Pretransplant Donor-specific Blood Transfusion Research Articles

Inhibitory and Stimulatory Effects of Cyclosporine A on the Development of Regulatory T Cells In Vivo

Regulatory T cells (Tregs) are increasingly recognized as playing a major role in nondeletional tolerance. To avoid rejection before tolerance is established, clinical trials of tolerance induction include immunosuppressive drugs early posttransplant. It is therefore essential that immunosuppressive protocols do not block Tregs generation. Tregs function has been shown to depend upon interleukin-2 signaling, but there are limited data available on how calcineurin inhibitors influence Tregs development and function in vivo. To study this, we used a previously established rat cardiac allograft model where donor-specific Tregs and tolerance are induced by pretransplant donor-specific blood transfusion (DSBT). In this model, we found that adjunction of 50 mg/kg cyclosporine (CsA) (not a lower dose, 10 mg/kg) at the time of DSBT (not at the time of transplantation) abrogates Tregs development and causes rejection. Interestingly, 10 mg/kg CsA given posttransplant (day 0-11) in the absence of pretransplant DSBT induced the development of Tregs and provoked a state of tolerance indistinguishable from the one induced by DSBT. Finally, DSBT given the day of transplantation did not promote tolerance, unless recipients also received a delayed short course (day 5-9) of 10 mg/kg CsA. Adjunction of high-dose CsA to pretransplant DSBT abrogates Tregs generation. On the contrary, a lower dose (10 mg/kg) of CsA promotes Tregs development either in synergy with perioperative DSBT (providing that a drug-free interval is respected) or by its own effect. These data provide new guidelines for a more tolerogenic use of calcineurin inhibitors in the clinic, particularly when immunomodulatory strategies aimed at inducing Tregs are applied.

Hepatic CCR7 lowCD62L lowCD45RC low allograft dendritic cells migrate to the splenic red pulp in immunologically unresponsive rats

Donor dendritic cells (DC) migrate into the recipient spleen after hepatic transplantation. Immunological unresponsiveness to rat hepatic allografts can be induced by prior donor-specific blood transfusion (DST). We investigated homing receptor phenotype and splenic distribution of donor DC after allografting and DST. Immunostaining revealed OX62+ cells in the splenic red pulp of animals receiving pre-transplant DST but only in the white pulp of untreated animals. Most OX62 cells were positive for OX76. There were two subsets of DC in the spleen, CD45RChighOX62+ and CD45RClowOX62+ cells. RT-PCR revealed that CD45RClowOX62+ cells expressed interleukin (IL)-10, while CD45RChighOX62+ cells expressed IL-2 and low levels of IL-10 mRNA. CD45RChighOX62+ cells strongly expressed CCR5 and CCR7, compared with weak expression in CD45RClowOX62+ cells. The Epstein-Barr virus-induced molecule 1 (EBI-1) ligand chemokine (ELC/MIP3beta) was expressed mainly within the splenic white pulp. Mucosal vascular addressin-cell adhesion molecule-1 (MAdCAM-1) was expressed in the marginal zone and white pulp, but expression of splenic MAdCAM-1 was down-regulated in DST-treated animals. L-selectin (CD62L), the ligand for MAdCAM-1, was strongly expressed on CD45RChighOX62+ cells but not on CD45RClowOX62+ cells. In conclusion, differential splenic migration of CCR5lowCCR7lowCD62Llow CD45RClow DC expressing Th2-type cytokines is associated with immunological unresponsiveness to rat hepatic allografts.

The effect of FTY 720 on engraftment in a model of spontaneous allograft acceptance.

Further development in organ transplantation requires the utilization of new immunosuppressive drugs that-in addition to being effective against rejection-do not block tolerance. We previously reported that FTY 720, a drug that alters lymphocyte trafficking, has marked anti-rejection properties. We now investigate how FTY 720 influences tolerance in a model of graft acceptance by donor-specific blood transfusion (DSBT). Two different transplant models--heart transplantation (Htx) and intestinal transplantation (Itx)--were studied. We performed orthotopic Itx and heterotopic Htx using fully mismatched inbred male RA (RT1(p)) and PVG (RT1(c)) rats as donors and recipients. Tolerance was induced by DSBT on pre-transplant day -12. To test the effect of FTY 720 on DSBT-induced tolerance, we administered FTY 720 orally prior to DSBT. Itx: control rats succumbed to rejection at 18+/-4 days. DSBT alone prolonged survival to 101.9+/-18 days (P<0.05 vs untreated). Long-term survivors were tolerant (acceptance of secondary donor-specific Htx). Adjunction of FTY 720 prior to DSBT reduced survival to 55.9+/-44.7 days (P<0.05). However, long-term survivors still accepted secondary donor-specific Htx. Htx: control rats survived 9+/-0.6 days. DSBT alone prolonged survival indefinitely (>120 days) and induced tolerance (acceptance of secondary donor-specific Htx). Unlike in Itx, adjunction of FTY 720 prior to DSBT did not reduce Htx survival. Acceptance of secondary donor-specific Htx was not influenced by FTY 720. In Itx, FTY 720 counteracts the beneficial effect of pre-transplant DSBT and triggers acute rejection of primary, but not secondary grafts. In Htx, however, FTY 720 allows full development of tolerance. The mechanisms by which FTY 720 causes rejection in primary intestinal but not in heart grafts need to be elucidated.

Studies investigating pretransplant donor-specific blood transfusion, rapamycin, and the CD154-specific antibody IDEC-131 in a nonhuman primate model of skin allotransplantation.

Anti-CD154 variably prolongs allograft survival in nonhuman primates. Rodent studies suggest that adding pretransplant donor-specific transfusion (DST) and/or rapamycin to anti-CD154 improves survival. The CD154-specific Ab IDEC-131 was tested alone and in combination with rapamycin for its ability to inhibit rhesus MLRs. The ability of the Ab to block endothelial activation was also assessed. IDEC-131 was then tested alone and in combination with DST and/or rapamycin for its ability to prevent rejection of full-thickness, MHC-mismatched rhesus skin allografts. Animals were monitored for donor-specific hyporesponsiveness by MLR and alloantibody determination. IDEC-131 modestly inhibited rhesus MLRs and inhibited CD154-dependent endothelial cell activation. Rapamycin combined with IDEC-131 additively inhibited MLRs. IDEC-131 modestly prolonged allograft survival when compared with no treatment, rapamycin alone, or DST plus rapamycin. Adding DST to IDEC-131 did not prolong survival beyond IDEC-131 alone. IDEC-131 plus rapamycin was effective in prolonging graft survival, although animals had episodes of acute rejection before graft demise. Therapy with IDEC-131, rapamycin, and DST induced long-term allograft survival without intermittent acute rejection. However, no evidence for MLR inhibition was seen, and most animals eventually developed alloantibody. All animals ultimately rejected their grafts after drug withdrawal. IDEC-131 modestly prolongs rhesus skin allograft survival. Rapamycin and rapamycin plus DST improves the efficacy of IDEC-131 in prolonging allograft survival. IDEC-131, rapamycin, and DST are a promising combination for clinical evaluation in allotransplantation.

Differential Splenic Migration of Dendritic Cells after Immunologic Unresponsiveness in Rat Hepatic Allografts Induced by Pretransplant Donor-Specific Transfusion

Background. Donor dendritic cells migrate into the recipient spleen after hepatic transplantation. We previously reported that immunologic unresponsiveness to rat hepatic allografts can be induced by prior donor-specific blood transfusion (DST). We investigated the phenotype and splenic distribution of donor dendritic cells after allografting and DST.Methods. Donor dendritic cells were identified with anti-rat dendritic cell (OX-62) and anti-donor class II MHC (RT1Ba) (OX-76) antibodies. The phenotype of dendritic cells was determined with antibodies to CD45RC, CD62L, and the maturation markers CD80 (B7-1) and CD86 (B7-2). The cytokine profile of sorted CD45RC+ OX-62+ and CD45RC− OX-62+ dendritic cells was analyzed by reverse transcription polymerase chain reaction (RT-PCR).Results. Pretransplant DST significantly prolonged rat hepatic allograft survival. Immunostaining revealed OX76+/OX-62+ cells in the splenic red pulp of animals receiving pretransplant DST and in the white pulp of untreated animals after transplantation. The ratio of splenic CD45RC− OX-62+ cells to CD45RC+ OX-62+ cells was significantly higher in DST recipients than in untreated animals. CD62L, CD80, and CD86 were lower on CD45RC− OX-62+ than CD45RC+ OX-62+ cells. RT-PCR revealed that sorted CD45RC− OX-62+ cells expressed interleukin (IL)-4 and IL-10. In contrast, sorted CD45RC+ OX-62+ cells expressed only IL-2 and interferon γ (IFN-γ).Conclusion. Differential splenic migration of CD45RC− dendritic cells is associated with immunologic unresponsiveness to rat hepatic allografts.

Posttransplant infusion of donor-specific blood induces immunological unresponsiveness in rat hepatic allografts.

We previously reported that pretransplant donor-specific blood transfusion (DST) induces CD45RC-CD4+ T cells, Th2-like effector cells, and prolongs rat hepatic allograft survival. Our study investigated the effects of posttransplant DST on rat hepatic allograft survival. Three days after transplantation, LEW (RT1(1)) recipient rats with ACI (RT1a) livers were injected i.v. with freshly heparinized donor-specific blood. The time kinetics of CD45RC-CD4+ and CD45RC+CD4+ T cell subsets in hepatic infiltrates were examined. Posttransplant DST significantly prolonged rat hepatic allograft survival. Interferon (IFN)-gamma, interleukin (IL)-12, and IL-18 mRNA levels in hepatic allografts of untreated recipients were significantly greater than in recipients treated with posttransplant DST. However, hepatic allografts of recipients treated with posttransplant DST showed significantly higher IL-4, IL-10, and transforming growth factor (TGF)-beta mRNA levels than untreated recipients. The ratio of CD45RC-CD4+ T cells to CD45RC+CD4+ T cells was significantly higher in hepatic allografts treated with posttransplant DST than in untreated animals. Immunostaining with anti-rat dendritic cell (OX-62) monoclonal antibody revealed that OX-62+ cells were distributed to the splenic red pulp of animals treated with posttransplant DST and to the splenic white pulp in untreated animals. Most OX62+ cells isolated from the spleen of recipients treated with posttransplant DST expressed donor RT1Ba class II major histocompatibility complex antigens, suggesting that OX-62+ cells were of donor origin. Posttransplant DST was associated with persistent infiltration of CD45RC-CD4+ T cells, Th2-like effector cells, in rat hepatic allografts, causing immunologic unresponsiveness and establishment of microchimerism in the spleen.

Donor-specific portal blood transfusion in intestinal transplantation: a prospective, preclinical large animal study.

Unlike in kidney and heart transplantation, the role of pretransplant donor-specific blood transfusion (DST) has not been studied prospectively in a large animal model of bowel transplantation. We investigated the impact of portal versus systemic DST on overall survival, rejection, graft-versus-host disease (GVHD), and infection after total (small and large) bowel transplantation in pigs. Mixed lymphocyte culture-reactive, outbred pigs underwent total enterectomy and orthotopic total bowel transplantation with portal vein graft drainage. One unit of donor blood was transfused via the portal or systemic circulation (according to a randomization protocol) before graft implantation was begun. We studied six groups, all of which underwent at least a total bowel transplant: group 1 (n=5) comprised nonimmunosuppressed control pigs with portal DST; group 2 (n=6), nonimmunosuppressed control pigs with systemic DST; group 3 (n=5), cyclosporine (CsA)-treated pigs with portal DST; group 4 (n=5), CsA-treated pigs with systemic DST; group 5 (n=5), tacrolimus-treated pigs with portal DST; and group 6 (n=5), tacrolimus-treated pigs with systemic DST. All immunosuppressed pigs received prednisone (2 mg/kg/day) and either CsA (to maintain levels between 250 and 350 ng/ml) or tacrolimus (to maintain levels between 10 and 30 ng/ml). Stomal biopsies and autopsies were obtained to study the incidence of rejection, GVHD, and infection. Portal DST and tacrolimus-based immunosuppression resulted in the highest survival rates. At 7, 14, and 28 days after transplantation, survival rates in group 5 were 100%, 100%, and 80%; in group 6, 100%, 60%, and 40%; and in group 3, 100%, 0%, and 0%, respectively. Only the combination of portal DST and tacrolimus prevented the occurrence of, and death from, rejection. Death from rejection at 7, 14, and 28 days in group 5 was 0%, 0%, and 0%; in group 6, 0%, 33%, and 67%; and in group 3, 0%, 100%, and 100%, respectively. Of note, if immunosuppression was used, the groups with portal (versus systemic) DST had a higher risk of death from infection but a lower risk of death from GVHD. Simultaneous immunologic events were noted more frequently in groups with systemic (versus portal) DST. Long-term survival was noted only in groups with tacrolimus-based immunosuppression and was more common for those with portal (versus systemic) DST. Portal DST at the time of total bowel transplantation and posttransplant immunosuppression with tacrolimus prevent rejection and significantly increase graft survival. The combination of portal antigen presentation and tacrolimus needs to be studied in clinical bowel transplantation.

Peripheral tolerance of an allograft in adult rats--characterization by low interleukin-2 and interferon-gamma mRNA levels and by strong accumulation of major histocompatibility complex transcripts in the graft.

Congenic LEW.1W(RT1.u) heart grafts in LEW.1A(RT1.a) recipient rats are rejected in 15 +/- 6 days. Tolerance (greater than 100 days) can be induced by pretransplant donor-specific blood transfusion. In this case, the graft is not rejected, although it is infiltrated by mononuclear cells specifically cytotoxic, in vitro, against allogeneic donor splenocytes. We studied the expression of MHC class I and class II antigens, IFN-gamma, and IL-2 mRNA in the rejected and tolerated grafts by Northern blotting and in situ hybridization. Our data show that both class I and class II mRNA accumulate in both types of graft, and that class I mRNA accumulation occurs more rapidly in the tolerated grafts. IFN-gamma and IL-2 mRNA accumulate to lower levels and with delayed kinetics in the tolerated grafts compared with the rejected ones, suggesting a role for these lymphokines in the mechanism of rejection/tolerance in this model. This hypothesis is also supported by the observation that IFN-gamma treatment abrogates the induction of tolerance in the recipients receiving pretransplant donor blood transfusion. Furthermore, we observed an uncoupling of the accumulation of IFN-gamma mRNA and of MHC class I and class II mRNA. Our data confirm that the mechanisms of tolerance in this model depend, in part, on alterations of the IL-2/IL-2R pathway of lymphocyte activation but also clearly indicate a decrease of IFN-gamma mRNA accumulation, suggesting that the defect involves several activation molecules.

Renal transplantation from HLA-haploidentical living-related donors: the effects of donor-specific blood transfusions and different immunosuppressive regimens.

One-hundred-nine HLA-haploidentical living related renal transplants have been retrospectively analysed to compare the effect of donor-specific blood transfusion (DST) and different immunosuppressive regimens on graft survival and acute rejection. The recipients were divided into four groups according to the immunosuppressive therapy. Group 1 (n = 44): conventional therapy with posttransplant azathioprine (AZP) + methylprednisolone (MP). Group 2 (n = 25): pretransplant DST + posttransplant AZP + MP. Group 3 (n = 12): triple-drug therapy with posttransplant AZP + MP + cyclosporine (CS). Group 4 (n = 25): pretransplant DST + posttransplant AZP + MP + CS. The five-year actuarial survival rates for groups 1, 2, 3 and 4 were 48%, 73%, 79%, and 89%, respectively. The graft survival rate in group 3 was significantly (p less than 0.01) better than that in group 1. The transfusion effect was reduced, and appears as a 10% improvement in the graft survival in the cyclosporin era compared with a 25% improvement at pre-cyclosporin era. Furthermore, the incidence of the first rejection episode was decreased in recipients that received DST. The present study revealed that DST, as pretransplant conditioning has a definite impact on rejection-free long-term graft survival in HLA-haploidentical living-related kidney recipients and the most favorable outcome in such patients could be achieved by DST pretreatment in conjunction with posttransplant triple-drug therapy including cyclosporine.

SMALL BOWEL TRANSPLANTATION IN RATS

The effects of pretransplant donor-specific blood transfusion on the survival of orthotopic small bowel transplants in rats were investigated in the fully allogeneic BN (Rt1n) to WAG (Rt1u) donor-host combination. Previous studies show that in this combination DSTs lead to permanent survival of heterotopically transplanted hearts, marked prolongation of kidney grafts, and moderate prolongation of pancreas grafts but have no effect on skin grafts. Without pretreatment, total small bowel grafts (+/- 45 cm) were rejected in 12.2 +/- 1.8 days (mean +/- SD), and 10-cm segments of jejunum or ileum in 11.2 +/- 4.0 and 11.6 +/- 0.5 days, respectively. Three DSTs given on days -21, -14, and -7 before transplantation had no effect on graft survival in any of the groups tested. Total small bowel grafts were rejected in 12.8 +/- 2.5 days, and 10-cm-long segments or jejunum or ileum in 17.0 +/- 7.2 days and 11.5 +/- 2.7 days, respectively. Graft-versus-host disease, which was mild and transient, occurred in 50% of the nontreated rats engrafted with a total small bowel, in 40% of the animals transplanted with an ileum segment, and in none of the rats that received a jejunal transplant. In the DST-pretreated groups, none of the animals transplanted with a total small bowel or ileum segment and 16.6% of the animals transplanted with a jejunum segment showed clinical signs of GVHD. When DST pretreatment was combined with cyclosporine, grafts did not survive any longer than with cyclosporine treatment alone. It is concluded that DSTs ameliorate GVHD but do not prolong the survival of small bowel allografts nor act additively with cyclosporine treatment.

ASSOCIATION OF DONOR-SPECIFIC BLOOD TRANSFUSION ENHANCEMENT OF RAT RENAL ALLOGRAFTS WITH ACCELERATED DEVELOPMENT OF ANTIIDIOTYPIC ANTIBODIES AND REDUCED ALLOANTIBODY RESPONSES

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

Donor-specific transfusion via the portal venous route induces prolongation of H-2-compatible but not H-2-incompatible cardiac graft survival

In the H-2-compatible donor-recipient combination (BALB/c----DBA/2), pretransplant donor-specific blood transfusion (DST) via the portal venous (PV) route significantly prolonged cardiac graft survival. DST via the intravenous (IV) route (systemic circulation) also showed a marked prolongation of heart tissue transplant survival in this model. In the H-2-incompatible combination (BALB/c----CBA/H), DST via the IV - but not via the PV - route resulted in accelerated graft rejection.

The role of drug timing and histoincompatibility barrier in prolongation of cardiac graft survival in mice treated with donor-specific blood and cyclophosphamide

In the H-2-compatible donor—recipient combination (BALB/c → DBA/2), pretransplant donor-specific blood transfusion (DST) significantly prolonged graft survival. Concomitant use of immunosuppression by cyclophosphamide (CY) brought about potentiation of DST effect, resulting in long-term graft survival. In contrast, in the ‘strong’ H-2-incompatible combination (BALB/c → CBA/H) pretreatment of the recipients with donor-specific blood resulted in hyperacute graft rejection or in impairment of drug-induced immunosuppression when DST was used with a single dose of CY. In this model however, combination of DST with both pre- and posttransplant CY immunosuppression interacted beneficially to produce significant donor-specific prolongation of graft survival.

Effects of a single donor-specific blood transfusion on the survival of rat cardiac allografts.

It is now well recognized that pre-transplant donor-specific blood transfusion (DST) has a beneficial effect on the survival of allografts. To determine the optimal interval between DST and transplantation, and to analyze the mechanisms of this effect, the survival of cardiac allografts to rats which received a single DST was examined. The cardiac allograft survival was found to be prolonged when the DST was performed 1 to 6 weeks before grafting. In addition, recipient rat sera collected 1 to 6 weeks after a single DST showed significant inhibition of a mixed lymphocyte reaction (MLR). This MLR inhibition correlated with prolongation of survival of histoincompatible rat cardiac allografts. It thus appears that a single DST given from 1 to 6 weeks before transplantation has a beneficial effect on allograft survival and that MLR inhibition may be essential for inducing the effect of transfusion on organ transplantation.

Effect of Pretransplant Donor-specific Blood Transfusion on the Survival of Living-related Kidney Allografts.

Effect of Pretransplant Donor-specific Blood Transfusion on the Survival of Living-related Kidney Allografts.

Immune reactivity in congenic mice after allogeneic or isogeneic transfusion.

Pretransplant donor-specific blood transfusion has a beneficial effect on subsequent skin graft survival in B10 congenic mice when donor and recipient differ at both the K and I regions of the H-2 complex, but has no effect on graft survival in donor-recipient combinations differing at the K region alone. Immune reactivity was studied in B10.A recipients after a single allogeneic transfusion from B10 donors differing either at both K and I regions or at K alone, or after a transfusion of isogeneic B10.A blood. Spleen cells were assayed for the ability to respond in mixed lymphocyte culture (MLC) and cell mediated lymphocytotoxicity (CML) assays against blood donor and third-party at 2, 9, 16, and 30 days after transfusion. MLC and CML responses were nonspecifically suppressed in recipients transfused with allogeneic blood from K or K and I region disparate donors. The responses of mice receiving K region disparate blood were partially recovered by 9 days posttransfusion and had returned to normal 16 days posttransfusion. Responses of mice receiving K and I region disparate blood remained suppressed 30 days post transfusion. Isogeneic transfusion induced a suppression of the MLC response. Two days posttransfusion, the MLC response was suppressed to a variety of stimulators representing disparity at K alone; K and I; or K, I, and D. The response to K region disparate stimulators had recovered 9 days after isogeneic transfusion, but the response to K and I or K, I, and D region disparate stimulators remained suppressed 30 days after transfusion. The suppression observed after allogeneic or isogeneic transfusion was not due to a shift in the day of peak response in the transfused mice. Kinetic studies showed that the day of peak MLC and CML responses in the transfused mice corresponded to that of normal controls, but the level of response was significantly lower in the transfused mice.

Role of IA-positive cells in the beneficial effect of donor blood transfusion and induction of suppressor cells in cardiac allotransplantation of rats.

An attempt was made to reveal the role of Ia-positive cells in the beneficial effect of pretransplant donor-specific blood transfusion (DST) and induction of suppressor cells in the cardiac allotransplantation of rats. Mean graft survival time (MST) of F344 (RT1 1v1) hearts transplanted in WKA (RT1k) rats was 6.5 +/- 0.8 days. Pretreatment of recipients with 1 ml DST 10 days before grafting prolonged it to 56.5 +/- 38.1 days. Plasma or erythrocytes did not prolong MST at all, whereas pretreatment with 1 X 10(7) lymphocytes prolonged MST significantly. B enriched cells separated on a nylon-wool column prolonged MST, but T-enriched cells did not. Treatment of lymphocytes with interspecies crossreactive monoclonal anti-Ia antibody and complement eliminated the ability of lymphocytes to prolong MST, suggesting an important role of Ia-positive cells in the beneficial effect of DST. The role of Ia-positive cells in prolonging graft survival, was analyzed by a cell and serum transfer experiment. MST of F344 heart allografts transplanted in sublethally irradiated (500 rads) WKA rats was 17.4 +/- 4.0 days. MSTs of allografts in rats irradiated and treated with serum or spleen cells of blood-conditioned rats were 23.2 +/- 1.6 (P less than 0.05) and 5.2 +/- 0.7 (NS) days, respectively. On the other hand, MSTs of the grafts in rats irradiated and treated with serum or spleen cells harvested from rats that had accepted grafts for 30 days were 18.7 +/- 1.9 (NS) and 63.8 +/- 29.9 (P less than 0.01) days, respectively. The results indicate that the initial role of a serum factor in prolonging graft survival induced by DST containing Ia-positive cells was followed by activating suppressor cells by the presence of the allograft.