Runx1 and Runx3 Are Involved in the Generation and Function of Highly Suppressive IL-17-Producing T Regulatory Cells

CD4+Foxp3+ T regulatory cells (Tregs) display phenotypic and functional plasticity that is regulated by cytokines and other immune cells. Previously, we determined that during co-culture with CD4+CD25− T cells and antigen presenting cells, Tregs produced IL-17. Here, we investigated the mechanisms underlying the differentiation of IL-17-producing Treg (Tr17) cells and their molecular and functional properties. We determined that during stimulation via TCR/CD3 and CD28, the combination of IL-1β and IL-2 was necessary and sufficient for the generation of Tr17 cells. Tr17 cells expressed Runx1 transcription factor, which was required for sustained expression of Foxp3 and RORγt and for production of IL-17. Surprisingly, Tr17 cells also expressed Runx3, which regulated transcription of perforin and granzyme B thereby mediating cytotoxic activity. Our studies indicate that Tr17 cells concomitantly express Foxp3, RORγt, Runx1 and Runx3 and are capable of producing IL-17 while mediating potent suppressive and cytotoxic function

Metabolic Targets for Improvement of Allogeneic Hematopoietic Stem Cell Transplantation and Graft-vs.-Host Disease

Utilization of the adaptive immune system against malignancies, both by immune-based therapies to activate T cells in vivo to attack cancer and by T-cell therapies to transfer effector cytolytic T lymphocytes (CTL) to the cancer patient, represent major novel therapeutic advancements in oncologic therapy. Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is a form of cell-based therapy, which replaces the HSC in the patient's bone marrow but also serves as a T-cell therapy due to the Graft-vs.-leukemia (GVL) effect mediated by donor T cells transferred with the graft. Allogeneic HSCT provides one potentially curative option to patients with relapsed or refractory leukemia but Graft-vs.-Host-Disease (GVHD) is the main cause of non-relapse mortality and limits the therapeutic benefit of allogeneic HSCT. Metabolism is a common cellular feature and has a key role in the differentiation and function of T cells during the immune response. Naïve T cells and memory T cells that mediate GVHD and GVL, respectively, utilize distinct metabolic programs to obtain their immunological and functional specification. Thus, metabolic targets that mediate immunosuppression might differentially affect the functional program of GVHD-mediating or GVL-mediating T cells. Components of the innate immune system that are indispensable for the activation of alloreactive T cells are also subjected to metabolism-dependent regulation. Metabolic alterations have also been implicated in the resistance to chemotherapy and survival of malignant cells such as leukemia and lymphoma, which are targeted by GVL-mediating T cells. Development of novel approaches to inhibit the activation of GVHD-specific naïve T cell but maintain the function of GVL-specific memory T cells will have a major impact on the therapeutic benefit of HSCT. Here, we will highlight the importance of metabolism on the function of GVHD-inducing and GVL-inducing alloreactive T cells as well as on antigen presenting cells (APC), which are required for presentation of host antigens. We will also analyze the metabolic alterations involved in the leukemogenesis which could differentiate leukemia initiating cells from normal HSC, providing potential therapeutic opportunities. Finally, we will discuss the immuno-metabolic effects of key drugs that might be repurposed for metabolic management of GVHD without compromising GVL

Activation of PI3K Is Indispensable for Interleukin 7–mediated Viability, Proliferation, Glucose Use, and Growth of T Cell Acute Lymphoblastic Leukemia Cells

Interleukin (IL)-7 is essential for normal T cell development. Previously, we have shown that IL-7 increases viability and proliferation of T cell acute lymphoblastic leukemia (T-ALL) cells by up-regulating Bcl-2 and down-regulating the cyclin-dependent kinase inhibitor p27kip1. Here, we examined the signaling pathways via which IL-7 mediates these effects. We investigated mitogen-activated protein kinase (MEK)–extracellular signal-regulated kinase (Erk) and phosphatidylinositol-3-kinase (PI3K)–Akt (protein kinase B) pathways, which have active roles in T cell expansion and have been implicated in tumorigenesis. IL-7 induced activation of the MEK–Erk pathway in T-ALL cells; however, inhibition of the MEK–Erk pathway by the use of the cell-permeable inhibitor PD98059, did not affect IL-7–mediated viability or cell cycle progression of leukemic cells. IL-7 induced PI3K-dependent phosphorylation of Akt and its downstream targets GSK-3, FOXO1, and FOXO3a. PI3K activation was mandatory for IL-7–mediated Bcl-2 up-regulation, p27kip1 down-regulation, Rb hyperphosphorylation, and consequent viability and cell cycle progression of T-ALL cells. PI3K signaling was also required for cell size increase, up-regulation of CD71, expression of the glucose transporter Glut1, uptake of glucose, and maintenance of mitochondrial integrity. Our results implicate PI3K as a major effector of IL-7–induced viability, metabolic activation, growth and proliferation of T-ALL cells, and suggest that PI3K and its downstream effectors may represent molecular targets for therapeutic intervention in T-ALL

Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector

Adaptive immunity depends on lymphocyte adhesion that is mediated by the integrin lymphocyte functional antigen 1 (LFA-1). The small guanosine triphosphatase Rap1 regulates LFA-1 adhesiveness through one of its effectors, Rap1-interacting adapter molecule (RIAM). We show that RIAM was recruited to the lymphocyte plasma membrane (PM) through its Ras association (RA) and pleckstrin homology (PH) domains, both of which were required for lymphocyte adhesion. The N terminus of RIAM inhibited membrane translocation. In vitro, the RA domain bound both Rap1 and H-Ras with equal but relatively low affinity, whereas in vivo only Rap1 was required for PM association. The PH domain bound phosphoinositol 4,5-bisphosphate (PI(4,5)P2) and was responsible for the spatial distribution of RIAM only at the PM of activated T cells. We determined the crystal structure of the RA and PH domains and found that, despite an intervening linker of 50 aa, the two domains were integrated into a single structural unit, which was critical for proper localization to the PM. Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P2

Mechanisms and consequences of agonist-induced talin recruitment to platelet integrin αIIbβ3

Platelet aggregation requires agonist-induced αIIbβ3 activation, a process mediated by Rap1 and talin. To study mechanisms, we engineered αIIbβ3 Chinese hamster ovary (CHO) cells to conditionally express talin and protease-activated receptor (PAR) thrombin receptors. Human PAR1 or murine PAR4 stimulation activates αIIbβ3, which was measured with antibody PAC-1, indicating complete pathway reconstitution. Knockdown of Rap1–guanosine triphosphate–interacting adaptor molecule (RIAM), a Rap1 effector, blocks this response. In living cells, RIAM overexpression stimulates and RIAM knockdown blocks talin recruitment to αIIbβ3, which is monitored by bimolecular fluorescence complementation. Mutations in talin or β3 that disrupt their mutual interaction block both talin recruitment and αIIbβ3 activation. However, one talin mutant (L325R) is recruited to αIIbβ3 but cannot activate it. In platelets, RIAM localizes to filopodia and lamellipodia, and, in megakaryocytes, RIAM knockdown blocks PAR4-mediated αIIbβ3 activation. The RIAM-related protein lamellipodin promotes talin recruitment and αIIbβ3 activity in CHO cells but is not expressed in megakaryocytes or platelets. Thus, talin recruitment to αIIbβ3 by RIAM mediates agonist-induced αIIbβ3 activation, with implications for hemostasis and thrombosis

Immunometabolic Regulations Mediated by Coinhibitory Receptors and Their Impact on T Cell Immune Responses

Host immunity provides wide spectrum protection that serves to eradicate pathogens and cancer cells, while maintaining self-tolerance and immunological homeostasis. Ligation of the T cell receptor (TCR) by antigen activates signaling pathways that coordinately induce aerobic glycolysis, mitochondrial activity, anabolic metabolism, and T effector cell differentiation. Activation of PI3K, Akt, and mTOR triggers the switch to anabolic metabolism by inducing transcription factors such as Myc and HIF1, and the glucose transporter Glut1, which is pivotal for the increase of glucose uptake after T cell activation. Activation of MAPK signaling is required for glucose and glutamine utilization, whereas activation of AMPK is critical for energy balance and metabolic fitness of T effector and memory cells. Coinhibitory receptors target TCR-proximal signaling and generation of second messengers. Imbalanced activation of such signaling pathways leads to diminished rates of aerobic glycolysis and impaired mitochondrial function resulting in defective anabolic metabolism and altered T cell differentiation. The coinhibitory receptors mediate distinct and synergistic effects on the activation of signaling pathways thereby modifying metabolic programs of activated T cells and resulting in altered immune functions. Understanding and therapeutic targeting of metabolic programs impacted by coinhibitory receptors might have significant clinical implications for the treatment of chronic infections, cancer, and autoimmune diseases

PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation

During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid β-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade

The Impact of Intermittent Umbilical Cord Occlusions on the Inflammatory Response in Pre-Term Fetal Sheep

Fetal hypoxic episodes may occur antepartum with the potential to induce systemic and cerebral inflammatory responses thereby contributing to brain injury. We hypothesized that intermittent umbilical cord occlusions (UCOs) of sufficient severity but without cumulative acidosis will lead to a fetal inflammatory response. Thirty-one chronically instrumented fetal sheep at ∼0.85 of gestation underwent four consecutive days of hourly UCOs from one to three minutes duration for six hours each day. Maternal and fetal blood samples were taken for blood gases/pH and plasma interleukin (IL)-1β and IL-6 levels. Animals were euthanized at the end of experimental study with brain tissue processed for subsequent counting of microglia and mast cells. Intermittent UCOs resulted in transitory fetal hypoxemia with associated acidemia which progressively worsened the longer umbilical blood flow was occluded, but with no cumulative blood gas or pH changes over the four days of study. Fetal arterial IL-1β and IL-6 values showed no significant change regardless of the severity of the UCOs, nor was there any evident impact on the microglia and mast cell counts for any of the brain regions studied. Accordingly, intermittent UCOs of up to three minutes duration with severe, but limited fetal hypoxemia and no cumulative acidemia, do not result in either a systemic or brain inflammatory response in the pre-term ovine fetus. However, fetal IL-1B and IL-6 values were found to be well correlated with corresponding maternal values supporting the placenta as a primary source for these cytokines with related secretion into both circulations. Female fetuses were also found to have higher IL-1β levels than males, indicating that gender may impact on the fetal inflammatory response to various stimuli

IL-6-174 G/C and -572 C/G Polymorphisms and Risk of Alzheimer’s Disease

Associations between interleukin 6 (IL-6) polymorphisms and Alzheimer’s disease (AD) remain controversial and ambiguous. The aim of this meta-analysis is to explore more precise estimations for the relationship between IL-6-174 G/C and -572 C/G polymorphisms and risk for AD. Electronic searches for all publications in databases PubMed and EMBASE were conducted on the associations between IL-6 polymorphisms and risk for AD until January 2012. Odds ratio (OR) and 95% confidence intervals (CIs) were calculated using fixed and random effects models. Twenty-seven studies were included with a total of 19,135 individuals, involving 6,632 AD patients and 12,503 controls. For IL-6-174 G/C polymorphism, the combined results showed significant differences in recessive model (CC vs. CG+GG: OR = 0.65, 95%CI = 0.52–0.82). As regards IL-6-572 C/G polymorphism, significant associations were shown in dominant model (CG+GG vs. CC: OR  = 0.73, 95% CI = 0.62–0.86) and in additive model (GG vs. CC, OR  = 0.66, 95% CI = 0.46–0.96). In conclusion, genotype CC of IL-6-174 G/C and genotype GG plus GC of IL-6-572 C/G could decrease the risk of AD