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.     

Recipient immature dendritic cells do not prolong allograft survival

Experimental studies on allogeneic transplantation have shown that recipient dendritic cells (DC) play a role in peripheral tolerance as well as in rejection of allografts. It is not known whether DCs exert their tolerogenic function in the graft or in recipient lymphoid tissue. To answer this question we created a chimeric heart model deprived of its own DCs and repopulated with recipient DCs. The rationale for this model was to observe whether recipient mature and immature DCs located in the graft attenuate recruitment and stimulation of recipient lymphocytes, subsequently prolonging graft survival. Vascularized bone marrow transplants from the prospective recipient to the lethally irradiated heart donor, which function for a period of 14 days, were used to replace donor DCs with prospective recipient either mature or immature DCs. Replacement of the donor heart with either of these cells did not prolong graft survival. The intragraft microchimerism did not mitigate the allogeneic rejection reaction.     

Donor DNA can be detected in recipient tissues during rejection of allograft

Abstract The main source of donor DNA in recipients of allograft are “passenger” cells. It is claimed that they are responsible for the posttransplantation microchimerism and prolongation of allograft survival. We have observed that besides cellular microchimerism, donor DNA can be found in the recipient tissues at the time of rejection of the allograft. In this study, we provide evidence for the presence in the recipient of both DNA in “passenger cells” and free DNA in tissues at the terminal stage of rejection. Male BN (RT1 n) rat heart or skin was transplanted to female LEW (RT1 1) rats followed by a vascularized bone marrow in a hind‐limb transplant. In another group, heart and skin were transplanted followed by immediate i. v. infusion of donor‐type bone marrow cells. CsA was given in a dose of 17 mg/kg body weight for 30 days, then the rats were followed up until day 100 unless rejection occurred earlier. LEW blood, spleen, mesenteric node and bone marrow cells were stained with moAb OX27 specific for BN but not LEW. Genomic male DNA was isolated and amplified with SRY oligonucleotide. At day 30 and day 100 cellular microchimerism was detected in blood, spleen, nodes and bone marrow cells. Donor DNA was detected in recipient skin, liver and heart extracts, as well as lymphoid organs, at the time of rejection of allograft, but not when the rats were maintained on CsA. Taken together, donor DNA was detected in recipient tissues at the time of heart or skin rejection. It appeared to be released from cells of rejecting grafts and not from “passenger” cells, representing only a minor cellular mass compared with the graft.     

Sensitization interval and administration method alter the effect of 15-deoxyspergualin on heart transplantation in sensitized recipient rats

We evaluated the effect of 15-deoxyspergualin (DSG) on accelerated rejection. Brown Norway rats (BN) served as organ donors and Lewis rats (LEW) as recipients. In an accelerated rejection model, after a LEW rat was sensitized with BN skin, a BN heart was transplanted. Various intervals between sensitization and heart transplantation were examined. The heart allografts in sensitized recipients were rejected earlier than those in unmodified recipients regardless of the sensitization interval. DSG (2.5 mg/kg per day), given to the recipients during the sensitization phase, significantly prolonged graft survival compared with the untreated hosts when the sensitization interval was short. When the recipients were treated with DSG after heart transplantation, heart graft survival was significantly prolonged regardless of the sensitization interval. Flow cytometric analysis and complement-dependent cytotoxicity tests revealed that DSG suppressed antidonor antibody formation and that postoperative administration of DSG significantly decreased the proliferation of B cells when the sensitization interval was short and the proliferation of class II antigen-positive cells when the sensitization interval was long.     

Cyclosporin A spares selectively lymphocytes with donor-specific suppressor characteristics

The effect of cyclosporin A (Cy A) on the host responses to heart allografts have been examined in rats following administration of the drug for 7 days after grafting. All grafts functioned greater than 100 days without rejection episodes in animals of major histocompatibility differences. Thymic or splenic lymphocytes (1 X 10(8) from LEW recipients of (LEW X BN)F1 hearts were transferred at varying periods into untreated LEW rats transplanted with (LEW X BN)F1 test hearts 24 hr later. Test grafts survived 12 to 16 days significantly (P less than 0.001) longer than in untreated animals (MST +/- SD = 7 +/- 0.3 days). Cells from normal LEW animals, Cy A-treated but ungrafted, and grafted but not treated animals, all failed to prolong test graft survival. Specificity of the effect was tested in vivo, using hearts from donor and third-party rats, and in vitro, using the mixed lymphocyte response (MLR). In vivo, thymocytes from treated LEW recipients of (LEW X WF)F1 grafts failed to prolong (LEW X BN)F1 test grafts; conversely, transferred thymocytes from LEW recipients of LEW X BN)F1 grafts failed to prolong (LEW X WF)F1 grafts. The MLR of lymphocytes from Cy A-treated rats was significantly decreased against donor lymphocytes but not against third-party lymphocytes. Additionally, both cellular and humoral immunity mounted by Cy A-treated recipients was depressed throughout the entire follow-up period. Prolonged heart graft survival after 7 days of Cy A treatment suggests emergence of cells with specific suppressor activity, which in turn may cause profound abrogation of host effector responses against vascularized organ allografts.     

Effect of recombinant interferon gamma and interleukin-2 and of a monoclonal antibody against interferon gamma on the rat immune response against heart allografts

We studied the effect of rat rec-IFN-gamma, human rec-IL-2, and an IgG1 monoclonal antibody (DB1) directed against rat IFN-gamma on allograft survival in the rat in various experimental conditions. The DB1 monoclonal antibody did not prolong heart allograft survival in the (LEW/BN)F1 to LEW combination, even when used at high doses (2 mg/rat x 9 days). Rec-IFN-gamma induced major histocompatibility antigen expression in vivo, but its administration had no effect on the graft survival either of untreated LEW recipients of (LEW x BN)F1 heart allografts or of donor blood-transfused LEW recipients. In addition, rec-IFN-gamma alone had no effect on graft survival in cyclosporine-treated rats. In contrast, rec-IL-2 shortened heart allograft survival both in untreated and in cyclosporine-treated recipients. Rec-IFN-gamma partially reversed the effects of rec-IL-2 in cyclosporine-treated rats. The data suggest that in vivo administration of IFN-gamma in allograft recipients may have a suppressor effect, in addition to the postulated augmenting effect on the immune response by increasing MHC antigen expression.     

Ex vivo perfusion with mitomycin C containing solution prolongs heart graft survival in rats

Mitomycin C (MMC) is an alkylating agent which suppresses allogeneic T-cell responses. We analyzed the effect of graft perfusion with MMC on transplant survival. Hearts from Brown-Norway (BN) rats were perfused ex vivo with MMC-containing solution, stored and implanted into Lewis (LEW) rats. In order to analyze the in vivo effect of MMC, recipients received MMC posttransplantation or were pretreated with MMC-incubated donor-derived peripheral blood mononuclear cells (PBMCs). The results show that MMC-perfusion significantly prolongs graft survival. Treatment of recipients with MMC has no effect, whereas MMC-treated donor PBMCs injected into the recipient prolong graft survival. Our findings indicate that the targeted perfusion of donor hearts with MMC-containing solution protects the graft from rejection.     

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.     

Infectious tolerance develops after intrathymic alloantigen-induced acceptance of rat heart allografts can be adoptively transferred

BACKGROUND: We have shown that intrathymic (IT) injection of alloantigen with antirat lymphocyte serum (ALS) treatment can induce donor-specific allograft acceptance. The purpose of this study was to investigate whether T-regulatory (T-reg) cells play a role in the maintenance of donor-specific heart graft tolerance that develops after IT injection of Lewis (LEW, RT1(l)) alloantigen into a Dark Agouti (DA, RT1(a)). METHODS: Na?ve DA rats were injected IT with 2.5 x10(7) LEW donor splenocytes and injected intraperitoneally with 1 mL ALS. Twenty-one days after pretreatment, a LEW or Brown Norway (BN, RT1(n)) heart was transplanted into a treated DA recipient. Splenocytes (1 x 10(8) or 5 x 10(7)) from a LEW heart-tolerant long-term survivor (LTS; >60 days) DA recipient were harvested and adoptively transferred (AT) into an irradiated (450 rad) na?ve DA rat 24 hours before transplanting a LEW heart. RESULTS: All LEW heart allografts were rejected by untreated DA rats in a mean survival time (MST) of 7.4 +/- 1.7 days (n=7). In contrast, 66.7% of LEW heart grafts into IT+ALS-pretreated DA recipients were accepted indefinitely (n=24). When either 1 x 10(8) (n=5) or 5 x 10(7) (n=5) splenocytes from a LEW heart graft-tolerant LTS (>60 days) DA recipient were AT into a new na?ve DA rat, all new LEW heart grafts were accepted indefinitely. CONCLUSIONS: The donor-specific tolerance that develops after IT+ALS-induced LEW heart acceptance by DA recipients can be transferred adoptively to new na?ve DA recipients, thus indicating that it is infectious tolerance.     

Cytotoxic T-cell elimination during anti-CD4-induced rat liver acceptance and rapid replacement of functional graft antigen-presenting cells.

In previous studies, we showed that primed T cells were eliminated in long-term survival Wistar Furth (WF) recipient rats with spontaneously accepted Lewis (LEW) liver graft and that the grafted liver lost the ability to elicit rejection reaction early after liver transplantation. We hypothesized that the same phenomenon may be observed in tolerant animals after immunosuppression in a rejector rat strain combination (WF-->LEW). Furthermore, we proposed the repopulation of liver allograft with host antigen-presenting cells rapidly after transplantation. Recipient LEW rats that underwent anti-CD4 therapy accepted the WF liver allografts after a transient rejection reaction. In tolerant animals, alloreactive CD8 T cell precursors were present, but primed T cells were absent. Intraperitoneal challenge with grafted WF liver homogenates obtained from recipient LEW rats on day 4 after transplantation did not induce transient rejection responses in long-term survival recipient LEW rats, a finding that differed from the results of experiments using normal WF liver homogenates. However, challenge with grafted WF liver homogenates, similar to those of normal LEW liver homogenates, induced rejection responses in long-term survival recipient WF rats with LEW liver allograft. Flow cytometric analysis confirmed that most of nonparenchymal cells in the grafted WF liver were recipient (LEW) genotype. These observations showed that the deletional mechanism of effector T cells also is observed in this setting, and professional donor antigen-presenting cells are replaced by those of recipient genotype within the graft during the early phase of transplantation.     

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.     

Transplantation of organs is transplantations of donor DNA: fate of DNA disseminated in recipient

Microchimerism after allogeneic organ transplantation has been widely documented using DNA identification techniques. However, the question as to whether the detected donor DNA is present in the surviving donor passenger cells, recipient macrophages phagocytizing rejected donor cells, or dendritic cells (DC) internalizing donor apoptotic bodies or cell fragments has not been answered. We provide evidence that allogeneic organ transplantation is followed not only by cellular microchimerism caused by release of graft passenger cells but also dissemination of donor DNA from the ischemic rejecting graft cells and its internalization in recipient DC. The high levels of donor DNA at the time of heart rejection were inversely proportional to the concentration of donor passenger cells detected with use of flow cytometry. Depending on the type of graft, the kinetics of DNA distribution in recipient tissues were different. Immunosuppressive drugs attenuated the rejection reaction and release of DNA from grafts. Allogeneic but not syngeneic donor DNA fragments were found in recipient splenic DC-enriched population. Interestingly, that donor DNA fragments could be detected in recipient tissue at high levels on day 30. This challenges the notion that fragments of DNA are immediately cleaved by cell plasmatic enzymes. The biologic significance of our findings is not clear. We speculate that donor DNA fragments in recipient DC may play a, so far unknown, role in the immunization/tolerance process to allogeneic antigens.     

Genetics of the blood transfusion effect on heart allografts in rats

Working with the recently available recombinant haplotypes of the rat major histocompatibility complex (MHC)--RT1, we investigated the effect of various types of blood transfusion (BT) on allograft prolongation, including blood identical for the whole RT1 haplotype with that of the donor or for only a part of it. One or two milliliters of donor blood significantly prolonged graft survival in the (LEW X BN)F1----LEW or the LEW X 1W----LEW X 1A combination. The optimal regimen consisted of two BTs given 15 and 7 days prior to grafting; BTs given at day -30 were ineffective. A BT given on the day of the operation was effective, but sequential BTs after grafting did not further increase graft survival. In the (LEW X BN)F1----LEW combination, blood from congenic LEW X 1N rats significantly prolonged graft survival, but third-party BTs were ineffective or had only a borderline effect when transfused (1 ml, 8 times) within the three months before transplantation. This showed the major role of the RT1 system as well as the specificity of the model. Although the survival of LEW X 1A heart grafts transplanted into LEW X 1W recipients could not be significantly prolonged by donor blood, with the reverse--and "weaker"--combination (LEW X 1W----LEW X 1A), 2 ml of donor blood led, in all cases, to greater than 100 days graft survival. In this last combination, third-party BT (LEW X 1N) was again totally ineffective. Blood from RT1-recombinant rats was used to test the role of the respective RT1.A, B, and C regions, in the enhancing effect. BTs from LEW X 1AR2 or LEW X 1WR2 recombinants--sharing, respectively, RT1.C and RT1.A with the graft donor--were only moderately effective, as compared with BTs from the graft donor. On the other hand, LEW X 1WR1 BTs--sharing the RT1.A and RT1.B regions with the graft donor--had a much more powerful effect on heart survival. The results strongly suggest that the RT1.B region (coding for Ia-like antigens) must be shared by the graft and blood donor in order to mediate a significant graft prolongation.     

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.     

Induction of indirect donor-specific hyporesponsiveness by transportal RT1-peptide pulse in rat skin transplantation

In the present study, we examined whether transportal pulse of class I major histocompatibility complex (MHC) allopeptides can induce indirect (non-chimeric) donor-specific hyporesponsiveness, using a high-responder rat skin transplantation model. Two donor-specific 8-amino acid peptides corresponding to residues 58-65 and 70-77 in the alpha(1) helical region of RT1.A(a) were synthesized. In order to test immunogenicity of these peptides, mixed lymphocyte reaction (MLR) was performed. Then, 100-microg portions of peptides were injected into recipient Lewis (LEW, RT1.A(l)) rats via the portal vein 14 days before skin transplantation. Skin allografts from August Copenhagen Irish (ACI, RT1(a)) or Wistar King A (WKA, RT1(k), third-party) donors were transplanted to LEW (RT1(l)) recipients. Transportal pulse of residues 58-65 and 70-77 prolonged graft survival significantly in ACI-to-LEW skin transplantation (17.6+/-0.40 and 18.0+/-0.45 days) compared with control (14.2+/-0.37 days). However, pulse of residues 106-113, a non-donor-specific control, did not prolong graft survival time (14.6+/-0.40 days) in the same combination. Regarding the third-party donor, residues 58-65 injected into LEW recipients had no effect on survival time of skin grafts (19.0+/-0.84 days) derived from WKA donors compared with the untreated WKA-to-LEW control (19.4+/-0.93 days). Transportal pulse of RT1.A(a) peptides induced donor-specific hyporesponsiveness even in a high-responder rat skin transplantation model. Our results suggest that graft enhancement by transportal exposure to donor cells may not be induced by a chimeric process but, instead, by an indirect mechanism not involving intervention of viable donor cells.     

Rejection following donor or recipient preoperative treatment with FTY720 in rat small bowel transplantation

BACKGROUND: Severe rejection of small bowel transplantation (SBTx) has been ascribed to abundant lymphoid tissues in the small intestine without well-established evidence. However, the role of donor lymphocytes in rejection is still unclear. The novel immunosuppressant, FTY720, is reported to transfer peripheral blood lymphocytes (PBLs) to lymphoid tissues such as mesenteric lymph nodes (MLNs) and Peyer patches (PP). In the present study, the number of donor lymphocytes in the graft was increased by FTY720, and the influence on rejection was studied in a rat model. Furthermore, the number of the PBL of recipient was decreased by FTY720 before SBTx and the effect on rejection was examined. MATERIALS AND METHODS: Orthotopic total SBTx was performed in Brown-Norway and Lewis rats. In the donor pretreatment study, FTY720 was administrated to donor rats 24 h prior to harvesting to increase the number of graft lymphocytes (FTY donor-pretreated group). In contrast, MLNs were surgically removed from the grafts to decrease the number of graft lymphocytes (MLN-resected group). In the recipient pretreatment study, FTY720 was administrated to recipient rats 24 h before SBTx to decrease recipient PBL (FTY group). In contrast, a subclinical dose of cyclosporine A (CsA) was administrated after SBTx (CsA group). Rats were administrated preoperative FTY720 combined with post-SBTx CsA (FTY+CsA group). Graft survival, pathology, lymphocyte count, and subtype were examined. RESULTS: In the donor pretreatment study, pretreatment with FTY720 did not enhance graft rejection. MLN resection did not prolong graft survival. In the recipient pretreatment study, FTY720 caused a significant reduction in the number of infiltrating lymphocytes in the graft, as well as the percentage of recipient CD4+ and CD25+ cells within the graft. FTY720 and CsA synergistically prolonged graft survival. CONCLUSION: SBTx rejection correlated with the number of recipient PBL, and not with the number of donor lymphocytes transplanted together with the graft. The pretreatment of the recipient with FTY720 was effective in the case of combined use of the low-dose postoperative CsA.     

Immunomodulation for intestinal transplantation by allograft irradiation, adjunct donor bone marrow infusion, or both

BACKGROUND: The passenger leukocytes in the intestine have a lineage profile that predisposes to graft-versus-host disease (GVHD) in some animal models and have inferior tolerogenic qualities compared with the leukocytes in the liver, other solid organs, and bone marrow. Elimination by ex vivo irradiation of mature lymphoid elements from the bowel allografts is known to eliminate the GVHD risk. We hypothesized that infusion of donor bone marrow cells (BMC) in recipients of irradiated intestine would improve tolerogenesis without increasing the risk of GVHD. METHODS: Orthotopic small intestine transplantation was performed with the GVHD-prone Lewis (LEW)-to-Brown Norway (BN) combination and the reverse GVHD-resistant BN-to-LEW model under a short course of tacrolimus treatment (1 mg/kg/day, days 0-13, 20, 27). Grafts were irradiated ex vivo, using a 137Cs source. In selected experimental groups, donor BMC (2.5 x 10(8)) were infused on the day of small intestine transplantation. RESULTS: The unmodified LEW intestine remained intact, whether transplanted alone or with adjunct donor BMC infusion, but all of the BN recipients died of GVHD after approximately 2 months. Intestinal graft irradiation (10 Gy) effectively prevented the GVHD and prolonged survival to 92.5 days, but all of the BN recipients died with chronic rejection of the LEW grafts, which was prevented by infusion of adjunct donor BMC without causing GVHD. In the GVHD-resistant reverse strain direction (BN-->LEW), all intestinal recipients treated for 27 days with tacrolimus survived > or =150 days without regard for graft irradiation or adjunct BMC, but chronic rejection was severe in the irradiated intestine, moderate in the unaltered graft, and least in the irradiated intestine transplanted with adjunct BMC. Mild arteritis in the 150 day allografts of both strain combinations (i.e., LEW--> BN and BN-->LEW) may have been irradiation associated, but this was prevented when weekly doses of tacrolimus were continued for the duration of the experiment rather than being stopped at 27 days. CONCLUSIONS: Recipients are protected from GVHD by irradiating intestinal allografts, but the resulting leukocyte depletion leads to chronic rejection of the transplanted bowel. The chronic rejection is prevented with adjunct donor BMC without causing GVHD. Although application of the strategy may be limited by the possibility of radiation injury, the results are consistent with the paradigm that we have proposed to explain organ-induced graft acceptance, tolerance, and chronic rejection.     

The combi-effect--reduced rejection of the heart by combined transplantation with the lung or spleen.

The term combi-effect was introduced to describe the phenomenon of a reduction in rejection of heart grafts after combined transplantation with the lung. In this study in rats we investigated whether the combi-effect was an immunological process and whether it could also be induced by combined transplantation of the heart with the spleen or with a lymphocyte-depleted spleen. Heart and spleen grafts were transplanted into the abdomen; left lungs were transplanted into the thorax of recipient rats. To deplete spleens of their lymphocytes, prospective donor rats were irradiated. Cyclosporine was injected once, on day 2 after transplantation. All heart allografts transplanted alone and treated with cyclosporine were rejected acutely (median survival time [MST] of 14.5 days). In contrast, after combined transplantation of a donor lung or spleen with the heart, almost all heart grafts survived indefinitely. Transplantation of a syngeneic lung or third-party spleen had little effect on heart graft survival (MST of 22.5 days and 26.5 days, respectively). Without cyclosporine treatment, combined transplantation with a donor lung or spleen hardly prolonged heart graft survival. Transplantation of a lymphocyte-depleted spleen with the heart induced a combi-effect in cyclosporine-treated rats that was somewhat weaker: only two of six hearts survived indefinitely. We conclude that in the combi-effect an immunological mechanism reduces rejection of the heart. This mechanism is probably generated by the lymphoid tissue (bronchus-associated lymphoid tissue in lung and white pulp in spleen) in the combined transplant.     

Efforts to enhance survival of limb allografts by prior administration of whole blood in rats using a new survival end- point

Injecting whole blood into the recipient before surgery can significantly prolong renal transplant survival in rats. Therefore, experiments were performed in rats to study the effects of prior administration of whole blood on the survival of limb allografts. Tests to quantitate survival of the allografts included monitoring the internal temperature of the leg, assaying serum creatine kinase levels, and testing for alloantibodies. Lewis recipients of (BN × LEW)F₁ limb transplants that received 1 ml of BN or (BN × LEW)F₁ whole blood before surgery had mean survival times that were longer compared with controls as measured by a 10 F change in temperature. In a test- retest experiment, decline of temperature proved to be a reliable quantitative determination of limb allograft survival since a difference of only 5.6% was observed in the mean number of days of graft survival between two separate groups of control Lewis recipients. Moreover, combined data demonstrated that control Lewis recipients of (BN × LEW)F₁ limb allografts averaged 24.0 days of graft survival based on a 10 F decline in temperature with a 95% confidence interval of ± 6.3 days. It is concluded that prior administration of whole blood can produce significant prolongation of survival in organ transplantation, but it is not as effective in enhancing survival of limb allografts. It is also concluded that internal temperature measurement of limb allografts is an easy, effective, and quantitative method of monitoring rejection.     

Heart allograft survival in rats following immunization with soluble peptide MHC class I donor antigens: evidence for the role of indirect recognition in rejection

Abstract The current series of experiments addressed the question of whether indirect priming with donor MHC antigens affects heart allograft survival. LEW (RT-1¹) rats were immunized with a mixture of two peptides corresponding to the variable region of MHC class I locus A^a antigen (α1 and α2 domain). The recipients were transplanted with a DA (RT1-1^a) heart 1 month after immunization, and graft survival was closely monitored by ECG. All peptide-treated recipients presented with anti-peptide antibodies at the time of transplantation and developed a strongly accelerated graft rejection. These findings indicated that indirect recognition of MHC I donor antigens promotes heart allograft rejection.