Negative feedback loop in T cell activation through IκB kinase-induced phosphorylation and degradation of Bcl10

BackgroundCD73 (ecto-5’-nucleotidase) is an ecto-nucleotidase that dephosphorylate AMP to form adenosine. Activation of adenosine signaling pathway in immune cells leads to the suppression of effector functions, down-regulate macrophage phagocytosis, inhibit pro-inflammatory cytokine release, as well as yield aberrantly differentiated dendritic cells producing pro-tumorigenic molecules.1 In the tumor microenvironment, adenosinergic negative feedback signaling facilitated immune suppression is considered an important mechanism for immune evasion of cancer cells.2 3 Combination of CD73 and anti-PD-1 antibody has shown promising activity in suppressing tumor growth. Hence, we developed AK119, an anti- human CD73 monoclonal antibody, and AK123,a bi-specific antibody targeting both PD-1 and CD73 for immune therapy of cancer.MethodsAK119 is a humanized antibody against CD73 and AK123 is a tetrameric bi-specific antibody targeting PD-1 and CD73. Binding assays of AK119 and AK123 to antigens, and antigen expressing cells were performed by using ELISA, Fortebio, and FACS assays. In-vitro assays to investigate the activity of AK119 and AK123 to inhibit CD73 enzymatic activity in modified CellTiter-Glo assay, to induce endocytosis of CD73, and to activate B cells were performed. Assay to evaluate AK123 activity on T cell activation were additionally performed. Moreover, the activities of AK119 and AK123 to mediate ADCC, CDC in CD73 expressing cells were also evaluated.ResultsAK119 and AK123 could bind to its respective soluble or membrane antigens expressing on PBMCs, MDA-MB-231, and U87-MG cells with high affinity. Results from cell-based assays indicated that AK119 and AK123 effectively inhibited nucleotidase enzyme activity of CD73, mediated endocytosis of CD73, and induced B cell activation by upregulating CD69 and CD83 expression on B cells, and showed more robust CD73 blocking and B cell activation activities compared to leading clinical candidate targeting CD73. AK123 could also block PD-1/PD-L1 interaction and enhance T cell activation.ConclusionsIn summary, AK119 and AK123 represent good preclinical biological properties, which support its further development as an anti-cancer immunotherapy or treating other diseases.ReferencesDeaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, Enjyoji K, Linden J, Oukka M, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 2007; 204:1257–65.Huang S, Apasov S, Koshiba M, Sitkovsky M. Role of A2a extracellular adenosine receptor-mediated signaling in adenosine-mediated inhibition of T-cell activation and expansion. Blood. 1997; 90:1600–10.Novitskiy SV, Ryzhov S, Zaynagetdinov R, Goldstein AE, Huang Y, Tikhomirov OY, Blackburn MR, Biaggioni I,Carbone DP, Feoktistov I, et al. Adenosine receptors in regulation of dendritic cell differentiation and function. Blood 2008; 112:1822–31.

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IκB kinase 2 but not NF-κB–inducing kinase is essential for effective DC antigen presentation in the allogeneic mixed lymphocyte reaction

Blood ◽

10.1182/blood-2002-06-1835 ◽

2003 ◽

Vol 101 (3) ◽

pp. 983-991 ◽

Cited By ~ 55

Author(s):

Evangelos Andreakos ◽

Clive Smith ◽

Claudia Monaco ◽

Fionula M. Brennan ◽

Brian M. Foxwell ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Antigen Presentation ◽

T Cell Activation ◽

Cell Activation ◽

Mixed Lymphocyte Reaction ◽

Dominant Negative ◽

Allogeneic Mixed Lymphocyte Reaction ◽

Iκb Kinase ◽

Antigen Presenting

AbstractAlthough dendritic cells (DCs) are the most potent antigen-presenting cells involved in numerous physiologic and pathologic processes, little is known about the signaling pathways that regulate DC activation and antigen-presenting function. Recently, we demonstrated that nuclear factor (NF)-κB activation is central to that process, as overexpression of IκBα blocks the allogeneic mixed lymphocyte reaction (MLR), an in vitro model of T-cell activation. In this study, we investigated the role of 2 putative NF-κB–inducing components, NF-κB–inducing kinase (NIK), and IκB kinase 2 (IKK2). Using an adenoviral gene transfer method to efficiently express dominant-negative (dn) forms of these molecules in monocyte-derived DCs, we found that IKK2dn but not NIKdn inhibited the allogeneic MLR. When DCs were fixed, this inhibitory effect of IKK2dn was lost, suggesting that IKK2 is involved in T-cell–derived signals that enhance DC antigen presentation during the allogeneic MLR period and does not have an effect on viability or differentiation state of DCs prior to coculture with T cells. One such signal is likely to be CD40 ligand (CD40L), as IKK2dn blocked CD40L but not lipopolysaccharide (LPS)–induced NF-κB activation, cytokine production, and up-regulation of costimulatory molecules and HLA-DR in DCs. In summary, our results demonstrate that IKK2 is essential for DC activation induced by CD40L or contact with allogeneic T cells, but not by LPS, whereas NIK is not required for any of these signals. In addition, our results support IKK2 as a potential therapeutic target for the down-regulation of unwanted immune responses that may occur during transplantation or autoimmunity.

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A Repressor of GATA-Mediated Negative Feedback Mechanism of T Cell Activation

The Journal of Immunology ◽

10.4049/jimmunol.172.1.170 ◽

2003 ◽

Vol 172 (1) ◽

pp. 170-177 ◽

Cited By ~ 14

Author(s):

Shi-Chuen Miaw ◽

Bok Yun Kang ◽

Ian Alexander White ◽

I-Cheng Ho

Keyword(s):

T Cell ◽

T Cell Activation ◽

Negative Feedback ◽

Cell Activation ◽

Feedback Mechanism ◽

Negative Feedback Mechanism

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Widespread JNK-dependent alternative splicing induces a positive feedback loop through CELF2-mediated regulation of MKK7 during T-cell activation

Genes & Development ◽

10.1101/gad.267245.115 ◽

2015 ◽

Vol 29 (19) ◽

pp. 2054-2066 ◽

Cited By ~ 42

Author(s):

Nicole M. Martinez ◽

Laura Agosto ◽

Jinsong Qiu ◽

Michael J. Mallory ◽

Matthew R. Gazzara ◽

...

Keyword(s):

Alternative Splicing ◽

T Cell ◽

T Cell Activation ◽

Positive Feedback ◽

Feedback Loop ◽

Cell Activation ◽

Positive Feedback Loop

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“A negative feedback loop mediated by the NR4A family of nuclear hormone receptors restrains expansion of B cells that receive signal one in the absence of signal two”

10.1101/2020.03.31.017434 ◽

2020 ◽

Author(s):

Corey Tan ◽

Ryosuke Hiwa ◽

James L. Mueller ◽

Vivasvan Vykunta ◽

Kenta Hibiya ◽

...

Keyword(s):

T Cell ◽

B Cells ◽

B Cell ◽

Negative Feedback ◽

Feedback Loop ◽

Hormone Receptors ◽

Receive Signal ◽

Negative Feedback Loop ◽

Orphan Nuclear Receptors ◽

T Cell Help

ABSTRACTAg stimulation (signal 1) triggers B cell activation and proliferation, and primes B cells to recruit, engage, and respond to T cell help (signal 2). However, failure to receive signal 2 within a defined window of time results in an abortive round of proliferation, followed by anergy or apoptosis. Although the molecular basis of T cell help has been extensively dissected, the mechanisms that restrain Ag-stimulated B cells, and enforce dependence upon co-stimulation, are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell receptor (BCR) stimulation, yet little is known about their function in humoral immune responses. Here we use germline and conditional loss-of-function mouse models to show that Nr4a1 and Nr4a3 play partially redundant roles to restrain both the survival and proliferation of B cells that receive signal 1 in the absence of co-stimulatory signals, and do so in part by repressing expression of BATF and consequently c-MYC. Correspondingly, Ab responses to TI-2 immunogens are enhanced in the absence of Nr4a1, but are unaltered in response to immunogens that incorporate co-stimulatory signals. Unexpectedly, we also identify a role for the NR4A family in restraining B cell access to T cell help by repressing expression of the T cell chemokines CCL3/4, as well as CD86 and ICAM1, and show that this is relevant under conditions of competition for limiting T cell help. Our studies collectively reveal a novel negative feedback loop mediated by the NR4A family that increases B cell dependence upon T cell help and restrains strongly Ag-activated B cell clones from monopolizing limiting amounts of T cell help. We speculate that this imposes B cell tolerance and dampens immunodominance to facilitate preservation of clonal diversity during an immune response.

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The molecular machinery for cAMP-dependent immunomodulation in T-cells

Biochemical Society Transactions ◽

10.1042/bst0340476 ◽

2006 ◽

Vol 34 (4) ◽

pp. 476-479 ◽

Cited By ~ 43

Author(s):

K. Taskén ◽

A.J. Stokka

Keyword(s):

T Cell ◽

Immune Responses ◽

Lipid Rafts ◽

T Cell Activation ◽

Negative Feedback ◽

Cell Activation ◽

Cell Receptor ◽

Membrane Microdomains ◽

Type I ◽

Anchoring Protein

cAMP inhibits Src-family kinase signalling by PKA (protein kinase A)-mediated phosphorylation and activation of Csk (C-terminal Src kinase). The PKA type I–Csk pathway is assembled and localized in membrane microdomains (lipid rafts) and regulates immune responses activated through the TCR (T-cell receptor). PKA type I is targeted to the TCR–CD3 complex during T-cell activation via an AKAP (A-kinase-anchoring protein) that serves as a scaffold for the cAMP–PKA/Csk pathway in lipid rafts of the plasma membrane during T-cell activation. Displacement of PKA by anchoring disruption peptides prevents cAMP/PKA type I-mediated inhibition of T-cell activation. These findings provide functional evidence that PKA type I regulation of T-cell responses is dependent on AKAP anchoring. Furthermore, we show that upon TCR/CD28 co-ligation, β-arrestin in complex with PDE4 (phosphodiesterase 4) is recruited to lipid rafts. The CD28-mediated recruitment of PDE4 to lipid rafts potentiates T-cell immune responses and counteracts the local, TCR-induced production of cAMP that produces negative feedback in the absence of a co-receptor stimulus. The specific recruitment of PDE4 thus serves to abrogate the negative feedback by cAMP which is elicited in the absence of a co-receptor stimulus.

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Negative feedback of T cell activation through inhibitory adapters and costimulatory receptors

Immunological Reviews ◽

10.1034/j.1600-065x.2003.00022.x ◽

2003 ◽

Vol 192 (1) ◽

pp. 143-160 ◽

Cited By ~ 46

Author(s):

Takashi Saito ◽

Sho Yamasaki

Keyword(s):

T Cell ◽

T Cell Activation ◽

Negative Feedback ◽

Cell Activation

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A Novel Feedback Mechanism by Ephrin-B1/B2 In T Cell Activation: Concentration-Dependent Switch From Costimulation to Inhibition

Blood ◽

10.1182/blood.v116.21.277.277 ◽

2010 ◽

Vol 116 (21) ◽

pp. 277-277

Author(s):

Hiroki Kawano ◽

Yoshio Katayama ◽

Kentaro Minagawa ◽

Manabu Shimoyama ◽

Mark Henkemeyer ◽

...

Keyword(s):

Cell Proliferation ◽

T Cells ◽

T Cell ◽

T Cell Activation ◽

Negative Feedback ◽

Knockout Mice ◽

Cell Activation ◽

Cytokine Production ◽

Signaling Molecule ◽

High Concentrations

Abstract Abstract 277 Eph is the largest known family of receptor tyrosine kinases, and bind to a cell surface-associated ligand, ephrin on neighboring cells upon direct cell-cell contact. The ensuing bidirectional signals have been recognized as a major form of contact-dependent cell communications, such as cell attraction and repulsion to control accurate spatial and temporal patterning in the development of the central nervous system. EphBs, EphB6 in particular, are expressed in T cells and its specific ligand, ephrin-B2 has been shown to act as a costimulatory molecule for the T cell receptor (TCR)-mediated cell proliferation. Recently, another remarkable feature of ephrins, a concentration-dependent transition from promotion to inhibition in axon growth has emerged in ephrin-As. Thus, we postulated that this type of ligand concentration dependent functional transition would be suitable for the delicate tuning of immune responses to avoid reckless drive. To figure this out, we carefully evaluated the costimulatory effects of ephrin-Bs by using murine primary T cells. Interestingly, low doses of solid phase ephrin-B1 as well as ephrin-B2 (at up to 5μ g/ml) costimulated, to the comparable level with anti-CD28, T cell proliferation induced by suboptimal concentration of immobilized anti-CD3 antibody, but high concentrations of ephrin-B1/B2 inhibited the TCR-mediated proliferation significantly (by approximately 70% reduction from the baseline at 20μ g/ml). The similar concentration-dependent transition from coactivation to inhibition was also observed under the optimal CD3 stimulation. The concentration-dependent biphasic effects, positively at low concentration and negatively at high concentration, by ephrin-B1/B2 in T cell activation were confirmed in the cytokine production such as TNF-α, IL-2, and IFN-γ. In contrast, ephrin-B3 showed steadily increasing stimulatory effect even in higher concentrations in proliferation and cytokine production. We speculated that these unique modulations were partly mediated by EphB6 because EphB6 transfected in HEK293T cells has been shown to exert biphasic effects in cell adhesion and migration in response to different concentrations of ephrin-B2. T cell derived from Ephb6 -/- mice showed decreased CD3-stimulated cell proliferation as reported previously. However, the unique comodulatory pattern by each ephrin-B was virtually preserved in Ephb6 -/- T cells. Since the functions of Eph family could be redundant, we further investigated by generating multiple EphB knockout mice lacking four genes, Ephb1, Ephb2, Ephb3 and Ephb6. Surprisingly, no further alteration was observed in T cells from the quadruple knockout mice compared to the Ephb6 single deficiency. We also confirmed that EphA4, an exception in EphA receptor family which binds ephrin-Bs, was not expressed in T cells by RT-PCR. Taken together with the fact that EphB5 does not exist in mammals, the unique comodification by ephrin-Bs might be regulated by EphB4. Next, we examined the cross-talk of EphB forward signaling with TCR pathway. The inhibitor of p38MAPK and p44/42MAPK significantly reduced the TCR-mediated proliferation, but did conserve the concentration-dependent effects of ephrin-B1/B2, suggesting the interference with EphB signaling in TCR signal transduction at the upstream of MAPKs which are important for cell growth and survival. Immuno-blot analyses revealed that high concentrations of ephrin-B1/B2, but not ephrin-B3, clearly inhibited the anti-CD3 induced phosphorylation of Lck and its downstream signaling molecules such as ZAP70, c-Raf, MEK1/2, Erk, and Akt, although the phosphorylation of CD3ζ was not inhibited by high concentrations of any ephrin-Bs. These data suggest that Eph signaling upon stimulation by high concentrations of ephrin-B1/B2 may engage in negative feedback to TCR signals via Lck. The present studies demonstrate that TCR-mediated primary T cell activation may be highly governed by EphB/ephrin-B axis with a complexity determined by the combination as well as the concentration of different ephrin-Bs expressed in immunological microenvironments. EphB-involved in negative feedback of T cell activation could be a novel therapeutic target to inhibit the most proximal TCR signaling molecule, Lck. The generation of strong signaling molecule which mimics ephrin-B1/B2 would be an effective strategy to control T cell mediated immune disorders. Disclosures: No relevant conflicts of interest to declare.

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The Protein Kinase C-Responsive Inhibitory Domain of CARD11 Functions in NF-κB Activation To Regulate the Association of Multiple Signaling Cofactors That Differentially Depend on Bcl10 and MALT1 for Association

Molecular and Cellular Biology ◽

10.1128/mcb.00418-08 ◽

2008 ◽

Vol 28 (18) ◽

pp. 5668-5686 ◽

Cited By ~ 43

Author(s):

Ryan R. McCully ◽

Joel L. Pomerantz

Keyword(s):

T Cell ◽

Protein Kinase ◽

T Cell Activation ◽

Cell Activation ◽

Coiled Coil ◽

Caspase 8 ◽

Tcr Signaling ◽

Iκb Kinase ◽

Inhibitory Domain ◽

Multiple Signaling

ABSTRACT The activation of NF-κB by T-cell receptor (TCR) signaling is critical for T-cell activation during the adaptive immune response. CARD11 is a multidomain adapter that is required for TCR signaling to the IκB kinase (IKK) complex. During TCR signaling, the region in CARD11 between the coiled-coil and PDZ domains is phosphorylated by protein kinase Cθ (PKCθ) in a required step in NF-κB activation. In this report, we demonstrate that this region functions as an inhibitory domain (ID) that controls the association of CARD11 with multiple signaling cofactors, including Bcl10, TRAF6, TAK1, IKKγ, and caspase-8, through an interaction that requires both the caspase recruitment domain (CARD) and the coiled-coil domain. Consistent with the ID-mediated control of their association, we demonstrate that TRAF6 and caspase-8 associate with CARD11 in T cells in a signal-inducible manner. Using an RNA interference rescue assay, we demonstrate that the CARD, linker 1, coiled-coil, linker 3, SH3, linker 4, and GUK domains are each required for TCR signaling to NF-κB downstream of ID neutralization. Requirements for the CARD, linker 1, and coiled-coil domains in signaling are consistent with their roles in the association of CARD11 with Bcl10, TRAF6, TAK1, caspase-8, and IKKγ. Using Bcl10- and MALT1-deficient cells, we show that CARD11 can recruit signaling cofactors independently of one another in a signal-inducible manner.

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