Single Donor-specific Transfusion Research Articles

Indirectly Activated Treg Allow Dominant Tolerance to Murine Skin-grafts Across an MHC Class I Mismatch After a Single Donor-specific Transfusion.

Tolerance induced in stringent animal transplant models using donor-specific transfusions (DST) has previously required additional immunological manipulation. Here, we demonstrate a dominant skin-allograft tolerance model induced by a single DST across an major histocompatibility class I mismatch in an unmanipulated B6 host. C57BL/6 (H-2) (B6) mice were injected intravenously with splenocytes from B6.C.H-2 (H-2k) (bm1) or F1 (B6 × bm1) mice before skin transplantation. Mice were transplanted 7 days postinjection with donor (bm1 or F1) and third-party B10.BR (H-2) skin grafts. B6 hosts acutely rejected skin grafts from B6.C.H-2 (bm1) and F1 (B6 × bm1) mice. A single transfusion of F1 splenocytes into B6 mice without any additional immune modulation led to permanent acceptance of F1 skin grafts. This graft acceptance was associated with persistence of donor cells long-term in vivo. The more rapid removal of DST bm1 cells than F1 cells was reduced by natural killer-cell depletion. Tolerant grafts survived an in vivo challenge with naive splenocytes. Both CD4CD25 and CD4CD25 T cells from F1 DST treated B6 mice suppressed alloproliferation in vitro. Tolerance was associated with expansion of peripheral Foxp3CD4CD25 regulatory T cells (Treg) and increased forkhead box P3 (Foxp3) expression in tolerant grafts. In tolerant mice, Foxp3 Treg arises from the proliferation of indirectly activated natural Foxp3 Treg (nTreg) and depletion of Foxp3 Treg abrogates skin-graft tolerance. This study demonstrates that the persistence of transfused semiallogeneic donor cells mismatched at major histocompatibility class I can enhance tolerance to subsequent skin allografts through indirectly expanded nTreg leading to dominant tolerance without additional immunological manipulation.

Long-term graft function of adult rat islets after transplantation in immunocompetent diabetic mice treated with donor specific splenocytes and anti-CD154 monoclonal antibody

Aim: 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 leeds to long-term islet allograft survival in mice. We hypothesized that this protocol could also induce long-term graft survival of adult rat islets in immunocompentent diabetic mice.

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.

Pretransplant blood transfusion without additional immunotherapy generates CD25+CD4+ regulatory T cells: a potential explanation for the blood-transfusion effect.

Preoperative blood transfusion has had a significant historic impact on graft outcome in clinical kidney transplantation, and the effect has been widely replicated in many experimental transplant models. Although the mechanisms underlying the blood-transfusion effect are poorly understood, one possibility is that preexposure to alloantigen results in the induction of regulatory cells with the capacity to control the effector arm of the immune response. Recent studies in autoimmune models have shown that T cells with regulatory function can be isolated from unmanipulated animals on the basis of CD25 expression, and we have recently shown that pretreatment of recipient mice with donor alloantigen combined with anti-CD4 antibody therapy generates CD25+CD4+ T cells that can prevent graft rejection. We therefore used this sensitive adoptive transfer mouse model to ask whether blood transfusion in the absence of any other treatment can also lead to the generation of alloreactive CD25+CD4+ regulatory T cells. Although a single donor-specific transfusion (DST) fails to induce dominant regulation, we demonstrate that pretreatment with multiple DSTs generates CD25+CD4+ T cells that are as effective as those that result from blood transfusion under anti-CD4 antibody cover. More importantly, our results show that these cells also develop following multiple transfusions of unrelated (random) blood. These results provide a basis for understanding the blood-transfusion effect in transplantation and, by providing a link between naturally occurring regulatory cells and those induced by alloantigen, may shed new light on the fundamental basis of the effect itself.

Skin allograft maintenance in a new synchimeric model system of tolerance.

Treatment of mice with a single donor-specific transfusion plus a brief course of anti-CD154 mAb uniformly induces donor-specific transplantation tolerance characterized by the deletion of alloreactive CD8+ T cells. Survival of islet allografts in treated mice is permanent, but skin grafts eventually fail unless recipients are thymectomized. To analyze the mechanisms underlying tolerance induction, maintenance, and failure in euthymic mice we created a new analytical system based on allo-TCR-transgenic hemopoietic chimeric graft recipients. Chimeras were CBA (H-2(k)) mice engrafted with small numbers of syngeneic TCR-transgenic KB5 bone marrow cells. These mice subsequently circulated a self-renewing trace population of anti-H-2(b)-alloreactive CD8+ T cells maturing in a normal microenvironment. With this system, we studied the maintenance of H-2(b) allografts in tolerized mice. We documented that alloreactive CD8+ T cells deleted during tolerance induction slowly returned toward pretreatment levels. Skin allograft rejection in this system occurred in the context of 1) increasing numbers of alloreactive CD8+ cells; 2) a decline in anti-CD154 mAb concentration to levels too low to inhibit costimulatory functions; and 3) activation of the alloreactive CD8+ T cells during graft rejection following deliberate depletion of regulatory CD4+ T cells. Rejection of healed-in allografts in tolerized mice appears to be a dynamic process dependent on the level of residual costimulation blockade, CD4+ regulatory cells, and activated alloreactive CD8+ thymic emigrants that have repopulated the periphery after tolerization.

Viral infection abrogates CD8(+) T-cell deletion induced by costimulation blockade.

Treatment with a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (mAb) prolongs skin allograft survival in mice. It is known that prolongation of allograft survival by this method depends in part on deletion of alloreactive CD8(+) T cells at the time of tolerance induction. Recent data suggest that infection with lymphocytic choriomeningitis virus (LCMV) abrogates the ability of this protocol to prolong graft survival. To study the mechanism by which viral infection abrogates allograft survival, we determined (1) the fate of tracer populations of alloreactive transgenic CD8(+) T cells and (2) the duration of skin allograft survival following treatment with DST and anti-CD154 mAb in the presence or absence of LCMV infection. We confirmed that treatment of uninfected mice with DST and anti-CD154 mAb leads to the deletion of alloreactive CD8(+) T cells and is associated with prolongation of skin allograft survival. In contrast, treatment with DST and anti-CD154 mAb in the presence of intercurrent LCMV infection was associated with the failure to delete alloreactive CD8(+) T cells and with the rapid rejection of skin allografts. The number of alloreactive CD8(+) cells actually increased significantly, and the cells acquired an activated phenotype. Interference with the deletion of alloreactive CD8(+) T cells mediated by DST and anti-CD154 mAb may in part be the mechanism by which viral infection abrogates transplantation tolerance induction.

NOD mice have a generalized defect in their response to transplantation tolerance induction.

A protocol consisting of a single donor-specific transfusion (DST) plus a brief course of anti-CD154 monoclonal antibody (anti-CD40 ligand mAb) induces permanent islet allograft survival in chemically diabetic mice, but its efficacy in mice with autoimmune diabetes is unknown. Confirming a previous report, we first observed that treatment of young female NOD mice with anti-CD154 mAb reduced the frequency of diabetes through 1 year of age to 43%, compared with 73% in untreated controls. We also confirmed that spontaneously diabetic NOD mice transplanted with syngeneic (NOD-Prkdc(scid)/Prkdc(scid)) or allogeneic (BALB/c) islets rapidly reject their grafts. Graft survival was not prolonged, however, by pretreatment with either anti-CD154 mAb alone or anti-CD154 mAb plus DST. In addition, allograft rejection in NOD mice was not restricted to islet grafts. Anti-CD154 mAb plus DST treatment failed to prolong skin allograft survival in nondiabetic male NOD mice. The inability to induce transplantation tolerance in NOD (H2g7) mice was associated with non-major histocompatibility complex (MHC) genes. Treatment with DST and anti-CD154 mAb prolonged skin allograft survival in both C57BL/6 (H2b) and C57BL/6.NOD-H2g7 mice, but it was ineffective in NOD, NOD.SWR-H2q, and NOR (H2g7) mice. Mitogen-stimulated interleukin-1beta production by antigen-presenting cells was greater in strains susceptible to tolerance induction than in the strains resistant to tolerance induction. The results suggest the existence of a general defect in tolerance mechanisms in NOD mice. This genetic defect involves defective antigen-presenting cell maturation, leads to spontaneous autoimmune diabetes in the presence of the H2g7 MHC, and precludes the induction of transplantation tolerance irrespective of MHC haplotype. Promising islet transplantation methods based on overcoming the alloimmune response by interference with costimulation may require modification or amplification for use in the setting of autoimmune diabetes.

Kinetics of induction of transplantation tolerance with a nondepleting anti-Cd4 monoclonal antibody and donor-specific transfusion before transplantation. A critical period of time is required for development of immunological unresponsiveness.

The combination of a depleting anti-Cd43 monoclonal antibody (mAb) and a single donor-specific transfusion before transplantation has been shown to induce operational transplantation tolerance in the majority of cardiac allograft recipients in a mouse model. To examine a protocol which might be more clinically relevant, we have modified this tolerance-inducing protocol by substituting the depleting with a nondepleting anti-Cd4 mAb. We show that this form of pretreatment can also induce immunologic unresponsiveness in most recipients (C3H/He, H2(k)), provided a critical period of time, in this case 28 days, is allowed between pretreatment and transplantation of a fully mismatched heart graft (H2(b)). When only 1 or 2 weeks were allowed between pretreatment and transplantation, only slight graft prolongation was obtained when compared with recipients receiving anti-Cd4 mAb alone, at these time points. Maintenance of tolerance in this model was due, at least in part, to active mechanisms as immunologic unresponsiveness to donor antigens could be transferred to naive syngeneic mice by splenocytes from recipients bearing long-term functioning grafts. These findings suggest that a population of regulatory cells develop after pretreatment with nondepleting anti-Cd4 mAb and donor-specific transfusion, and that it takes at least 1 month for these cells to expand and effectively drive the recipient's immune system toward immunologic unresponsiveness.

Donor-recipient microchimerism is not required for tolerance induction following recipient pretreatment with donor-specific transfusion and anti-CD4 antibody. Evidence of a clear role for short-term antigen persistence.

There is considerable current interest in the possibility that long-term graft acceptance in clinical solid-organ transplantation might be dependent upon the development of a microchimeric state between the donor and recipient. This possibility has been prompted by the observation that in some transplant patients cells of donor origin can be detected in peripheral sites such as the skin, and it has been proposed that these cells play an essential role in maintaining graft survival. The hypothesis that peripheral microchimerism is an absolute requirement for the long-term survival of solid-organ allografts was tested in a well-characterized model of transplantation tolerance in which adult recipient mice are pretreated 28 days before transplant with a single donor-specific transfusion under the cover of a depleting anti-CD4 antibody. Mice pretreated with this protocol accept donor-specific cardiac allografts (MST > 100 days) but reject those of a third party (MST 16.5 days). The protocol leads to operational tolerance in the long term in that donor-specific skin grafts show prolonged survival while those from a third party strain are rejected acutely. Since peripheral blood contains haematopoietic stem cells we speculated that the success of the anti-CD4/DST protocol might be dependent on the development of microchimerism. To address this possibility the DST was irradiated before administration under anti-CD4 antibody cover in order to prevent donor stem cells in the transfusion from establishing a microchimeric state in the recipient animals. Mice in this group rejected their grafts acutely (MST 12 days), suggesting indeed that stem cells might be very important in the success of this model. However, when the protocol was modified by giving three additional doses of irradiated whole blood to increase the possibility that recipient T cells would be engaged during antibody-induced immunocompromise, graft prolongation was restored (MST > 100 days). These results demonstrate that persistence of donor antigen at the time of anti-CD4 antibody treatment is critical for the induction of unresponsiveness in this model and show that microchimerism is not an absolute requirement for long-term graft survival.

INDEFINITE ALLOGRAFT SURVIVAL INDUCED BY THE COMBINATION OF MULTIPLE DONOR-SPECIFIC TRANSFUSIONS, CYCLOSPORINE, AND AN ANTI-T CELL MONOCLONAL ANTIBODY IN A PROTOCOL RELEVANT TO CADAVERIC ORGAN TRANSPLANTATION THE IMPORTANCE OF PROLONGED POSTTRANSPLANT CYCLOSPORINE COVERAGE

A single donor specific transfusion (DST) 24 hr pretransplant and Cyclosporine (CsA) are synergistic in inducing prolongation of allograft survival. This synergy was further potentiated by giving 3 additional DSTs at weekly intervals posttransplant, or by the additional administration of a small dose of the anti-T cell monoclonal antibody OX-52 24 hr pretransplant to the above protocol. This study determined whether indefinite survival can be achieved in the ACI-to-Lewis heart allograft model by combining an anti-T cell monoclonal antibody, 4 DSTs given once 24 hr pretransplant and weekly 3 times posttransplant, and CsA. CsA was given daily for 28 days or 60 days posttransplant. The dose consisted of CsA 5 mg/kg/day starting 24 hr pretransplant and continuing until day 7 posttransplant and followed by 2.5 mg/kg/day for either 21 more days (total CsA days = 28 days) or 53 days (total CsA days = 60 days). The anti-T cell monoclonal antibody OX52 (200 micrograms i.v.) was given once 24 hr pretransplant. DST (1 ml) was given 24 hr pretransplant and on days 7, 14, and 21 posttransplant. Low-dose CsA for 28 days never induced indefinite allograft survival. CsA for 60 days, however, resulted in the occasional indefinite allograft survival (> 1 year) even in this difficult model. The addition of DST 24 hr pre- and posttransplant on days 7, 14, and 21 to both 28 days and 60 days of CsA further prolonged allograft survival. The best survival was seen in the group given DST 24 hr pre- and on days 7, 14, and 21 posttransplant and 60 days of CsA, with all the allografts surviving beyond 100 days of CsA, with all the allografts surviving beyond 100 days and more than 50% surviving indefinitely (> 1 year). The addition of a single dose of OX52 24 hr pretransplant to the multiple DSTs and 28 days of CsA protocol significantly improved indefinite allograft survival but its influence was less dramatic when given to the multiple DSTs and 60 days of CsA protocol. This beneficial interaction between CsA, DST, and an anti-T cell MoAb offers a clinically applicable protocol for both living donor and cadaveric organ transplantation in inducing donor-specific hyporesponsiveness, and further investigations are warranted.

Effects of pre- and postengraftment donor-specific transfusions and cyclosporine on the enhancement of experimental allograft survival

The tolerogenic effects of immediate pretransplant donor-specific transfusion (DST) with cyclosporine (CsA) have been well described. The purpose of this study was to determine if these effects could be improved upon by the administration of post-transplant DSTs. When added to a 29-day course of CsA, a single DST given 24 hr pretransplant improved graft survival compared to CsA alone (84.9 ± 12.3 vs 40.0 ± 8.8 days; P < 0.05). The administration of an additional DST on post-transplant Days 7, 14, and 21 further improved this survival to 152 ± 28 days, with 45% of grafts surviving >200 days, until sacrifice. The donor specificity of this effect was demonstrated by the increased survival of second ACI grafts transplanted into Lewis recipients with existing “tolerant” ACI allografts (long-term survivors, or LTS); third-party Buffalo rat hearts transplanted into LTS rats in a similar manner were rejected normally. Loss of graft antigenicity was not seen, as retransplanted ACI hearts obtained from LTS Lewis rats were rejected in a first-order manner. From this we conclude that (1) the addition of multiple post-transplant DSTs improve the enhancement seen with preoperative DST and CsA, (2) loss of graft antigenicity does not contribute to this improved enhancement, and (3) this effect appears to be donor antigen specific.

ELIMINATION OF ACUTE GVHD AND PROLONGATION OF RAT PANCREAS ALLOGRAFT SURVIVAL WITH DST, CYCLOSPORINE, AND SPLEEN TRANSPLANTATION

Allogeneic spleen transplantation has been shown to have a tolerizing effect on pancreas allograft survival in rats. In this study we examined the effect of blood transfusions and cyclosporine administration on both rat pancreas allograft survival and acute graft-versus-host disease in BN recipients of Lewis pancreas-spleen allografts. We found that in this strain combination significant pancreas allograft prolongation occurred when the spleen was included en bloc with the pancreas graft. However, 50% of these recipients developed GVHD and died. A single donor-specific transfusion delivered to BN recipients of Lewis pancreas and pancreas-spleen allografts did not extend graft survival, but precluded the development of GVHD. A short, 6-day, and long, 26-day, course of CsA to recipients of pancreas and pancreas-spleen allografts extended graft and animal survival times, although not indefinitely. All pancreas-spleen recipients of both CsA protocols died following acute GVHD. Combined DST and CsA (short and long-course) administration in pancreas-only allograft recipients was deleterious to graft survival, compared with CsA administration alone. However, in pancreas-spleen recipients, combined DST and CsA had an additive effect. Moreover, in DST and long-course CsA-treated pancreas-spleen recipients, indefinite graft survival occurred with no signs of acute GVHD. The beneficial effect of the spleen allograft on pancreas graft survival was not dependent upon GVHD, since splenic-induced prolongation of pancreas graft survival was observed in the F1-donor to parent-recipient combination.

Augmentation of donor-specific transfusion and cyclosporine effects with dietary linoleic acid.

Increased prostaglandin production is a possible mechanism for the immunosuppressive effects of both cyclosporine and blood transfusions. Therefore, dietary supplementation with linoleic acid, a prostaglandin precursor, combined with either modality could act synergistically. Intraabdominal cardiac allografts were performed from Buffalo rat donors to Lewis recipients. Transplant recipients received a single donor-specific transfusion, low-dose cyclosporine (CsA, 1 mg/kd/d x 7 days), dietary supplementation with linoleic acid (LA, 16% of total calories) or a combination of the three modalities. CsA, DST or LA alone significantly prolonged allograft survival. Both CsA and LA acted synergistically with DST in further prolongation of survival--however, animals receiving all three modalities achieved 100% long-term survival. Augmentation of transfusion- and cyclosporine-induced immunosuppression with dietary prostaglandin precursor is possible.

The effect of donor specific transfusions and dietary fatty acids on rat cardiac allograft survival

A rat heterotopic cardiac transplant model was used to study the effect of dietary lipids on the immune response. Animals receiving linoleic acid (LA), oleic acid (OA), and fish oil (FO) enriched diets showed significant prolongation of allograft survival when compared to the control diet fed animals. When LA was given to animals who had received a single donor specific transfusion (DST) augmentation of the beneficial DST effect was observed, while the OA and FO fed groups showed no differences from control DST animals. Dietary regulation of the immune response, possibly through manipulation of arachidonic acid metabolism, is implied.