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The clinical response to immune checkpoint blockade (ICB) correlates with tumor-infiltrating cytolytic T lymphocytes (CTLs) prior to treatment. However, many of these inflamed tumors resist ICB through unknown mechanisms. We show that tumors with transcription elongation deficiencies (TEdef+), which we previously reported as being resistant to ICB in mouse models and the clinic, have high baseline CTLs. We show that high baseline CTLs in TEdef+ tumors result from aberrant activation of the nucleic acid sensing-TBK1-CCL5/CXCL9 signaling cascade, which results in an immunosuppressive microenvironment with elevated regulatory T cells and exhausted CTLs. ICB therapy of TEdef+ tumors fail to increase CTL infiltration and suppress tumor growth in both experimental and clinical settings, suggesting that TEdef+, along with surrogate markers of tumor immunogenicity such as tumor mutational burden and CTLs, should be considered in the decision process for patient immunotherapy indication. [Display omitted] • Aberrant NAS in TEdef+ tumors triggers TBK1-mediated release of pro-inflammatory chemokine • CD8+ T cell infiltration is increased in ICB-resistant TEdef+ tumors • TEdef+ tumors attract regulatory T cells and promote CD8+ T cell exhaustion • Inert inflammation in TEdef+ human tumors is associated with ICB therapy resistance Modur et al. show that tumors with transcription elongation deficiencies (TEdef+) resist ICB despite having a high baseline of tumor-infiltrating cytolytic T lymphocytes (CTLs), resulting from aberrant activation of TBK1-induced inert inflammation with elevated regulatory T cells and exhausted CTLs, making TEdef+ important in patient immunotherapy indication. [ABSTRACT FROM AUTHOR]

The multidomain protein Trio regulates among others neuronal outgrowth and axonal guidance in vertebrates and invertebrates. Trio contains two Dbl-homology/pleck- strin homology (DH/PH) tandem domains that activate several RhoGTPases. Here, we present the x-ray structure of the N-terminal DH/PH, hereafter TrioN, refined to 1.7-A resolution. We show that the relative orientations of the DH and PH domains of TrioN and free Dbs are similar. However, this relative orientation is dissimilar to Dbs in the Dbs/Cdc42 structure. In vitro nucleotide exchange ex- periments catalyzed by TrioN show that RhoG is ∼3× more efficiently exchanged than Rac and support the conclusion that RhoG is likely the downstream target of TrioN. Residues 54 and 69, which are not conserved between the two GTPases, are responsible for this specificity. Dot-blot assay reveals that the TrioN-PH domain does not detectably bind phosphatidylinositol 3,4-bisphosphate, PtdIns(3,4)P2, or other phospholipids. This finding is supported by our three-dimensional structure and affinity binding experiments. Interestingly, the presence of RhoG but not Rac or a C-terminal-truncated RhoG mutant allows TrioN to bind PtdIns(3,4)P2 with a micromolar affinity constant. We conclude the variable C-terminal basic tail of RhoG specifically assists the recruitment of the TrioN-PH domain to specific membrane-bound phospholipids. Our data suggest a role for the phosphoinositide 3-kinase, PI 3-kinase, in modulating the Trio/RhoG signaling pathway. [ABSTRACT FROM AUTHOR]

A recent study shows that the protumorigenic guanine nucleotide exchange factor (GEF) Vav1 functions as a tumor suppressor in T cell acute lymphoblastic leukemia (T-ALL) through its ability to complex with the Cbl-b ubiquitin ligase and the intracellular domain of Notch1 (ICN1) and to promote ICN1 degradation. Vav1can act as a double-edged sword in tumorigenesis. [ABSTRACT FROM AUTHOR]

Background Mitochondrial metabolism is known to be important for T-cell activation. However, its involvement in effector T-cell differentiation has just begun to gain attention. Importantly, how metabolic pathways are integrated with T-cell activation and effector cell differentiation and function remains largely unknown. Objective We sought to test our hypothesis that RhoA GTPase orchestrates glycolysis for T H 2 cell differentiation and T H 2-mediated allergic airway inflammation. Methods Conditional RhoA-deficient mice were generated by crossing RhoA flox/flox mice with CD2-Cre transgenic mice. Effects of RhoA on T H 2 differentiation were evaluated based on in vitro T H 2-polarized culture conditions and in vivo in ovalbumin-induced allergic airway inflammation. Cytokine levels were measured by using intracellular staining and ELISA. T-cell metabolism was measured by using the Seahorse XF24 Analyzer and flow cytometry. Results Disruption of RhoA inhibited T-cell activation and T H 2 differentiation in vitro and prevented the development of allergic airway inflammation in vivo , with no effect on T H 1 cells. RhoA deficiency in activated T cells led to multiple defects in metabolic pathways, such as glycolysis and oxidative phosphorylation. Importantly, RhoA couples glycolysis to T H 2 cell differentiation and allergic airway inflammation through regulating IL-4 receptor mRNA expression and T H 2-specific signaling events. Finally, inhibition of Rho-associated protein kinase, an immediate downstream effector of RhoA, blocked T H 2 differentiation and allergic airway inflammation. Conclusion RhoA is a key component of the signaling cascades leading to T H 2 differentiation and allergic airway inflammation at least in part through control of T-cell metabolism and the Rho-associated protein kinase pathway. [ABSTRACT FROM AUTHOR]

Background In our previous study, Activin B induced actin stress fiber formation and cell migration in Bone marrow-derived mesenchymal stem cells (BMSCs) in vitro. However, the underlying molecular mechanisms are not well studied. RhoA is recognized to play a critical role in the regulation of actomyosin cytoskeletal organization and cell migration. Methods Pull-down assay was performed to investigate the activity of RhoA. The dominant-negative mutants of RhoA (RhoA(N19)) was used to determine whether RhoA has a role in Activin B-induced cytoskeleton organization and cell migration in BMSCs. Cytoskeleton organization was examined by fluorescence Rhodamine-phalloidin staining, and cell migration by transwell and cell scratching assay. Western blot was carried out to investigate downstream signaling cascade of RhoA. Inhibitor and siRNAs were used to detect the role of downstream signaling in stress fiber formation and/or cell migration. Results RhoA was activated by Activin B in BMSCs. RhoA(N19) blocked Activin B-induced stress fiber formation and cell migration. ROCK inhibitor blocked Activin B-induced stress fiber formation but enhanced BMSCs migration. Activin B induced phosphorylation of LIMK2 and Cofilin, which was abolished by ROCK inhibition. Both of siRNA LIMK2 and siRNA Cofilin inhibited Activin B-induced stress fiber formation. Conclusions RhoA regulates Activin B-induced stress fiber formation and migration of BMSCs. A RhoA-ROCK-LIMK2-Cofilin signaling node exists and regulates actin stress fiber formation. RhoA regulates Activin B-induced cell migration independent of ROCK. General significance Better understanding of the molecular mechanisms of BMSCs migration will help optimize therapeutic strategy to target BMSCs at injured tissues. [ABSTRACT FROM AUTHOR]

Extracellular matrix (ECM) actively participates in normal cell regulation and in the process of tumor progression. The Rho GTPase Cdc42 has been shown to regulate cell-ECM interaction in conventional two-dimensional culture conditions by using dominant mutants of Cdc42 in immortalized cell lines that may introduce nonspecific effects. Here, we employ three-dimensional culture systems for conditional gene targeted primary mouse embryonic fibroblasts that better simulate the reciprocal and adaptive interactions between cells and surrounding matrix to define the role of Cdc42 signaling pathways in ECM organization. Cdc42 deficiency leads to a defect in global cell-matrix interactions reflected by a decrease in collagen gel contraction. The defect is associated with an altered cell-matrix interaction that is evident by morphologic changes and reduced focal adhesion complex formation. The matrix defect is also associated with a reduction in synthesis and activation of matrix metalloproteinase 9 (MMP9) and altered fibronectin deposition patterning. A Cdc42 mutant rescue experiment found that downstream of Cdc42, p21-activated kinase (PAK), but not Par6 or WASP, may be involved in regulating collagen gel contraction and fibronectin organization. Thus, in addition to the previously implicated roles in intracellular regulation of actin organization, proliferation, and vesicle trafficking, Cdc42 is essential in ECM remodeling in three dimensions. [ABSTRACT FROM AUTHOR]

Methamphetamine (METH) is a highly addictive psychostimulant that strongly activates dopamine receptor signaling in the nucleus accumbens (NAc). However, how dopamine D 1 and D 2 receptors (D 1 Rs and D 2 Rs, respectively) as well as downstream signaling pathways, such as those involving Rac1 and Cdc42, modulate METH-induced behavioral and structural plasticity is largely unknown. Using NAc conditional D 1 R and D 2 R deletion mice, Rac1 and Cdc42 mutant viruses, and a series of behavioral and morphological methods, we assessed the effects of D 1 Rs and D 2 Rs on Rac1 and Cdc42 in modulating METH-induced behavioral and structural plasticity in the NAc. D 1 Rs and D 2 Rs in the NAc consistently regulated METH-induced conditioned place preference, locomotor activation, and dendritic and spine remodeling of medium spiny neurons but differentially regulated METH withdrawal–induced spatial learning and memory impairment and anxiety. Interestingly, Rac1 and Cdc42 signaling were oppositely modulated by METH, and suppression of Rac1 signaling and activation of Cdc42 signaling were crucial to METH-induced conditioned place preference and structural plasticity but not to locomotor activation. D 1 Rs activated Rac1 and Cdc42 signaling, while D 2 Rs inhibited Rac1 signaling but activated Cdc42 signaling to mediate METH-induced conditioned place preference and structural plasticity but not locomotor activation. In addition, NAc D 1 R deletion aggravated METH withdrawal–induced spatial learning and memory impairment by suppressing Rac1 signaling but not Cdc42 signaling, while NAc D 2 R deletion aggravated METH withdrawal–induced anxiety without affecting Rac1 or Cdc42 signaling. D 1 Rs and D 2 Rs differentially regulate Rac1 and Cdc42 signaling to modulate METH-induced behavioral plasticity and the structural remodeling of medium spiny neurons in the NAc. [ABSTRACT FROM AUTHOR]

Asthma is a chronic airway inflammation in which Th2 and Th17 cells play critical roles in its pathogenesis. We have reported that atypical protein kinase (PKC) λ/ι is a new regulator for Th2 differentiation and function. However, the role of PKCλ/ι for Th17 cells remains elusive. In this study, we explored the effect of PKCλ/ι on Th17 cells in the context of ex vivo cell culture systems and an in vivo murine model of allergic airway inflammation with the use of activated T cell-specific conditional PKCλ/ι-deficient mice. Our findings indicate that PKCλ/ι regulates Th17 cells. The secretion of Th17 effector cytokines, including IL-17, IL-21 and IL-22, were inhibited from PKCλ/ι-deficient T cells under non-skewing or Th17-skewing culture conditions. Moreover, the impaired Th17 differentiation and function by the PKCλ/ι-deficiency was associated with the downregulation of Stat3 and Rorγt, key Th17 transcription factors. We developed a model of Th17 and neutrophil-involved allergic airway inflammation by intratracheal inoculation of house dust mites. PKCλ/ι-deficiency significantly inhibited airway inflammations. The infiltrating cells in the lungs and bronchoalveolar lavage fluids were significantly reduced in conditional PKCλ/ι-deficient mice. Th17 effector cytokines were reduced in the bronchoalveolar lavage fluids and lungs at protein and mRNA levels. Thus, PKCλ/ι emerges as a critical regulator of Th17 differentiation and allergic airway hyperresponsiveness. [ABSTRACT FROM AUTHOR]

Abstract: Repeated exposure to cocaine can induce persistent alterations in the brain''s reward system, including increases in the number of dendrites and spine density on medium-sized spiny neurons (MSNs) in the nucleus accumbens (NAc). The structural remodeling of dendrites and spines in the NAc is thought to play a critical role in cocaine addiction. MSNs in the NAc can be classified by expression of either D1 or D2 dopamine receptors, which are localized to the direct and indirect pathway, respectively. It is unknown whether the dendritic changes induced by repeated cocaine treatment occur in MSNs of the direct or indirect pathway. Because the traditional Golgi-Cox impregnation of neurons precludes identifying particular subpopulations of MSNs, we performed dendritic morphology analysis after biocytin-labeling and Golgi-Cox impregnation. We found that the biocytin staining MSNs showed higher dendritic spine density and higher number of dendrites than that in Golgi impregnation group. In addition, we found that the increasing spine density induced by repeated cocaine treatment in female mice was higher than that in male mice. Next we used biocytin staining and dynorphin/D2 receptor colocalization to determine which cell type(s) displayed dendritic changes after repeated cocaine treatment. We found that cocaine-induced changes in dendritic parameters occurred in MSNs of both the direct (D1-expressing) and indirect (D2-expressing) pathways. [Copyright &y& Elsevier]

Abstract: Activation of RhoA prevents NGF-induced outgrowth and causes retraction of neurites in neuronal cells, including PC12 cells. Despite its inhibitory effect on neurite outgrowth, NGF activates GTP loading of and effector binding to RhoA, setting up an apparent contradiction. According to the molecular switch hypothesis of GTPase function GTP-loading of RhoA should be sufficient to activate its effectors uniformly. However, when monitoring NGF-induced binding of GTP-RhoA to multiple targets, we noted differential interactions with its effectors. We found that NGF elicits a protein kinase A-mediated phosphorylation of RhoA on serine188, which renders it unable to bind to Rho-associated kinase (ROK), whereas it retains the ability to interact with other RhoA targets including rhotekin, mDia-1 and PKN. We show in vitro and in vivo that phosphorylation of serine188 represents an additional switch, capable of directing signals among effector pathways. In the context of PC12 cell differentiation, NGF-induced phosphorylation of RhoA on serine188 prevents it from interacting with ROK, which would otherwise block neurite outgrowth. Transfection of RhoAS188A mutant into PC12 cells prevents NGF-induced neurite outgrowth, just like constitutively activated RhoA14V does, indicating the requirement of this phosphorylation site. Replacement of serine188 with the phosphomimetic glutamate residue in RhoAV14/S188E selectively impairs interaction with ROK and when transfected into PC12 cells restores NGF-induced neurite outgrowth. Therefore, phosphorylation of serine188 may serve as a novel secondary switch of RhoA capable of overriding GTP-binding-elicited effector activation to a subset of targets such as ROK, which interact with the C-terminus of RhoA. [Copyright &y& Elsevier]