Antigenicity of fresh and cryopreserved rat valve allografts

Aortic valve allografts have demonstrated excellent clinical performance, but the importance of antigenic differences between donor and recipient is largely unknown. To determine the antigenicity of aortic valve grafts, rat aortic valves with a short portion of thoracic aorta were transplanted into the abdominal aorta of recipient rats. Valves were used immediately after harvest (fresh) or following cryopreservation. Three weeks after this procedure, the recipient rats received a skin graft from a rat of a strain syngeneic to that of the aortic valve donor. Additional groups of rats were subjected to sham operation (sham) followed three weeks later by skin grafting. Recipient rats were of the Lewis strain. Donor rats were of the Lewis, F344 (weakly allogeneic, RT1-compatible, non-RT1-incompatible), LBN F1 (moderately allogeneic, one-haplotype-identical and one-haplotype-incompatible at both the RT1 and non-RT1 loci), or BN (strongly allogeneic, RT1 and non-RT1-incompatible) strain. Time to skin graft rejection was measured. Among rats receiving the F344 grafts, the time to skin graft rejection (mean +/- SD) was sham: 9.1 +/- 1.0 days, fresh: 7.1 +/- 1.2 days, cryopreserved: 6.9 +/- 0.7 days. Among rats receiving the LBN F1 grafts, the corresponding times were sham: 7.8 +/- 0.8 days, fresh: 5.6 +/- 0.5 days, cryopreserved: 5.4 +/- 0.5 days. Among rats receiving the BN grafts, the corresponding times were sham: 7.1 +/- 0.3, fresh: 4.5 +/- 1.0 days, and cryopreserved: 4.3 +/- 0.7 days. Significant differences (P less than 0.05) existed between sham and fresh and between sham and cryopreserved, but not between fresh and cryopreserved. Significant differences (P less than 0.05) also existed between each histocompatibility grouping. It is concluded that aortic valve allografts in rats are antigenic and produce recipient sensitization. Cryopreservation does not diminish this sensitization. The degree of antigenicity is related to the degree of histoincompatibility between donor and recipient. Both RT1 and non-RT1 antigens appear to play a role in this process.     

Initiation of rejection of established islet allografts by third-party thyroid allografts and splenic dendritic cells

Due to concerns of cross-reactivity between renal and islet allografts in initiation of rejection, we determined the ability of donor-specific and third-party splenic dendritic cells (DCs) and thyroids (whole-organ transplant) to initiate rejection of established islet allografts. Purified islets from neonatal F-344 (RT1Lv1) rats were transplanted bilaterally under the kidney capsule of Wistar-Furth (W/F, RT1u) rats without immunosuppression. The islet allografts were not rejected by 21 days posttransplantation. On day 22, freshly isolated or cultured DCs were injected intraperitoneally into the host. Both freshly isolated and cultured donor-specific (F-344) and some third-party (Buffalo, RT1b; ACI, RT1a) DCs initiated rejection of the islets as indicated by lymphocyte infiltration and destruction of the allograft. DCs, whether freshly isolated or cultured for 8 days from the recipient strain (W/F) and one third-party rat (Brown Norway, RT1n), did not initiate rejection. Splenic DCs from the Lewis (RT1l) rat, which has the same class I and II antigen haplotype as F-344 islet donor rats, also initiated rejection. Only 10(3)-10(4) DCs isolated from the spleen of donor rats were required to initiate rejection of the allograft. In a parallel series of W/F rats with islet allografts, a thyroid (half lobe) from the islet donor strain (F-344), recipient strain (W/F), or third-party rat (Buffalo, Brown Norway, or ACI) was inserted under the kidney capsule at 22 days post-islet transplantation. At 35 days, all thyroids and most islet allografts exhibited active or complete rejection after thyroid transplant from Buffalo, F-344, or ACI rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of storage at 4 degrees C in a nutrient medium on antigenic properties of rat aortic valve allografts

The effects of prolonged storage at 4 degrees C in nutrient medium on the antigenic properties of aortic valve allografts are unknown. Lewis rats received heterotopic aortic valve allografts from Brown Norway donors. Valves were transplanted immediately after harvest (fresh), or after antibiotic sterilization and storage in a nutrient medium at 4 degrees C for 3, 7, 14, or 21 days. Additional rats underwent sham laparotomy (sham). All recipient rats received Brown Norway skin grafts 3 weeks after valve transplant or sham procedure. Time to skin graft rejection for all groups was as follows: fresh (n = 10), 4.5 +/- 0.9 days; sham (n = 10), 7.1 +/- 0.3 days; 3-day (n = 10), 4.9 +/- 0.3 days; 7-day (n = 10), 5.2 +/- 0.4 days; 14-day (n = 10), 6.1 +/- 0.7 days; and 21-day (n = 10), 6.0 +/- 0.6 days. Significant differences existed between the sham group and each of the transplanted groups. No significant differences existed between the fresh group and either the 3-day or 7-day groups. The difference between the fresh group and the 14-day group approached significance (0.05 less than p less than 0.10), and the difference between the fresh and 21-day groups was significant (p less than 0.05). Additional valves not used for transplantation were studied with scanning electron microscopy. The valves preserved in nutrient medium exhibited a progressive loss of endothelium as compared with fresh valves. Storage of aortic valve allografts in a nutrient medium at 4 degrees C is associated with a progressive attenuation of antigenic response.(ABSTRACT TRUNCATED AT 250 WORDS)     

Decellularization reduces the immune response to aortic valve allografts in the rat

OBJECTIVES: Cryopreserved valve allografts used in congenital cardiac surgery are associated with a significant cellular and humoral immune response. This might be reduced by removal of antigenic cellular elements (decellularization). The aim of this study was to determine the immunologic effect of decellularization in a rat allograft valve model. METHODS: Brown Norway and Lewis rat aortic valves were decellularized with a series of hypotonic and hypertonic buffers, protease inhibitors, gentle detergents (Triton X-100), and phosphate-buffered saline. Valves were implanted into Lewis rats in syngeneic and allogeneic combinations. Cellular (CD3 and CD8) infiltrates were assessed with morphometric analysis, and the humoral response was assessed with flow cytometry. RESULTS: Morphometric analysis identified a significant reduction in CD3 + cell infiltrates (cells per square millimeter of leaflet tissue) in decellularized allografts compared with that seen in nondecellularized allografts at 1 (79 +/- 29 vs 3310 +/- 223, P < .001), 2 (26 +/- 11 vs 109 +/- 20, P = .004), and 4 weeks (283 +/- 122 vs 984 +/- 145, P < .001). Anti-CD8 staining confirmed the majority of infiltrates were cytotoxic T cells. Flow cytometric mean channel fluorescence intensity identified a negative shift (abrogated antibody formation) for decellularized allografts compared with nondecellularized allografts at 2 (19 +/- 1 vs 27 +/- 3, P = .033), 4 (35 +/- 2 vs 133 +/- 29, P = .001), and 16 weeks (28 +/- 2 vs 166 +/- 54, P = .017). CONCLUSIONS: Decellularization significantly reduces the cellular and humoral immune response to allograft tissue. This could prolong the durability of valve allografts and might prevent immunologic sensitization of allograft recipients.     

Study of the local graft versus host reaction in the rat. I. Evidences for a donor-related "rat effect"

The local graft versus host reaction (GVHR) observed in the hybrid F1 LBN rat was chosen to evaluate the immunologic effect induced by allogeneic transfusion of Brown Norway blood to the Lewis rat. Study of the kinetics of this GVHR with immunized as well as with nonimmunized cells shows an "individual rat effect" which is shown by a variability of the popliteal lymph node weight from one recipient to another. When spleen cells from different Lewis donors are pooled this effect disappears which indicates that it is related to the donor's rather than to the F1 recipient's cells. To even out variation between individual donors, cells from a number of donors should be pooled to allow better interpretation of experimental protocols using this GVHR.     

Treatment of streptozotocin-induced diabetes mellitus in rats by transplantation of islet cells from two major histocompatibility complex disparate rats in combination with intra bone marrow injection of allogeneic bone marrow cells

BACKGROUND: We have established a new method for the transplantation of allogeneic pancreatic islets obtained from two different rat strains in combination with a newly developed bone marrow transplantation (BMT) method in which bone marrow cells (BMCs) are directly injected into the bone marrow cavity (intra bone marrow BMT [IBM-BMT]). METHODS: Streptozotocin-induced diabetic Brown Norway (BN: RT1A(n)) rats were injected with fludarabine, irradiated with 5.0 Gy x 2, and BMCs from two allogeneic rat strains, Fischer 344 (F344: RT1A(1)) and PVG (PVG: RT1A(c)), were then directly injected into the bone marrow cavity (IBM-BMT). Simultaneously, approximately 600 pancreatic islets (PIs) from F344 and PVG rats were mixed and transplanted into the liver by way of the portal vein. RESULTS: All the recipients thus treated showed normoglycemia 30 days after the treatment. Hematolymphoid cells were completely reconstituted with the two donor-type cells, and immunologic tolerance to F344 and PVG major histocompatibility complex (MHC) determinants were induced. CONCLUSIONS: The transplantation of PIs from two MHC-disparate donors was completely achieved in combination with IBM-BMT, resulting in the improvement of blood glucose levels and the amelioration of diabetes mellitus.     

A new method of bone marrow transplantation leads to extension of skin allograft survival

Tolerance induction through allogeneic bone marrow transplantation is an alternative method to chronic immunosuppression in maintaining long-term allograft survival. In this article, we introduce a new method of bone marrow allotransplantation, which preserves its natural microenvironment and does not require marrow processing or recipient conditioning. A total of 43 skin graft transplantations were performed in nine experimental groups between isogeneic [Lewis to Lewis (LEW, RT1(1))] and allogeneic [Lewis x Brown Norway (LBN --> F1, RT1(1+n)) to Lewis] rats under 35-day protocol of alphabeta T-cell receptor (TCR) monoclonal antibody (mAb) and cyclosporine (CsA) protocol. Monotherapies combined with "crude" bone marrow transplantation resulted in extended survival up to 21 days under CsA and up to 10 days under alphabeta-TCR mAb protocol. The use of combined protocol of alphabeta-TCRmAb/CsA with crude bone marrow transplantation resulted in the extension of skin allograft survival up to 65 days (P < .05). This new simple method of "crude" bone marrow allotransplantation without recipient conditioning is a promising, minimally invasive technique with a potential for direct clinical application.     

Cross-reactivity of organs in allograft rejection. Comparison of effect of thyroid allografts on established islet allografts

The effect of allotransplantation of thyroid or islet allografts into rats with established islet allografts was studied to determine the cross-reactivity of the thyroid and islets in allograft rejection. Islets obtained from cultured neonatal rat (F344) pancreas explants were transplanted bilaterally underneath the kidney capsule of Wistar-Furth rats. After 21 days these allografts did not exhibit signs of rejection. Thyroid (half lobe) from either F344 or Brown Norway rats was transplanted underneath the capsule of the remaining kidney. Transplant of the thyroid from F344 rats resulted in immediate rejection of the islet transplant, whereas transplant of the thyroid from Brown Norway rats was without effect on the islet allograft. This indicates that the thyroid contains immunocompetent cells (cells that present antigen or induce recognition of antigen) that are capable of initiating rejection of established islet allografts. The cytotoxic T-lymphocytes that result are specific for the organ bearing the immunocompetent cells at time of transplantation.     

非清髓性方案在诱导大鼠后肢移植免疫耐受中的应用

目的 探讨基于淋巴细胞毒性相关抗原4-抗体重组腺病毒(AdCTLA4-Ig)的非清髓性方案在造血干细胞嵌合体诱导复合组织异体移植免疫耐受中的作用.方法 以近交系Brown Norway(RT1n)大鼠为供体,Lewis(RT11)大鼠为受体.以后肢移植当天记为day 0.实验分4组,A组:受体直接给予同种异体后肢移植,移植前不进行非清髓件预处理,移植后连续100 d,每天仅给予低剂量环胞素A(CsA),8 mg/kg腹腔注射.B组:受体先给予非清髓性预处理,移植前第33天至移植后第100天,每日用免疫抑制剂三联方案腹腔注射雷帕霉素(RAPA,0.2 mg/kg)+麦考(MMF,20 mg/kg)+甲泼尼龙(MP,10 mg/kg),在移植当日及移植前及移植后第30天分3次尾静脉注射AdCTLA4-Ig(5×109 PFU/d),后肢移植前30 d接受单次3 Gy(照射率0.5 Gy/min)低强度全身照射,不予骨髓移植(BMT).C组:受体预处理方案同B组,移植前30 d,在低强度全身照射后4 h内给予单次尾静脉注射供体骨髓细胞(100×106 cells).D组:受体预处理方案及BMT方法同C组,但大鼠后肢移植供体为第三方动物WF大鼠.在后肢移植后第100天开始,B、C及D组均停止免疫抑制剂三联方案,每日仅给予低剂量CsA(8 mg/kg),连续100 d,直至大鼠后肢移植物发生排异反应而坏死.通过外周血嵌合率检测、移植物抗宿主病检测、后肢移植物存活情况观察、移植物组织病理学检查与评价及混合淋巴细胞反应对免疫耐受状态进行分析评价.结果 C组外周血嵌合率移植当口为(38.8±10.6)%,并长期保持稳定,移植后第300天为(29.3±11.9)%,均未发生移植物抗宿主病,停止免疫抑制剂三联方案后移植物存活>200 d,A、B、D组均发生免疫排异,后肢移植物分别存活(8±2)、(18±3)及(20±2)d,与C组相比,差异有统计学意义(P<0.01).C组移植物病理学检查显示无毛囊炎及血管周围炎等慢性免疫排异现象,混合淋巴细胞反应显示为供体特异性免疫耐受状态.结论 基于AdCTLA4-Ig的非清髓性BMT方案可以诱导长期稳定的造血干细胞嵌合体状态,并可以诱导受体对大鼠后肢移植物的供体特异性部分性免疫耐受.     

Passive transfer of cytomegalovirus by cardiac and renal organ transplants in a rat model

Passive transfer of latent rat cytomegalovirus (R-CMV) infection by means of vascularized organ transplants was examined in inbred rat strains. LEWIS (LEW) rats 4-5 weeks old were infected with RCMV and used as donors at 5 months of age when the infection had become latent. Well-perfused LEW hearts and kidneys were transplanted into unmodified or 500-rad x-irradiated syngeneic or allogeneic Brown Norway (BN) recipients; recipients were sacrificed 3 weeks after transplantation, and RCMV virus from various organs was quantitated by means of a plaque assay. Passive transfer of latent infection could be accomplished with renal allografts (60%) and renal isografts (40%). When BN hosts were x-irradiated LEW renal allografts invariably transferred the latent infection (100%); cardiac allografts rarely did so (8%). X-irradiation of syngeneic hosts did not enhance the capacity of LEW kidneys to transfer the latent infection. The latent infection could not be transferred with thoracic duct lymphocytes. Results show the passive transfer of latent infection with well-perfused vascularized organ allografts to be a relative organ-specific phenomenon.     

Prevention of allograft heart valve failure in a rat model

OBJECTIVE: Allograft heart valves are commonly used in cardiac surgery. Despite mounting evidence that these valves are immunogenic, leading to premature failure, current clinical practice does not attempt to minimize or control such a response. The objective of this study was to evaluate immune modulatory approaches to ameliorate allograft valve failure in a rat model. METHOD: Aortic valve grafts were implanted infrarenally into Lewis rat recipients (n = 32). There were 4 transplant groups: syngeneic grafts (Lewis to Lewis), untreated allografts (Brown Norway to Lewis), allograft recipients treated with cyclosporine (INN: ciclosporin) (10 mg/kg per day for 7 or 28 days), and allograft recipients treated with anti-alpha4 integrin and anti-beta2 integrin monoclonal antibodies for 7 days. At 7 and 28 days the valves were examined for structural integrity and cellular infiltration. RESULTS: Both cyclosporine and anti-alpha4/beta2 integrin treatment resulted in significant reduction in leaflet infiltration by macrophages (ED1(+)), T cells (CD3(+)), and CD8(+) T cells at 7 days with preservation of structural integrity when compared with control allografts. Twenty-eight days after implantation, daily treatment with cyclosporine preserved leaflet structural integrity and inhibited cellular infiltration. However, a short course of cyclosporine (7 days) failed to prevent destruction of the valves at 28 days. CONCLUSIONS: Immune modulatory approaches aimed at T-cell activation or trafficking decrease leaflet cellular infiltration and prevent allograft valve structural failure. However, short-course therapy does not appear to be sufficient and must be maintained to allow long-term preservation of leaflet structural integrity (28 days).     

Hyperhomocyst(e)inemia Induces Accelerated Transplant Vascular Sclerosis in Syngeneic and Allogeneic Rat Cardiac Transplants

Chronic rejection (CR) and transplant vascular sclerosis (TVS) cause the majority of graft failures in cardiac transplantation. Hyperhomocyst(e)inemia [hH(e)] is associated with human TVS without a proven causal relationship. This study investigated the effect of hH(e) on graft survival and TVS in allogeneic and syngeneic rat cardiac transplants. Lewis recipients of heterotopic F344 heart allografts, received normal or hH(e)-inducing (folate, methionine) diets [controls: syngeneic transplanted [+/- hH(e), + CsA] and nontransplanted rats [+/- hH(e), +/- CsA]]. Serial plasma homocyst(e)ine [H(e)] levels were measured. TVS was assessed in clinically rejected grafts and a subset of pre-rejection normal diet allografts (day 64) (neointimal index, NI). The hH(e) diet elevated plasma H(e) levels. When compared with normal diet controls (n = 9), hH(e) diet allografts (n = 9) had decreased time to onset of CR (40 +/- 9 vs. 72 +/- 10d, p = 0.02), and graft failure (64 +/- 10 vs. 107 +/- 12d, p = 0.009). hH(e) diet allografts at rejection (n = 9, 64d) had more severe TVS (NI = 68 +/- 2) than both time-matched normal diet allografts (NI = 49 +/- 6, n = 8, 64d, p <0.001) and normal diet allografts at rejection (NI = 58 +/- 5, n = 9, 107d, p = 0.007). hH(e) induced TVS in syngeneic grafts (NI=50 +/- 3, n = 10 vs. NI = 5 +/- 3, n = 10, 130d, p <0.001). hH(e) accelerated rejection and increased the severity of TVS in allogeneic cardiac transplants, and induced TVS in syngeneic cardiac transplants.     

Allograft Conduit Wall Calcification in a Model of Chronic Arterial Graft Rejection

A bstract Early allograft vascular wall degeneration has emerged as a major important complication in young patients. To explain this mechanism, we reviewed studies on explants of allograft valved conduits implanted heterotopically into the infrarenal aorta in inbred rats (LEW;RT1, and CAP-RT1°C). The following strain combinations (isografts and allografts) were used: syngeneic, LEW-> LEW, strongly allogeneic, and CAP > LEW (RT1- and non-RT1-incompatible). Second-set skin grafting was performed 3 weeks after the heterotrophic implant to test for immunogenicity and presensitization. The animals (LEW) were sacrificed serially on days 20, 30, 50, and 100 for immunofluorescence and SEM studies. Endothelial disruption was observed on day 30, while valve leaflets appeared normal. Humoral allograft rejection was demonstrated and associated with production of antibodies (IgG) against the endothelial cells and around the smooth muscle cells, and in areas of smooth cell necrosis, through 100 days. Neointimal repopulation by host cells and migrated smooth muscle cells was also observed in both viable and allovital grafts. Allovital grafts demonstrated more disorganized collagen and elastic fibers, as well as calcific degeneration in the media and neointima on day 50; the viable conduits showed such structural changes on day 100. In conclusion, vascular walls of allovital conduits calcified earlier than the viable conduits without discernible calcification of the valves. There is therefore evidence to prove causative relationships between cellular viability, immune response, and fibroproliferative calcific degeneration in allograft vascular conduits.     

Efficacy of transient treatment with FK506 in the early phase on cyclophosphamide-induced bone marrow chimerism and transplant tolerance across MHC barriers

BACKGROUND: The use of mixed allogeneic bone marrow chimerism to induce donor-specific transplantation tolerance has been extensively demonstrated. In the present study, we assessed the effect of combined use of a short course of FK506 and a single-dose cyclophosphamide (CYP) on the induction of tolerance and development of GVHD after allogeneic BMT. MATERIALS AND METHODS: Lewis rat (RT1(l)) recipients received BMT from Brown Norway (RT1(n)) donors on the next day after injection of CYP at a dose of 200 mg/kg. The recipients were further treated with no FK506 (n = 8), 0.3 mg/kg/day FK506 on days 10-16 (n = 6), or the same dose of FK506 on days 0-6 (n = 6). In a subgroup of animals, heterotopic heart transplantation was performed to investigate transplantation tolerance. RESULTS: Six of eight recipient rats that did not receive FK506 died of severe GVHD, while high levels of chimerism were induced. Recipients of FK506 in the later phase developed mild transient GVHD around 2 to 3 weeks after BMT and recovered thereafter; however, the level of chimerism was significantly decreased (2.8 +/- 2.3% on day 100). Treatment with FK506 in the early phase completely prevented the development of GVHD and induced stable allogeneic chimerism in the long-term (13.8 +/- 8.3% on day 100). These recipients with stable chimerism accepted subsequent BN heart allografts indefinitely (>200 days x 5), while rejecting third-party (BUF) heart allografts by day 12. CONCLUSIONS: Early transient FK506 promotes the induction of stable bone marrow chimerism without GVHD after BMT with CYP pretreatment. The timing of treatment with FK506 is critical with a view to preventing GVHD and inducing stable long-lasting chimerism.     

巨噬细胞在小肠移植急性排斥反应期的极化状态及意义

目的  探究小肠移植术后发生急性排斥反应（AR）时巨噬细胞极化状态的改变。 方法  将6只Brown Norway（BN）大鼠和24只Lewis大鼠分为假手术组（6只Lewis大鼠）、同基因组（Lewis→Lewis,供受体各6只）和异基因组（BN→Lewis,供受体各6只）。对各组大鼠术后7 d的移植肠组织进行苏木素-伊红（HE）染色和脱氧核糖核酸末端转移酶介导的 dUTP 缺口末端标记（TUNEL）法检测,观察其病理学表现和细胞凋亡情况;采用酶联免疫吸附试验（ELISA）检测血清中M1和M2型巨噬细胞极化相关细胞因子表达水平;利用免疫荧光技术检测各组移植肠组织中M1和M2型巨噬细胞表面标志物并进行共定位计数分析。 结果  HE染色和TUNEL检测结果显示假手术组与同基因组肠上皮形态结构正常,未见明显凋亡小体;异基因组大鼠术后7 d移植肠组织上皮层绒毛结构破坏严重,隐窝数量减少,凋亡小体增多,炎症细胞浸润肠壁全层,呈现中-重度AR。ELISA结果显示异基因组受体鼠血清中M1型巨噬细胞极化相关细胞因子肿瘤坏死因子（TNF）-α、干扰素（IFN）-γ和白细胞介素（IL）-12表达水平高于假手术组和同基因组,同基因组中M2型巨噬细胞极化相关细胞因子IL-10和转化生长因子（TGF）-β表达水平高于假手术组和异基因组,差异均有统计学意义（均为P<0.05）。免疫荧光结果显示异基因组移植肠组织中M1型巨噬细胞计数多于假手术组和同基因组,同基因组M2型巨噬细胞计数多于假手术组和异基因组,差异均有统计学意义（均为P<0.05）。 结论  小肠移植术后发生AR的移植物中,大量巨噬细胞浸润肠壁全层,以M1型为主并分泌大量促炎因子,调控巨噬细胞极化方向是治疗小肠移植术后AR的潜在方法。     

Cyclosporine abrogation of second-set rejection: dependence upon major histocompatibility complex disparity

The purpose of these experiments was to evaluate the use of cyclosporine (CyS) in preventing accelerated rejection in donor-specific presensitized hosts by comparing its efficacy in donor-recipient strain combinations that were either haploidentical or completely mismatched at the major histocompatibility complex (MHC). Lewis X Brown Norway F1 (LBN) (Rt1(1)n) secondary heart allograft survival was prolonged indefinitely in CyS-treated Lewis (Rt1(1] recipients while ACI (Rt1a) grafts were ultimately rejected despite maintenance use of CyS. However, graft survival was significantly prolonged in these latter experiments with mean survival times (MSTs) of 29.4 +/- 32.1 days (CyS 10 mg/kg/day) and 19.4 +/- 21.1 days (CyS 15 mg/kg/day) compared to both untreated second-set controls (MST of 3.9 +/- 0.8 days, P less than 0.05), and untreated primary graft recipients (MST of 6.9 +/- 0.4 days, P less than 0.05). An attempt to identify suppressor cells in the long-term Lewis recipients of LBN hearts using an adoptive transfer experiment was unsuccessful when the spleen donors were still receiving CyS. Conversely, in a control experiment using spleens from CyS prolonged primary graft recipients in which splenectomy was performed after cessation of CyS, subsequent adoptive transfer did significantly prolong test heart graft survival in three of seven rats suggesting that suppressor cells may have been present. These data suggest that CyS is an effective immunosuppressant in presensitized hosts when MHC disparity is incomplete but that it cannot indefinitely prevent rejection in donor-recipient combinations that are completely mismatched at the MHC. Moreover, they suggest that splenic suppressor cells may not be present in animals concurrently receiving CyS.     

Long-term renal allograft survival in rats preimmunized with donor strain RT1.B antigens

Rat renal allograft survival was enhanced by active immunization with donor strain RT1.B (Ia) antigens. Lewis (LEW) rats (16) were immunized with Brown Norway (BN) lymphocyte extracts containing RT1.B, but not RT1.A antigens, prior to receiving (LEW X BN)F1 renal allografts. Group 1 (8 rats) was immunized with lymphocyte membrane fragments group 2(8 rats) was primed with lymphocyte supernatant extract. Longterm survivors (greater than 60 days; 12 animals) had a mean blood urea nitrogen of 75 +/- 31 mg% and serum creatinine of 2.0 +/- 0.8 mg% at one month. Death occurred in 90% of control allograft recipients within 10 days. Anti-BN RT1.B but not RT1.A antibodies were detected in sera from actively enhanced rats following immunization and at day 7 posttransplantation. We conclude that preimmunization with cell extracts containing donor RT1.B antigens has a protective effect on the allograft, and that the phenomenon of active immunologic enhancement can be produced without immunization to RT1.A antigens.     

Active enhancement of rat kidney allografts. Effect of pretreatment with prednisolone and donor-specific antigen.

(Lewis x Brown Norway) F1 hybrid rat kidney allografts were transplanted to bilaterally nephrectomized Lewis recipients pretreated in various ways. The mean survival time of untreated controls was 16.1 +/- 1.7 days. All rats pretreated with 1.67 g/kg of semi-soluble Brown Norway spleen extract and 5 mg/kg of prednisolone on days 15, 8, and 1 before transplantation survived indefinitely. Pretreatment with semi-soluble or soluble extract alone prolonged survival modestly (36.5 +/- 13.6 and 30.8 +/- 5.6 days, respectively), but the former induced indefinite survival in two of eight animals. Prednisolone on its own failed to bring about prolongation of survival and the combined use of soluble extract and prednisolone did not reveal a synergistic effect. Cytotoxic antibody titres in animals showing indefinite survival were very low, and there was no correlation between antibody titres and prolonged survival. It is assumed that the pretreatment with semi-soluble extract and prednisolone inhibited the formation of cytotoxic antibodies as well as cell-mediated immunity, and encouraged the formation of enhancing antibodies. To study the cellular and humoral reactivity of five prolonged survived kidney recipients, 1st and 2nd donor-specific skin grafts were carried out. The humoral and cell-mediated responses were somewhat delayed in these recipients but otherwise normal except for the absence of the second-set phenomenon.     

Intrathymic immunomodulation in sensitized rat recipients of cardiac allografts: requirements for allorecognition pathways.

BACKGROUND: Intrathymic injection of alloantigen in the form of donor cells, soluble major histocompatibility complex (MHC) molecules, or MHC allopeptides induces donor-specific tolerance in a variety of acute allograft rejection models. We have previously shown that a single intrathymic injection of donor spleen cells into pre-sensitized rats abrogates accelerated (circa 24-hour) rejection and prolongs the survival of cardiac allografts to about 7 days. The present study was designed to investigate the mechanisms by which intrathymic administration of donor cells modifies the course of accelerated rejection. METHODS: Lewis RT1(1) (LEW) rats sensitized by transplantation with Wistar-Furth RT1(u) (WF) skin grafts received WF cardiac allografts 7 days later-a classic model of accelerated rejection. At the time of skin challenge, however, certain animals received intrathymic cell suspensions (either allogeneic or syngeneic) or donor-derived class I and/or class II MHC peptides. RESULTS: Control animals (sensitized by skin grafts but receiving no other treatment) rejected cardiac allografts within 24 hours. Intrathymic injection of WF splenocytes at the time of skin transplantation abrogated rejection at 24 hours and prolonged cardiac allograft survival to 6.6+/-0.6 days (p<0.001), whereas intrathymic administration of syngeneic (LEW) or allogeneic third party Brown Norway RT1(n) cells was ineffective in this regard. Intrathymic injection of gamma-irradiated donor cells marginally extended cardiac allograft survival to 3.0+/-0.9 days (p< 0.001), but the grafts were still rejected in an accelerated fashion. Intrathymic injection of donor-derived class I and/or class II MHC allopeptides at the same time period also failed to prolong cardiac allograft survival beyond 3 days. In the group receiving unmodified donor cells, elevated immunoglobulin M (IgM) and immunoglobulin G (IgG) allo-antibodies were found at the time of cardiac transplantation; this pattern was not observed with any other treatment. CONCLUSION: The superiority of non-modified donor spleen cells over gamma-irradiated donor cells or donor specific allopeptides in modifying the course of accelerated cardiac rejection suggests that direct allorecognition is the dominant pathway initiating rejection in sensitized transplant recipients. Marked alterations in the antidonor IgM and IgG responses are associated with successful abrogation of accelerated rejection by thymic immunomodulatory mechanisms.     

Facial tissue allograft transplantation

A hemifacial allograft transplant model was used to investigate the rationale for development of functional tolerance across an MHC barrier. Thirty hemiface transplantations were performed in five groups of six Lewis (RT1(1)) rat recipients each. Isografts were performed in group 1. Transplants were obtained from semiallogenic LBN(RT1(1+n)) in group 2 and from fully allogenic ACI(RT1(a)) in group 3 donors, which served as allograft rejection controls. Group 4 grafts using LBN donors and group 5 using ACI donors in addition received CsA monotherapy (16 mg/kg/d for 1 week) and maintained at 2 mg/kg/d. Signs of graft rejection were sought daily. Isograft controls survived indefinitely. All nontreated allografts were rejected within 5 to 8 days posttransplant. Eighty-three percent of face-transplant recipients from LBN donors and 67% from ACI donors did not show any signs of rejection up to 270 days and 200 days, respectively. Flow cytometry at day 63 in LBN recipients showed the presence of donor-specific chimerism for MHC class I RT1(n) antigens, namely 3.39% CD4/RT1(n); 1.01% CD8/RT1(n) T-lymphocytes; and 3.54% CD45RA/RT1(n) B-lymphocytes. In ACI recipients the chimerism test revealed 10.55% CD4/RT1(a) and 4.59% of CD8/RT1(a) T-lymphocytes. MLR assay at day 160 posttransplant revealed suppressed responses against LBN donor antigens in group 4, but moderate reactivity to ACI donor antigens in group 5. Functional tolerance toward hemifacial allograft transplants induced across MHC barrier using a CsA monotherapy protocol was associated with the presence of donor-specific chimerism in T- and B-cell subpopulations.