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Chronic hepatitis B virus (HBV) infection affects 300 million patients worldwide 1,2 , in whom virus-specific CD8 T cells by still ill-defined mechanisms lose their function and cannot eliminate HBV-infected hepatocytes 3-7 . Here we demonstrate that a liver immune rheostat renders virus-specific CD8 T cells refractory to activation and leads to their loss of effector functions. In preclinical models of persistent infection with hepatotropic viruses such as HBV, dysfunctional virus-specific CXCR6 + CD8 T cells accumulated in the liver and, as a characteristic hallmark, showed enhanced transcriptional activity of cAMP-responsive element modulator (CREM) distinct from T cell exhaustion. In patients with chronic hepatitis B, circulating and intrahepatic HBV-specific CXCR6 + CD8 T cells with enhanced CREM expression and transcriptional activity were detected at a frequency of 12-22% of HBV-specific CD8 T cells. Knocking out the inhibitory CREM/ICER isoform in T cells, however, failed to rescue T cell immunity. This indicates that CREM activity was a consequence, rather than the cause, of loss in T cell function, further supported by the observation of enhanced phosphorylation of protein kinase A (PKA) which is upstream of CREM. Indeed, we found that enhanced cAMP-PKA-signalling from increased T cell adenylyl cyclase activity augmented CREM activity and curbed T cell activation and effector function in persistent hepatic infection. Mechanistically, CD8 T cells recognizing their antigen on hepatocytes established close and extensive contact with liver sinusoidal endothelial cells, thereby enhancing adenylyl cyclase-cAMP-PKA signalling in T cells. In these hepatic CD8 T cells, which recognize their antigen on hepatocytes, phosphorylation of key signalling kinases of the T cell receptor signalling pathway was impaired, which rendered them refractory to activation. Thus, close contact with liver sinusoidal endothelial cells curbs the activation and effector function of HBV-specific CD8 T cells that target hepatocytes expressing viral antigens by means of the adenylyl cyclase-cAMP-PKA axis in an immune rheostat-like fashion., (© 2024. The Author(s).)

Liver sinusoidal endothelial cells (LSECs) are liver-resident antigen (cross)-presenting cells that generate memory CD8 T cells, but metabolic properties of LSECs and LSEC-primed CD8 T cells remain understudied. Here, we report that high-level mitochondrial respiration and constitutive low-level glycolysis support LSEC scavenger and sentinel functions. LSECs fail to increase glycolysis and co-stimulation after TLR4 activation, indicating absence of metabolic and functional maturation compared with immunogenic dendritic cells. LSEC-primed CD8 T cells show a transient burst of oxidative phosphorylation and glycolysis. Mechanistically, co-stimulatory IL-6 signaling ensures high FOXO1 expression in LSEC-primed CD8 T cells, curtails metabolic activity associated with T cell activation, and is indispensable for T cell functionality after re-activation. Thus, distinct immunometabolic features characterize non-immunogenic LSECs compared with immunogenic dendritic cells and LSEC-primed CD8 T cells with memory features compared with effector CD8 T cells. This reveals local features of metabolism and function of T cells in the liver., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Nonalcoholic steatohepatitis (NASH) is a manifestation of systemic metabolic disease related to obesity, and causes liver disease and cancer 1,2 . The accumulation of metabolites leads to cell stress and inflammation in the liver 3 , but mechanistic understandings of liver damage in NASH are incomplete. Here, using a preclinical mouse model that displays key features of human NASH (hereafter, NASH mice), we found an indispensable role for T cells in liver immunopathology. We detected the hepatic accumulation of CD8 T cells with phenotypes that combined tissue residency (CXCR6) with effector (granzyme) and exhaustion (PD1) characteristics. Liver CXCR6 + CD8 T cells were characterized by low activity of the FOXO1 transcription factor, and were abundant in NASH mice and in patients with NASH. Mechanistically, IL-15 induced FOXO1 downregulation and CXCR6 upregulation, which together rendered liver-resident CXCR6 + CD8 T cells susceptible to metabolic stimuli (including acetate and extracellular ATP) and collectively triggered auto-aggression. CXCR6 + CD8 T cells from the livers of NASH mice or of patients with NASH had similar transcriptional signatures, and showed auto-aggressive killing of cells in an MHC-class-I-independent fashion after signalling through P2X7 purinergic receptors. This killing by auto-aggressive CD8 T cells fundamentally differed from that by antigen-specific cells, which mechanistically distinguishes auto-aggressive and protective T cell immunity.

Background & Aims: Liver sinusoidal endothelial cells (LSECs) are prominent liver-resident antigen (cross-)presenting cells. LSEC cross-priming of naïve CD8 T cells does not require CD4 T cell help in contrast to priming by dendritic cells (DC) but leads to the formation of memory T cells that is preceded by transient Granzyme B (GzmB) expression. Here we provide evidence for a so far unrecognized CD4 T helper cell function in LSEC-induced CD8 T cell activation., Methods: Naïve CD8 T cells and differentiated T helper 1 (T h 1) cells were stimulated by antigen-presenting LSEC, and GzmB expression in CD8 T cells was determined by flow cytometry. To identify molecular pathways mediating this GzmB expression, mechanistic proof-of-concept experiments were conducted using stimulatory anti-CD3 antibody together with Hyper-IL-6., Results: We demonstrate that LSECs simultaneously function in antigen co-presentation to CD8 and CD4 T cells. Such co-presentation revealed a function of T h 1 cells to increase GzmB expression in CD8 T cells after LSEC but not DC cross-priming. IL-2 released from T h 1 cells was required but not sufficient for rapid GzmB induction in CD8 T cells. T cell receptor together with IL-6 trans-signaling was necessary for IL-2 to mediate rapid GzmB induction., Conclusions: Our findings indicate that LSECs can serve as a platform to facilitate CD4-CD8 T cell crosstalk enhancing the immune function of LSECs to cross-prime CD8 T cells. IL-6 trans-signaling-mediated responsiveness for IL-2 inducing sustained GzmB expression in CD8 T cells reveals unique mechanisms of CD4 T cell help and CD8 T cell differentiation through liver-resident antigen-presenting cells., Lay Summary: Our findings demonstrate that LSEC co-present antigen to CD8 and CD4 T cells and thereby enable CD4 T cell help for LSEC-priming of CD8 T cells. This CD4 T cell help selectively enhances the rapid upregulation of GzmB and effector function of LSEC-primed CD8 T cells thereby enhancing functional differentiation towards CD8 effector T cells., (Copyright © 2016 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion.

Immune control of infections with viruses or intracellular bacteria relies on cytotoxic CD8(+) T cells that use granzyme B (GzmB) for elimination of infected cells. During inflammation, mature antigen-presenting dendritic cells instruct naive T cells within lymphoid organs to develop into effector T cells. Here, we report a mechanistically distinct and more rapid process of effector T cell development occurring within 18 hr. Such rapid acquisition of effector T cell function occurred through cross-presenting liver sinusoidal endothelial cells (LSECs) in the absence of innate immune stimulation and known costimulatory signaling. Rather, interleukin-6 (IL-6) trans-signaling was required and sufficient for rapid induction of GzmB expression in CD8(+) T cells. Such LSEC-stimulated GzmB-expressing CD8(+) T cells further responded to inflammatory cytokines, eliciting increased and protracted effector functions. Our findings identify a role for IL-6 trans-signaling in rapid generation of effector function in CD8(+) T cells that may be beneficial for vaccination strategies., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Development of CD8(+) T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1(+) memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Viruses can escape cytotoxic T cell (CTL) immunity by avoiding presentation of viral components via endogenous MHC class I antigen presentation in infected cells. Cross-priming of viral antigens circumvents such immune escape by allowing noninfected dendritic cells to activate virus-specific CTLs, but they remain ineffective against infected cells in which immune escape is functional. Here, we show that cross-presentation of antigen released from adenovirus-infected hepatocytes by liver sinusoidal endothelial cells stimulated cross-primed effector CTLs to release tumor necrosis factor (TNF), which killed virus-infected hepatocytes through caspase activation. TNF receptor signaling specifically eliminated infected hepatocytes that showed impaired anti-apoptotic defense. Thus, CTL immune surveillance against infection relies on two similarly important but distinct effector functions that are both MHC restricted, requiring either direct antigen recognition on target cells and canonical CTL effector function or cross-presentation and a noncanonical effector function mediated by TNF., (Copyright © 2012 The Authors. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Cross-presentation is an important function of immune competent cells, such as dendritic cells (DCs), macrophages, and an organ-resident liver cell population, i.e., liver sinusoidal endothelial cells (LSECs). Here, we characterize in direct comparison to DCs the distinct dynamics and kinetics of cross-presentation employed by LSECs, which promote tolerance induction in CD8 T cells. We found that LSECs were as competent in cross-presenting circulating soluble antigen ex vivo as DCs at a per-cell basis. However, antigen uptake in vivo was 100-fold more pronounced in LSECs, indicating distinct mechanisms of cross-presentation. In contrast to mannose-receptor-mediated antigen uptake and routing into stable endosomes dedicated to cross-presentation in DCs, we observed distinct antigen-uptake and endosomal routing with high antigen turnover in LSECs that resulted in short-lived cross-presentation. Receptor-mediated endocytosis did not always lead to cross-presentation, because immune-complexed antigen taken up by the Fc-receptor was not cross-presented by LSECs, indicating that induction of CD8 T cell tolerance by LSECs is impaired in the presence of preexisting immunity., Conclusion: These results provide a mechanistic explanation how organ-resident LSECs accommodate continuous scavenger function with the capacity to cross-present circulating antigens using distinct kinetics and dynamics of antigen-uptake, routing and cross-presentation compared to DCs.

Unlabelled: Peripheral CD8 T-cell tolerance can be generated outside lymphatic tissue in the liver, but the course of events leading to tolerogenic interaction of hepatic cell populations with circulating T-cells remain largely undefined. Here we demonstrate that preferential uptake of systemically circulating antigen by murine liver sinusoidal endothelial cells (LSECs), and not by other antigen-presenting cells in the liver or spleen, leads to cross-presentation on major histocompatibility complex (MHC) I molecules, which causes rapid antigen-specific naïve CD8 T-cell retention in the liver but not in other organs. Using bone-marrow chimeras and a novel transgenic mouse model (Tie2-H-2K(b) mice) with endothelial cell-specific MHC I expression, we provide evidence that cross-presentation by organ-resident and radiation-resistant LSECs in vivo was both essential and sufficient to cause antigen-specific retention of naïve CD8 T-cells under noninflammatory conditions. This was followed by sustained CD8 T-cell proliferation and expansion in vivo, but ultimately led to the development of T-cell tolerance., Conclusion: Our results show that cross-presentation of circulating antigens by LSECs caused antigen-specific retention of naïve CD8 T-cells and identify antigen-specific T-cell adhesion as the first step in the induction of T-cell tolerance.

Unlabelled: Liver sinusoidal endothelial cells (LSEC) are unique organ-resident antigen-presenting cells capable of cross-presentation and subsequent tolerization of naïve CD8(+) T cells. We investigated the molecular mechanisms underlying this tolerance induction in naive CD8(+) T cells. MHC class I-restricted antigen presentation by LSEC led to initial stimulation of naïve CD8(+) T cells, which up-regulated CD69, CD25, CD44, and programmed death (PD)-1 and proliferated similar to dendritic cell (DC)-activated CD8(+) T cells. Importantly, cognate interaction with naïve CD8(+) T cells triggered increased expression of co-inhibitory B7-H1 but not co-stimulatory CD80/86 molecules exclusively on LSEC but not DC. This matured phenotype of B7-H1(high) CD80/86(low) was critical for induction of CD8(+) T cell tolerance by LSEC: B7-H1-deficient LSEC, that failed to interact with PD-1 on stimulated T cells, were incapable of inducing CD8(+) T cell tolerance. Moreover, increased costimulation via CD28 interfered with tolerance induction, indicating that the noninducible low expression levels of CD80/86 on LSEC supported B7-H1-dependent tolerance induction. LSEC-tolerized CD8(+) T cells had a distinctive phenotype from naïve and activated T cells with CD25(low), CD44(high), CD62L(high). They also expressed the homeostatic cytokine receptors CD127, CD122, and high levels of Bcl-2, indicating survival rather than deletion of tolerant CD8(+) T cells. On adoptive transfer into congenic animals, tolerized CD8(+) T cells failed to show specific cytotoxicity in vivo., Conclusion: Cognate interaction of LSEC with naïve CD8(+) T cells elicits a unique tolerogenic maturation of LSEC and permissiveness of T cells for tolerogenic signals, demonstrating that LSEC-induced tolerance is an active and dynamic process.

Background: Although STAT5 promotes survival of hematopoietic progenitors, STAT5-/- mice develop mild neutrophilia., Methodology/principal Findings: Here, we show that in STAT5-/- mice, liver endothelial cells (LECs) autonomously secrete high amounts of G-CSF, allowing myeloid progenitors to overcompensate for their intrinsic survival defect. However, when injected with pro-inflammatory cytokines, mutant mice cannot further increase neutrophil production, display a severe deficiency in peripheral neutrophil survival, and are therefore unable to maintain neutrophil homeostasis. In wild-type mice, inflammatory stimulation induces rapid STAT5 degradation in LECs, G-CSF production by LECs and other cell types, and then sustained mobilization and expansion of long-lived neutrophils., Conclusion: We conclude that STAT5 is an ambivalent factor. In cells of the granulocytic lineage, it exerts an antiapoptotic function that is required for maintenance of neutrophil homeostasis, especially during the inflammatory response. In LECs, STAT5 negatively regulates granulopoiesis by directly or indirectly repressing G-CSF expression. Removal of this STAT5-imposed brake contributes to induction of emergency granulopoiesis.

Development of tumor-specific T cell tolerance contributes to the failure of the immune system to eliminate tumor cells. Here we report that hematogenous dissemination of tumor cells followed by their elimination and local removal of apoptotic tumor cells in the liver leads to subsequent development of T cell tolerance towards antigens associated with apoptotic tumor cells. We provide evidence that liver sinusoidal endothelial cells (LSEC) remove apoptotic cell fragments generated by induction of tumor cell apoptosis through hepatic NK1.1+ cells. Antigen associated with apoptotic cell material is processed and cross-presented by LSEC to CD8+ T cells, leading to induction of CD8+ T cell tolerance. Adoptive transfer of LSEC isolated from mice challenged previously with tumor cells promotes development of CD8+ T cell tolerance towards tumor-associated antigen in vivo. Our results indicate that hematogenous dissemination of tumor cells, followed by hepatic tumor cell elimination and local cross-presentation of apoptotic tumor cells by LSEC and subsequent CD8+ T cell tolerance induction, represents a novel mechanism operative in tumor immune escape.

After ingestion, oral antigens distribute systemically and provoke T cell stimulation outside the gastrointestinal tract. Within the liver, scavenger liver sinusoidal endothelial cells (LSEC) eliminate blood-borne antigens and induce T cell tolerance. Here we investigated whether LSEC contribute to oral tolerance. Oral antigens were efficiently cross-presented on H-2K(b) by LSEC to naive CD8 T cells. Cross-presentation efficiency in LSEC but not dendritic cells was increased by antigen-exposure to heat or low pH. Mechanistically, cross-presentation in LSEC requires endosomal maturation, involves hsc73 and proteasomal degradation. H-2K(b)-restricted cross-presentation of oral antigens by LSEC in vivo induced CD8 T cell priming and led to development of CD8 T cell tolerance in two independent experimental systems. Adoptive transfer of LSEC from mice fed with antigen (ovalbumin) into RAG2-/- knockout mice, previously reconstituted with naive ovalbumin-specific CD8 T cells, prevented development of specific cytotoxicity and expression of IFN-gamma in CD8 T cells. Using a new transgenic mouse line expressing H-2K(b) only on endothelial cells, we have demonstrated that oral antigen administration leads to tolerance in H-2K(b)-restricted CD8 T cells. Collectively, our data demonstrate a participation of the liver, in particular scavenger LSEC, in development of CD8 T cell tolerance towards oral antigens.

Kupffer cells and liver sinusoidal endothelial cells (LSEC) clear portal venous blood from gut-derived bacterial degradation products such as lipopolysaccharide (LPS) without inducing a local inflammatory reaction. LPS tolerance was reported for Kupffer cells, but little is known whether sensitivity of LSEC toward LPS is dynamically regulated. Here, we demonstrate that LSEC react to LPS directly as a function of constitutive Toll-like receptor 4 (TLR4)/CD14 expression but gain a LPS-refractory state upon repetitive stimulation without loss of scavenger activity. LPS tolerance in LSEC is characterized by reduced nuclear localization of nuclear factor-kappaB upon LPS rechallenge. In contrast to monocytes, however, TLR4 surface expression of LSEC is not altered by LPS stimulation and thus does not account for LPS tolerance. Mechanistically, LPS tolerance in LSEC is linked to prostanoid production and may account for cross-tolerance of LPS-treated LSEC to interferon-gamma stimulation. Functionally, LPS tolerance in LSEC results in reduced leukocyte adhesion following LPS rechallenge as a consequence of decreased CD54 surface expression. Furthermore, LPS tolerance is operative in vivo, as we observed by intravital microscopy-reduced leukocyte adhesion to LSEC and improved sinusoidal microcirculation in the liver after repetitive LPS challenges. Our results support the notion that LPS tolerance in organ-resident scavenger LSEC contributes to local hepatic control of inflammation.

Adenoviral vectors are known to transduce hepatocytes in normal liver tissue with high efficiency. The aim of this study was to investigate whether sinusoidal endothelial cells, which separate hepatocytes from the bloodstream in the sinusoidal lumen, are permissive for infection by adenoviruses. We show here that microvascular liver sinusoidal endothelial cells are not infected by adenovirus type 5 in vivo or in vitro unless high MOIs are used. In contrast, macrovascular endothelial cells from aorta are efficiently infected by adenovirus type 5. In addition, Kupffer cells, similar to sinusoidal endothelial cells, are not infected by adenovirus type 5. Liver sinusoidal endothelial cells do not express the integrin receptor alpha(v)beta3, which is required for efficient infection by adenoviruses. Our results demonstrate that hepatocytes are the main cell population of the liver that is infected by adenovirus type 5.

Background & Aims: Murine liver sinusoidal endothelial cells (LSECs) constitutively express accessory molecules and can present antigen to memory Th1 CD4(+) T cells. Using a T-cell receptor transgenic mouse line, we addressed the question whether LSECs can prime naive CD4(+) T cells., Methods: Purified LSECs were investigated for their ability to induce activation and differentiation of naive CD4(+) T cells in comparison with bone marrow-derived antigen-presenting cells and macrovascular endothelial cells. Activation of T cells was determined by cytokine production. LSECs were further studied for expression of interleukin (IL)-12 by reverse-transcription polymerase chain reaction, and the unique phenotype of LSECs was determined by flow cytometry., Results: We provide evidence that antigen-presenting LSECs can activate naive CD62Lhigh CD4(+) T cells. Activation of naive CD4(+) T cells by LSECs occurred in the absence of IL-12. In contrast, macrovascular endothelial cells from aorta could not activate naive CD4(+) T cells. The unique functional characteristics of microvascular LSECs together with a unique phenotype (CD4(+), CD11b+, CD11c+, CD80(+), CD86(+)) make these cells different from macrovascular endothelial cells. Furthermore, LSECs did not require in vitro maturation to activate naive CD4(+) T cells. Most importantly, LSECs failed to induce differentiation toward Th1 cells, whereas conventional antigen-presenting cell populations induced a Th1 phenotype in activated CD4(+) T cells. Upon restimulation, CD4(+) T cells, which were primed by antigen-presenting LSECs, expressed interferon gamma, IL-4, and IL-10, which is consistent with a Th0 phenotype. Exogenous cytokines (IL-1beta, IL-12, or IL-18) present during T-cell priming by antigen-presenting LSECs could not induce a Th1 phenotype, but neutralization of endogenously produced IL-4 during T-cell priming led to a reduced expression of IL-4 and IL-10 by CD4(+) T cells upon restimulation. The addition of spleen cells to cocultures of LSECs and naive CD4(+) T cells during T-cell priming led to differentiation of T cells toward a Th1 phenotype., Conclusions: The ability of antigen-presenting LSECs to induce cytokine expression in naive CD4(+) T cells and their failure to induce differentiation toward a Th1 phenotype may contribute to the unique hepatic microenvironment that is known to promote tolerance.

Endotoxin is physiologically present in portal venous blood at concentrations of 100 pg/ml to 1 ng/ml. Clearance of endotoxin from portal blood occurs through sinusoidal lining cells, i.e., Kupffer cells, and liver sinusoidal endothelial cells (LSEC). We have recently shown that LSEC are fully efficient APCs. Here, we studied the influence of endotoxin on the accessory function of LSEC. Incubation of Ag-presenting LSEC with physiological concentrations of endotoxin lead to >/=80% reduction of the accessory function, measured by release of IFN-gamma from CD4+ T cells. In contrast, conventional APC populations rather showed an increase of the accessory function after endotoxin treatment. Inhibition of the accessory function in LSEC by endotoxin was not due to lack of soluble costimulatory signals, because neither supplemental IL-1beta, IL-2, IFN-gamma, or IL-12 could rescue the accessory function. Ag uptake was not influenced by endotoxin in LSEC. However, we found that endotoxin led to alkalinization of the endosomal/lysomal compartment specifically in LSEC but not in bone marrow macrophages, which indicated that Ag processing, i.e., proteolytic cleavage of protein Ags into peptide fragments, was affected by endotoxin. Furthermore, endotoxin treatment down-regulated surface expression of constitutively expressed MHC class II, CD80, and CD86. In conclusion, it is conceivable that endotoxin does not alter the clearance function of LSEC to remove gut-derived Ags from portal blood but specifically affects Ag processing and expression of the accessory molecules in these cells. Consequently, Ag-specific immune responses by CD4+ T cells are efficiently down-regulated in the hepatic microenvironment.

Clostridium difficile toxin B exhibits cytotoxic activity that is characterized by the disruption of the microfilamental cytoskeleton. Here we studied whether the GTP-binding Rho protein, which reportedly participates in the regulation of the actin cytoskeleton, is involved in the toxin action. Toxin B treatment of Chinese hamster ovary cells reveals a time- and concentration-dependent decrease in the ADP-ribosylation of Rho by Clostridium botulinum C3 exoenzyme in the cell lysate. Disruption of the microfilament system induced by C. botulinum C2 toxin or cytochalasin D does not cause impaired ADP-ribosylation of Rho. Toxin B exhibits its effects on Rho not only in intact cells but also when added to cell lysates. Besides endogenous Rho, RhoA-glutathione S-transferase (Rho-GST) fusion protein added to cell lysate showed decreased ADP-ribosylation after toxin B treatment. Immunoblot analysis reveals identical amounts of Rho-GST and no change in molecular mass after toxin B treatment compared with controls. ADP-ribosylation of Rho-GST purified from toxin B-treated cell lysate is inhibited, indicating a modification of Rho itself. Finally, transfection of rhoA DNA under the control of a strong promoter into cells protects them from the activity of toxin B. Altogether, the data indicate that C. difficile toxin B acts directly or indirectly on Rho proteins to inhibit ADP-ribosylation and suggest that the cytotoxic effect of toxin B involves Rho.