Sung Hoon Cho

Vital roles of mTOR complex 2 in Notch-driven thymocyte differentiation and leukemia

Rictor is essential in Notch-driven T-ALL pathogenesis.

PARP-14, a member of the B aggressive lymphoma family, transduces survival signals in primary B cells

Poly(ADP-ribos)ylation is one of the longest-known but most enigmatic posttranslational modifications transducing specific signals. The enzyme responsible for the majority of poly(ADP-ribose) polymerization in cells, PARP-1, promotes DNA repair but also mediates a caspase-independent form of apoptosis in response to stressors such as irradiation. However, the biologic function of most other PARPs is not known. Macro-PARPs constitute one branch of the large family of PARP-like proteins also designated as B aggressive lymphoma proteins (BAL1, 2a/2b, 3, or PARP-9, PARP-14, and PARP-15). To elucidate biologic role(s) of a BAL-family macro-PARP, we analyzed mice deficient in PARP-14, a binding partner of the IL-4-induced transcription factor Stat6. We show here that PARP-14 plays a fundamental role mediating protection against apoptosis in IL-4-treated B cells, including that after DNA damage, and mediates IL-4 effects on the levels of gene products that regulate cell survival, proliferation, and lymphomagenesis. Collectively, the results establish that PARP-14 mediates regulation of gene expression and lymphocyte physiology by IL-4 and has a function distinct from PARP-1. Furthermore, the findings suggest mechanisms by which BAL-family proteins might influence pathologic processes involving B lymphocytes.

Glycolytic rate and lymphomagenesis depend on PARP14, an ADP ribosyltransferase of the B aggressive lymphoma (BAL) family

Poly(ADP-ribose)polymerase (PARP)14—a member of the B aggressive lymphoma (BAL) family of macrodomain-containing PARPs—is an ADP ribosyltransferase that interacts with Stat6, enhances induction of certain genes by IL-4, and is expressed in B lymphocytes. We now show that IL-4 enhancement of glycolysis in B cells requires PARP14 and that this process is central to a role of PARP14 in IL-4–induced survival. Thus, enhancements of AMP-activated protein kinase activity restored both IL-4–induced glycolytic activity in Parp14−/− B cells and prosurvival signaling by this cytokine. Suppression of apoptosis is central to B-lymphoid oncogenesis, and elevated macro-PARP expression has been correlated with lymphoma aggressiveness. Strikingly, PARP14 deficiency delayed B lymphomagenesis and reversed the block to B-cell maturation driven by the Myc oncogene. Collectively, these findings reveal links between a mammalian ADP ribosyltransferase, cytokine-regulated metabolic activity, and apoptosis; show that PARP14 influences Myc-induced oncogenesis; and suggest that the PARP14-dependent capacity to increase cellular metabolic rates may be an important determinant of lymphoma pathobiology.

Collaborator of Stat6 (CoaSt6)-associated poly(ADP-ribose) polymerase activity modulates Stat6-dependent gene transcription.

The transcription factor Stat6 plays a critical role in interleukin-4-dependent gene activation. To mediate this function, Stat6 recruits canonical transcriptional co-activators including the histone acetyl transferases CREB-binding protein and NCoA-1 and other proteins such as a p100 co-factor. However, much remains unknown regarding the constituents of Stat6 enhancer complexes, and the exact molecular events that modulate Stat6-dependent gene activation are not fully understood. Recently, we identified a novel co-factor, CoaSt6 (collaborator of Stat6), which associates with Stat6 and enhances its transcriptional activity. Sequence homologies place CoaSt6 in a superfamily of poly(ADP-ribosyl)polymerase (PARP)-like proteins. We have demonstrated here that PARP enzymatic activity is associated with CoaSt6, and this function of CoaSt6 can append ADP-ribose to itself and p100. Further, we show that a catalytically inactive mutant of CoaSt6 was unable to enhance Stat6-mediated transcription of a test promoter. Consistent with these findings, chemical inhibition of PARP activity blocked interleukin-4-dependent transcription from target promoters in vivo. Taken together, we have identified a CoaSt6-associated PARP activity and provided evidence for a role of poly(ADP ribosyl)ation in Stat-mediated transcriptional responses involving a novel PARP.

Requirement for Rictor in homeostasis and function of mature B lymphoid cells

The mammalian target of rapamycin (mTOR), an essential serine/threonine kinase, functions in biochemically distinct multiprotein complexes, but little is known about roles of the complexes in B cells. The acutely rapamycin-sensitive mTOR complex 1 (mTORC1) is defined by a core subunit Raptor, whereas mTORC2 lacks Raptor and, instead, has Rictor and SIN1 as distinct essential components. We now show that homeostasis and function of B cells require Rictor. Conditional deletion of Rictor before lymphoid specification impaired generation of mature follicular, marginal zone, and B1a B lymphocytes. Induced inactivation in adult mice caused cell-autonomous defects in B lymphoid homeostasis and antibody responses in vivo, along with affecting plasma cells in bone marrow. Survival of B lymphocytes depended on Rictor, which was vital for normal induction of prosurvival genes, suppression of proapoptotic genes, nuclear factor κB induction after B-cell receptor stimulation, and B-cell activating factor-induced nuclear factor κB2/p52 generation. Collectively, the findings provide evidence that mTOR signaling affects survival and proliferation of mature B lymphocytes, and establish Rictor as an important signal relay in B-cell homeostasis, fate, and functions.

Germinal centre hypoxia and regulation of antibody qualities by a hypoxia response system.

Germinal centres (GCs) promote humoral immunity and vaccine efficacy. In GCs, antigen-activated B cells proliferate, express high-affinity antibodies, promote antibody class switching, and yield B cell memory. Whereas the cytokine milieu has long been known to regulate effector functions that include the choice of immunoglobulin class, both cell-autonomous and extrinsic metabolic programming have emerged as modulators of T-cell-mediated immunity. Here we show in mice that GC light zones are hypoxic, and that low oxygen tension () alters B cell physiology and function. In addition to reduced proliferation and increased B cell death, low impairs antibody class switching to the pro-inflammatory IgG2c antibody isotype by limiting the expression of activation-induced cytosine deaminase (AID). Hypoxia induces HIF transcription factors by restricting the activity of prolyl hydroxyl dioxygenase enzymes, which hydroxylate HIF-1α and HIF-2α to destabilize HIF by binding the von Hippel–Landau tumour suppressor protein (pVHL). B-cell-specific depletion of pVHL leads to constitutive HIF stabilization, decreases antigen-specific GC B cells and undermines the generation of high-affinity IgG, switching to IgG2c, early memory B cells, and recall antibody responses. HIF induction can reprogram metabolic and growth factor gene expression. Sustained hypoxia or HIF induction by pVHL deficiency inhibits mTOR complex 1 (mTORC1) activity in B lymphoblasts, and mTORC1-haploinsufficient B cells have reduced clonal expansion, AID expression, and capacities to yield IgG2c and high-affinity antibodies. Thus, the normal physiology of GCs involves regional variegation of hypoxia, and HIF-dependent oxygen sensing regulates vital functions of B cells. We propose that the restriction of oxygen in lymphoid organs, which can be altered in pathophysiological states, modulates humoral immunity.

STAT4 and T-bet Are Required for the Plasticity of IFN-γ Expression across Th2 Ontogeny and Influence Changes in Ifng Promoter DNA Methylation

CD4+ T cells developing toward a Th2 fate express IL-4, IL-5, and IL-13 while inhibiting production of cytokines associated with other Th types, such as the Th1 cytokine IFN- γ. IL-4–producing Th2 effector cells give rise to a long-lived memory population committed to reactivation of the Th2 cytokine gene expression program. However, reactivation of these effector-derived cells under Th1-skewing conditions leads to production of IFN-γ along with IL-4 in the same cell. We now show that this flexibility (“plasticity”) of cytokine expression is preceded by a loss of the repressive DNA methylation of the Ifng promoter acquired during Th2 polarization yet requires STAT4 along with T-box expressed in T cells. Surprisingly, loss of either STAT4 or T-box expressed in T cells increased Ifng promoter CpG methylation in both effector and memory Th2 cells. Taken together, our data suggest a model in which the expression of IFN-γ by Th2-derived memory cells involves attenuation of epigenetic repression in memory Th2 cells, combined with Th1-polarizing signals after their recall activation.

B Cell-Intrinsic and -Extrinsic Regulation of Antibody Responses by PARP14, an Intracellular (ADP-Ribosyl)Transferase

The capacity to achieve sufficient concentrations of Ag-specific Ab of the appropriate isotypes is a critical component of immunity that requires efficient differentiation and interactions of Ag-specific B and Th cells along with dendritic cells. Numerous bacterial toxins catalyze mono(ADP-ribosyl)ation of mammalian proteins to influence cell physiology and adaptive immunity. However, little is known about biological functions of intracellular mammalian mono(ADP-ribosyl)transferases, such as any ability to regulate Ab responses. poly-(ADP-ribose) polymerase 14 (PARP14), an intracellular protein highly expressed in lymphoid cells, binds to STAT6 and encodes a catalytic domain with mammalian mono(ADP-ribosyl)transferase activity. In this article, we show that recall IgA as well as the STAT6-dependent IgE Ab responses are impaired in PARP14-deficient mice. Whereas PARP14 regulation of IgE involved a B cell–intrinsic process, the predominant impact on IgA was B cell extrinsic. Of note, PARP14 deficiency reduced the levels of Th17 cells and CD103+ DCs, which are implicated in IgA regulation. PARP14 enhanced the expression of RORα, Runx1, and Smad3 after T cell activation, and, importantly, its catalytic activity of PARP14 promoted Th17 differentiation. Collectively, the findings show that PARP14 influences the class distribution, affinity repertoire, and recall capacity of Ab responses in mice, as well as provide direct evidence of the requirement for protein mono-ADP-ribosylation in Th cell differentiation.

Complementing T Cells’ Functions: Bringing in Metabolism Matters

Components of the complement system act directly on T cells to alter conventional and regulatory T cell subsets. In this issue of Immunity, Kolev, Dimeloe, Le Friec et al. (2015) provide evidence of a mechanism by which the complement stimulates sustained mTORC1 activation and regulates cellular metabolism.

B Cell–Intrinsic mTORC1 Promotes Germinal Center–Defining Transcription Factor Gene Expression, Somatic Hypermutation, and Memory B Cell Generation in Humoral Immunity

B lymphocytes migrate among varied microenvironmental niches during diversification, selection, and conversion to memory or Ab-secreting plasma cells. Aspects of the nutrient milieu differ within these lymphoid microenvironments and can influence signaling molecules such as the mechanistic target of rapamycin (mTOR). However, much remains to be elucidated as to the B cell–intrinsic functions of nutrient-sensing signal transducers that modulate B cell differentiation or Ab affinity. We now show that the amino acid–sensing mTOR complex 1 (mTORC1) is vital for induction of Bcl6—a key transcriptional regulator of the germinal center (GC) fate—in activated B lymphocytes. Accordingly, disruption of mTORC1 after B cell development and activation led to reduced populations of Ag-specific memory B cells as well as plasma cells and GC B cells. In addition, induction of the germ line transcript that guides activation-induced deaminase in selection of the IgG1 H chain region during class switching required mTORC1. Expression of the somatic mutator activation-induced deaminase was reduced by a lack of mTORC1 in B cells, whereas point mutation frequencies in Ag-specific GC-phenotype B cells were only halved. These effects culminated in a B cell–intrinsic defect that impacted an antiviral Ab response and drastically impaired generation of high-affinity IgG1. Collectively, these data establish that mTORC1 governs critical B cell–intrinsic mechanisms essential for establishment of GC differentiation and effective Ab production.