Allogeneic hematopoietic cell transplantation (HCT) is effective therapy for hematologic malignancies through T cellmediated GVL effects. However, HCT benefits are frequently offset by the destructive GVHD, which is also induced by donor T cells. Naive Th can differentiate into Th1 and Th17 subsets and both can mediate GVHD after adoptive transfer into an allogeneic host. Here we tested the hypothesis that blockade of Th1 and Th17 differentiation is required to prevent GVHD in mice. T cells with combined targeted disruption of T-bet and ROR␥t have defective differentiation toward Th1 and Th17 and skewed differentiation toward Th2 and regulatory phenotypes, and caused ameliorated GVHD in a major MHC-mismatched model of HCT. GVL effects mediated by granzyme-positive CD8 T cells were largely preserved despite T-bet and ROR␥t deficiency. These data indicate that GVHD can be prevented by targeting Th1 and Th17 transcription factors without offsetting GVL activity. (Blood. 2011;118(18):5011-5020) IntroductionSeparation of GVHD from GVL effects is the major challenge of allogeneic hematopoietic cell transplantation (HCT) that is used for the treatment of hematologic malignancies. On Ag stimulation, T-cell precursors can differentiate into distinct functional cell subsets including Th1 and Th17 cells. Understanding the role of each subset in the development of GVHD is critical to develop effective therapy and improve HCT outcome.The cytokine storm caused by the conditioning regimen and Th1-cell cytokines is key to initiating the inflammatory cascade and amplifying immune responses that cause GVHD. 1-3 However, studies using IFN-␥ gene knockout (KO) mice as donors showed that deficiency of IFN-␥ is paradoxically associated with more severe acute GVHD. 4,5 Our group and others found that Th17 cells can augment GVHD in some circumstances, 6,7 and in vitro-generated Th17 cells alone are sufficient to mediate lung and skin GVHD. 8 IFN␥ blockade promotes Th17 differentiation, while IL-17 blockade promotes Th1 differentiation and each blockade alone is ineffective for preventing GVHD,9 suggesting that Th1 and Th17 cells are mutually inhibitory, and that each Th type alone is sufficient to induce GVHD.The transcription factor T-bet is required for the differentiation of Th1 cells 10 and ROR␥t is necessary for Th17 cells. 11 Therefore, we hypothesized that targeted disruption of both T-bet and ROR␥t factors would block Th1 and Th17 differentiation and prevent GVHD. In the current study, we used mice deficient for T-bet, ROR␥t, or both as T-cell donors to test T-bet and ROR␥t as targets to prevent GVHD after allogeneic HCT. Methods Mice Abs and flow cytometryThe following Abs were used for cell-surface staining: anti-CD4-FITC, or -allophycocyanin (L3T4), anti-CD8␣-FITC, -allophycocyanin, -allophycocyanin-cy7 or -Alexa Fluor 700(Ly-2), anti-CD45.1-FITC, or -allophycocyanin (A20), anti-B220-PE (RA3-6B2), anti-H-2K b -FITC, -PE, or -biotin (AF6), purchased from eBioscience; anti-CD4-Pacific Blue (RM4-5) purchased from BD Bioscience...

Drug-induced liver injury (DILI) is a major health issue, as it remains difficult to predict which new drugs will cause injury and who will be susceptible to this disease. This is due in part to the lack of animal models and knowledge of susceptibility factors that predispose individuals to DILI. In this regard, liver eosinophilia has often been associated with DILI, though its role remains unclear. We decided to investigate this problem in a murine model of halothane-induced liver injury (HILI). When female Balb/cJ mice were administered halothane, eosinophils were detected by flow cytometry in the liver within 12 hours and increased thereafter proportionally to liver damage. Chemokines, CCL11 and CCL24, which are known to attract eosinophils, increased in response to halothane-treatment. The severity of HILI was decreased significantly when the study was repeated in wild-type mice made deficient in eosinophils with a depleting antibody and in eosinophil lineage-ablated ΔdblGata−/− mice. Moreover, depletion of neutrophils by pretreating animals with Gr-1 antibody prior to halothane administration failed to reduce the severity of HILI at antibody concentrations that did not affect hepatic eosinophils. Immunohistochemical staining for the granule protein, major basic protein, revealed that eosinophils accumulated exclusively around areas of hepatocellular necrosis. Conclusion: Our findings indicate that eosinophils have a pathologic role in HILI in mice and suggest that they may contribute similarly in many clinical cases of DILI.

CD28 costimulation is required for the generation of naturally derived regulatory T cells (nTregs) in the thymus through lymphocyte-specific protein tyrosine kinase (Lck) signaling. However, it is not clear how CD28 costimulation regulates the generation of induced Tregs (iTregs) from naive CD4 T-cell precursors in the periphery. To address this question, we induced iTregs (CD25 ؉ Foxp3 ؉ ) from naive CD4 T cells (CD25 ؊ Foxp3 ؊ ) by T-cell receptor stimulation with additional transforming growth factor␤ (TGF␤) in vitro, and found that the generation of iTregs was inversely related to the level of CD28 costimulation independently of IL-2. Using a series of transgenic mice on a CD28-deficient background that bears wild-type or mutated CD28 in its cytosolic tail that is incapable of binding to Lck, phosphoinositide 3-kinase (PI3K), or IL-2-inducible T-cell kinase (Itk), we found that CD28-mediated Lck signaling plays an essential role in the suppression of iTreg generation under strong CD28 costimulation. Furthermore, we demonstrate that T cells with the CD28 receptor incapable of activating Lck were prone to iTreg induction in vivo, which contributed to their reduced ability to cause graft-versushost disease. These findings reveal a novel mechanistic insight into how CD28 costimulation negatively regulates the generation of iTregs, and provide a rationale for promoting T-cell immunity or tolerance by regulating Tregs through targeting CD28 signaling. (Blood. 2011; 117(11):3096-3103) IntroductionRegulatory T cells (Tregs) play an essential role in the maintenance of immunologic tolerance to prevent autoimmune disease. The development of Tregs in the thymus requires Foxp3, a member of the group of transcription factors characterized by their winged helix-forkhead DNA-binding domain. 1 Although it is widely accepted that natural Tregs (nTregs) develop in the thymus, compelling evidence indicates that Tregs with an identical phenotype can be induced in the periphery from CD4 ϩ non-Treg precursors under certain conditions. For example, all CD4 ϩ cells from RAG Ϫ/Ϫ T-cell receptor (TCR)-transgenic (Tg) mice are CD25 Ϫ , but a small proportion of these cells convert to a CD25 ϩ Treg phenotype after adoptive transfer into antigen-bearing mice or mice that have been administered a tolerizing dose of peptide antigen. 2,3 Furthermore, de novo generation of CD4 ϩ CD25 ϩ Tregs from CD4 ϩ CD25 Ϫ cells can also occur in thymectomized mice. 4 Such Tregs that are induced in the periphery are called induced Tregs (iTregs). Although our understanding of the microenvironment for iTreg development in vivo is still limited, it is clear that TCR stimulation, transforming growth factor␤ (TGF␤), and interleukin-2 (IL-2) are required for their development. [5][6][7][8] A crucial regulator of Tregs is the CD28 receptor, a dominant costimulatory molecule for T-cell activation. The first clue to the critical role of the CD28 family in nTreg function was the observation that prevention of CD28 ligation with CTLA4-Ig exacerbated autoimmune dis...

Clinical evidence suggests that many cases of serious idiosyncratic drug-induced liver injury are mediated by the adaptive immune system in response to hepatic drug-protein adducts, also referred to as "drug-induced allergic hepatitis"; but detailed mechanistic proof has remained elusive due to the lack of animal models. We have hypothesized that drug-induced allergic hepatitis is as rare in animals as it is in humans due at least in part to the tolerogenic nature of the liver. We provide evidence that immune tolerance can be overcome in a murine model of halothane-induced liver injury initiated by trifluoroacetylated protein adducts of halothane formed in the liver. Twenty-four hours after female Balb/cJ mice were initially treated with halothane, perivenous necrosis and an infiltration of CD11b Gr-1 high cells were depleted from the liver with Gr-1 antibody treatment, enhanced liver injury was observed at 9 days after halothane rechallenge. Toxicity was associated with increased serum levels of interleukin-4 and immunoglobulins G1 and E directed against hepatic trifluoroacetylated protein adducts, as well as increased hepatic infiltration of eosinophils and CD41 T cells, all features of an allergic reaction. When hepatic CD41 T cells were depleted 5 days after halothane rechallenge, trifluoroacetylated protein adduct-specific serum immunoglobulin and hepatotoxicity were reduced. Conclusion: Our data provide a rational approach for developing animal models of drug-induced allergic hepatitis mediated by the adaptive immune system and suggest that impaired liver tolerance may predispose patients to this disease. (HEPATOLOGY 2015;62:546-557)