The effects of perioperative portal venous inoculation with donor lymphocytes on renal allograft survival in the rat. II. Phenotypic and functional analyses of graft-infiltrating cells

Phenotype, donor-specific cytolytic activity, and helper activity to release cytokines of cells infiltrating within renal allografts of hosts rendered unresponsive by perioperative administration of donor lymphocytes via the portal vein (p.v.) were investigated in order to analyze the mechanism of prolongation of allograft survival. Graft-infiltrating cells (GIC) were obtained from Lewis (LEW, RT-1l) hosts inoculated perioperatively with 1 x 10(8) donor Brown-Norway (BN, RT-1n) lymphocytes p.v., a group that displays prolonged renal allograft survival (MST: 22.2 +/- 5.3 days, n = 10) compared with an uninoculated control group (MST: 7.8 +/- 0.6 days, n = 10, P less than 0.01). The percentages of cytotoxic/suppressor T cells (OX-8+) and Ia-positive cells (OX-6+) in GIC (23.1 +/- 4.4% and 9.0 +/- 2.0%, respectively) and in spleen cells (7.5 +/- 2.6% and 8.5 +/- 1.1%, respectively) from p.v.-inoculated LEW hosts on day 6 postgrafting were significantly lower than those of uninoculated control recipients (GIC: OX-8; 39.4 +/- 8.2%, OX-6; 23.0 +/- 1.9%. SP cell: OX-8; 21.6 +/- 9.9%, OX-6; 12.7 +/- 0.4%, P less than 0.05). Cytolytic activity of GIC from tolerant hosts on day 6 postgrafting toward donor blastoid lymphocytes was significantly decreased (19.0 +/- 1.2% at E/T = 50), compared with that from control allografts during ongoing rejection (51.5 +/- 5.3%, P less than 0.01). The amounts of in vitro cytokine production of GIC from tolerant hosts after mitogen stimulation were remarkably decreased (IL-2: 8.7 +/- 1.4 U/ml, IL-3: 15.4 +/- 0.6 U/ml, and BSF-2: 24.6 +/- 3.5 U/ml) than those of uninoculated control hosts during ongoing rejection (IL-2: 19.6 +/- 2.9 U/ml, IL-3: 22.2 +/- 2.7 U/ml, and BSF-2: 67.5 +/- 13.2 U/ml, P less than 0.05). These results demonstrated that activation of both Tc cells and Th cells was inhibited in the spleen and in situ in renal allografts following administration of donor lymphocytes through the portal vein.     

Pretransplant portal venous administration of donor antigen and portal venous allograft drainage synergistically prolong rat cardiac allograft survival

The effect of antigen given through the portal vein (PV) before transplantation or continuous drainage of a graft into the PV results in moderate prolongation of allograft survival. This study examines these treatment modalities further. Pretransplant donor antigen as 25 x 10(6) ultraviolet B-irradiated (12,000 joules/m2) donor spleen cells was given 7 days before heart transplantation through either the PV or systemic venous (IV) routes. On day 0, Lewis-to-Buffalo rat cardiac allografts were drained either into the PV or IV. Pretransplant PV donor antigen administration (p less than 0.005), but not by IV administration, significantly prolonged cardiac allograft survival across the strong RT 1 rat histoincompatibility barrier. Similarly PV, but not IV, drainage of the graft prolonged graft survival (p less than 0.005). Pretransplant IV antigen administration had no additive effect on PV drainage graft survival. In contrast, when pretransplant PV donor antigen was combined with PV drainage, 11 of 14 allografts (p less than 0.001) continued to function, free of rejection, after 150 days. Therefore for rat cardiac transplants a clearly synergistic graft-prolonging effect results when pretransplant PV donor antigen is combined with PV drainage of the allograts. These data clarify the potent tolerogenic effects of alloantigen not only administered into the PV but also continuously shed intraportally so that it is first processed by the liver.     

Effects of portal venous administration with allogenic cells on renal allograft survival in the rat

The effects of administration of donor lymphocytes via portal vein (PV) on capacity of alloreactivity and renal allograft survival were investigated in comparison with those of intra-venous (IV) administration in the rats. Orthotopic renal transplantations were performed from Brown-Norway (BN, RT-In) to Lewis (LEW, RT-11) male rats. Donor lymphocytes were prepared from BN or third party DA(RT-1a) rat spleens and lymph nodes and injected via PV or IV to LEW rats on the day of transplantation (day 0). Untreated LEW hosts rejected BN grafts at 7.8 +/- 0.6 days (n = 10). IV administration of 1 x 10(8) BN cells to LEW rats caused a slight prolongation of BN graft survival to 10.4 +/- 3.1 days (n = 9, p less than 0.05), whereas PV inoculation of the same number of BN cells further prolonged graft survival to 28.9 +/- 9.2 days (n = 9, p less than 0.01). This effect was antigen specific; the administration of 1 x 10(8) third party DA cells via PV to LEW rats did not prolong survival of BN graft (MST = 7.4 +/- 0.8, n = 6). Serum from tolerant recipients had significant antigen specific suppressor effect (70.6%) on the MLR proliferative reaction of LEW responder cells toward donor BN cells, but not third party DA cells. Spleen cells from these recipients did not show any suppressive effect. These results demonstrate that PV administration of donor lymphoid cells to recipients results in rapidly inducible and long-lasting immunologic tolerance specific to donor alloantigen, and that this tolerance is mediated by serum factor induced in hosts, but not by suppressor cells.     

Factors determining prolongation of rat heart allograft survival by perioperative injection of donor spleen cells

Previously, we have shown that a perioperative injection of donor mononuclear cells in combination with cyclosporine treatment on day 2 after transplantation prolongs heart allograft survival in rats. In this study we determined whether the efficacy of this treatment was influenced by the same factors that have been shown to affect the efficacy of preoperative administration of donor cells. The effect of the following factors were investigated: dosage and repetition of the donor cell injection, viability of the donor cells, immunosuppressive drugs other than cyclosporine, and the rat strain combination. We found that there was an optimal dosage of donor cells; dosages of 4 x 10(7) or 1 x 10(8) cells gave the best heart graft survival. Repetition of the donor cell injection was not useful. Reducing viability of the cells by irradiation did not abrogate the prolonged graft survival, whereas killing of the cells did. Methylprednisolone, azathioprine, or cyclophosphamide in combination with the perioperative donor cell injection did not prolong heart graft survival in comparison with treatment with the drug only. The efficacy of this treatment was also influenced by the rat strain combination. In some combinations, this treatment prolonged graft survival, whereas in others an effect was absent or undetectable. Importantly, this treatment never adversely affected graft survival. We conclude that the efficacy of this treatment is influenced by similar factors as found for preoperative treatment with donor cells. A major advantage of this treatment over preoperative blood transfusions is that it avoids sensitization.     

Intravenous and portal venous administration of modified donor antigen prolongs rat parathyroid allograft survival

The effect of pretransplant (day -6) systemic intravenous or portal venous immunization with modified donor antigen combined with cyclophosphamide treatment (75 mg/kg on day -4) on rat parathyroid allograft survival was evaluated. Systemic intravenous preimmunization of Buffalo recipients with 10(8) untreated Lewis donor spleen cells plus cyclophosphamide resulted in 100% accelerated rejection of Lewis parathyroid allografts (mean survival time, 7.3 +/- 0.9 days vs 10.8 +/- 1.1 days for controls). Portal venous administration of untreated cells plus cyclophosphamide reduced accelerated rejection to 40% but could not prolong graft survival (10.8 +/- 2.7 days). Intravenous or portal venous preimmunization with heat-inactivated cells (45 degrees C for 60 minutes) plus cyclophosphamide also did not prolong graft survival, with accelerated rejection occurring in 20% and 40% of recipients, respectively. In contrast, preimmunization by either route with ultraviolet B-irradiated cells (UVB; 12,000 joule/m2) plus cyclophosphamide significantly prolonged graft survival (intravenous = 22.2 +/- 6.0 days and portal venous = 21.4 +/- 7.2 days; p less than 0.005), with no accelerated rejection. Preimmunization with UVB cells combined with cyclophosphamide was synergistic, because neither treatment alone prolonged allograft survival (UVB cell preimmunization only = 10.8 +/- 1.3 days; cyclophosphamide only = 12.6 +/- 2.6 days). The effect of UVB preimmunization was donor specific because third-party Wistar-Furth UVB cells had no effect on Lewis graft survival (12.5 +/- 2.9 days). We conclude that pretreatment with UVB-modified donor antigen plus cyclophosphamide induces allospecific immune hyporesponsiveness and prolongs parathyroid allograft survival.     

Extensive prolongation of rat renal allograft survival following donor or nonspecific transfusions and concomitant immunosuppressant

We report here a marked beneficial effect upon rat renal allograft survival transplanted across a strong histocompatibility barrier (BN----LEW) by pretransplant concomitant donor-strain blood transfusion (DST) and CsA treatment. Comparisons between recipient groups treated with pretransplant nonspecific blood (NST) and concomitant cyclosporine (CsA) or azathioprine (Aza) administration were also made. LEW recipients receiving only a BN renal allograft survived for a geometric average time of 8.9 days. Recipients receiving 1 ml of donor blood at weekly intervals, each week for three weeks prior to transplantation, demonstrated a geometric mean survival time (GMST) of 40.5 days. Recipients receiving this same regimen and concurrent CsA cover (5 mg/kg/day) starting 7 days prior to the first transfusion with discontinuation 5 days prior to transplantation showed extensive prolongation (greater than 100 days). Recipients treated with only CsA cover survived for a GMST of 34.4 days. LEW recipients receiving 1 ml of nonspecific blood at weekly intervals (DA, BUF, WKY, respectively) each week for 3 weeks prior to transplantation were prolonged to 27.7 days. Recipients treated with this same regimen while under CsA cover also demonstrated extended prolongation (greater than 100 days). Recipients receiving multiple donor blood transfusions under Aza (2 mg/kg/day) cover demonstrated lesser prolongation (22.8 days). Recipients receiving the multiple nonspecific blood protocol under Aza cover showed similar prolongation (38.6 days). Recipients treated only with Aza did not show prolonged survival (9.3 days). These differences in survival were considered significant among the 9 transplant groups as determined by ANOVA (P less than 0.001). The majority of recipient groups showed relatively poor renal function over their life spans, independent of whether prolongation occurred. Yet, renal function in the NST or particularly the DST groups covered by pretransplant CsA, demonstrated the best renal function in our laboratory over many years of investigations using the BN----LEW combination. In conclusion, there was a dramatic synergistic beneficial effect of prior multiple DST or NST specific to CsA, as opposed to another immunopharmacologic agent, Aza.     

The effects of perioperative administration of donor lymphocytes via portal vein in rat skin transplant model]

The effects of perioperative portal venous (P.V.) administration of donor lymphocytes on skin allograft survival were investigated in rat skin transplant model. Heterotopic skin transplantations were performed form Brown-Norway (BN, RT-1n) to Lewis (LEW, RT-1(1] male rats. P.V. administration of donor BN lymphocytes (1 x 10(8] resulted in significant prolongation of BN skin graft survival (MST = 13.4 +/- 3.9 days, p less than 0.05) compared with I.V. administration of same number of donor lymphocytes (8.6 +/- 1.2 days) or with PV administration of third party DA (RT-1a) rat's lymphocytes (7.4 +/- 0.8 days) or with untreated controls (9.0 +/- 1.4 days). These results suggested that this effect was antigen specific. P.V. administration of donor lymphocytes prevented recipient which received BN skin graft form developing delayed-type hypersensitivity responses to donor antigen. Serum from LEW recipients which induced unresponsiveness by PV administration with donor BN lymphocytes had significant antigen specific suppressor effect (77.0 +/- 5%) on the MLR proliferative reaction of LEW responder cells toward donor BN cells, but not third party DA stimulation. Moreover, this immunological unresponsiveness was transferable by the serum in kidney transplant model. These results indicate that PV administration of donor lymphocytes induces recipient's unresponsiveness to donor alloantigen in rat skin transplant model, and this effect is transferable by the suppressor factor in the serum.     

The combined effect of perioperative donor spleen cells or KCl-extracted antigen and cyclosporine on renal allograft survival in the rat

The effects of using perioperative cyclosporine in conjunction with pretreatment with donor spleen cells or 3M KCl solubilized extracts of donor antigen were investigated in a LEW-to-DA rat renal allograft model. Cyclosporine given orally in a dose of 10 mg/kg/day around the time of transplantation (days -1, 0, +1), did not prolong renal allograft survival (median survival time [MST]--10 days). However when used in combination with pretreatment with either 10(8) donor spleen cells (1 day before transplantation), or 10(5) donor spleen cells (7 days before transplantation), pretreatment regimens that were in themselves ineffective, DA recipients accepted Lewis renal allografts indefinitely (MST greater than 100 days). Soluble antigen was prepared by 3M KCl extraction from donor spleen cells. Absorption assays were used to quantify the amount of class I major histocompatibility complex antigen in the preparation, and amounts of antigen equivalent to that expressed by 10(6)-10(8) donor spleen cells were used for pretreatment. These soluble antigen preparations given either 1 or 7 days before transplantation with or without perioperative cyclosporine did not prolong allograft survival of either homozygous or heterozygous donors (MST 10 days).     

Requirement for both MHC and non-MHC antigens residing on the same erythrocyte for donor erythrocyte-mediated prolongation of rat renal allograft survival

Transfusions of highly purified LEW erythrocytes (E) administered to BN recipients prior to insertion of LEW kidneys markedly prolonged the survival of these allografts (greater than 35 days). Administration of E from syngeneic (BN), third-party (PVG), and MHC-congenic LEW.1N or BN.1L rats did not improve LEW kidney graft survival to the same extent (less than 14 days). BN.1L E were shown to carry at least the same quantity of LEW MHC antigens on their surface as LEW E, thus the failure to prolong LEW kidney graft survival is due to the absence of LEW non-MHC antigens from BN.1L E. Attempts to substitute for this deficiency by mixing LEW.1N E to BN.1L E prior to transfusion failed to restore the beneficial effect, demonstrating that donor E-mediated prolonged renal allograft survival requires the presence of both MHC and non-MHC alloantigens on the same E.     

The effect of transfusions and donor pretreatment on canine renal allograft survival

Allograft survival is influenced by a complexity of known and unknown factors. The effect of several types of transfusion on renal allograft survival has been studied in mongrel dogs. Nontreated and drug-pretreated kidneys were transplanted. Preoperative and peroperative cell suspensions of unrelated dogs were transfused to graft recipients. All recipients received low-dose postoperative immunosuppression. One peroperative transfusion of 100 ml third-party blood enhanced graft survival of nontreated kidneys significantly. A transfusion of 100 ml blood 14 days before transplantation led to variable results, whereas a transfusion of cells obtained from a third party spleen, given 14 days before transplantation, did not prolong graft survival of nontreated kidneys at all. The administration of procarbazine hydrochloride and methylprednisolone to the donor before procuring the graft increased survival of canine renal allografts significantly. A combination of donor drug pretreatment and 100-ml peroperative third-party blood led to graft survival comparable to that obtained by either treatment. Pretreatment of the recipient with spleen cells 14 days before transplantation even abrogated the effect of donor drug pretreatment completely. These results show that preoperative transfusions cannot successfully be combined with donor drug pretreatment as commonly practiced in man. This is a possible explanation for the conflicting data on donor pretreatment in man. Furthermore this study gives evidence for the effectiveness of a peroperative blood transfusion on kidney graft survival.     

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.     

The immunosuppressive action of suppressor cells from antigen-cyclosporine-treated hosts on renal allograft survival

Systemic adoptive transfer was employed to assess the immunosuppressive efficacy of antigen-specific suppressor T (Ts) cells purified from recipients treated with 3M KCl-extracted donor histocompatibility antigen (Ag) and cyclosporine (CsA). Suppressor cells were obtained from Wistar-Furth (WFu, RT-1u) hosts treated with a single i.v. injection of 5 mg 3M KCl-extracted donor Buffalo (Buf, RT-1b) antigen combined with a three-day course of CsA, a group that displays prolonged renal allograft survival (MST 23.2 +/- 10.2 days) compared with animals treated with CsA alone (MST 12.2 +/- 2.4 days). These noncytolytic, OX-8 phenotype, 800-rad-resistant/1500-rad-sensitive, nylon-wool-nonadherent and cyclophosphamide-sensitive suppressor T cells (1 X 10(6)) were adoptively transferred ten days after transplantation into virgin, secondary syngeneic hosts-thereby prolonging Buf graft survival from 7.2 to 17.5 days. The suppressor effect was immunologically specific; adoptive transfer did not prolong the survival of third-party Brown-Norway (BN) grafts (MST 10.4 +/- 3.1 days) compared with the nontreated control group (MST 11.0 +/- 2.9 days). The potency of Ts cells purified from Ag-CsA-treated hosts to transfer unresponsiveness into normal secondary WFu hosts (MST 17.5 +/- 8.0 days) was stronger than that of Ts cells from hosts treated with CsA only (MST 10.6 +/- 2.6 days). Moreover, in vitro stimulation of monoclonal-antibody-purified Ts cells by irradiated donor Buf spleen cells potentiated the in vivo induced suppressor activity, leading to an MST of 38.1 +/- 32.6 days; indeed 3 of 12 animals (25%) displayed permanent unresponsiveness. Furthermore, Ts cells from Ag-CsA-treated hosts displayed a synergistic effect with a three-day course of CsA administration into the secondary hosts (MST 24.2 +/- 8.0 days) compared with animals only treated with CsA (MST 12.2 +/- 2.4 days, P less than 0.001). Moreover, the combination of the Ag-CsA regimen with Ts cells administered one day after transplantation caused even greater prolongation of graft survival (MST 34.2 +/- 14.2 days) compared with Ag-CsA-treated hosts (MST 23.2 +/- 10.2 days, P less than 0.025). Thus adoptively transferred antigen-specific suppressor T cells may be explored to intensify the specific immunosuppressive effect of the Ag-CsA regimen to achieve long-term unresponsiveness.     

Prolongation of renal allograft survival in the rat treated with amniotic fluid.

To assess the role of amniotic fluid (AMF) in the maintenance of pregnancy, immunosuppressive effects of AMF were studied in vivo, and the mechanisms of suppressor activity were analyzed immunologically in vitro in the rat. Female Lewis (LEW, RT-1l) rats mated with Brown-Norway (BN, RT-1n) rats for 14 days were sacrificed and cell-free AMF was obtained. AMF was diafiltered with PBS (PH 7.2) and reconstituted to 2 OD units measured at 280 nm. Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.2 days (n = 10). Five days of intravenous inoculation of AMF into LEW hosts remarkably enhanced BN graft survivals (MST = 20.3 +/- 4.4 days, n = 12) compared with controls (P less than 0.01), and slightly prolonged third-party DA (RT-1a) graft survivals (MST = 9.4 +/- 0.8 days, n = 7) compared with control LEW hosts engrafted with a DA kidney (MST = 7.6 +/- 0.2 days, n = 6). Five days of intravenous inoculation of pregnant sera into LEW hosts had no effect on BN graft survival. The AMF suppressed the proliferative response of LEW lymphocytes against not only irradiated BN stimulator cells but also irradiated third-party DA stimulators. The AMF also suppressed allokiller T cell generation of normal LEW lymphocytes against BN cells by 70.1% and 51.3%, and against DA cells by 64.9% and 38.9% at concentrations of 25% and 12.5%, respectively (P less than 0.01). To dissect the immunosuppressive activity of AMF, the effect of AMF on cytokine production and interleukin 2 (IL-2) receptor expression of concanavalin A-stimulated lymphocytes were investigated. AMF suppressed interferon and IL-2 production. Interestingly, however, AMF did not suppress interleukin 3 (IL-3) and interleukin 6 (IL-6) production, as well as IL-2 receptor expression. These results demonstrated that rat AMF displayed a strong immunosuppression in vivo as well as in vitro, and that AMF might play an important role in the maintenance of pregnancy.     

Lung allograft rejection in the rat. II. Specific immunological properties of lung grafts

The immunological mechanism of lung allograft rejection was studied in inbred rats, in order to explain the rapid progress of the rejection response against RT1-incompatible lung grafts. Histological appearances of the graft and of the recipient's spleen were studied, migration patterns of graft and recipient lymphocytes were assessed, and titers of circulating alloantibodies were determined. Histologically, we discriminated four phases of the rejection response in lung grafts: sequentially the latent, vascular, alveolar, and destruction phases. Early in the vascular phase, recipient lymphocytes primarily infiltrated the bronchus-associated lymphoid tissue (BALT) of the graft, causing a local immune response. Concurrent with these local rejection phenomena in the graft, a strong systemic immune response developed in the recipient's spleen, presumably induced by the great number of lymphocytes that migrated from the graft's BALT into the recipient's lymphoid tissues. We conclude that BALT facilitates a fast and intensive interaction between lung graft and recipient that is likely to accelerate the induction of the rejection response both locally in the graft and systemically in the recipient's lymphoid organs.