The induction of SHP-1 degradation by TAOK3 ensures the responsiveness of T cells to TCR stimulation.

Thousand-and-one-amino acid kinase 3 (TAOK3) is a serine and threonine kinase that belongs to the STE-20 family of kinases. Its absence reduces T cell receptor (TCR) signaling and increases the interaction of the tyrosine phosphatase SHP-1, a major negative regulator of proximal TCR signaling, with the kinase LCK, a component of the core TCR signaling complex. Here, we used mouse models and human cell lines to investigate the mechanism by which TAOK3 limits the interaction of SHP-1 with LCK. The loss of TAOK3 decreased the survival of naïve CD4+ T cells by dampening the transmission of tonic and ligand-dependent TCR signaling. In mouse T cells, Taok3 promoted the secretion of interleukin-2 (IL-2) in response to TCR activation in a manner that depended on Taok3 gene dosage and on Taok3 kinase activity. TCR desensitization in Taok3-/- T cells was caused by an increased abundance of Shp-1, and pharmacological inhibition of Shp-1 rescued the activation potential of these T cells. TAOK3 phosphorylated threonine-394 in the phosphatase domain of SHP-1, which promoted its ubiquitylation and proteasomal degradation. The loss of TAOK3 had no effect on the abundance of SHP-2, which lacks a residue corresponding to SHP-1 threonine-394. Modulation of SHP-1 abundance by TAOK3 thus serves as a rheostat for TCR signaling and determines the activation threshold of T lymphocytes.

A novel, ataxic mouse model of ataxia telangiectasia caused by a clinically relevant nonsense mutation.

Ataxia Telangiectasia (A-T) and Ataxia with Ocular Apraxia Type 1 (AOA1) are devastating neurological disorders caused by null mutations in the genome stability genes, A-T mutated (ATM) and Aprataxin (APTX), respectively. Our mechanistic understanding and therapeutic repertoire for treating these disorders are severely lacking, in large part due to the failure of prior animal models with similar null mutations to recapitulate the characteristic loss of motor coordination (i.e., ataxia) and associated cerebellar defects. By increasing genotoxic stress through the insertion of null mutations in both the Atm (nonsense) and Aptx (knockout) genes in the same animal, we have generated a novel mouse model that for the first time develops a progressively severe ataxic phenotype associated with atrophy of the cerebellar molecular layer. We find biophysical properties of cerebellar Purkinje neurons (PNs) are significantly perturbed (e.g., reduced membrane capacitance, lower action potential [AP] thresholds, etc.), while properties of synaptic inputs remain largely unchanged. These perturbations significantly alter PN neural activity, including a progressive reduction in spontaneous AP firing frequency that correlates with both cerebellar atrophy and ataxia over the animal's first year of life. Double mutant mice also exhibit a high predisposition to developing cancer (thymomas) and immune abnormalities (impaired early thymocyte development and T-cell maturation), symptoms characteristic of A-T. Finally, by inserting a clinically relevant nonsense-type null mutation in Atm, we demonstrate that Small Molecule Read-Through (SMRT) compounds can restore ATM production, indicating their potential as a future A-T therapeutic.

Intranasal Application of Lactococcus lactis W136 Is Safe in Chronic Rhinosinusitis Patients With Previous Sinus Surgery.

Objective: Modulation of the dysbiotic gut microbiome with "healthy" bacteria via a stool transplant or supplementation is increasingly practiced, however this approach has not been explored in the nasal passages. We wished to verify whether Lactococcus lactis W136 (L. lactis W136) bacteria could be safely applied via irrigation to the nasal and sinus passages in individuals with chronic rhinosinusitis (CRS) with previous undergone endoscopic sinus surgery, and whether this was accompanied by bacterial community flora modification. Study Design: Prospective open-label pilot trial of safety and feasibility. Setting: Academic tertiary hospital center. Subjects and Methods: Twenty-four patients with CRS refractory to previous medical and surgical therapy received a 14-day course of BID sinus irrigations containing 1.2 × 109 CFU live L. lactis W136. Patients were monitored for safety using questionnaire, sinus endoscopy, otoscopy, UPSIT-40 smell testing, and endoscopically-obtained conventional sinus culture and a swab for 16S microbiome profiling. Results: All 24 patients receiving at least one treatment successfully completed treatment. L. lactis W136 probiotic treatment was safe, with no major adverse events or new infections. Treatment was associated with improvement in sinus symptoms, QOL, and mucosal scores, which remained improved during the subsequent 14-day observation period. Microbiome changes associated with treatment were limited to an increase of the pathobiont Dolosigranulum pigrum, a bacteria identified as potentially beneficial in the upper airways. Subgroup analysis suggested differences in microbiomes and responses for CRSsNP and CRSwNP phenotypes, but these did not attain significance. Conclusion: Intranasal irrigation of live L. lactis W136 bacteria to patients with refractory chronic rhinosinusitis was safe, and was associated with effects on symptoms, mucosal aspect and microbiome composition. Intranasal bacteria may thus find a role as a treatment strategy for CRS. Clinical Trials Registration: www.ClinicalTrials.gov. identifier: NCT04048174.

TAOK3, a Regulator of LCK-SHP-1 Crosstalk during TCR Signaling.

Signaling from the T cell receptor for antigen turns on the physiological response of a T cell. The canonical TCR signaling pathway relies on early activation of the Src kinase LCK. This step initiates a cascade of events that lead not only to the phenotypic changes that characterize effector T cells but also to the activation of negative regulatory mechanisms that stop early TCR signaling. These mechanisms ensure qualitative and quantitative fine-tuning of T cell activation. The tyrosine phosphatase SHP-1 is a key player in the downregulation of LCK activation. In this review, we focus on the crosstalk between LCK and SHP-1 and, based on recent data, we introduce the putative kinase TAOK3 as an important regulator of this crosstalk. Given the widespread expression of TAOK3 and SHP-1, we propose that the function of TAOK3 extends beyond T cells and may be fundamental in the regulation of early signaling from receptors that utilize Src kinases.

TAOK3 Regulates Canonical TCR Signaling by Preventing Early SHP-1-Mediated Inactivation of LCK.

Activation of LCK is required for canonical TCR signaling leading to T cell responses. LCK activation also initiates a negative feedback loop mediated by the phosphatase SHP-1 that turns off TCR signaling. In this article, we report that the thousand-and-one amino acid kinase 3 (TAOK3) is a key regulator of this feedback. TAOK3 is a serine/threonine kinase expressed in many different cell types including T cells. TAOK3-deficient human T cells had impaired LCK-dependent TCR signaling resulting in a defect in IL-2 response to canonical TCR signaling but not to bacterial superantigens, which use an LCK-independent pathway. This impairment was associated with enhanced interaction of LCK with SHP-1 after TCR engagement and rapid termination of TCR signals, a defect corrected by TAOK3 reconstitution. Thus, TAOK3 is a positive regulator of TCR signaling by preventing premature SHP-1-mediated inactivation of LCK. This mechanism may also regulate signaling by other Src family kinase-dependent receptors.

Notch activation is required for downregulation of HoxA3-dependent endothelial cell phenotype during blood formation.

Hemogenic endothelium (HE) undergoes endothelial-to-hematopoietic transition (EHT) to generate blood, a process that requires progressive down-regulation of endothelial genes and induction of hematopoietic ones. Previously, we have shown that the transcription factor HoxA3 prevents blood formation by inhibiting Runx1 expression, maintaining endothelial gene expression and thus blocking EHT. In the present study, we show that HoxA3 also prevents blood formation by inhibiting Notch pathway. HoxA3 induced upregulation of Jag1 ligand in endothelial cells, which led to cis-inhibition of the Notch pathway, rendering the HE nonresponsive to Notch signals. While Notch activation alone was insufficient to promote blood formation in the presence of HoxA3, activation of Notch or downregulation of Jag1 resulted in a loss of the endothelial phenotype which is a prerequisite for EHT. Taken together, these results demonstrate that Notch pathway activation is necessary to downregulate endothelial markers during EHT.

Suppression of CYP1 members of the AHR response by pathogen-associated molecular patterns.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that triggers a broad response, which includes the regulation of proinflammatory cytokine production by monocytes and macrophages. AHR is negatively regulated by a set of genes that it transcriptionally activates, including the AHR repressor (Ahrr) and the cytochrome P450 1 (Cyp1) family, which are critical for preventing exacerbated AHR activity. An imbalance in these regulatory mechanisms has been shown to cause severe defects in lymphoid cells. Therefore, we wanted to assess how AHR activation is regulated in monocytes and macrophages in the context of innate immune responses induced by pathogen-associated molecular patterns (PAMPs). We found that concomitant stimulation of primary human monocytes with PAMPs and the AHR agonist 6-formylindolo(3,2-b)carbazole (FICZ) led to a selective dose-dependent inhibition of Cyp1 family members induction. Two other AHR-dependent genes [Ahrr and NADPH quinone dehydrogenase 1 (Nqo1)] were not affected under these conditions, suggesting a split in the AHR regulation by PAMPs. This down-regulation of Cyp1 family members did not require de novo protein production nor signaling through p38, ERK, or PI3K-Akt-mammalian target of rapamycin (mTOR) pathways. Furthermore, such a split regulation of the AHR response was more apparent in GM-CSF-derived macrophages, a finding corroborated at the functional level by decreased CYP1 activity and decreased proinflammatory cytokine production in response to FICZ and LPS. Collectively, our findings identify a role for pattern recognition receptor (PRR) signaling in regulating the AHR response through selective down-regulation of Cyp1 expression in human monocytes and macrophages.

Stomatin-like protein 2 deficiency results in impaired mitochondrial translation.

Mitochondria translate the RNAs for 13 core polypeptides of respiratory chain and ATP synthase complexes that are essential for the assembly and function of these complexes. This process occurs in close proximity to the mitochondrial inner membrane. However, the mechanisms and molecular machinery involved in mitochondrial translation are not fully understood, and defects in this process can result in severe diseases. Stomatin-like protein (SLP)-2 is a mainly mitochondrial protein that forms cardiolipin- and prohibitin-enriched microdomains in the mitochondrial inner membrane that are important for the formation of respiratory supercomplexes and their function. Given this regulatory role of SLP-2 in processes closely associated with the mitochondrial inner membrane, we hypothesized that the function of SLP-2 would have an impact on mitochondrial translation. 35S-Methionine/cysteine pulse labeling of resting or activated T cells from T cell-specific Slp-2 knockout mice showed a significant impairment in the production of several mitochondrial DNA-encoded polypeptides following T cell activation, including Cytb, COXI, COXII, COXIII, and ATP6. Measurement of mitochondrial DNA stability and mitochondrial transcription revealed that this impairment was at the post-transcriptional level. Examination of mitochondrial ribosome assembly showed that SLP-2 migrated in sucrose-density gradients similarly to the large ribosomal subunit but that its deletion at the genetic level did not affect mitochondrial ribosome assembly. Functionally, the impairment in mitochondrial translation correlated with decreased interleukin-2 production in activated T cells. Altogether, these data show that SLP-2 acts as a general regulator of mitochondrial translation.

Staphylococcus aureus Downregulates IP-10 Production and Prevents Th1 Cell Recruitment.

Staphylococcal superantigens cause toxic shock syndrome, which is characterized by massive T cell activation and a predominant Th1 profile of cytokine production. However, superantigen-producing Staphylococcus aureus strains are often part of the human nasal microbiome, and this carrier state has often been associated with some type 2 immune responses such as chronic sinusitis with polyps and atopic dermatitis. We have previously reported that the S. aureus cell wall downregulates the human T cell response to superantigens through a TLR2-dependent, IL-10-mediated mechanism. In this study, we show that S. aureus also regulates the profile of superantigen-induced T cell recruitment. The staphylococcal superantigen SEE induced the production of Th1 cell-recruiting chemokines, including IP-10, through an IFN-γ-dependent mechanism. Such an induction was suppressed by the concomitant presence of S. aureus The downregulation of IP-10 by S. aureus was mediated by components of its cell wall, but was not due to peptidoglycan-induced IL-10 production. Instead, S. aureus triggered activation of MAPKs p38 and ERK, as well as inhibition of STAT1 signaling in monocytes, altogether contributing to the downregulation of IP-10 and other Th1 cell-recruiting chemokines (e.g., CXCL9 and CXCL11). These effects translated into inhibition of superantigen-induced Th1 cell recruitment. Taken together, our data may explain why colonization of superantigen-producing S. aureus can induce, under some circumstances, mucosal type 2 immune responses.

Topical probiotics as a therapeutic alternative for chronic rhinosinusitis: A preclinical proof of concept.

INTRODUCTION: Patients with chronic rhinosinusitis (CRS) have been shown to manifest a high inflammatory phenotype, with a sinus microbiome deficient in gram-positive bacteria. Gram-positive bacteria are capable of downregulating proinflammatory host responses via an interleukin (IL) 10 mediated response and may represent a potential therapeutic alternative for CRS. We wanted to (i) immunoprofile the IL-10 induction capacity of two gram-positive probiotic strains and (ii) verify the tolerance of these strains by the sinus epithelium. METHODS: A peripheral blood mononuclear cell (PBMC) challenge model was used to document probiotic induction of IL-10 and tumor necrosis factor (TNF) alpha responses at various bacterial dilutions. Epithelial cell tolerance was demonstrated by using a primary epithelial cell model derived from patient biopsy specimens (six patients total [three with CRS and three controls]). After an incubation period with either a live or a heat-killed probiotic strain, cell viability was assessed by using light microscopy. RESULTS: Both probiotic strains induced high IL-10 secretion in PBMCs, with differing profiles of TNF alpha production. Microscopic evaluation after probiotic incubation demonstrated intact cell viability for all cell cultures. CONCLUSION: We identified well-tolerated, nonpathogenic, "generally recognized as safe" status gram-positive probiotics with anti-inflammatory properties. Topical probiotics represented a potential novel topical therapeutic strategy for CRS relevant for further clinical evaluation.

Senataxin suppresses the antiviral transcriptional response and controls viral biogenesis.

The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders.

Uncoupling of pro- and anti-inflammatory properties of Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacterium that is carried by a quarter of the healthy human population and that can cause severe infections. This pathobiosis has been linked to a balance between Toll-like receptor 2 (TLR2)-dependent pro- and anti-inflammatory responses. The relationship between these two types of responses is unknown. Analysis of 16 nasal isolates of S. aureus showed heterogeneity in their capacity to induce pro- and anti-inflammatory responses, suggesting that these two responses are independent of each other. Uncoupling of these responses was corroborated by selective signaling through phosphoinositol 3-kinase (PI3K)-Akt-mTOR and extracellular signal-regulated kinase (ERK) for the anti-inflammatory response and through p38 for the proinflammatory response. Uncoupling was also observed at the level of phagocytosis and phagosomal processing of S. aureus, which were required solely for the proinflammatory response. Importantly, the anti-inflammatory properties of an S. aureus isolate correlated with its ability to modulate T cell immunity. Our results suggest the presence of anti-inflammatory TLR2 ligands in the staphylococcal cell wall, whose identification may provide templates for novel immunomodulatory drugs.

The role of IL-10 in microbiome-associated immune modulation and disease tolerance.

Current research on the microbiome of humans and other species is revealing a fundamental role for the interaction between the microbiota and the immune system in determining the health status of the host. In these studies, the cytokine interleukin-10 (IL-10) is emerging as an important player. We present here an overview of the developments in the field emphasizing how the microbiota composition and its interplay with immune cells affect the health of the host through changes in IL-10 production. In addition, we explore the function that IL-10-producing immune cells may have on the qualitative and quantitative changes in the microbiota and thus influence the balance between microbial commensalism and pathogenicity. In the last section of this review, we present a summary of the strategies that target IL-10 for therapeutic purposes using probiotics, purified proteins or biologicals.

Fibroblast growth factor-23 and calcium phosphate product in young chronic kidney disease patients: a cross-sectional study.

BACKGROUND: Fibroblast growth factor-23 (FGF-23), a novel marker of bone disease in chronic kidney disease (CKD) has been shown to correlate with vascular calcifications. We aimed to describe the effect of the calcium phosphate product (Ca*P) on FGF-23 concentrations in children and young adults without confounding cardiovascular disease. METHODS: Pediatric and young adult patients with CKD stages I-V were recruited in this cross sectional study to measure FGF-23, cystatin C, vitamin D-metabolites and other serum markers of bone metabolism. FGF-23 levels were determined with an enzyme-linked immunosorbent assay. The association between FGF-23 and (Ca*P) was assessed using non-parametric methods. Patients were divided into two age groups, less than 13 years of age and greater than 13 years of age. RESULTS: This cross-sectional study measured serum FGF-23, in 81 patients (42 females, 51.9%) at London Health Sciences Centre, aged 2 to 25 years, with various stages of CKD (Cystatin C estimated glomerular filtration rate, eGFR=10.7-213.0 ml/min). For the whole entire group of patients, FGF-23 levels were found to correlate significantly with age (Spearman r= 0.26, p=0.0198), Cystatin C eGFR (Spearman r=-0.40 p=0.0002), CKD stage (Spearman r=0.457, p<0.0001), PTH (Spearman r=0.330, p=0.0039), ionized calcium (Spearman r=-0.330, p=0.0049), CysC (Spearman r= 0.404, p=0.0002) and 1,25-dihydroxyvitamin D (Spearman r=-0.345, p=0.0034) concentrations. No significant correlation was found between FGF-23 levels and calcium phosphate product (Spearman r= 0.164, p=0.142). Upon classification of patients into two age groups, less than 13 years of age and more than 13 years of age, correlational results differed significantly. FGF-23 correlated with CysC eGFR( Spearman r= -0.633, p<0.0001), CKD stage (Spearman r=0.731, p<0.0001), phosphate (Spearman r= 0.557, p<0.0001), calcium phosphate product (Spearman r=0.534, p<0.0001), 125(OH)2 Vit D (Spearman r=-0.631, p<0.0001), PTH (Spearman r= 0.475, p=0.0017) and ionized calcium (Spearman r= -0.503, p=0.0015) only in the older group. The relationship between FGF-23 and Ca*P for the older group could be expressed by the exponential model FGF-23= 38.15 e0.4625Ca*P. CONCLUSION: Abnormal values of FGF-23 in adolescents and young adults with CKD correlate with Ca* P in the absence of vascular calcifications, and may serve as a biomarker for the risk of cardiovascular calcifications.

Stomatin-like protein 2 binds cardiolipin and regulates mitochondrial biogenesis and function.

Stomatin-like protein 2 (SLP-2) is a widely expressed mitochondrial inner membrane protein of unknown function. Here we show that human SLP-2 interacts with prohibitin-1 and -2 and binds to the mitochondrial membrane phospholipid cardiolipin. Upregulation of SLP-2 expression increases cardiolipin content and the formation of metabolically active mitochondrial membranes and induces mitochondrial biogenesis. In human T lymphocytes, these events correlate with increased complex I and II activities, increased intracellular ATP stores, and increased resistance to apoptosis through the intrinsic pathway, ultimately enhancing cellular responses. We propose that the function of SLP-2 is to recruit prohibitins to cardiolipin to form cardiolipin-enriched microdomains in which electron transport complexes are optimally assembled. Likely through the prohibitin functional interactome, SLP-2 then regulates mitochondrial biogenesis and function.

A modulatory interleukin-10 response to staphylococcal peptidoglycan prevents Th1/Th17 adaptive immunity to Staphylococcus aureus.

Toll-like receptor (TLR) 2 on antigen-presenting cells (APCs) enables these cells to recognize peptidoglycan-embedded lipopeptides and glycopolymers in the Staphylococcus aureus cell wall and mount an inflammatory response to this microbe. TLR2 signalling can also modulate immunity to S. aureus by inducing an interleukin (IL)-10 response in APCs. What determines the balance between proinflammatory and modulatory responses to S. aureus is unknown. We show that the modulatory IL-10 response preferentially occurs upon CD14- and CD36-independent TLR2 signaling, triggering PI3K activation, and is restricted to monocytes and monocyte-derived macrophages (MΦs). In contrast, monocyte-derived dendritic cells (DCs) produce mostly IL-12 and IL-23. The differential APC polarization induced by staphylococcal peptidoglycan translates into differential T helper responses: MΦs primarily trigger IL-10 and weak IL-17 responses, whereas DCs trigger a robust Th1/Th17 response. Exploitation of TLR2 signalling plasticity by S. aureus may explain the wide range of outcomes of human encounters with this microbe.

Impaired GFR is the most important determinant for FGF-23 increase in chronic kidney disease.

OBJECTIVES: It is unclear whether fibroblast growth factor-23 (FGF-23) increases in response to phosphate accumulation or to decrease clearance in chronic kidney disease (CKD) as is the case with other low molecular weight proteins such as cystatin C (CysC). DESIGN AND METHODS: This cross-sectional study measured serum FGF-23, CysC, and other serum markers of bone metabolism in 69 patients, aged 18 months-24 years, with various stages of CKD (eGFR=11-214mL/min). RESULTS: FGF-23 levels were significantly correlated with CysC and parathyroid hormone levels (PTH) on univariate non-linear regression analysis. In multivariate linear regression analysis, log (CysC) (ß=0.660, p<0.0001), log (PTH) (ß=0.038, p=0.37), and phosphate (ß=0.222, p=0.028) explained 69.1% of the variance of FGF-23. CONCLUSIONS: CysC had the largest unique contribution to FGF-23 variance in this model, supporting the hypothesis that renal clearance may be the most responsible factor for elevated FGF-23 levels in early stages of CKD.

Tumor suppression by phospholipase C-beta3 via SHP-1-mediated dephosphorylation of Stat5.

Given its catalytic activity to generate diacylglycerol and inositol 1,4,5-trisphosphate, phospholipase C (PLC) is implicated in promoting cell growth. However, we found that PLC-beta3-deficient mice develop myeloproliferative disease, lymphoma, and other tumors. The mutant mice have increased numbers of hematopoietic stem cells with increased proliferative, survival, and myeloid-differentiative abilities. These properties are dependent on Stat5 and can be antagonized by the protein phosphatase SHP-1. Stat5-dependent cooperative transformation by active c-Myc and PLC-beta3 deficiency was suggested in mouse lymphomas in PLC-beta3(-/-) and in Emicro-myc;PLC-beta3(+/-) mice and human Burkitt's lymphoma cells. The same mechanism for malignant transformation seems to be operative in other human lymphoid and myeloid malignancies. Thus, PLC-beta3 is likely a tumor suppressor.

Receptor-interacting protein-2 deficiency delays macrophage migration and increases intracellular infection during peritoneal dialysis-associated peritonitis.

BACKGROUND: Early upregulation of receptor-interacting protein-2 (RIP2) expression during peritoneal dialysis (PD)-associated peritonitis correlates with a favorable clinical outcome, while failure to upregulate RIP2 correlates with a protracted course. We noticed that patients who do not upregulate RIP2 during PD-associated peritonitis have more peritoneal macrophages during the early phase of infection. METHODS: To study the mechanism behind this observation, we examined the role of RIP2 in the immune response to bacterial challenge in a mouse model of acute peritonitis. We injected RIP2(+/+) and RIP2(-/-) mice intraperitoneally with a Staphylococcus epidermidis cell free-preparation, and peritoneal cells were isolated 3, 6 and 24 h after challenge. RESULTS: Surprisingly, RIP2(-/-) mice had a comparable influx of inflammatory leukocytes, but had a significantly higher number of peritoneal macrophages at 3 h, indicating delayed emigration of these cells. No significant differences were seen at later times suggesting that migration was delayed but not inhibited. In addition, RIP2(-/-) macrophages were more permissive to intracellular infection by Staphylococcus aureus, indicating that, in the absence of RIP2, resident peritoneal macrophages could become reservoirs of bacteria. CONCLUSION: These findings provide a mechanism for the observation that upregulation of RIP2 expression is required for rapid resolution of peritonitis, by decreasing intracellular infection and by regulating the migration of antigen-presenting cells in the early stages of an inflammatory response.

Molecular determinants of inverse agonist activity of biologicals targeting CTLA-4.

Ligation of CD28 or CTLA-4 with some biologicals can activate T cells due to an unexpected superagonist or inverse agonist activity, respectively. The risk of such an outcome limits the therapeutic development of these reagents. Thus, identifying the molecular determinants of superagonist/inverse agonist properties for biologicals targeting costimulatory/inhibitory receptors has not only fundamental value but also important therapeutic implications. In this study, we show that ligation of CTLA-4 with either soluble B7.1 Ig (but not B7.2 Ig) or with a recombinant bispecific in-tandem single chain Fv known as 24:26 induces TCR-independent, T cell activation. Such an inverse agonist activity requires CD28 expression and high CTLA-4 expression and is not seen when CTLA-4 is ligated by membrane-bound B7.1 or B7.2. At the molecular level, the inverse agonist activity of B7.1 Ig or 24:26 correlates with their ability to induce the formation of unique dimer-based, CTLA-4 oligomers on the T cell surface and involves CTLA-4 signaling through its cytoplasmic domain. Our results provide a potential mechanism to explain and to predict inverse agonist activity for CTLA-4 ligands.