Prolongation of rat hepatic allografts by donor splenocyte infusion and peritransplant cyclosporine treatment

This study examined the effect of pretransplant infusion of donor splenocytes and peritransplant Cyclosporine (CsA) treatment on Wistar orthotopic liver allograft survival in recipient SD rats. Treatment with either donor splenocytes or CsA significantly prolonged the survival of recipients. When pretransplant infusion of donor splenocytes combined with peritransplant use of CsA, 4/7 animals survived indefinitely (greater than 150 days). T cell subsets classification in the peripheral blood of long survivors by means of McAbs (MRC W/OX) and flow cytometer were significantly different from those in controls. The percentage of W3/25+ cells was lower and that of OX-8+ cells was higher with a decreased ratio of W3/25+/OX-8+. These results suggested the important role of suppressor T-lymphocytes in the induction of specific immunologic unresponsiveness to rat hepatic allografts.     

Prolongation of allograft survival by repeated cycles of donor antigen and cyclosporine in rat kidney transplantation

Combination therapy with a short course of cyclosporine (CsA) on the day prior to, to day of, and the day after transplantation and one dose of 5 mg 3M-KCl-extracted donor-soluble antigen (Ag) prolongs the survival of Buffalo (Buf, RT1b) kidney allografts in Wistar-Furth (WFu, RTu) inbred rats because of the induction of specific suppressor cells. Four systems were utilized to demonstrate suppressor cell activity in vivo. First, pooled lymphocytes from CsA-Ag-treated hosts suppressed the capacity of admixed, syngeneic WFu cells to display an in vivo mixed lymphocyte culture reaction toward donor Buf, but not third-party Brown-Norway (BN, RT1n), hosts. Second, systemic adoptive transfer two days prior to, or on the day of, transplantation of 5 x 10(8) putative suppressor cells harvested ten days after combined Ag-CsA treatment prolonged graft survival slightly but significantly from 7 to 9 days in virgin, secondary hosts. Third, admixture of 5 x 10(8) cells from Ag-CsA-treated hosts vitiated the capacity of 5 x 10(8) virgin WFu spleen cells to restore the capacity of recipients sublethally irradiated with 500 rads to reject. Buf allografts at 7.9 days rather than 16.7 days. Fourth, i.p. administration of low-dose cyclosphophamide (CY) 7 days after transplantation, a regimen known to inhibit suppressor cells, reduced the capacity of the Ag-CsA regimen to prolong graft survival. Two additional cycles of CsA therapy at 10-day intervals administered in an attempt to maintain T suppressor dominance over T helper cells prolonged median graft survival to 65 days. Similar prolongation was not achieved using donor blood transfusion as the immunogen, or using cycles of CsA alone. These findings suggest that 3M KCl donor antigen amplifies the induction of specific suppressor cells, and that CsA by virtue of helper T cell inhibition facilitates the establishment of suppressor cell dominance, eventually leading to host unresponsiveness.     

Synergistic effect of donor-specific blood transfusion and a short course of deoxyspergualin in rat kidney transplantation

Deoxyspergualin (DSG), an analogue of spergualin produced by B. laterosporus, has a strong immunosuppressive effect in various transplantation models. We have investigated the mechanism of donor-specific prolongation of survival time in rat kidney grafting by donor-specific blood transfusion (DST) and a short course of DSG. Lewis (LEW) kidney allografts were transplanted into fully allogeneic BN rats. Fresh, whole LEW blood 1.0 ml, was injected i.v. into BN rats 2 days prior to transplantation. Then, DSG, 6 mg/kg per day, was administered by i.m. injection on days 0, 1, and 2 after transplantation. The recipients were divided into five groups: group 1 (n=6) no treatment: group 2 (n=6) DST only; group 3 (n=7) DSG only; group 4 (n=7) DST and DSG; and group 5 (n=6), third party (ACI rats) blood transfusion and DSG. Lymphocytes (cervical lymph nodes) and serum were harvested from BN recipients on day 7 postgrafting. For suppressor cell assays, lymphocytes from BN recipients in each group were added as a third cell to the mixed lymphocyte reaction (MLC) between nontransplanted BN lymphocytes (responder) and LEW or other third party (PVGC, ACI, WKA rats) lymphocytes (stimulator). Antidonor lymphocytotoxic antibody (ADLA) was checked by microcytotoxicity assays. Median survival times (MST) for each group were: group 1, 10 days; group 1, 10 days; group 3, 13 days; group 4, 75 days; and group 5, 13 days. Remarkable prolongation of MST was only noted in group 4. In the suppressor cell assay, group 4 showed significant suppression (40%; P<0.05); the other groups did not show any suppression. This suppressive activity in group 4 was effective only during the MLC between BN and LEW, not during the MLC of third party-BN combinations. Thus, suppressor cells from DST/DSG-treated BN recipients appear to be donor-specific. In the microcytotoxicity assay, the only group that showed any ADLA was group 2, which was not treated with DSG. These results clearly show that both induction of donor-specific suppressor cells and inhibition of ADLA production are associated with the remarkable donor-specific prolongation of kidney allograft survival in DST/DSG-treated recipients.     

Long-term prolongation of cardiac allografts by subtherapeutic levels of cyclosporine in rats conditioned with pretransplant blood transfusions and cyclosporine

This study was aimed at ascertaining whether long-term graft survival was achievable with short term cyclosporine (CsA) therapy or with subtherapeutic doses of CsA in rats conditioned with blood transfusions (BT) combined with CsA. Previous studies had shown that donor-specific transfusions combined with a short course of CsA interacted synergistically, resulting in considerable prolongations of ACI and BUF grafts in LEW hosts receiving no postoperative treatment. The donor-specific depression of alloreactivity was confirmed in the present study by showing a depression of mixed-lymphocyte reaction (MLR) reactivity as well as of humoral antidonor responses in BT-CsA conditioned rats. The effects of postoperative CsA were then studied in recipients conditioned with BT-CsA or BT alone. ACI and BUF cardiac graft survival in LEW hosts conditioned with BT and treated with a five-day postoperative course of CsA (20 mg/kg/day) were indistinguishable from graft survival in untransfused hosts (ACI: 35.6 +/- 15.5 vs. 38.8 +/- 7.4; BUF: 58.4 +/- 39.8 vs. 48.0 +/- 21.7) indicating no interaction between BT and CsA under these conditions. In contrast, the effect of a post-operative five-day course of CsA (10 mg/kg/day) was extended by conditioning the recipients with donor-specific BT and CsA (ACI:41.7 +/- 7.0 vs. 27.4 +/- 11.6; P less than 0.05). More remarkably, a thirty-day course of subtherapeutic doses of CsA (2.5 mg/kg/day) resulted in long-term prolongation (greater than 100 days) of ACI grafts in a large proportion of hosts conditioned with donor-specific BT and CsA, while the majority of controls conditioned with nonspecific BT and CsA or CsA alone rejected their grafts within three weeks (P less than 0.01). The possible mechanisms of this phenomenon are discussed.     

Minimal sensitization and excellent renal allograft outcome following donor-specific blood transfusion with a short course of cyclosporine

A new protocol of donor-specific blood transfusion under cyclosporine coverage was developed and examined for immunologic consequences and clinical efficacy in recipients of one- or zero-HLA-haplotype-matched renal allografts. Between 1985 and 1989, 75 recipients were transfused with 100 ml of stored whole blood at 1, 8, and 15 days of its storage from either one-HLA-haplotype-matched related donors (n = 65, 33 from their parents, 30 from siblings, and 2 from offspring) or from zero-HLA-haplotype-matched donors (n = 10, 7 from spouses and 3 from siblings). During DST, all recipients received cyclosporine, 6 mg/kg/day, starting a day before and finishing a week after DST (23 days). Recipients were monitored by donor-specific mixed lymphocyte culture responses before and after DST, and serially for antibodies by fluorescence activated cell sorter analysis and by standard complement-dependent lymphocytotoxicity assay. Following DST with CsA, only 3 of 75 patients (4%) were sensitized against the blood donor. This rate is considerably lower, albeit statistically not significantly, compared with the 10% rate found in 30 recipients who had received DST without CsA in our previous study. Repeat MLC studied one to two months after DST (the day before transplant) were significantly increased compared with pre-DST (stimulation index: mean +/- SEM; 10.3 +/- 1.4 to 15.8 +/- 2.8, P = 0.004, and relative response: 40.9 +/- 5.1% to 49.8 +/- 5.5%, P = 0.003). Since the stimulation index with controls did not change after DST (23.4 +/- 2.9 to 26.2 +/- 3.3), enhanced MLC responses appear to be donor-specific. The changes in MLC responses did not correlate with the number of blood transfusion received prior to DST, the number of rejection episodes, or graft outcome. Fifty-seven recipients underwent a kidney transplant from their one-HLA-haplotype-matched blood donors within two to three months after DST. All 10 recipients of zero-haplotype-matched donors were also successfully transplanted from their respective blood donors. The graft survival rates were at least 90% at two years in both groups. In conclusion: (1) 100 ml of stored whole-blood DST, three times at weekly intervals with a short course of CsA is minimally sensitizing but effective in enhancing graft survival; (2) this protocol could be used in donor-recipient pairs who do not share a haplotype; and (3) DST with CsA elicits augmentation of donor-specific MLC responses.     

负载供肾抗原的致耐受性树突状细胞延长大鼠移植肾存活时间

目的探讨负载供肾抗原的致耐受性受者树突状细胞(DCs)对移植肾存活时间的影响。方法以BN大鼠为供者,Lewis大鼠为受者,Wistar大鼠为无关供者。将受者的骨髓分离培养,未经其它处理得到的DCs,将其作为DCs1;DCs1经CTLA-4Ig基因重组的复制缺陷型腺病毒(AdvCTLA-4Ig)转染后作为DCs2;DCs1先经BN大鼠供肾抗原负载后再进行AdvCTLA-4Ig转染,作为DCs3。将受者随机分为DCs1、DCs2、DCs3及对照组,每组6只。术前24h,前3组分别注入1×106个DCs1、DCs2和DCs3,对照组不注射DCs。肾移植后观察各组移植肾存活时间,术后第20d检测受者脾细胞对负载供肾抗原及无关供者肾脏抗原DCs的反应。结果DCs1、DCs2,DCs3和对照组的移植肾存活时间分别为(8.1±0.8)d、(8.8±0.9)d、(62.1±13.7)d和(8.6±0.9)d。DCs1、DCs2组与对照组比较,差异无统计学意义(P>0.05),DCs3组与对照组比较,移植肾存活时间显著延长(P<0.01)。肾移植术后,DCs3组脾细胞对负载供肾抗原的DCs反应明显低于对照组(P<0.01),而对负载无关供者抗原的DCs反应与对照组比较,差异无统计学意义(P>0.05)。结论负载供肾抗原的受者DCs经CTLA-4Ig基因修饰后具有致耐受性,可以抑制受者对供者抗原的免疫反应,明显延长移植肾的存活时间。     

Prolongation of skin allograft survival by combined feeding of donor spleen cells and cyclosporine in mice

BACKGROUND: Oral immune tolerance is a method for inducing donor-specific immunotolerance and prolonging graft survival. OBJECTIVES: We studied the effect of feeding donor spleen cells in combination with cyclosporine (CsA) on skin allograft survival in mice. METHODS: Tail skins from BALB/c (H-2d) female mice were transplanted onto C57BL/6 (H-2b) female mice. The animals were divided into four groups, each with eight mice: group I, untreated controls; group II, treated with spleen cells; group III, treated with CsA; and group IV, treated with spleen cells and CsA. All grafts were inspected daily. Rejection was diagnosed when the graft loss was >80% to 90%. The immune responses of C57BL/6 toward donor mice were examined by delayed-type hypersensitivity (DTH). RESULTS: Survival times of allogeneic skin grafts in groups I, II, III, and IV were 9.9 +/- 0.6, 13.1 +/- 0.6, 14.7 +/- 0.9, and 20.0 +/- 0.7 days, respectively. When compared with group I, the survival times of groups II, III, and IV were prolonged significantly (P < .01). The survival time for group IV was prolonged significantly compared with groups II and III (P < .01). The DTH responses of group IV were decreased significantly in contrast to groups II and III (P < .01). CONCLUSIONS: Feeding donor spleen cells prolonged the survival of skin allografts in mice; combination with CsA led to further prolongation of skin allograft survival.     

Evidence that pretransplant donor blood transfusion prevents rat renal allograft dysfunction but not the in situ cellular alloimmune or morphologic manifestations of rejection

The effects of preoperative donor-specific blood transfusion (DSBT) on the physiologic, morphologic, and immunologic aspects of allograft responsiveness were evaluated in a rat renal transplant model, using the ACI (RT1a) into PVG (RT1c) high-responder strain combination. Indefinite graft survival (mean greater than 63 days) could be induced by DSBT administration alone. In comparison, animals receiving autologous blood transfusion (ABT) all died within 7 days posttransplantation. As assessed by clearance of inulin and paraaminohippurate, renal allograft function in DSBT-pretreated recipients at 6 days was equivalent to that of isograft recipients, and in contrast to the significant reduction seen in ABT treated rats. Likewise, thromboxane B2 (TXB2) production by ex-vivo-perfused allografts from DSBT-treated recipients was comparable to that of isografts, and significantly lower than that of allografts from ABT-treated rats. A significant inverse correlation was found between renal TXB2 production and inulin clearance. Despite these substantial differences in renal function and eicosanoid metabolism, morphologic evaluation of renal allografts from DSBT-enhanced and ABT-rejecting recipients at comparable time points showed equivalent histologic manifestations of rejection. In addition, immunohistologic labeling of renal allograft sections and fluorescence-activated cell sorter analysis of cells eluted from allografts showed the same phenotype and pattern of infiltrating T cell subsets in both groups. Specific antidonor cytotoxic T lymphocyte precursor (pCTL) frequencies of cells eluted from kidney grafts were equivalent in DSBT and ABT-pretreated animals, and both groups expressed significantly higher (but equivalent) pCTL frequencies in the kidneys than spleens. Comparisons of the lysis of PVG.R1 (RT1.Aa on a PVG background) and ACI targets indicated that cytotoxic responses from effector cells freshly eluted from DSBT and ABT kidneys were primarily directed against allogeneic class I major histocompatibility complex (MHC) specificities, whereas several long term T cell lines generated from 6-day kidney transplants of both groups expressed a predominant W3/25+ (T helper) phenotype and cytotoxic activity against donor specificities other than RT1.Aa class I MHC. Specific antidonor proliferative T lymphocyte (pPTL) precursor frequencies of cells eluted from renal allografts were also equivalent for both DSBT- and ABT-treated recipients, and the range of pPTL frequencies for allograft cell eluates was similar to that in spleens, regardless of the source of the transfusion.(ABSTRACT TRUNCATED AT 400 WORDS)     

Different patterns of donor MHC antigen induction in rat kidney allografts following active and passive enhancement

We compare the expression of donor class I and class II major histocompatibility complex antigens in DA kidney grafts transplanted to PVG recipients treated by different protocols of donor-specific immunosuppression. MHC expression was evaluated using donor-specific antibodies and assays by immunohistology and quantitative absorption analysis. PVG recipients were either untreated or treated by (A) twice-weekly intravenous injections of 0.5 ml DA blood for 12 weeks; (B) 0.5 ml DA blood intravenously at 7 days pregraft; (C) as for (B), but with the addition or oral cyclosporine at 10 mg/kg/day from the day of grafting; and (D) passive enhancement with DA anti-PVG serum. Grafts were assessed at 3, 5, and 7 days after transplantation. In untreated controls at day 3, there is a periarteriolar leukocyte infiltrate, weak or absent class II induction, but strong class I induction. Class II induction in untreated controls is maximal at day 5. We confirm that active enhancement by blood transfusion, even using the intensive protocol of twice-weekly transfusions for 3 months, results in accelerated leukocyte infiltration and accelerated donor class I and class II MHC induction. At day 3, there is an intense, diffuse leukocyte infiltration and maximal class II induction. Cyclosporine treatment of blood-transfused recipients reduced the leukocyte infiltration and MHC induction to levels seen in untreated controls--i.e., the accelerated MHC induction caused by the transfusion was partially reversible by cyclosporine. In passively enhanced recipients, leukocyte infiltration and class I MHC induction were similar to untreated controls. However, class II induction was much delayed, not being evident until day 7.     

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

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

Long-term results of donor-specific blood transfusion with cyclosporine in living related kidney transplantation

Donor-specific blood transfusion (DST) was introduced to achieve better graft survival. However, its benefits are controversial considering the immunosuppression of cyclosporine (CYA) or tacrolimus (Tac), and its long-term effects have not been well discussed. Of the 40 patients who received DST with CYA, 3 (7.5%) became cross-match positive. Of the 37 patients with negative cross-match, 34 patients received a one-haplotype-matched kidney and were compared to patients with one-haplotype-matched kidney transplant without preoperative DST (n = 13). Acute rejection within 3 months after transplant was 29.4% in the DST group, and 15.4% in the non-DST group. All rejection episodes were steroid resistant in the non-DST group. If the graft survival rates were calculated excluding non-immunological graft loss, graft survival rate was 91.0 and 72.8% at 5 and 10 years in the DST group, and 83.3% at 5 and 10 years in the non-DST group, respectively. The two graft survival lines converged 7 years and 7 months after transplantation. No beneficial effect of DST was statistically evident under CYA immunosuppression. In terms of the severity of acute rejection or the onset of chronic rejection, DST induced a small benefit, however, which seemed to disappear within 8 years after transplantation.