The effects of perioperative portal venous inoculation with donor lymphocytes on renal allograft survival in the rat. I. Specific prolongation of donor grafts and suppressor factor in the serum

In order to investigate the in vivo functional role of the liver in the immune responses in organ transplantation, effects of perioperative portal venous p.v. administration of donor lymphocytes on renal allograft survival were tested in the rat kidney transplant model. Donor lymphocytes were prepared from BN (BN, RT-1n) or third-party DA (RT1a) rat spleens and lymph nodes and injected p.v. or intravenously to Lewis (LEW, RT-1l) hosts on the day of transplantation (day 0). Untreated LEW hosts rejected BN renal grafts at 7.8 +/- 0.6 days (n = 10). Intravenous administration of 1 x 10(8) BN cells to LEW hosts on day 0 caused a slight, but not significant, prolongation of renal allograft survival (MST = 9.5 +/- 3.0 days, n = 13, NS), whereas portal venous inoculation of 1 x 10(8) BN cells on day 0 remarkably prolonged renal graft survival to 22.2 +/- 5.3 (n = 10, P less than 0.01). The prolongation of graft survival was antigen-specific; the administration of 1 x 10(8) DA cells p.v. to LEW hosts did not prolong the survival of BN renal grafts (MST = 7.4 +/- 0.8, n = 5). Spleen cells from p.v. treated LEW hosts 10 days after transplantation had no suppressor effect on the one-way MLC reaction of normal LEW responder cells toward donor BN or third-party DA stimulators. On the other hand, when serum from p.v.-treated LEW hosts was added to MLC at a concentration of 3 per cent of total volume, it suppressed the MLC reaction toward donor BN cells by 71.6 per cent, but not toward third-party DA stimulators (-8.5 per cent suppression, NS). Histological examination of p.v.-treated LEW hosts at 10 days after transplantation revealed that the liver had normal lobular architecture without expansion of portal tracts and infiltration of inflammatory cells. On the other hand, the transplanted kidney demonstrated a moderate mononuclear cell infiltration around the artery without an interstitial hemorrhage. Moreover, adoptive transfer of the serum from p.v.-treated LEW rats into the virgin secondary LEW hosts significantly prolonged the graft survival of BN kidneys from 7.8 days to 18.9 +/- 5.5 days (P less than 0.01), but not third-party DA graft survivals (MST = 7.5 +/- 0.6 days), indicating that an antigen-specific tolerogenic factor was released into the circulation through the process of allogeneic cells in the liver.     

The use of spleen transplantation and cyclosporine treatment to induce unresponsiveness to pancreatic islet transplantation in rats

One way of achieving permanent survival of allografted islets in the rat has been to first transplant a kidney of the same strain as the islets and induce acceptance of the kidney using cyclosporin A treatment. Rats bearing long-surviving renal allografts will then accept islets of the same strain without further immunosuppression. Such an approach may be effective for combined renal and islet grafting, but transplantation of the kidney as well as islets may not always be desirable, and the question arises as to whether another organ could be used to achieve the same effect. Auxiliary spleen transplants were performed from LEW to DA rats, followed by cyclosporine 10 mg/kg for 14 days. Twelve rats with long-surviving grafts were then made diabetic with streptozotocin and given LEW islets placed under the kidney capsule, 6 rats being given a further course of cyclosporine 10 mg/kg for 7 days after islet transplantation. Rats that did not reject their islets then underwent removal of the spleen transplant, and if this did not result in rejection islet function was proven by removal of the kidney bearing the islets. The results show that spleen allograft rejection can be prevented by cyclosporine treatment and that 50% of animals with long-surviving spleen allografts will accept subsequent islet allografts from rats of the same donor strain. The acceptance of islet allografts can be increased to 100% by a further short course of cyclosporine treatment. It is concluded that spleen allografts can be used to produce unresponsiveness to islets in rats.     

Differential response of kidney and pancreas rejection to cyclosporine immunosuppression.

Immunological interferences between kidney and pancreas transplants were investigated in a genetically defined rat model of combined kidney and pancreas transplantation. Kidney and whole-pancreas grafts were transplanted microsurgically either as individual grafts or in a combined technique. Whole pancreas grafts were grafted into streptozotocin diabetic recipients (55 mg/kg bodyweight i.v.) three days after induction of diabetes. The exocrine secretion was suppressed by duct ligation. Rejection of the grafts was defined by recurrence of diabetes in pancreas-grafted recipients and renal failure after kidney transplantation. There were marked differences in the efficacy of identical short-term cyclosporine immunosuppression (15 mg/kg intramuscularly for 14 days): DA kidneys survived indefinitely in LEW rats (MST greater than 100 days), while DA pancreas allografts underwent prolonged but not permanent survival (P less than 0.01) either as individual grafts (MST 27.3 +/- 1,9 days) or when transplanted simultaneously together with the kidney (44 +/- 16 days) (P less than 0.01). LEW rats carrying a DA kidney for 100 days also rejected a subsequent donor-specific pancreas transplant within 30 days. The histological alterations in the kidney were more pronounced than after cyclosporine-induced DA kidney long-term survival alone. By contrast to the rejecting subsequently transferred pancreas, a metachronous second DA kidney was permanently accepted (greater than 100 days) without further immunosuppression after removal of the first graft, while unrelated LEW. 1U kidneys were acutely rejected. In summary, the results indicate that there are not only quantitative differences of kidney and pancreas allograft survival but also differences concerning the state of immunological unresponsiveness induced by identical cyclosporine immunosuppression. While CsA induces donor-specific immunological unresponsiveness after kidney transplantation, pancreas transplants are all eventually rejected after some differential prolongation of survival. Further investigations on the effects of different MHC and minor alloantigens may provide more insight into the complex immunological situation of individual and combined kidney and pancreas transplantation.     

The role of T suppressor cells in the maintenance of spontaneously accepted orthotopic rat liver allografts.

Orthotopic liver allografts from BN donors to LEW recipients are spontaneously accepted, and the recipients develop donor-specific immunological unresponsiveness. This unresponsiveness may be mediated by suppressor T cells. Immunomagnetically purified splenic T cells from LEW rats bearing BN liver grafts were shown to adoptively transfer suppression of skin, heart, and kidney graft rejection in a donor-specific manner, prolonging the survival of BN but not third-party DA grafts. However, the suppressor T cells were sessile, being resident in the spleen but not present in thoracic duct lymph. The presence of a nonrecirculating suppressor T cell in rats spontaneously accepting liver transplants is strongly suggestive of an important function in the maintenance of donor-specific unresponsiveness, although the contribution of other possible mechanisms of unresponsiveness has not been investigated.     

Direct evidence of a functional separation of alloreactive T lymphocytes from bystander cells infiltrating rat allografts. Interleukin 2 receptor-positive cells reacting with the monoclonal antibody ART 18 mediating second-set rejection

In this study we examined the functional capacity of unseparated, IL-2R positive and IL-2R negative leukocytes infiltrating BN rat hearts or kidneys grafted into allogeneic LEW rats. Upon adoptive transfer into syngeneic LEW recipients, splenocytes or day-3 graft infiltrate cells of either cardiac or renal transplants were ineffective to alter BN cardiac test graft survival (controls 7.8 +/- 0.8 day). However, adoptive transfer of day-5 heart infiltrate cells resulted in a delay of test graft rejection (9.4 +/- 0.7 day, P less than 0.001), while day-5 kidney-graft-infiltrating cells produced second set rejection (6.2 +/- 0.5, P less than 0.001). Specificity controls of day-5 cells infiltrating DA heart or kidney grafts rejected at 7.8 +/- 0.8 or 7.7 +/- 0.5 days. Following separation into IL-2R positive and negative subpopulations by use of the mAB ART 18, IL-2R positive but not IL-2R negative cells caused second set rejection in both the renal and the cardiac model (6.2 +/- 0.4, respectively, 6.3 +/- 0.5 days, P less than 0.001 or P less than 0.005). Furthermore, in the kidney model IL-2R positive nylon-wool nonadherent cells also caused second set rejection (6.2 +/- 0.4, P less than 0.005) suggesting that IL-2R positive T cells present in the graft at maximal infiltration are the mediators of rejection. Thus, it appears that these cells can be phenotypically and functionally separated from bystander cells.     

Induction of transplantation tolerance in rats by spleen allografts. II. Evidence that W3/25+ T suppressor/inducer and OX8+ T suppressor/effector cells are required to mediate specific unresponsiveness

The results presented in this report demonstrate that T cells, isolated from AGUS rats bearing long-term (WAG X AGUS)F1 spleen allografts adoptively transferred to irradiated AGUS recipients could not mediate the rejection of WAG hearts but rejected PVG. A hearts in acute fashion. Further, unresponsive T cells were able to suppress the capacity of adoptively transferred (40 X 10(6) normal T cells to reject WAG but not PVG.A heart allografts. We also studied the role of W3/25+ and OX8+ T cells subsets in the maintenance of unresponsiveness. Isolated W3/25+ or OX8+ unresponsive T cells were not able to mediate acute rejection, but were less effective in prolonging WAG allograft survival than the unresponsive whole T cell population, suggesting that both W3/25+ Ts1 and OX8+ Ts2 subsets were required for effective suppression in vivo. When, however, unresponsive W3/25+ T cells were infused simultaneously with normal OX8+ T cells, they could produce indefinite survival of WAG heart allografts. These results indicate that the unresponsive state induced by (WAG X AGUS)F1 spleen allografts transplanted to AGUS rats is maintained by the interaction of W3/25+ T suppressor/inducer and OX8+ T suppressor/effector cells.     

Splenectomy and renal allograft survival in the rat

The effect of splenectomy on renal allograft survival is not clear. In the rat, spleens isolated from recipients with functioning grafts have been shown to be a major source of cells that are capable of suppressing the rejection response (suppressor T lymphocytes). Thus the removal of the spleen in these allograft recipients could be detrimental to renal allograft survival. This study investigates this hypothesis, and looks for the presence of suppressor cells in other lymphoid organs apart from the spleen. In the rat renal allograft model, donor Lewis spleen cells given to DA recipients intravenously 1 week before transplantation of a Lewis kidney leads to indefinite allograft survival (median survival time (MST) greater than 100 days). Splenectomy before or after pretreatment with donor spleen cells failed to abrogate this effect (MST greater than 100 days). Experiments were performed in which cells or serum were prepared from long-term surviving splenectomized animals which had already been pretreated and transplanted, and then were injected into untreated recipients (adoptive transfer experiments). This was done to determine if cells capable of suppressing graft rejection were present in lymphoid organs outside the spleen in these splenectomized recipients. Thus the IV transfer of 10(8) lymph node cells harvested from splenectomized DA recipients with a long-term surviving LEW graft (LTS), into untreated but lightly irradiated (200 rad) DA recipients resulted in indefinite survival of a fresh Lewis kidney (MST greater than 100 days). In contrast, adoptive transfer of normal DA lymph node cells was ineffective (MST 13 days). Thus splenectomy is not necessarily detrimental to graft survival, as cells capable of preventing graft rejection are found in other lymphoid organs, such as lymph nodes, in splenectomized recipients.     

Role of antibody in rat renal allograft rejection. II. Failure to enhance renal allografts by sensitization to donor class I antigens presented by donor strain erythrocytes

BN rats were immunized with one or three doses of 1 X 10(8) highly purified LEW erythrocytes (LEW-E) yielding IgM antibody (IgM-BN rats) and IgG antibody (IgG-BN rats) to LEW class I antigens, respectively. LEW kidneys transplanted into IgM-BN rats elicited cytotoxic T cell responses and lymphocytotoxic antibody responses comparable to those elicited by LEW renal grafts in unmodified BN rats. However, LEW kidneys were rejected by IgM-BN hosts in a slightly delayed fashion compared with controls (mean rejection times (MRTs), 9.4 versus 7.1 days); delayed rejection was associated with the absence of anti-LEW IgG hemagglutinins from the recipients' blood and the absence of vasculonecrotic lesions from rejected renal grafts. LEW kidneys inserted into IgG-BN rats were rejected in a slightly accelerated fashion compared with controls (MRT, 6.6 days). Lymphocytotoxins developed in IgG-BN recipients of LEW kidneys in a fashion similar to that of controls, but cytotoxic T cell responses were delayed up to the 6th day after transplantation. These observations confirm our previous finding that cytotoxic T cells do not play a decisive role in acute rejection in this model. The association observed between delayed or accelerated rejection of LEW kidneys by BN rats sensitized with LEW-E and the absence or presence of anti-donor IgG hemagglutinins in the blood of these recipients after transplantation suggests an important role for IgG anti-donor class I antibodies in the rejection of LEW renal allografts by BN rats.     

Chronic renal allograft rejection. Selective involvement of the glomerular endothelium in humoral immune reactivity and intravascular coagulation

To study immune reactive and thrombotic mechanisms involved in chronic renal allograft rejection, Lewis rat kidneys were transplanted into bilaterally nephrectomized Brown Norway recipients tolerant of LEW erythrocyte antigens. Such BN rats fail to produce anti class I MHC alloantibodies after insertion of a LEW kidney. The LEW renal allografts experience a transient rejection episode without proteinuria followed by the development of chronic rejection, clinically characterized by glomerular proteinuria in the presence of stable renal function. Immunohistological studies of such chronically rejected LEW renal allografts showed the occurrence of glomerular and interstitial infiltration of predominantly monocytes and T cells. CD4-positive T cells dominated over CD8-positive T cells in the chronically rejected LEW renal grafts. IgG deposition was found deposited throughout the renal vasculature--this in contrast to IgM, which was observed only in the glomerular vasculature. Glomerular antibodies were not directed to endothelial class II MHC antigens, and showed only weak complement fixation as demonstrated by C3 staining. Selective glomerular IgM deposition was associated with vascular (platelet-containing) thrombi, and focal and segmental fibrinoid necrosis. In contrast, acutely rejected LEW renal grafts in unmodified BN recipients showed IgM deposition as well as thrombus formation throughout the entire renal vasculature. The results demonstrate that the antibody response to endothelial--and, in particular, glomerular endothelial non-MHC antigens--may bring about chronic vascular renal allograft rejection. How the formation of glomerular thrombotic lesions may be assisted by endothelial reactivity to cytokines from local immune reactive cells is discussed.     

A detailed analysis of the potential of water-soluble classical class I MHC molecules for the suppression of kidney allograft rejection and in vitro cytotoxic T cell responses

Water-soluble classical (RT1-A) class I MHC molecules were purified from aqueous extracts of DA strain liver. Following monoclonal antibody affinity, lentil lectin affinity, and gel filtration chromatography, 600 micrograms of soluble RT1-A class I molecules with antigen activity equivalent to 1.3 x 10(11) nucleated DA spleen cells (greater than 500 DA spleens) was obtained. Both PVG and LEW strain recipients of DA kidney allografts were pretreated with intravenous injections of the DA soluble class I molecules, in doses with antigen activity equivalent to 10(8) nucleated DA spleen cells. Three protocols of pretreatment were used: twice-weekly injections for 4-5 weeks, with grafting 3 or 4 days after the last injection; a single injection 7 days pregraft; or a single injection 1 day pregraft. The PVG and LEW rats received the soluble class I pretreatment either alone or in combination with suboptimal doses (2 mg/kg/day) of cyclosporine after grafting, making a total of 12 experimental groups treated with soluble class I antigen. In no case did treatment with soluble class I antigen elicit an antibody response in prospective graft recipients; influence kidney graft survival in any way; or enhance or suppress the antibody response to the kidney graft. The soluble DA class I MHC molecules were tested in vitro for their effect on the generation and effector function of allospecific PVG and LEW anti DA RT1-A class I cytotoxic T cells and TNP specific, self RT1-Aa restricted cytotoxic T cells. Concentrations up to 5 micrograms/ml (10(-7) M), equivalent to 10(9) nucleated DA spleen cells/ml, were without any effect. We conclude that monomeric forms of water-soluble classical class I molecules are poor immunogens--and, at doses conventionally used for active enhancement, do not influence cytotoxic T cell responses and have little potential for donor-specific immunosuppression.     

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.     

Allograft rejection in CD4+ T cell-reconstituted athymic nude rats--the nonessential role of host-derived CD8+ cells.

PVG-rnu/rnu nude rats reject fully allogenic renal (DA) and skin (BN, AO) allografts after the adoptive transfer of naive CD4+ T cells alone, but rejection is accompanied by the accumulation of many nude-derived CD8+ leukocytes within the graft. In addition, mononuclear cells infiltrating the rejecting renal grafts in these animals display cytotoxic activity in vitro against specific and third-party alloantigens. In this investigation we have treated CD4+ T cell-restored nude rats bearing renal or skin allografts with the mAb MRC OX8 to deplete the host of CD8+ cells. In vivo treatment with OX8 completely eliminated CD8+ cells from rejecting grafts of both kidney and skin, but it did not prevent graft rejection, nor did OX8 treatment abolish the cytotoxic effector cells found in nude rat spleen or in graft-infiltrating cells (GIC) of rejecting renal allografts. The nature of the cytotoxic activity was examined with anti-CD3 mAb 1F4, which was shown to block conventional CD8+ Tc killing in vitro but did not inhibit allogeneic target cell lysis by spleen cells from nude rats. The cytotoxic activity found in GIC of rejecting allografts was not inhibited by anti-CD3 mAb, suggesting that these cytotoxic effector cells were CD3-CD8- and were of extrathymic origin. We conclude that non-thymus-derived CD8+ GIC are not essential for allograft rejection in CD4+ T cell-restored nude rats.     

Cytokine and T cell receptor gene expression at the site of allograft rejection.

Intragraft cytokine and T cell receptor gene expression was analyzed in rejecting renal allografts by polymerase chain reaction (PCR). Message for IL-1 beta, IL-6, and TNF-alpha was detected in nephrectomy tissue with pathological evidence of acute or chronic rejection. Similarly, mRNA for both IL-6 and TNF-alpha was present in renal biopsies from acute rejecting kidneys. IL-2R, IL-4, and IL-5 mRNA was present in both rejecting and rejected kidney allografts, indicating that these cytokines may play a role in ongoing renal allograft rejection. Conversely, IL-2, IL-7, and IFN-gamma message was detected infrequently. In order to address the diversity of T cells in rejecting kidneys, we have analyzed the clonality of the TcR present within the allograft tissue. Rearranged TcR genes were identified in all allografts examined (n = 16) indicating the presence of T cells bearing the alpha/beta TcR. We have determined that there is a heterogeneous infiltration of T cells in the rejected allograft with TcR representing x = 7.47 +/- 2.4 families rearranged in samples obtained from nephrectomies, whereas x = 5.33 +/- 0.58 families were detected in samples obtained from biopsy tissue. These data indicate that (1) cytokines are produced locally which may contribute to graft cell destruction, (2) the heterogeneity of intragraft T cells during kidney allograft rejection may exist because nonspecific lymphocytes have been recruited to the site by locally produced cytokines or because T cells are responding to multiple epitopes or multiple donor antigens. Detection of intragraft cytokines and TcR may prove useful in elucidating the mechanism of rejection and therefore lead to improved immunosuppression.     

Mediation of the induction of immunologic unresponsiveness following antigen pretreatment by a CD4 (W3/25+) T cell appearing transiently in the splenic compartment and subsequently in the TDL

We have previously demonstrated in a rat renal allograft model, using adoptive transfer studies, that a single blood transfusion results in the generation of blood donor-specific W3/25+ (CD4) T suppressor cells detectable in the thoracic duct lymph, but not in the spleen, 7 days after transfusion. In this study, again using the LEW-to-DA strain combination, we have investigated the time of appearance of suppressor activity in different lymphoid compartments in vivo and in vitro following a single blood transfusion. Cells (5 x 10(7) unfractionated TDL or 1 x 10(8) unfractionated spleen cells) were harvested from DA rats on days 1-6 after a single LEW blood transfusion and were adoptively transferred into syngeneic (DA), lightly irradiated (200 rads) hosts. The following day these animals received a kidney allograft (LEW or PVG). Specific suppressor activity, as demonstrated by the prolongation of survival of LEW, but not PVG, renal allografts was noted in the spleen 4 days after transfusion (MST greater than 100 days). Suppressor cells were not found in the TDL at this stage--but 6 days after transfusion suppressor activity could be detected in the TDL (median survival time [MST]greater than 100 days), while at this time no such activity was demonstrable in the spleen. The suppressor activity in the spleen on day 4 after transfusion was shown to be mediated by a W3/25+ (CD4) T cell. The proliferative response in a mixed lymphocyte culture of DA spleen cells, prepared 4 days after transfusion, or TDL cells prepared 6 days after transfusion, to LEW stimulator cells was significantly suppressed when compared with the response of cells harvested from untreated controls. In contrast, the proliferative response of spleen cells examined 6 days after transfusion, when no suppressor activity was detected in vivo, was significantly augmented in comparison with untreated controls. Thus we have demonstrated that a DST results in the generation of W3/25-positive (CD4) T cells capable of suppressing renal allograft rejection that appear transiently in the splenic compartment (4 days after transfusion) and subsequently in the TDL (six days after transfusion).     

负向免疫调节树突状细胞对心脏移植大鼠免疫状态的影响

目的 探讨经体外光化学法(PUVA)处理的供者脾淋巴细胞与受者树突状细胞(DC)共培养后,对移植受者体液免疫、细胞免疫及移植物排斥反应的影响.方法 以DA大鼠为供者,LEW大鼠为受者,建立大鼠腹部异位心脏移植模型.分离供者脾淋巴细胞(SP),制备经PUVA处理的供者脾淋巴细胞(PUVA-SP).在体外分别将供者PUVA-SP和SP与受者骨髓来源的未成熟DC共培养,得到PUVA-SP-DC及SP-DC,流式细胞仪检测上述DC表型.根据受者心脏移植术前1周静脉输注成分的不同,将受者随机分为3组:(1)对照组(n=7):单纯输注磷酸盐缓冲液(PBS);(2)SP-DC组(n=8):输注Sp-DC 5×106个;(3)PUVA-SP-DC组(n=8):输注PUVA-SP-DC 5×106个.每日观察各组移植心的存活状况.移植后第6天,检测受者血清中抗供者特异性IgG水平;通过混合淋巴细胞反应(MLR)检测受者脾脏T淋巴细胞对供者抗原刺激的增殖反应;比较各组受者脾脏体积的大小.结果 供者脾淋巴细胞经PUVA处理后细胞凋亡率为81.93%.正常LEW大鼠DC共刺激分子CD80和CD86阳性率分别为(3.5±0.27)%和(13.0±0.58)%,受者DC与供者SP混合培养后,其CD80和CD86的表达水平为(16.6±0.72)%和(36.5±0.87)%,后者明显高于前者(P<0.01);受者DC与供者PUVA-SP混合培养后,其CD86和CD80的表达率分别为(3.9±0.12)%和(13.4±0.59)%,与正常LEW大鼠DC相当(P>0.05).PUVA-SP-DC组的受者抗供者特异性IgG水平明显低于SP-DC组及对照组(P<0.01).PUVA-SP-DC组受者T淋巴细胞对供者抗原的刺激反应指数为1.66±0.29,明显低于SP-DC及对照组(7.28±0.38、4.19±0.16,P<0.01);而其对无关供者抗原的刺激反应指数为4.37±0.11,与SP-DC及对照组相当(4.51±0.40、4.36±0.14,P>0.05).PUVA-SP-DC组的移植心存活时间比其他两组明显延长(P<0.01),而且其脾脏体积最小.结论 PUVA-SP-DC能够特异性的下调移植受者对供者抗原的细胞免疫及体液免疫反应,从而明显延长移植物存活时间.     

Specific unresponsiveness to fully allogeneic kidney allografts in rats induced by procarbazine hydrochloride and antilymphocyte serum

A short course of procarbazine hydrochloride (PCH; 50 mg/kg) and antilymphocyte serum (ALS; 5 ml/kg), administered to Lewis (LEW;RT1(1] rats in the first week following transplantation of Brown Norway (BN;RT1n) kidneys, substantially prolonged allograft survival and induced long-term survival in 62% of the grafts. The two agents acted synergistically, in that neither of them administered alone had much effect. Graft recipients did not produce detectable cytotoxic antibodies and antigen-reactive cells injected i.v. were not diverted to the liver, thus showing that neither antibodies nor immune complexes are likely to mediate the unresponsiveness. Spleen cells from graft-bearing recipients failed to cause graft-versus-host responses (GVHR) in both (LEW X BN)F1 and (LEW X DA)F1 hybrids, but they specifically suppressed the GVHR given by normal syngeneic cells to donor strain (BN) antigens. This suppression was specific because the response against third-party antigens (DA; RT1a) was unaffected. Adoptive transfer of spleen and thymus cells from PCH-ALS-treated LEW rats bearing healthy BN kidneys caused a profound prolongation of BN graft survival in sublethally irradiated LEW recipients. This transfer was specific and mediated by W3/13+ (T) lymphocytes. It is concluded that a limited regimen of PCH and ALS given in the first postoperative week incites the generation of specific suppressor T lymphocytes and that this form of immunosuppression, even without preoperative donor antigen, is an effective way of prolonging kidney allograft survival.     

Indium-labeled presensitized T cells for diagnosis of graft rejection.

The aim of this experiment was to test a safe, noninvasive method for necessary, accurate diagnosis of early allograft rejection. Heart-lung allograft was performed heterotopically using Brown Norway (BN) rats as the donor and Lewis (LEW) rats as the recipient. T cell suspensions were prepared from lymphnodes of specifically sensitized LEW rats that had acutely rejected full-thickness BN skin graft. Cell count was adjusted 50 x 10(6) cells/ml. The suspension was incubated in vitro with 111I oxide (1 m Ci-ml). An aliquot of labeled cell suspension containing 40 x 10(6) cells and a total radioactivity of 200 mCi was administered intravenously to each animal 3 and 6 days after heart-lung transplant. The traffic of T cells was followed in vivo and in isolated organs under large field view gamma camera. The gamma camera revealed radioactivity on the graft starting Postoperative Day 5 when the heart was actively beating; no radioactivity was revealed at the site of the isografted organs. The histology showed mild to moderate cellular infiltration parallel to the grade of radioimaging intensity. The injection of indium-labeled presensitized T cells was able to detect the rejection process in an early phase when there are no clinical symptoms of rejection and/or the rejection cascade can be reversed. These results suggest that a similar method can be used in human organ transplantation for early diagnosis of rejection.     

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.     

Transfer of "infectious" cardiac allograft tolerance induced by donor-specific transfusion

BACKGROUND: "Infectious tolerance" has been defined as the tolerance induced in a new recipient by the adoptive transfer of cells from a recipient accepting an allograft after anti-CD4 and anti-CD8 monoclonal antibody treatment. A clear understanding of the mechanisms responsible for graft acceptance after donor-specific blood transfusion (DST) has remained elusive. We examined the development and "infectious" nature of immunologic changes resulting in indefinite survival of LEW to DA rat cardiac allografts after DST alone without the need for antibody. METHODS: One hundred x 10(6) LEW splenocytes (SC) as DST were injected intravenously into DA recipients 7 days before LEW cardiac transplantation. Subsequently, 100 x 10(6) SC harvested from a DA recipient 30, 60, or 100 days after graft acceptance were adoptively transferred into lightly gamma-irradiated (450 rad) na?ve DA recipients 24 hours before a second LEW cardiac allograft. Subsequent graft function was determined. RESULTS: Adoptive transfer of SC from the DST-treated DA rats 30 days after LEW heart transplant acceptance into na?ve gamma-irradiated DA rats failed to transfer tolerance to LEW cardiac allografts. However, SC from DA rats bearing LEW hearts for more than 60 days induced indefinite tolerance to all LEW hearts. This infectious tolerance could be adoptively transferred again to a second DA recipient. CONCLUSIONS: DST-generated regulatory cells can downregulate na?ve lymphocytes to promote allograft acceptance. This tolerance can be expanded and serially transferred to a subsequent na?ve cardiac recipient.