Anti-ceramide single-chain variable fragment mitigates radiation GI syndrome mortality independent of DNA repair.

After 9/11, threat of nuclear attack on American urban centers prompted government agencies to develop medical radiation countermeasures to mitigate hematopoietic acute radiation syndrome (H-ARS) and higher-dose gastrointestinal acute radiation syndrome (GI-ARS) lethality. While repurposing leukemia drugs that enhance bone marrow repopulation successfully treats H-ARS in preclinical models, no mitigator potentially deliverable under mass casualty conditions preserves GI tract. Here, we report generation of an anti-ceramide 6B5 single-chain variable fragment (scFv) and show that s.c. 6B5 scFv delivery at 24 hours after a 90% lethal GI-ARS dose of 15 Gy mitigated mouse lethality, despite administration after DNA repair was complete. We defined an alternate target to DNA repair, an evolving pattern of ceramide-mediated endothelial apoptosis after radiation, which when disrupted by 6B5 scFv, initiates a durable program of tissue repair, permitting crypt, organ, and mouse survival. We posit that successful preclinical development will render anti-ceramide 6B5 scFv a candidate for inclusion in the Strategic National Stockpile for distribution after a radiation catastrophe.

Fusion of lysosomes to plasma membrane initiates radiation-induced apoptosis.

Diverse stresses, including reactive oxygen species (ROS), ionizing radiation, and chemotherapies, activate acid sphingomyelinase (ASMase) and generate the second messenger ceramide at plasma membranes, triggering apoptosis in specific cells, such as hematopoietic cells and endothelium. Ceramide elevation drives local bilayer reorganization into ceramide-rich platforms, macrodomains (0.5-5-µm diameter) that transmit apoptotic signals. An unresolved issue is how ASMase residing within lysosomes is released extracellularly within seconds to hydrolyze sphingomyelin preferentially enriched in outer plasma membranes. Here we show that physical damage by ionizing radiation and ROS induces full-thickness membrane disruption that allows local calcium influx, membrane lysosome fusion, and ASMase release. Further, electron microscopy reveals that plasma membrane "nanopore-like" structures (∼100-nm diameter) form rapidly due to lipid peroxidation, allowing calcium entry to initiate lysosome fusion. We posit that the extent of upstream damage to mammalian plasma membranes, calibrated by severity of nanopore-mediated local calcium influx for lysosome fusion, represents a biophysical mechanism for cell death induction.

Organoids Reveal That Inherent Radiosensitivity of Small and Large Intestinal Stem Cells Determines Organ Sensitivity.

Tissue survival responses to ionizing radiation are nonlinear with dose, rather yielding tissue-specific descending curves that impede straightforward analysis of biologic effects. Apoptotic cell death often occurs at low doses, while at clinically relevant intermediate doses, double-strand break misrepair yields mitotic death that determines outcome. As researchers frequently use a single low dose for experimentation, such strategies may inaccurately depict inherent tissue responses. Cutting edge radiobiology has adopted full dose survival profiling and devised mathematical algorithms to fit curves to observed data to generate highly reproducible numerical data that accurately define clinically relevant inherent radiosensitivities. Here, we established a protocol for irradiating organoids that delivers radiation profiles simulating the organ of origin. This technique yielded highly similar dose-survival curves of small and large intestinal crypts in vivo and their cognate organoids analyzed by the single-hit multi-target (SHMT) algorithm, outcomes reflecting the inherent radiation profile of their respective Lgr5+ stem cell populations. As this technological advance is quantitative, it will be useful for accurate evaluation of intestinal (patho)physiology and drug screening. SIGNIFICANCE: These findings establish standards for irradiating organoids that deliver radiation profiles that phenocopy the organ of origin.See related commentary by Muschel et al., p. 927.

Targeting acid sphingomyelinase with anti-angiogenic chemotherapy.

Despite great promise, combining anti-angiogenic and conventional anti-cancer drugs has produced limited therapeutic benefit in clinical trials, presumably because mechanisms of anti-angiogenic tissue response remain only partially understood. Here we define a new paradigm, in which anti-angiogenic drugs can be used to chemosensitize tumors by targeting the endothelial acid sphingomyelinase (ASMase) signal transduction pathway. We demonstrate that paclitaxel and etoposide, but not cisplatin, confer ASMase-mediated endothelial injury within minutes. This rapid reaction is required for human HCT-116 colon cancer xenograft complete response and growth delay. Whereas VEGF inhibits ASMase, anti-VEGFR2 antibodies de-repress ASMase, enhancing endothelial apoptosis and drug-induced tumor response in asmase+/+, but not in asmase-/-, hosts. Such chemosensitization occurs only if the anti-angiogenic drug is delivered 1-2h before chemotherapy, but at no other time prior to or post chemotherapy. Our studies suggest that precisely-timed administration of anti-angiogenic drugs in combination with ASMase-targeting anti-cancer drugs is likely to optimize anti-tumor effects of systemic chemotherapy. This strategy warrants evaluation in future clinical trials.

Adenoviral transduction of human acid sphingomyelinase into neo-angiogenic endothelium radiosensitizes tumor cure.

These studies define a new mechanism-based approach to radiosensitize tumor cure by single dose radiotherapy (SDRT). Published evidence indicates that SDRT induces acute microvascular endothelial apoptosis initiated via acid sphingomyelinase (ASMase) translocation to the external plasma membrane. Ensuing microvascular damage regulates radiation lethality of tumor stem cell clonogens to effect tumor cure. Based on this biology, we engineered an ASMase-producing vector consisting of a modified pre-proendothelin-1 promoter, PPE1(3x), and a hypoxia-inducible dual-binding HIF-2α-Ets-1 enhancer element upstream of the asmase gene, inserted into a replication-deficient adenovirus yielding the vector Ad5H2E-PPE1(3x)-ASMase. This vector confers ASMase over-expression in cycling angiogenic endothelium in vitro and within tumors in vivo, with no detectable enhancement in endothelium of normal tissues that exhibit a minute fraction of cycling cells or in non-endothelial tumor or normal tissue cells. Intravenous pretreatment with Ad5H2E-PPE1(3x)-ASMase markedly increases SDRT cure of inherently radiosensitive MCA/129 fibrosarcomas, and converts radiation-incurable B16 melanomas into biopsy-proven tumor cures. In contrast, Ad5H2E-PPE1(3x)-ASMase treatment did not impact radiation damage to small intestinal crypts as non-dividing small intestinal microvessels did not overexpress ASMase and were not radiosensitized. We posit that combination of genetic up-regulation of tumor microvascular ASMase and SDRT provides therapeutic options for currently radiation-incurable human tumors.

Anti-ceramide antibody prevents the radiation gastrointestinal syndrome in mice.

Radiation gastrointestinal (GI) syndrome is a major lethal toxicity that may occur after a radiation/nuclear incident. Currently, there are no prophylactic countermeasures against radiation GI syndrome lethality for first responders, military personnel, or remediation workers entering a contaminated area. The pathophysiology of this syndrome requires depletion of stem cell clonogens (SCCs) within the crypts of Lieberkühn, which are a subset of cells necessary for postinjury regeneration of gut epithelium. Recent evidence indicates that SCC depletion is not exclusively a result of DNA damage but is critically coupled to ceramide-induced endothelial cell apoptosis within the mucosal microvascular network. Here we show that ceramide generated on the surface of endothelium coalesces to form ceramide-rich platforms that transmit an apoptotic signal. Moreover, we report the generation of 2A2, an anti-ceramide monoclonal antibody that binds to ceramide to prevent platform formation on the surface of irradiated endothelial cells of the murine GI tract. Consequently, we found that 2A2 protected against endothelial apoptosis in the small intestinal lamina propria and facilitated recovery of crypt SCCs, preventing the death of mice from radiation GI syndrome after high radiation doses. As such, we suggest that 2A2 represents a prototype of a new class of anti-ceramide therapeutics and an effective countermeasure against radiation GI syndrome mortality.

Antitumor efficacy following the intracellular and interstitial release of liposomal doxorubicin.

pH-triggered lipid-membranes designed from biophysical principles are evaluated in the form of targeted liposomal doxorubicin with the aim to ultimately better control the growth of vascularized tumors. We compare the antitumor efficacy of anti-HER2/neu pH-triggered lipid vesicles encapsulating doxorubicin to the anti-HER2/neu form of an FDA approved liposomal doxorubicin of DSPC/cholesterol-based vesicles. The HER2/neu receptor is chosen due to its abundance in human breast cancers and its connection to low prognosis. On a subcutaneous murine BT474 xenograft model, superior control of tumor growth is demonstrated by targeted pH-triggered vesicles relative to targeted DSPC/cholesterol-based vesicles (35% vs. 19% decrease in tumor volume after 32 days upon initiation of treatment). Superior tumor control is also confirmed on SKBR3 subcutaneous xenografts of lower HER2/neu expression. The non-targeted form of pH-triggered vesicles encapsulating doxorubicin results also in better tumor control relative to the non-targeted DSPC/cholesterol-based vesicles (34% vs. 41% increase in tumor volume). Studies in BT474 multicellular spheroids suggest that the observed efficacy could be attributed to release of doxorubicin directly into the acidic tumor interstitium from pH-triggered vesicles extravasated into the tumor but not internalized by cancer cells. pH-triggered liposome carriers engineered from gel-phase bilayers that reversibly phase-separate with lowering pH, form transiently defective interfacial boundaries resulting in fast release of encapsulated doxorubicin. Our studies show that pH-triggered liposomes release encapsulated doxorubicin intracellularly and intratumorally, and may improve tumor control at the same or even lower administered doses relative to FDA approved liposomal chemotherapy.

Accelerated hematopoietic toxicity by high energy (56)Fe radiation.

PURPOSE: There is little information on the relative toxicity of highly charged (Z) high-energy (HZE) radiation in animal models compared to γ or X-rays, and the general assumption based on in vitro studies has been that acute toxicity is substantially greater. METHODS: C57BL/6J mice were irradiated with (56)Fe ions (1 GeV/nucleon), and acute (within 30 d) toxicity compared to that of γ rays or protons (1 GeV). To assess relative hematopoietic and gastrointestinal toxicity, the effects of (56)Fe ions were compared to γ rays using complete blood count (CBC), bone marrow granulocyte-macrophage colony forming unit (GM-CFU), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis in bone marrow, and intestinal crypt survival. RESULTS: Although onset was more rapid, (56)Fe ions were only slightly more toxic than γ rays or protons with lethal dose (LD)(50/30) (a radiation dose at which 50% lethality occurs at 30-day) values of 5.8, 7.25, and 6.8 Gy, respectively, with relative biologic effectiveness for (56)Fe ions of 1.25 and 1.06 for protons. CONCLUSIONS: (56)Fe radiation caused accelerated and more severe hematopoietic toxicity. Early mortality correlated with more profound leukopenia and subsequent sepsis. Results indicate that there is selective enhanced toxicity to bone marrow progenitor cells, which are typically resistant to γ rays, and bone marrow stem cells, because intestinal crypt cells did not show increased HZE toxicity.

Ceacam1 separates graft-versus-host-disease from graft-versus-tumor activity after experimental allogeneic bone marrow transplantation.

BACKGROUND: Allogeneic bone marrow transplantation (allo-BMT) is a potentially curative therapy for a variety of hematologic diseases, but benefits, including graft-versus-tumor (GVT) activity are limited by graft-versus-host-disease (GVHD). Carcinoembryonic antigen related cell adhesion molecule 1 (Ceacam1) is a transmembrane glycoprotein found on epithelium, T cells, and many tumors. It regulates a variety of physiologic and pathological processes such as tumor biology, leukocyte activation, and energy homeostasis. Previous studies suggest that Ceacam1 negatively regulates inflammation in inflammatory bowel disease models. METHODS: We studied Ceacam1 as a regulator of GVHD and GVT after allogeneic bone marrow transplantation (allo-BMT) in mouse models. In vivo, Ceacam1(-/-) T cells caused increased GVHD mortality and GVHD of the colon, and greater numbers of donor T cells were positive for activation markers (CD25(hi), CD62L(lo)). Additionally, Ceacam1(-/-) CD8 T cells had greater expression of the gut-trafficking integrin α(4)ß(7), though both CD4 and CD8 T cells were found increased numbers in the gut post-transplant. Ceacam1(-/-) recipients also experienced increased GVHD mortality and GVHD of the colon, and alloreactive T cells displayed increased activation. Additionally, Ceacam1(-/-) mice had increased mortality and decreased numbers of regenerating small intestinal crypts upon radiation exposure. Conversely, Ceacam1-overexpressing T cells caused attenuated target-organ and systemic GVHD, which correlated with decreased donor T cell numbers in target tissues, and mortality. Finally, graft-versus-tumor survival in a Ceacam1(+) lymphoma model was improved in animals receiving Ceacam1(-/-) vs. control T cells. CONCLUSIONS: We conclude that Ceacam1 regulates T cell activation, GVHD target organ damage, and numbers of donor T cells in lymphoid organs and GVHD target tissues. In recipients of allo-BMT, Ceacam1 may also regulate tissue radiosensitivity. Because of its expression on both the donor graft and host tissues, this suggests that targeting Ceacam1 may represent a potent strategy for the regulation of GVHD and GVT after allogeneic transplantation.

Alpha particles induce apoptosis through the sphingomyelin pathway.

The sphingomyelin pathway involves the enzymatic cleavage of sphingomyelin to produce ceramide, a second messenger that serves as a key mediator in the rapid apoptotic response to various cell stressors. Low-linear energy transfer (LET) γ radiation can initiate this pathway, independent of DNA damage, via the cell membrane. Whether short-ranged, high-LET α particles, which are of interest as potent environmental carcinogens, radiotherapies and potential components of dirty bombs, can act through this mechanism to signal apoptosis is unknown. Here we show that irradiation of Jurkat cells with α particles emitted by the ²²5Ac-DOTA-anti-CD3 IgG antibody construct results in dose-dependent apoptosis. This apoptosis was significantly reduced by pretreating cells with cholesterol-depleting nystatin, a reagent known to inhibit ceramide signaling by interfering with membrane raft coalescence and ceramide-rich platform generation. The effects of nystatin on α-particle-induced apoptosis were related to disruption of the ceramide pathway and not to microdosimetry alterations, because similar results were obtained after external irradiation of the cells with a broad beam of collimated α particles using a planar ²4¹Am source. External irradiation allowed for more precise control of the dosimetry and geometry of the irradiation, independent of antibody binding or cell internalization kinetics. Mechanistically consistent with these findings, Jurkat cells rapidly increased membrane concentrations of ceramide after external irradiation with an average of five α-particle traversals per cell. These data indicate that α particles can activate the sphingomyelin pathway to induce apoptosis.

Regulation of ceramide synthase-mediated crypt epithelium apoptosis by DNA damage repair enzymes.

Acute endothelial cell apoptosis and microvascular compromise couple gastrointestinal tract irradiation to reproductive death of intestinal crypt stem cell clonogens (SCCs) following high-dose radiation. Genetic or pharmacologic inhibition of endothelial apoptosis prevents intestinal damage, but as the radiation dose is escalated, SCCs become directly susceptible to an alternate cell death mechanism, mediated via ceramide synthase (CS)-stimulated de novo synthesis of the proapoptotic sphingolipid ceramide, and p53-independent apoptosis of crypt SCCs. We previously reported that ataxia-telangiectasia mutated deficiency resets the primary radiation lethal pathway, allowing CS-mediated apoptosis at the low-dose range of radiation. The mechanism for this event, termed target reordering, remains unknown. Here, we show that inactivation of DNA damage repair pathways signals CS-mediated apoptosis in crypt SCCs, presumably via persistent unrepaired DNA double-strand breaks (DSBs). Genetic loss of function of sensors and transducers of DNA DSB repair confers the CS-mediated lethal pathway in intestines of sv129/B6Mre11(ATLD1/ATLD1) and C57BL/6(Prkdc/SCID) (severe combined immunodeficient) mice exposed to low-dose radiation. In contrast, CS-mediated SCC lethality was mitigated in irradiated gain-of-function Rad50(s/s) mice, and epistasis studies order Rad50 upstream of Mre11. These studies suggest unrepaired DNA DSBs as causative in target reordering in intestinal SCCs. As such, we provide an in vivo model of DNA damage repair that is standardized, can be exploited to understand allele-specific regulation in intact tissue, and is pharmacologically tractable.

The use of pH-triggered leaky heterogeneities on rigid lipid bilayers to improve intracellular trafficking and therapeutic potential of targeted liposomal immunochemotherapy.

During endocytosis, pH-triggered release of encapsulated therapeutics from delivery carriers may accelerate their intracellular trafficking increasing therapeutic efficacy. To improve the therapeutic potential of targeted immunochemotherapy using anti-HER2/neu liposomal doxorubicin, we exploit the formation of leaky heterogeneities on rigid lipid bilayers to extensively release doxorubicin during endocytosis. We have previously demonstrated that pH-dependent formation of phase-separated lipid heterogeneities on the plane of a bilayer membrane increases the permeability of bilayers when they are composed of lipid pairs with rigid non-matching acyl chain lengths. This was suggested to be due to defective packing among lipids residing at the interfaces of lipid domains. Here we design nanometer-size antiHER2/neu-labeled PEGylated vesicles composed of lipid pairs with longer non-matching acyl chain lengths (n=18 and 21). These vesicles exhibit superior killing efficacy of cancer cells compared to established liposome formulations, and their killing efficacy is similar to the effect of combined free doxorubicin and free antiHER2/neu antibody. Other transport-related properties such as liposome blood circulation times, and specific binding and internalization by cancer cells are unaffected. These results demonstrate the potential of vesicles with pH-triggered leaky heterogeneities to increase the therapeutic potential of targeted immunochemotherapy.

Cytolytic T cells induce ceramide-rich platforms in target cell membranes to initiate graft-versus-host disease.

Alloreactive donor cytolytic T lymphocytes play a critical role in pathophysiology of acute graft-versus-host disease (GVHD). As GVHD progression involves tumor necrosis factor superfamily receptor activation, and as apoptotic signaling for some tumor necrosis factor superfamily receptors might involve acid sphingomyelinase (ASMase)-mediated ceramide generation, we hypothesized that ASMase deletion would ameliorate GVHD. Using clinically relevant mouse models of acute GVHD in which allogeneic bone marrow and T cells were transplanted into asmase+/+ and asmase(-/-) hosts, we identify host ASMase as critical for full-blown GVHD. Lack of host ASMase reduced the acute inflammatory phase of GVHD, attenuating cytokine storm, CD8+ T-cell proliferation/activation, and apoptosis of relevant graft-versus-host target cells (hepatocytes, intestinal, and skin cells). Organ injury was diminished in asmase(-/-) hosts, and morbidity and mortality improved at 90 days after transplantation. Resistance to cytolytic T lymphocyte-induced apoptosis was found at the target cell membrane if hepatocytes lack ASMase, as hepatocyte apoptosis required target cell ceramide generation for formation of ceramide-rich macrodomains, sites concentrating proapoptotic Fas. These studies indicate a requirement for target cell ASMase in evolution of GVHD in liver, small intestines, and skin and provide potential new targets for disease management.

Bax and Bak do not exhibit functional redundancy in mediating radiation-induced endothelial apoptosis in the intestinal mucosa.

PURPOSE: To address in vivo the issue of whether Bax and Bak are functionally redundant in signaling apoptosis, capable of substituting for each other. METHODS AND MATERIALS: Mice were exposed to whole-body radiation, and endothelial cell apoptosis was quantified using double immunostaining with TUNEL and anti-CD31 antibody. Crypt survival was determined at 3.5 days after whole-body radiation by the microcolony survival assay. Actuarial animal survival was calculated by the product-limit Kaplan-Meier method, and autopsies were performed to establish cause of death. RESULTS: Radiation exposure of Bax- and Bak-deficient mice, both expressing a wild-type acid sphingomyelinase (ASMase) phenotype, indicated that Bax and Bak are both mandatory, though mutually independent, for the intestinal endothelial apoptotic response. However, neither affected epithelial apoptosis at crypt positions 4-5, indicating specificity toward endothelium. Furthermore, Bax deficiency and Bak deficiency each individually mimicked ASMase deficiency in inhibiting crypt lethality in the microcolony assay and in rescuing mice from the lethal gastrointestinal syndrome. CONCLUSIONS: The data indicate that Bax and Bak have nonredundant functional roles in the apoptotic response of the irradiated intestinal endothelium. The observation that Bax deficiency and Bak deficiency also protect crypts in the microcolony assay provides strong evidence that the microvascular apoptotic component is germane to the mechanism of radiation-induced damage to mouse intestines, regulating reproductive cell death of crypt stem cell clonogens.

A partial allelotyping of urothelial carcinoma of bladder in the Chinese.

The cancer spectrum and genetic pathways underlying tumorigenesis vary among different ethnic populations due to genetic background and/or environment discrepancies. We have implied in our previous study that the genetic alterations found in bladder cancers of Chinese patients differ from those of Caucasians. We performed the present study to explore the genetic pathways of the urothelial carcinoma (UC) in Chinese patients. We carried out a partial allelotyping of Chinese UC on chromosome arms commonly deleted in Caucasian UC, and compared the allelo-typing between Chinese and Caucasian UC. Forty-five Chinese UC specimens were allelotyped using 30 microsatellite markers on 18 chromosome arms. The most frequent regions of loss of heterozygosity found included 9q (54.1%), 17p (51.2%), 9p (48.8%), 18q (42.2%), 3p (41.9%), 16q (33.5%) and 11p (30.0%). Compared with UC from the UK and US, the LOH frequencies on most chromosome arms in this study are higher, with statistically significant differences on 3p, 16q and 18q. Our results suggest that both consensus and different alterations exist between Chinese and Caucasian UC, indicating that genetic alterations of cancer can vary between different ethnic populations due to genetic and/or etiological discrepancies.

Insulin-like growth factor-I enhances lymphoid and myeloid reconstitution after allogeneic bone marrow transplantation.

BACKGROUND: Prolonged immunodeficiency after allogeneic bone marrow transplantation (allo BMT) results in significant morbidity and mortality from infection. Previous studies in murine syngeneic BMT models have demonstrated that posttransplantation insulin-like growth factor (IGF)-I administration could enhance immune reconstitution. METHODS: To analyze the effects of IGF-I on immune reconstitution and graft-versus-host disease (GVHD) after allo BMT, we used murine models for MHC-matched and -mismatched allo BMT. Young (3-month-old) recipient mice received 4 mg/kg per day of human IGF-I from days 14 to 28 by continuous subcutaneous administration. RESULTS: IGF-I administration resulted in increased thymic precursor populations (triple negative-2 and triple negative-3) as determined on day 28 but had no effect on overall thymic cellularity. In the periphery, the numbers of donor-derived splenic CD3+ T cells were increased and these cells had an improved proliferative response to mitogen stimulation. IGF-I treatment also significantly increased the numbers of pro-, pre-, and mature B cells and myeloid cell populations in the spleens of allo BMT recipients on day 28. The administration of IGF-I in combination with interleukin 7 had a remarkable additive effect on B-cell, but not on T-cell, lymphopoiesis. Finally, we tested the effects of IGF-I administration on the development of GVHD in three different MHC-matched and -mismatched models and found no changes in GVHD morbidity and mortality. CONCLUSION: IGF-I administration can enhance lymphoid and myeloid reconstitution after allo BMT without aggravating GVHD.

Donor T cell-derived TNF is required for graft-versus-host disease and graft-versus-tumor activity after bone marrow transplantation.

Previous studies in murine bone marrow transplantation (BMT) models using neutralizing anti-tumor necrosis factor (TNF) antibodies or TNF receptor (TNFR)-deficient recipients have demonstrated that TNF can be involved in both graft-versus-host disease (GVHD) and graft-versus-leukemia (GVL). TNF in these GVHD and GVL models was thought to be primarily produced by activated monocytes and macrophages, and the role of T cell-derived TNF was not determined. We used TNF(-/-) mice to study the specific role of TNF produced by donor T cells in a well-established parent-into-F1 hybrid model (C57BL/6J-->C3FeB6F1/J). Recipients of TNF(-/-) T cells developed significantly less morbidity and mortality from GVHD than recipients of wild-type (wt) T cells. Histology of GVHD target organs revealed significantly less damage in thymus, small bowel, and large bowel, but not in liver or skin tissues from recipients of TNF(-/-) T cells. Recipients of TNF(-/-) T cells which were also inoculated with leukemia cells at the time of BMT showed increased mortality from leukemia when compared with recipients of wt cells. We found that TNF(-/-) T cells do not have intrinsic defects in vitro or in vivo in proliferation, IFN-gamma production, or alloactivation. We could not detect TNF in the serum of our transplant recipients, suggesting that T cells contribute to GVHD and GVL via membrane-bound or locally released TNF.

T cells require TRAIL for optimal graft-versus-tumor activity.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a member of the TNF superfamily that exhibits specific tumoricidal activity against a variety of tumors. It is expressed on different cells of the immune system and plays a role in natural killer cell-mediated tumor surveillance. In allogeneic hematopoietic-cell transplantation, the reactivity of the donor T cell against malignant cells is essential for the graft-versus-tumor (GVT) effect. Cytolytic activity of T cells is primarily mediated through the Fas-Fas ligand and perforin-granzyme pathways. However, T cells deficient for both Fas ligand and perforin can still exert GVT activity in vivo in mouse models. To uncover a potential role for TRAIL in donor T cell-mediated GVT activity, we compared donor T cells from TRAIL-deficient and wild-type mice in clinically relevant mouse bone-marrow transplantation models. We found that alloreactive T cells can express TRAIL, but the absence of TRAIL had no effect on their proliferative and cytokine response to alloantigens. TRAIL-deficient T cells showed significantly lower GVT activity than did TRAIL-expressing T cells, but no important differences in graft-versus-host disease, a major complication of allogeneic hematopoietic cell transplantation, were observed. These data suggest that strategies to enhance TRAIL-mediated GVT activity could decrease relapse rates of malignancies after hematopoietic cell transplantation without exacerbation of graft-versus-host disease.