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Simian virus SV40 large T Antigen expression in the islets of Langerhans of transgenic mice results in beta-cell hyperproliferation, onset of new blood vessel formation and the development of highly vascularized solid tumors. Angiogenesis in the RIPTag mouse model, as well as in human cancer, is a hallmark of multistage tumorigenesis and precedes the development of solid tumors. In our study, intravital microscopy was used to monitor changes in the blood vessel phenotype, microcirculation and leukocyte adhesion during the progression from normal islets to angiogenic islets and solid tumors. In RIP1-Tag5 mice, an aberrant microangioarchitecture becomes apparent in early stages during spontaneous tumor development. Notably, the transition from normal to angiogenic islets is characterized by an increase in vessel diameter rather than vessel numbers. Thus, dilatation of existing vessels precedes vessel sprouting. Once initiated, neovascularization in angiogenic islets results in loss of vessel hierarchy and differentiation. Solid insulinomas display a higher vessel density and even more dramatic vessel heterogeneity as revealed by local "hot spots" of neovascularization and irregular vessel diameters. Strikingly, profound changes in the microangioarchitecture are already observed in early angiogenic islets suggesting that key features of the angiogenic vasculature are established prior to the expansion of tumor mass. Moreover, adhesion of leukocytes was found to be dramatically decreased in both angiogenic islets and solid tumors and correlates with morphological alterations of the vasculature. Thus, vessel transformation and reduced leukocyte-endothelium interactions are not exclusively features of solid tumors but represent early events during tumorigenesis.

In both the MHC class II and class I pathways of antigen presentation, accessory molecules influence formation of MHC–peptide complexes. In the MHC class II pathway, DM functions in the loading and editing of peptides; recent work demonstrated that it is acting not only in late endosomal compartments but also in recycling compartments and on the surface of B cells and immature dendritic cells. DM activity is modulated by another accessory molecule, DO, but this modulation is mainly operative in B cells, where it may lead to preferential activation of B cells producing high-affinity antibodies. In the MHC class I pathway of antigen presentation, recent in vivo experiments with knockout mice confirmed the role of tapasin in antigen presentation and indicate that it acts as a peptide editor and as a chaperone for TAP and the MHC class I heavy chain. In the class I loading complex, calreticulin and the thiol-dependent oxidoreductase ER60/ERp57 appear to support the function of tapasin in an as-yet-unknown fashion. The picture emerges that DM and tapasin have analogous functions in shaping the peptide repertoire presented by the respective MHC class II and class I molecules.

Major histocompatibility complex (MHC) class I molecules present peptides derived from nuclear and cytosolic proteins to CD8+ T cells. These peptides are translocated into the lumen of the endoplasmic reticulum (ER) to associate with class I molecules. Two MHC-encoded putative transporter proteins, TAP1 and TAP2, are required for efficient assembly of class I molecules and presentation of endogenous peptides. Expression of TAP1 and TAP2 in a mutant cell line resulted in the delivery of an 11-amino acid oligomer model peptide to the ER. Peptide translocation depended on the sequence of the peptide, was adenosine triphosphate (ATP)-dependent, required ATP hydrolysis, and was inhibited in a concentration-dependent manner.

Unmethylated cytosine-phosphorothioate-guanine (CpG) containing oligodeoxynucleotides (CpG-ODN) are known to act as adjuvants and powerful activators of the innate immune system. We investigated the therapeutic effect of CpG-ODN on a variety of established mouse tumors including AG104A, IE7 fibrosarcoma, B16 melanoma, and 3LL lung carcinoma. These tumors are only weakly immunogenic and notoriously difficult to treat. Repeated peritumoral injection of CpG-ODN resulted in complete rejection or strong inhibition of tumor growth, whereas systemic application had only partial effects. The CpG-ODN-induced tumor rejection was found to be mediated by both NK and tumor-specific CD8+ T cells. Comparison of parental tumors and variants rendered more antigenic by transfection with tumor Ags suggested that the efficiency of the CpG-ODN therapy correlated with the antigenicity of the tumors. Peritumoral CpG-ODN treatment was even effective in a situation where the immune system was tolerant for the tumor Ag, as shown by breakage of tolerance and tumor elimination. These results suggest that peritumoral application of CpG-ODN acts locally by inducing NK cells, and also leads to efficient presentation of tumor Ags and stimulation of CD8+ effector and memory T cells, thus providing a powerful antitumor therapy that can be also applied without knowledge of the tumor Ag.

SPECIFIC AIMSIn this study, we investigated the role of ER60 (ERp57/GRP58) in the formation of disulfide bonds within the major histocompatibility (MHC) class I heavy chain. We also examined the role of calnexin in recruiting ER60 into early folding complexes with the nascent class I heavy chain (HC).PRINCIPAL FINDINGS1. ER60/ERp57 forms disulfide-bonded intermediates with class I heavy chainMouse EL4 cells were metabolically labeled for 15 min with [35S] cysteine/methionine. After labeling, the cells were washed in PBS containing N-ethyl-maleimide (NEM), a membrane-permeable alkylating agent, and lysed in 1% digitonin lysis buffer containing NEM to trap unreacted cysteines. Protein complexes containing ER60 were isolated by immunoprecipitation using anti-ER60 sera. To identify a disulfide-bonded intermediate, the complexes were resolved on a 2-dimensional gel system using nonreducing gels in the first dimension. Proteins were resolved on 8% polyacrylamide gels in capillary tubes. The gels were then extru...

Antigen presentation on MHC class I molecules is a key event for CD8+T cell development as well as initiation and maintenance of immunological responses against intracellular microorganisms. For a cellular immune response to be initiated, peptides derived from invading pathogens need to be generated, loaded onto MHC class I molecules in the endoplasmic reticulum (ER) and displayed at the cell surface for CD8+ T cell recognition1,2. The generation of antigenic peptides is usually achieved by proteolytic degradation in the cytosol by the proteasome. Peptides are then selectively transported into the ER lumen by the transporter associated with antigen presentation (TAP) and loaded onto “peptide-receptive” class I molecules in a process assisted by several molecular chaperones and accessory proteins.

CYTOTOXIC T lymphocytes recognize fragments (peptides) of protein antigens presented by major histocompatibility complex (MHC) class I molecules. In general, the peptides are derived from cytosolic proteins and are then transported to the endoplasmic reticulum where they assemble with the MHC class I heavy chains and β2-microglobulin to form stable and functional class I molecules1–4. The proteases involved in the generation of these peptides are unknown. One candidate is the proteasome, a non-lysosomal proteinase complex abundantly present in the cytosol. Proteasomes have several proteolytically active sites and are complexes of high relative molecular mass (Mr about 600K), consisting of about 20–30 subunits with Mrs between 15 and 30K (refs 5–10). Here we show that at least one of these subunits is encoded by the mouse MHC in the region between the K locus and the MHC class II region, and inducible by interferon-γ. This raises the intriguing possibility that the MHC encodes not only the MHC class I molecules themselves but also proteases involved in the formation of MHC-binding peptides.

Expression of mouse major histocompatibility complex (MHC) class I molecules in different cell lines derived from Syrian hamsters has revealed antigen presentation deficiencies of some H2 allelic products in two cell lines (BHK and NIL-2) which were overcome by transient expression of the rat transporter associated with antigen processing (TAP; Lobigs et al. 1995). Here we show that in both cell lines the endogenous MHC class I cell surface expression was completely down-regulated. Lymphokine treatment induced endogenous and recombinant mouse MHC class I cell surface expression to levels similar to that in other Syrian hamster cell lines competent for antigen presentation through transduced H2 molecules. Accordingly, constitutive downregulation of expression of accessory molecules of the MHC class I pathway can reveal differences between H2 class I alleles in antigen presentation not encountered when the expression levels are augmented. In addition to the differential expression of MHC class I pathway genes, two cell lines representing competent (FF) and defective (BHK) antigen presentation phenotypes for mouse class I MHC restriction elements demonstrated substantial sequence polymorphism in Tap1 but not Tap2. Cytokine-treated FF or BHK cells and human TAP-deficient T2 cells transfected with FF or BHK TAP1 in combination with FF TAP2 differed in their preference for C-terminal peptide residues, as shown by an in vitro peptide transport assay. Thus, polymorphic residues in TAP1 can influence the substrate selectivity of the Syrian hamster peptide transporter.

HLA-DM (DM) functions as a peptide editor by catalyzing the release of class II-associated invariant chain peptides (CLIP) and other unstable peptides, thus supporting the formation of stable class II-peptide complexes for presentation. To investigate the general features that determine the DM susceptibility of HLA-DR1/peptide complexes, we generated a large DM-sensitive peptide repertoire from an M13 bacteriophage display library using a novel double selection protocol: we selected bacteriophage capable of binding to DR1 molecules and, subsequently, we enriched DR1-bound bacteriophage susceptible to elution by purified DM molecules. Sequence and mutational analyses of the DR1/DM double-selected peptides revealed that the amino acids Gly and Pro play a destabilizing role in the dissociation kinetics of DR1 ligands. This observation was confirmed also in natural peptide sequences such as CLIP 89-101, HA 307-319 and bovine collagen II (CII) 261-273. Our results demonstrate that DM susceptibility does not only depend on the number and nature of anchor residues, or the peptide length. Instead, less obvious sequence characteristics play a major role in the DM editing process and ultimately in the composition of peptide repertoires presented to T cells.

The effect of peptide length on the stability of peptide-HLR-DR1 (DR1) complexes was analyzed using two peptide series of increasing length, each containing a 7mer core with five DR1-binding anchors, extended stepwise with Ala residues at the N- and C-terminus, respectively. The Ala extensions, although did not affect binding affinity, significantly increased the half lives of peptide-DR1 complexes (from 1.5 h up to 10 h) in live antigen presenting cells (APC). Flanking residues from position -2 to 0 and 8 to 11 were involved in the affinity-independent increase of complex stability. The shortest (8mer and 9mer) peptides, with in vivo half lives of2.5 h, were unable to form stable complexes with DR1 in presence of HLA-DM (DM) molecules, and were poor competitors of antigen presentation. Longer peptides were resistant to DM-mediated unloading, and were efficient competitors of antigen presentation. Thus, DM appears to limit short peptides in establishing biologically relevant DR occupancy, despite their high binding affinity. In APC, stable complexes can form only with high affinity peptides of9 residues, and the longevity of complexes seems to depend on full of occupation of the binding site.

In B cells, the non-classical human leukocyte antigens HLA-DO (DO) and HLA-DM (DM) are residents of lysosome-like organelles where they form tight complexes. DM catalyzes the removal of invariant chain-derived CLIP peptides from classical major histocompatibility complex (MHC) class II molecules, chaperones them until peptides are available for loading, and functions as a peptide editor. Here we show that DO preferentially promotes loading of MHC class II molecules that are dependent on the chaperone activity of DM, and influences editing in a positive way for some peptides and negatively for others. In acidic compartments, DO is engaged in DR-DM-DO complexes whose physiological relevance is indicated by the observation that at lysosomal pH DM-DO stabilizes empty class II molecules more efficiently than DM alone. Moreover, expression of DO in a melanoma cell line favors loading of high-stability peptides. Thus, DO appears to act as a co-chaperone of DM, thereby controlling the quality of antigenic peptides to be presented on the cell surface.

Publisher Summary This chapter reviews the generation and TAP-mediated transport of peptides for major histocompatibility complex class I molecules. The translocation of peptides into the endoplasmic reticulum (ER) is achieved by the transporter associated with antigen processing (TAP transporter), which belongs to a large family of membrane translocators containing an ATP-binding cassette. The peptide transporter TAP is thus a crucial link among the peptide generation machinery in the cytosol, the proteasome, and the peptide-presenting MHC class I molecule in the lumen of the ER. The peptide translocation mechanism enables T lymphocytes to continuously probe for foreign microbes that have invaded the cell and propagate in the cytosol. TAP also allows the immune system to sense the presence of peptides generated by mutational events in the course of tumorigenesis. The environmental pathogens have been the major evolutionary force driving the development of the antigen presentation pathway because most tumors arise rather late in life and do not affect a whole population, as in the case of infectious agents.

HLA-DM (DM) is a non-classical major histocompatibility complex (MHC) class II molecule that interacts with classical MHC II molecules in acidic compartments. During this association DM is supposed to catalyze the release of invariant chain (II)-derived CLIP peptides thereby rendering the peptide binding groove accessible for antigenic peptide loading. However, in situations of peptide scarcity the fate of these DM:DR complexes is not known. We could show that DR molecules incubated at lysosomal pH in the absence of peptide rapidly undergo functional inactivation and aggregation. In the presence of DM, however, empty DR molecules were shown to be stabilised and kept receptive for peptide loading, with the degree of the stabilising effect of DM varying for different DR alleles. In addition, in lysosomal compartments a considerable fraction of DM was found to be stably associated with empty DR alpha beta dimers thereby preserving their functionality. Upon encounter with antigenic peptide the DM-associated DR molecules could be rapidly loaded, whereupon they did no longer bind to DM. Thus, DM seems to act as a dedicated class II-specific chaperone that rescues uncharged alpha beta dimers. In view of the suggested shortage of self-peptides in the loading compartment, empty class II molecules that are kept receptive for loading by the chaperone function of DM may enable the antigen processing system to respond promptly to the challenge by newly entering antigens.

Considerable progress has been made in the field of major histocompatibility complex (MHC) class II-restricted antigen presentation. The analysis of mutant cell lines defective in antigen presentation revealed a central role for the nonclassical MHC class II molecule HLA-DM. Cell biological and biochemical characterization of HLA-DM provided deeper insight into the molecular mechanisms underlying the loading process: HLA-DM accumulates in acidic compartments, where it stabilizes classical class II molecules until a high-stability ligand occupies the class II peptide binding groove. Thus, HLA-DM prevents empty alpha beta dimers from functional inactivation at low endosomal/lysosomal pH in a chaperone-like fashion. In the presence of peptide ligands, HLA-DM acts as a catalyst for peptide loading by releasing CLIP, the residual invariant chain-derived fragment by which the invariant chain is associated with the class II molecules during transport from the endoplasmic reticulum to the loading compartments. Finally, there is accumulating evidence that HLA-DM functions as a peptide editor that removes low-stability ligands, thereby skewing the class II peptide repertoire toward high-stability alpha beta: peptide complexes presentable to T cells.

The endoplasmic reticulum (ER)-resident stress protein gp96 induces a major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response against antigens present in the cells from which it has been prepared. In this study, photoreactive peptides were translocated into the ER by the transporter associated with antigen processing (TAP). These peptides can be cross-linked specifically to gp96. Thus, we provide the first evidence that gp96 binds TAP-translocated peptides which have been implicated in the induction of specific CTL responses after immunization with gp96 (Srivastava, P. K. et al., Immunogenetics 1994. 39: 93).

Loading of classical major histocompatibility complex (MHC) class II molecules with antigen-derived peptides is fast, efficient and highly selective in vivo, quite in contrast to in vitro findings with isolated class II proteins and synthetic peptides. Do accessory proteins speed up the loading process in antigen-presenting cells? Here, a model is presented in which the nonclassical MHC class II molecule HLA-DM plays a pivotal role as a chaperone, catalyst and editor during epitope selection.

Antigen recognition and discrimination by T lymphocyte are essential in initiating appropriate immune responses. The mechanisms underlying exquisite sensitivity and specificity of antigen discrimination are not fully elucidated but involved physical intercellular interactions between T cell and antigen-presenting cell (APC) including dendritic cells (DCs) and B cells. Using single cell force spectroscopy (SCFS), we have successfully probe mechanical interactions between DC and T cells in response to a panel of altered peptide ligands (APLs). In the presence of different type of APLs, DC:T conjugate displays different ranges of mechanical forces that match their corresponding T cell responsiveness. Strong cell-cell interaction forces are contributed not only by surface adhesion molecules including TCR:pMHC, CD28:B7 and ICAM-1:LFA-1 interaction pairs, but also dependent on the integrity of membrane cholesterol and cytoskeleton dynamics. As compared to B:T interactions, stronger DC:T cell-cell interactions are likely to provide a mechanically stable environment that induces potent functional T cell activation, suggesting that the measurement of mechanical forces could be a significant predictor of T cell functional activation efficacy. Our SCFS studies provide mechanical insights into the adhesive roles of cell surface proteins in regulating APC:T cell-cell interactions. Functional roles of TLR ligands in regulating APC:T interactions and T-cell responses will be discussed.

Efficient regulatory T cell (Treg) depletion leads to tumor rejection in mice. The goal of this study was to understand the mechanisms involved in this rejection process. For the selective and efficient depletion of Tregs, we used the FoxP3.LuciDTR-4 mouse model where Tregs express, among others, the human diphtheria toxin receptor. Our previous studies show that the depletion of more than 90% of Tregs in FoxP3.LuciDTR-4 mice promotes the infiltration of immune cells and subsequent rejection of ovalbumin (OVA)-expressing B16 melanomas. To unravel the mechanisms involved, we characterized the tumor-infiltrating immune populations and performed a cytokine profile on tumors at the onset of rejection. The analysis revealed a change in the population composition of the tumor-infiltrating leucocytes. There was an increase in T cell numbers, especially CD8+ T cells, and dendritic cells in tumors in rejection. Moreover, the amount of B cells was drastically reduced in tumors being rejected while they were enriched in tumors in progression. Tumor-infiltrating myeloid cells also differed phenotypically between tumors in rejection and tumors in progression. Inflammatory cytokines related to the type 1 response such as IFN-γ, TNF and IL-12 were significantly more expressed in tumors in rejection as measured at mRNA and protein levels. On the other hand, the expression of some cytokines related to the type 2 response such as IL-5 and IL-10 and of the inhibitory cytokine TGF-β did not change between tumors in rejection and in progression. We also determined that the concentration of molecules related to the M1 macrophage phenotype such as inducible nitric oxide synthase (iNOS), interferon regulatory factor 5 (IRF5), CXCL9 and CXCL10 was significantly higher in tumors in rejection; however, the concentration of most of the molecules related to the M2 macrophage phenotype such as CD163, macrophage mannose receptor 1 (MRC1), Fizz-1 and PPAR-γ was not different between tumors in rejection and tumors in progression. Interestingly, angiogenesis drivers like VEGF and angiotensin 1 (ANG1) were found to be significantly less expressed in tumors in rejection, a fact that correlates with the normalization of the tumor vasculature previously described to be promoted by Treg depletion. We conclude that Treg depletion provokes a strong change in the tumor microenvironment, not only in terms of the elevated number of immune infiltrating cells but also in terms of the enrichment of certain subpopulations. Furthermore, this change is associated with a striking alteration of the cytokine and chemokine profile that becomes proinflammatory, supports mainly a Type 1-related response and inhibits angiogenesis pathways. Citation Format: Oscar C. Salgado, Rafael Carretero, Xingrui Li, Ibrahim M. Sektioglu, Natalio Garbi, Günter J. Hämmerling. Tumor rejection driven by efficient Treg depletion is associated with a strong change in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B79.

Human cytomegalovirus (HCMV) genes expressed in the early phase of infection mediate the destabilization of nascent major histocompatibility complex (MHC) class I molecules in infected cells and thus prevent antigen presentation to CD8+ T lymphocytes. We report that HCMV genes interfere with the MHC class I pathway of antigen presentation by at least two mechanisms. Firstly, permissive infection of fibroblasts is characterized by a continuous decline in the capacity to translocate peptides from the cytosol into the endoplasmic reticulum (ER) by the transporter associated with antigen processing (TAP). Inactivation of peptide transport is operative despite augmented TAP expression during HCMV infection. Secondly, TAP molecules fail to associate with MHC class I heavy chains indicating that HCMV early gene expression also interferes with MHC class I maturation. A temperature-sensitive mutant of HCMV, ts9, which lacks 15 kb of DNA encoding the genes US1–US15 of HCMV, had lost the capacity to interfere with MHC class I assembly and to inhibit the peptide translocation function of TAP. One of the genes deleted in ts9, US11, which was reported to downregulate the expression of MHC class I molecules, does not affect peptide transport by TAP. Therefore, we conclude that HCMV encodes at least two early gene functions that interfere with the MHC class I antigen presentation pathway.

The most important mechanism for the establishment of self tolerance is deletion of self-reactive thymocytes during naturation in the thymus (reviewed in 1). In addition to this central thymic tolerance, there is also a requirement for peripheral tolerance induction of T cells that have not been deleted in the thymus, possibly because the antigen was not present in the thymus or was present in amounts insufficient for deletion. Since soluble peripheral antigens can be taken up by major histocompatibility complex (MHC) class II positive cells in the thymus and induce thymic tolerance (2), peripheral tolerance induction appears to be particularly important for CD8+ cells, because soluble antigens usually do not enter the MHC class I pathway. Detailed knowledge of tolerance induction in peripheral T cells is essential for therapeutic intervention in autoimmune diseases, organ transplantation, or in attempts to break nonresponsiveness to tumor antigens.

Since the pioneering work of Townsend and colleagues we know that class I molecules of the major histocompatibility complex (MHC) present antigenic peptides at the cell surface mostly originating from endogenously synthesized proteins that have no access to the secretory compartment.1–3 Following their formation through the activity of cytosolic proteases, such peptide epitopes need to be transported across the membrane of the endoplasmic reticulum (ER) in order to associate with the peptide binding groove of newly synthesized class I molecules. This topological problem prompted the search for a cell-biological mechanism mediating vectorial peptide transport into the ER. Five years ago, genetic approaches provided a satisfying explanation with the discovery of putative transporter molecules in the ER membrane that are now called TAP (“transporters associated with antigen processing”).

During pregnancy a semiallogeneic fetus survives despite the presence of maternal T cells specific for paternally inherited histocompatibility antigens. A mouse transgenic for a T cell receptor recognizing the major histocompatibility (MHC) antigen H-2Kb was used to follow the fate of T cells reactive to paternal alloantigens. In contrast to syngeneic and third-party allogeneic pregnancies, mice bearing a Kb-positive conceptus had reduced numbers of Kb-reactive T cells and accepted Kb-positive tumor grafts. T cell phenotype and responsiveness were restored after delivery. Thus, during pregnancy maternal T cells acquire a transient state of tolerance specific for paternal alloantigens.

The invariant chain (Ii) prevents binding of ligands to major histocompatibility complex (MHC) class II molecules in the endoplasmic reticulum and during intracellular transport. Stepwise removal of the Ii in a trans-Golgi compartment renders MHC class II molecules accessible for peptide loading, with CLIP (class II-associated Ii peptides) as the final fragment to be released. Here we show that CLIP can be subdivided into distinct functional regions. The C-terminal segment (residues 92-105) of the CLIP-(81-105) fragment mediates inhibition of self- and antigenic peptide binding to HLA-DR2 molecules. In contrast, the N-terminal segment CLIP-(81-98) binds to the Staphylococcus aureus enterotoxin B contact site outside the peptide-binding groove on the alpha 1 domain and does not interfere with peptide binding. Its functional significance appears to lie in the contribution to CLIP removal: the dissociation of CLIP-(81-105) is characterized by a fast off-rate, which is accelerated at endosomal pH, whereas in the absence of the N-terminal CLIP-(81-91), the off-rate of C-terminal CLIP-(92-105) is slow and remains unaltered at low pH. Mechanistically, the N-terminal segment of CLIP seems to prevent tight interactions of CLIP side chains with specificity pockets in the peptide-binding groove that normally occurs during maturation of long-lived class II-peptide complexes.

Prior to their association with major histocompatibility complex (MHC) class I molecules, peptides generated from cytosolic antigens need to be translocated by the MHC-encoded peptide transporter (TAP) into the lumen of the endoplasmic reticulum (ER). While class I molecules possess well-known binding characteristics for peptides, the fine specificity of TAP for its peptide substrates has not been analyzed in detail. Previously, we have studied the effect of amino acid variations at the N-terminal, the C-terminal, and the penultimate residue on the efficiency of peptide translocation. Using permeabilized cells, we have shown that TAP pre-selects peptides in an allele- and species-specific manner, for which only the C-terminal residue is crucial. This finding is confirmed in the present study by using microsomes containing different TAP. The influence of amino acid substitutions at positions 2 to 7 of 9-residue model peptides on TAP-dependent peptide translocation is systematically examined. Only a few amino acid substitutions at these positions affect the efficiency of peptide translocation significantly, e.g. Pro at position 2 or 3 negatively influences transport whereas Glu at positions 6 and 7 enhances transport. The differences in translocation by the rat TAP alleles a or u, mouse TAP and human TAP are, however, minor for the peptide with internal substitutions used in this study. These results show that the C-terminal residue essentially governs the species-specific substrate specificity of TAP.

In normal antigen-presenting cells, newly synthesized major histocompatibility complex (MHC) class II molecules associate with the invariant chain (Ii) glycoprotein in the endoplasmic reticulum (ER). They are loaded with peptides only after proteolytic removal of the Ii in post-Golgi endocytic vesicles. Since the Ii inhibits peptide binding to MHC class II molecules, this association could protect MHC class II molecules from being loaded with endogenous peptides early after biosynthesis. If this were an important function of the Ii in vivo, MHC class II molecules synthesized in cells lacking the Ii should be loaded efficiently with short endogenous peptides in the ER; such peptides are known to be present there due to TAP-mediated import from the cytosol. To examine this possibility, we have studied peptide loading in HeLa transfectants expressing murine H-2Ak MHC class II molecules either alone or together with an excess of Ii. Endogenous peptides could readily be extracted from conformationally intact Ak alpha beta dimers of biosynthetically labeled Ii+ cells, whereas peptide loading was greatly (95%) diminished in the absence of Ii. Significant amounts of sodium dodecyl sulfate-(SDS) stable 55-kDa peptide: Ak complexes were only found in the Ii+ transfectants. In the absence of Ii, the MHC class II molecules instead formed stable complexes with long (20 and 50 kDa) polypeptides. Known Ak-binding peptides bound stably to Ak molecules on Ii- cells, could be co-purified with them, and were resistant to release in SDS, suggesting that poor recovery of endogenous peptides was not due to decreased stability of Ak:peptide complexes in the absence of Ii. We conclude that protection of MHC class II molecules from endogenous short peptides does not appear to be a quantitatively important function of the Ii molecule, because peptide loading is inefficient in its absence.

Antigen-specific immunosuppression requires an understanding of the parameters that control peripheral T cell tolerance. A liver-specific inducible promoter was used to drive the expression of the major histocompatibility complex antigen Kb in transgenic mice. Minute amounts of Kb, expressed exclusively on hepatocytes, induced tolerance by partial down-regulation of the T cell receptor (TCR) on the self-reactive CD8+ cells. Contact of these tolerant T cells with high concentrations of Kb after induction led to complete down-regulation of TCR. Thus, tolerant T cells are susceptible to further tolerogenic signals and reach different levels of tolerance depending on antigen dose.

Tolerant T cells are characterized by their partial or full resistance to activation by antigen. We investigated whether tolerant T cells were still receptive to further tolerogenic signals. T cells expressing a transgenic T cell receptor (TCR) specific for the major histocompatibility complex (MHC) class I molecule Kb were deleted in mice carrying Kb but not in mice expressing the mutant Kb-molecule Kbm1 [TCR (H-2bm1 × k) mice]. These T cells were tolerant in vivo but could be activated in vitro by the Kb antigen. This in vitro reactivity was abolished after the tolerant T cells encountered Kb-positive cells that had been intravenously injected. Furthermore, in TCR (H-2bm1 × k), mice expressing Kb only on hepatocytes, no T lymphocytes bearing the transgenic TCR could be found in the periphery, indicating that the additional contact with Kb on hepatocytes led to deletion of the tolerant T cells. These findings demonstrate that tolerance induction can be a multi-step process.

The accurate discrimination of the immune system between pathogens and self-determinants is based on specific recognition events by receptors on B and T lymphocytes. The enormous diversity of the receptor repertoire is achieved by random rearrangement of DNA segments. In this process specificities for non-self as well as for self-determinants are created. T lymphocytes with high affinity for self-antigens are physically eliminated when they develop in the thymus provided the respective self-antigen is present on bone marrow derived cells (von Boehmer, 1990). Clonal deletion appears to be highly sensitive. Even thymocytes with an avidity below the required threshold for activation in the periphery are deleted (Pircher et al., 1991; Knobloch et al., 1992). On the other hand, thymocytes are not deleted but rendered functionally incompetent (clonal anergy) when the self-antigens are exclusively expressed on thymic epithelial cells (Ramsdell et al., 1989; Salaun et al., 1990; Hoffmann et al., 1992; Schonrich et al., 1992a).

Publisher Summary Mature T lymphocytes can be rendered tolerant of antigens expressed outside the thymus. Depending on the tolerogenic signals, T cells reach various levels of tolerance that are distinct because of their capacity for reactivation. Tolerance induction can occur in one step or in several consecutive steps. Distinct mechanisms of extrathymic tolerance differ in their efficiency in protecting against the breakdown of self-tolerance and against autoimmunity. The finding that different tissues can induce different levels of tolerance could explain why some organs are more susceptible to autoimmune diseases than others. Strategies to improve immunosuppression in allograft transplantation and autoimmune diseases should concentrate on suitable methods for eliminating the circulating T cell pool that is responsible for graft rejection or the onset of an autoimmune disease. Because allograft-specific or autoreactive T cells newly exported from the thymus would undergo peripheral tolerance induction, specific immunosuppression could be achieved this way.

The murine antigen-processing-defective mutant cell line RMA-S is leaky in the presentation of certain endogenously synthesized minor histocompatibility and viral antigens to major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTL). The viral antigens include influenza virus nucleoprotein, vesicular stomatitis virus (VSV) nucleocapsid and Rauscher murine leukemia virus (MuLV) antigen. Here we demonstrate Sendai virus antigen presentation by the HAM2 (murine TAP2, transporter associated with antigen presentation type 2)-defective RMA-S cell line and compare antigen presentation after restoration of the defect by murine TAP1/2 gene transfection. Kinetic studies revealed that RMA-S cells required 2-3 h longer incubation and approximately 10 times higher doses of Sendai virus to reach the same level of killing as the RMA parental line. After transfection of RMA-S cells with the murine TAP1/2 gene, Sendai virus antigen presentation was restored to levels of the RMA wild-type line with regard to time of virus infection and dose of virus needed for sensitizing target cells. The presentation of Sendai virus antigen in RMA-S cells was sensitive to brefeldin A (BFA), suggesting that the presentation was mediated via the endogenous pathway. Our findings confirmed leakiness of antigen presentation in RMA-S cells and extended it to Sendai virus. The results underscored the role for intact expression of the TAP 1/2 molecules for efficient MHC class I-mediated antigen presentation.

Publisher Summary This chapter discusses the mechanisms of peripheral tolerance. It appears that nature has developed multiple backup systems to take care of the potentially autoreactive cells that have not been physically deleted in the thymus. The finding that different tissues can induce different levels of tolerance could explain why some organs are more susceptible to autoimmune diseases than others. Peripheral tolerance can be mediated by several distinct mechanisms. These include the existence of clonotype-positive but in vivo functionally inert T cells, downregulation of the TCR that is reversible by MLR, and downregulation of the TCR not reversible by MLR but reversible by stimulation with anti-CD2.

The establishment and maintenance of self tolerance is based on multiple events in the thymus and periphery, resulting in either deletion of self-reactive T cells or induction of nonresponsiveness. Here, Bernd Arnold and colleagues propose that, depending on the tolerogenic signals, peripheral T cells can reach different levels of tolerance with regard to their capacity for reactivation. In addition, it appears that tolerant T cells are still susceptible to further tolerogenic signals, driving them into a deeper state of tolerance. Thus, induction of T-cell tolerance can be viewed as a multistep mechanism.

DURING cell differentiation in the thymus, self-reactive T cells can be generated. The majority of these seem to be deleted after intrathymic encounter with the relevant autoantigen1. As all self antigens are unlikely to be present in the thymus, some autoreactive T cells may escape censorship. Here we study the fate of these cells using transgenic mice expressing the class I molecule H–2Kb(Kb) in the insulin-producing β -cells of the pancreas2,3. These mice were crossed with mice transgenic for genes encoding a Kb-specific T-cell antigen receptor (TCR)4 which could be detected using a clonotype-specific monoclonal antibody5. Although T cells expressing the highest level of transgenic TCR were deleted intrathymi-cally in double-transgenic mice, Kb-specific T cells were detected in the periphery. These cells caused the rejection of Kb-expressing skin grafts, but ignored islet Kb antigens even after priming. But when double-transgenic mice were crossed with transgenic mice expressing the lymphokine interleukin-2 in the pancreatic β-cells6, there was a rapid onset of diabetes. These results indicate that autoreactive T cells that ignore self antigens may cause autoimmune diabetes when provided with exogenous 'help' in the form of interleukin-2.

Thymocytes can be rendered tolerant by non-deletional mechanisms upon interaction with major histocompatibility complex (MHC) antigens on thymic epithelium. Whether the epithelial cells in the cortex or medulla could mediate this effect was not clear so far. To address this question, a transgenic mouse was generated in which the bovine keratin IV promoter was used to control expression of the alloantigen Kb. In the periphery the Kb transgene was expressed on a subset of keratinocytes. In the thymus expression was restricted to a subpopulation of medullary epithelial cells. No expression was found in the cortex. Such a tissue distribution has been reported for the keratin IV molecule demonstrating the faithfulness of the promoter used here. To follow the fate of the Kb-reactive thymocytes, this mouse was mated with another transgenic mouse expressing an anti-Kb T cell receptor (TcR). In the double-transgenic mice the CD8+CD4− thymocytes were not deleted but they were found to be anergic as assayed by their failure to be activated in vitro by either Kb-positive spleen cells or by cross-linked anti-TcR antibodies. These observations establish that expression of an MHC class I antigen in the thymic medullary epithelium is sufficient to induce anergy in the mature CD8+CD4− thymocyte population.

The T-cell repertoire is formed during T lymphocyte maturation in the thymus. There is, however, increasing evidence that there are additions to, and modifications of, this repertoire by extrathymic events. Most importantly, mature peripheral T cells are not only susceptible to activation signals but also to tolerance induction upon encounter of extrathymic antigen. An understanding of these inactivation processes should result in strategies for specific immunosuppression in organ transplantation and autoimmune diseases.

It has frequently been suggested that loss of beta 2-microglobulin (beta 2m) in tumor cells may lead to malignant progression due to escape from immunological recognition. Here, we directly tested the role of beta 2m expression in tumorigenicity. A beta 2 m loss mutant (C4.4-25-), selected from the murine lymphoma EL-4, showed a marked reduction in tumorigenicity as compared with EL-4 in normal C57B1/6 (B6) mice. The reduced tumorigenicity was directly related to beta 2 m expression. Transfection of an intact murine beta 2m gene markedly increased the tumorigenic potential. The reduced tumorigenicity of C4.4-25- compared with beta 2m transfected cells was observed also in athymic B6 nu/nu mice, but was abolished in B6 mice depleted of natural killer (NK) 1.1-positive cells. These results show that restoration of beta 2m expression can promote tumorigenicity and demonstrate for the first time that induction of major histocompatibility complex class I expression by transfection can lead to escape from NK cells in vivo.

Recent evidence suggests that endogenously produced antigenic peptides are required for assembly of major histocompatibility complex class I chains with beta 2-microglobulin and transport to the cell surface. The RMA-S mutant cells are thought to be defective in intracellular peptide loading to class I molecules and, therefore, devoid of class I surface expression. Here we report that at physiological temperature (37 degrees C) "empty" class I molecules appear at the cell surface of RMA-S cells where they can be trapped with H-2 antibodies. In the absence of the stabilizing ligand, the class I molecules rapidly alter their conformation but remain at the cell surface as demonstrated with a rabbit antiserum. Such denatured H-2 molecules can also be found on normal wild-type RMA cells. However, their amount is strongly reduced after culture of RMA cells with a class I binding peptide. These findings indicate that empty class I molecules appear at the surface not only on mutant but also on normal cells, suggesting that in normal cells the supply with peptides is limited.

The introduction of cloned genes into the germline of mice has been proven to be a powerful tool to investigate the role of the respective gene products within the immune system. Here we summarize the transgenic mouse models that have been established with major histocompatibility complex (MHC) class-I genes. Foreign class-I alleles can be expressed in transgenic mice according to their normal expression patterns as authentic self molecules and can function in T-cell responses in the same way as endogenous class-I molecules. Since this is also true for most of the introduced human HLA class-I alleles, there is great interest in establishing mouse models for HLA-linked diseases. A new field of experimental approaches concerning self-tolerance has been opened by tissue specific expression of MHC antigens under specific promoters. Besides negative selection in the thymus, peripheral mechanisms could be identified that induce and maintain self-tolerance.

In a previous report we described how cross-immunizations of pairs of transgenic mice expressing different HLA class I antigens led to the production of antibodies directed exclusively at polymorphic epitopes. This was ascribed to self-tolerance of HLA that prevents immune responses to monomorphic epitopes and focuses responses on polymorphic ones. In the present report we extend our findings and demonstrate that immunizations of class I transgenic mice with HLA transfected mouse fibrosarcoma as well as with human lymphoblastoid cells also preferentially yield antibodies to polymorphic epitopes. This was the case whether or not immunizations were carried out across locus barriers [e.g., Tg(HLA-A *0201) or Tg(HLA-Cw*0301) transgenic mice immunized with HLA-B27 transfectants] or within the same locus [e.g., Tg(HLA-B*1302) transgenic mice immunized with HLA-B27 transfectants or B27-expressing lymphoblastoid cells]. Use of an extended immunization protocol with four or more booster injections favored antibodies of IgG isotype with affinities high enough to lyse normal peripheral blood lymphocytes (PBLs) in complement-dependent cytotoxicity assays and to immunoprecipitate HLA antigens. The specificities covered by the monoclonal antibodies (mAbs) could be either broad or narrow, depending on the genetic distance of the HLA antigens or alleles involved. For instance, a Tg(HLA-B*1302) transgenic mouse immunized with B27 produced both broad B7/B27-specific antibodies, Bw4-specific antibodies, and one antibody reacting with all B alleles except B13 and with some C alleles. On the other hand, a Tg(HLA-B*1302) transgenic mouse immunized with Bw47 transfectants responded narrowly with an antibody to Bw60 and Bw47. Thus it appears that by choosing appropriate recipient mice and closely related or more distant HLA antigens, antibodies of a programmed specificity can be generated.

Discordant differentiation antigen pattern in a case of Richter's syndrome with monoclonal idiotype expression and immunoglobulin gene rearrangement

Several reports have suggested that an inverse correlation exists between major histocompatibility complex class I expression and the susceptibility to natural killer (NK)-mediated lysis. For example, the increased class I expression induced by interferon-gamma was always accompanied by an increased resistance to NK lysis. Likewise, class I loss variants were often more NK susceptible than their normal counterparts. To investigate whether the inverse correlation between class I expression and NK susceptibility was fortuitous or whether the class I molecules were directly responsible for this effect we resorted to gene transfection studies. From the murine thymoma line EL4 and H-2Db- and Kb-negative variant S3 was selected. This variant was highly susceptible to NK lysis. S3 was found to have a defect in beta 2-microglobulin gene expression. Therefore, restoration of Db and Kb expression could be achieved by transfection with the beta 2-microglobulin gene. This resulted in a strong decrease in susceptibility to NK lysis to the level of the H-2+ parental EL4. Transfection with class II genes had no effect. Blocking of the class I molecules on the H-2+ cells with anti-H-2b F(ab')2 fragments increased the susceptibility to NK cells to the level of the H-2- variant S3. These data demonstrate that the class I molecules on the targets are directly responsible for regulation of NK susceptibility but the mechanism is not clear. Possibly the class I molecules interfere with the unknown NK target structures.