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-c 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.

Cross-presentation of soluble Ag on MHC class I molecules to naive CD8 T cells by liver sinusoidal endothelial cells (LSECs) leads to induction of T cell tolerance that requires interaction between coinhibitory B7-H1 on LSECs and programmed cell death-1 on CD8 T cells. In this study, we investigate whether cross-presentation of high as well as low Ag concentrations allowed for LSEC-induced tolerance. Ag concentration directly correlated with the cross-presentation capacity of murine LSECs and thus strength of TCR stimulation. Although LSEC cross-presentation at low-Ag concentrations resulted in tolerance, they induced differentiation into effector T cells (CTL) at high-Ag concentrations. CTL differentiation under these conditions was not caused by increased expression of costimulatory CD80/86 on cross-presenting LSECs but was determined by early IL-2 release from naive CD8 T cells. B7-H1 signals from LSECs and TCR avidity reciprocally controlled early T cell release of IL-2 and CTL differentiation. B7-H1 expression directly correlated with cross-presentation at low- but not high-Ag concentrations, indicating an imbalance between TCR and coinhibitory signals regulating T cell release of IL-2. Exogenous IL-2 overrode coinhibitory B7-H1–mediated signals by LSECs and induced full CTL differentiation. Our results imply that LSEC-mediated T cell tolerance can be broken in situations where T cells bearing high-avidity TCR encounter LSECs cross-presenting high numbers of cognate MHC class I peptide molecules, such as during viral infection of the liver. Furthermore, we attribute a novel costimulatory function to IL-2 acting in a T cell autonomous fashion to promote local induction of immunity in the liver even in the absence of CD80/86 costimulation.

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 crosspresentation of apoptotic tumor cells by LSEC and subsequent CD8 + T cell tolerance induction, represents a novel mechanism operative in tumor immune escape. IntroductionThe liver is often a site of tumor metastasis once tumor cells reach the circulation. In particular for tumors arising in the gastrointestinal tract, the liver represents the first vascular bed that allows tumor seeding or promotes tumor elimination. The molecular mechanisms involved in tumor cell adhesion within the liver have been partly elucidated and are critical in the initiation of metastasis [1]. There is mutual interaction between liver sinusoidal cells and tumor cells, which involves the mannose receptor, release of IL-1b and IL-18 as well as endothelial cell up-regulation of adhesion molecules such as VCAM-1 [2], which all favor the development of hepatic metastasis.At the same time, hepatic sinusoidal cell populations contribute to tumor defense. Kupffer cells as the hepatic macrophage population have the capacity to kill tumor cells efficiently through phagocytosis [3] and induction Immunomodulation * These authors contributed equally to this work. ** These authors are co-senior authors.Correspondence: Percy A. Knolle [7]. These cells efficiently eliminate metastasizing tumor cells through induction of apoptosis through TRAIL, CD95L or the perforin/granzyme B pathway [8][9][10][11]. The hepatic overall capacity to eliminate tumor cells seems to be linked to the physiological role of the liver to eliminate gut-derived material from portal venous blood, because the hepatic activity of tumor cell killing is reduced in germ-free mice [12]. Furthermore, the liver is known to remove activated T cells from the circulation. Bone marrowderived as well as organ-resident hepatic cell populations attract circulating activated T cells employing CD54/CD106 and induce T cell apoptosis [13][14][15]. This similarity in retention and elimination of tumor cells and activated T cells suggests that not only may similar molecular mechanisms be employed, but also t...

CD28 and CTLA-4 are the critical costimulatory receptors that predominantly determine the outcome of T cell stimulation, with CD28 promoting positive costimulation and CTLA-4 inducing inhibitory signals. Blockage of the B7-CD28/CTLA-4 pathway leads to transplantation tolerance. However, the exact mechanism of the inhibitory function of CTLA-4 remains elusive. Here, we investigated the influence of CTLA-4 expression on CD28 using CTLA-4-transfected Jurkat T cells as well as primary T cells. Up-regulation of CTLA-4 induced abrogation of IL-2 production, indicating an anergic phenotype of CTLA-4(high) T cells. Besides the negative signaling function of CTLA-4, we show for the first time that CTLA-4 expression promotes the down-regulation of CD28 on the T cell surface as a result of enhanced internalization and degradation of CD28. These data suggest that apart from the established competition for B7.1 and B7.2 by CTLA-4, inhibition of T cells by CTLA-4 might be additionally explained by reduction of CD28 on the cell surface, which might impede T cell response to stimulation. Our data provide a previously unrecognized mechanism for T cell regulation.

Only recently have natural antigens for CD1d-dependent, invariant Va14 + natural killer T (iNKT) cells been identified. Similar data for CD1d-independent and CD8 + NKT cell populations are still missing. Here, we show that the MHC class I-restricted CD8 + TCRtransgenic mouse lines OT-I, P14 and H-Y contain a significant proportion of transgenic CD8 + NK1.1 + T cells. In liver, most of NK1.1 + T cells express CD8aa homodimers. Transgenic NKT cells did not bind invariant Va14-to-Ja18 TCR rearrangement (Va14i)-specific CD1d/a-galactosylceramide tetramers and the frequency of iNKT cells was severely reduced. The activated cell surface phenotype and the distribution of transgenic NKT cells in vivo were similar to that reported for iNKT cells. The OT-I and P14 CD8 + NKT cells recognized their cognate antigen in the context of H2-K b and produced cytokines shortly after TCR stimulation. Importantly, transgenic NKT cells exerted immediate antigen-specific cytotoxicity in vitro and in vivo. Our results demonstrate the presence of transgenic CD8 + NKT cells in MHC class I-restricted TCR-transgenic animals, which are endowed with rapid antigen-specific effector functions. These data imply that experiments studying naive T cell function in TCR-transgenic animals should be interpreted with caution, and that such animals could be utilized for studying CD8 + NKT cell function in an antigen-specific manner.

Monomeric and dimeric soluble major histocompatibility complex (MHC) molecules down-regulate activated T cells in an antigen-specific manner in vitro. This property could be exploited to modulate alloresponses in vivo but has remained difficult to demonstrate. Here, intraperitoneal infusion of a Lewis-derived rat MHC class I molecule, RT1.A(l)-Fc, in Dark Agouti (RT1.A(a)) recipient rats prolonged cardiac graft survival, which led to permanent engraftment. This effect was mediated by T cell impairment of target cell lysis by CD8+ T cells and down-regulation of interferon-gamma production by CD4+ T cells. The binding of the dimeric MHC allowed ex vivo visualization of alloreactive T cells in peripheral blood, splenocytes, and allografts, revealing low frequency of alloreactive CD8+ T cells after establishment of permanent engraftment of cardiac allografts. Thus, these data show the potential of dimeric MHC molecules to promote graft survival and allow visualization of alloreactive T cells.

Key words: soluble HLA; DNMT-1; cancer vaccine; tumor antigen; immunotherapyThe preferred approach for the elimination of tumor cells is to elicit a specific antitumor CTL response that will select tumor cells presenting the appropriate T-cell epitopes. 1-4 Human clinical trials for a variety of malignant diseases showed that T-cell therapy was effective and curative for some individuals. 5,6 A number of TAAs have been identified, mainly melanoma antigens, such as Melan-A and MART-1. Studies were initiated to vaccinate tumor patients against these antigens, and their ability to induce antitumor T-cell immunity has been shown in clinical studies. 7 Many of the recently identified antigens are nonmutated differentiation antigens overexpressed in tumors, e.g., a peptide derived from protein tyrosinase that was shown to successfully induce the attack and lysis of melanoma cells from cancer patients. 8 Several strategies have evolved to identify novel TAAs, e.g., cloning of epitopes recognized by CTLs 9 and SEREX technology, 10 where a patient's antibodies are used to identify tumor antigens from a cDNA expression library made from mRNA of tumor specimens.Conventionally, endogenous peptides have been eluted from affinity-purified HLA, such as HLA-A*0201, followed by analysis and sequencing using HPLC combined with tandem mass spectrometry. 11,12 HLA class I molecules are integral membrane glycoproteins; however, the presence of a soluble form of these molecules was demonstrated decades ago. 13,14 Similar to membrane-bound MHC, sMHC class I molecules bind peptides which are 8 -10 amino acids long, with a few exceptions having a length of 6, 11 and 12 amino acids. 15 Purification of MHC molecules is complicated due to their unavoidable contamination with cell debris and detergents after cell lysis. We recently identified HLA-restricted, tumor-specific antigens presented by the sMHC class I molecule by transfecting human tumor cell lines with truncated genes of HLA-A2. After purification of the secreted sHLA by affinity chromatography, peptides were eluted and sequenced. 15 Novel peptides unique to the sMHC variants were identified and screened for tumor-relevant precursor proteins. Detection of these peptides on sMHC indicates that these peptides are properly processed and displayed on transfected sMHC molecules similarly to those displayed on membrane-bound MHC molecules. Peptides recovered from sHLA molecules form similar patterns to those of membrane-bound HLA and include known MHC peptides. 15 Therefore, the repertoire of MHC peptides recovered from sMHC molecules is a good representation of the naturally processed and displayed peptides of transfected cells. Peptides detected this way should be presented at many copy numbers per cell and are likely to be bound with relatively high affinity to HLA.Use of sHLA as a source of peptides enabled us to identify large numbers of MHC peptides presented by human cancer cells and to screen these for peptides possibly derived from TAAs as candidates for tumor vaccination. 15 ...