Connie M. Krawczyk

The Energy Sensor AMPK Regulates T Cell Metabolic Adaptation and Effector Responses In Vivo

Naive T cells undergo metabolic reprogramming to support the increased energetic and biosynthetic demands of effector T cell function. However, how nutrient availability influences T cell metabolism and function remains poorly understood. Here we report plasticity in effector T cell metabolism in response to changing nutrient availability. Activated T cells were found to possess a glucose-sensitive metabolic checkpoint controlled by the energy sensor AMP-activated protein kinase (AMPK) that regulated mRNA translation and glutamine-dependent mitochondrial metabolism to maintain T cell bioenergetics and viability. T cells lacking AMPKα1 displayed reduced mitochondrial bioenergetics and cellular ATP in response to glucose limitation in vitro or pathogenic challenge in vivo. Finally, we demonstrated that AMPKα1 is essential for T helper 1 (Th1) and Th17 cell development and primary T cell responses to viral and bacterial infections in vivo. Our data highlight AMPK-dependent regulation of metabolic homeostasis as a key regulator of T cell-mediated adaptive immunity.

CD28-dependent Activation of Protein Kinase B/Akt Blocks Fas-mediated Apoptosis by Preventing Death-inducing Signaling Complex Assembly

The T cell costimulatory molecule CD28 is important for T cell survival, yet both the signaling pathways downstream of CD28 and the apoptotic pathways they antagonize remain poorly understood. Here we demonstrate that CD4+ T cells from CD28-deficient mice show increased susceptibility to Fas-mediated apoptosis via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. Protein kinase B (PKBα/Akt1) is an important serine/threonine kinase that promotes survival downstream of PI3K signals. To understand how PI3K-mediated signals downstream of CD28 contribute to T cell survival, we examined Fas-mediated apoptosis in T cells expressing an active form of PKBα. Our data demonstrate that T cells expressing active PKB are resistant to Fas-mediated apoptosis in vivo and in vitro. PKB transgenic T cells show reduced activation of caspase-8, BID, and caspase-3 due to impaired recruitment of procaspase-8 to the death-inducing signaling complex (DISC). Similar alterations are seen in T cells from mice which are haploinsufficient for PTEN, a lipid phosphatase that regulates phosphatidylinositol-3,4,5-trisphosphate (PIP3) and influences PKBα activity. These findings provide a novel link between CD28 and an important apoptosis pathway in vivo, and demonstrate that PI3K/PKB signaling prevents apoptosis by inhibiting DISC assembly.

Review series on helminths, immune modulation and the hygiene hypothesis: Mechanisms underlying helminth modulation of dendritic cell function

Dendritic cells (DCs) play a central role in activating CD4 T (T helper, Th) cells. As a component of their response to pathogen‐associated stimuli, DCs produce cytokines and express surface molecules that provide important cues to modulate the effector functions of responding Th cells. Much is known of how DCs respond to, and influence immune response outcome to, bacterial and viral pathogens. However, relatively little is understood about how DCs respond to helminth parasites. This is an area of considerable interest since it impacts our understanding of the initiation of Th2 responses, which are stereotypically associated with helminth infections, and the regulation of allergic and autoimmune pathologies which evidence suggests are less severe or absent in individuals infected with helminths. This review attempts to summarize our understanding of the effects of helminth products on dendritic cell biology.

Functional Plasticity in Memory T Helper Cell Responses

Following activation, naive CD4+ Th cells can differentiate to selectively produce either the Th1 lineage-specific cytokine IFN-γ or the Th2 cytokine IL-4 and, in so doing, lose the capacity to produce cytokines of the alternative lineage. Lineage commitment of murine CD4+ T cells has largely been considered to be absolute with little flexibility to produce cytokines of the opposing lineage. In this study, we demonstrate that cells within Th2 memory populations can produce IFN-γ if reactivated in vivo in the context of an innate response that favors Th1 cell development. Likewise, cells within Th1 memory populations produce IL-4 when challenged under conditions that promote Th2 responses. Both effector and unpolarized central memory cells retain the potential to produce cytokines that were not made during the primary response. These findings reveal that both effector and central memory Th1 and Th2 cells possess the capacity to respond to environmental cues to produce pathogen-appropriate cytokines of the opposing lineage.

Th2 cell hyporesponsiveness during chronic murine schistosomiasis is cell intrinsic and linked to GRAIL expression

Chronic infections are associated with progressively declining T cell function. Infections with helminth parasites, such as Schistosoma mansoni, are often chronic and characterized by the development of strong Th2 responses that peak during the acute stage of infection and then decline despite ongoing infection; this minimizes Th2-dependent immunopathology during the chronic stage of infection. We sought to understand the basis for the decline in Th2 responses in chronic schistosomiasis. Using IL-4 reporter mice (mice that express EGFP as a reporter for Il4 gene expression) to identify Th2 cells, we found that Th2 cell numbers plateaued during acute infection and remained constant thereafter. However, the percentages of Th2 cells proliferating during late infection were strikingly lower than those during acute infection. Th2 cell hyporesponsiveness was evident within 10 d of initiation of the Th2 response and became progressively ingrained thereafter, in response to repeated Ag stimulation. Gene expression analyses implicated the E3-ubiquitin ligase gene related to anergy in lymphocytes (GRAIL) in the hyporesponsive state. Consistent with this, suppression of GRAIL expression using retrovirally delivered siRNA prevented the development of hyporesponsiveness induced by repeated Ag stimulation in vitro or in vivo. Together, these data indicate that the decline in Th2 cell responsiveness during chronic schistosomiasis is the net result of the upregulation of GRAIL expression in response to repeated Ag stimulation.

Th2 Differentiation Is Unaffected by Jagged2 Expression on Dendritic Cells

Expression of the Jagged Notch ligands by dendritic cells (DCs) has been suggested to play a role in instructing Th2 responses. Supporting this hypothesis, we found that Jagged2 but not Jagged1 expression, correlates with the ability of DCs to induce Th2 responses. Jagged2 expression is up-regulated in response to the helminth soluble Schistosoma mansoni egg Ag, which conditions DCs to induce Th2 responses, and is markedly down-regulated following exposure to TLR agonists that generally promote Th1 responses. Conversely, Jagged1 expression is markedly induced by TLR ligation. Despite these correlations, suppression of expression of Jagged2 using retrovirally delivered small interfering RNA failed to affect the ability of DCs to induce Th2 cell differentiation either in vitro or in vivo. Moreover, retrovirally induced expression of Jagged2 did not enhance the ability of DCs to induce Th2 cell responses. Our data indicate that Jagged2 expression by DCs is not sufficient or required for Th2 cell differentiation.

Cbl-3-deficient mice exhibit normal epithelial development

ABSTRACT Cbl family proteins are evolutionarily conserved ubiquitin ligases that negatively regulate signaling from tyrosine kinase-coupled receptors. The mammalian cbl family consists of c-Cbl, Cbl-b, and the recently cloned Cbl-3 (also known as Cbl-c). In this study, we describe the detailed expression pattern of murine Cbl-3 and report the generation and characterization of Cbl-3-deficient mice. Cbl-3 exhibits an expression pattern distinct from those of c-Cbl and Cbl-b, with high levels of Cbl-3 expression in epithelial cells of the gastrointestinal tract and epidermis, as well as the respiratory, urinary, and reproductive systems. Cbl-3 expression was not detected in nonepithelial cells, but within epithelial tissues, the levels of Cbl-3 expression varied from undetectable in the alveoli of the lungs to very strong in the cecum and colon. Despite this restricted expression pattern, Cbl-3-deficient mice were viable, healthy, and fertile and displayed no histological abnormalities up to 18 months of age. Proliferation of epithelial cells in the epidermises and gastrointestinal tracts was unaffected by the loss of Cbl-3. Moreover, Cbl-3 was not required for attenuation of epidermal growth factor-stimulated Erk activation in primary keratinocytes. Thus, Cbl-3 is dispensable for normal epithelial development and function.

NF-κB Couples Protein Kinase B/Akt Signaling to Distinct Survival Pathways and the Regulation of Lymphocyte Homeostasis In Vivo

Protein kinase B (PKBα/Akt1) a PI3K-dependent serine-threonine kinase, promotes T cell viability in response to many stimuli and regulates homeostasis and autoimmune disease in vivo. To dissect the mechanisms by which PKB inhibits apoptosis, we have examined the pathways downstream of PKB that promote survival after cytokine withdrawal vs Fas-mediated death. Our studies show that PKB-mediated survival after cytokine withdrawal is independent of protein synthesis and the induction of NF-κB. In contrast, PKB requires de novo gene transcription by NF-κB to block apoptosis triggered by the Fas death receptor. Using gene-deficient and transgenic mouse models, we establish that NF-κB1, and not c-Rel, is the critical signaling molecule downstream of the PI3K-PTEN-PKB signaling axis that regulates lymphocyte homeostasis.

Pharmacological characterization of MK-7246, a potent and selective CRTH2 (chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells) antagonist.

The chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells (CRTH2) is a G protein-coupled receptor that has been reported to modulate inflammatory responses in various rodent models of asthma, allergic rhinitis and atopic dermatitis. In this study, we describe the biological and pharmacological properties of {(7R)-7-[[(4-fluorophenyl)sulfonyl](methyl)amino]-6,7,8,9-tetrahydropyrido[1,2-a]indol-10-yl}acetic acid (MK-7246), a novel synthetic CRTH2 antagonist. We show that MK-7246 1) has high affinity for the human, monkey, dog, rat, and mouse CRTH2, 2) interacts with CRTH2 in a reversible manner, 3) exhibits high selectivity over all prostanoid receptors as well as 157 other receptors and enzymes, 4) acts as a full antagonist on recombinant and endogenously expressed CRTH2, 5) demonstrates good oral bioavailability and metabolic stability in various animal species, 6) yields ex vivo blockade of CRTH2 on eosinophils in monkeys and sheep, and 7) significantly blocks antigen-induced late-phase bronchoconstriction and airway hyper-responsiveness in sheep. MK-7246 represents a potent and selective tool to further investigate the in vivo function of CRTH2.

LKB1 and AMPK: central regulators of lymphocyte metabolism and function.

When T cells encounter foreign antigen and appropriate costimulatory signals from professional antigen‐presenting cells (APCs), they initiate a coordinated program of rapid proliferation and differentiation, leading to the development of activated T cells with specific effector functions tailored toward pathogen clearance or control. One of the fundamental programs that underpin T‐cell proliferation and function is the regulation of cellular metabolism. Recent efforts to identify the signal transduction pathways that regulate T‐cell metabolism have led to the identification of liver kinase B1 (LKB1) and AMP‐activated protein kinase (AMPK) as key regulators of T‐cell metabolism. LKB1 and AMPK are part of an evolutionarily conserved signal transduction pathway that monitors cellular energy status. AMPK senses bioenergetic fluctuations in cells and works in concert with LKB1 to maintain cellular energy homeostasis by promoting catabolic pathways of ATP production and limiting processes that consume ATP. Recent data indicate that LKB1 and AMPK can influence diverse aspects of T‐cell biology beyond metabolism, including T‐cell development, peripheral T‐cell homeostasis, and T‐cell effector function. In this review, we focus on the regulation of lymphocyte metabolism by this energy‐sensing pathway and discuss its influence on T‐cell function.