Neuroblastoma RAS viral oncogene homolog (N-RAS) deficiency aggravates liver injury and fibrosis.

Progressive hepatic damage and fibrosis are major features of chronic liver diseases of different etiology, yet the underlying molecular mechanisms remain to be fully defined. N-RAS, a member of the RAS family of small guanine nucleotide-binding proteins also encompassing the highly homologous H-RAS and K-RAS isoforms, was previously reported to modulate cell death and renal fibrosis; however, its role in liver damage and fibrogenesis remains unknown. Here, we approached this question by using N-RAS deficient (N-RAS-/-) mice and two experimental models of liver injury and fibrosis, namely carbon tetrachloride (CCl4) intoxication and bile duct ligation (BDL). In wild-type (N-RAS+/+) mice both hepatotoxic procedures augmented N-RAS expression in the liver. Compared to N-RAS+/+ counterparts, N-RAS-/- mice subjected to either CCl4 or BDL showed exacerbated liver injury and fibrosis, which was associated with enhanced hepatic stellate cell (HSC) activation and leukocyte infiltration in the damaged liver. At the molecular level, after CCl4 or BDL, N-RAS-/- livers exhibited augmented expression of necroptotic death markers along with JNK1/2 hyperactivation. In line with this, N-RAS ablation in a human hepatocytic cell line resulted in enhanced activation of JNK and necroptosis mediators in response to cell death stimuli. Of note, loss of hepatic N-RAS expression was characteristic of chronic liver disease patients with fibrosis. Collectively, our study unveils a novel role for N-RAS as a negative controller of the progression of liver injury and fibrogenesis, by critically downregulating signaling pathways leading to hepatocyte necroptosis. Furthermore, it suggests that N-RAS may be of potential clinical value as prognostic biomarker of progressive fibrotic liver damage, or as a novel therapeutic target for the treatment of chronic liver disease.

Novel Loss-of-Function KCNA5 Variants in Pulmonary Arterial Hypertension.

Reduced expression and/or activity of Kv1.5 channels (encoded by KCNA5) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Likewise, genetic variants in KCNA5 have been found in patients with PAH, but their functional consequences and potential impact on the disease are largely unknown. Herein, this study aimed to characterize the functional consequences of seven KCNA5 variants found in a cohort of patients with PAH. Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with wild-type or mutant Kv1.5 cDNA. Flow cytometry, Western blot, and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells. KCNA5 variants (namely, Arg184Pro and Gly384Arg) found in patients with PAH resulted in a clear loss of potassium channel function as assessed by electrophysiological and molecular modeling analyses. The Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with Arg184Pro or Gly384Arg variants decreased apoptosis of human pulmonary artery smooth muscle cells compared with the wild-type cells, demonstrating that KCNA5 dysfunction in both variants affects cell viability. Thus, in addition to affecting channel activity, both variants were associated with impaired apoptosis, a crucial process linked to the disease. The estimated prevalence of dysfunctional KCNA5 variants in the PAH population analyzed was around 1%. The data indicate that some KCNA5 variants found in patients with PAH have critical consequences for channel function, supporting the idea that KCNA5 pathogenic variants may be a causative or contributing factor for PAH.

HIV and Schistosoma Co-Exposure Leads to Exacerbated Pulmonary Endothelial Remodeling and Dysfunction Associated with Altered Cytokine Landscape.

HIV and Schistosoma infections have been individually associated with pulmonary vascular disease. Co-infection with these pathogens is very common in tropical areas, with an estimate of six million people co-infected worldwide. However, the effects of HIV and Schistosoma co-exposure on the pulmonary vasculature and its impact on the development of pulmonary vascular disease are largely unknown. Here, we have approached these questions by using a non-infectious animal model based on lung embolization of Schistosoma mansoni eggs in HIV-1 transgenic (HIV) mice. Schistosome-exposed HIV mice but not wild-type (Wt) counterparts showed augmented pulmonary arterial pressure associated with markedly suppressed endothelial-dependent vasodilation, increased endothelial remodeling and vessel obliterations, formation of plexiform-like lesions and a higher degree of perivascular fibrosis. In contrast, medial wall muscularization was similarly increased in both types of mice. Moreover, HIV mice displayed an impaired immune response to parasite eggs in the lung, as suggested by decreased pulmonary leukocyte infiltration, small-sized granulomas, and augmented residual egg burden. Notably, vascular changes in co-exposed mice were associated with increased expression of proinflammatory and profibrotic cytokines, including IFN-γ and IL-17A in CD4+ and γδ T cells and IL-13 in myeloid cells. Collectively, our study shows for the first time that combined pulmonary persistence of HIV proteins and Schistosoma eggs, as it may occur in co-infected people, alters the cytokine landscape and targets the vascular endothelium for aggravated pulmonary vascular pathology. Furthermore, it provides an experimental model for the understanding of pulmonary vascular disease associated with HIV and Schistosoma co-morbidity.

CD3G or CD3D Knockdown in Mature, but Not Immature, T Lymphocytes Similarly Cripples the Human TCRαß Complex.

The human αß T-cell receptor (TCR) is composed of a variable heterodimer (TCRαß) and three invariant dimers (CD3γε, CD3δε, and ζζ/CD2472). The role of each invariant chain in the stepwise interactions among TCR chains along the assembly is still not fully understood. Despite the high sequence homology between CD3γ and CD3δ, the clinical consequences of the corresponding immunodeficiencies (ID) in humans are very different (mild and severe, respectively), and mouse models do not recapitulate findings in human ID. To try to understand such disparities, we stably knocked down (KD) CD3D or CD3G expression in the human Jurkat T-cell line and analyzed comparatively their impact on TCRαß assembly, transport, and surface expression. The results indicated that TCR ensembles were less stable and CD3ε levels were lower when CD3γ, rather than CD3δ, was scarce. However, both defective TCR ensembles were strongly retained in the ER, lacked ζζ/CD2472, and barely reached the T-cell surface (<11% of normal controls) in any of the CD3 KD cells. This is in sharp contrast to human CD3γ ID, whose mature T cells express higher levels of surface TCR (>30% vs. normal controls). CD3 KD of human T-cell progenitors followed by mouse fetal thymus organ cultures showed high plasticity in emerging immature polyclonal T lymphocytes that allowed for the expression of significant TCR levels which may then signal for survival in CD3γ, but not in CD3δ deficiency, and explain the immunological and clinical disparities of such ID cases.

Human Invariant Natural Killer T Cells Respond to Antigen-Presenting Cells Exposed to Lipids from Olea europaea Pollen.

BACKGROUND: Allergic sensitization might be influenced by the lipids present in allergens, which can be recognized by natural killer T (NKT) cells on antigen-presenting cells (APCs). The aim of this study was to analyze the effect of olive pollen lipids in human APCs, including monocytes as well as monocyte-derived macrophages (Mϕ) and dendritic cells (DCs). METHODS: Lipids were extracted from olive (Olea europaea) pollen grains. Invariant (i)NKT cells, monocytes, Mϕ, and DCs were obtained from buffy coats of healthy blood donors, and their cell phenotype was determined by flow cytometry. iNKT cytotoxicity was measured using a lactate dehydrogenase assay. Gene expression of CD1A and CD1D was performed by RT-PCR, and the production of IL-6, IL-10, IL-12, and TNF-α cytokines by monocytes, Mϕ, and DCs was measured by ELISA. RESULTS: Our results showed that monocytes and monocyte-derived Mϕ treated with olive pollen lipids strongly activate iNKT cells. We observed several phenotypic modifications in the APCs upon exposure to pollen-derived lipids. Both Mϕ and monocytes treated with olive pollen lipids showed an increase in CD1D gene expression, whereas upregulation of cell surface CD1d protein occurred only in Mϕ. Furthermore, DCs differentiated in the presence of human serum enhance their surface CD1d expression when exposed to olive pollen lipids. Finally, olive pollen lipids were able to stimulate the production of IL-6 but downregulated the production of lipopolysaccharide- induced IL-10 by Mϕ. CONCLUSIONS: Olive pollen lipids alter the phenotype of monocytes, Mϕ, and DCs, resulting in the activation of NKT cells, which have the potential to influence allergic immune responses.

TCR signal strength controls thymic differentiation of discrete proinflammatory γδ T cell subsets.

The mouse thymus produces discrete γδ T cell subsets that make either interferon-γ (IFN-γ) or interleukin 17 (IL-17), but the role of the T cell antigen receptor (TCR) in this developmental process remains controversial. Here we show that Cd3g(+/-) Cd3d(+/-) (CD3 double-haploinsufficient (CD3DH)) mice have reduced TCR expression and signaling strength on γδ T cells. CD3DH mice had normal numbers and phenotypes of αß thymocyte subsets, but impaired differentiation of fetal Vγ6(+) (but not Vγ4(+)) IL-17-producing γδ T cells and a marked depletion of IFN-γ-producing CD122(+) NK1.1(+) γδ T cells throughout ontogeny. Adult CD3DH mice showed reduced peripheral IFN-γ(+) γδ T cells and were resistant to experimental cerebral malaria. Thus, TCR signal strength within specific thymic developmental windows is a major determinant of the generation of proinflammatory γδ T cell subsets and their impact on pathophysiology.

Inmunodeficiencias congénitas del receptor de antígeno de los linfocitos T / Congenital T-cell receptor immunodeficiencies

Las inmunodeficiencias humanas del TCR son enfermedades autosómicas recesivas con baja prevalencia, caracterizadas por un defecto de expresión del TCR asociado a una linfopenia T selectiva (más leve en el caso de CD3γ, TCRα o CD247, o grave en el caso de CD3δ o CD3¿). La ausencia congénita de componentes del TCR tiene un impacto diferencial en el desarrollo y función de los linfocitos T, que depende de la cadena del TCR afectada y de la especie, siendo en algunos casos diferente en los pacientes humanos en comparación con los modelos en ratones. El estudio del inmunofenotipo mediante citometría de flujo, junto con los estudios moleculares, proporciona información esencial para el diagnóstico y el tratamiento, que continúa siendo a día de hoy el trasplante de progenitores hematopoyéticos en los casos asociados a inmunodeficiencia grave (AU)

T-cell receptor (TCR) immunodeficiencies of humans are low-prevalence autosomal recessive diseases characterized by impaired surface TCR expression and selective T lymphopenia (milder in CD3γ, TCRα or CD247 deficiency, and severe in individuals lacking CD3δ or CD3¿). The congenital absence of TCR components has a differential impact on T-cell development and function depending on the affected TCR chain and on the species, with human patients being, in some cases, rather different from mouse counterparts. The study of the immunophenotype by flow cytometry, along with molecular analyses, provides essential information for diagnosis and treatment, which is still to date the transplant of hematopoietic progenitors in severe immunodeficiency associated cases(AU)

N-ras couples antigen receptor signaling to Eomesodermin and to functional CD8+ T cell memory but not to effector differentiation.

Signals from the TCR that specifically contribute to effector versus memory CD8⁺ T cell differentiation are poorly understood. Using mice and adoptively transferred T lymphocytes lacking the small GTPase N-ras, we found that N-ras-deficient CD8⁺ T cells differentiate efficiently into antiviral primary effectors but have a severe defect in generating protective memory cells. This defect was rescued, although only partly, by rapamycin-mediated inhibition of mammalian target of rapamycin (mTOR) in vivo. The memory defect correlated with a marked impairment in vitro and in vivo of the antigen-mediated early induction of T-box transcription factor Eomesodermin (Eomes), whereas T-bet was unaffected. Besides N-ras, early Eomes induction in vitro required phosphoinositide 3-kinase (PI3K)-AKT but not extracellular signal-regulated kinase (ERK) activation, and it was largely insensitive to rapamycin. Consistent with N-ras coupling Eomes to T cell memory, retrovirally enforced expression of Eomes in N-ras-deficient CD8⁺ T cells effectively rescued their memory differentiation. Thus, our study identifies a critical role for N-ras as a TCR-proximal regulator of Eomes for early determination of the CD8⁺ T cell memory fate.

Human CD3γ, but not CD3δ, haploinsufficiency differentially impairs γδ versus αß surface TCR expression.

BACKGROUND: The T cell antigen receptors (TCR) of αß and γδ T lymphocytes are believed to assemble in a similar fashion in humans. Firstly, αß or γδ TCR chains incorporate a CD3δε dimer, then a CD3γε dimer and finally a ζζ homodimer, resulting in TCR complexes with the same CD3 dimer stoichiometry. Partial reduction in the expression of the highly homologous CD3γ and CD3δ proteins would thus be expected to have a similar impact in the assembly and surface expression of both TCR isotypes. To test this hypothesis, we compared the surface TCR expression of primary αß and γδ T cells from healthy donors carrying a single null or leaky mutation in CD3G (γ+/-) or CD3D (δ+/-, δ+/leaky) with that of normal controls. RESULTS: Although the partial reduction in the intracellular availability of CD3γ or CD3δ proteins was comparable as a consequence of the mutations, surface TCR expression measured with anti-CD3ε antibodies was significantly more decreased in γδ than in αß T lymphocytes in CD3γ+/- individuals, whereas CD3δ+/- and CD3δ+/leaky donors showed a similar decrease of surface TCR in both T cell lineages. Therefore, surface γδ TCR expression was more dependent on available CD3γ than surface αß TCR expression. CONCLUSIONS: The results support the existence of differential structural constraints in the two human TCR isotypes regarding the incorporation of CD3γε and CD3δε dimers, as revealed by their discordant surface expression behaviour when confronted with reduced amounts of CD3γ, but not of the homologous CD3δ chain. A modified version of the prevailing TCR assembly model is proposed to accommodate these new data.

H-ras and N-ras are dispensable for T-cell development and activation but critical for protective Th1 immunity.

The small guanine nucleotide binding proteins of the Ras family, including in mammals the highly homologous H-ras, N-ras, and K-ras isoforms, are rapidly activated on ligation of the T-cell antigen receptor (TCR), but whether each isoform plays specific roles in T cells is largely unknown. Here, we show, with the use of mice specifically lacking H-ras or N-ras, that these isoforms are dispensable for thymocyte development and mature T-cell activation. By contrast, CD4⁺ T cells from Ras-deficient mice exhibited markedly decreased production of the Th1 signature cytokine IFN-γ early after TCR stimulation, concomitantly with impaired induction of the Th1-specific transcription factor T-bet. Accordingly, Ras-deficient mice failed to mount a protective Th1 response in vivo against the intracellular parasite Leishmania major, although they could be rendered resistant to infection if a Th1-biased milieu was provided during parasite challenge. Collectively, our data indicate that the TCR recruits distinct Ras isoforms for signal transduction in developing and mature T cells, thus providing a mechanism for differential signaling from the same surface receptor. Furthermore, we demonstrate for the first time that H-ras and N-ras act as critical controllers of Th1 responses, mostly by transmitting TCR signals for Th1 priming of CD4⁺ T cells.

A natural anti-T-cell receptor monoclonal antibody protects against experimental autoimmune encephalomyelitis.

The therapeutic potential of natural anti-T-cell receptor (TCR) antibodies is largely unknown. We investigated whether passive administration of C1-19, a novel natural anti-TCRVß8 monoclonal antibody, could interfere with the development of EAE. Treatment with C1-19 prevented myelin basic protein (MBP)-induced EAE in Vß8-sufficient B10.PL but not in Vß8-deficient SJL mice. Furthermore, C1-19 reduced disease severity when administrated shortly after disease onset. These protective effects of C1-19 correlated with a Th2 bias of the cytokine response, in the absence of T-cell deletion or anergy. Together, these findings indicate that natural anti-TCR antibodies could function as therapeutic tools in autoimmune inflammatory diseases.

Cancer abolishes the tissue type-specific differences in the phenotype of energetic metabolism.

Nowadays, cellular bioenergetics has become a central issue of investigation in cancer biology. Recently, the metabolic activity of the cancer cell has been shown to correlate with a proteomic index that informs of the relative mitochondrial activity of the cell. Within this new field of investigation, we report herein the production and characterization of high-affinity monoclonal antibodies against proteins of the "bioenergetic signature" of the cell. The use of recombinant proteins and antibodies against the mitochondrial beta-F1-ATPase and Hsp60 proteins and the enzymes of the glycolytic pathway glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase M2 in quantitative assays provide, for the first time, the actual amount of these proteins in normal and tumor surgical specimens of breast, lung, and esophagus. The application of this methodology affords a straightforward proteomic signature that quantifies the variable energetic demand of human tissues. Furthermore, the results show an unanticipated finding: tumors from different tissues and/or histological types have the same proteomic signature of energetic metabolism. Therefore, the results indicate that cancer abolishes the tissue-specific differences in the bioenergetic phenotype of mitochondria. Overall, the results support that energetic metabolism represents an additional hallmark of the phenotype of the cancer cell and a promising target for the treatment of diverse neoplasias.

Differential antibody binding to the surface alphabetaTCR.CD3 complex of CD4+ and CD8+ T lymphocytes is conserved in mammals and associated with differential glycosylation.

We have previously shown that the surface alphabeta T cell antigen receptor (TCR).CD3 complex borne by human CD4(+) and CD8(+) T lymphocytes can be distinguished using mAbs. Using two unrelated sets of antibodies, we have now extended this finding to the surface alphabetaTCR.CD3 of seven additional mammalian species (six non-human primates and the mouse). We have also produced data supporting that differential glycosylation of the two main T cell subsets is involved in the observed TCR.CD3 antibody-binding differences in humans. First, we show differential lectin binding to human CD4(+) versus CD8(+) T lymphocytes, particularly with galectin 7. Second, we show that certain lectins can compete differentially with CD3 mAb binding to human primary CD4(+) and CD8(+) T lymphocytes. Third, N-glycan disruption using swainsonine was shown to increase mAb binding to the alphabetaTCR.CD3. We conclude that the differential antibody binding to the surface alphabetaTCR.CD3 complex of primary CD4(+) and CD8(+) T lymphocytes is phylogenetically conserved and associated with differential glycosylation. The differences may be exploited for therapeutic purposes, such as T cell lineage-specific immunosuppression of graft rejection. Also, the impact of glycosylation on CD3 antibody binding requires a cautious interpretation of CD3 expression levels and T cell numbers in clinical diagnosis.

Different composition of the human and the mouse gammadelta T cell receptor explains different phenotypes of CD3gamma and CD3delta immunodeficiencies.

The gammadelta T cell receptor for antigen (TCR) comprises the clonotypic TCRgammadelta, the CD3 (CD3gammaepsilon and/or CD3deltaepsilon), and the zetazeta dimers. gammadelta T cells do not develop in CD3gamma-deficient mice, whereas human patients lacking CD3gamma have abundant peripheral blood gammadelta T cells expressing high gammadelta TCR levels. In an attempt to identify the molecular basis for these discordant phenotypes, we determined the stoichiometries of mouse and human gammadelta TCRs using blue native polyacrylamide gel electrophoresis and anti-TCR-specific antibodies. The gammadelta TCR isolated in digitonin from primary and cultured human gammadelta T cells includes CD3delta, with a TCRgammadeltaCD3epsilon(2)deltagammazeta(2) stoichiometry. In CD3gamma-deficient patients, this may allow substitution of CD3gamma by the CD3delta chain and thereby support gammadelta T cell development. In contrast, the mouse gammadelta TCR does not incorporate CD3delta and has a TCRgammadeltaCD3epsilon(2)gamma(2)zeta(2) stoichiometry. CD3gamma-deficient mice exhibit a block in gammadelta T cell development. A human, but not a mouse, CD3delta transgene rescues gammadelta T cell development in mice lacking both mouse CD3delta and CD3gamma chains. This suggests important structural and/or functional differences between human and mouse CD3delta chains during gammadelta T cell development. Collectively, our results indicate that the different gammadelta T cell phenotypes between CD3gamma-deficient humans and mice can be explained by differences in their gammadelta TCR composition.

Overlapping functions of human CD3delta and mouse CD3gamma in alphabeta T-cell development revealed in a humanized CD3gamma-mouse.

Humans lacking the CD3gamma subunit of the pre-TCR and TCR complexes exhibit a mild alphabeta T lymphopenia, but have normal T cells. By contrast, CD3gamma-deficient mice are almost devoid of mature alphabeta T cells due to an early block of intrathymic development at the CD4(-)CD8(-) double-negative (DN) stage. This suggests that in humans but not in mice, the highly related CD3delta chain replaces CD3gamma during alphabeta T-cell development. To determine whether human CD3delta (hCD3delta) functions in a similar manner in the mouse in the absence of CD3gamma, we introduced an hCD3delta transgene in mice that were deficient for both CD3delta and CD3gamma, in which thymocyte development is completely arrested at the DN stage. Expression of hCD3delta efficiently supported pre-TCR-mediated progression from the DN to the CD4(+)CD8(+) double-positive (DP) stage. However, alphabetaTCR-mediated positive and negative thymocyte selection was less efficient than in wild-type mice, which correlated with a marked attenuation of TCR-mediated signaling. Of note, murine CD3gamma-deficient TCR complexes that had incorporated hCD3delta displayed abnormalities in structural stability resembling those of T cells from CD3gamma-deficient humans. Taken together, these data demonstrate that CD3delta and CD3gamma play a different role in humans and mice in pre-TCR and TCR function during alphabeta T-cell development.