Immunohistologic profile of cells infiltrating acutely rejecting and long-surviving rat cardiac allografts.
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Biomedical subjects
D Araneda
Publications and source records attributed to D Araneda.
Two phenotypically distinct populations of T cells have suppressor capabilities simultaneously in the maintenance phase of immunologic enhancement.
The events leading to immunologic enhancement in LEW rats immunized actively with Brown Norway (BN) rat spleen cells and passively with LEW anti-BN hyperimmune serum 11 and 10 days before receiving (LEW X BN)F1 cardiac allografts, respectively, have been studied. Cellular suppressor mechanisms developing during the induction phase of this phenomenon have recently been shown to be mediated by W3/25+ T cells in an antigen-specific manner. The present study suggests that the late maintenance phase of immunologic enhancement is mediated in vivo by simultaneously present separate donor-specific T cell subpopulations of W3/25+ and OX8+ phenotypes. Splenocyte subsets from grafted recipients greater than 100 days after transplantation were adoptively transferred into unmodified syngeneic LEW rats that received a specific test allograft 24 hr later, or into B recipients bearing indefinitely surviving heart grafts. Test graft survival was prolonged significantly in the first group and not altered in the second. Indeed, nonoverlapping W3/25+ and OX8+ cell fractions were separately responsible for suppression. However, when suppressor activity was tested in vitro in a three-component coculture mixed lymphocyte reaction, no suppression by T cells was obtained; this lack of correlation between in vivo and in vitro results has also been noted by other investigators in different systems. Thus, in the maintenance phase of actively and passively induced immunologic enhancement, interplay between two phenotypically distinct T cells with suppressor characteristics, but not putative cell-surface blocking factors, seems to prevent development of an alloreactive response and mediate host unresponsiveness.
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Selective immunosuppression with anti-interleukin 2 receptor-targeted therapy: helper and suppressor cell activity in rat recipients of cardiac allografts.
(LEW X BN)F1 cardiac allografts are rejected within 8 days in unmodified LEW rats. ART18, a mouse anti-rat IgG1 monoclonal antibody which binds specifically in vitro to the interleukin 2 receptor (IL 2R) molecule expressed primarily on activated T cells, prolongs allograft survival in a dose-dependent fashion to ca. 3 weeks (p less than 0.001) after being administered for 10 days after transplantation. This effect was related to the specificity of the antibody for IL 2R, as therapy with ART62 (a monoclonal antibody recognizing MHC class I antigen but not binding the rat IL 2R) was ineffectual. Suppressor activity was detected in spleen cells of ART18-treated grafted hosts: in vivo, splenic T suppressor/cytotoxic fraction adoptively transferred into normal LEW improved donor-specific but not third-party test graft survival (17 days, vs. 8 days, respectively, p less than 0.001); in vitro, mixed lymphocyte reaction was profoundly but nonspecifically inhibited (less than 5% of test mixed lymphocyte reaction, p less than 0.001 as compared to acutely rejecting controls). In contrast, splenic T helper (Th) cells from ART18-treated hosts were functionally depressed, as noted by their passive transfer into immunologically anergic B recipients of cardiac allografts (rejection in ca. 40 days, vs. ca. 13 days after transfer of Th from specifically sensitized rats). ART18 treatment also resulted in diminished elaboration of IL 2 as compared to normal (p less than 0.005) or acutely rejecting hosts (p less than 0.001); however, a remarkable increase in the production of IL 3 occurred (p less than 0.001). These results demonstrate that IL 2R-targeted therapy of immunocompetent graft recipients produces a selective immune defect in which donor-specific T suppressor cells are spared, but Th cells attenuated or destroyed. Decreased elaboration of IL2 concomitantly augments the release of IL 3, a lymphokine which might play a role in suppressor effect in vivo. In addition, IL 2R-targeted therapy of the immunodeficient graft recipients abrogates the capacity of alloactivated T cells to re-establish acute immune responsiveness.
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Synergy between cyclosporine and other immunosuppressive regimens in experimental organ transplantation.
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Development of suppressor lymphocytes during acute rejection of rat cardiac allografts and preservation of suppression by anti-IL-2-receptor monoclonal antibody.
Suppressor activity was investigated in rats undergoing acute rejection of heterotopic cardiac allografts. Spleen cells were harvested at 7 days from LEW rats rejecting (LEW x BN)F1 heart grafts and fractionated into their T, T suppressor/cytotoxic, and T helper subpopulations. Transfer of alloimmune unseparated spleen cells to syngeneic recipients of (Lew x BN)F1 test grafts accelerated rejection from 8 to 6.5 days (P less than 0.01). Graft survival was prolonged to about 15 days (P less than 0.005) after transfer of the splenic T suppressor/cytotoxic fraction. Treatment of test graft recipients with ART-18, a mouse antirat monoclonal antibody directed against the rat interleukin 2 receptor on the surface of activated lymphocytes, increased graft survival to about 3 weeks (P less than 0.005), and to about 23 days (P less than 0.005) when test graft recipients were treated with ART 18 following transfer of alloimmune unseparated spleen cells. In contrast, ART-18 treatment of test graft recipients already injected with T suppressor/cytotoxic cells had no additive effect. Increased production of endogenous interleukin 2 occurred concomitantly with the onset of rejection in these animals; interleukin 2 release declined during the late stages of rejection when suppressor activity had increased. Similarly, in T-cell-depleted (B) rats, allograft rejection could be produced by immune reconstitution with sensitized lymphocytes, but could be significantly delayed by prior transfer of suppressor cells. These data document the presence of potent suppressor activity in the acutely rejecting host and suggest that the suppressor mechanisms are inhibited less than effector mechanisms by interleukin-2-receptor-targeted therapy.
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Cyclosporine and experimental skin allografts: long-term survival in rats treated with low maintenance doses.
Although cyclosporine (CsA) is a powerful immunosuppressive agent in organ transplantation, its efficacy in skin transplantation has not been examined completely. We have tested it as primary immunosuppression in a rat skin allograft model. Histoincompatible Brown-Norway skin grafts are rejected in untreated Lewis hosts within 9 +/- 1 days but survive for 22 +/- 3, 34 +/- 2, or 41 +/- 8 days after 7, 14, or 21 days of CsA treatment (15 mg/kg per day subcutaneously), respectively (p less than 0.001). Animals treated daily for 4 weeks died from drug toxicity; however, an initial 2-week course followed by a low maintenance dose (15 mg/kg every fourth day) produced indefinite (greater than 150 days) graft acceptance without side effects. The long-surviving grafts were supple, grew long hair, and showed normal histology. When the drug was stopped at any time during this maintenance period, early signs of rejection (hair loss, epidermal breakdown, and localized ulceration) occurred, which could be reversed completely by a short CsA "pulse" (15 mg/kg per day for 7 days). These experimental data support the potential application of CsA immunosuppression in human skin allotransplantation.
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Behavior of helper T lymphocytes in cyclosporine-mediated long-term graft acceptance in the rat.
(LEW X BN)F1 cardiac allografts are rejected acutely (7 days) in unmodified LEW rats, yet survive indefinitely following cyclosporine (CsA) treatment (15 mg/kg im for 7 days) or in T-cell-deprived (B) recipients. Using these models, the function of T helper cells (Th) in the maintenance phase of CsA-mediated long-term graft survival was examined. With monoclonal antibody immunoaffinity fractionation techniques, Th (W3/25+OX8-) were separated from spleens of CsA-treated hosts 3-4 weeks after grafting (CsA-Th), from specifically sensitized (s-Th), or from normal ungrafted (n-Th) rats. Adoptive transfer of 60 X 10(6) CsA-Th into B recipients produced rejection of donor-specific, but not third-party grafts in 21 +/- 7 days, comparable to s-Th (17 +/- 4 days), but faster than n-Th (4-5 weeks, P less than 0.025). CsA-Th recombined with T cytotoxic/suppressor phenotype (CsA-Tc/s, OX8+W3/25-) in numbers contained in 100 X 10(6) CsA-T cells were ineffectual, even when supplemented with exogenous interleukin 2-rich conditioned medium (IL-2CM); in contrast 100 X 10(6) s-T cells + IL-2CM inevitably caused acute rejection in B hosts (11 +/- 3 days). Increasing numbers of Th incrementally to 100 X 10(6) augmented the effectiveness of s-Th (rejection in 13 +/- 2 days), but did not improve potency of CsA-Th (20 +/- 2 days). Suppressor activity produced by small numbers of contaminating CsA-Tc/s (c. 0.4%, 4-5 X 10(5) cells in 100 X 10(6) CsA-Th) accounted for extended graft survival in B recipients, as this small number of CsA-Tc/s transferred into untreated syngeneic rats increased test graft survival to c. 16 days (P less than 0.001). IL-2 production by spleen cells, depressed during CsA treatment, returned to normal levels 2-3 weeks following drug withdrawal, whereas transfer of CsA-Th into B recipients induced a shift of IL-2 levels from dramatically depressed to normal, findings suggesting normal IL-2 production by CsA-Th. This report demonstrates that an unresponsive state in CsA-treated animals is achieved despite the presence of fully potent donor-specific Th. Active suppressor activity plays a critical role in the maintenance phase of graft survival in rats treated transiently with CsA.
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T suppressor lymphocytes reverse ongoing acute allograft rejection.
(LEW X BN)F1 cardiac allografts are rejected within 8 days in untreated LEW recipients. At the critical time point of 5 days after transplantation, the obviously rejecting grafts are enlarged and maximally infiltrated by host cells as shown by 111In-labeled lymphocyte tracer studies. However, when such hearts were retransplanted back to naive (LEW X BN)F1 secondary hosts, they survive indefinitely, showing that even late rejection is reversible in the absence of sustained host immunological drive. Attempts were then made to abrogate this advanced immune responsiveness using Cyclosporine (CsA). CsA therapy (15 mg/kg/day for 7 days) starting from day 5 produced indefinite graft survival, similar as if initiated at the time of operation. Addition of exogenous IL-2, which drives the proliferation of Tc, could not reverse this effect. Serial changes in phenotype of lymphocyte subpopulations infiltrating both acutely rejecting and indefinitely functioning cardiac allografts in unmodified and CsA treated hosts, respectively, were then studied. Ratio of Th:Tc/s cells in acutely rejecting grafts was 1.6 by day 3; it inverted abruptly to 0.7 by day 5-6, suggesting predominance of Tc/s during the later stages of allograft rejection. Similarly, treatment with CsA produced a transient depression of Th, with recovery of original Th:Tc/s ratio during the next 2-3 weeks. Adoptive transfer experiments were then performed to investigate the functional significance of these findings.(ABSTRACT TRUNCATED AT 250 WORDS)
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Phenotype, activation status, and suppressor activity of host lymphocytes during acute rejection and after cyclosporine-induced unresponsiveness of rat cardiac allografts.
Serial changes in phenotype, cell cycle, and functional behavior of lymphocyte subpopulations occurring both during acute rejection in unmodified hosts and in long-surviving heterotopic cardiac allografts in rats treated with cyclosporine (CsA) were studied. Using flow cytometry, RNA and DNA content of cells was examined during various phases of cell activation. In animals acutely rejecting their grafts, numbers of cells infiltrating the grafts and in host spleen in G1 phase (higher RNA content) increased, starting from day 3, and peaked by 5-6 days posttransplantation, and numbers of cells in S/G2/M phase (higher DNA content) remained stable. Similar, although slightly delayed changes were noted in CsA-treated recipients. The ratio of T helper (Th) to T suppressor/cytotoxic (Ts/c) phenotype cells infiltrating acutely rejecting grafts by day 3, was 1.6; it inverted abruptly to 0.7 by days 5-6, suggesting a preponderance of Ts/c during the later stages of allograft rejection. Ratio inversion occurred slightly later in host spleen and later in peripheral blood. Similarly, treatment with CsA produced a transient depression of Th, with recovery of the Th: Ts/c ratio during weeks 3-4 following transplantation. Adoptive transfer studies were then performed to investigate the functional significance of the T cell subsets. Survival of test grafts was prolonged significantly (ca. 14 days, P less than 0.001) when cells infiltrating grafts and spleen were transferred during inversion of Th:Ts/c; before that period, test graft survival was shortened in a second-set manner. These experiments suggest that suppressor cells may be responsible for resolution of acute rejection, as well as for host unresponsiveness seen after CsA treatment, and they represent an important homeostatic host mechanism following immunological stimulation.
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Cyclosporine and experimental skin allografts. II. Indefinite survival and development of specific immunologic unresponsiveness.
Immunological unresponsiveness toward skin allografts was studied in cyclosporine (CsA)-treated rats. BN skin grafts survive about 22 days and about 34 days in LEW hosts following 7 or 14 days of daily CsA treatment (15 mg/kg/day), respectively; in unmodified hosts grafts are rejected by 9 days. Indefinite (greater than 100 days) survival can, however, be produced by administering maintenance 15 mg/kg CsA every fourth day, following an initial course of the agent for 14 days. Early signs of graft rejection (hair loss, localized epidermal breakdown, and ulcerations) occurring in some animals were reversed by a CsA "pulse" (15 mg/kg/day) for 7 days, reduced gradually to the maintenance dose. CsA was equally effective when started as late as 4 days after grafting, but ineffectual when started after day 4. Once BN grafts were rejected, the agent could not prevent second-set rejection of donor-specific grafts, but significantly prolonged the survival of third-party (WF) skins. Survival of original BN grafts was unchanged by the placement of second BN grafts during both the inductive and maintenance phases; these second grafts survived as long as the original grafts. In contrast, secondary third-party (WF) grafts were promptly rejected; their destruction did not influence survival of the original grafts. Thus, indefinite survival of rat skin allografts is feasible with low maintenance doses of CsA. Graft rejection at later stages can be reversed by resuming daily therapy. Host unresponsiveness is stable and specific both during the early inductive and later maintenance phases.
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Host-graft relationship: the systemic nature of allograft rejection.
The interdependence between immunologic events occurring within acutely rejecting rat cardiac allografts and those in host lymphoid tissues were studied. To evaluate cellular dynamics of allograft infiltration, 111In-labeled thoracic duct lymphocytes from Lewis rats were administered intravenously daily (0 to 7 days after transplantation) to (Lewis X BN)F1 heart-grafted unmodified Lewis rats, sacrificed 24 hours later. Accumulation of thoracic duct lymphocytes in the allografts peaked 4 to 5 days after transplantation. To evaluate whether these changes at the graft site were sufficient to carry on the rejection response in the absence of a sustained host immunologic drive, acutely rejecting (Lewis X BN)F1 cardiac allografts were retransplanted serially at days 1 through 5 into normal syngeneic animals. All these regrafts survived greater than 100 days. Neither infusion of interleukin-2-conditioned medium (100 IU for 7 days intravenously) into regrafted hosts nor preoperative perfusion of the retransplanted hearts with interleukin-2-conditioned medium (300 IU) could complete the rejection process. Using flow cytometry analysis, we then assessed the phenotypic alterations of the mononuclear cells infiltrating the graft. The ratio of T helper: T cytotoxic/suppressor cells, which at day 3 was 1.57, inverted abruptly to 0.67 by days 5 to 6. After retransplantation a dramatic depression in T-lymphocyte subsets occurred, particularly affecting the T cytotoxic/suppressor phenotype. Trafficking studies revealed that the T cells that left the regrafts migrated mainly to spleen and mesenteric lymph nodes and away from bone marrow and peripheral blood of the syngeneic secondary recipients. Finally, histologic manifestations of acute rejection at days 4 to 5 virtually reversed themselves after regrafting. These studies emphasize the systemic nature of the rejection cascade, which depends fully on the host lymphoid system; even late changes in the graft microenvironment are not sufficient to produce final immunologic destruction.
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Lack of evidence for an active role for natural killer cells in acute rejection of organ allografts.
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Restoration of allograft responsiveness in B rats. IV. The divergent migratory behavior of lymphocyte populations mediating cardiac allograft rejection.
The T lymphocyte-deprived (B) rat, produced by X-radiation and bone marrow reconstitution of adolescent thymectomized animals, exhibits a true immunological deficit and are unable to reject histoincompatible heterotopic cardiac allografts. A comprehensive survey of lymphocyte traffic in B recipients was performed to correlate the differential potency of specifically sensitized lymphocyte populations mediating re-establishment of immune responsiveness toward the graft, with their migratory and recirculatory behavior. 111In-oxine-labeled thoracic duct lymphocytes (TDL) were retained in the peripheral blood and migrated from nonlymphoid organs to lymph nodes of B recipients in higher proportion than any other lymphoid population, particularly splenic lymphocytes (SL). Although all cell groups but TDL were sequestered in the spleen in equal and relatively large numbers, no differences were found between the lymphocyte populations tested in their capacity to accumulate in the grafts. In contrast, an increased avidity in the allograft of 125IUdR-labeled TDL and lymph node (LNL) lymphoblasts, as compared to 125IUdR-labeled SL, resembles closely the results of functional studies of the differential potency of adoptively transferred cells. We assume that specific cellular interactions induced by the accumulated 125IUdR-labeled cells invoke nonspecific mechanisms for the recruitment of other uncommitted 111In-labeled lymphocytes which recirculate between blood and lymph and localize indiscriminately in the allograft amplifying its rejection. The latter lymphocytes can be "armed" by adherent cells residing in the lymphoid organs of graft recipients, particularly spleen, and subsequently increase the penetration of the foreign tissue. When radiolabeled lymphocytes were traced in B recipients experiencing rejection of their allografts following transfer of sensitized cells plus lymphokine, their migration patterns as well as blastogenic response in B hosts were similar to those observed during acute rejection of cardiac allografts in unmodified hosts. Thus the similarities between the rejection network brought by alloimmune cells into otherwise unresponsive animals and immunocompetent animals able to reject their grafts are stressed.
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Interactions between T lymphocyte subsets supported by interleukin 2-rich lymphokines produce acute rejection of vascularized cardiac allografts in T cell deprived rats.
A series of adoptive transfer studies were performed to define both the role of lymphocyte subpopulations and of lymphokines in the reestablishment of immunologic responsiveness towards vascularized organ allografts in T cell-deprived B rats. Acute rejection of otherwise indefinitely surviving cardiac allografts (10.9 +/- 2.9 days) occurred when sensitized T cells were adoptively transferred with a course of partially purified, lectin-free IL 2-conditioned medium (IL 2CM), whereas rats receiving T cells alone rejected their grafts at 19.8 +/- 4.6 days (p = 0.025). Rejection is mediated primarily by alloactivated T helper cells highly enriched for W3/25 phenotype. The relationship between the mean allograft survival time and the cell number transferred was of exponential order. In contrast to rejection occurring after the transfer of intact T cells, that produced by W3/25+ cells was independent of the administration of exogenous IL 2CM. W3/25+ T helper cells were also found to be less potent in mediating graft rejection than were whole T cells when limited cell numbers were transferred. The OX8+ T cytotoxic/suppressor cell subpopulation, even when supplied with a course of IL 2CM, was unable to bring about rejection, but induced transient graft enlargement in about 50% of the animals. Recombination of individual subsets caused acute rejection at a rate comparable to that of unseparated T cells when IL 2CM was supplied (10.8 +/- 1.0 days), in contrast to those rats which received the recombined inoculum in the absence of IL 2CM (16.8 +/- 2.2 days, p = 0.025). Lymphocyte migration studies revealed a more vigorous homing of OX8+ cells to the allograft as compared to W3/25+ cells. However, to produce maximal accumulation of transferred cells in the graft, both T cell subsets supplemented by lymphokines must be introduced together into T cell-deprived hosts.
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Restoration of allograft responsiveness in B rats. II. Requirements for lymphoid populations and lymphokine.
B rats, produced by lethal x-irradiation of thymectomized animals that are then reconstituted with bone marrow cells from syngeneic, thymectomized, and thoracic-duct-drained donors, are unable to reject histoin-compatible heterotopic cardiac allografts. In these studies, we have continued to investigate the role of immune competent cells and lymphokine in restoring acute allograft rejection in these animals, noting that adoptive transfer of 10(8) sensitized splenocytes (sSL) plus Interleukin-2-rich conditioned medium, IL-2 (CM) will reproducibly produce acute responsiveness toward long-standing, well-functioning, heart grafts. We have now noted that sensitized lymph node cells or thoracic duct lymphocytes can also reverse the unresponsive state, with IL-2 rich conditioned supernatants increasing the effectiveness of small numbers of cells from these populations in inducing allograft rejection. Furthermore, the major cellular components within the spleen have been separated using antibody techniques, and their individual role in the rejection reaction has been assessed. Although 10(8) sSL plus IL-2 (CM) can produce acute rejection in a time comparable to that occurring in unmodified recipients (n = 20, MST +/- SD = 8.4 +/- 1.3 days), neither the T cell fraction (6 x 10(7] of 10(8) SL + IL-2 (CM) (n = 10, MST +/- SD = 17.4 +/- 7.3 days), nor addition of the adherent cell fraction to the T cell component (MST +/- SD = 18 +/- 2 days) or addition of the B cell fraction (MST +/- SD = 15.5 +/- 2.1 days) could restore acute responsiveness. However, increasing the numbers of T cells to 10(8) and transferring concomitantly with IL-2 (CM) again produced acute rejection (n = 4, MST +/- SD = 9 +/- 1.1 days). These data suggest that T cells form the essential element of transfer in the B rat, although other cells present in SL appear necessary to provide an optimal milieu. Our experiments also suggest that this response is independent of the route of administration or of higher doses of IL-2. Finally, using a dual allograft model, it appears that graft destruction is determined by the specific sensitivity of transferred SL; IL-2 (CM) being a nonspecific factor in the response.
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Migration patterns of lymphocytes in untreated and immunologically manipulated recipients of organ allografts.
We have investigated the migration patterns of normal LEW rat splenic lymphocytes (SLs) radiolabeled in vitro with [2-3H]adenosine and adoptively transferred i.v. into LEW hosts bearing LBNF1 heterotopic cardiac allografts. Twenty-four hours after cell transfer, the animals were killed and the radioactivity of all lymphoid and nonlymphoid tissues measured in a beta counter. The following experimental groups were studied: group 1, untreated recipients acutely rejecting their grafts at 7 days after transplantation; group 2, actively and passively enhanced recipients bearing long-term surviving grafts, at 7, 14 to 18, and 25 days; group 3, cyclosporin A-treated recipients; and group 4, B rats, each bearing indefinitely surviving grafts, at 7 and 20 to 30 days. In group 1, 28% of recoverable activity was found in spleen and 22% in mesenteric and peripheral lymph nodes. In animals with well functioning grafts of groups 2 and 3, SLs accumulated in both organs equally (25 to 27%). In animals in group 4, SLs migrated primarily to lymph nodes (30%) and away from spleen (20%). Sequestration in nonlymphoid tissues of animals experiencing graft rejection was higher than those with prolonged or indefinitely surviving hearts. By using mouse anti-rat monoclonal antibodies, the quantitative relationship between T cell subpopulations in transferred cell suspensions and in lymphoid organs of grafted hosts was also assessed. Lymphocyte migration patterns are influenced dramatically by the immunological status of recipients of vascularized organ allografts.
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