Silke Hegenbarth

Induction of cytokine production in naive CD4+ T cells by antigen-presenting murine liver sinusoidal endothelial cells but failure to induce differentiation toward Th1 cells

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.

Tolerogenic maturation of liver sinusoidal endothelial cells promotes B7-homolog 1-dependent CD8(+) T cell tolerance

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‐H1high CD80/86low 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 CD25low, CD44high, CD62Lhigh. 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. (HEPATOLOGY 2007.)

Development and functional consequences of LPS tolerance in sinusoidal endothelial cells of the liver

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‐κB 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‐γ 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.

Cross-presentation of oral antigens by liver sinusoidal endothelial cells leads to CD8 T cell tolerance.

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‐2Kb 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‐2Kb‐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‐γ in CD8 T cells. Using a new transgenic mouse line expressing H‐2Kb only on endothelial cells, we have demonstrated that oral antigen administration leads to tolerance in H‐2Kb‐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.

Systemic antigen cross-presented by liver sinusoidal endothelial cells induces liver-specific CD8 T-cell retention and tolerization†

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‐2Kb 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. (HEPATOLOGY 2009.)

Liver sinusoidal endothelial cells veto CD8 T cell activation by antigen‐presenting dendritic cells

The liver is known to induce tolerance rather than immunity through tolerogenic antigen presentation or elimination of effector T cells. In particular, hepatic dendritic cells (DC) are known to be little immunogenic for CD8 T cells. Here, we investigated whether this peculiar phenotype resulted from interaction with resident hepatic cell populations. Contact of DC with liver sinusoidal endothelial cells (LSEC) but not hepatocytes or B cells vetoed antigen‐presenting DC to fully activate naive CD8 T cells. This MHC‐independent regulatory effect of LSEC on DC function was not connected to soluble mediators but required physical contact. Because interaction with third‐party LSEC still allowed antigen‐presenting DC to stimulate expression of initial activation markers on naive CD8 T cells and to stimulate activated CD8 T cells, we hypothesize that LSEC controlled the DC costimulatory function. Indeed, contact with LSEC led to reduced DC expression levels of CD80/86 or IL‐12, but supplementation of these signals failed to rescue the ability to prime naive CD8 T cells, indicating involvement of further molecules. Taken together, our results reveal a novel principle operative in hepatic tolerance induction, in which LSEC not only tolerize T cells themselves but also suppress neighboring APC normally capable of inducing T cell immunity.

Cross-presentation of antigens from apoptotic tumor cells by liver sinusoidal endothelial cells leads to tumor-specific CD8+ T cell tolerance

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.

Liver-Primed Memory T Cells Generated under Noninflammatory Conditions Provide Anti-infectious Immunity

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.

TNF-induced target cell killing by CTL activated through cross-presentation.

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.

Distinct kinetics and dynamics of cross-presentation in liver sinusoidal endothelial cells compared to dendritic cells.

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. (HEPATOLOGY 2009.)