Donor-specific Transfusion Research Articles

Total lymphoid irradiation as an immunomodulator in laryngeal transplantation

Problem: Transplant research continues to seek immunosuppressive regimens that minimize recipient morbidity and do not promote tumor growth. Several animal studies have demonstrated long-term donor-specific allograft tolerance after posttransplant total lymphoid irradiation (TLI) when used in conjunction with anti-lymphocyte globulin (ALG) and donor-specific transfusion (DST). The goals of the current study were as follows: (1) to assess the ability of posttransplant TLI to induce allograft tolerance in an established murine laryngeal transplant model, (2) to compare postoperative TLI regimens with varying doses of radiation and anti-lymphocyte globulin, and (3) to evaluate the ability of sirolimus to augment tolerance when used in combination with a posttransplant TLI regimen. Methods: Using 8 treatment arms containing 10 animals each, recipient rats received 1 of 4 postoperative regimens: (1) 1,000 rads of TLI in 5 fractions, ALG and DST, (2) 2,400 rads of TLI in 10 fractions, ALG and DST, (3) 2,400 rads of TLI in 10 fractions, ALG, DST, and intraperitoneal sirolimus, and (4) ALG only. Recipient groups were assessed at 15 or 30 days after the completion of the assigned treatment regimen. Laryngeal allografts were graded for histologic rejection level using an established scale. Results: Both 1,000 and 2,400 rads of TLI were well tolerated by recipient animals. Laryngeal allograft preservation was significantly better using 2,400 rads of TLI when compared to 1,000-rad regimens. The addition of sirolimus did not augment graft preservation. All 2,400-rad TLI regimens were superior to ALG alone, with only mild to moderate rejection observed on average at 30 days after treatment. No treatment regimen yielded consistent allograft preservation on histologic analysis. Conclusion: Posttransplant total lymphoid irradiation regimens significantly reduce the severity of allograft rejection at 15 and 30 days. However, consistent histologic evidence of allograft tolerance was not observed. Significance: At this point, TLI has limited applicability to human laryngeal transplantation. Support: This study was funded by the Cleveland Clinic Department of Otolaryngology and Communicative Disorders and a matching Cleveland Clinic Research Programs Council grant.

Critical Role for CD8+ T Cells in Allograft Acceptance Induced by DST and CD40/CD154 Costimulatory Blockade

Donor-specific transfusion (DST) and CD40/CD154 costimulation blockade is a powerful immunosuppressive strategy which prolongs survival of many allografts. The efficacy of DST and anti-CD154 mAb for prolongation of hepatocellular allograft survival was only realized in C57BL/6 mice that have both CD4- and CD8-dependent pathways available (median survival time, MST, 82 days). Hepatocyte rejection in CD8 KO mice which is CD4-dependent was not suppressed by DST and anti-CD154 mAb treatment (MST, 7 days); unexpectedly DST abrogated the beneficial effects of anti-CD154 mAb for suppression of hepatocyte rejection (MST, 42 days) and on donor-reactive alloantibody production. Hepatocyte rejection in CD4 KO mice which is CD8-dependent was suppressed by treatment with DST and anti-CD154 mAb therapy (MST, 35 days) but did not differ significantly from immunotherapy with anti-CD154 mAb alone (MST, 32 days). Induction of hepatocellular allograft acceptance by DST and anti-CD154 mAb immunotherapy was dependent on host CD8+ T cells, as demonstrated by CD8 depletion studies in C57BL/6 mice (MST, 14 days) and CD8 reconstitution of CD8 KO mice (MST, 56 days). These studies demonstrate that both CD4+ and CD8+ T-cell subsets contribute to induction of hepatocellular allograft acceptance by this immunotherapeutic strategy.

PRE-TRANSPLANT DONOR-SPECIFC TRANSFUSIONS COMBINED WITH CYCLOSPORINE BUT NOT WITH RAPAMYCIN INDUCE TOLERANCE TO MHC CLASS I-MISMATCHED CARDIAC ALLOGRAFTS IN MINIATURE SWINE

P1125 Aims: To evaluate the effect of pretransplant donor-specific transfusion combined with cyclosporine A and rapamycin on tolerance induction to cardiac allografts in a clinically relevant large-animal model. Methods: Heterotopic cardiac transplants were performed across a major histocompatibility complex (MHC) class I barrier in MHC-inbred miniature swine. Experimental animals were treated with two donor-specific transfusions (DSTs), each containing 1.4x108 viable peripheral blood mononuclear cells, 14 and 7 days prior to heart transplantation. All animals received a 12-day course of cyclosporine (CyA) (trough level 300-800 ng/ml) or rapamycin (RPM) (trough level 10-20 ng/ml) starting on post-operative day (POD) 0. Control groups received CyA or RPM alone, DST alone or no treatment. Cell mediated lympholysis (CML) assays were performed at regular intervals. T cell priming and activation induced cell death (AICD) were assessed by carboxyfluoroscein succinimidyl ester (CFSE) proliferation assays and Annexin V staining. Results: Untreated (n=2) and DST-only (n=2) treated control animals rejected between POD 6 and 8. Animals treated with CyA (n=3) or RPM (n=3) alone exhibited graft survival to 53, 52 and 59 days or 41, 50 and 53 days, respectively. In contrast, DST-pretreatment combined with CyA (n=3) led to stable graft function for >200 days in all animals. Grafts in recipients treated with pretransplant DST and RPM (n=4) survived 52, 45, 100 and >200 days. Like the controls, long-term acceptors in the DST+CyA group and DST+RPM group mostly maintained peripheral CML response against donor and third party antigen. Following DSTs, the donor-specific proliferative response of CD8+ recipient T cells was significantly increased on CFSE assay (p=0.011), and a significant number of recipient CD8+ T cells underwent apoptosis (10.1% on POD 0 versus 5.2% on POD –14; p=0.04). Conclusions: The combination of pre-transplant DST and a short course of CyA consistently prolongs the survival of MHC class I disparate cardiac allografts in miniature swine. The sustained peripheral anti-donor CML response and the fact that alloreactive T cells were primed by the DST prior to transplantation suggest an active regulatory mechanism rather than peripheral deletion. In contrast, rapamycin did not demonstrate with the tolerogenic effect of pretransplant DST potentially because it failed to achieve appropriate regulatory cells.

ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION, MIXED CHIMERISM AND TOLERANCE IN LIVING RELATED DONOR RENAL ALLOGRAFT RECIPIENTS

P911 Aims: We designed a prospective, randomized control clinical trial to evaluate the efficacy and safety of achieving mixed chimerism associated tolerance protocol in living related donor (LRD) renal allograft recipients. Methods: Sixty six consecutive patients divided in two equal groups of 33 patients with end stage renal disease, were enrolled for transplantation after negative lymphocytotoxicity cross-matching (LCM). Both groups {treated (Tn) and control (Cn)} were well-balanced in their clinical, lab parameters and donor HLA match profile. Tn underwent thymic transplantation of donor renal tissue, 2 donor specific transfusions, low intensity conditioning and high dose hematopoietic stem cell transplantation (HSCT) before renal transplantation. Conditioning regimen included low dose target specific irradiation (to abdominal and inguinal lymph nodes, bone marrow(BM) of thoracolumbar vertebrae and part of pelvis on alternate days, 100 rads x 4), anti-T cell antibodies (15 mg/kg body weight (BW)), cyclophosphamide (10 mg/kg BW x 2 consecutive days) and cyclosporine (CsA) (3 mg/kg BW/day). Unfractionated HSCT procured from donor marrow was administered in recipient’s BM, portal and peripheral circulation within 24 hours of achieving CD 4+/ CD 8 + T- cell count < 10% of normal. This was supplemented by peripherally mobilized stem cells, with mean total dose of 20 x 10 8 cells/kg BW of recipient administered peripherally. Renal transplantation was performed after negative LCM. Donor specific cytotoxic antibodies were eliminated with intravenous immunoglobulins and plasmapheresis before renal transplantation. Mixed chimerism was evaluated before and after transplantation at monthly intervals in patients with donors of opposite gender by FISH technique. Both groups received CsA and prednisolone for immunosuppression, Cn received mycofenolate mofetil/ Azathioprine in addition. Rejection was treated with standard anti-rejection treatment. Immunosuppression was withdrawn 6 months after renal transplantation in patients with consistent positive chimerism. Clinical tolerance was defined by achieving stable allograft function for more than 100 days without immunosuppression confirmed by allograft biopsy. Results: Over a mean follow-up of 210 days, all Tn patients had stable allograft function with mean serum creatinine (SCr) of 1.20 mg/dl, no rejection/ CMV infections/ graft and patient loss. Low level donor-specific cytotoxic antibodies were observed in all of them. CsA toxicity was noted in 10 (30.3%) patients. Persistent mixed hematopoietic chimerism was noted in all 21 patients irrespective of donor-recipient HLA matching (mean 0.5 % before and 1 ± 0.3 % after transplantation). All 4 patients on drug withdrawal have developed donor-specific tolerance with a mean follow up of 129.8 days. Other Tn patients are in the process of being weaned off immunosuppression. Mean SCr of Cn was 1.45 mg/dl over a mean follow up of 216 days. Acute rejection was observed in 17 (51.5 %) patients, no CMV infection / patient loss was noted and 1(3.03%) graft was lost in Cn. No graft Vs host disease was observed in Tn. Conclusions: We have achieved mixed hematopoietic chimerism associated tolerance with high dose HSCT, intrathmic donor renal tissue transplantation and minimum conditioning without any adverse effects.

GITR ACTIVATION IN DONOR SPECIFIC TRANSFUSION ACCELERATES ALLOGRAFT REJECTION

P1016 Aims: Glucocorticoid-induced tumour necrosis factor receptor family-related gene (GITR) is expressed at high levels by CD25+CD4+ regulatory T cells. We made murine transplantation model of which graft survival was prolonged by donor specific transfusion and investigate the function of CD25+CD4+ and CD25-CD4+ T cells under the impact of GITR activation. We studied how GITR ligation effects on antigen reactive T cells during the induction of specific unresponsiveness in vivo. Methods: We examined the impact of GITR ligation on cardiac allograft rejection. We made donor specific transfusion, DST model that BALB/c mice heart graft was transplanted to BL6 mice 7days after 107 BALB/c spleen cells were infused to BL6. Then we observed the graft survival in the presence and absence of GITR activation using agonistic GITR antibody, DTA-1, or control rat Ig. To monitor the fate of T cells in the presence and absence of GITR activation we investigated the absolute number and activation status of CD4+Vb6+ T cells responding to Mls1a. BALB/c mice were treated with either DTA-1 or control rat Ig before administration of DBA/2 spleen cells (DST). The absolute number and activation status of CD4+Vb6+ T cells present in the spleen and LN was determined by FACS on days 2 – 7 inclusive and on day 14 after DST. Results: DST 107 BALB/c spleen cells 7 days before transplantation can consistently prolong the survival of BALB/c heart grafts in BL6 mice compared with naïve BL6 mice. Administration of DTA-1 the day before DST not only prevented graft prolongation but also triggered accelerated rejection of the BALB/c heart allografts. The accelerated rejection response confirmed that the DTA-1 has dual effects on T cells responding to alloantigen in vivo. At later time points, day 6, 7 and 14, there was a decline in the absolute number of T cells responding to Mls1a superantigen reactive T cells. Treatment with either DTA-1 or control rat Ig alone in the absence of DST did not change the absolute number of CD4+Vb6+ present in the spleen or LN over the time course studied compared to the number present in untreated adult BALB/c mice. Conclusions: GITR activation at the time of alloantigen infusion triggers accelerated graft rejection. GITR agonists can therefore augments immune responsiveness in vivo by simultaneously targeting the immunoregulatory and effector arms of the immune response.

A RANDOM TRIAL OF MIZORIBINE VERSUS MYCOPHENOLATE MOFETIL IN TACROLIMUS-BASED RENAL TRANSPLANTATION

A61 Aims: Mycophenolate mofetile (MMF) and Mizoribine (MZ) are immunosuppressive drugs that impair de novo purine synthesis. Some recent papers reported the efficacy and safety of MZ with Cyclosporine (CsA) and those of MMF with Tacrolimus (FK506) in renal transplantation. However, there have been no reports comparing MZ and MMF with the FK506-based immunosuppression for renal transplant recipients. This study was designed to investigate the safety and efficacy of MZ and FK506 compared to MMF and FK506 for renal transplant recipients. Methods: From November 1999 to February 2003, there were 37 recipients (28 male, 9 female) entered into this trial. All patients received donor specific blood transfusion (DST) prior to transplantation, and they were transplanted from living donors. From the day of transplantation, 5mg/kg/day of Deoxyspergualin (DSG) was administered for one week, followed by MZ or MMF. MZ or MMF was administered on 5mg/kg/day, 1g b.i.d., respectively; 18 patients were in the MZ group (14 male, 4 female), and 19 patients were in the MMF group (14 male, and 5 female). Results: Rejection (biopsy proven): CMV Infection: There were four cases of CMV infection (4/18: 22%) in the MZ group; however, the MMF group had seven cases of CMV infection (7/19: 38%). Polyoma Infection: Each group had one case of polyoma infection (the MZ group 1/18: 6%; the MMF group 1/19: 5%). Gastrointestinal disorder: There was more diarrhea in the MMF group (4 cases, 21%) than in the MZ group (1 case, 6%). Crossover: Three cases were forced to change from MZ to MMF due to steroid-resistant acute rejection. Conclusions: 1. Although this was a small-scale study, there were no significant differences between the MZ and MMF groups in the frequency of acute rejection. 2. There was more CMV infection in the MMF group than in the MZ group. 3. Further investigation is required, however, MZ and MMF are both useful immunosuppressive agents.Figure

CD25+CD4+ REGULATORY T CELLS GENERATED IN VIVO PREVENT ISLET ALLOGRAFT REJECTION BY BLOCKING THE RECRUITMENT OF EFFECTOR CELLS INTO THE GRAFT

O178* The concept of CD25+CD4+ regulatory T cells (T-reg) is well established in various autoimmune and transplantation models and a growing body of evidence suggests that operational transplantation tolerance depends on active T cell regulation mediated by these T-regs. Previously, we have demonstrated that T-reg generated in vivo prior to transplant by pre-treatment with donor-specific blood transfusion (DST) plus anti-CD4 monoclonal antibody can regulate donor-specific allograft rejection in a CTLA-4 and IL-10 dependent manner. Aim: The aim of this study was to ask whether T-reg generated by the same protocol are capable of preventing the rejection of a life-sustaining islet allograft, and if so, explore the mechanisms involved. Methods: T cell depleted CBA (H-2k) recipients were rendered diabetic by streptozotocin (blood glucose >20 mmol/L). These mice were then reconstituted with 1x105 CD45RBhigh CD4+ effector cells from naïve mice with or without co-transfer of 4x105 CD25+CD4+ from cell donors tolerized with nondepleting anti-CD4 antibody on days –28 and –27 plus a C57BL/6 (H-2b) DST on day –27. The naïve CD45RBhigh CD4+ cells used as effectors in this model co-express human CD52 as a transgene which allows them to be distinguished phenotypically from putative T-regs following adoptive transfer. On day 0, reconstituted, diabetic animals were transplanted with 400 C57BL/6 islets under the kidney capsule. Graft function was assessed by daily blood glucose measurements and rejection was defined as a blood glucose level ≥14.5 mmol/L on two consecutive days. Cellular infiltration was quantified by immunofluorescence using anti-hCD52-FITC. Four separate planes were analysed with an average of 4 high power fields (hpf) examined per plane. Results and Conclusion: Mice reconstituted with CD45RBhigh CD4+ effector cells alone rejected their grafts acutely (MST=18, n=7). This was completely prevented by co-transfer of CD25+CD4+ T cells isolated from tolerized animals (MST>70, n=10). These animals remained normoglycemic (8-13 mmol/L, normal range 8-12 mmol/L). In the absence of CD25+CD4+ T cells, CD45RBhigh CD4+ effector cells rapidly infiltrated the graft site and accumulated until day 15. However, in the presence of CD25+CD4+ T cells, at day 5 there was a 7-fold decrease in the number of infiltrating effector cells (CD45RBhigh only 3.66 ± 0.33; CD45RBhighplus CD25+ 0.47 ± 0.15 cells/hpf, p<0.0001; syngeneic control 0.57 ± 0.2). To our knowledge, this is the first demonstration that CD25+CD4+ T cells generated in vivo by immunomodulation can protect a life-sustaining, functional primary islet allograft. The effect of regulation on the recruitment of effector cells into the graft provides an insight into one of the mechanisms in which regulatory T cells can prevent allograft rejection and confirms that the generation of CD25+CD4+ regulatory cells should be considered as an important strategy for providing operational tolerance in clinical transplantation.

“PROPE” TOLERANCE AFTER INTESTINAL TRANSPLANTATION IN MAN.

P608 Aims: Current protocols for Intestinal Transplantation (Itx) use heavy immunosuppression (IS) but this causes infection, lymphoma and drug toxicity. “Prope” or “almost” tolerance refers to a state of graft acceptance maintained by low, non-toxic immunosuppression. To establish prope tolerance, we developed an immuno-modulatory protocol including: 1) Donor-Specific Blood Transfusion (DSBT induces regulatory cells); 2) low-dose calcineurin-inhibitor (CI) (high-dose CI prevents DSBT-induced regulatory cells generation); and 3) no steroids bolus (shown to break tolerance in a DSBT-induced tolerance model). Methods: 2 females (55 and 57 years old) with short gut and liver failure received an Itx in addition to a new liver. 300cc donor whole blood was transfused in the recipient after reperfusion. For induction IS, no iv steroids bolus were given, but only 2 doses of anti-IL-2 RA (20mg Simulect days1/4). Patients received postTx maintenance IS with very low Tac levels for this type of Tx (15ng/ml 1stmth; 5-10ng/ml 2nd-3rdmth; ∼5ng/ml thereafter), low-dose azathioprine (1mg/kg 1st–3rd mth; .5mg/kg thereafter) and low-dose steroids (medrol 8mgx2 daily tapered to 2mgx2 by the 3rdmth). Standard anti-bacterial, -fungal and -viral prophylaxis was given. Patients were monitored for rejection, GVHD, infection and PTLD. Protocol biopsies were taken from the ileal stomy. Results: Despite exposure to low IS, clinical, endoscopic and histologic signs of rejection were absent. Chimerism (donor HLA-type cells in peripheral blood) was transiently identified at day 28 in patient 1 but was self-limiting. GVHD was absent in both patients. Under this low IS, patients remained free of systemic opportunistic infections, lymphoma (no increased copies of EBV genome by PCR), and drug toxicity. Both patients are nutritionally independent and well 3 and 1.5 year postTx. Last follow-up biopsies show normal intestinal mucosa. Conclusions: The herein described immuno-modulatory protocol allowed development of “prope” tolerance and restored nutritional independence in 2 consecutive Itx recipients, a form of Tx that usually requires profound IS. Maintenance IS in these patients is less than after kidney Tx or in certain patients with chronic inflammatory bowel disease. Mechanisms of graft acceptance in these patients - in particular the development of regulatory cells - are under investigation.

DEOXYSPERGUALIN PROPHYLAXIS WITH TACROLIMUS FURTHER IMPROVES LONG-TERM GRAFT SURVIVAL IN LIVING-RELATED RENAL-TRANSPLANT RECIPIENTS TRANSFUSED WITH DONOR-SPECIFIC BLOOD

Abstract Background We examined whether deoxyspergualin (DSG) prophylaxis in combination with cyclosporine (CsA)- or tacrolimus (Tac)-based immunosuppression augments the effect of donor-specific blood transfusions (DSTs) to improve long-term survival of living-related renal-transplants. Methods From May 1985 to January 1998, 176 patients received DST from one-haplotype-identical donors prior to kidney transplantation. Group A (n = 64, 1985 to 1989) received CsA, prednisolone (PSL), antilymphocyte globulin (ALG), and azathioprine (AZA). Group B (n = 89, 1989 to 1996) received CsA, PSL, ALG, and DSG. Group C (n = 23, 1996 to 1998) received Tac, PSL, ALG, and DSG, with DSG followed by AZA. Rejection episodes were classified as acute rejection (AR, within the first 3 months) or late acute rejection (LAR, from 4 months to 1 year). Results Five-year graft survivals were 73.4%, 88.8%, and 91.3% for groups A, B, and C, respectively. The incidence of AR was 34%, 30%, and 13%, and that of LAR was 23%, 26%, and 30% for groups A, B, and C, respectively. There was no significant difference in the incidence of AR or LAR among the three groups. However, an elevated serum creatinine (sCr) ≥1 mg/dL was observed in 73%, 15%, and 0% of patients during AR, and in 53%, 30%, and 14% during LAR for groups A, B, and C, respectively. These results suggest that the severity of AR or LAR was lowest among group C, contributing to the improved long-term graft survival in these patients. Conclusions DSG prophylaxis with Tac-based immunosuppression further improves long-term graft survival among living-related renal-transplant recipients treated with DST by decreasing the severity of acute rejection episodes.

HIGH-DOSE CALCINEURIN INHIBITOR BLOCKS THE GENERATION OF REGULATORY CELLS, WHEREAS LOW-DOSE PROMOTES THEIR DEVELOPMENT.

O181* Aims: Regulatory cells (RC) are increasingly recognized as playing a major role in non-deletional forms of tolerance and seem to depend upon IL2-signaling. For safety reasons, clinical trials of tolerance induction (transfusion of donor-specific hematopoietic cells, stem cells etc.) include immunosuppressive (IS) drugs in the early postTx period. It is therefore essential that IS protocols do not block RC generation. The effects of calcineurin inhibitors (dose/timing) on RC generation is not elucidated yet. Methods: We developed a model of RC-based tolerance to cardiac allografts via preTx donor-specific blood transfusion (DSBT) in a fully mismatched RA-to-PVG rat combination. Results: Adoptive transfer from tolerant-to-naïve recipients demonstrated that donor-specific RC operate in this model. We then exposed DSBT-treated rats to high (50mg/kg) and low (10mg/kg) dose of Cyclosporine A (CsA) and we found that high-dose CsA given at the time of DSBT abrogated the development of RC and caused rejection. High-dose CsA at the time of Tx did reduce - but did not abrogate - the tolerogenic effect of DSBT. In stark contrast with high-dose, low-dose CsA did not affect DSBT-induced tolerance. On the contrary, low-dose CsA alone given postTx (day 0-11) in DSBT-untreated rats induced tolerance and allowed the development of RC that could adoptively transfer tolerance in second set naïve recipients. Finally, DSBT given the day of Tx did not promote tolerance in this model, unless recipients received a delayed and subtherapeutic course of low-dose CsA (day 5-9) (n=6 in each experiment, p<.01=significant). Conclusions: High-dose calcineurin inhibitor abrogates RC generation whereas low-dose (alone or in synergy with DSBT) promotes their development. These data have important implications with regard to a more judicious use of calcineurin inhibitors in the clinic, particularly when immunomodulatory strategies based upon RC generation are applied. Sustained and profound (but not delayed and mild) inhibition of IL2-signaling prevents RC generation.

Transplant: immunology and treatment of rejection

Transplant: immunology and treatment of rejection

Portal donor-specific blood transfusion and mycophenolate mofetil allow steroid avoidance and tacrolimus dose reduction with sustained levels of chimerism in a pig model of intestinal transplantation.

In a pig model of intestinal transplantation, we previously showed that hepatic conditioning through portal donor-specific blood transfusion (pDSBT), high-dose tacrolimus (TAC), and steroids prevented rejection and increased survival Our current study tests a protocol of pDSBT, short-term mycophenolate mofetil (MMF), and low-dose TAC to eliminate the use of steroids, reduce TAC dosage, and increase the level of chimerism in the peripheral blood. Four groups of outbred, mixed lymphocyte culture (MLC)-reactive pigs underwent bowel transplants and pDSBT. Immunosuppression (group 1, high-dose TAC and steroids; group 2, low-dose TAC and MMF; group 3, low-dose TAC, MMF, and aminoguanidine; group 4, low-dose TAC, MMF, and arginine) was discontinued after 28 days. RNA was extracted from intestinal graft and native liver biopsies for cytokine measurements. Chimerism levels were determined using a Q-PCR analysis. Pig survival and death rates due to rejection did not significantly differ between the four groups. Chimerism levels determined by Q-PCR analysis were not different until day 28. After discontinuation of immunosuppression, we noted a trend (P = 0.15) toward higher mean chimerism levels on day 60 for groups 2, 3, and 4 (9%) vs. group 1 (0.5%). Tissue cytokine and serum nitrate levels did not significantly differ between the four groups. Attempts to modify nitric oxide synthase activity offered no added benefit. The combination of pDSBT, MMF, and low-dose TAC (vs. high-dose TAC and steroids) allowed sustained levels of mixed chimerism to develop after discontinuation of immunosuppression.

PROLONGED SURVIVAL OF NEONATAL PORCINE ISLET XENOGRAFTS IN MICE TREATED WITH A DONOR-SPECIFIC TRANSFUSION AND ANTI-CD154 ANTIBODY1

Combined treatment with a single donor-specific transfusion (DST) and a brief course of anti-mouse CD154 monoclonal antibody (mAb) to induce co-stimulation blockade leads to long-term murine islet allograft survival. The authors hypothesized that this protocol could also induce long-term survival of neonatal porcine islet cell clusters (NPCC) in chemically diabetic immunocompetent mice and allow their differentiation into functional insulin-producing cells. Pancreata from 1- to 3-day-old pigs were collagenase digested and cultured for 8 days. NPCC were recovered and transplanted into the renal subcapsular space. Recipients included chemically diabetic nonobese diabetic (NOD)-scid and C57BL/6 mice that were otherwise untreated, treated with anti-CD154 mAb alone, or treated with DST plus anti-CD154 mAb. Plasma glucose concentration and body weight were measured, and xenografts were examined histologically. NPCC fully differentiated and restored normoglycemia in four of five diabetic NOD-scid recipients but were uniformly rejected by diabetic C57BL/6 recipients. Anti-CD154 mAb monotherapy restored normoglycemia in 4 of 10 (40%) NPCC-engrafted, chemically diabetic C57BL/6 mice, but combined treatment with DST and anti-CD154 mAb restored normoglycemia in 12 of 13 (92%) recipients. Reversal of diabetes required 5 to 12 weeks. Surviving grafts were essentially free of inflammatory infiltrates 15 weeks after transplantation. Combination therapy with a single DST and a brief course of anti-mouse CD154 mAb without maintenance immunosuppression permits survival and differentiation of NPCC in diabetic C57BL/6 mice. Successful grafts were associated with durable restoration of normoglycemia and the absence of graft inflammation.

In vivo helper functions of alloreactive memory CD4+ T cells remain intact despite donor-specific transfusion and anti-CD40 ligand therapy.

Memory T cells have specific properties that are beneficial for rapid and efficient protection from pathogens previously encountered by a host. These same features of memory T cells may be deleterious in the context of a transplanted organ. Consistent with this contention is the accumulating evidence in experimental transplantation that previously sensitized animals are resistant to the effects of costimulatory blockade. Using a model of murine cardiac transplantation, we now demonstrate that alloreactive memory CD4(+) T cells prevent long-term allograft survival induced through donor-specific cell transfusion in combination with anti-CD40 ligand Ab (DST/anti-CD40L). We show that memory donor-reactive CD4(+) T cells responding through the direct or indirect pathways of allorecognition provide help for the induction of antidonor CD8(+) T effector cells and for Ab isotype switching, despite DST/anti-CD40L. The induced pathogenic antidonor immunity functions in multiple ways to subsequently mediate graft destruction. Our findings show that the varied functions of alloreactive memory CD4(+) T cells remain intact despite DST/anti-CD40L-based costimulatory blockade, a finding that will likely have important implications for designing approaches to induce tolerance in human transplant recipients.

Clinical Tolerance: the end of the beginning.

Clinical Tolerance: the end of the beginning.

Amelioration of hyperglycemia in streptozotocin-induced diabetic mice with fetal pancreatic allografts: prevention of rejection by donor specific transfusion in conjunction with CTLA4Ig.

Fetal pancreas has been considered as an alternative donor source for islet transplantation since it has potent capacity for beta cell differentiation and proliferation. However, prevention of fetal pancreatic allograft rejection can be hardly achieved compared with adult islet allografts. The aim of the study is to determine whether donor specific transfusion (DST) in conjunction with CTLA4Ig has any favorable effect on prevention of fetal pancreatic allograft rejection in mice. BALB/c splenocytes (SPC, 1 x 10) were injected iv into C57BL/6 mice in conjunction with CTLA4Ig (ip, 50 microgram, day 0, 2, and 4). Fourteen days later, the mice were made diabetic with streptozotocin (STZ, iv) and donor specific or third party pancreatic allografts were transplanted beneath the kidney capsule. Morphologically, it was found that rejection of fetal pancreatic allografts can be prevented at 30 days after transplantation only when donor specific allografts were grafted into the mice treated with DST in conjunction with CTLA4Ig. Functionally, 3 out of 9 diabetic mice became normoglycemic by 120 days after transplantation of fetal pancreatic allografts. DST in conjunction with CTLA4Ig can have a favorable effect on prevention of fetal pancreatic allograft rejection resulting in amelioration of STZ-induced diabetes in mice.

Intestinal graft versus native liver cytokine expression in a rat model of intestinal transplantation: effect of donor-specific cell augmentation

Introduction Immunomodulation by portal vein delivery of donor antigen reduces intestinal graft rejection. We investigated the impact of portal venous donor-specific cell augmentation (blood versus bone marrow) on cytokine expression in intestinal grafts versus native livers. Methods Ten groups of intestinal transplants (brown Norway male to Lewis female rats) varied by (1) the type of donor-specific cell augmentation and (2) the use and dose of tacrolimus-based immunosuppression. Tissue samples for histologic analysis and cytokine mRNA analysis were obtained at designated time points. Results Without immunosuppression, no type of cell augmentation reduced the rate of rejection. With immunosuppression, outcome was significantly better after portal donor-specific blood transfusion (versus bone marrow infusion). Irrespective of the type of cell augmentation, severe rejection caused strong intragraft expression of IL-1α, IL-1β, IFN-γ, and TNF-α; liver expression mainly involved TNF-α. Of note, nonimmunosuppressed, cell-augmented rats showed hardly any differences in cytokine expression in their grafts versus significant increases in their native livers. With immunosuppression, bone marrow infusion (versus blood transfusion) increased intragraft cytokine expression of IL-1α, IL-1β, IFN-γ, as well as TNF-α, and liver expression of IL-1β. Conclusions (1) Rejection and donor-specific cell augmentation independently caused differences in intragraft versus native liver cytokine expression after intestinal transplants. (2) Portal donor-specific blood transfusion (versus bone marrow infusion) lowered the incidence of rejection and diminished intragraft cytokine up-regulation. (3) In our study, TNF-α appeared to be the cytokine most strongly associated with rejection.

Pre-transplant donor-specific transfusions combined with cyclosporine induce tolerance to MLC class I-mismatched cardiac allografts in miniature swine

Pre-transplant donor-specific transfusions combined with cyclosporine induce tolerance to MLC class I-mismatched cardiac allografts in miniature swine

Earlier Low-Dose TBI or DST Overcomes CD8+ T-Cell-Mediated Alloresistance to Allogeneic Marrow in Recipients of Anti-CD40L

Treatment with a single injection of anti-CD40L (CD154) monoclonal antibody (mAb) and fully mismatched allogeneic bone marrow transplant (BMT) allows rapid tolerization of CD4+ T cells to the donor. The addition of in vivo CD8 T-cell depletion leads to permanent mixed hematopoietic chimerism and tolerance. We now describe two approaches that obviate the requirement for CD8 T-cell depletion by rapidly tolerizing recipient CD8 T cells in addition to CD4 cells. Administration of donor-specific transfusion (DST) to mice receiving 3 Gy total body irradiation (TBI), BMT and anti-CD40L mAb on day 0 uniformly led to permanent mixed chimerism and tolerance, compared with only 40% of mice receiving similar treatment without DST. In the absence of DST, moving the timing of 3 Gy TBI to day –1 or day –2 instead of day 0 led to rapid (by 2 weeks) induction of CD8+ cell tolerance, and also permitted uniform achievement of permanent mixed chimerism and donor-specific tolerance in recipients of anti-CD40L and BMT on day 0. These nontoxic regimens overcome CD8+ and CD4+ T-cell-mediated alloresistance without requiring host T-cell depletion, permitting the induction of permanent mixed chimerism and tolerance.

Break of tolerance via donor-specific blood transfusion by high doses of steroids: a differential effect after intestinal transplantation and heart transplantation

Tolerance requires active mechanisms. How immunosuppressors affects tolerance is poorly understood. Methods RA (RT1 p)/PVG (RT1 c) rats were used as donor/recipient. Intestinal and heart transplant model were selected as highly and poorly immunogenic organs. Studied groups were 1, rejecting control; 2, received peritransplant steroids; 3, donor-specific blood transfusion (DSBT); 4, DSBT plus peritransplant steroids; and 5, DSBT+periDSBT Ste. Results Intestinal transplant recipients in group 1 died on posttransplant day (d) 18. In group 2, steroids did not change survival (17 days, P > .05 versus group 1). With DSBT (group 3), all rats survived >75 days, whereas with steroids those in group 4 survived 59 days ( P > .05 vs group 3) and group 5 survived 51 days ( P < .05 versus group 3). Survivors in group 2 were tolerant as evidenced by acceptance of secondary donor-specific (not third-party) graft. However, 100% and 33% of donor-specific secondary grafts were rejected in groups 4 and 5 ( P < .05 and P > .05 versus group 3). In heart transplants, steroid treatment had no effect on graft survival (group 1 9 days; group 2 9 days; P > .05). DSBT (group 3) induced 100% tolerance (primary: >100 days, secondary: 100%). Unlike in intestinal transplantation, adjunction peritransplant steroids (group 4) allowed 100% of primary and 83% of secondary graft acceptance ( P > .05 versus group 3). In group 5, (DSBT+periDSBT steroids) acceptance of primary and secondary grafts tended to be reduced (primary: 77 days; P > .05 versus group 3; secondary: 67%, P > .05 versus group 3). Conclusion Steroid induction did not prolong graft survival after either intestinal or heart transplant. Adjunction of steroids to a DSBT tolerogenic regimen caused rejection of primary and secondary grafts, particularly after intestinal transplantation. Routine use of steroids in the clinics must be reconsidered, particularly when immunogenic organs are transplanted and when immunomodulation is applied.