Antibody and complement mediated injury in transplants following sensitization by allogeneic blood transfusion

BACKGROUND: Many patients on the waiting list for transplants are sensitized from previous blood transfusions, pregnancy, or transplants. We investigated the role of complement in acute and chronic pathology in hearts transplanted to sensitized rats. METHODS: Blood was transfused from allogeneic PVG.R8 rats or control isogeneic PVG.1U rats to C6-sufficient and -deficient PVG.1U rats. Three weeks later hearts were transplanted from PVG.R8 donors and low-dose cyclosporin A was initiated. RESULTS: Allogeneic but not isogeneic blood transfusion elicited strong immunoglobulin (Ig) M, IgG1 and IgG2b alloantibody responses. Sensitization caused accelerated acute rejection of cardiac allografts by C6-sufficient recipients (4 days). In contrast, allografts functioned over 40 days in all C6-deficient recipients, but sensitization caused increased interstitial fibrosis and chronic vasculopathy. Circulating alloantibodies were associated with deposits of C4d on the vascular endothelium together with pericapillary accumulation of neutrophils and macrophages in the grafts. In contrast, T cells accumulated in periarterial lymphatics that did not have C4d deposits. CONCLUSIONS: Presensitization by allogeneic blood transfusion causes accelerated acute graft rejection in the presence of the complete complement cascade. In the absence of C6, macrophages colocalized with deposits of C4d and T cells accumulated in the periarterial lymphatics.     

Accelerated graft arteriosclerosis in cardiac transplants: complement activation promotes progression of lesions from medium to large arteries

BACKGROUND: A critical role for the terminal components of complement (C5b-C9) has been demonstrated previously in acute allograft rejection with the use of C6-deficient PVG congenic rat strains. The C6 deficiency prevents the formation of membrane attack complex (MAC) by C5b-C9. Hearts transplanted from PVG.1A (RT1a) rats are rejected acutely (7-9 days) by fully MHC-incompatible C6-sufficient PVG.1L (RT11) recipients, but they survive significantly longer in untreated C6-deficient PVG.1L recipients (19 to >60 days). METHODS: To investigate the contribution of MAC to chronic rejection and accelerated graft arteriosclerosis (AGA) in long-term cardiac allografts, hearts were transplanted heterotopically from PVG.1A donors to C6-sufficient and C6-deficient PVG.1L hosts that were treated with cyclosporine 15 mg/kg/day for 14 days after cardiac grafting. Alloantibody responses in hosts were measured by flow cytometry at 4, 8, 12, and 16 weeks after transplantation. Vigorously contracting grafts were removed at 60 days (n=5) and at 90-128 days (n=12) after surgery for morphological evaluation. Computerized planimetry measurements were made in complete cross-sections of grafts on all assessable arteries larger than 16 microns in diameter. RESULTS: The survival of most (six of seven) cardiac allografts in C6-deficient recipients was prolonged by cyclosporine treatment to greater than 90 days. In contrast, 14 of 25 hearts that were transplanted to C6-sufficient recipients were rejected between 21 and 84 days with severe vascular injury. AGA, defined as smooth muscle cells forming a neointima inside the internal elastic lamina and luminal compromise, affected a greater percentage of arteries in C6-sufficient than in C6-deficient recipients. AGA developed earlier and more frequently in arteries of medium (<100 micron) diameter than those of large diameter in both C6-sufficient and C6-deficient recipients. Serial sections demonstrated the lesions in medium arteries to be located adjacent to the smooth muscle sphincters at the junction of arteriolar branches. CONCLUSIONS: These results demonstrate that MAC promotes the pathogenesis of AGA in long-term cardiac allografts.     

IgG alloantibody responses to donor-specific blood transfusion in different rat strain combinations as a predictor of renal allograft survival.

Donor-specific blood transfusion prolongs the survival of fully allogeneic ACI (RT1a) renal allografts in PVG (RT1c) recipients from 7-10 days to greater than 100 days. We have observed significant differences in the alloantibody (Ab1) responses to ACI renal allografts in control and DSBT-treated PVG recipients: DSBT is associated with decreased IgG and IgM alloantibody circulating in serum, deposited in the allograft, and produced in culture by splenocytes. In the present studies the effects of DSBT on alloantibody production and renal allograft survival were extended to examine other recipient strains: F344 (RT1lv1), BN (RT1n), W/F (RT1u) and LEW (RT1l). Animals of each recipient strain were injected i.v. with 0.5 ml of ACI blood alone or followed by a renal allograft. Studies on the kinetics of IgM and IgG alloantibody responses were performed by flow cytometry on lymphocytes from donor ACI, PVG, and PVG.R1 (RT1.Aa class I MHC antigen on PVG background) rats. In F344 and PVG rats, DSBT from ACI rats elicited a transient IgM response that peaked at day 7 and was not followed by a switch to IgG. In control PBS transfused F344 recipients, an ACI renal allograft stimulated both IgM and IgG alloantibody production. DSBT pretreatment significantly decreased circulating IgG alloantibody following ACI renal transplantation and prolonged graft survival in F344 recipients. In DSBT-treated F344 recipients that rejected ACI renal allografts acutely, small amounts of IgG (5-12 mode channel shift) were detected in sera harvested 7 days after transplantation, whereas almost no IgG was detected in the sera from DSBT treated F344 rats that accepted their renal allografts indefinitely. In contrast, DSBT alone from ACI to BN, W/F, or LEW strains elicited a transient IgM response that peaked at day 7 and was followed by a strong IgG response that peaked on days 10-14 and remained high through day 21. DSBT failed to prolong ACI renal allograft survival in any of these strains (survival less than 11 days in control and DSBT rats). The alloantibody response to DSBT in all five recipient strains examined was directed primarily to RT1.Aa class I MHC antigens, as determined by binding studies on lymphocytes from ACI, PVG and PVG.R1 rats and alloantibody blocking studies using biotinylated rat monoclonal antibodies to distinct epitopes of the RT1.Aa antigen. The relative magnitude of blocking of R2/10P and R2/15S binding by sera from BN, W/F, and LEW rats was: control allograft recipients greater than DSBT pretreated allograft recipients greater than DSBT alone.(ABSTRACT TRUNCATED AT 400 WORDS)     

Allochimeric class I MHC molecules prevent chronic rejection and attenuate alloantibody responses.

BACKGROUND: We have shown that treatment with molecularly engineered, allochimeric [alpha1 hl/u]-RT1.Aa class I MHC antigens bearing donor-type Wistar-Furth (WF, RT1.Au) amino acid substitutions for host-type ACI (RTI.Aa) sequences in the alpha1-helical region induces donor-specific tolerance to cardiac allografts in rat recipients. This study examined the effect of allochimeric molecules on the development of chronic rejection. METHODS: Allochimeric [alpha1 hl/u]-RT1.Aa class I MHC antigenic extracts (1 mg) were administered via the portal vein into ACI recipients of WF hearts on the day of transplantation in conjunction with subtherapeutic oral cyclosporine (CsA, 10 mg/kg/day, days 0-2). Control groups included recipients of syngeneic grafts and ACI recipients of WF heart allografts treated with high-dose CsA (10 mg/kg/day, days 0-6). RESULTS: WF hearts in ACI rats receiving 7 days of CsA exhibited myocardial fibrosis, perivascular inflammation, and intimal hyperplasia at day 80. At day 120, these grafts displayed severe chronic rejection with global architectural disorganization, ventricular fibrosis, intimal hyperplasia, and progressive luminal narrowing. In contrast, WF hearts in rats treated with [alpha1 hl/u]-RT1.Aa molecules revealed only mild perivascular fibrosis, minimal intimal thickening, and preserved myocardial architecture. Alloantibody analysis demonstrated no IgM alloantibodies in all groups. An attenuated, but detectable, anti-WF IgG response was present in recipients receiving allochimeric molecules, with IgG1 and IgG2a subclasses predominating. Immunohistochemical analysis of allografts demonstrated minimal T cell infiltration and IgG binding to vascular endothelium. CONCLUSION: Treatment with allochimeric molecules prevents the development of chronic rejection. Such effect may be in part caused by deviation of host alloantibody responses.     

Alloantibody-associated chronic rejection of MHC class I-disparate heart grafts is modulated by intravenous soluble donor alloantigen

Increasing evidence suggests that there may be a causal relationship between the development of donor-specific alloantibodies and chronic allograft vasculopathy (CAV). PVG.RT1(u) rat heart allografts spontaneously undergo chronic rejection when transplanted into unmodified PVG.R8 congenic recipients that differ only at the classical MHC class I RT1.A locus. Here we show that development of vasculopathy in this experimental model is associated with production of a strong anti-A(u) antibody response. Perioperative intravenous administration of recombinant soluble RT1.A(u) heavy chain that is sequence identical to donor MHC class I, or chimaeric A(u/a) (donor/recipient) protein had a variable effect resulting generally in either sensitisation and accelerated rejection, or abrogation of alloantibody and attenuation of chronic rejection. These findings highlight the potential for soluble donor MHC class I alloantigen given at the time of heart transplantation to influence alloantibody production and graft outcome.     

Terminal complement components mediate release of von Willebrand factor and adhesion of platelets in arteries of allografts

BACKGROUND: Both humoral and cellular immune responses can cause arterial injury in organ transplants, but the manifestations of these different inflammatory mechanisms have not been dissected fully. The present study was designed to define the effects of the terminal complement components on arterial injury in vivo. METHODS: The authors have developed congenic rat strains with a C6 deficiency. The absence of C6 terminates the cascade of complement after C5 cleavage and prevents the assembly of the membrane attack complex. Hearts were transplanted from PVG.1A (RT1) rats to major histocompatibility complex-incompatible C6-deficient (C6-) or C6-sufficient (C6+) PVG.1U (RT1) rats. RESULTS: PVG.1A (C6-) cardiac grafts were rejected acutely (6-7 days) by untreated PVG.1U (C6+) recipients but survived significantly longer in PVG.1U (C6-) recipients (8 to >30 days). Arteries of cardiac allografts in C6+ recipients demonstrated extensive endothelial injury evidenced by release of von Willebrand factor (vWF) and accompanied by platelet aggregation. In contrast, vWF was retained in Weibel-Palade storage granules of arterial endothelial cells in cardiac allografts that were rejected by C6- recipients. In the absence of C6, intimal alterations were limited to lifting of endothelial cells from supporting stroma by infiltrating mononuclear cells, duplicating the clinical lesion described as endotheliitis or intimal arteritis. Delaying graft rejection with a short course of cyclosporine did not decrease vWF release and platelet aggregation in PVG.1U (C6+) recipients. CONCLUSIONS: Mononuclear cell infiltration of the arterial intima occurs in the absence of C6, but C6 deficiency limits the release of vWF from arterial endothelial cells.     

Association of donor-specific blood transfusion enhancement of rat renal allografts with accelerated development of antiidiotypic antibodies and reduced alloantibody responses

Pretransplant donor-specific blood transfusion (DSBT) has been shown to enhance renal allograft survival in man and indefinitely prolong renal transplants among various MHC-disparate rat strains. Using PVG (RT1c) recipients and ACI (RT1a) donor-strain rats, DSBT alone was found to elicit complement-dependent cytotoxic IgM antibody (Ab) to donor class I (RT1.Aa) alloantigens that peaked at 7 days. An enzyme-linked immunosorbent assay was developed to measure host Ab against allospecific (idiotypic) determinants on the anti-RT1.Aa monoclonal Ab R2/10P, R2/15S, and YR1/100. Following DSBT alone, antiidiotypic Ab were detected in the circulation within 7-11 days after transfusion. Transplantation of a donor strain kidney in the presence of antiidiotypic Ab at day 7 or 11 post-DSBT resulted in enhanced graft survival and a rapid decline in circulating alloantibody, such that by days 4-6 posttransplantation little IgM or IgG alloantibody was detected. In contrast, all 6 PVG rats that were transplanted 4 days after DSBT (prior to development of detectable antiidiotypic Ab) rejected their grafts within 30 days, and 4 of 6 showed elevated alloantibody titers within 3 days posttransplantation. Control PVG rats receiving autologous blood transfusion (ABT) alone developed no alloantibody response but developed high titers of donor-specific alloantibody by 6 days posttransplantation, at the time of irreversible rejection. ABT alone did not elicit antiidiotypic Ab and ABT pretreated graft recipients developed antiidiotypic Ab only after the onset of rejection at day 4. In both DSBT and ABT groups, the antiidiotypic Ab were primarily IgM, IgG1, and IgG2c. These findings indicate that DSBT induces production of cytotoxic alloantibodies followed by an antiidiotypic Ab response at days 7-11, during which time transplanted renal allografts are not rejected and there is a reduction in circulating alloantibody. In contrast, renal allografts placed in DSBT-treated rats prior to antiidiotypic Ab development (less than or equal to 4 days) or in ABT-treated rats that do not develop any antiidiotypic Ab, elicit a rapid rise in alloantibody and are rejected.     

IgM and IgG alloantibody responses to MHC class I and II following rat renal allograft rejection. Effects of transplantectomy and posttransplantation blood transfusion.

Renal allograft rejection in rats and humans is a potent inducer of alloantibody to donor major histocompatibility complex antigens. Alloantibody in such presensitized recipients can cause hyperacute rejection of subsequent renal allografts. In order to characterize alloantibody production in rats presensitized by renal graft rejection, ACI (RT1a) kidneys were transplanted into untreated fully allogeneic PVG (RT1c) recipients and allowed to reject while one native kidney remained in situ for host survival. Serum samples collected at weekly intervals were analyzed by flow cytometry for IgM and IgG antibody binding to ACI lymphoid target cells. The specificity of alloantibody responses was assessed by (1) differential binding to congenic rat strain target cells expressing only donor class I (PVG.R1) versus both donor class I and II (PVG.1A) antigens, (2) differential binding to unseparated donor lymphoid target cells versus lymphoid target cells depleted of class II MHC antigen-expressing cells, and (3) specific blocking of monoclonal antibodies to donor class I (R2/10P, R2/15S) or class II (F17.23.2) epitopes. Alloantibody responses to both donor class I and II MHC antigens were detected. The initial IgM response to donor class I MHC antigens peaked at the time of rejection, followed by a steady decline to relatively low levels by 4 weeks posttransplantation. The IgM response to donor class II MHC antigens was found to be cyclical with apparent peaks at day 7 and 5-6 weeks. The IgG response to donor class I and class II MHC antigens reached maximum by 5-6 weeks before slowly decreasing. IgM and IgG alloantibody specific for class I and class II MHC antigens could be detected through 19 weeks posttransplantation. The effects on circulating alloantibody of two manipulations, posttransplantation donor specific blood transfusion and allograft removal, were examined in this model. The alloantibody responses to class I MHC antigens were not affected by giving DSBT weekly beginning at day 14 after transplantation. However, posttransplantation DSBT eliminated the second peak of IgM alloantibody to class II MHC antigens seen approximately 5-6 weeks posttransplantation and also decreased circulating IgG specific for class II antigens. Transplantectomy at day 5-7 days after transplantation had no apparent effect on circulating IgM or IgG alloantibody through 7 weeks posttransplantation. These data indicate that in a fully allogeneic rat renal allograft model alloantibody responses are elicited to both class I and II MHC donor antigens, but that the kinetics and regulation of the responses to class I differ from those to class II alloantigens.(ABSTRACT TRUNCATED AT 400 WORDS)     

IgM and IgG alloantibody production by splenocytes and deposition in rat renal allografts are decreased by donor-specific blood transfusion

Untreated PVG (RT1c) rats reject ACI (RT1a) renal grafts in 6-8 days. Autologous (PVG) blood transfusions (ABT) do not alter ACI allograft rejection, but donor-specific blood transfusions (DSBT) 7 or 11 days prior to transplantation usually results in indefinite graft survival. We have reported previously that DSBT is associated with the development of antiidiotypic antibody and reduced circulating cytotoxic alloantibodies in this model. To further define the effects of DSBT on the alloantibody responses to renal allografts, we examined PVG rats that received DSBT or ABT prior to ACI renal transplantation. Antibody production by cells in the spleen was investigated by tissue culture techniques; circulating antibody titers were measured by antibody binding to target ACI lymphoblasts with flow cytometry; and antibodies bound to the ACI allograft were recovered by hypertonic acid elution and quantitated by flow cytometry and ELISA. Seven days after DSBT alone, circulating IgM alloantibodies to ACI reached peak titers. After renal allografting, serum IgM alloantibody titers decreased in DSBT-pretreated rats and little IgG could be detected. In contrast, renal allografts in ABT-pretreated rats elicited high titers of IgM and moderate titers of IgG in the circulation by 5-7 days posttransplantation. Spleens harvested one week posttransplantation from ABT-pretreated rats produced high titers (16-32) of IgM and IgG antibodies to ACI antigens, but no such antibody production was detected in spleens cultured from DSBT-pretreated rats. In addition, 4-32-fold more IgM and IgG was eluted from kidneys removed 6-7 days after grafting to ABT-treated rats than from allografts in DSBT-treated rats. IgG2a was the predominant subclass of IgG that bound to target ACI cells. No IgA was detected in graft eluates from any rat. Polyacrylamide gel electrophoresis demonstrated that the eluates contained predominantly IgM and IgG without significant contamination by other serum proteins. These data suggest that DSBT decreases the levels of IgM and IgG normally produced in the spleen and deposited in the graft following renal transplantation. Because IgM fixes complement and IgG (especially IgG2a) triggers ADCC, the reduced deposition of IgM and IgG in the graft may be of particular importance in DSBT enhancement.     

Evidence that LS-2616 (linomide) causes acute rejection of rat allografts protected by cyclosporine but not of long-term surviving allografts

The immunomodulator LS-2616 (Linomide) induces rejection of cyclosporine-protected rat cardiac allografts. The aim of this study was to characterize this rejection in the presence of CsA and to test LS-2616 in other models of permanent graft acceptance in the rat. PVG rat hearts were transplanted heterotopically to Wistar/Kyoto (Wi/Ky) rat recipients on day 0. The recipients were treated orally on days 0-9 with CsA (10-40 mg/kg) and/or with LS-2616 (2.5-160 mg/kg) starting at different times (day -7 -+5) until the day of complete rejection. The addition of LS-2616 (day -1--stop) to CsA (10 mg/kg) resulted in a dose-dependent antagonism of the immunosuppressive effect of CsA with daily doses of 2.5-160 mg/kg. Furthermore, the results were similar, irrespective of whether LS-2616 treatment (160 mg/kg) was started on day -7, -1, +1, +3, or +5. LS-2616 (160 mg/kg) pretreatment of the recipient for 7 days before transplantation was considerably less effective. CsA (20 mg/kg) for 14 days after a PVG to DA transplantation resulted in permanent graft survival. This was not abrogated by LS-2616. Neither was rejection induced in long-term surviving grafts of RT1.C incompatible Lewis recipients. Our data suggest that LS-2616 activates already stimulated and sensitized T cells that are otherwise controlled by CsA.     

Identification of early tolerance regulator genes induced by allochimeric therapy using microarray-based genomewide scan

We have demonstrated that peri- or postoperative delivery of allochimeric [a1h(u)]-RT1.A(a) class I major histocompatibility complex molecules with donor-type (RT1A(u)) immunogenic epitopes presented in recipient-type (RT1A(a)) sequences induced donor-specific tolerance in ACI (RT1a) recipients of WF (RT1u) heart allografts. A genomic scan during the early posttransplant period was performed to elucidate the underlying operative mechanisms. A rat genome study after transplantation was carefully designed using Affymetrix Rat Genome 230 2.0 Array. The allochimeric treatment group is 3-day cyclosporine (CsA)-treated ACI recipients that accepted Wistar Furth RT1u cardiac allografts with postoperative dosage of allochimeric molecules, while the control is 3-day CsA-treated ACI recipients of WF cardiac allografts. All the samples were harvested 5 days after heart transplant as the early stage of tolerance detection. Following array data normalization and modeling, we compared the above two treatment groups and identified a total of 250 tolerance regulator genes induced by allochimeric molecules only.     

C4d deposition and cellular infiltrates as markers of acute rejection in rat models of orthotopic lung transplantation

BACKGROUND: C4d is a useful marker of antibody-mediated rejection in cardiac and renal transplants, but clinical studies examining correlations between circulating alloantibodies, C4d deposition, and rejection in lung transplants have yielded conflicting results. METHODS: We studied circulating alloantibody levels and C4d deposition in two rat models of lung transplantation: Brown Norway (BN) to Wistar-Kyoto (WKY) and PVG.R8 to PVG.1U lung allografts. The availability of C6 deficient (C6-) and C6 sufficient (C6+) PVG 1U rats allowed evaluation of the effects of the terminal complement components on graft injury and C4d deposition. RESULTS: The lung allografts had histologic features resembling human posttransplant capillaritis, characterized by neutrophilic infiltration of alveoli, edema, and hemorrhage. Immunoperoxidase stains on cross sections of allografts showed intense, diffuse, C4d deposition in a continuous linear pattern on the vascular endothelium. C4d deposits were found in both BN to WKY and PVG R8 to 1U allografts, whereas no staining was detectable in WKY to WKY isografts or native lungs. Complement deposition was associated with vascular disruption in C6+, but not in C6- recipients. The presence of circulating donor-specific alloantibodies was verified by flow cytometry. Cell-specific staining revealed perivascular accumulation of macrophages and T lymphocytes whereas neutrophils were sequestered in the intravascular and alveolar capillary compartments. CONCLUSIONS: The deposition of C4d on vascular endothelium as well as the coincident presence of alloantibodies is consistent with previous findings in antibody-mediated rejection of renal and cardiac transplants. Furthermore, the histological features of our allografts support the concept that posttransplant capillaritis is a form of humoral rejection.     

Zinc chloride-mediated reduction of apoptosis as an adjunct immunosuppressive modality in cardiac transplantation.

BACKGROUND: Zinc (Zn) blocks caspase-3 activation in cardiac allografts and therefore may synergistically decrease apoptosis along with cyclosporine (CsA), which inhibits mitochondrial release of cytochrome c. Simultaneous treatment of rat recipients of heterotopic heart transplants with zinc chloride (ZnCl(2)) thus may allow lower doses of CsA for immunosuppression. METHODS: PVG (RT1(c)) rat hearts were transplanted heterotopically into the abdomen of ACI (RT1(a)) rats. Group 1 (n = 15) rats received no treatment. Group 2 rats (n = 8) received 2 mg/kg/day CsA (sub-therapeutic dose) by oral gavage. Group 3 rats (n = 9) received 2 mg/kg/day oral CsA in addition to 1 mg/kg/day sub-cutaneous ZnCl(2) delivered by osmotic pump. All rats were imaged using Annexin V-bound (99m)Technetium ((99m)Tc-Annexin V) on post-operative Day 4 and subsequently killed. Annexin V avidly binds apoptotic cells in vivo. Region of interest per whole body (WB) data were calculated using the images. The allograft survival study was conducted with n = 11, 6, and 5 in control, CsA, and CsA+Zn groups, respectively. Finally, percentages of allografts that reached tolerance were measured in both CsA-only and CsA+Zn groups (n = 8 each). RESULTS: Zinc chloride had an additive effect with CsA on apoptotic blockade and graft survival. The regions of interest per WB uptake of (99m)Tc-Annexin V were 2.43% +/- 0.37%, 2.08% +/- 0.52%, and 1.49% +/- 0.29%*, and acute survivals were 6.4 +/- 1.7, 7.2 +/- 2.1, and 11.2 +/- 2.5* days for control, CsA, and CsA+Zn groups, respectively (*p < 0.001 vs controls). In addition, 87.5% of allografts became tolerant and survived for 90 days in the CsA+Zn group compared with only 37.5% in the CsA-only group (p = 0.049). CONCLUSION: Zinc-mediated reduction of apoptosis served as an effective adjunct immunosuppressive therapy to CsA in a rat model of cardiac transplantation.     

Immunohistological observations of rat fetal pancreas allografts transplanted into unmodified and cyclosporine-treated recipients

Administration of CsA (15 mg/kg/day) prolonged the survival of DA (RT1a) rat fetal pancreas transplanted to the renal subcapular site of both PVG (RT1c) and Lewis (RT1(1] recipients. Sections of fetal pancreas examined 40 days after transplantation into allogeneic CsA-treated recipients showed growth and development of the fetal pancreas tissue, and the presence of numerous insulin-containing islets. CsA treatment prevented the induction of MHC antigen within allografts. Whereas at day 4, both rejecting and CsA treated grafts showed donor class I MHC expression on duct epithelium and islet cells, only rejecting grafts displayed class I MHC induction on acinar cells. Rejecting grafts showed strong induction of class II MHC antigen expression on duct epithelium from day 4 onward but this was completely prevented by CsA treatment. Islet cells in both rejecting and CsA treated allografts remained class II-negative throughout. CsA also resulted in a reduction in the day 6 cellular infiltrate of allografts (median area leukocyte infiltrate reduced from 43% to 10%) with a marked decrease in the number of MRC OX-8-positive cells. These results show a favorable effect of CsA on rat fetal pancreas allografts with a reduction in MHC antigen expression within the graft and prolonged survival of insulin-rich endocrine tissue.     

Cyclosporine and skin allografts for the treatment of thermal injury. I. Extensive graft survival with low-level long-term administration and prolongation in a rat burn model

The hypothesis tested in the present and accompanying study is that an effective treatment for severe burns involves early excision of necrotic tissue followed by skin allografting and cyclosporine (CsA) immunosuppressive therapy. LEW (RT1) rats served as recipients of thermal injury and/or skin allografts. BN x LEW F1 (LBN, RT1(l+n)) rats served as skin donors. LEW burn recipients received a hot water (90 degrees C for 10 sec) 30% body surface area (BSA) full-thickness burn. As expected, LEW recipients treated with CsA (25 mg/kg/day for 20 days) demonstrated significant graft prolongation compared with controls (P less than 0.005). Skin graft survival was similarly prolonged in LEW recipients undergoing burn injury, primary wound excision, and CsA administration compared with burn-skin allograft controls (P less than 0.001). Mortality was not increased in the thermal injury-CsA-treated recipients compared with burn controls. A final experiment was initiated to investigate how low-level long-term (greater than 100 days) maintenance CsA treatment influenced skin allograft survival for possible future consideration in burn trauma. Recipients receiving skin allografts plus CsA (20 days, 8mg/kg/day, followed by every other day thereafter) did not reject their grafts. However, a possible early sign of rejection (a single small ulcerative lesion) was noted in five of these long-term CsA-treated animals at a mean of 34 +/- 11 (SD) days. The lesion in these animals did not progress any further during CsA administration. Histopathologic study of selected animals removed from the CsA maintenance regimen for greater than 50 days following long-term administration revealed a number of interesting chronic lesions similar to those previously reported in the skin component of composite tissue (limb) allografts following long-term low-level CsA intervention. In conclusion, CsA was very successful in preventing rejection of skin allografts in a rat burn model without apparent adverse effects.     

Are corneal cells susceptible to antibody-mediated killing in corneal allograft rejection?

PURPOSE: The precise role of antibodies in corneal transplantation is controversial. Clinical and experimental evidence both supports and refutes the contribution of donor-derived alloantibody in corneal allograft rejection. Accordingly, we prospectively evaluated the presence of donor-derived alloantibody in two high-risk donor-host combinations. We also evaluated the ability of this alloantibody to kill corneal epithelial, keratocytes, and endothelial cells in complement-dependent and complement-independent fashions. METHODS: C3H/Hej (H-2(k)) and Balb/c (H-2(d)) corneal grafts were transplanted orthotopically to CB6F1 (H-2(b/d)) and C57BL/6 (H-2(b)) recipients, respectively. These two donor-host combinations represent disparity at the entire MHC and multiple minor histocompatibility loci. Four objectives were addressed. First, we wished to determine if there was a correlation between the appearance of donor-specific serum IgG antibody and corneal graft rejection. Second, we evaluated the effect of passive transfer of hyperimmune donor-specific antibody on corneal allograft rejection. Third, we examined the capacity of donor-specific alloantibody to mediate complement-dependent cytolysis of corneal cells. Finally, we determined the capability of donor-specific alloantibody to mediate apoptosis of corneal cells. RESULTS: The presence of donor-specific serum IgG alloantibodies did not correlate with corneal graft rejection. One hundred percent of CB6F1 and C57BL/6 hosts rejected their C3H and Balb/c orthotopic corneal allografts, respectively. However, two of these seven CB6F1 hosts and one C57BL/6 host did not produce donor-specific IgG alloantibody that was significantly different from naive donors. Passive transfer of hyperimmune allo-antiserum prior to corneal transplantation did not increase the incidence, severity, or tempo of corneal allograft rejection in either donor-host combination. Hyperimmune allo-antiserum produced complement-mediated lysis of C3H corneal endothelial but not C3H corneal epithelial cells in the C3H-CB6F1 donor-host combination. Interestingly, all three corneal cell layers were vulnerable to complement-mediated cytolysis in the Balb/c-C57BL/6 donor-host combination. Additionally, Balb/c corneal epithelial, keratocytes, and endothelial cells were vulnerable to complement-independent, antibody induced apoptosis. CONCLUSIONS: Corneal graft rejection does not appear to correlate with the production of IgG alloantibody and can occur in the absence of donor-specific IgG alloantibody. Antibody-mediated killing of the corneal endothelium can occur in a complement-dependent or complement-independent fashion.     

Frequency of alloantigen-specific T cytotoxic cells in high- and low-responder recipients of class I MHC-disparate heart allografts

Presentation of an isolated class I antigeneic disparity to PVG.1U (RT1u) high-responder recipients resulted in rapid PVG.R8 (RT1.AaBuDuCu) heart allograft rejection with a median survival time (MST) of 8.25 days, but indefinite PVG.R1 (RT1.AaBcDcCc) heart allograft survival in PVG (RT1c) low-responder recipients. (PVG.R8xPVG.1U)F1 heart allografts, which express half the RT1.Aa-encoded molecules present on PVG.R8 hearts, were rejected within an MST of 14.75 days by PVG.1U recipients. Despite these in vivo differences, normal PVG.1U rats display a frequency of anti-RT1.Aa-directed T cytotoxic (fTc) cells of 1:990 in spleen and 1:1240 in LN, which are similar to the fTc found in low-responder PVG rats of 1:950 in spleen and 1:1423 in LN. On day 5 postgrafting, PVG.1U recipients of PVG.R8 hearts showed an increased fTc within graft-infiltrating T cells to 1:151, and within spleen to 1:527, but the fTc remained unchanged in LN (1:1310). Either 30 or 100 days after PVG.R8 heart allograft rejection, PVG.1U rats displayed an increased fTc in spleen (1:405) but the fTc in LN remained unchanged (1:1165). In contrast, low-responder PVG recipients that bore functional PVG.R1 heart allografts revealed no change in the splenic fTc on day 5 (1:2073) or on day 14 (1:1246) postgrafting. Additional in vitro experiments revealed further differences between high and low responders; both purified sensitized W3/25+ (CD4) and OX8+ (CD8) T cells obtained from the high PVG.1U, but not the low PVG responder strain, proliferated well in MLR culture in response to class I alloantigens. These findings suggest that the W3/25+ T cell population may participate in the induction of rejection in high-responder PVG.1U rats.     

ICOS-Ig 融合蛋白联合亚剂量环孢素A诱导小鼠移植心脏长期存活

目的 探讨可诱导共刺激分子-Ig融合蛋白(ICOS-Ig)联合亚剂量环孢素A(CsA)对小鼠移植心脏存活时间的影响及其机制.方法 自行构建ICOS-Ig.以Balb/c小鼠为供者,C57BL/6小鼠为受者,套管法制备小鼠颈部心脏移植模型,然后将模型分为5组:(1)未处理组,不做任何处理;(2)对照IgG组,移植当天以及术后第2、4、6天腹腔注射IgG 250 μg;(3)IcoS-Ig组,移植当天以及术后第2、4、6天腹腔注ICOSIg250 μg;(4)CsA组,移植当天以及术后第1～7天腹腔注射CsA 10mg/kg;(5)ICOS-Ig+CsA组,同时给予ICOS-Ig和CsA,使用时间和剂量同前.术后观察移植心脏存活时间,观察移植后第7天移植心脏的病理变化,并进行供、受者混合淋巴细胞反应(MLR),测定受者血清中供者特异性的同种抗体水平.结果 各组小鼠移植心脏存活时间分别为:未处理组(8.5±1.5)d,对照IgG(8.00.8)d,ICOSIg(29.57.7)d.CsA处理组(21.0±5.0)d,ICOS-Ig+CsA组移植心脏存活时间均超过50d,6只(6/9)移植心脏存活时间>100d,ICOS-Ig+CsA组与其他4组比较,差异均有统计学意义(P<0.01).移植后7d,未处理组及对照IgG组心肌明显变性,纤维断裂,间质水肿,肌束间及血管周围有大量炎症细胞浸润,而ICOS-Ig组和CsA组心肌无明显变性,间质略水肿,血管周围有少量淋巴细胞浸润,ICOS-Ig+CsA组的病理改变明显I(X)S-Ig组和CsA组.移植后7d,ICOS-Ig组和CsA组的脾脏淋巴细胞对同种抗原刺激反应比未处理组和对照IgG组明显降低(P<0.05),而ICOS-Ig+CsA组的抑制作用明显强于ICOS-Ig组和CsA组(P<0.05).移植后7d,ICOS-Ig组和CsA组受者血清中针对特异性供者的抗体水平明显低于未处理组和对照IgG组(P<0.05),ICOS-Ig+CsA组的抗体水平明显低于ICOS-Ig组和CsA组(P<0.05).结论 ICOS-Ig可以降低受者对供免疫反应性,延长移植心脏的存活时间,联合亚剂量CsA可使异体移植心脏长期存活.     

Increased risk of antibody-mediated rejection of reduced-size liver allografts

Because of the shortage of liver allografts in children, transplantation of reduced-size liver allografts from adult cadaveric donors or living, related donors is being done more frequently. Reduced-size liver allografts may be used in cases of ABO incompatibility and T-cell warm cross-match positivity. This experimental study in inbred rats was undertaken to determine if reduced-size liver allografts are more sensitive to antibody-mediated rejection than full-size liver allografts. Brown-Norway (BN) (RT1(n)) rats were sensitized by three successive skin grafts at 10-day intervals. Then orthotopic Lewis (LEW) (RT1(1)) liver grafts were transplanted into these BN rats. Full-size liver allografts were compared with reduced-size liver allografts (70% of donor liver). Control groups were composed of full-size and/or reduced-size isografts. Titers of specific antibodies were assayed using a complement-dependent assay before and after orthotopic liver transplantation. Histological and immunofluorescence studies (IgG, IgM, C(3), and fibrinogen deposits) were assessed. Recipients of reduced-size liver allografts died of hyperacute rejection at 36.6 +/- 4.1 h, significantly earlier than recipients receiving full-size liver allografts, which died of accelerated acute rejection at 259.2 +/- 25.2 h (P < 0.001). Either full-size or reduced-size isograft recipients survived indefinitely. A decrease in the titers of donor-specific antibodies was observed in both groups of animals. Slight deposits of IgG, IgM, C(3), and fibrinogen were observed in recipients of reduced-size liver allografts, whereas larger deposits were observed in recipients of full-size liver allografts. Our data demonstrate that there is an increased risk of antibody-mediated rejection of reduced-size liver allografts in sensitized recipients. This may have important clinical implications for partial liver grafting in cases of ABO incompatibility and T-cell warm cross-match positivity.     

Down-regulation of the immune response to histocompatibility antigens and prevention of sensitization by skin allografts by orally administered alloantigen.

The effects of oral administration of major histocompatibility antigens on the alloimmune response have not been investigated. Lymphocytes from inbred LEW (RT1u) rats that were pre-fed allogeneic WF (RT1l) splenocytes exhibited significant antigen specific reduction of the mixed lymphocyte response in vitro and delayed-type hypersensitivity response in vivo, when compared with unfed controls. In an accelerated allograft rejection model, LEW rats were presensitized with BN (RT1n) skin allografts 7 days before challenging them with (LEW x BN)F1 or BN vascularized cardiac allografts. While sensitized control animals hyperacutely reject their cardiac allografts within 2 days, animals prefed with BN splenocytes maintained cardiac allograft survival to 7 days, a time similar to that observed in unsensitized control recipients. This phenomenon was antigen-specific, as third-party WF grafts were rejected within 2 days. Immunohistologic examination of cardiac allografts harvested on day 2 from the fed animals had markedly reduced deposition of IgG, IgM, C3, and fibrin. In addition, there were significantly fewer cellular infiltrates of total white blood cells, neutrophils, macrophages, T cells, IL-2 receptor-positive T cells, and mononuclear cells with positive staining for the activation cytokines IL-2 and IFN-g. On day 6 posttransplant, the grafts from fed animals showed immunohistologic changes typical of acute cellular rejection usually seen in unsensitized rejecting controls. Feeding allogeneic splenocytes prevents sensitization by skin grafts and transforms accelerated rejection of vascularized cardiac allografts to an acute form typical of unsensitized recipients. Oral administration of alloantigen provides a novel approach to down-regulate the specific systemic alloimmune response against histocompatibility antigens.