A Comparative Analysis of the Safety, Efficacy, and Cost of Islet Versus Pancreas Transplantation in Nonuremic Patients With Type 1 Diabetes.

Few current studies compare the outcomes of islet transplantation alone (ITA) and pancreas transplantation alone (PTA) for type 1 diabetes (T1D). We examined these two beta cell replacement therapies in nonuremic patients with T1D with respect to safety, graft function and cost. Sequential patients received PTA (n = 15) or ITA (n = 10) at our institution. Assessments of graft function included duration of insulin independence; glycemic control, as measured by hemoglobin A1c; and elimination of severe hypoglycemia. Cost analysis included all normalized costs associated with transplantation and inpatient management. ITA patients received one (n = 6) or two (n = 4) islet transplants. Mean duration of insulin independence in this group was 35 mo; 90% were independent at 1 year, and 70% were independent at 3 years. Mean duration of insulin independence in PTA was 55 mo; 93% were insulin independent at 1 year, and 64% were independent at 3 years. Glycemic control was comparable in all patients with functioning grafts, as were overall costs ($138 872 for ITA, $134 748 for PTA). We conclude that with advances in islet isolation and posttransplant management, ITA can produce outcomes similar to PTA and represents a clinically viable option to achieve long-term insulin independence in selected patients with T1D.

Clinical grade manufacturing of human alloantigen-reactive regulatory T cells for use in transplantation.

Regulatory T cell (Treg) therapy has the potential to induce transplantation tolerance so that immunosuppression and associated morbidity can be minimized. Alloantigen-reactive Tregs (arTregs) are more effective at preventing graft rejection than polyclonally expanded Tregs (PolyTregs) in murine models. We have developed a manufacturing process to expand human arTregs in short-term cultures using good manufacturing practice-compliant reagents. This process uses CD40L-activated allogeneic B cells to selectively expand arTregs followed by polyclonal restimulation to increase yield. Tregs expanded 100- to 1600-fold were highly alloantigen reactive and expressed the phenotype of stable Tregs. The alloantigen-expanded Tregs had a diverse TCR repertoire. They were more potent than PolyTregs in vitro and more effective at controlling allograft injuries in vivo in a humanized mouse model.

Induction of high levels of allogeneic hematopoietic reconstitution and donor-specific tolerance without myelosuppressive conditioning.

Donor-specific tolerance induced by bone marrow transplantation (BMT) would allow organ allografting without chronic immunosuppressive therapy. However, the toxicity of conditioning regimens used to achieve marrow engraftment has precluded the clinical use of BMT for tolerance induction. We have developed a BMT strategy that achieves alloengraftment without toxic or myelosuppressive host conditioning. B6 mice received depleting anti-CD4 and anti-CD8 monoclonal antibodies, local thymic irradiation, and a high-dose (174 x 10(6)) of major histocompatibility (MHC)-mismatched B10.A bone marrow cells (BMCs) divided over days 0 through 4. High levels of donor cells were observed among white blood cells (WBCs) of all lineages. Permanent, multilineage mixed chimerism; donor-specific skin-graft tolerance; and in vitro tolerance were observed in most animals. Large numbers of donor class II(high) cells were detected in thymuses of long-term chimeras, and their presence was associated with intrathymic deletion of donor-reactive host thymocytes. The treatment was not associated with significant myelosuppression, toxicity, or graft-versus-host disease (GVHD). Thus, high levels of allogeneic stem-cell engraftment can be achieved without myelosuppressive host conditioning. As stem-cell mobilization and in vitro culture techniques have increased the feasibility of administering high doses of hematopoietic cells to humans, this approach brings hematopoietic cell transplantation closer to clinical use for the induction of central deletional T-cell tolerance.

Islet transplantation in type 1 diabetics using an immunosuppressive protocol based on the anti-LFA-1 antibody efalizumab.

The applicability of islet transplantation as treatment for type 1 diabetes is limited by renal and islet toxicities of currently available immunosuppressants. We describe a novel immunosuppressive regimen using the antileukocyte functional antigen-1 antibody efalizumab which permits long-term islet allograft survival while reducing the need for corticosteroids and calcineurin inhibitors (CNI). Eight patients with type 1 diabetes and hypoglycemic unawareness received intraportal allogeneic islet transplants. Immunosuppression consisted of antithymocyte globulin induction followed by maintenance with efalizumab and sirolimus or mycophenolate. When efalizumab was withdrawn from the market in mid 2009, all patients were transitioned to regimens consisting of mycophenolate and sirolimus or mycophenolate and tacrolimus. All patients achieved insulin independence and four out of eight patients became independent after single-islet transplants. Insulin independent patients had no further hypoglycemic events, hemoglobin A1c levels decreased and renal function remained stable. Efalizumab was well tolerated and no serious adverse events were encountered. Although long-term follow-up is limited by discontinuation of efalizumab and transition to conventional imunnosuppression (including CNI in four cases), these results demonstrate that insulin independence after islet transplantation can be achieved with a CNI and steroid-free regimen. Such an approach may minimize renal and islet toxicity and thus further improve long-term islet allograft survival.

Human albumin preserves islet mass and function better than whole serum during pretransplantation islet culture.

OBJECTIVE: Human islet transplant protocols frequently include a brief period of islet culture before transplantation. Some investigators have suggested that medium supplementation with human serum might quench collagenase activity and provide better culture conditions when compared with human albumin. We studied the effect of whole serum on islet count, islet equivalence, insulin secretion, and DNA content in human islets. METHODS: Adult human islets isolated from a single pancreas with purity >50% were cultured in identical 150 islet equivalent samples at 37 degrees C using CMRL 1066-based islet medium (Mediatech) supplemented with either 0.5% human albumin or 10% human AB serum. Prior to culture and after 3 days, islets were assessed in vitro using dithizone staining (n = 4), insulin release after static glucose stimulation (n = 8), and DNA content (n = 8). RESULTS: After 3 days, islet mass (defined by the number of islets and islet equivalents counted after dithizone staining) was better preserved in islets cultured in 0.5% human albumin. Although the stimulation index and total DNA content were similar between groups, islets cultured in human albumin demonstrated greater absolute insulin secretion (p = .02) and insulin secretion per cell (p = .02). CONCLUSIONS: When used to supplement CMRL 1066-based islet culture medium, human albumin preserves islet mass and secretory capacity better than whole human serum. Human serum offers no advantage in islet preservation or function.

Separate regulation of peripheral hematopoietic and thymic engraftment.

Long-term multilineage chimerism, indicating pluripotent hematopoietic stem cell engraftment, was achieved in an Ly5-congenic strain combination without irradiation or other host conditioning when a large number of donor marrow cells (1.4-2x10(8)) was administered. However, the initial (2-4 weeks posttransplantation) percentages of T and B lymphocytes of donor origin were markedly lower than those of myeloid lineages. Steady-state levels of donor and host repopulation of all lineages were reached by 7 to 15 weeks posttransplantation and remained relatively constant for at least 41 weeks. B cell chimerism was similar to that seen in myeloid lineages at steady state. In contrast, long-term donor representation in the T cell lineage was much lower than in the B cell or myeloid lineages. Host treatment with depleting anti-T cell monoclonal antibodies increased the donor contribution to early T cell repopulation, but long-term T cell chimerism was still significantly lower in all lymphohematopoietic tissues, including the thymus, than B cell or myeloid cell chimerism. Pretreatment of hosts with 3.5 Gy of local irradiation to the thymic region further increased the donor contribution to initial T cell repopulation, which equaled that of other lineages at 4 to 7 weeks. However, donor representation in the T cell lineage declined by the time steady-state chimerism was attained and was lower than donor representation in the B cell or myeloid lineages. A higher dose of thymic irradiation (7 Gy) led to a reduction in this discrepancy, so that levels of donor thymopoiesis and hematopoiesis in other lineages were similar by 23 to 27 weeks posttransplantation. The differential contribution of adult donor marrow to long-term, steady-state thymopoiesis vs. hematopoiesis in other lineages under certain conditions in this competitive repopulation assay suggests that functionally distinguishable progenitors are responsible for these activities.

IL-2 inhibits early increases in serum gamma interferon levels associated with graft-versus-host-disease.

We have recently demonstrated that a short course of high-dose IL-2 administered to lethally irradiated mice leads to marked protection from early and late GVHD mortality, especially when T cell-depleted (TCD) host-type bone marrow cells (BMC) are also administered. IL-2 inhibits the GVHD-inducing activity of donor CD4+ cells without inhibiting their graft-vs.-leukemia effects. Since CD4+ T-lymphocytes produce a variety of cytokines, some of which have recently been implicated in the pathogenesis of GVHD, we have studied the possible effect of IL-2 administration on serum levels of various cytokines. Acute GVHD was induced in lethally irradiated B10 mice by bone marrow transplantation (BMT) with MHC-mismatched allogeneic (A/J) BMC and splenocytes. TCD B10 (host-type) BMC were coadministered to maximize the protective effect of IL-2. Serum cytokine levels were compared in recipients of these inocula with or without a protective course of IL-2 treatment. A marked increase in serum IFN-gamma levels was noted on days 3 through 5 post-BMT in GVHD mice compared with syngeneic BMT control recipients. This GVHD-induced rise in serum IFN-gamma was markedly inhibited in IL-2-protected mice. Murine IL-2 levels were only slightly increased in sera of GVHD mice, and were not influenced by treatment with human recombinant IL-2. Serum levels of the monokines TNF-alpha and IL-1 alpha showed variable early elevations in GVHD mice with or without IL-2 treatment, and were not different from levels observed in syngeneic controls. Serum levels of IFN-gamma, IL-1 alpha, and TNF-alpha all declined markedly by day 7 to 8 post-BMT, when GVHD mortality begins. Administration of neutralizing anti-IFN-gamma mAb did not attenuate and tended to accelerate GVHD mortality, and administration of exogenous IFN-gamma did not overcome the protective effect of IL-2 against GVHD. Together, our results indicate that GVHD is associated with high serum levels of several proinflammatory cytokines in the first week post-BMT, but that these levels decline by the time when GVHD mortality begins. IL-2 specifically inhibits the GVHD-associated production of IFN-gamma, but this inhibition in itself does not explain and may even mitigate the protective effect of IL-2 against early GVHD mortality. However, the demonstration that IL-2 markedly inhibits the production of a GVHD-associated cytokine raises the possibility that alterations in the production of as yet undefined cytokines may be responsible for IL-2-induced GVHD protection.

Inhibition of graft-versus-host disease by interleukin-2 treatment is associated with altered cytokine production by expanded graft-versus-host-reactive CD4+ helper cells.

In a fully MHC plus multiple minor antigen-mismatched murine bone marrow transplantation (BMT) model, we have demonstrated that a short course of high dose IL-2, begun on the day of BMT, protects against graft-versus-host disease (GVHD). This inhibitory effect is directed against donor CD4+ cells. To determine whether the mechanism of IL-2-induced GVHD protection involves clonal deletion or anergy of host-reactive donor T helper cells (Th), we performed limiting dilution analyses to measure the frequency of activated Th that reacted to donor, host, and third-party antigens in GVHD control and IL-2-protected mice. Marked and specific expansion of host-reactive Th was observed to a similar extent in GVHD control and IL-2-protected mice by day 5 after BMT, and the number of these cells in the spleen increased by several orders of magnitude between days 3 and 5 after BMT, which suggests that recirculation from other tissues occurred in this period. A high proportion (approximately 80%) of donor T cells expressed CD25 in both GVHD control and IL-2-protected mice on day 4 after BMT, which suggests a high level of bystander T cell activation. Since marked quantitative differences in the GVH response were not observed between GVHD control and IL-2-protected mice, we assessed both groups for qualitative differences in the Th response. Spleen cells isolated in the first 8 days after BMT were cultured with host-type, donor-type, or third-party stimulators or without stimulators, and cytokines were measured in supernatants harvested at 24 hr. GVHD was associated with marked increases in supernatant IFN-gamma levels from day 3 to day 6 after BMT, and with increases in IL-2 levels compared with naive A/J controls or syngeneic BMT controls stimulated with host antigens. Production of these cytokines was specifically induced by host-type antigens. Supernatants from spleens of IL-2-treated mice showed delayed kinetics of IFN-gamma production, and tended to contain higher levels of IL-4 in response to host antigen compared with GVHD controls on days 2 and 4 after BMT. Both IL-4 and IFN-gamma were produced almost exclusively by CD4+ cells in spleens of GVHD control and IL-2-protected mice on day 4. However, no consistent difference was observed between the groups in supernatant IL-2 or IL-10 levels, ruling out a simple Th1 to Th2 switch.(ABSTRACT TRUNCATED AT 400 WORDS)

Absence of host B7 expression is sufficient for long-term murine vascularized heart allograft survival.

CD28 antagonists have been shown to promote long-term graft survival and induce donor-specific tolerance. In this study, the role of CD28/B7 costimulation and the relative importance of host versus donor B7 expression in allograft rejection was assessed in a murine abdominal vascularized heterotopic heart transplant model. Wild-type, CD28-deficient, or B7-1/B7-2-deficient C57BL/6 (B6) mice were grafted with allogeneic wild type or B7-1/B7-2-deficient hearts. The results demonstrate allogeneic heart grafts survive long-term in mCTLA4Ig-treated B6 and untreated B7-1/B7-2-deficient B6 recipients but not CD28KO B6 mice. B7-1/B7-2KO B6 recipients treated with anti-CD28 (PV-1) or recombinant human IL-2 rejected the heart transplants indicating that these mice are immunologically competent to reject grafts if costimulatory signals are supplied or bypassed. Finally, there was no difference in rejection between normal animals transplanted with wild-type versus B7-1/B7-2-deficient hearts. These results support a critical role for B7-expressing host antigen presenting cells in the rejection of heart allografts in mice and differences among B7KO and CD28KO animals.

The role of CD8 and CD4 T cells in intestinal allograft rejection: a comparison of monoclonal antibody-treated and knockout mice.

BACKGROUND: The relative contribution of CD8 and CD4 T cells to allograft rejection remains an unresolved issue. Experimental results suggest that the relative importance of these T-cell subsets may vary depending on the model used and the organ studied. We have previously shown that treatment of murine recipients of intestinal allografts with a depleting anti-CD8 or a depleting anti-CD4 monoclonal antibody (mAb) significantly inhibited allograft rejection. This study was undertaken to further examine the contribution of CD8 and CD4 T cells to the rejection of intestinal allografts. METHODS: Intestinal allografts from B6C3F1/J (C57BL/6 x C3H/HeJ) mice were transplanted into C57BL/6 recipients. Recipient groups included mice with an acquired deficiency in CD8 or CD4 T cells caused by treatment with depleting mAb or mice genetically deficient in CD8 or CD4 T cells as a result of disruption of the genes encoding major histocompatibility complex (MHC) class I, MHC class II, CD8, or CD4. In all cases, rejection was assessed histologically at predetermined time points. In some recipient groups, graft function was also assessed using a maltose absorption assay. RESULTS: Rejection, assessed between days 10 and 28 after transplantation, was significantly inhibited in mice deficient in CD8 or CD4 T cells after treatment with depleting mAb. In contrast, mice genetically deficient in either CD8 T cells (MHC class I or CD8 knockouts) or CD4 T cells (MHC class II or CD4 knockouts) rejected intestinal allografts promptly. Both histologic and functional evaluation of anti-CD8 mAb-treated mice on day 60 showed that the inhibition of rejection persisted even after the return of a substantial number of CD8 T cells. Although intestinal allografts from anti-CD8 mAb-treated mice displayed little to no evidence of rejection on day 60 after transplantation, these mice were able to reject both donor and third-party skin grafts. CONCLUSIONS: These results demonstrate that the inhibition of intestinal allograft rejection associated with mAb treatment is not attributable solely to depletion of CD8 or CD4 T cells. Furthermore, anti-CD8 mAb administration did not induce donor-specific tolerance or cause nonspecific immune suppression, as indicated by the skin-grafting experiments. Our findings suggest that at least some depleting mAbs mediate their protective effect on allograft rejection via an alternative mechanism such as the induction of a regulatory cell population(s).

IL-2-induced GVHD protection is not inhibited by cyclosporine and is maximal when IL-2 is given over a 25 h period beginning on the day following bone marrow transplantation.

We have recently demonstrated, in a fully MHC-mis-matched murine bone marrow transplantation (BMT) model, that administration of a short course of high-dose interleukin 2 (IL-2) markedly delays the onset of graft-versus-host disease (GVHD) without compromising alloengraftment or the graft-versus-leukemia (GVL) effect of allogeneic T cells. Early timing of IL-2 administration and high dose were shown to be critical to achieve this protective effect. Although a 2.5 day course of IL-2, begun on the day of BMT, was found to confer marked protection without observable toxicity, shorter courses and a higher dose of IL-2 than 5 x 10(4) Cetus units per treatment have not been previously evaluated in this model. We now demonstrate that administration of a three-treatment course of IL-2 over a 25 h period beginning 15 h following BMT is sufficient to provide maximal GVHD protection, that increasing the IL-2 dose beyond 5 x 10(4) units per treatment does not further improve the level of GVHD protection, and that further division of IL-2 treatments to achieve more constant tissue levels does not result in improved GVHD protection. We also demonstrate that IL-2 is still protective when administered in combination with cyclosporine. These results suggest that IL-2 administered in a sufficient short course to avoid toxicity might have the potential to achieve effective GVHD prophylaxis in humans, even if given in combination with cyclosporine.

Human islet viability and function is maintained during high-density shipment in silicone rubber membrane vessels.

BACKGROUND: The shipment of human islets (IE) from processing centers to distant laboratories is beneficial for both research and clinical applications. The maintenance of islet viability and function in transit is critically important. Gas-permeable silicone rubber membrane (SRM) vessels reduce the risk of hypoxia-induced death or dysfunction during high-density islet culture or shipment. SRM vessels may offer additional advantages: they are cost-effective (fewer flasks, less labor needed), safer (lower contamination risk), and simpler (culture vessel can also be used for shipment). METHOD: IE were isolated from two manufacturing centers and shipped in 10-cm(2) surface area SRM vessels in temperature- and pressure-controlled containers to a distant center after at least 2 days of culture (n = 6). Three conditions were examined: low density (LD), high density (HD), and a microcentrifuge tube negative control (NC). LD was designed to mimic the standard culture density for IE preparations (200 IE/cm(2)), while HD was designed to have a 20-fold higher tissue density, which would enable the culture of an entire human isolation in 1-3 vessels. Upon receipt, islets were assessed for viability (measured by oxygen consumption rate normalized to DNA content [OCR/DNA)]), quantity (measured by DNA), and, when possible, potency and function (measured by dynamic glucose-stimulated insulin secretion measurements and transplants in immunodeficient B6 Rag(+/-) mice). Postshipment OCR/DNA was not reduced in HD vs LD and was substantially reduced in the NC condition. HD islets exhibited normal function postshipment. Based on the data, we conclude that entire islet isolations (up to 400,000 IE) may be shipped using a single, larger SRM vessel with no negative effect on viability and ex vivo and in vivo function.

Interleukin-2 inhibits graft-versus-host disease-promoting activity of CD4+ cells while preserving CD4- and CD8-mediated graft-versus-leukemia effects.

We have recently shown that a short course of high-dose interleukin-2 (IL-2) can markedly inhibit the graft-versus-host disease (GVHD)-promoting activity of donor CD4+ T cells. The difficulty in dissociating GVHD-promoting from graft-versus-leukemia (GVL) effects of alloreactive donor T cells currently prevents clinical bone marrow transplantation (BMT) from fulfilling its full potential. To test the capacity of IL-2 treatment to promote such a dissociation, we have developed a new murine transplantable acute myelogenous leukemia model using a class II major histocompatibility complex-positive BALB/c Moloney murine leukemia virus-induced promonocytic leukemia, 2B-4-2. BALB/c mice receiving 2.5 x 10(5) 2B-4-2 cells intravenously 1 week before irradiation and syngeneic BMT died from leukemia within 2 to 4 weeks after BMT. Administration of syngeneic spleen cells and/or a 2.5-day course of IL-2 treatment alone did not inhibit leukemic mortality. In contrast, administration of non-T-cell-depleted fully allogeneic B10 (H-2b) spleen cells and T-cell-depleted B10 marrow led to a significant delay in leukemic mortality in IL-2-treated mice. In these animals GVHD was inhibited by IL-2 treatment. GVL effects were mediated entirely by donor CD4+ and CD8+ T cells. Remarkably, IL-2 administration did not diminish the magnitude of the GVL effect of either T-cell subset. This was surprising, because CD4-mediated GVHD was inhibited in the same animals in which CD4-mediated GVL effects were not reduced by IL-2 treatment. These results suggest a novel mechanism by which GVHD and GVL effects of a single unprimed alloreactive T-cell subset can be dissociated; different CD4 activities promote GVHD and GVL effects, and the former, but not the latter activities are inhibited by treatment with IL-2.

Interleukin-12 inhibits graft-versus-host disease through an Fas-mediated mechanism associated with alterations in donor T-cell activation and expansion.

We have recently made the paradoxical observation that a single injection of recombinant murine interleukin-12 (IL-12) on the day of bone marrow transplantation (BMT) inhibits graft-versus-host disease (GVHD) in lethally irradiated mice receiving fully major histocompatability complex (MHC)-mismatched bone marrow and spleen cells. We have now examined the mechanism of this effect of IL-12 on acute GVHD. By day 4 post-BMT, IL-12-treated mice showed marked reductions in splenic donor CD4(+) and CD8(+) T cells compared with GVHD controls. Expression of the early activation markers IL-2R alpha chain (CD25) and CD69 on splenic donor CD4(+) cells was considerably higher at early time points (36 and 72 hours post-BMT) in IL-12-treated mice compared with GVHD controls. However, the later, GVHD-associated increase in CD25 and very late antigen-4 (VLA-4) expression on donor T cells was greatly depressed in IL-12-protected mice compared with GVHD controls. The marked GVHD-associated expansion of host-reactive T helper cells by day 4 was also completely inhibited in the IL-12-treated group. Expression of Fas was increased on donor CD4 cells of IL-12-treated mice compared with those of controls on days 3 through 7 post-BMT. Furthermore, the ability of IL-12 to protect against GVHD was at least partially dependent on the ability of donor cells to express functional Fas molecules. We conclude that IL-12 treatment at the time of BMT markedly perturbs the activation of alloreactive donor CD4(+) T cells that play a critical role in the pathogenesis of acute GVHD. We hypothesize that these perturbations culminate in Fas-dependent apoptosis of donor T cells, thus impeding their expansion and their GVHD-promoting activity.

Interleukin-12 inhibits murine graft-versus-host disease.

Interleukin-12 (IL-12) is a potent immunostimulatory cytokine and an inducer of type-1 T-helper cell activity and of cytotoxic T lymphocyte and natural killer cell function. We report here the paradoxical observation that a single injection of 4,900 IU of recombinant murine IL-12 inhibits acute graft-versus-host disease (GVHD) in a fully major histocompatibility complex (MHC) plus multiple minor antigen-mismatched bone marrow transplantation (BMT) model (A/J-->B10). The protective effect was enhanced by administration of T-cell-depleted host-type BM cells, and complete donor-type lymphohematopoietic reconstitution was observed in most animals. Treatment with a protective course of IL-12 led to increased serum interferon-gamma (IFN-gamma) levels as compared with those for GVHD controls at early time points, when IFN-gamma was produced predominantly by host-type natural killer cells, but led to almost complete inhibition of the later GVHD-associated increase in serum IFN-gamma levels, when IFN-gamma is produced predominantly by CD4+ T cells. Furthermore, IL-12 treatment was associated with marked alterations in the kinetics of donor T-cell expansion. Reductions in donor CD4+ and CD8+ T cells were observed in the spleen on day 4 post-BMT, but a marked increase in donor CD8+ cells was observed on day 7. Unlike broadly immunosuppressive methods for inhibiting GVHD, which are associated with loss of antileukemic effects, IL-12 has the potential to mediate antileukemic effects of its own; therefore, the GVHD-inhibitory effects of IL-12 described here suggest a potential application for this cytokine in clinical BMT.

Interleukin-12 preserves the graft-versus-leukemia effect of allogeneic CD8 T cells while inhibiting CD4-dependent graft-versus-host disease in mice.

We have recently demonstrated that a single injection of 4,900 IU of interleukin-12 (IL-12) on the day of bone marrow transplantation (BMT) markedly inhibits acute graft-versus-host disease (GVHD) in a fully major histocompatibility complex plus minor antigen-mismatched BMT model (A/J --> B10, H-2(a) --> H-2(b)), in which donor CD4(+) T cells are required for the induction of acute GVHD. We show here that donor CD8-dependent graft-versus-leukemia (GVL) effects against EL4 (H-2(b)) leukemia/lymphoma can be preserved while GVHD is inhibited by IL-12 in this model. In mice in which IL-12 mediated a significant protective effect against GVHD, marked GVL effects of allogeneic T cells against EL4 were observed. GVL effects against EL4 depended on CD8-mediated alloreactivity, protection was not observed in recipients of either syngeneic (B10) or CD8-depleted allogeneic spleen cells. Furthermore, we analyzed IL-12-treated recipients of EL4 and A/J spleen cells which survived for more than 100 days. No EL4 cells were detected in these mice by flow cytometry, tissue culture, adoptive transfer, necropsies, or histologic examination. Both GVL effects and the inhibitory effect of IL-12 on GVHD were diminished by neutralizing anti-interferon-gamma (IFN-gamma) monoclonal antibody. This study demonstrates that IL-12-induced IFN-gamma production plays a role in the protective effect of IL-12 against GVHD. Furthermore, IFN-gamma is involved in the GVL effect against EL4 leukemia, demonstrating that protection from CD4-mediated GVHD and CD8-dependent anti-leukemic activity can be provided by a single cytokine, IFN-gamma. These observations may provide the basis for a new approach to inhibiting GVHD while preserving GVL effects of alloreactivity.

Dose and timing of interleukin (IL)-12 and timing and type of total-body irradiation: effects on graft-vs.-host disease inhibition and toxicity of exogenous IL-12 in murine bone marrow transplant recipients.

Paradoxically, a single injection of recombinant murine interleukin (IL)-12 on the day of bone marrow transplantation (BMT) inhibits graft-vs.-host disease (GVHD) while preserving graft-vs.-leukemia (GVL) effects in lethally irradiated mice receiving fully MHC-mismatched bone marrow and spleen cells. These protective effects are mediated by interferon (IFN)-gamma, whose early secretion is induced by IL-12 treatment. We investigated the relationship of IL-12 dose and timing of administration, as well as timing and type of total-body irradiation (TBI), with the ability of IL-12 to inhibit GVHD or mediate toxicity. The results show that a relatively low dose of IL-12 (as little as 50 U in a single injection) can mediate significant GVHD protection. The timing of IL-12 administration, however, is a critical factor. IL-12 administered 1 hour before BMT was most protective, but protection was still observed when it was administered 1-12 hours after BMT. Delaying IL-12 administration to 36 hours post-BMT completely obviated its protective effect. Administration of a second IL-12 injection 6 days after BMT negated the protective effect of an initial injection at the time of BMT. While IL-12 protection was evident when TBI was administered by 137Cs-irradiator in one or two fractions on day -1 or day 0, the use of an X-irradiator to deliver TBI on day -1 was associated with marked IL-12 toxicity. Whereas the protective effect of IL-12 against GVHD depended on donor-derived IFN-gamma, toxicity depended on the ability of host cells to produce IFN-gamma. Careful studies are warranted to test the effects of IL-12 in the context of BMT with various conditioning regimens in large animal preclinical models before this novel approach to GVHD protection can be applied clinically.