Thymectomy impairs but does not uniformly abrogate long-term acceptance of semi-identical liver allograft in inbred miniature Swine temporarily treated with FK506

BACKGROUND: Long-term acceptance of semi-identical orthotopic liver transplants (OLTs) in inbred swine is induced by a 12-day course of FK506. To study whether acceptance is attributable to central or peripheral immune mechanisms, the effect of complete thymectomy was determined. METHODS: Total thymectomy was performed in 15 swine 3 to 4 weeks before OLT. Twelve of these animals received a 12-day course of FK506 after OLT, and three animals did not receive immunosuppression. Five additional nonthymectomized pigs received OLT and a FK506 regimen. Graft survival, liver function, histology, and cellular and humoral responses were assessed. RESULTS: Nonthymectomized, FK506-treated animals uniformly showed long-term acceptance of OLT and developed stable donor unresponsiveness. Of the 12 thymectomized, FK506-treated pigs, seven died of non-immunologic causes within 3 postoperative months, and five maintained their OLT for more than 6 months (range 180-450 days). Among these survivors, two developed a complete anti-donor response (mixed lymphocyte reaction [MLR], cell-mediated lymphocytotoxicity [CML], and immunoglobulin [IgG] antibodies) and eventually rejected their OLT at postoperative day 180. The three remaining pigs kept their liver allografts up to 450 days and developed a donor-specific unresponsiveness (a transient anti-donor MLR was observed during the follow-up but never an anti-donor CML or IgG antibodies). All three thymectomized, untreated animals rejected their allografts acutely and displayed a complete anti-donor response (MLR, CML, and IgG antibodies). CONCLUSIONS: Complete thymectomy before OLT impaired but did not uniformly abrogate long-term acceptance of semi-identical OLT, suggesting that peripheral immune mechanisms may be sufficient to induce long-term acceptance of liver allografts in some recipients.     

A 12-day course of FK506 allows long-term acceptance of semi-identical liver allograft in inbred miniature swine

BACKGROUND: Spontaneous tolerance to liver allograft has been reported previously in outbred pig models, but the lack of genetic background did not allow to analyze the impact of the major histocompatibility complex (MHC) on tolerance induction. A model of semi-identical liver allograft was therefore developed in inbred miniature swine in order to mimic the clinical situation of living related liver transplant (parent into infant) and to study a protocol for inducing tolerance to liver allograft. METHODS: SLAdd (class Id/d, class IId/d) pigs received orthotopic liver allograft from heterozygous SLAcd (class Ic/d, class IIc/d) miniature swine. Eight animals did not receive immunosuppression. Fourteen SLAdd animals had a 12-day course of FK506 and were divided in two subgroups. In subgroup FK-1, six pigs received a daily intramuscular injection of FK506 at 0.1-0.4 mg/kg, in order to reach daily trough levels between 7 and 20 ng/ml; in subgroup FK-2, eight additional animals received two daily injections of FK506 at 0.05 mg/kg regardless of the daily trough levels. Graft survival, liver biological tests, histology, cellular and humoral immune responses, as well as detection of microchimerism were assessed in all groups. RESULTS: All untreated animals rejected their allograft and died within 28.1 +/- 9.5 days. These rejector animals developed a significant anti-donor cellular and humoral immune response. No peripheral or lymphoid tissue microchimerism was detected in this group. In contrast, long-term survival was obtained in five FK-treated animals (112, 154, 406, 413, and 440 days), whereas several pigs died with a normal allograft function from either overimmunosuppression or intercurrent causes. All FK-treated pigs developed a specific anti-donor unresponsiveness in both cell mediated lymphocytotoxicity and mixed lymphocyte reaction and did not develop anti-donor alloantibodies. The study of the anti-donor immune response by mixed lymphocyte reaction, during the first postoperative week, demonstrated a specific anti-donor unresponsiveness in the peripheral blood from the first posttransplant day. Although microchimerism was detectable in the peripheral blood for several postoperative weeks (maximum 10 weeks) in FK-treated animals, donor cells or DNA were not detected during the long-term follow-up in peripheral blood or lymphoid tissues. CONCLUSIONS: Spontaneous tolerance to semi-identical orthotopic liver allograft did not occur, whereas a 12-day course of FK506 allowed long-term graft acceptance. All FK-treated animals developed in vitro signs of specific immune unresponsiveness and transient peripheral microchimerism. The specific anti-donor cellular unresponsiveness occurred on the first postoperative day after surgery and was of long-term duration. The study of the early immunological events in this model could be of major importance regarding clinical living related liver transplantation.     

Spontaneous acceptance or rejection of orthotopic liver transplants in outbred and partially inbred miniature swine.

BACKGROUND: Results of clinical liver transplantation have shown that rejection and loss of human liver allografts occurs despite immunosuppression. Because genetic disparity and liver immunogenicity remain a matter of controversy, we reexamined the fate of outbred liver allografts without immunosuppression and used partially inbred miniature swine, in which the genetics of major histocompatibility complex (MHC) antigens have been characterized and can be controlled. METHODS: Orthotopic liver transplantation was performed between pairs of outbred domestic farm pigs and between pairs of inbred miniature swine with genetically defined major histocompatibility (SLA) loci. A passive splenic and vena caval to jugular vein shunt with systemic heparinization prevented hypotension during the anhepatic phase. Immunological responses were monitored by mixed lymphocyte culture (MLC), CML, skin graft rejection, liver biopsies, and serial serum chemistries. RESULTS: Median survival of technically successful liver allografts between pairs of outbred pigs (n=20) was 38 days and between partially inbred swine matched at the SLA locus (n=17) was 79 days. MLC responsiveness did not correlate with the development of rejection. Five of 20 (25%) outbred pigs and 6 of 17 (35%) MHC matched inbred miniature swine survived more than 100 days. In the long-term survivors, donor, but not third party, MHC matched skin graft survival times were prolonged. In contrast, all SLA-mismatched inbred recipients (n=26) died rapidly from massive liver rejection, with a median survival time of 9 days. In these rejecting animals, the marked MLC responsiveness to donor lymphocytes evident pretransplant diminished rapidly after transplantation, but an undiminished PHA responsiveness and a blunted third party MLC response persisted. CONCLUSION: The length of survival and the degree and incidence of rejection were similar in outbred pigs and in SLA-matched inbred miniature pigs, indicating that the outbred animals were, therefore, probably closely related and shared relevant genes. However, survival was significantly shortened and liver allograft rejection was accelerated in SLA-mismatched inbred swine. These results indicate that major histocompatibility differences play an important role in the rejection of liver allografts, as is true for other vascularized grafts in the unimmunosuppressed recipient. The development of liver allograft rejection across non-MHC differences is variable and, when present, appears to be a chronic process.     

Kidney‐Induced Cardiac Allograft Tolerance in Miniature Swine is Dependent on MHC‐Matching of Donor Cardiac and Renal Parenchyma

Kidney allografts possess the ability to enable a short course of immunosuppression to induce tolerance of themselves and of cardiac allografts across a full‐MHC barrier in miniature swine. However, the renal element(s) responsible for kidney‐induced cardiac allograft tolerance (KICAT) are unknown. Here we investigated whether MHC disparities between parenchyma versus hematopoietic‐derived “passenger” cells of the heart and kidney allografts affected KICAT. Heart and kidney allografts were co‐transplanted into MHC‐mismatched recipients treated with high‐dose tacrolimus for 12 days. Group 1 animals (n = 3) received kidney and heart allografts fully MHC‐mismatched to each other and to the recipient. Group 2 animals (n = 3) received kidney and heart allografts MHC‐matched to each other but MHC‐mismatched to the recipient. Group 3 animals (n = 3) received chimeric kidney allografts whose parenchyma was MHC‐mismatched to the donor heart. Group 4 animals (n = 3) received chimeric kidney allografts whose passenger leukocytes were MHC‐mismatched to the donor heart. Five of six heart allografts in Groups 1 and 3 rejected <40 days. In contrast, heart allografts in Groups 2 and 4 survived >150 days without rejection (p < 0.05). These data demonstrate that KICAT requires MHC‐matching between kidney allograft parenchyma and heart allografts, suggesting that cells intrinsic to the kidney enable cardiac allograft tolerance.     

Posttransplant lymphoproliferative disorder after liver transplantation in miniature swine.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) is a well-known, life-threatening complication of immunosuppressive therapy, occurring in both adult and pediatric transplant recipients. METHODS: To study the effect of major histocompatibility complex on tolerance induction to primarily vascularized liver allograft, a semi-identical miniature swine model was developed to mimic the clinical situation of parent-into-infant liver transplantation. Long-term acceptance of semi-identical liver allograft was obtained by a transient course of FK506. In a subgroup of six animals, three developed a lethal PTLD. These animals were studied by histology and immunohistochemistry and the anti-donor cellular immune response was assessed. In addition, the possible viral origin of the proliferative process was evoked. RESULTS: Histology and immunohistochemistry revealed an abnormal B-cell proliferation in many organs of swine suffering from PTLD. Evidence of human Epstein-Barr virus, cytomegalovirus, and adenovirus was not evidenced, but a porcine virus responsible for a respiratory and reproductive syndrome (PRRS) was identified in the lymphoid tissue of these animals. In mixed lymphocyte reaction, a significant antiself immune response confirmed an infection by a virus. CONCLUSIONS: This is the first report suggesting that PRRS virus might provoke PTLD in immunosuppressed miniature swine after orthotopic liver transplantation. Whether PTLD could be induced by injection of the PRRS virus in immunosuppressed animals, a pig model of PTLD might be developed and would represent an interesting preclinical model for testing anti-PTLD therapies.     

Long-term acceptance of renal allografts following prenatal inoculation with adult bone marrow

BACKGROUND: The aim was to investigate if intravascular in utero injection of adult bone marrow into swine fetuses could lead to macrochimerism and tolerance to the donor. METHODS: Outbred Yorkshire sows and boars screening negative for MHC allele SLA of MGH miniature swine were bred. A laparotomy was performed on the sows at 50 days gestation to expose the uterus. Bone marrow harvested from SLA miniature swine was T-cell depleted and injected intravascularly into seventeen fetuses. Flow cytometry was performed to detect donor cells (chimerism) in the peripheral blood after birth. Mixed lymphocyte reactions (MLR) and cell-mediated lympholysis (CML) assays were used to assess the response to donor MHC. Previously frozen skin grafts from the bone marrow donor were placed on the offspring from the first litter. Donor-matched renal transplant from SLA donors were performed on chimeric swine, with and without a short 12-day course of cyclosporine, and one nonchimeric littermate. RESULTS: Nine inoculated offspring demonstrated donor cell chimerism in the peripheral blood and lymphohematopoietic tissues. All animals with detectable chimerism within the first three weeks were consistently nonreactive to donor MHC in vitro. Animals challenged with donor skin grafts displayed prolonged graft survival without producing antidonor antibodies. All chimeric animals accepted donor-matched kidney allografts, even one without cyclosporine. The kidney in the nonchimeric littermate rejected by day 21. CONCLUSIONS: Transplantation of allogeneic adult bone marrow into immunocompetent fetal recipients resulted in chimerism. In utero inoculation led to operational tolerance to the donor's major histocompatibility antigens and long-term acceptance to organ allografts.     

Recipient bone marrow engraftment in donor tissue after long-term tolerance to a composite tissue allograft

BACKGROUND: An important component of a composite tissue limb allograft (CTA) is the vascularized bone marrow and bone marrow stroma, which when transplanted could create immediate marrow space and engraftment. We have previously demonstrated that tolerance to musculoskeletal allografts can be achieved with a 12-day course of cyclosporine without the presence of long-term peripheral donor cell chimerism. The objective of this study was to determine the fate of the donor bone marrow after transplantation of a limb allograft in a miniature swine model. METHODS: CTAs from donor swine were heterotopically transplanted into six MHC-matched, minor-antigen-mismatched recipients, and a 12-day course of cyclosporine was given. Previous animals transplanted without cyclosporine rejected their grafts in less than 42 days. A non-MHC-linked marker, pig allelic antigen (PAA), was used to distinguish host and donor cells. Three PAA- animals received PAA+ CTAs, and three PAA+ animals received PAA- CTAs. Bone marrow was harvested from the donor limb grafts and the recipient and analyzed by flow cytometry and histology. Thymus, spleen, and mesenteric lymph nodes were also harvested from the recipient swine and evaluated for the presence of donor cells by flow cytometry. RESULTS: All animals receiving cyclosporine demonstrated permanent tolerance to their allografts. Donor bone marrow cells were present in all grafts at the time of transplantation and during the immediate postoperative period. By 48 weeks, donor cells were no longer detectable within the marrow space of the allograft. In long-term animals host bone marrow cells replaced donor cells in the graft marrow space. No evidence of donor cell engraftment was found in recipient animals. CONCLUSION: This study demonstrates that in long-term tolerant recipients of musculoskeletal allografts there is no evidence of persistent donor bone marrow cells in the hematopoietic tissues of the graft or the host. Rather, the recipient's bone marrow cells and lymphocytes repopulate the donor marrow space of the graft.     

The effect of thymectomy on tolerance induction and cardiac allograft vasculopathy in a miniature swine heart/kidney transplantation model.

BACKGROUND: We have previously demonstrated that MHC class I disparate hearts transplanted into miniature swine treated with a short course of cyclosporine developed florid cardiac allograft vasculopathy (CAV) and were rejected within 55 days. However, when a donor-specific kidney is cotransplanted with the heart allograft, recipients become tolerant to donor antigen and accept both allografts long-term without the development of CAV. In the present study, we have investigated the role of the host thymus in the induction of tolerance and prevention of CAV in heart/kidney recipients. METHODS: Total thymectomies were performed in six animals (postoperative day [POD]-21), which on day 0 received either an isolated MHC class I disparate heart allograft (n=3) or combined class I disparate heart and kidney allografts (n=3), followed in both cases by a 12-day course of cyclosporine (POD 0-11). Graft survival and the development of CAV in these thymectomized recipients were compared to the same parameters in non-thymectomized, cyclosporine-treated recipients bearing either class I disparate heart allografts (n=5) or heart and kidney allografts (n=4). RESULTS: In the group of animals bearing isolated class I disparate heart allografts, the thymectomized recipients rejected their allografts earlier (POD 8, 22, 27) than the non-thymectomized recipients (POD 33,35,45,47,55). The donor hearts in both the thymectomized and non-thymectomized animals developed florid CAV. In the group of animals bearing combined class I disparate heart and kidney allografts, the nonthymectomized recipients accepted both donor organs long term with no evidence of CAV. In contrast, none of the thymectomized heart/kidney recipients survived >100 days, and they all developed the intimal proliferation of CAV. CONCLUSION: Thymic-dependent mechanisms are necessary for the induction of acquired tolerance and prevention of CAV in porcine heart/kidney recipients.     

The graft helps to define the character of the alloimmune response

Introduction: In mice, kidney and liver allografts may be spontaneously accepted, whereas cardiac and skin allografts in the same strain combinations are rapidly rejected. The reasons for this dichotomy in murine response outcomes remains to be determined. Methods and results: When DBA/2 (H-2d) cardiac allografts were placed in C57BL/6 (H-2b) recipients, they were rejected within 10 days, unless the allograft recipients were transiently treated with gallium nitrate (GN), at which time the allografts were accepted for > 150 days. The cardiac allograft rejector mice displayed DBA/2-reactive DTH responses, whereas the cardiac allograft acceptor mice displayed both TGFbeta- and IL10-mediated inhibition of DTH responses. In contrast, DBA/2 kidney allografts placed at the same location in C57BL/6 mice were spontaneously accepted without immunosuppression. These kidney allograft acceptor mice displayed TGFbeta-mediated, but not IL10-mediated inhibition of donor-reactive DTH responses. Conclusions: In the DBA/2-> C57B1/6 strain combination, cardiac allografts induce pro-inflammatory immunity and allograft rejection, while kidney allografts induce anti-inflammatory immunity and allograft acceptance despite the fact that both organs display the same strong MHC disparities and are implanted at the same location. Anti-inflammatory immunity and allograft acceptance are displayed by cardiac allograft recipients when they are transiently treated with select immunosuppressants. Thus, multiple immune response options are available to the organ allograft recipient, and the choice is determined, to some degree, by the allograft, itself.     

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

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

Induction of tolerance to heart transplants by simultaneous cotransplantation of donor kidneys may depend on a radiation-sensitive renal-cell population

BACKGROUND: To determine the mechanism by which cotransplantation of a donor kidney and heart allograft induces tolerance to both organs in miniature swine, we examined the renal elements responsible for tolerance induction. METHODS: Recipients received 12 days of cyclosporine, and transplants were performed across a major histocompatibility complex (MHC) class I mismatch. Group 1 animals received heart transplants (n=5); group 2 animals received heart and kidney allografts with no other manipulation (n=4); group 3 animals received heart transplants and donor-specific renal parenchymal cells (n=4); group 4 animals received heart and kidney allografts from lethally irradiated donors (n=7); group 5 animals received irradiated hearts and nonirradiated kidneys (n=2); group 6 animals received nonirradiated hearts and peripheral blood leukocytes from swine MHC matched to recipients and becoming tolerant to donor antigen (n=2); group 7 animals received nonirradiated hearts and donor-specific peripheral blood monocyte cells (PBMC) (n=2). RESULTS: Animals in group 1 developed vasculopathy and fulminant rejection by day 55. Animals in group 2 never developed vascular lesions. Parenchymal kidney cell infusion (group 3) did not prolong cardiac survival. Animals in group 4 developed arteriopathy by postoperative day (POD) 28. Group 5 recipients accepted allografts without vascular lesions. Adoptive transfer of leukocytes from tolerant swine (group 6) prolonged cardiac graft survival as much as 123 days, whereas donor PBMC infusion (group 7) did not affect cardiac survival or development of arteriopathy. CONCLUSIONS: Radiosensitive elements in kidney allograft may be responsible for tolerance induction and prevention of chronic vascular lesions in recipients of simultaneous heart and kidney allografts.     

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.     

Humoral immune responses during acute rejection in rat lung transplantation

The detailed responses of humoral immunity during acute rejection remain obscure in lung transplantation (LTx). In order to clarify the reactions of alloantibodies (allo-Abs) during acute rejection, we demonstrated the time-course of changes in anti-donor Ab reaction in the peripheral blood and deposition in the grafts using a rat LTx model. Lewis (LEW) rats served as recipients for Brown Norway (BN) lung allografts (MHC fully incompatible combination). The left lung was transplanted orthotopically using a cuff technique. Syngeneic transplants (LEW to LEW) served as control. No immunosuppression therapy was administered in this model. We evaluated the alloreactivity against donor in rat recipients by detecting allo-Abs with a flow cytometric cross-match (FCXM) technique. Recipient serum samples were incubated with donor lymphocytes and stained with anti-rat immunoglobulin (Ig), to determine the titers of circulating allo-Abs in the peripheral blood with a three-color FCXM technique. We also examined the deposition of anti-donor Abs (IgG and IgM) in the grafts with an immunofluorescent method. All allografts were completely rejected and lost their aeration within 6 days after LTx. Strong allo-Abs responses of both IgG and IgM were observed in the peripheral blood during acute rejection. The level of IgM allo-Abs had already significantly increased on day 2 at the time of mild rejection; however, IgG Abs did not elevate until day 6, when the grade of rejection was severe. Circulating IgM levels started decreasing on day 8, whereas IgG Abs continued elevating. On the other hand, no evident deposition of allo-Abs in the grafts was observed until day 6. We have shown in this study that circulating IgM allo-Abs was detected at the time of mild allograft rejection, interestingly, before evident deposition in the graft. It might be suggested that allograft rejection progressed without antibody deposition until severe rejection.     

Tolerance to composite tissue allografts across a major histocompatibility barrier in miniature swine

BACKGROUND: Tolerance to composite tissue allografts might allow the widespread clinical use of reconstructive allotransplantation if protocols to achieve this could be rendered sufficiently nontoxic. The authors investigated whether tolerance could be generated in miniature swine to composite tissue allografts across a major histocompatibility (MHC) barrier. A clinically relevant tolerance protocol involving hematopoietic cell transplantation without the need for irradiation or myelosuppressive drugs was tested. METHODS: Seven recipient animals were transiently T-cell depleted and a short course of cyclosporine was initiated. Twenty-four hours later, a donor hematopoietic cell transplant consisting of cytokine-mobilized peripheral blood mononuclear cells or bone marrow cells and a heterotopic limb transplant were performed. In vitro anti-donor responsiveness was assessed by mixed-lymphocyte reaction and cell-mediated lympholysis assays. Acceptance of the limb allografts was determined by gross and histologic appearance. Chimerism in the peripheral blood and lymphohematopoietic organs was assessed by flow cytometry. RESULTS: All seven experimental animals accepted the musculoskeletal elements but rejected the skin of the allografts. All but one of the animals displayed donor-specific unresponsiveness in vitro. The animals that received cytokine mobilized-peripheral blood mononuclear cells showed chimerism but had clinical evidence of graft-versus-host disease (GVHD). None of the animals that received bone marrow cells showed stable chimerism and none developed GVHD. CONCLUSIONS: This protocol can achieve tolerance to the musculoskeletal elements of composite tissue allografts across an MHC barrier in miniature swine. Stable chimerism does not appear to be necessary for tolerance and may not be desirable because of the risk of GVHD.     

Irradiation before and donor splenocyte infusion immediately after transplantation induce tolerance to lung,but not heart allografts in miniature swine

Solid organs may differ in their potential to induce and maintain a state of donor‐specific tolerance. Previously, we induced stable immunological tolerance in a lung transplantation model in miniature swine. Here, we wished to transfer this established protocol into a heart transplantation model in miniature swine. Heterotopic heart transplantation (HTX) was performed in four and left‐sided lung transplantation (LTX) in seven minipigs from gender‐ and SLA‐mismatched donors. All recipients received nonmyeloablative irradiation, donor splenocyte infusion and intravenous pharmacologic immunosuppression for 28 postoperative days. All transplanted hearts were rejected within 95 days. In contrast, four animals of the LTX group developed stable tolerance surviving beyond 500 days, and three further animals rejected 119, 239 and 360 days post‐transplantation. In both groups, peripheral blood donor leucocyte chimerism peaked 1 h after reperfusion of the allograft. Importantly, the early chimerism level in the LTX group was significantly higher compared to the HTX group and remained detectable throughout the entire observation period. In conclusion, lungs and hearts vary in their potential to induce a state of tolerance after transplantation in a protocol with pre‐operative recipient irradiation and donor splenocyte co‐transplantation. This could be due to differential early levels of passenger leucocyte chimerism.     

Humoral Rejection of Organ Allografts

In recent years, the deleterious clinical consequences of recipient de novo alloantibody production against HLA antigens from human organ allografts have been extensively investigated. In kidney transplantation, the identification of the complement C4d fragment in peritubular capillaries as a specific marker for humoral rejection has helped to define and characterize distinct clinical alloantibody-mediated syndromes. This knowledge is relevant for patient management as new therapeutic strategies to remove and control anti-donor antibody production, particularly in the setting of acute humoral rejection, have been reported. For recipients of nonrenal organ allografts such as heart transplant recipients, de novo anti-HLA alloantibody may also be important, although more studies are needed before clear guidelines can be proposed.     

The antibody crossmatch in liver transplantation

Six hundred sixty-seven first, second, and third orthotopic liver allografts in 520 patients were reviewed to determine the effect of recipient panel-reactive antibody (PRA) and donor-recipient antibody crossmatch on 2-year patient and liver allograft survival rates. Neither a high panel-reactive antibody nor a positive crossmatch for donor-specific preformed antibody was associated with decreased patient or liver allograft survival for primary grafts or retransplants. Two patients have been given kidney transplants immediately after a liver allograft from a donor with whom each patient had an initial strongly positive donor-specific antibody crossmatch. The liver apparently removed or neutralized circulating anti-donor antibody, since the renal allografts functioned promptly and did not experience hyperacute rejection.     

Monitoring antidonor alloantibodies as a predictive assay for renal allograft tolerance/long-term observations in nonhuman primates

BACKGROUND: In an effort to define reliable assays that might predict postimmunosuppressant-withdrawal development of chronic rejection (CR), despite conditioning for tolerance induction, we evaluated various immunological responses in nonhuman primate renal allograft recipients. METHODS: Fourteen Cynomolgus monkeys received low dose total body irradiation, thymic irradiation, antithymocyte globulin, and peritransplant CD154 blockade, followed by a one-month course of cyclosporine. Recipients underwent major histocompatibility complex mismatched kidney transplantation with donor bone marrow infusion (Group A, n=8), without donor cell infusion (Group B, n=2), or with donor splenocyte infusion (Group C, n=4). RESULTS: All Group A recipients developed mixed chimerism and four of them survived long-term without rejection. The remaining four rejected their kidney allografts either chronically or acutely. All recipients in Groups B and C failed to develop chimerism and rejected their allografts. Among various in vitro assays, detection of anti-donor alloantibody (ADA) by flow cytometry (FCM) was the most relevant to long-term outcome. All five recipients that developed both anti-T cell and B cell IgG ADA in Groups A, B and C, developed histological evidence of CR within 200 days of the appearance of ADA. One of two recipients that developed only anti-B cell IgG ADA eventually developed CR over two years following discontinuation of immunosuppression and 1.5 years after ADA development. Another recipient with very low anti-B cell ADA has never developed CR. CONCLUSION: ADA monitoring with FCM assay appears to be useful in predicting the failure of tolerance prior to the development of functional or histologic abnormalities of the renal allograft.     

Detection of regulatory cells as an assay for allograft tolerance in miniature swine.

BACKGROUND: There is currently a great need for an in vitro assay to assess the presence of tolerance following allotransplantation to determine whether immunosuppressive medications can be discontinued. Our laboratory has recently developed an assay involving coculture inhibition of cell-mediated lympholysis that correlates with tolerance to allografts in swine leukocyte antigen (SLA) Class I-mismatched miniature swine. The potential for clinical application of this assay may depend on 2 important factors: (1) whether the assay can be used in the presence of immunosuppression; and (2) whether frozen-stored naive responder cells can be utilized. METHODS: Long-term tolerant MGH miniature swine that had accepted SLA Class I-mismatched kidney transplants after a 12-day course of cyclosporine or tacrolimus were studied. Two long-term tolerant and 2 naive control animals were treated with a clinically relevant dose of cyclosporine for 2 weeks (trough level 100 to 400 ng/ml) to simulate the ongoing "chronic" immunosuppression used in human recipients of allografts. Cells from tolerant or naive, recipient-matched animals were stimulated for 6 days with donor or third-party SLA. These primed cells were then cocultured with naive unstimulated recipient major histocompatibility complex (MHC)-matched responders and irradiated stimulators. Responder cells were tested both fresh and frozen. RESULTS: Suppression of cytotoxic responses of naive responder cells was observed in all coculture assays using cells from tolerant animals primed against donor antigen in vitro, but not in assays using similarly primed cells from naive animals. Responder cells from tolerant animals receiving immunosuppression had a suppressive activity similar to that from cells of the same animals not receiving immunosuppression. Similar suppression was also observed in coculture assays using either fresh or frozen naive responder cells. CONCLUSIONS: This coculture assay appears to correlate with the presence of tolerance under conditions applicable to the clinical setting. The assay appears to identify peripheral regulatory mechanisms of tolerance in allogeneic transplant recipients, and therefore may provide an approach for determining an appropriate timepoint at which to test withdrawal of immunosuppressive medications.