Differential effects of interleukin 2 receptor-targeted therapy on heart and kidney allografts in rats. Depression of effectiveness of ART-18 monoclonal antibody treatment by uremia

ART-18, a mouse antirat IL-2R mAb inhibits IL-2 binding and IL-2-dependent T cell growth. Although both (LEW x BN)F1 kidney and heart allografts survive ca. 3 weeks in ART-18-treated LEW rats (acute rejection occurs within 10 days, P less than 0.001), the host responses against the two organs vary. In the heart model, the splenic CD4:CD8 ratio as determined by FMF was similar both in untreated and treated animals, but decreased significantly in kidney recipients conditioned with ART-18. In both mAb-modulated animal groups, splenocytes inhibited test MLR and prolonged test cardiac allograft survival in a donor-specific fashion upon adoptive transfer, suggesting that ART-18 mediates "sparing" of Ts. However, both CD4+ and CD8+ cells from kidney-grafted hosts conferred suppression in vivo; only the CD8+ subset was effective in the heart model. Immunohistologically, IL-2R+ cells were absent in the heart grafts of treated hosts; a significant proportion of the kidney cell infiltrate remained IL-2R+ despite continuous mAb administration. Although ART-18 therapy prolonged renal graft survival significantly, function was poor and the rats remained uremic. However, when one of the native kidneys was retained and the rat continued to enjoy normal renal function, IL-2R+ cells were abolished from the graft infiltrate, as shown by FMF and immunohistology. Thus, ART-18 treatment influences host responses differentially against kidney and heart allografts (modulation and depletion of IL-2R+ cells, respectively) despite increasing their survival comparably. The uremic state in the kidney model prevents elimination of infiltrating IL-2R+ mononuclear cells by a mAb directed specifically against them.     

Evidence that therapeutic strategies targeted at CD4+ cells modulate accelerated rejection of cardiac allografts in sensitized rats by different mechanisms.

(LEW x BN)F1 cardiac allografts are rejected within 36 hr in LEW rats presensitized with BN skin grafts 7 days earlier (acute rejection occurs within 8 days). We have previously described the effects of individual CD4 (BWH-4), CD25 (IL-2R, ART-18) mAbs, and CsA therapeutic regimens upon cardiac allograft survival in sensitized hosts. The present studies were designed to probe an adjunctive use of ART-18 or CsA upon BWH-4-mediated suppression of accelerated graft injury. Sequential therapy with BWH-4 and ART-18 in the sensitization phase (days -7 to -1) and effector phase (from day 0, the day of cardiac transplant), respectively, prolonged graft survival additively to c. 22 days. Treatment with BWH-4 markedly diminished host humoral response against ART-18 preparation. BWH-4 given in concert with subtherapeutic dose of CsA produced graft survival comparable to that induced by mAb alone (c. 13 days) with concomitant decreased host anti-BWH-4 response. None of the combined regimens affected the frequency of circulating CD4+ cells, as compared with that exerted by BWH-4 monotherapy. Thus this study defines principles and some mechanistic aspects of optimal immunosuppressive strategies potentiating the effects of CD4-targeted therapy.     

Agonistic and antagonistic interactions of anti-interleukin 2 receptor monoclonal antibodies in rat recipients of cardiac allografts

A panel of five mouse mAbs recognizing 4 distinct epitopes (R1-4) of the rat 55kD IL-2R molecule were tested for their influence on acute rejection (8 days) of (LEWxBN)F1 cardiac allografts in LEW hosts. IL-2R1 targeted therapy with ART-18 (IgG1, inhibits IL-2-dependent responses) prolonged graft survival to ca.21 days. IL-2R2 is recognized by ART-65 and ART-75, mAbs that do not inhibit T cell growth. Treatment with ART-65 (IgG1) but not ART-75 (IgG2a) abrogated acute rejection (ca. 16 days and 9 days, respectively). ART-35, an anti-IL-2R3 mAb (IgG1, does not inhibit T cell function) extended graft survival marginally to ca. 12 days. Finally, therapy with OX-39, (anti-R4 IgG1 mAb, inhibits IL-2 binding, but not IL-2-driven growth) was completely ineffectual. Simultaneous targeting of two IL-2R epitopes increased the therapeutic index synergistically (ART-18 [R1] + ART-65 [R2]--60% permanent graft acceptance), additively (ART-75 [R1] + ART-35 [R3]--graft survival ca. 18 days), or did not improve further graft survival at all (ART-18 [R1] + OX-39 [R4]--graft survival ca. 18 days). Thus, the cellular targeting patterns and isotype of mAbs are crucial: (1) targeting at functionally distinct epitopes controls rejection most effectively; (2) IgG1 and IgG2b mAbs are more influential in vivo than IgG2a, data supported by the studies employing the family of ART-18 isotype switch variants. Treatment with anti-IL-2R mAb did not depress Ts activity as tested both in vitro and in vivo. Sparing of putative Ts by mAb is also shown by thymectomy of graft recipients before or during ART-18 therapy, which shortened graft survival to 13-15 days; thymectomy after ART-18 therapy did not influence graft survival. However, infusion of IFN-gamma recreated classic acute rejection within 10 days in otherwise longstanding cardiac allografts in ART-18 treated hosts. Upregulation of MHC class II antigen and IL-2R expression seems to be primarily responsible for this striking biological effect of IFN-gamma in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)     

Modulation of accelerated rejection of cardiac allografts in sensitized rats by anti-interleukin 2 receptor monoclonal antibody and cyclosporine therapy.

LBNF1 cardiac allografts (Tx) are rejected within 36 hr in LEW rats sensitized with BN skin Tx 7 days earlier, compared to 8-day rejection in unmodified hosts. Treatment with cyclosporine or ART-18, an anti-interleukin 2 receptor (IL-2[R]) mAb monoclonal antibody, abrogates accelerated rejection and prolongs mean Tx survival to 42 days and 16 days, respectively. ART-18 given in concert with subtherapeutic dose of CsA extends survival to 25 days. These studies were designed to dissect the early mechanisms leading to ART-18 and/or CsA-mediated abrogation of accelerated Tx injury. No effect of concomitant mAb administration upon CsA through levels was noted in Tx recipients conditioned with both modalities. The beneficial effect of ART-18+CsA treatment was also unrelated to CsA-induced diminished host responses to mAb, as shown by ELISA. In the biodistribution studies 125I-labeled ART-18 accumulated preferentially into host lymphoid tissues and Tx itself and away from the blood. In animals that were concomitantly given "cold" CsA, the clearance of labeled ART-18 from the blood increased further, as did mAb sequestration into the Tx. The sensitized hosts developed high titers of complement-dependent cytotoxic (CDC) antibodies, which peaked at the time of actual Tx loss. ART-18 or CsA alone inhibited CDC, whereas combined therapy decreased further the humoral effects of sensitization. The cell-mediated lymphocytotoxicity assay revealed a similar pattern. CsA, ART-18, and combination therapy each modulated the deposition of IgG, IgM, and C3 in Tx, as shown by immunohistology. However, only CsA or ART-18+CsA therapy abolished or markedly decreased elaboration of IL-2, interferon gamma, and tissue necrosis factor alpha. In conclusion: (1) the adjunctive low dose of CsA potentiates the inhibitory effects of ART-18 upon humoral and cellular responses, leading to accelerated rejection of cardiac Tx in presensitized rats; (2) the synergistic interaction between both modalities that results in the inhibition of lymphokine production is critical and correlates with long-term Tx survival; (3) the biodistribution patterns of mAb are important--an increased blood level of mAb does not necessarily translate into its higher therapeutic efficacy.     

Persistence of stable intragraft cell chimerism in rat liver allografts after drug-induced tolerance.

BACKGROUND: Drug-induced tolerance of rat liver allografts is well documented. We analyzed cellular events during immunosuppressive therapy on day (d) 10 and in the late phase (d 100) after transplantation to assess for characteristics in the intrahepatic leukocyte (IHL) population in the phase of tolerance. METHODS: Lewis rats served as recipients of Dark Agouti rat livers. Temporary immunosuppression with either cyclosporine (CsA) monotherapy (3 mg/kg/d) or triple therapy that consisted of a subtherapeutic CsA dosage (0.25 mg/kg/d) and monoclonal antibodies directed against the interleukin-2 receptor (IL-2R, CD25) and the intercellular adhesion molecule-1 (ICAM-1, CD54) was administered from postoperative d 0 to d 13. Cell migration and cell activation within liver grafts was assessed by standard histology and flow cytometry. IHL apoptosis was detected by terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling (TUNEL). RESULTS: Both CsA monotherapy and triple therapy prolonged liver allograft survival to more than 100 d and led to the induction of donor-specific tolerance. Untreated recipients rejected their allografts within 14 d. In both groups, donor-specific IHLs initially dropped to 18% to 25% on d 10, but they rebounded to as much as 40% on d 100 as a common characteristic of both groups. Within this population, donor-specific T cells were dominant. In both groups, increased numbers of activated (IL-2R+) CD8+ T lymphocytes were present on d 100. No accumulation of apoptotic IHL was observed on d 100. Their proportion was unchanged in the triple therapy group and slightly decreased in the CsA group compared to the syngeneic controls. CONCLUSIONS: The present study reveals that tolerant liver allografts are repopulated by donor-specific T lymphocytes. This phenomenon is independent of the type of applied immunosuppression. The persistence of activated CD8+ T cells in the phase of proven donor-specific tolerance on d 100 indicates that liver tolerance is associated with the state of a permanent intragraft immune activation. It seems that the coexistence of donor cells with infiltrating recipient cells within liver grafts, termed intrahepatic cell chimerism, is characteristic for tolerated liver allografts.     

Evidence that monoclonal antibodies against the 55kD subunit of the rat IL-2 receptor do not inhibit the development of suppressor cells generated in mixed lymphocyte culture

Although the ability of Ts to prevent allograft rejection has been well established, their intrinsic characteristics and dependence upon lymphokines remain poorly defined. The cells from unmodified LEWxBN bulk 5-day rat MLR inhibit both proliferation in test MLR and generation of CTL, as well as prolonging the survival of donor-specific test cardiac allografts following adoptive transfer. We have examined the effects of a panel of mAb directed against functionally distinct epitopes on the p55 subunit of rat IL-2R on the generation and in vitro/in vivo activity of MLR-generated Ts. ART-18 (which blocks IL-2-dependent T cell growth) was the only mAb from the panel that profoundly suppressed alloreactive T cell proliferation in primary MLR (47.5%). However, the generation of Ts was never affected by any mAb (% suppression in test MLR = 40-60%). Neither ART-18 nor ART-65 (which does not affect T cell proliferation) interfered with the efficacy of Ts to inhibit CTL generation in fresh bulk MLR. Adoptive transfer of cells (3-10 x 10(6] from ART-18 or ART-65-modulated MLR into naive LEW rats prolonged (LEW x BN)F1 test cardiac allograft survival to 11-13 days (P less than 0.05 as compared with acutely rejecting hosts). All in vitro and in vivo effects exerted by MLR-generated cells were antigen-specific. In unmodified MLR, Ts were IL-2R+ (ca. 50% of total blasts), as shown by cell separation using magnetic beads. In contrast, in MLR with ART-18 added, Ts were primarily IL-2R- (ca. 10% of blasts). Thus, antirat p55 subunit IL-2R mAb do not inhibit MLR-generated Ts functionally operative in vitro and in vivo. IL-2R- Ts precursors requiring lymphokine(s) other than IL-2 may differentiate into IL-2-dependent Ts effectors. Such divergent IL-2 requirements for Ts growth in vitro may explain the Ts-sparing effects in allograft recipients treated with anti-IL-2R mAb.     

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.     

Cyclosporine and anti-interleukin 2 receptor monoclonal antibody therapy suppress accelerated rejection of rat cardiac allografts through different effector mechanisms

Increasing numbers of sensitized patients are either precluded from receiving an allograft or experience accelerated rejection which may be refractory to conventional therapy. Using a rat model, we have shown that accelerated (24 hr) rejection of LBN cardiac Tx in LEW rats sensitized with BN skin grafts 7 days earlier, could be prevented by treatment with cyclosporine (15 mg/kg/day x7 days, Tx survival about 42 days) or ART-18, an anti-IL-2R mAb (300 micrograms/kg/day x10 days i.v., Tx survival about 16 days). In this study, we evaluated intragraft mechanisms responsible for these effects by immunoperoxidase localization of relevant humoral mediators (IgG, IgM, C3, cross-linked fibrin), graft infiltrating cells (GIC), and associated cytokines (IL-2, IFN-g, tumor necrosis factor [TNF], or cytokine receptors (IL-2R). Tx rejected in fulminating fashion by 24 hr in sensitized hosts showed extensive and progressive endothelial deposition of IgG, C3, and fibrin from 2 hr, followed by an influx of neutrophils at 3 hr, and peak numbers of GIC by 18 hr (88.8 +/- 20.3 leukocytes/field). At 18 hr, GIC consisted of neutrophils (26%), T cells (20%, greater than 90% of which were OX-8+), and monocytes/macrophages (53%), whereas B cells were absent. By 18 hr, up to 20% of GIC were IFN-g+, 10% were IL-2R+, and 10% were IL-2+, consistent with labeling of 20% of cells with OX-22. Widespread endothelial and mononuclear cell labeling for TNF and the procoagulant molecular tissue factor (TF) were also noted. In contrast to untreated grafts, CsA treatment essentially abolished intragraft Ig, C3, and fibrin deposition. Moreover, despite dense cell infiltration at 24 hr (total GIC 55.3 +/- 13.4/field), analysis of CsA-treated Tx showed markedly decreased neutrophils (0.5%), with increased T cells (35%) and similar proportions of macrophages (66%). In addition to the reduction in neutrophils, Ig and C3, fewer IL-2R+ (6%) and OX-22+ (3%) cells, considerably less TNF and TF, and almost no IL-2+ or IFN-g+ GIC (less than 1%) were detected. Surprisingly, ART-18 treatment also greatly decreased but did not abolish endothelial deposition of C3, IgG, or IgM, whereas widespread endothelial and mononuclear labeling for fibrin, TNF, and TF remained. In addition, GIC (about 54.8 +/- 16.1/field) contained only moderately reduced numbers of neutrophils (31%) and the proportions of T cells (27%) and macrophages (49%) were generally comparable to those of rejecting Tx in untreated rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

环孢素对共刺激阻断剂延长移植肾存活效应的影响

目的:探讨环孢素(CsA)对共刺激阻断剂CTLA4Ig延长移植肾存活效应的影响。方法:肾移植大鼠分为对照组(第1组)、CTLA4Ig组(第2组)、CsA CTLA4Ig组(第3组)、CTLA4Ig IL-2组(第4组)和CsA CTLA4Ig IL-2组(第5组),观察术后血肌酐(Scr)、移植肾病理改变、移植肾存活时间。结果:与第1组、第4组相比,第2组、第3组、第5组移植肾存活时间显著延长(P<0.01),其中,第3组移植肾存活时间最长(66.1±10.6)d;术后15天,第2组Scr显著低于第3组、第5组(P<0.05);术后30天,第3组、第5组Scr显著低于第2组(P<0.01);术后30天,第3组、第5组移植肾淋巴细胞浸润明显少于第2组。结论:CsA可增强CTLA4Ig延长移植肾存活的效应,对外源性IL-2逆转CTLA4Ig的效应具有抵抗作用。     

Synergy between cyclosporine and anti-IL-2 receptor monoclonal antibodies in rats. Functional studies of heart and kidney allografts

Although cyclosporine has improved results of organ transplantation, treatment regimens using multiple agents are being evaluated both experimentally and clinically in attempts to diminish its often profound nephrotoxicity; some therapies act synergistically by differential inhibition of distinct steps of the rejection cascade. The effects on graft function of a full dose or a subclinical dose of CsA, ART-18, a monoclonal antibody (mAb) directed against the IL-2 receptor expressed on activated host cells, and a combination of low-dose CsA and ART-18, have been tested in rat recipients of both heart and kidney allografts. Renal graft function was assessed by several classic techniques; heart function by isolated perfusion methods. Full-dose CsA and combination treatment were most effective in both organ graft systems, with at least one-third of grafts surviving indefinitely. At seven days after transplantation, glomerular filtration rates and renal plasma flow of all grafted recipients were decreased as compared with normal; at 14 days, function in the best treatment groups had improved toward that of isografts. Similarly, cardiac output and stroke work index of best treatment groups were comparable to that of isografts. These functional studies complement previously reported immunological and immunohistological findings stressing that synergy occurs between subclinical doses of CsA and anti-IL-2-R mAb in two rat organ graft systems.     

Immunohistological observations of rat fetal pancreas allografts transplanted into unmodified and cyclosporine-treated recipients

Administration of CsA (15 mg/kg/day) prolonged the survival of DA (RT1a) rat fetal pancreas transplanted to the renal subcapular site of both PVG (RT1c) and Lewis (RT1(1] recipients. Sections of fetal pancreas examined 40 days after transplantation into allogeneic CsA-treated recipients showed growth and development of the fetal pancreas tissue, and the presence of numerous insulin-containing islets. CsA treatment prevented the induction of MHC antigen within allografts. Whereas at day 4, both rejecting and CsA treated grafts showed donor class I MHC expression on duct epithelium and islet cells, only rejecting grafts displayed class I MHC induction on acinar cells. Rejecting grafts showed strong induction of class II MHC antigen expression on duct epithelium from day 4 onward but this was completely prevented by CsA treatment. Islet cells in both rejecting and CsA treated allografts remained class II-negative throughout. CsA also resulted in a reduction in the day 6 cellular infiltrate of allografts (median area leukocyte infiltrate reduced from 43% to 10%) with a marked decrease in the number of MRC OX-8-positive cells. These results show a favorable effect of CsA on rat fetal pancreas allografts with a reduction in MHC antigen expression within the graft and prolonged survival of insulin-rich endocrine tissue.     

1alpha,25-dihydroxyvitamin D3 shows strong and additive immunomodulatory effects with cyclosporine A in rat renal allotransplants.

BACKGROUND: Vitamin D3 and its metabolites have long been found to exert immunosuppressive effects both in vivo and in vitro. The present study investigated the effect of 1alpha,25-dihydroxycholecalciferol (1,25DHC) on vascularized renal allografts in rats. METHODS: Three days prior to transplantation, two groups of animals were subjected to 1,25DHC (1 microg/kg/day IP) and a low calcium diet, which was continued until the end of the experiments. Recipient organs were removed and single allografts were transplanted in a high responder strain combination (ACI --> Lewis). Following transplantation, low-dose cyclosporine A (3.2 mg/kg/day CsA) administration was started in two experimental groups of recipients (one group receiving 1,25 DHC additionally) whereas the control allograft recipients received no immunosuppression (control III). Graft survival and renal function was monitored until death or the end of experiments and allograft rejection was assessed histologically using the Banff classification. RESULTS: 1,25DHC significantly prolonged allograft survival in comparison to control III (9.6 +/- 1 vs. 5.7 +/- 0.2 days; P=0.009). In addition, a combination of 1,25DHC and low-dose CsA increased allograft survival compared to CsA administration alone (24 +/- 0.9 vs. 13 +/- 0.3 days; P=0.008). 1,25DHC preserved renal creatinine clearance and decreased proteinuria in comparison to control III, and the combination of 1,25DHC and low-dose CsA again showed an additive effect on preservation of renal function. 1,25DHC and low-dose CsA both decreased interleukin (IL)-2 and IL-12 expression levels in serum and allografts, and a combination treatment produced the strongest attenuation of IL-2 and IL-12 expression. In addition, 1,25DHC increased IL-4 and IL-10 expression levels in allografts, whereas CsA alone did not alter IL-4 and IL-10 expression. In contrast, combination of 1,25DHC and low-dose CsA showed a significant increase in IL-10 expression levels whereas IL-4 expression was not elevated. CONCLUSION: Monotherapy with 1,25DHC significantly prolongs survival of renal allografts and preserves graft function in rats. A combination of 1,25DHC and CsA caused an additive effect on graft survival with differential regulation of pro- and anti-inflammatory cytokines, as compared to 1,25DHC administration alone.     

T suppressor cells induced by transfusions and cyclosporine. Studies in the murine cardiac allograft model

Pregraft transfusion combined with immunosuppression at the time of grafting improves the survival of clinical and experimental allografts. The mechanisms responsible for this effect were investigated in the murine model of cardiac transplantation, combining transfusions 7 to 30 days prior to transplantation with cyclosporine 100 mg/kg, 7 to 20 days pregraft or on days 0, 4, and 6 after grafting. Pregraft DST, third-party blood, and CsA all improved graft survival in the BALB/c-to-CBA donor-recipient combination. In animals treated with DST at 14 days pregrafting, 4/9 grafts survived for greater than 100 days. In those given C57BL/6 blood, or CsA on days 0, 4, 6 postgraft, 1/9 grafts survived for greater than 100 days. When 10(7) spleen cells from DST-treated CBA mice with long-surviving BALB/c heart grafts were transferred to naive CBA mice that then received a BALB/c heart 24 hr later, the transferred cells prolonged graft survival, with all grafts functioning at greater than 40 days, and 4/7 at greater than 100 days. Selective removal of T cells from the spleen cell population prior to transfer showed that L3T4+ T cells, but not Ly-2+ T cells, were required to maintain BALB/c allografts. Combining a short course of CsA with DST was more effective than either treatment alone. The most effective combined treatment was DST at day -14 with 100 mg/kg CsA given on days 0, 4, and 6 postgrafting (8/10 grafts survived greater than 100 days). This treatment also induced splenic suppressor T cells of the L3T4+ Ly-2- phenotype. These results clearly show that L3T4+ splenic T suppressor cells are induced by donor-specific blood transfusion with or without CsA treatment, and that these cells play a role in maintaining long-term tolerance to allografts in the mouse heart transplant model.     

Indefinite survival of rat parathyroid allografts without postoperative immunosuppression

Methods that avoid long-term immunosuppression must be developed for human parathyroid allotransplantation to be feasible. Pretransplant treatment of the graft to eliminate passenger cells is one such method. An alternative approach is short-term treatment of the recipients with cyclosporine (CsA). In this study, parathyroid glands from Lewis X Brown Norway rats were cultured for 1 week and treated with antiserum directed against class II major histocompatibility complex antigens. Treated glands were transplanted into hypocalcemic Wistar-Furth recipients that previously received 30 mg/kg of CsA once a day for 3 days before transplantation. At 280 days after transplantation, 67% of the recipients had functional parathyroid allografts. Control rats (no CsA; fresh, untreated glands) rejected these grafts within 28 days. Control rats given 3 days of CsA and transplanted with fresh, untreated glands had functional grafts for greater than 56 days (median survival, 80.5 days). Prolongation of allograft survival with short-term, preoperative CsA demonstrates the efficacy of immunosuppression given at the time of antigen presentation. This course of CsA is even more effective when the recipient receives a graft whose passenger cells are eliminated.     

Prolongation of survival of fully allogeneic cardiac grafts and generation of regulatory cells by a histamine receptor 2 antagonist

BACKGROUND: The effects of histamine on immunologic responses via the histamine receptor 2 (HR2) have been studied, but few investigations explored the immunomodulatory role of histamine in vivo. We examined whether the HR2 antagonist ranitidine affects the alloimmune response in a murine model of cardiac transplantation. METHODS: CBA (H-2k) recipients were given no treatment or one intravenous injection of ranitidine on the day of transplantation of a heart from C57BL/10 (H-2b) donors. Survival of the allografts was recorded. The effect of the ranitidine treatment on cell proliferation and cytokine production was assessed by mixed leukocyte culture and enzyme-linked immunosorbent assays. An adoptive transfer study was conducted to determine whether regulatory cells were generated. The effect on graft survival of adding FK506 to the ranitidine treatment was also examined. RESULTS: CBA recipients given ranitidine (60 mg/kg) had prolonged graft survival (median survival time [MST], 87 days). Ranitidine treatment also suppressed the proliferation of splenocytes and production of interleukin (IL)-2 and up-regulated IL-10 production. Adoptive transfer of splenocytes and CD4 cells from ranitidine-treated allograft recipients induced significant prolongation of allograft survival in naive secondary recipients (MST, 71 and >100 days, respectively). CBA recipients given both ranitidine and FK506 (0.1 mg/kg/day for 14 days) had indefinite survival of cardiac allografts (MST, >100 days). CBA recipients treated with FK506 alone rejected the allografts (MST, 27 days). CONCLUSION: In our model, ranitidine treatment induced significantly prolonged survival of fully allogeneic cardiac grafts, generated CD4 regulatory cells, and indefinite survival when combined with FK506 (0.1 mg/kg/day).     

Induction of donor-specific unresponsiveness to rat cardiac allograft by donor leukocytes and cyclosporine

Many recent reports have emphasized the importance of donor antigens in the induction of allograft tolerance. This study examines the effect of pretransplant infusion of 10(8) donor leukocytes (DL) combined with peritransplant cyclosporine (CsA) on W/F cardiac allograft survival in Lewis rats. Peritransplant recipient treatment consisted of CsA 20 mg/kg given i.m. on days 0, +1, and +2 relative to heart transplantation. Lewis recipients, 5-8 per group, were pretreated with 10(8) DL with or without peritransplant CsA. A single DL transfusion on day -3 or day -7 prior to transplantation significantly prolonged the mean survival time (MST) of W/F hearts from 7.0 +/- 0.9 days in controls to 12.2 +/- 4.5 days and 12.4 +/- 1.0 days (P less than 0.01), respectively. Two DL infusions on days -7 and -3 or on days -14 and -7 prolonged the MST to 10.6 +/- 1.3 days (P less than 0.02) and 16.4 +/- 2.8 days (P less than 0.001), respectively. The administration of peritransplant CsA alone significantly prolonged W/F heart allograft survival to 43.1 +/- 2.7 days. When pretransplant DL transfusion on day -3 was combined with CsA treatment, 4/8 animals maintained their grafts indefinitely (greater than 100 days). Similarly, DL infusion on day -7 with peritransplant CsA led to indefinite graft survival in 3/5 animals. Administration of DL on days -7 and -3 combined with CsA resulted in indefinite graft survival (greater than 100 days) in 4/6 animals. Transfusion of DL on day -3 alone or in combination with peritransplant CsA, had no effect on a third-party (ACI) heart allograft survival prolongation compared with appropriate controls. To define the underlying mechanisms responsible for donor-specific unresponsiveness in this model, pooled sera and unseparated spleen cells were passively transferred from recipients of long-term cardiac allografts to syngeneic rats receiving donor-type (W/F) or third-party (ACI) cardiac allografts. Transfer of serum (1 ml on days 0, and 1, 0.5 ml on days +2, +3, and +4) from ungrafted recipients of DL on days -14 and -7 led to significant donor graft survival of 9.8 +/- 0.4 days (P less than 0.02) in unmodified hosts. Similarly, passive transfer of serum obtained at 20 and 100 days after transplantation significantly prolonged the MST of donor-type hearts in syngeneic untreated hosts to 11.3 +/- 0.8 and 10.0 +/- 1.1 days, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)