Injection of mitomycin-C-treated spleen cells induces donor-specific unresponsiveness to cardiac allografts in rats

BACKGROUND: In this study, preoperative mitomycin-C- (MMC) treated donor-specific transfusion (DST) was examined for its ability to induce unresponsiveness to cardiac allografts in rats. METHODS: DA (RT1a) rats were used as donors, BUF (RT1b) or WS (RT1k) rats as recipients, and Lew (RT1l) rats as third party donors. BUF or WS rats were given i.v. injection of DA spleen cells (SPCs) suspension (5x10(7)/l ml) with or without MMC treatment 10 days before cardiac transplantation. Delayed-type hypersensitivity and complement-dependent cytotoxicity assays were carried out in these animals separately to examine in vivo immunosuppressive effect. Suppressor assay was also examined to determine in vitro immunosuppressive effects in allogeneic mixed leukocyte culture. RESULTS: In the full allogeneic DA-to-BUF rat strain combination, preoperative i.v. administration of MMC-treated donor SPCs led to a significant prolongation of graft survival over the control (110+/-66 versus 7.2+/-0.8 days: P<0.01), although administration of nontreated donor SPCs did not (9.3+/-1.0 days). This beneficial effect of MMC treatment was also seen in the DA-to-WS rat combination (31+/-16 days versus donor-specific transfusion alone; 11+/-1.5 days or untreated control; 12+/-1.5 days; P<0.05). However, injection of third party DA SPCs in the Lew-to-BUF combination induced no significant prolongation of cardiac allograft survival compared with the untreated control (11+/-0.6 versus 11+/-2.0 days; NS), indicating that this prolongation effect was induced in an antigen-specific manner. The immunosuppressive effect was also secured for both delayed-type hypersensitivity response and anti-donor cytotoxic antibody production. Moreover, addition of MMC-treated SPCs to mixed lymphocyte culture led to antigen-specific suppression. CONCLUSIONS: Preoperative i.v. injection of MMC-treated donor SPCs is promising for inducing unresponsiveness in rat cardiac allograft model.     

UVB pretreatment of rat bone marrow allografts. Prevention of GVHD and induction of allochimerism and donor-specific unresponsiveness

Ultraviolet B irradiation has been used to pretreat blood and islets to prevent subsequent graft rejection. In this study the optimal dose of UVB irradiation of bone marrow was determined in syngeneic recipients and was subsequently applied to in-vitro treatment of bone marrow allografts. UVB pretreatment of donor bone marrow inoculum led to complete prevention of GVHD in allogeneic rat recipients without major marrow or other toxicity. Long-standing recipients of allogeneic UVB-BM became stable adult chimeras. The recipients of allogeneic BM were populated by donor-type peripheral blood lymphocytes and did not reject host or donor-type heart grafts. The BM allograft recipients were immunocompetent as measured by their ability to normally reject third-party cardiac allografts. We suggest that the prevention of GVHD and induction of stable chimerism in adult recipients of allogeneic UVB-BM may be mediated by suppressor mechanisms.     

B7-H4 induces donor-specific tolerance in mouse islet allografts

Negative cosignaling molecules play an important role in regulating T-cell responses to alloantigen stimulation. We recently reported that adenoviral-mediated transduction of islet allografts with B7-H4 inhibits allograft rejection. In this study, we investigate the mechanism for B7-H4-induced prolongation of mouse islet allograft survival. Streptozotocin-induced diabetic C57BL/6 mice were rendered normoglycemic by renal subcapsular implants of B7-H4-transduced BALB/c islets. Grafts and spleens were removed after days 2, 10, and 60 (n = 8 each) for characterization of kinetics of Foxp3 and interleukin 10 (IL-10) expression. Mixed lymphocyte reaction (MLR) was done at day 60. Ten mice were subjected to nephrectomy at 60 days and then five were implanted with secondary BALB/c islets and five were given third-party CBA/J islets. An increase in Foxp3 and IL-10 mRNA expression was detected in recipients' spleens at day 60 and this was associated with increased quantities of Foxp3(+) cells. Splenocytes at day 60 showed hyporesponsiveness during MLR to alloantigen stimulation. Proliferation was partially restored after CD25(+) T-cell depletion. Secondary BALB/c islets survived for 79 ± 29 days compared with 21 ± 3.6 days for CBA/J islets (p < 0.001). Local expression of B7-H4 induces long-term unresponsiveness to donor-specific alloantigens, and is associated with T regulatory cells, suggesting the development of tolerance.     

Induction of donor-specific unresponsiveness to cardiac allografts in rats by pretransplant anti-CD4 monoclonal antibody therapy

In the present report a monoclonal antibody designated OX-38 directed against the rat CD4 molecule was tested for its ability to prolong the survival of heterotopic vascularized rat heart allografts transplanted across major histocompatibility barriers. Fluorescence-activated cell-sorter analysis showed that administration of OX-38 selectively depleted 80-95% of CD4+ cells from peripheral blood of treated rats. The immunosuppressive effects of OX-38 in vivo were verified by suppression of an antibody response against OX-38 itself as a heterologous protein immunogen. Recipient rats received OX-38 antibody as a single agent given in pretransplant regimens. Nine of 12 treated rats have maintained heterotopic abdominal heart allografts for greater than 175 days. Control rats that did not receive antibody therapy rejected their grafts within 14 days. Rats that maintained heart allografts for greater than 100 days accepted second donor strain hearts but rejected third-party heart grafts transplanted into the femoral space. Anti-CD4-induced allograft unresponsiveness persisted for at least 90 days following surgical removal of donor tissue and retransplantation of a second donor-matched heart. These results indicated that transient, pretransplant therapy with monoclonal antibodies directed against the CD4+ lymphocyte induced specific, long-lasting unresponsiveness to fully MHC-mismatched cardiac allografts in rats without additional immunosuppression.     

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

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

Mechanisms of immunologic unresponsiveness induced by ultraviolet-irradiated donor-specific blood transfusions and peritransplant cyclosporine

Recipient pretreatment with UV-B irradiated donor-specific blood transfusions (UV-DST) combined with peritransplant cyclosporine on days 0, +1, and +2 leads to permanent cardiac allograft survival in the ACI-to-Lewis rat strain combination. This study investigates the mechanisms of immunologic unresponsiveness induced by UV-DST and CsA by examining several in vitro and in vivo parameters in long-term cardiac allograft recipients. The results of the in vitro studies demonstrate that thoracic duct lymphocytes (TDL) of treated and allografted Lewis rats respond less in a mixed lymphocyte reaction to donor splenic lymphocytes (SpL) by 69%, 75%, and 73% (P less than 0.001) at 30, 50, and 100 days after transplantation, respectively, compared with controls, while the response to a third-party (W/F) SpL is unimpaired. In coculture experiments, the TDL from treated recipients specifically suppressed the response of unmodified Lewis TDL to ACI SpL by 59% and 40% (P less than 0.01) at 30 and 50 days after transplantation, respectively, while responses to W/F SpL were suppressed by only 3-6%. The sera obtained from ungrafted rats transfused with UV-DST suppressed the MLR between unmodified Lewis TDL and ACI SpL by 31% (P less than 0.05) while the sera from UV-DST and CsA-treated and allografted rats specifically suppressed the MLR by 75%, 80% (P less than 0.001) and 37% (P less than 0.01) at 10, 30, and 50 days after transplantation, respectively. In vivo adoptive transfer of 10(4) donor-type dendritic cells (DC) into recipients of beating cardiac allografts at 40 or 60 days after transplantation led to rapid and acute allograft rejection, while the adoptive transfer of 10(8) unseparated SpL obtained at 50 days after transplantation from treated Lewis recipients to syngeneic naive hosts led to a modest but significant prolongation of ACI test cardiac allografts. The transfer of serum from treated and allografted recipients at 10, 30, and 50 days after transplantation led to specific and significant prolongation of test grafts in syngeneic naive hosts. These findings suggest that the mechanisms underlying the in vivo immunologic unresponsiveness induced by pretreatment with UV-DST and peritransplant CsA include inactivation without elimination of class II-antigen presenting cells (APC), generation of specific serum suppressor factor(s) and/or antiidiotypic antibody, and induction of donor-specific suppressor cells.     

Induction of donor-specific unresponsiveness to rat cardiac allograft by donor leukocytes and cyclosporine

Many recent reports have emphasized the importance of donor antigens in the induction of allograft tolerance. This study examines the effect of pretransplant infusion of 10(8) donor leukocytes (DL) combined with peritransplant cyclosporine (CsA) on W/F cardiac allograft survival in Lewis rats. Peritransplant recipient treatment consisted of CsA 20 mg/kg given i.m. on days 0, +1, and +2 relative to heart transplantation. Lewis recipients, 5-8 per group, were pretreated with 10(8) DL with or without peritransplant CsA. A single DL transfusion on day -3 or day -7 prior to transplantation significantly prolonged the mean survival time (MST) of W/F hearts from 7.0 +/- 0.9 days in controls to 12.2 +/- 4.5 days and 12.4 +/- 1.0 days (P less than 0.01), respectively. Two DL infusions on days -7 and -3 or on days -14 and -7 prolonged the MST to 10.6 +/- 1.3 days (P less than 0.02) and 16.4 +/- 2.8 days (P less than 0.001), respectively. The administration of peritransplant CsA alone significantly prolonged W/F heart allograft survival to 43.1 +/- 2.7 days. When pretransplant DL transfusion on day -3 was combined with CsA treatment, 4/8 animals maintained their grafts indefinitely (greater than 100 days). Similarly, DL infusion on day -7 with peritransplant CsA led to indefinite graft survival in 3/5 animals. Administration of DL on days -7 and -3 combined with CsA resulted in indefinite graft survival (greater than 100 days) in 4/6 animals. Transfusion of DL on day -3 alone or in combination with peritransplant CsA, had no effect on a third-party (ACI) heart allograft survival prolongation compared with appropriate controls. To define the underlying mechanisms responsible for donor-specific unresponsiveness in this model, pooled sera and unseparated spleen cells were passively transferred from recipients of long-term cardiac allografts to syngeneic rats receiving donor-type (W/F) or third-party (ACI) cardiac allografts. Transfer of serum (1 ml on days 0, and 1, 0.5 ml on days +2, +3, and +4) from ungrafted recipients of DL on days -14 and -7 led to significant donor graft survival of 9.8 +/- 0.4 days (P less than 0.02) in unmodified hosts. Similarly, passive transfer of serum obtained at 20 and 100 days after transplantation significantly prolonged the MST of donor-type hearts in syngeneic untreated hosts to 11.3 +/- 0.8 and 10.0 +/- 1.1 days, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

The effects of donor-specific blood transfusion enhancement of rat renal allografts on host NK cell responses.

Donor-specific blood transfusion (DSBT) given 1-2 weeks prior to transplantation prolongs the survival of fully allogeneic ACI (RT1a) renal allografts in PVG (RT1c) recipients from 6-8 days to greater than 100 days. We have previously demonstrated that ACI kidneys transplanted to autologous blood transfusion (ABT)- or DSBT-pretreated PVG recipients stimulated an increase in CD8+ (OX8+) cells in the peripheral blood by 6 days after transplantation. To determine whether this increase represents a general expansion of the entire CD8+ population or only a subpopulation of CD8+ cells, subset analysis was performed on peripheral blood lymphocytes depleted of cells reactive with monoclonal antibodies against rat alpha beta T cell receptor (TCR), CD8, or NK cells (R7.3, OX8, or 3.2.3, respectively). Phenotypic studies of PBL depleted of CD8+ cells demonstrated that all 3.2.3+ NK cells coexpressed CD8; depletion of 3.2.3+ PBL revealed a second subpopulation of CD8+3.2.3- cells comprised predominantly of alpha beta TCR+ T cells. In naive PVG rats the prevalence of these two CD8+ subpopulations was approximately equal. Both ABT- and DSBT-pretreated renal allograft recipients demonstrated a significant and equivalent expansion of the CD8+ cell subpopulation that coexpresses the 3.2.3 NK marker. In contrast, the second subpopulation of CD8+3.2.3- cells did not change significantly after allografting. There were also no differences between DSBT and ABT pretreated rats in activity of PBL against the NK targets YAC-1 and Doxie at 6 days after renal transplantation, though the level of activity was modestly increased compared with naive controls. These findings indicate that renal transplantation in the rat is associated with a significant increase in PBL with the NK phenotype (CD8+3.2.3+) and a modest increase of NK activity, but that DSBT enhancement does not affect this NK cell response.     

Evidence of donor-specific cellular suppressor activity in donor-specific cell-mediated lympholysis unresponsiveness in renal transplant patients

Twenty patients with well-functioning kidney grafts from one-haplotype-mismatched related donors, were studied 1-10 years after transplantation (A). Another group of six patients were studied at various times after transplantation (B). The presence of donor-specific transplantation tolerance, using mixed lymphocyte culture (MLC) and cell-mediated lympholysis (CML) tests was investigated, as well as the possible existence of cells with suppressive activity. All recipients were transfused prior to transplantation and treated with conventional immunosuppression. The patients in group A showed MLC reactivity against donor and third-party cells, indicating a allogeneic response capacity. The CML activity against the donor was low, however, and remained low also following removal of adherent cells. The CML activity toward third-party cells was within the normal range of unmatched individuals. In group B, two of six recipients and high postoperative CML activity against the donor. Both recipients showed clinical signs of rejection. In the remaining four recipients, the antidonor CML reactivity one week after transplantation was lower than the preoperative level. The decrease was even more pronounced at 12 months, although the reactivity against third-party cells was unaltered. The CML reactivity from unrelated fourth-party individuals toward donors was suppressed when cells from recipients with long-term functioning kidneys were added to the cell cultures. The results suggest the presence of a donor-specific cellular suppressor mechanism underlying the donor-specific CML unresponsiveness in recipients with long-term-functioning kidney allografts.     

Induction of specific unresponsiveness (tolerance) to skin allografts by intrathymic donor-specific splenocyte injection in antilymphocyte serum-treated mice.

Although chronic immunosuppression has been extremely successful in clinical organ transplantation, it is associated with severe complications such as opportunistic infections, spontaneous neoplasms, drug toxicities, metabolic complications, and the inability to control rejection. We therefore have investigated the ability of allogeneic donor lymphoid cells to produce specific tolerance following intrathymic (IT) injection into allograft recipients. Groups of B6AF1 mice received ALS on days -1 and +2 relative to C3H/He skin grafts on day 0; experimental groups received 1, 5, or 10 x 10(7) syngeneic (B6AF1) or allogeneic (C3H) spleen cells (SPCs) by IT injection on day +7. IT injection of C3H splenocytes significantly prolonged allograft survival at all cell doses tested when compared with ALS controls. The best survival was obtained following IT injection of 5 x 10(7) C3H cells (median survival time [MST] = 132 days; ALS controls = 21.5 days), with 8 of 13 skin grafts surviving longer than 100 days. IT injection of syngeneic splenocytes or third-party DBA/2 splenocytes did not prolong allograft survival beyond that observed in ALS controls. C3H spleen cells injected IT into ALS treated mice on day 0 relative to grafting of C3H skin also produced significant allograft survival (1, 5, or 10 x 10(7) SPCs = MSTs of 75, 47, and 35, respectively) but the results were inferior to those obtained by 5 x 10(7) SPCs IT on day +7. Spleen cells (1 or 5 x 10(7)) injected intraperitoneally or intravenously prolonged allograft survival beyond that seen in ALS controls but were inferior to IT injection at all doses and times studied. Bone marrow, thymocytes, or lymph node cells (5 x 10(7) cells) were substituted for SPCs for IT injection. IT injection of BM, LN or thymocytes all significantly prolonged graft survival over ALS controls. However none of these cell types was as effective as IT splenocytes. Eight B6Af1 recipients of IT splenocytes bearing C3H skin grafts for > 100 days received a second C3H skin graft as well as a simultaneous third-party B10.AKM skin graft. All rejected third-party grafts in normal first-set fashion. Three tolerated both 1st and 2nd C3H grafts without any sign of rejection; 1 rejected the 2nd C3H graft while tolerating the 1st graft; and 4 rejected the 2nd C3H graft in an attenuated fashion but also rejected the 1st graft at the same pace.(ABSTRACT TRUNCATED AT 400 WORDS)