The PI3K inhibitor GDC-0941 combines with existing clinical regimens for superior activity in multiple myeloma.

The phosphatidylinositol 3'-kinase (PI3K) pathway is dysregulated in multiple myeloma (MM); we therefore tested a highly selective class I PI3K inhibitor, GDC-0941, for anti-myeloma activity. Functional and mechanistic studies were first performed in MM cell lines, then extended to primary MM patient samples cultured in vitro. GDC-0941 was then assessed as a single agent and in various combinations in myeloma tumor xenograft models. We show p110 α and ß are the predominant PI3K catalytic subunits in MM and that a highly selective class I PI3K inhibitor, GDC-0941, has robust activity as a single agent to induce cell cycle arrest and apoptosis of both MM cell lines and patient myeloma cells. Mechanistic studies revealed an induction of cell cycle arrest at G0/G1, with decreased phospho-FoxO1/3a levels, decreased cyclin D1 and c-myc expression, and an increase in the cell cycle inhibitor, p27kip. Induction of apoptosis correlated with increased expression of the pro-apoptotic BH3-only protein BIM, cleaved caspase 3 and cleaved poly (ADP-ribose) polymerase (PARP). In vitro, GDC-0941 synergized with dexamethasone (Dex) and lenalidomide (combination index values of 0.3-0.4 and 0.4-0.8, respectively); in vivo GDC-0941 has anti-myeloma activity and significantly increases the activity of the standard of care agents in several murine xenograft tumor models (additional tumor growth inhibition of 37-53% (Dex) and 22-72% (lenalidomide)). These data provide a clear therapeutic hypothesis for the inhibition of PI3K and provide a rationale for clinical development of GDC-0941 in myeloma.

Duration and strength of extracellular signal-regulated kinase signals are altered during positive versus negative thymocyte selection.

During thymocyte development, high-affinity/avidity TCR engagement leads to the induction of negative selection and apoptosis, while lower TCR affinity-avidity interactions lead to positive selection and survival. To elucidate how these extracellular interactions are translated into intracellular signals that distinguish between positive and negative selection, we developed a culture system in which naive double-positive thymocytes were either induced to differentiate along the CD8(+) lineage pathway or were triggered for clonal deletion. Using this system, we show that sustained low level activation of extracellular signal-regulated kinases (ERKs) promotes positive selection, whereas strong but transient ERK activation is coupled with negatively selecting stimuli. Importantly, similar ERK activation profiles were demonstrated during positive selection for strong agonist ligands presented at low concentrations or weak agonist ligands. This is consistent with the affinity/avidity model and a role for strong or weak agonists during positive selection. Surprisingly, the addition of a pharmacological inhibitor which blocks ERK activation prevented the induction of negative selection. These data suggest that the duration and strength of the TCR signal is involved in discriminating between positive and negative selection.

Regulation of T cell activation, anxiety, and male aggression by RGS2.

Regulators of G protein signaling (RGS) proteins accelerate the GTPase activity of Galpha protein subunits in vitro, negatively regulating G protein-coupled receptor signaling. The physiological role of mammalian RGS proteins is largely unknown. The RGS family member rgs2 was cloned as an immediate early response gene up-regulated in T lymphocytes after activation. To investigate the role of RGS2 in vivo, we generated rgs2-deficient mice. We show that targeted mutation of rgs2 in mice leads to reduced T cell proliferation and IL-2 production, which translates in an impaired antiviral immunity in vivo. Interestingly, rgs2(-/-) mice also display increased anxiety responses and decreased male aggression in the absence of cognitive or motor deficits. RGS2 also controls synaptic development and basal electrical activity in hippocampal CA1 neurons. Thus, RGS2 plays an important role in T cell activation, synapse development in the hippocampus, and emotive behaviors.

Degree of ERK activation influences both positive and negative thymocyte selection.

Considerable evidence suggests that the ERK pathway is required for positive but not negative thymocyte selection. Here, we report that ERK is highly activated in double-positive (DP) thymocytes expressing an MHC class I-restricted TCR (P14) in response to negatively selecting conditions, whereas ligands that trigger positive selection induced weaker ERK activation. Biochemical evidence also shows that death by neglect is associated with a further reduction in ERK activation. These findings are consistent with the affinity / avidity model of thymocyte selection. To further examine the role of ERK in negative selection we used the MEK-1 inhitibor, PD98059, a specific pharmacological inhibitor of the ERK pathway. Biochemical data demonstrated a reduction of ERK activity by PD98059 in the presence of the negatively selecting ligand. Analysis of P14 TCR-transgenic fetal thymic lobes cultured with PD98059 under negatively selecting conditions showed impaired clonal deletion of DP thymocytes and a concomitant increase in positive selection of functional mature, TCR(hi) transgenic T cells. This demonstrates that altering ERK activity switched negative to positive selection. Contrary to previous reports that show an exclusive role for ERK signaling in positive selection, our data demonstrate that negative selection is also sensitive to the degree of ERK activation.

Negative regulation of lymphocyte activation and autoimmunity by the molecular adaptor Cbl-b.

The signalling thresholds of antigen receptors and co-stimulatory receptors determine immunity or tolerance to self molecules. Changes in co-stimulatory pathways can lead to enhanced activation of lymphocytes and autoimmunity, or the induction of clonal anergy. The molecular mechanisms that maintain immunotolerance in vivo and integrate co-stimulatory signals with antigen receptor signals in T and B lymphocytes are poorly understood. Members of the Cbl/Sli family of molecular adaptors function downstream from growth factor and antigen receptors. Here we show that gene-targeted mice lacking the adaptor Cbl-b develop spontaneous autoimmunity characterized by auto-antibody production, infiltration of activated T and B lymphocytes into multiple organs, and parenchymal damage. Resting cbl-b(-/-) lymphocytes hyperproliferate upon antigen receptor stimulation, and cbl-b(-/-) T cells display specific hyperproduction of the T-cell growth factor interleukin-2, but not interferon-gamma or tumour necrosis factor-alpha. Mutation of Cbl-b uncouples T-cell proliferation, interleukin-2 production and phosphorylation of the GDP/GTP exchange factor Vav1 from the requirement for CD28 co-stimulation. Cbl-b is thus a key regulator of activation thresholds in mature lymphocytes and immunological tolerance and autoimmunity.

Signals involved in thymocyte positive and negative selection.

Extensive research has focused upon understanding how thymocytes distinguish between interactions that lead to positive or negative selection. Various intracellular pathways that are activated after TCR engagement are outlined in this review, and their contribution to thymocyte selection is discussed. Although thymocyte fate is generally governed by a quantitative/avidity model, this largely reflects the interactions that occur at the cell surface. Therefore, we outline possible models of how different intercellular interactions are translated into intracellular signals that diverge and lead to thymocyte survival or death.

Selection of the T cell repertoire.

Advances in gene technology have allowed the manipulation of molecular interactions that shape the T cell repertoire. Although recognized as fundamental aspects of T lymphocyte development, only recently have the mechanisms governing positive and negative selection been examined at a molecular level. Positive selection refers to the active process of rescuing MHC-restricted thymocytes from programmed cell death. Negative selection refers to the deletion or inactivation of potentially autoreactive thymocytes. This review focuses on interactions during thymocyte maturation that define the T cell repertoire, with an emphasis placed on current literature within this field.

Degree of TCR internalization and Ca2+ flux correlates with thymocyte selection.

Recent evidence suggests that TCR down-regulation directly reflects the number of TCRs that have engaged MHC/peptide ligand complexes. Here, we examined the influence of defined peptides on thymic selection based on their ability to induce differential TCR internalization. Our results demonstrate that there is a direct correlation: peptides that induce strong TCR down-regulation are most efficient at mediating negative selection, whereas peptides that induce suboptimal TCR internalization are more efficient at triggering positive selection. As a consequence of suboptimal TCR internalization, a proportion of TCR complexes that remain on the cell surface may be able to relay continual signals required for survival and differentiation. In addition, we show that the magnitude of Ca2+ influx set by these peptides reflects the hierarchy of TCR down-regulation and correlates with positive vs negative selection of transgenic thymocytes. Together, our data suggest that T cell selection is mediated by differing intensities of the same TCR-mediated signal, rather than by distinct signals.

Vav regulates peptide-specific apoptosis in thymocytes.

The protooncogene Vav functions as a GDP/GTP exchange factor (GEF) for Rho-like small GTPases involved in cytoskeletal reorganization and cytokine production in T cells. Gene-targeted mice lacking Vav have a severe defect in positive and negative selection of T cell antigen receptor transgenic thymocytes in vivo, and vav-/- thymocytes are completely resistant to peptide-specific and anti-CD3/anti-CD28-mediated apoptosis. Vav acts upstream of mitochondrial pore opening and caspase activation. Biochemically, Vav regulates peptide-specific Ca2+ mobilization and actin polymerization. Peptide-specific cell death was blocked both by cytochalasin D inhibition of actin polymerization and by inhibition of protein kinase C (PKC). Activation of PKC with phorbol ester restored peptide-specific apoptosis in vav-/- thymocytes. Vav was found to bind constitutively to PKC-theta in thymocytes. Our results indicate that peptide-triggered thymocyte apoptosis is mediated via Vav activation, changes in the actin cytoskeleton, and subsequent activation of a PKC isoform.

The inositol polyphosphate 5-phosphatase ship is a crucial negative regulator of B cell antigen receptor signaling.

Ship is an Src homology 2 domain containing inositol polyphosphate 5-phosphatase which has been implicated as an important signaling molecule in hematopoietic cells. In B cells, Ship becomes associated with Fcgamma receptor IIB (FcgammaRIIB), a low affinity receptor for the Fc portion of immunoglobulin (Ig)G, and is rapidly tyrosine phosphorylated upon B cell antigen receptor (BCR)-FcgammaRIIB coligation. The function of Ship in lymphocytes was investigated in Ship-/- recombination-activating gene (Rag)-/- chimeric mice generated from gene-targeted Ship-/- embryonic stem cells. Ship-/-Rag-/- chimeras showed reduced numbers of B cells and an overall increase in basal serum Ig. Ship-/- splenic B cells displayed prolonged Ca2+ influx, increased proliferation in vitro, and enhanced mitogen-activated protein kinase (MAPK) activation in response to BCR-FcgammaRIIB coligation. These results demonstrate that Ship plays an essential role in FcgammaRIIB-mediated inhibition of BCR signaling, and that Ship is a crucial negative regulator of Ca2+ flux and MAPK activation.

Peptide-induced T cell receptor down-regulation on naive T cells predicts agonist/partial agonist properties and strictly correlates with T cell activation.

Recent experiments defining T cell agonists, partial agonists and antagonists have suggested that the T cell can discriminate between subtle differences in interactions leading to T cell activation. To further understand the complexities of T cell activation, we have analyzed the requirements for the induction of a variety of effector functions using naive T cells and a variety of altered peptide ligands. Using a strong agonist peptide, massive T cell receptor (TCR) down-regulation correlated with a wide range of effector functions that were all induced above the same threshold peptide concentration. Interestingly, the kinetics of TCR down-regulation correlated with the concentration of the peptide, whereas the maximal degree of TCR down-regulation correlated with the induction of all monitored effector functions. A selected group of altered peptide ligands was also examined that were able to render target cells susceptible for lysis by effector cytotoxic T lymphocytes. The extent of TCR down-regulation induced by these peptides corresponded to the induction of a subset of effector functions. These studies have shown that the extent of TCR down-regulation defines the strength of TCR-mediated "signal 1" which correlates with the spectrum of effector functions activated within the T cell. Thus, activation of different T cell functions requires the triggering of distinct numbers of TCR. The different parameters that influence TCR down-regulation define important distinctions between our results and previously reported findings with T cell clones and may outline decisive parameters for the consequences of T cell activation in vivo.

Four types of Ca2+ signals in naive CD8+ cytotoxic T cells after stimulation with T cell agonists, partial agonists and antagonists.

Stimulation of T cells via the T cell receptor (TCR) leads to an increase intracellular in free Ca2+ levels ([Ca2+]i) and the activation of the MAP kinase signaling pathway. This study analyzes for the first time Ca2+ fluxes in naive cytotoxic T cells stimulated with full agonists, partial agonists, or antagonists. Four different types of Ca2+ responses could be observed. Full agonists triggered a strong and sustained increase in [Ca2+]i. In contrast, partial T cell agonists induced either a strong but transient Ca2+ flux or very low to no increases in [Ca2+]i, while T cell antagonists failed to induce any measurable Ca2+ flux. The ability of peptides to induce elevated [Ca2+]i perfectly paralleled their ability to trigger TCR internalization and T cell activation. Thus, stimulation of naive cytotoxic T cells with a panel of defined altered peptide ligands reveals a consistent picture, where Ca2+ fluxes predict agonist, partial agonist and antagonist properties of peptides.

Biochemical and genetic defects in the DNA-dependent protein kinase in murine scid lymphocytes.

The scid gene product has been identified as the 460-kDa catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs p460), a member of the phosphatidylinositol 3-kinase family. DNA-PK activity is undetectable in scid cells, but the molecular basis for this defect has not been identified. Here we report that expression of p460 in scid lymphocyte precursors is detectable but is reduced at least 10-fold relative to that in wild-type lymphocytes. In addition, we show that the scid mutation disturbs p460 nuclear association, presumably affecting its role in DNA repair pathways. To examine the molecular basis for our observations, we used a degenerate PCR strategy to clone the C-terminal p460 kinase domain from wild-type and scid thymocytes. Northern (RNA) analysis with these probes revealed normal steady-state p460 mRNA levels in scid cells, suggesting that the reduced abundance of p460 protein is due to a posttranscriptional defect. Sequence comparisons identified a single-base-pair alteration in the scid C-terminal p460 kinase domain, resulting in a premature stop codon. This mutation is predicted to truncate p460 by approximately 8 kDa, but it preserves the conserved motifs required for kinase activity in members of the phosphoinositidyl 3-kinase family. Despite a computed molecular weight alteration of less than 2%, we were able to visualize this difference by Western blot (immunoblot) analysis of wild-type and scid p460. These data demonstrate that the scid DNA-PKes mutation is not a null allele and suggest a molecular rationale for the well-described leakiness of the scid phenotype.

Affinity maturation without germinal centres in lymphotoxin-alpha-deficient mice.

Affinity maturation by somatic hypermutation is thought to occur within germinal centres. Mice deficient in lymphotoxin-alpha (LT alpha-/- mice) have no lymph nodes or Peyer's patches, and fail to form germinal centres in the spleen. We tested whether germinal centres are essential for maturation of antibody responses to T-cell-dependent antigens. LT alpha-/- mice immunized with low doses of (4-hydroxy-3-nitrophenyl)acetyl-ovalbumin (NP-OVA) showed dramatically impaired production of high-affinity anti-NP IgG1. However, LT alpha-/- mice immunized with high doses of NP-OVA, even though they failed to produce germinal centres, manifested a high-affinity anti-NP IgG1 response similar to wild-type mice. Furthermore, when LT alpha-/- mice were multiply immunized with high doses of NP-OVA, the predominantly expressed anti-NP VH gene segment VH186.2 showed somatic mutations typical of affinity maturation. Thus, B-cell memory and affinity maturation are not absolutely dependent on the presence of germinal centres.

Mice deficient in IL-1beta manifest impaired contact hypersensitivity to trinitrochlorobenzone.

Mice rendered deficient in IL-1 beta by gene targeting in embryonic stem cells develop and grow normally in a protected laboratory environment. Endotoxin-stimulated peritoneal macrophages from IL-1beta-deficient mice showed normal synthesis and cellular release of IL-1alpha after treatment with 5 mM ATP demonstrating that IL-1beta is not necessary for expression and release of the IL-1alpha isoform. Mice deficient in IL-1beta showed unaltered sensitivity to endotoxic shock, with or without pretreatment with D-galactosamine. In contrast, IL-1beta-deficient mice showed defective contact hypersensitivity responses to topically applied trinitrochlorobenzene (TNCB). This defect could be overcome either by application of very high doses of sensitizing antigen, or by local intradermal injection of recombinant IL-1beta immediately before antigen application. These data demonstrate an essential role for IL-1beta in contact hypersensitivity and suggest that IL-1beta acts early during the sensitization phase of response. They suggest an important role for IL-1beta in initiation of the host of response at the epidermal barrier.

Markedly impaired humoral immune response in mice deficient in complement receptors 1 and 2.

Complement receptor 1 (CR1, CD35) and complement receptor 2 (CR2, CD21) have been implicated as regulators of B-cell activation. We explored the role of these receptors in the development of humoral immunity by generating CR1- and CR2-deficient mice using gene-targeting techniques. These mice have normal basal levels of IgM and of IgG isotypes. B- and T-cell development are overtly normal. Nevertheless, B-cell responses to low and high doses of a T-cell-dependent antigen are impaired with decreased titers of antigen-specific IgM and IgG isotypes. This defect is not complete because there is still partial activation of B lymphocytes during the primary immune response, with generation of splenic germinal centers and a detectable, although reduced, secondary antibody response. These data suggest that certain T-dependent antigens manifest an absolute dependence on complement receptors for the initiation of a normally robust immune response.

Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers.

In mice deficient in either lymphotoxin-alpha (LT-alpha) or the type I tumor necrosis factor (TNF) receptor, but not the type II TNF receptor, germinal centers failed to develop in peripheral lymphoid organs. Germinal center formation was restored in LT-alpha-deficient mice by transplantation of normal bone marrow, indicating that the LT-alpha-expressing cells required to establish this lymphoid structure are derived from bone marrow.

Absence of lymph nodes in lymphotoxin-alpha(LT alpha)-deficient mice is due to abnormal organ development, not defective lymphocyte migration.

Mice homozygous for a targeted null mutation of lymphotoxin-alpha (LT alpha) are born without lymph nodes (LN) or Peyer's patches (PP) and with altered splenic architecture. To investigate the mechanism of failed LN organogenesis, we transferred bone marrow (BM) from Thy 1.2 LT alpha-deficient or Thy 1.2 wild type mice to lethally irradiated 8-12-week-old Thy 1.1 wild type recipients. Six to 10 weeks later, reconstitution of LN and spleen with Thy 1.2 cells was similar whether the BM was derived from LT alpha-deficient or wild type donors. In contrast, reconstitution of irradiated LT alpha-deficient mice with wild type BM did not induce the development of detectable LN, although reconstitution of the spleen occurred appropriately. The expression and regulation of the lymphocyte adhesion molecule L-selectin from the LT alpha-deficient mice appeared normal. These data indicate that LT alpha-dependent interactions must occur during development in order for LN genesis to take place; however, lymphocyte expression of LT alpha is not required for these cells to home to existing LN structures.

Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin.

Mice rendered deficient in lymphotoxin (LT) by gene targeting in embryonic stem cells have no morphologically detectable lymph nodes or Peyer's patches, although development of the thymus appears normal. Within the white pulp of the spleen, there is failure of normal segregation of B and T cells. Spleen and peripheral blood contain CD4+CD8- and CD4-CD8+ T cells in a normal ratio, and both T cells subsets have an apparently normal lytic function. Lymphocytes positive for immunoglobulin M are present in increased numbers in both the spleen and peripheral blood. These data suggest an essential role for LT in the normal development of peripheral lymphoid organs.