Chuanmin Wang

Tolerance to rat liver allografts. IV. Acceptance depends on the quantity of donor tissue and on donor leukocytes

Liver allografts in some rat strains are often spontaneously accepted across a complete major histocompatibility barrier without the requirement for immunosuppression while other nonliver allografts are rejected. In previous studies, we have shown that spontaneous acceptance is dependent on liver passenger leukocytes. Depletion of passenger leukocytes by donor irradiation allows rejection, with DA recipients of irradiated PVG livers having a median survival time (MST) of 16 days. Here we show that, in this model, spontaneous acceptance is reconstituted by intravenous injection of donor leukocytes. Intravenous injection of 3-5x10(7) PVG liver leukocytes significantly prolonged DA survival time (MST=96 days, P=0.026), as did 5x10(7) spleen leukocytes (MST>100 days, P=0.002). Deletion of T cells from the reconstituting inoculum reduced survival time (MST=78 days, P=0.039), whereas deletion of B cells or monocytes/macrophages had no effect on survival time. In contrast, PVG hearts are regularly rejected by DA recipients, and PVG liver or spleen leukocytes, even at doses of greater than 3x10(8) cells/recipient, were unable to induce heart acceptance. To investigate the possibility that acceptance of the irradiated liver but not the heart might be due to the large mass of the liver, two kidneys and two hearts of PVG origin were transplanted to each DA recipient together with 1.5x10(8) PVG leukocytes. These organs survived for greater than 200 days, thereby showing that a large mass of donor tissue, in association with donor leukocytes, leads to acceptance of organs that are rejected if transplanted singly. It appears likely that spontaneous liver transplant tolerance is a high-dose or activation-associated immune phenomenon.

Evidence that apoptosis of activated T cells occurs in spontaneous tolerance of liver allografts and is blocked by manipulations which break tolerance

BACKGROUND Fully allogeneic liver grafts from piebald virol glaxo to dark agouti rats are spontaneously tolerated, whereas kidney transplants between these strains are rejected. Liver tolerance is broken by donor irradiation or peritransplant corticosteroid treatment of recipient rats, both of which interfere with the activation of recipient cells. METHODS In this study we used a combination of immunohistochemical staining, reverse transcription-polymerase chain reaction, and terminal deoxynucleotide transferase-mediated dUTP nick end labeling and Annexin-V apoptosis assays to compare donor cell migration, cytokine profiles, and leukocyte apoptosis in grafts and lymphoid organs from tolerant liver and rejecting kidney recipients. We then examined the effect on apoptosis of treatments which abrogate liver tolerance. RESULTS Liver transplantation in this tolerant strain combination is accompanied by rapid migration of many passenger leukocytes to the recipient spleen and lymph node, concurrent with a marked but transient increase in the amount of mRNA for the cytokines interleukin-2 and interferon-gamma. Apoptotic cells appear promptly in the spleen, their numbers reaching a peak 2 days earlier than has been previously shown for the graft infiltrate. Both CD4+ and CD8+ T cells undergo apoptosis and apoptotic cells are most concentrated among CD25+ T cells. In contrast, renal transplant rejection is associated with limited donor cell migration to lymphoid tissues and significantly less up-regulation of interleukin-2 and interferon-gamma in the spleen. Few apoptotic cells are detected in spleen or graft infiltrate during rejection, whereas apoptotic renal tubular and glomerular cells are found from day 5. Either recipient steroid treatment or donor irradiation significantly reduced the number of apoptotic cells in liver graft infiltrates and recipient spleen. CONCLUSIONS Taken together, these findings suggest that a mechanism akin to activation-induced cell death, with apoptosis of alloreactive recipient cells may be responsible for the induction of spontaneous liver transplant tolerance.

A short course of methylprednisolone immunosuppression inhibits both rejection and spontaneous acceptance of rat liver allografts.

BACKGROUND The effects of immunosuppressive drugs on transplant tolerance have not been extensively studied, although their effect on rejection is well established. METHODS We examined the effects of a short course of treatment with the immunosuppressive drug methylprednisolone (MP) on the survival of PVG liver allografts in Dark Agouti (DA) recipients that accepted the livers and in Lewis recipients that rejected the livers. Infiltration of liver allografts was examined by immunohistochemical staining of liver sections, and apoptosis was measured by terminal deoxynucleotide transferase-mediated dUTP nick end labeling. RESULTS A 5-day course of MP (days 0 to 4) led to rejection of four of six livers (mean survival time [MST] 99 days) in DA recipients compared with long-term survival (MST >100 days) in untreated animals. Delayed administration of MP (days 3 to 7) exacerbated rejection in DA recipients, and all eight animals rejected the graft (MST 68.5 days). Treatment of Lewis recipients with MP did not significantly prolong survival when administered from days 0 to 4 (MST 13 days), although delay of administration improved the outcome. Treatment from days 3 to 7 resulted in an MST of 21 days, whereas treatment from days 7 to 11 resulted in an MST of 41.5 days. MP treatment from day 3 to day 7 reduced T cells and interleukin 2 receptor-expressing cells but increased the numbers of apoptotic cells infiltrating both DA and Lewis strain allografts. CONCLUSIONS These results show that immunosuppression with MP inhibits both spontaneous tolerance and rejection of liver allografts in a rat model and question the efficacy of administering MP to all liver allograft recipients from the time of transplantation.

Spontaneous acceptance of liver transplants in rodents: Evidence that liver leucocytes induce recipient T-cell death by neglect

In many animal models transplanted livers are not rejected, even when there is a complete MHC mismatch between the donor and recipient and the recipient is not immunosuppressed. This distinguishes liver transplants from other organs, such as kidneys and hearts, which are rapidly rejected in mismatched individuals. Acceptance of transplanted livers in a rat model is not due to the absence of an immune response to the liver and there is a rapid, abortive response that is ultimately exhausted. Donor leucocytes transferred with the liver appear to be responsible for both liver acceptance and the abortive activation of the recipients T cells. The immune mechanism of liver transplant acceptance appears to be due to ‘death by neglect’ in which T cells are activated to express IL‐2 and IFN‐γ mRNA in the recipient lymphoid tissues, but not at adequate levels within the graft. Subsequently the activated T cells die leading to specific clonal deletion of liver donor‐reactive T cells. These findings have important implications for liver transplant patients as immunosuppressive drugs that are given to prevent rejection can also interfere with this form of tolerance. In addition, it might be possible to modify the immunosuppressive drug treatment of transplant patients to promote the process of death by neglect of recipient alloreactive T cells.

Posttransplant Administration of Donor Leukocytes Induces Long-Term Acceptance of Kidney or Liver Transplants by an Activation-Associated Immune Mechanism

Donor leukocytes play a dual role in rejection and acceptance of transplanted organs. They provide the major stimulus for rejection, and their removal from the transplanted organ prolongs its survival. Paradoxically, administration of donor leukocytes also prolongs allograft survival provided that they are administered 1 wk or more before transplantation. Here we show that administration of donor leukocytes immediately after transplantation induced long-term acceptance of completely MHC-mismatched rat kidney or liver transplants. The majority of long-term recipients of kidney transplants were tolerant of donor-strain skin grafts. Acceptance was associated with early activation of recipient T cells in the spleen, demonstrated by a rapid increase in IL-2 and IFN-γ at that site followed by an early diffuse infiltrate of activated T cells and apoptosis within the tolerant grafts. In contrast, IL-2 and IFN-γ mRNA were not increased in the spleens of rejecting animals, and the diffuse infiltrate of activated T cells appeared later but resulted in rapid graft destruction. These results define a mechanism of allograft acceptance induced by donor leukocytes that is associated with activation-induced cell death of recipient T cells. They demonstrate for the first time that posttransplant administration of donor leukocytes leads to organ allograft tolerance across a complete MHC class I plus class II barrier, a finding with direct clinical application.

Quantitation of fibroblast activation protein (FAP)-specific protease activity in mouse, baboon and human fluids and organs

The protease fibroblast activation protein (FAP) is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAPs unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20‐ and 1.3‐fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal‐infected skin of unhealthy baboons. FAP activity was 14‐ to 18‐fold greater in cirrhotic than in non‐diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

Conversion of pancreas allograft rejection to acceptance by liver transplantation

BACKGROUND Liver allografts of PVG(RT1c)-->DA(RT1a) are spontaneously accepted, whereas pancreas, heart, and kidney allografts are rejected. Our previous studies have shown that simultaneous liver and pancreas transplantation prevents pancreas allograft rejection. The aim of this study was to examine the effect of liver transplantation on subsequent pancreas allografts and on ongoing pancreas rejection. METHODS Heterotopic, duct-ligated segmental pancreas grafts were transplanted into streptozotocin-induced diabetic recipients (60 mg/kg body weight i.p.) with or without orthotopic liver grafting at different times. Experimental design was as follows. Group 1 received PVG-->PVG pancreas syngrafts (n=6); group 2, PVG-->DA pancreas allografts (n=7); groups 3-5, PVG-->DA pancreas allografts followed by liver transplantation on day 2 (n=6), on day 4 (n=5), and on day 6 (n=5), respectively, group 6, PVG-->DA pancreas allografts after liver transplantation at 4 weeks (n=6). RESULTS The results showed that pancreas allografts in group 2 were rejected from postoperative day 7 to 13. Liver transplantation prevented subsequent pancreas allograft rejection in group 6. Ongoing pancreas rejection was reversed by liver transplantation with subsequent graft acceptance in groups 3-5. Significant graft-infiltrating lymphocyte apoptosis was demonstrated at 2 weeks in pancreas transplants associated with liver grafting. Graft-versus-host disease was not detected in the pancreas recipients. CONCLUSIONS We conclude that pancreas allografts in the PVG-->DA combination are rejected rapidly with median survival time of 9 days. Liver transplantation can protect subsequent pancreas grafts from rejection and reverse ongoing pancreas graft rejection with subsequent pancreatic acceptance. Graft-infiltrating lymphocyte apoptosis may be associated with the process of graft acceptance.

Infiltrating Foxp3(+) regulatory T cells from spontaneously tolerant kidney allografts demonstrate donor-specific tolerance.

Foxp3+ regulatory T cells (Tregs) have an essential role in immune and allograft tolerance. However, in both kidney and liver transplantation in humans, FOXP3+ Tregs have been associated with clinical rejection. Therefore, the role and function of graft infiltrating Tregs have been of great interest. In the studies outlined, we demonstrated that Foxp3+ Tregs were expanded in tolerant kidney allografts and in draining lymph nodes in the DBA/2 (H‐2d) to C57BL/6 (H‐2b) mouse spontaneous kidney allograft tolerance model. Kidney allograft tolerance was abrogated after deletion of Foxp3+ Tregs in DEpletion of REGulatory T cells (DEREG) mice. Kidney allograft infiltrating Foxp3+ Tregs (K‐Tregs) expressed elevated levels of TGF‐β, IL‐10, interferon gamma (IFN‐γ), the transcriptional repressor B lymphocyte‐induced maturation protein‐1 (Blimp‐1) and chemokine receptor 3 (Cxcr3). These K‐Tregs had the capacity to transfer dominant tolerance and demonstrate donor alloantigen‐specific tolerance to skin allografts. This study demonstrated the crucial role, potency and specificity of graft infiltrating Foxp3+ Tregs in the maintenance of spontaneously induced kidney allograft tolerance.

Spontaneous acceptance of mouse kidney allografts is associated with increased Foxp3 expression and differences in the B and T cell compartments.

Spontaneous acceptance of organ allografts can identify novel mechanisms of drug-free transplantation tolerance. Spontaneous acceptance occurs in both mouse kidney transplants and rat liver transplants however the early immune processes of mouse kidney acceptance have not been studied. Acceptance of C57BL/6 strain kidney allografts in fully MHC-incompatible B10.BR recipients was compared with rejection (REJ) of heart allografts in the same strain combination. Graft infiltrate and antibody deposition were examined by immunohistochemical staining. Expression of mRNA was measured by quantitative real-time PCR. Apoptosis was examined by TUNEL staining. The majority of kidney allografts were accepted long-term and induced tolerance (TOL) of donor-strain skin grafts, showing that acceptance was not due to immune ignorance. There was an extensive infiltrate of T cells in the TOL kidney that exceeded the level in REJ hearts but subsequently declined. The main differences were deposition of IgG2a antibody in REJ that was absent in TOL, more B cells infiltrating TOL kidneys and a progressive increase in the ratio of CD8:CD4 cells during rejection. There was also significantly greater Foxp3 mRNA expression in TOL. Kidneys from RAG-/- donors were accepted, showing that donor lymphocytes were not necessary for acceptance. Neutralising antibodies to TGF-β administered from day 0 to day 6 did not prevent TOL. On the basis of cytokine expression and apoptosis there was no evidence for immune deviation or deletion as mechanisms of acceptance. In accord with the findings of spontaneous acceptance of liver allografts in rats, the main difference between mouse kidney TOL and heart REJ was in the B cell compartment. The major difference to rat liver allograft acceptance was that apoptosis of infiltrate did not appear to play a role. Instead, increased Foxp3 expression in TOL kidneys implies that regulatory T cells might be important.

Blocking indoleamine dioxygenase activity early after rat liver transplantation prevents long-term survival but does not cause acute rejection.

In a well-characterized rat model of liver transplantation, Piebald Virol Glaxo strain livers are accepted long term in fully mismatched Dark Agouti recipients (tolerance; TOL), but rejected in Lewis recipients (rejection; REJ). Spontaneous tolerance induction is associated with increased interferon-&ggr; expression, and we examined the role of the interferon-&ggr;-inducible immunomodulatory enzyme indoleamine dioxygenase (IDO) in this model. On day 3 after transplantation, IDO expression in the spleen of TOL recipients was significantly greater than in REJ. The B-cell population accounted for this early IDO increase. Intragraft expression of IDO increased to the same extent in both TOL and REJ. IDO inhibition for 7 days after transplantation reduced survival, but did not cause acute rejection of the liver in the TOL model. In conclusion, the differential IDO expression by B lymphocytes in the spleen of TOL recipients is not critical for preventing acute rejection.