Daniel R. Wahl

Manipulating the Bioenergetics of Alloreactive T Cells Causes Their Selective Apoptosis and Arrests Graft-Versus-Host Disease

Bioenergetic properties differentiate alloreactive T cells from other proliferating cells and can be exploited to arrest GVHD in mice. A Xenophobe’s Guide to Treating Graft-Versus-Host Disease Immune cells are in essence xenophobes—they distinguish and then attack cells that are foreign to the body. This prejudice is helpful in the context of infection or cancer; the immune system raises destructive responses directed against stranger cells that have been infected or transformed while remaining calm in the presence of healthy cells recognized as “self.” However, when immune cells are transferred to a new host, for example, by bone marrow transplantation, these cells see the patient’s own tissues as foreign and attack, resulting in graft-versus-host disease (GVHD). Gatza et al. have found a way to differentiate these so-called alloreactive donor cells from non-alloreactive donor cells and host immune cells and thus provide a target for preventing GVHD. Cells can generate energy in the form of adenosine triphosphate (ATP) through either aerobic glycolysis or oxidative phosphorylation (OXPHOS). In general, proliferating lymphocytes preferentially use aerobic glycolysis, which produces significantly fewer ATP molecules than OXPHOS, but little is known about the specific metabolic requirements of proliferating alloreactive T cells. Gatza et al. found that after bone marrow transplantation, alloreactive T cells, but not other proliferating T cells and bone marrow cells, up-regulated both aerobic glycolysis and OXPHOS to meet their increased energy demand. Relative to other proliferating cells, alloreactive T cells produced higher concentrations of acylcarnitines—fatty acid oxidation intermediates that transport fatty acids into the mitochondrial matrix. Further highlighting their altered metabolism, alloreactive T cells could be specifically killed by Bz-423, a small-molecule inhibitor of the mitochondrial F1F0-ATPase, and Bz-423 decreased the severity of GVHD in mice without impairing transplant engraftment and bone marrow reconstitution. Thus, bioenergetic differences may provide targets for order-maintaining therapeutics that stop alloreactive T cells from attacking perceived foreigners in the transplanted immune cells’ new neighborhood. Cells generate adenosine triphosphate (ATP) by glycolysis and by oxidative phosphorylation (OXPHOS). Despite the importance of having sufficient ATP available for the energy-dependent processes involved in immune activation, little is known about the metabolic adaptations that occur in vivo to meet the increased demand for ATP in activated and proliferating lymphocytes. We found that bone marrow (BM) cells proliferating after BM transplantation (BMT) increased aerobic glycolysis but not OXPHOS, whereas T cells proliferating in response to alloantigens during graft-versus-host disease (GVHD) increased both aerobic glycolysis and OXPHOS. Metabolomic analysis of alloreactive T cells showed an accumulation of acylcarnitines consistent with changes in fatty acid oxidation. Alloreactive T cells also exhibited a hyperpolarized mitochondrial membrane potential (ΔΨm), increased superoxide production, and decreased amounts of antioxidants, whereas proliferating BM cells did not. Bz-423, a small-molecule inhibitor of the mitochondrial F1F0 adenosine triphosphate synthase (F1F0-ATPase), selectively increased superoxide and induced the apoptosis of alloreactive T cells, which arrested established GVHD in several BMT models without affecting hematopoietic engraftment or lymphocyte reconstitution. These findings challenge the current paradigm that activated T cells meet their increased demands for ATP through aerobic glycolysis, and identify the possibility that bioenergetic and redox characteristics can be selectively exploited as a therapeutic strategy for immune disorders.

Outcomes After Stereotactic Body Radiotherapy or Radiofrequency Ablation for Hepatocellular Carcinoma

PURPOSE Data guiding selection of nonsurgical treatment of hepatocellular carcinoma (HCC) are lacking. We therefore compared outcomes between stereotactic body radiotherapy (SBRT) and radiofrequency ablation (RFA) for HCC. PATIENTS AND METHODS From 2004 to 2012, 224 patients with inoperable, nonmetastatic HCC underwent RFA (n = 161) to 249 tumors or image-guided SBRT (n = 63) to 83 tumors. We applied inverse probability of treatment weighting to adjust for imbalances in treatment assignment. Freedom from local progression (FFLP) and toxicity were retrospectively analyzed. RESULTS RFA and SBRT groups were similar with respect to number of lesions treated per patient, type of underlying liver disease, and tumor size (median, 1.8 v 2.2 cm in maximum diameter; P = .14). However, the SBRT group had lower pretreatment Child-Pugh scores (P = .003), higher pretreatment alpha-fetoprotein levels (P = .04), and a greater number of prior liver-directed treatments (P < .001). One- and 2-year FFLP for tumors treated with RFA were 83.6% and 80.2% v 97.4% and 83.8% for SBRT. Increasing tumor size predicted for FFLP in patients treated with RFA (hazard ratio [HR], 1.54 per cm; P = .006), but not with SBRT (HR, 1.21 per cm; P = .617). For tumors ≥ 2 cm, there was decreased FFLP for RFA compared with SBRT (HR, 3.35; P = .025). Acute grade 3+ complications occurred after 11% and 5% of RFA and SBRT treatments, respectively (P = .31). Overall survival 1 and 2 years after treatment was 70% and 53% after RFA and 74% and 46% after SBRT. CONCLUSION Both RFA and SBRT are effective local treatment options for inoperable HCC. Although these data are retrospective, SBRT appears to be a reasonable first-line treatment of inoperable, larger HCC.

Distinct metabolic programs in activated T cells: opportunities for selective immunomodulation

For several decades, it has been known that T‐cell activation in vitro leads to increased glycolytic metabolism that fuels proliferation and effector function. Recently, this simple model has been complicated by the observation that different T‐cell subsets differentially regulate fundamental metabolic pathways under the control of distinct molecular regulators. Although the majority of these data have been generated in vitro, several recent studies have documented the metabolism of T cells activated in vivo. Here, we review the recent data surrounding the differential regulation of metabolism by distinct T‐cell subsets in vitro and in vivo and discuss how differential metabolic regulation might facilitate T‐cell function vis‐à‐vis proliferation, survival, and energy production. We further discuss the important therapeutic implications of differential metabolism across T‐cell subsets and review recent successes in exploiting lymphocyte metabolism to treat immune‐mediated diseases.

Characterization of the Metabolic Phenotype of Rapamycin-Treated CD8+ T Cells with Augmented Ability to Generate Long-Lasting Memory Cells

Background Cellular metabolism plays a critical role in regulating T cell responses and the development of memory T cells with long-term protections. However, the metabolic phenotype of antigen-activated T cells that are responsible for the generation of long-lived memory cells has not been characterized. Design and Methods Using lymphocytic choriomeningitis virus (LCMV) peptide gp33-specific CD8+ T cells derived from T cell receptor transgenic mice, we characterized the metabolic phenotype of proliferating T cells that were activated and expanded in vitro in the presence or absence of rapamycin, and determined the capability of these rapamycin-treated T cells to generate long-lived memory cells in vivo. Results Antigen-activated CD8+ T cells treated with rapamycin gave rise to 5-fold more long-lived memory T cells in vivo than untreated control T cells. In contrast to that control T cells only increased glycolysis, rapamycin-treated T cells upregulated both glycolysis and oxidative phosphorylation (OXPHOS). These rapamycin-treated T cells had greater ability than control T cells to survive withdrawal of either glucose or growth factors. Inhibition of OXPHOS by oligomycin significantly reduced the ability of rapamycin-treated T cells to survive growth factor withdrawal. This effect of OXPHOS inhibition was accompanied with mitochondrial hyperpolarization and elevation of reactive oxygen species that are known to be toxic to cells. Conclusions Our findings indicate that these rapamycin-treated T cells may represent a unique cell model for identifying nutrients and signals critical to regulating metabolism in both effector and memory T cells, and for the development of new methods to improve the efficacy of adoptive T cell cancer therapy.

Anaplerotic Metabolism of Alloreactive T Cells Provides a Metabolic Approach To Treat Graft-Versus-Host Disease

T-cell activation requires increased ATP and biosynthesis to support proliferation and effector function. Most models of T-cell activation are based on in vitro culture systems and posit that aerobic glycolysis is employed to meet increased energetic and biosynthetic demands. By contrast, T cells activated in vivo by alloantigens in graft-versus-host disease (GVHD) increase mitochondrial oxygen consumption, fatty acid uptake, and oxidation, with small increases of glucose uptake and aerobic glycolysis. Here we show that these differences are not a consequence of alloactivation, because T cells activated in vitro either in a mixed lymphocyte reaction to the same alloantigens used in vivo or with agonistic anti-CD3/anti-CD28 antibodies increased aerobic glycolysis. Using targeted metabolic 13C tracer fate associations, we elucidated the metabolic pathway(s) employed by alloreactive T cells in vivo that support this phenotype. We find that glutamine (Gln)-dependent tricarboxylic acid cycle anaplerosis is increased in alloreactive T cells and that Gln carbon contributes to ribose biosynthesis. Pharmacological modulation of oxidative phosphorylation rapidly reduces anaplerosis in alloreactive T cells and improves GVHD. On the basis of these data, we propose a model of T-cell metabolism that is relevant to activated lymphocytes in vivo, with implications for the discovery of new drugs for immune disorders.

Apoptotic Signaling Activated by Modulation of the F0F1-ATPase: Implications for Selective Killing of Autoimmune Lymphocytes

7-Chloro-5-(4-hydroxyphenyl)-1-methyl-3-(napthalen-2-ylmetyl)-4,5,-dihydro-1H-benzo[b][1,4]diazepin-2(3H)-one (Bz-423) is a proapoptotic 1,4-benzodiazepine that potently suppresses disease in the murine model of lupus by selectively killing pathogenic lymphocytes. In MRL/MpJ-Faslpr (MRL-lpr) mice, Bz-423 overcomes deficient expression of the Fas death receptor and hyperactivation of antiapoptotic phosphatidylinositol 3-kinase (PI3K)-Akt signaling to specifically kill pathogenic CD4+ T cells. Bz-423 binds to the oligomycin-sensitivity-conferring protein component of the mitochondrial F0F1-ATPase, which modulates the enzyme leading to formation of superoxide by the mitochondrial respiratory chain. Scavenging this reactive oxygen species blocks all subsequent components of the apoptotic cascade. To gain insight into how apoptotic signaling activated by Bz-423-induced superoxide contributes to the selective depletion of MRL-lpr CD4+ T cells, we characterized the death mechanism in a CD4+ T cell leukemia line (Jurkat). Although Bz-423-induced superoxide indirectly inactivates Akt, this response is not required for T cell death. Apoptosis instead results from parallel increases in levels of the proapoptotic Bcl-2 proteins Noxa and Bak leading to specific activation of Bak, mitochondrial outer membrane permeabilization, and a commitment to apoptosis. By directly up-regulating proteins that trigger loss of mitochondrial outer membrane integrity, Bz-423 bypasses defective Fas function and antiapoptotic PI3K-Akt signaling in MRL-lpr CD4+ T cells. Moreover, because disease-associated abnormalities should sensitize autoreactive CD4+ T cells to transcriptional up-regulation of Noxa by redox signals and to Bak-dependent apoptosis, the apoptotic mechanism elucidated in Jurkat cells provides important clues into the cell-type- and disease-selective effects of Bz-423 in MRL-lpr mice.

Cost-effectiveness of Stereotactic Body Radiation Therapy versus Radiofrequency Ablation for Hepatocellular Carcinoma: A Markov Modeling Study.

Purpose To assess the cost-effectiveness of stereotactic body radiation therapy (SBRT) versus radiofrequency ablation (RFA) for patients with inoperable localized hepatocellular carcinoma (HCC) who are eligible for both SBRT and RFA. Materials and Methods A decision-analytic Markov model was developed for patients with inoperable, localized HCC who were eligible for both RFA and SBRT to evaluate the cost-effectiveness of the following treatment strategies: (a) SBRT as initial treatment followed by SBRT for local progression (SBRT-SBRT), (b) RFA followed by RFA for local progression (RFA-RFA), (c) SBRT followed by RFA for local progression (SBRT-RFA), and (d) RFA followed by SBRT for local progression (RFA-SBRT). Probabilities of disease progression, treatment characteristics, and mortality were derived from published studies. Outcomes included health benefits expressed as discounted quality-adjusted life years (QALYs), costs in U.S. dollars, and cost-effectiveness expressed as an incremental cost-effectiveness ratio. Deterministic and probabilistic sensitivity analysis was performed to assess the robustness of the findings. Results In the base case, SBRT-SBRT yielded the most QALYs (1.565) and cost

Integrating chemoradiation and molecularly targeted therapy.

197 557. RFA-SBRT yielded 1.558 QALYs and cost

2-Hydoxyglutarate: D/Riving Pathology in gLiomaS

193 288. SBRT-SBRT was not cost-effective, at

Glioblastoma Therapy Can Be Augmented by Targeting IDH1-Mediated NADPH Biosynthesis

558 679 per QALY gained relative to RFA-SBRT. RFA-SBRT was the preferred strategy, because RFA-RFA and SBRT-RFA were less effective and more costly. In all evaluated scenarios, SBRT was preferred as salvage therapy for local progression after RFA. Probabilistic sensitivity analysis showed that at a willingness-to-pay threshold of