

In this study, cellular requirements for rejection are examined by the use of adoptive transfer assays in the ACI to Lewis cardiac allograft model. The findings show that adoptive transfer of 1 x 10(8) spleen cells (SpL), 5 x 10(7) T-cells, and 2 x 10(7) helper T-cells (W3/25+) obtained from normal, nonsensitized donors restores acute ACI graft rejection in sublethally irradiated (750 rad) Lewis recipients. In contrast, reconstitution with 2 x 10(7) cytotoxic T-cells (0X8+) does not restore first-set graft rejection. Reconstitution of the irradiated recipients with either W3/25+ or 0X8+ T-cells obtained from specifically sensitized syngeneic donors resulted in acute rejection. The W3/25+ T-cell subset was significantly more potent (P less than 0.01) in effecting rejection on a per-cell basis. Adoptive transfer of SpL, T-cells, and 0X8+ T-cells obtained from sensitized rats led to accelerated cardiac allograft rejection in the naive secondary recipients while W3/25+ T-cells did not. This study suggests that although the W3/25+ T-cells alone have the capacity to initiate first-set graft rejection, both W3/25+ and 0X8+ subsets appear to be critical to the completion of rejection of heart allografts. We also examined the capacity of adoptively transferred B-cells from sensitized donors to influence graft rejection. Our findings suggest that while B-cells fail to restore the capacity for graft rejection in irradiated recipients, they can, however, present MHC antigens to the secondary naive host thus causing allosensitization which results in accelerated rejection of a subsequent graft.

Abrogation of alloreactive spleen cell-induced second-set skin graft rejection in mice with donor-specific bone marrow cells.

To study the mechanism of induction of specific unresponsiveness to allografts in animals treated with antilymphocyte serum and donor bone marrow cells, we examined the effect of donor BMC on second-set graft rejection responses caused by antidonor-sensitized spleen cells (SSC) in mice bearing donor ear skin grafts. Anti-C3H SSC were obtained from skin-grafted, ALS-treated B6AF1 mice after rejection of their grafts. The second-set rejections of C3H skin grafts were assessed in B6AF1 mice following adoptive i.v. transfer of 30 x 10(6) SSC one day after grafting. Median survival time (MST) of C3H skin grafts in the group injected with SSC was 7 days, which is significantly lower than an MST of 11 days observed in the control group, which exhibited a first-set graft rejection response. Addition of 50 x 10(6) C3H BMC to 30 x 10(6) SSC abrogated the second-set rejection of C3H skin grafts (MST = 11 days). This abrogation effect of BMC is strain-specific, since BMC of a third-party strain (DBA/2) failed to abrogate the second-set rejection responses caused by anti-C3H SSC. Of the different C3H lymphoid cells tested for abrogation of the second-set graft rejection, BMC were the most effective. Splenocytes were more effective than thymocytes, which showed a partial effect. Lymph node cells had no effect. Our data suggest that unresponsiveness to allografts in animals treated with the ALS/bone marrow protocol may result from the inactivation of graft-reactive cells by donor BMC.

Attempts to break perimetamorphically induced skin graft tolerance by treatment of Xenopus with cyclophosphamide and interleukin-2.

The maintenance of skin allotolerance induced by perimetamorphic application of MHC-disparate skin to isogeneic Xenopus is investigated. Removal of the perimetamorphically applied first-set graft after 4 weeks did not, in general, completely break allotolerance; however, many second-set semi-allogeneic grafts, applied up to 14 weeks after first-set removal, were no longer maintained in perfect condition. Skin allografts tolerated for up to 42 weeks continued to express donor histocompatibility antigens, as indicated by their survival times when transplanted back to the original donor or recipient strain. Treatment with human recombinant IL-2 (rIL-2), shown elsewhere to be an effective immunoregulatory lymphokine for Xenopus in vivo, failed to cause long-term-tolerated 1st-set allografts, or newly-applied 2nd-set grafts, to be rejected. In contrast, cyclophosphamide (CyP) treatment led to acute (less than 4 weeks) destruction of both 1st- and 2nd-set allografts; breaking of tolerance was regularly seen when donor and host differed by two MHC haplotypes, but occurred infrequently in semiallogeneic combinations. The experiments suggest that skin-induced allotolerance is maintained by an immunosuppressive mechanism, that is CyP-sensitive but resistant to rIL-2 treatment.

Facilitation of Skin Allograft Survival by Blood Leucocyte Extracts

Fresh and frozen-stored mitochondrial, microsomal, and endoplasmic reticulum extracts of blood leucocytes act as potent alloantigens in human recipients. Similar results were obtained with freshly prepared extracts consisting of mixtures of all cytoplasmic fractions; storage of such mixtures at--20 C, followed by 1-2 hr thawing at 37 C abrogated their capacity to induce allograft sensitivity in 45 of 47 recipients. Donor-specific skin allografts and grafts from other sources exhibited significant attenuations in the tempo and intensity of rejection, ranging from first-set rejection to chronic rejection and/or to prolongations in allograft survival. In contrast with the 66.2% rejection rate of first-set skin grafts at 10 days in 71 normal subjects, only 29% of skin grafts from the leucocyte donor applied to 24 recipients of 0.1 to 9 Transplantation Antigen (T.A.) units of pooled cytoplasmic mixtures were rejected by that time. Only 17.4% similar grafts in 23 recipients of 25 to 515 T.A. units were rejected at 10 days. Seventeen skin grafts placed on recipients of 45 to 140 T.A. units of the same cytoplasmic preparation exhibited a slow rejection characterized by progressive shrinkage and eventual disappearance, with no evidence of hemorrhagic necrosis (chronic skin graft rejection). These results support the possibility that the attenuations in allograft reactivity observed in patients with end-stage renal disease after blood transfusions may be related to the leucocyte components of such transfusions. The capacity of blood transfusions to decrease reactivity to renal allografts in uremic patients maintained on hemodialysis stands in contrast with the ability of such transfusions to sensitize normal human recipients to donor-specific skin allografts. The differential effect may be related to the immunosuppressed state(s) documented in the uremic population, and/or the use of immunosuppressive drug therapy in such patients at the time of transplantation and thereafter.

Definition, genetics, and possible significance of a newly defined endothelial antigen in the rat.

The purpose of the present experiments was to define a non-major-histocompatibility-complex (MHC) endothelial antigen system in the rat and to study the genetics of these antigens as well as their significance in renal transplantation. Several MHC-identical rat strain combinations underwent reciprocal immunization using spleen and lymph node cells and complete Freund's adjuvant. In one combination (MAXX anti-BN) alloantibodies were found against antigens on peritubular and venous endothelium of the kidney from the immunizing strain as well as from two other strains. Preliminary results suggested that the endothelial antigen is present on monocytes but not on nonstimulated T and B lymphocytes. With kidneys from 7 MHC-congenic lines it was shown that the endothelial antigens are encoded outside the MHC-region. The antigen seems to be expressed as a dominant trait. In an F2 population of 32 animals, segregation of the endothelial antigen or antigens appeared to be independent of the MHC, AgF, and tubular basement membrane antigens--as well as the locus for albinism. Transplantation of MHC-identical but endothelial-antigen-incompatible kidneys into nonimmunized recipients did not provoke acute rejection. Pretransplant immunity against donor endothelial antigens was, however, associated with accelerated acute rejection. The rejection was donor-specific because third-party MHC-incompatible but endothelial-antigen-compatible kidneys were rejected like first-set grafts. This model shows that graft rejection in presensitized recipients of an MHC-identical kidney can be mediated through immunity against non-MHC antigens.

Prolonged survival of major histocompatibility complex-disparate skin allografts transplanted to the metamorphosing frog, xenopus laevis.

Perimetamorphic larval and postmetamorphic froglet Xenopus laevis were grafted with adult skin which differed from the host by one or two major histocompatbility complex (MHC) haplotypes and multiple minor H loci. In family studies, long-term survival of one-MHC haplotype-disparate grafts was observed at frequencies from 10 to 100% when perimetamorphic F1 progeny were grafted with skin from their field-collected, presumably MHC heterozygous parents. When donor and host differed by two MHC haplotypes, fewer metamorphosing recipients retained intact grafts. Recipients transplanted with first-set grafts as postmetamorphic froglets rejected their transplants in all protocols. Rejection of oneor two-MHC haplotype-disparate allografts by animals grafted during the perimetamorphic period was chronic and variable, whereas skin graft rejection by animals grafted after metamorphosis was more acute. Studies using partially inbred strains of Xenopus, whose MHC haplotypes are genetically defined, confirmed results from family studies. The incidence of allograft survival on perimetamorphic recipients was 93% when donor and host differed by one-MHC haplotype and 30% when transplantation was across a two-MHC haplotype barrier. Animals in one family that were first-set grafted during metamorphosis and did not completely reject their grafts during the more than 2 years that they were observed, promptly rejected third-party tests grafts that were transplanted 28 months after the initial transplantation procedure. Changes in the animals' lymphoid and endocrine systems are known to occur at metamorphosis. Acceptance of allografts by animals grafted during metamorphosis may reflect their physiological changes or it may be a by-product of immunological mechanisms necessary for self-tolerance that have been postulated to be present at this time in development.

THE INFLUENCE OF TEMPERATURE ON THE KINETICS OF ALLOGRAFT REACTIONS IN A TROPICAL SPONGE AND A REEF CORAL

Many tropical sponges and reef-building corals demonstrate highly discriminating transplantation immunity when grafted with allogeneic tissue. The speed of rejection changes seasonally, and therefore the role of temperature was investigated. Replicate parabiotic allografts of a Hawaiian sponge, Callyspongia diffusa, and a reef-coral, Montipora verrucosa, were exposed to three different temperature regimens: approximately 23°, 25°, and 27°C. The primary allograft reactions were extremely temperature dependent in both species; the median reaction times (MRT) for the first-set grafts at summer temperatures (27°C) were approximately half the winter temperature (23°C) MRTs. Significantly accelerated second-set reactions in the coral were similarly affected by temperature; however, maximally accelerated second-set reactions in the sponge were similar at all three temperatures. The primary mean reaction times for replicate grafts varied significantly among the individual genotypic combinations brought into parabiosis. Furthermore, different genotypic combinations responded to temperature changes with qualitatively distinctive reaction trends as well as different reaction times. Parabiotic allografts mimic intraspecific interactions that are a consequence of natural contact through growth of sedentary marine animals. The influence of temperature on the outcome of intraspecific competition suggests that temperature fluctuations could maintain high levels of genetic polymorphism within individual populations of sponges and corals.

The basis of graft rejection in the earthworms Lumbricus terrestris and Eisenia foetida

Body wall grafts have been exchanged between Lumbricus terrestris and Eisenia foetida unicolor, and the survival times of second-set grafts and a limited number of third-set grafts have been compared with those of first-set grafts. Survival of grafts has also been studied following implantation. The results do not support the hypothesis that second-set rejection is accelerated due to an immune response as in higher vertebrates. Evidence is presented that Eisenia are individually different and that the differential graft survival may be due to tissue incompatibility, survival times depending on the degree of difference in the environment presented by the recipient.

The cellular basis of allograft rejection in vivo. I. The cellular requirements for first-set rejection of heart grafts.

The nature of the cells required for first-set graft rejection in vivo was examined by using an adoptive transfer system to restore heart-graft rejection in irradiated rats. Highly purified inocula of peripheral T lymphocytes were shown to quantitatively account for the restorative ability of adoptively transferred cells. These T cells were shown to be long-lived small lymphocytes which are not recently derived from the thymus during adult life. They belong to the pool of T cells which constantly recirculate from blood to lymph as shown by their rapid appearance in the lymph of iradiated syngeneic rats after intravenous injection. Neither B lymphocytes nor antibodies in the circulation or in the graft itself are required for first-set graft rejection.

Histocompatibility antigens controlled by the I region of the murine H-2 complex. I. Mapping of H-2A and H-2C loci.

Skin grafts were reciprocally exchanged in pairs of congenic lines identical in all genes except those located in the central portion of the H-2 complex. Seven such lines were tested: 6R, B10.AQR, A.TL, A.TH, 7R, 9R, and B10.HTT. In all donor-recipient combinations at least some grafts were rejected. In combinations differing at the IA subregion (and other central H-2 regions or subregions), all first-set grafts were rejected within 3 wk after transplantation, and all second-set grafts were rejected within 10 days. In combinations differing at the IC subregion (and other central regions, but not at the IA subregion) between 60 and 100% of first-set grafts were rejected, but some grafts survived for over 100 days. Most of the second-set grafts were rejected within 1 mo after grafting. This behavior of skin grafts indicated the presence of two histocompatibility loci in the I region, a strong one and a weak one. This conclusion was confirmed by genetic mapping which placed the strong locus in the IA subregion and the weak locus in the IC subregion. We designate the former locus H-2A and the latter H-2C. The same strain combinations used for the skin grafting were also used for determination of the capacity of I-region antigens to function as targets in the in vitro cell-mediated lymphocytotoxicity (CML) assay. Spleen cells from mice presensitized in vivo by skin grafting were restimulated in vitro and tested against 51Cr-labeled concanavalin A or lipopolysaccharide blasts. The testing revealed the presence in the I region of two loci coding for CML-target antigens. The two loci comapped with the H-2A and H-2C loci and were most likely identical to them. As in the skin grafting test, in the CML test, the H-2A antigens evoked stronger response than the H-2C antigens. Rejection of skin grafts across the H-2A and H-2C loci was accompanied by the production of Ia antibodies. Direct cytotoxic and absorption tests with Ia antibodies directed against antigens coded for by the IC subregion revealed the presence of IaC antigens on epidermal cells. We suggest that the products of Ia loci might function as transplantation antigens.

Characteristics of cell-mediated immunity and memory in annelids.

To search for the phylogenesis of foreign tissue graft rejection we have utilized the common garden earthworm extensively. Whereas earthworms never destroy self-tissue or autografts, they are fully capable of rejecting foreign or not-self tissue allografts (1–5) or xenografts (6). At 15°C, single first-set xenografts exchanged between Lumbricus terrestris and Eisenia foetidaare destroyed at approximately 25–35 days. After a first-set graft is destroyed at 15°C, immunologic memory is demonstrable by regrafting the hosts with a second transplant from the original donor of the first-graft. Both positive and negative memory are demonstrable. Positive memory occurs when second-set transplants are rejected significantly faster than first-sets. By contrast, a lesser percentage of worms have grafts that show prolonged survival indicating negative memory. However, if repeat second-sets are performed at 15°C, five days after transplanting a first-set, during the induction phase of the immune response, there is no dissociation into positive and negative memory. Instead, both first- and second-set grafts are destroyed faster than a single graft (7). It appears therefore that at the evolutionary level of annelid worms, foreign transplant rejection is specific and the mechanism includes a memory component. Memory is one characteristic of adaptive immunity as defined for vertebrates (8).

The rate of formation and the composition of lymph from primary and secondary renal allografts.

Summary The rate of formation and composition of lymph from renal allografts has been studied in normal and sensitized ewes and related to the histopathological appearance of the graft at various times during its life. Analysis of the volume of lymph being formed, its protein content, and the types and numbers of cells appearing in the lymph showed that pathological events within the graft were reflected closely in changes in the lymph. In first-set allografts large numbers of lymphocytes migrated from the blood into the graft. Many of these cells underwent blast transformation and left the graft by way of the lymph. During the first 24–48 hr the blood capillaries of the graft were very permeable to red cells and plasma proteins, but after this time the protein content of the lymph fell, the red cells disappeared, and the rate of lymph flow increased many-fold. This phase correlated with the appearance of lymphoid cell emboli in the small vessels of the graft. Around 160 hr there was a precipitous fall in lymph flow and the numbers of red cells and the protein content of the lymph rose. This terminal phase was related to the appearance of antibody in the blood and to the widespread destruction of the vasculature of the graft. Renal grafts rejected in an accelerated fashion produced significantly less lymph than first-set grafts. The permeability of the blood capillaries of grafts installed in sensitized recipients remained high throughout, and large numbers of red cells and polymorphonuclear leucocytes appeared in the lymph from the outset. When antibody was present in the blood of the recipient, the traffic of lymphocytes into the graft was greatly reduced and amounted to about 1–5% of the number of cells migrating through primary allografts. Although antibody had a pronouced effect on the migration of lymphocytes into the graft, it did not seem to affect the subsequent trans-formation of lymphocytes into blast cells, and the proportion of blast cells to lymphocytes was similar to that seen in primary grafts.

Active enhancement of rat renal allografts with soluble splenic antigen.

Renal transplantation was performed between inbred rats after pretreatment with i.v. soluble splenic antigen from the donor strain. A variety of dosage and timing of therapy was utilized. The most effective pretreatment was 0.5 mg of antigen administered 1 day prior to transplantation. A similar pretreatment dosage given over 14 days was also effective. Cyclophosphamide (CP) did not increase the effectiveness of antigen pretreatment. Graft-vcrsus-host (GVH) response was increased in those groups of rats that demonstrated increased renal allograft survival when studied at the time of transplantation. Spleen cells obtained from long surviving rats lost this increased reactivity and donor strain skin grafts placed in these long surviving rats were rejected as first-set grafts.

ADAPTATION OF SKIN ALLOGRAFTS IN MICE TREATED WITH ANTILYMPHOCYTE SERUM

SUMMARY Mice treated with antilymphocyte serum (ALS) at the time of placement of a firstset skin allograft were grafted additionally with second-set and third-party skin 2 weeks later. The survival of first-set grafts was consistently prolonged beyond the time of rejection of the second-set grafts. Such “adaptation” of first-set grafts was either diminished or abolished in presensitized mice or in mice given additional donor antigen in the form of splenic cells at the time of first-set grafting. Neither enhancing antibody nor residual leukocytes in the graft were thought to be significant factors in facilitating adaptation. The timing of immunosuppressive treatment with respect to the period of vascularization of the allograft by host endothelium may be an important determinant of adaptation.

OBSERVATIONS ON THE PASSIVE TRANSFER OF TRANSPLANTATION IMMUNITY AND DELAYED HYPERSENSITIVITY WITH LYMPHOID CELLS IN MILLIPORE CHAMBERS.

Experiments were done to determine whether antigen or a transfer factor escaped from millipore chambers containing viable cells with subsequent immunization of the recipient mice. Homologous nonsensitized and isologous sensitized lymphoid cells enclosed in millipore chambers were transferred to recipient mice. After appropriate time intervals, the hosts were tested by skin homografts. There was no evidence that recipients were immunized by an escape from the chambers of homologous cells, of antigen carried over with sensitized cells, or of a transfer factor. In mice given 650 r total-body irradiation, a dose sufficient to prolong the MST of first-set grafts by 6 days, accelerated rejection of test skin hornografts was achieved by lymphoid cells sensitized to homologous tissues and enclosed in millipore chambers. That leakage of cells might occur, but in numbers too small to effect immunization of the host, was indicated by the deaths of 5 of 53 recipients of virulent leukemia cells placed in chambers. In guinea pigs, tuberculin and contact chemical sensitivity could not be transferred by cells sensitized to tubercle bacilli or dinitrofluorobenzene and contained in cell-impenetrable millipore chambers. These data corroborated the conclusion that transplantation immunity on the one hand and tuberculin and contact sensitivity on themore » other hand are mediated by different immunologic mechanisms. (auth)« less

Effect of the graft-versus-host reaction on the immunological responsiveness of the mouse

Graft-versus-host reaction has been induced in unirradiated adult ( C 57 BL x CBA ) F 1 hybrid mice by intravenous injection of parent-line spleen cells, and the capacity of the injected animals to react to first-set and second-set grafts of A -line skin, and to Salm. typhi H antigen, has been investigated. Injection of CBA cells resulted in little or no loss of weight, a low mortality, little or no impairment of the recipient’s capacity to react to A skin or to the bacterial antigen when encountered for the first time, and no loss of pre-existing immunity to A skin. Injection of (40 to 100) x 10 6 C 57 BL cells on the other hand resulted in severe loss of weight and diarrhoea, often culminating in death. Surviving non-immunized animals reacted feebly to the bacterial antigen and some were slower than normal in rejecting first-set grafts of A skin. Pre-existing immunity to A skin was lost except in the special case when the spleen cell donor had itself been immunized against A skin. It is concluded that severe graft-versus-host reaction depresses the host’s immunological reactivity, but that the foreign cells may enable the host to muster a variety of immunological reactions by proxy.

Effect of the graft-versus-host reaction on the immunological responsiveness of the mouse

Graft-versus-host reaction has been induced in unirradiated adult (C57BLxCBA)F1hybrid mice by intravenous injection of parent-line spleen cells, and the capacity of the injected animals to react to first-set and second-set grafts ofA-line skin, and toSalm. typhiH antigen, has been investigated. Injection ofCBAcells resulted in little or no loss of weight, a low mortality, little or no impairment of the recipient’s capacity to react toAskin or to the bacterial antigen when encountered for the first time, and no loss of pre-existing immunity toAskin. Injection of (40 to 100) x 106C57BLcells on the other hand resulted in severe loss of weight and diarrhoea, often culminating in death. Surviving non-immunized animals reacted feebly to the bacterial antigen and some were slower than normal in rejecting first-set grafts ofAskin. Pre-existing immunity toAskin was lost except in the special case when the spleen cell donor had itself been immunized againstAskin. It is concluded that severe graft-versus-host reaction depresses the host’s immunological reactivity, but that the foreign cells may enable the host to muster a variety of immunological reactions by proxy.