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Long-bone growth by endochondral ossification is cooperatively accomplished by chondrocyte proliferation, hypertrophic differentiation, and appropriate secretion of collagens, glycoproteins, and proteoglycans into the extracellular matrix (ECM). Before folding and entering the secretory pathway, ECM macromolecules in general are subject to extensive posttranslational modification, orchestrated by chaperone complexes in the endoplasmic reticulum (ER). ERp57 is a member of the protein disulfide isomerase (PDI) family and facilitates correct folding of newly synthesized glycoproteins by rearrangement of native disulfide bonds. Here, we show that ERp57-dependent PDI activity is essential for postnatal skeletal growth, especially during the pubertal growth spurt characterized by intensive matrix deposition. Loss of ERp57 in growth plates of cartilage-specific ERp57 knockout mice (ERp57 KO) results in ER stress, unfolded protein response (UPR), reduced proliferation, and accelerated apoptotic cell death of chondrocytes. Together this results in a delay of long-bone growth with the following characteristics: (1) enlarged growth plates; (2) expanded hypertrophic zones; (3) retarded osteoclast recruitment; (4) delayed remodeling of the proteoglycan-rich matrix; and (5) reduced numbers of bone trabeculae. All the growth plate and bone abnormalities, however, become attenuated after the pubertal growth spurt, when protein synthesis is decelerated and, hence, ERp57 function is less essential.

Many aspects of an immune response are regulated by dendritic cells (DCs). DCs play key proinflammatory roles by sensing microbial invaders and promoting the activation of innate immune cells such as NK cells. In addition, DCs are required for the initiation and maintenance of adaptive T-cell responses against invading pathogens. Moreover, DCs also fulfill important anti-inflammatory functions: they induce peripheral T-cell tolerance by silencing self-reactive T cells and supporting the homeostasis of regulatory T cells (Tregs). A study in this issue of the European Journal of Immunology reveals that CD80/CD86 expression on DCs contributes to the maintenance of the peripheral pool of Tregs. This Commentary discusses current findings on this topic, focusing on the role of DCs in the homeostatic control of Tregs and myeloid cells, and the potential consequences for T-cell activation.

Dendritic cells (DC) are known to support the activation of natural killer (NK) cells. However, little is known about the role for DC in NK-cell homeostasis. In order to investigate this question, a novel bacterial artificial chromosome transgenic mouse model was generated in which the diphtheria toxin receptor is expressed under the CD11c promoter. In these mice efficient DC depletion can be achieved over prolonged periods of time by multiple injections of diphtheria toxin. We show here that NK cells require DC for full acquisition of effector function in vivo in response to the bacterial-derived TLR ligand CpG. Importantly, DC were found to play an instrumental role for maintaining normal homeostasis of NK cells. This is achieved by IL-15 production by DC, which supports the homeostatic proliferation of NK cells.

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.

Peptide loading onto MHC class II molecules takes place in endosomal compartments along the endocytic pathway. There, loading is facilitated by the catalytic function of the accessory molecule H2-M, which helps to exchange the invariant chain-derived CLIP peptide in the groove of class II molecules for antigenic peptide. H2-O is another accessory molecule specific to the class II pathway, which is found tightly associated with H2-M and selectively expressed in B cells. Using stable H2-O ribozyme-antisense transfectants, H2-O overexpressing murine B cell lines, and H2-O-transgenic mice, we investigated the effects of H2-O on antigen presentation. The results show that presentation of a variety of exogenous protein antigens to a panel of T cell hybridomas depended on the levels of H2-O in the antigen-presenting B cells. Thus, increased H2-O expression downmodulated, whereas reduced H2-O levels, enhanced presentation. Presentation of endogenous antigen was also diminished by H2-O. Despite the pronounced effects on antigen presentation, the mass spectrometric profiles of peptides eluted from Ab molecules were very similar in cells expressing different H2-O levels. The intracellular location of H2-O inhibitory activity was investigated with the drug chloroquine, which prevents acidification of the endocytic pathway. The observations indicate that H2-O predominantly inhibits antigen presentation in early endosomal compartments. Thus, H2-O appears to skew peptide loading to late endosomal/lysosomal compartments. This may favor presentation of antigens taken up by the B cell receptor.

Tapasin is a member of the MHC class I loading complex where it bridges the TAP peptide transporter to class I molecules. The main role of tapasin is assumed to be the facilitation of peptide loading and optimization of the peptide cargo. Here, we describe another important function for tapasin. In tapasin-deficient (Tpn(-/-)) mice the absence of tapasin was found to have a dramatic effect on the stability of the TAP1/TAP2 heterodimeric peptide transporter. Steady-state expression of TAP protein was reduced more than 100-fold from about 3 x 10(4) TAP molecules per wild-type splenocyte to about 1 x 10(2) TAP per Tpn(-/-) splenocyte. Thus, a major function of murine tapasin appears to be the stabilization of TAP. The low amount of TAP moleculesin Tpn(-/-) lymphocytes is likely to contribute to the severe impairment of MHC class I expression. Surprisingly, activation of Tpn(-/-) lymphocytes yielded strongly enhanced class I expression comparable to wild-type levels, although TAP expression remained low and in the magnitude of several hundred molecules per cell. The high level of class I on activated Tpn(-/-) cells depended on peptides generated by the proteasome as indicated by blockade with the proteasome-specific inhibitor lactacystin. Lymphocyte activation induced an increase in ubiquitinated proteins that are cleaved into peptides by the proteasome. These findings suggest that in the presence of a large peptide pool in the cytosol, a small number of TAP transporters is sufficient to translocate enough peptides for high class I expression. However, these class I molecules were less stable than those of wild-type cells, indicating that tapasin is not only required for stabilization of TAP but also for optimization of the spectrum of bound peptides.

Summary: The versatility of the bacteriophage Cre/LoxP system is dependent on the availability of a spectrum of tissue-specific Cre transgenic mice to address a host of biological questions. In this paper, we report on the generation of an inducible Tie2Cre transgenic mouse line that facilitates gene targeting exclusively in endothelial cells. The temporal manner of recombination is feasible through the use of a Cre-estrogen receptor fusion protein ERT2 and was, in practical terms, achieved by feeding the animals the estrogen antagonist tamoxifen orally for 5 weeks. High efficiency of recombination was found in the vast majority of endothelial cell populations examined, as monitored by an EGFP reporter mouse line. Critically, no EGFP expression was observed in any uninduced mice. This inducible Cre line will be a very beneficial asset to investigating the role of endothelial specific genes in the adult mouse and to induce transgenes in the endothelium in an extremely efficient manner. genesis 33:191–197, 2002. © 2002 Wiley-Liss, Inc.

Simian virus SV40 large T Antigen expression in the islets of Langerhans of transgenic mice results in β-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. © 2002 Wiley-Liss, Inc.

The use of the Cre/loxP system has greatly empowered the field of gene targeting. Here we describe the successful establishment of a novel knock-in EGFP reporter mouse line to monitor Cre-induced recombination in the vast majority of cell types. The value of this reporter mouse line is demonstrated by the use of a novel Tie2Cre transgenic mouse line that facilitates gene targeting in endothelial and hematopoietic cells. High efficiency of recombination was found in all endothelial cells and in the majority of hematopoietic cells but was absent in other tissues. Furthermore, in the second generation, the Tie2Cre mouse can be used to get 100% recombination of one allele, whilst allowing tissue specific in the second, therefore offering excellent efficiency.

HLA-DM is known to catalyze the exchange of class II-associated invariant chain (Ii) peptide (CLIP) for cognate peptide during biosynthesis. In DM-negative cells HLA-DR3 molecules have been shown to predominantly present CLIP and to lack the DR3-specific mAb epitope 16.23, which has led to the assumption that CLIP prevents binding of mAb 16.23. In the present study we show that CLIP does not prohibit 16.23 epitope expression, but that the formation of this epitope is directly influenced by interactions of the DR molecule with Ii and DM. Detergent solubilized DR3 from wild-type as well as DM(-) cells bound CLIP in a 16.23(+) mode. On cells, however, neither CLIP nor antigenic peptide bound to DR3 in a 16.23(+) conformation, unless HLA-DM was expressed. Thus, HLA-DM appears to alter the conformation of DR3 in a peptide-independent fashion. Since in DM-deficient cells that also lack Ii, DR3 molecules assembled in a 16.23(+) conformation, we conclude that during biosynthesis Ii and DM exert opposing conformational constraints, characterized by suppressing or releasing 16.23 epitope expression. These results imply that DR3/peptide complexes, including DR3/ CLIP, can exist in two conformations depending on previous interaction with DM, but independent of the nature of the peptide bound. We show that these naturally occurring class II conformers can be selectively recognized by T cells.

Peptide binding to classical major histocompatibility complex (MHC) class II molecules is known to be determined by the properties of the class II peptide binding groove but recently it turned out to be co-controlled by the activity of the non-classical MHC molecules HLA-DM and HLA-DO: HLA-DM functions as a mediator of peptide exchange. In addition, HLA-DM is a chaperone for MHC class II molecules in endosomal and lysosomal loading compartments because it stabilizes the empty MHC class II peptide binding groove and keeps it receptive for peptide loading until appropriate peptide ligands are captured. Since HLA-DM favors the generation of high-stability peptide-MHC class II complexes by releasing low-stability peptide ligands, DM activity affects the peptide repertoire presented on the cell surface of antigen-presenting cells. HLA-DO is expressed mainly in B cells and binds tightly to HLA-DM thereby modulating its activity. Together, HLA-DM and HLA-DO are critical factors in shaping the MHC class II-associated self or foreign peptide repertoire of antigen presenting cells and, hence, govern initiation or prevention of an immune response.

Unlike the main TCR alphabeta T cell lineage in which deletion occurs at the CD4+ CD8+ double-positive (DP) stage upon TCR engagement by antigen in the thymus, some T cells appear to require such engagement for their selection, either in the thymus or extrathymically. We used a transgenic TCR (tgTCR) model which, as we previously showed, led to selection upon expression of the corresponding antigen H-2Kb (Kb) in the thymus, of tgTCR/CD3(lo) CD4- CD8- double-negative (DN) thymocytes that expressed the NK1.1 marker (NK T cells) (Curnow, S. J., et al., Immunity 1995. 3: 427). We now report that antigen expression on medullary epithelial cells of the thymus failed to select the NK T cells, whereas its expression on thymocytes did, although tgTCR DP thymocyte development was affected under both conditions. Antigen expression on hepatocytes (Alb-Kb mice) did not perturb tgTCR DP thymocyte development. No enrichment in tgTCR NK T cells was detected in the periphery, except for the liver of the Alb-Kb/tgTCR mice. When reconstitution of thymectomized and irradiated H-2k hosts expressing or not Kb was performed with bone marrow from tgTCR H-2k mice, an enrichment in tgTCR+ NK T cells was found in the liver, but not in the spleen, of the hosts which expressed Kb, either selectively on hepatocytes or ubiquitously. Surprisingly, the majority of the hepatic tgTCR+ NK T cells also expressed the CD8 alpha/beta heterodimer. These results indicate that thymus-independent NK T cells with unique phenotypic characteristics can be selected upon antigen encounter in the liver.

T lymphocytes with self-destructive capacity are often found in healthy individuals, indicating efficient control mechanisms that prevent chronic autoimmune diseases. Since naive T lymphocytes do not circulate through extralymphoid tissues the concept has emerged that peripheral T cells ignore tissue-specific antigens unless they are presented by professional antigen-presenting cells in the lymphoid compartments. However, this view pays attention only to experiments performed in adult animals. This report reviews the evidence that tissues of neonatal mice, in contrast to adults, exhibit high accessibility for naive T cells, thereby allowing the direct contact with tissue-specific self-antigens on parenchymal cells during neonatal life and tolerance induction to such self-antigens. In mouse bone marrow chimeras generated at different ages, recent thymic emigrants were tolerized to a major histocompatibility class I antigen expressed on keratinocytes only during a neonatal period and not during adulthood. Blockade of T-cell migration neonatally prevented tolerance induction. The neonatally induced tolerance is maintained during adulthood, apparently by a dominant regulatory mechanism. Thus, parenchymal cells and T-cell migration in the neonate contribute to the control of autoreactive T cells.

Summary: Establishment of self-tolerance prevents autoaggression against organ-specific self-antigens. This beneficial effect, however, may In turn be responsible for tumor immune evasion. Thus, dissecting the mechanisms leading to the breakdown of self-tolerance in autoimmune diseases might provide insights for successful antitumor immune therapies. In a variety of animal models, organ- or tumor-specific immunity has been described, focusing on antigen-specific T-cell activation. Here, we discuss two trans -genic mouse models which demonstrate that both autoaggression and tumor rejection require more than activated, self-reactive T cells. TCR transgenic mice, which are tolerant to a liver-specific MHC class I antigen, Kb, can be activated to reject Kbb-positive grafts, but fail to attack Kb-expressing liver. However, autoaggression occurs when activated T cells are combined with “conditioning” of the target organ by irradiation or infection with a liver-specific pathogen. Similarly, in a mouse model of islet cell carcinoma, neither co-stimulatory tumor cells nor highly activated antitumor lymphocytes provoke an effective immune response against the tumor. Instead, a combination of activated lymphocytes and irradiation is required for lymphocyte infiltration into solid tumors. Both model systems provide evidence that although activated antigen-specific lymphocytes are a prerequisite for autoaggression, effector cell extravasation and appropriate interaction with the target organ/tumor are equally important. Thus, we propose that the organ/tumor microenvironment is a critical parameter in determining the effectiveness of an anti-self immune response.

The assembly of newly synthesized MHC class I molecules within the endoplasmic reticulum and their association with the transporter associated with antigen processing (TAP) is a process involving the chaperones calnexin and calreticulin. Using peptide mapping by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry to identify a new component, we now introduce a third molecular chaperone, the thiol-dependent reductase ER-60 (ERp57/GRP58/ERp61/HIP-70/Q2), into this process. ER-60 is found in MHC class I heavy chain complexes with calnexin that are generated early during the MHC class I assembly pathway. The thiol reductase activity of ER-60 raises the possibility that ER-60 is involved in the disulfide bond formation within heavy chains. In addition, ER-60 is part of the late assembly complexes consisting of MHC class I, tapasin, TAP, calreticulin and calnexin. In a beta2-microglobulin (beta2m)-negative mouse cell line, S3, ER-60-calnexin-heavy chain complexes are shown to bind to TAP, suggesting that beta2m is not required for the association of MHC class I heavy chains with TAP.

Mice were immunized i.p. with soluble or heat-denatured protein antigens [ovalbumin, beta-galactosidase, or recombinant E7 protein of human papilloma virus type 16 (HBV)]. Heat-denatured (100 degrees C) preparations of these proteins were able to induce cytotoxic T lymphocytes (CTL) that recognize cells expressing the respective genes, whereas native protein was either inefficient or required up to 30-fold higher doses. If the heat-treated proteins were separated into aggregated and soluble fractions by ultracentrifugation, only the aggregated fractions were able to induce specific CTL; this is probably because of the easier access to one of the major histocompatibility complex class I loading pathways for exogenous antigen. Addition of the adjuvant aluminium hydroxide (alum) to aggregated proteins abolished their ability to induce CTL; thus, a condition leading to a strong antibody response appeared to inhibit CTL induction. Interestingly, immunization with heat-denatured ovalbumin plus alum increased the IgM/IgG1 ratio compared to immunization with native ovalbumin and alum. Immunization of B6 mice transgenic for an HLA-A2/H-2K(b) hybrid gene with heat-denatured, recombinant HPV 16-E7 protein induced D(b)-restricted CTL specific for the peptide 49-57 of E7, indicating that this epitope is immunodominant over any A2-restricted E7 epitope in these mice. A whole influenza virus preparation heated to 100 degrees C or even autoclaved was still able to induce virus-specific CTL and BALB/c spleen cells heated to 100 degrees C could still cross-prime minor H-specific CTL in B6 mice, although with lower efficiency than fresh spleen cells. Thus, aggregated proteins can be considered as components for future vaccines.

Antigenic peptide loading of classical major histocompatibility complex (MHC) class II molecules requires the exchange of the endogenous invariant chain fragment CLIP (class II associated Ii peptide) for peptides derived from antigenic proteins. This process is facilitated by the non-classical MHC class II molecule HLA-DM (DM) which catalyzes the removal of CLIP. Up to now it has been unclear whether DM releases self-peptides other than CLIP and thereby modifies the peptide repertoire presented to T cells. Here we report that DM can release a variety of peptides from HLA-DR molecules. DR molecules isolated from lymphoblastoid cell lines were found to carry a sizeable fraction of self-peptides that are sensitive to the action of DM. The structural basis for this DM sensitivity was elucidated by high-performance size exclusion chromatography and a novel mass spectrometry binding assay. The results demonstrate that the overall kinetic stability of a peptide bound to DR determines its sensitivity to removal by DM. We show that DM removes preferentially those peptides that contain at least one suboptimal side chain at one of their anchor positions or those that are shorter than 11 residues. These findings provide a rationale for the previously described ligand motifs and the minimal length requirements of naturally processed DR-associated self-peptides. Thus, in endosomal compartments, where peptide loading takes place, DM can function as a versatile peptide editor that selects for high-stability MHC class II-peptide complexes by kinetic proofreading before these complexes are presented to T cells.

The heterodimeric transporter associated with antigen processing (TAP1/TAP2) translocates peptides from the cytosol into the endoplasmic reticulum where loading of major histocompatibility complex class I molecules takes place. TAP transporters from different species are known to exhibit distinct transport specificities with regard to the C-terminal amino acid (aa) of peptides. Thus, human TAP (hTAP), and rat TAP (rTAP) containing the rTAP2a allele are rather promiscuous, whereas mouse TAP (mTAP), and rTAP containing the rTAP2a allele are restrictive and select against peptides with C-terminal small polar/hydrophobic or positively charged aa. The structural basis for this selectivity is not clear. To assess the relative contribution of the TAP1 and TAP2 subunits to transport specificity, we have constructed and analyzed interspecies TAP hybrids and point mutants of hTAP2 expressed in Sf9 insect cells and in TAP-deficient T2 cells. Transport assays with 20 C-terminal variants of the peptide RYWANATRSX showed that: first, transport specificity with regard to C-terminal aa is mainly influenced by TAP2, but TAP1 can also contribute. Second, the selective transport of peptides with C-terminal positively charged aa is critically controlled by the amino-terminal region (1-361) on the TAP2 chain, while transport of peptides with C-terminal small polar/hydrophobic aa is determined by residues located within as well as outside the region 1-361. Third, a single point mutation in hTAP2 (374A-->D) resulted in a drastic alteration of the transport pattern. These results indicate that both TAP1 and TAP2 contribute to efficient peptide transport and that single point mutations in hTAP2 are able to alter the peptide transport specificity. This opens the possibility that naturally occurring mutations in one of the hTAP subunits may alter epitope selection in vivo.

The major histocompatibility complex (MHC)-encoded transporters associated with antigen processing (TAP) translocate peptides from the cytosol into the lumen of the endoplasmic reticulum (ER) where they associate with MHC class I molecules. The length of class I-binding peptides is usually 8-11 amino acids, but examples of significantly longer peptides have been described. The preferred lengths and upper and lower size limits for peptides translocated by TAP have not been determined in detail because in the currently used test systems, peptides are subject to proteolytic degradation. In the present study, three sets of individual peptides or partially randomized peptide libraries ranging between 6 and 40 residues were used that contained a radiolabeled tyrosine and a consensus sequence for ER-specific N-glycosylation at opposite ends, thus ensuring that only nondegraded peptides were monitored in the transport/glycosylation assay. For three different transporters, rat TAP1/2a, rat TAP1/2u and hTAP, the most efficient ATP-dependent transport was observed for peptides with 8-12 amino acids. Hexamers and longer peptides of up to 40 amino acids were also translocated, albeit less efficiently. For two of the three sets of peptides analyzed, rat TAP1/2a showed a less stringent length selection than rat TAP1/2u and human TAP. The superior transport of the decamer of the TNKT.. Y series was not due to faster degradation or less efficient glycosylation of shorter or longer length variants. A binding assay with TAP-containing microsomes revealed a high affinity for the radiolabeled decamer (KD = 580 nM), while other length variants were clearly inferior in their binding affinities. Thus, TAP binds and preferentially translocates peptides with a length suitable for binding to MHC class I molecules, but peptides that are considerably longer may also be substrates. About 10(5) peptide binding sites per cell equivalent of microsomes were determined, providing an estimate for the number of TAP complexes in the ER membrane.

Unlike class I histocompatibility (MHC) antigens, most newly synthesized MHC class II molecules fail to be loaded with peptides in the endoplasmic reticulum (ER), binding instead to the invariant chain glycoprotein (Ii). Ii blocks the class II peptide binding groove until the class II:Ii complexes are transported to endosomes where Ii is removed by proteolysis, thus permitting loading with endosomal short peptides (approximately 12-25 amino acids). Ligands from which the groove is protected by Ii have not yet been identified; theoretically they could be short peptides or longer polypeptides (or both), because the class II groove is open at both ends. Here we show that in Ii- deficient cells, but not in cells expressing large amounts of Ii, a substantial fraction of class II alpha beta dimers forms specific, SDS-resistant 1:1 complexes with a variety of polypeptides. Different sets of polypeptides bound to H-2Ak, Ek, Ed and HLA-DR1 class II molecules; for Ak, a major species of Mr 50 kDa (p50) and further distinct 20 and 130 kDa polypeptides were detectable. Class II binding of p50 was characterized in detail. Point mutations within the Ak antigen binding groove destabilized the p50:class II complexes; a mutation outside the groove had no effect. A short segment of p50 was sufficient for association with Ak. The p50 polypeptide was synthesized endogenously, bound to Ak in a pre-Golgi compartment, and was transported to the cell surface in association with Ak. Thus, Ii protects the class II groove from binding endogenous, possibly misfolded polypeptides in the ER. The possibility is discussed that polypeptide binding is an ancestral function of the MHC antigen binding domain.

The major histocompatibility complex (MHC)-encoded transporter associated with antigen processing (TAP) delivers cytosolic peptides to the lumen of the endoplasmic reticulum (ER) for presentation by MHC class I molecules. For the rat, it has been demonstrated that TAP polymorphism results in the selection of different sets of peptides, the nature of the C terminus being of particular importance. Here, we investigated whether TAP polymorphism in mice and humans has functional consequences for transport of peptide sets variable at the C-terminal residues. Using cell lines of H-2d, H-2k, and H-2dxk haplotype and a panel of human lymphoblastoid cell lines expressing eight different TAP alleles, we detected species-specific transport patterns, but no significant influence of TAP polymorphism on peptide selection. In addition, peptides with different core sequences were translocated to the same extent by different TAP. These results suggest that a major contribution of human TAP polymorphism to disease progression and autoimmunity is not very likely.

The protein products of the Tap (Transporter associated with antigen processing) 1 and 2 genes are presumed to deliver peptides across the endoplasmic reticulum (ER) for assembly with major histocompatibility complex (MHC) class I molecules. The antigen processing-defective cell line RMA-S (H-2b) has a premature stop in the Tap 2 gene and probably therefore fails to deliver peptides into the ER, which leads to a low level of cell surface MHC class I molecules. Transfection of a Tap 2 gene restores to RMA-S both MHC class I molecule expression and the ability to present influenza viral antigens. We investigated the ability of RMA-S cells transfected with a Tap 2 gene to process and present alloantigens, Sendai and Rauscher viral antigens to allogeneic and virus-specific cytotoxic T lymphocytes. We found that allogeneic peptides as well as Rauscher and Sendai viral peptides can be processed and presented by RMA-S but at reduced levels. Transfection of a Tap 2 gene of mouse (BALB/c, H-2d) or rat origin into RMA-S increased the presentation of Sendai viral antigens and partially restored the presentation of allogeneic antigens. The already low level of Rauscher viral peptides presented by RMA-S is not elevated by transfection of either Tap 2 gene into RMA-S. This indicates a differential effect of transfection of a Tap 2 gene of rat or allogeneic mouse origin into RMA-S on viral antigen processing.

We have cloned and analysed the second mouse MHC-linked proteasome subunit, designated MC13, which appears to be homologous to the human RING10 proteasome protein. The isolated cDNA has an ORF encoding a protein of 276 amino acids with a molecular weight of ca. 30 kDa. Sequence alignment reveals that the subunit MC13 and several other mammalian proteasome subunits are encoded by a second proteasome gene family. This second gene family encodes subunits of the beta-type, reveals striking sequence similarities with the beta-subunit of archaebacterial proteasomes and is related to, but distinct from, the genes encoding the so-called alpha-type subunits.

The influence of the class II-associated invariant chain (Ii) on the presentation of the protein antigens hen egg-white lysozyme (HEL) and ribonuclease A (RNase) was investigated. For this purpose the Ii- rat-2 fibroblasts were transfected with I-Ak genes with or without Ii. Transfectants expressing Ii were superior in the presentation of the complete HEL protein to a panel of I-Ak-restricted T hybridomas characterized by distinct specificities for different HEL peptides and by different sensitivities to antigen concentration. There appeared to be a correlation between the antigen-presenting capacity and the amount of Ii, in that transfectants expressing large amounts of Ii were the best antigen presentors. The presentation of synthetic HEL peptides was not influenced by Ii. In contrast to the findings with HEL, the presentation of RNase by the same set of transfectants was clearly independent of Ii. Both antigens, HEL and RNase, required processing in the chloroquine-sensitive compartment. However, only the presentation of HEL but not of RNase could be efficiently blocked by brefeldin A. These data confirm that presentation of HEL depends on de novo synthesized class II molecules, whereas the presentation of RNase seems to be predominantly mediated by a pool of pre-existing class II molecules whose interaction with endocytosed antigen does not depend on Ii. These results suggest different mechanisms for the presentation of HEL and RNase and they raise the possibility that different antigens intersect the class II pathway at distinct intracellular locations.

The invariant (Ii) chain is a membrane-spanning glycoprotein found intracellularly associated with class II major histocompatibility complex (MHC) molecules. Using hybrid-selected translation and the Ii-specific monoclonal antibody In-1, we have isolated a cDNA clone (pIi-5) coding for most of the Ii chain. Sequence analysis of this clone reveals an open reading frame encoding 169 amino acid residues. The protein is rich in methionine and contains two potential N-glycosylation sites. No stretch of uncharged amino acid residues, characteristic for a membrane-spanning segment, is found close to the COOH-terminal end. There is one, however, close to the NH2-terminal end. As it is know that approximately 20 amino acid residues of Ii chain are exposed on the cytoplasmic side, we conclude that the Ii chain spans the membrane exposing the NH2 terminus on the cytoplasmic side and the COOH terminus on the luminal side.

Alloantiserum and hybridoma-derived anti-Ia antibodies were investigated for their idiotypic similarity using 4 monoclonal anti-idiotypes raised against monoclonal anti-Ia. With one anti-idiotype, idiotypic cross-reactivity could be found between three independently derived monoclonal BALB/c anti-Ia antibodies, all of which were directed against the same serological determinant, Ia.2. The respective idiotype, tentatively designated Ia.2 idiotope, could also be demonstrated on anti-Ia.2 antibodies in BALB/C alloantisera, but not on anti-Ia against other specificities. The expression of the Ia.2 idiotope is linked to the Igh allotype; the use of Ig-recombinant mice allowed us to map the Igh-Ia.2 locus to the left of the T15 marker in the Igha V region.

B10.BR mice received i.v. increasing doses of recombinant interferon-gamma (rIFN-gamma) on three consecutive days. Using an immunoperoxidase technique the distribution of H-2K/D antigens was studied in frozen tissue sections of thirteen organs (kidney, liver, pancreas, esophagus, stomach, small intestine, colon, lungs, heart, brain, thymus, lymph node and spleen). Class I antigens were shown to be induced or enhanced in almost every organ after exposure to IFN-gamma. This effect was particularly conspicuous for renal tubular cells, hepatocytes, bronchiolar epithelial cells, gastric mucous cells, thymic cortical lymphocytes and capillary endothelial cells in heart and kidney. Neurons, glial cells, gastric chief and parietal cells, and pancreas cells were not inducible. The findings show that i.v. application of IFN-gamma leads to strong induction or enhancement of major histocompatibility complex class I antigens in a wide variety of tissues.

It is well established that a large fraction of murine cytotoxic T cells can recognize allogeneic major histocompatibility complex (MHC) antigens, and that the majority of this response are not restricted by H-2 antigens of the responding host. In contrast, the murine response against the xenogeneic HLA class I antigens is relatively weak. Moreover, a large proportion of the responding murine T cells do not recognize the HLA antigen per se but only in an H-2-restricted manner, probably as an HLA peptide bound to H-2. Considerable evidence suggests that in mice the T cell repertoire is selected by thymic H-2 antigens. Therefore, we asked the question whether in transgenic mice expressing an HLA class I antigen the T cell repertoire would be shaped toward a more effective recognition of other HLA alleles. Normal C57BL/6 (B6) and HLA-Cw3-transgenic B6 mice were immunized with a B6-derived cell line transfected with HLA-A2. The resulting A2-specific CTL were tested on L cells transfected with either A2 alone, which should identify the H-2-unrestricted CTL, and on L cells transfected with A2 plus H-2b genes, which should identify the sum of H-2b-restricted and unrestricted CTL. Both bulk culture and limiting dilution experiments showed that the CTL precursor frequencies for A2-specific CTL were not increased in the transgenic mice, and that both strains produced comparable proportions of H-2b-restricted and of unrestricted A2-specific CTL. The B6.Cw3 mice seemed to respond less well to HLA-A2 than the normal B6 mice, suggesting the possibility of tolerance for peptides shared by the Cw3 and A2 molecules. In conclusion, the T cells in the B6.Cw3 transgenic mice did not seem to be selected towards a stronger and more unrestricted recognition of an allo-HLA antigen. The possible reasons are discussed.

MHC class I antigens play a crucial role in immunological functions, e.g. transplant and tumor rejection and antigen presentation. Whereas class I antigens are normally expressed on most adult tissues, albeit in varying amounts, embryonic as well as many tumor cells are characterized by the absence of major histocompatibility complex class I antigens on their cell surfaces. In this study the mechanism controlling the lack of class I expression was analyzed at the level of the chromatin structure. Five DNase I hypersensitive sites were determined at the H-2 D-locus of cell lines constitutively expressing class I genes. Two of them (DH1, located at the TATA box/transcription start site, and DH2, located at the enhancer/interferon response sequence) were absent in the fibrosarcoma IC9 in which expression of the silent Dk class I gene was not inducible. DH1 and DH2 remained absent even after fusion with class-I-positive cells. However, transfected Dk genes were expressed in IC9, and both DH1 and DH2, which were probably derived from the transfected gene, became detectable. In tumor cells expressing class I genes only after treatment with IFN-gamma (e.g. the lung carcinoma CMT64.5) or after in vitro differentiation (F9 embryonal carcinoma cells), DH1 and DH2 were already present before induction of class I expression. However, the intensity of the band indicative of DH2 was reduced in undifferentiated and differentiated F9 cells and in untreated CMT cells.

We have used the 3-Methylcholanthrene induced T-10 fibrosarcoma tumour cell system (H-2b xH-2k)F1 to elucidate the possible correlation between metastatic potential, expression of individual H-2 antigens and susceptibility to NK cells. Transfection of the non-metastatic and NK sensitive IC9 cells (Db+, Dk-, Kb-, Kk-) with the H-2Dk gene, altered the metastatic phenotype of the parental cells, yet had no effect on the susceptibility of these tumour cells to lysis by NK and did not elicit a specific CTL response in syngeneic hosts. Variants of the metastatic and NK resistant IE7 clone (Db+, Dk+, Kb-, Kk-), lacking H-2Dk, were selected by treatment with monoclonal anti H-2Dk antibodies and complement. These variants were sensitive to NK and poorly or non metastatic. Retransfection of 'Dk' 'loss' variants with the H-2Dk gene, resulted in the isolation of several clones expressing a wide range of metastatic phenotypes but maintained sensitivity to NK. These results indicate that the H-2D region of the MHC and or closely linked genes may be involved in the complex interrelationship between target susceptibility to NK and metastasis.

This study describes the generation of permanent T cell tissue culture lines by cell fusion techniques. The AKR strain-derived T cell tumor BW 5147 was hybridized in the presence of polyethylene glycol with various T cell populations isolated from antigen-sensitized mice. Surface analysis of resulting hybrid cell lines showed expression of both Thy-1.2 and H-2 antigens which are characteristic for the lymphocyte used for fusion. In contrast, in hybrids derived from spleen cells neither expression of Ig nor of B cell-typicyl Ia determinants was found suggesting either preferential hybridization of BW 5147 cells with T lymphocytes or extinction of B cell markers in hybrid cells. These hybrid lines which may display the immunological properties of the T cell population chosen are presently investigated for their antigen reactivity.

The B6.C-H-2bm12 has been examined serologically with a new set of reagents and several complementation studies were performed to determine the extent of the mutation. The results show that: (a) the mutation has also affected the site(s) bound by xenogeneic anti-Ia antibodies; (b) the IJb region was not affected; (c) complementation studies with stains bearing a, b, d, k and s haplotypes did not complement bm12 for the expression of the lost I-Ab specificities, suggesting a structural (rather than regulatory) gene alteration in bm12; (d) H-2 haplotypes b and bm12 could complement d to establish the Ia.22 specificity, indicating that Ia-1 and Ae are separate genes in the I-A subregion. In addition, an antibody to the gained specificity on bm12 is described.

The expression of Ia (immune response region-associated) antigens on the surface of lymphocyte subpopulations with defined immunological function has been investigated by negative selection of subpopulations with anti-Ia sera and complement. Ia determinants were found on both unprimed (IgM) and on primed (IgG) antibody-forming precursor cells. No Ia antigens were detected on the surface of helper T cells. In contrast, suppressor T cells were sensitive to treatment with anti-Ia sera and complement demonstrating the presence of Ia determinants on this T cell subpopulation.

The cell-mediated cytotoxic response against autologous cells modified with the sulfhydryl reagent I-AED [N-iodo-acetyl-N-(5-sulfonic-1-naphthyl) ethylene diamine] has been described by Levy, R. B., Shearer, G. M., Richardson, J. C. and Henkart, P. A., [J. Immunol. 1981. 127: 523.]. We have established two H-2Db- and eight H-2Kb-restricted, AED -specific long-term cytotoxic T lymphocyte (CTL) clones of C57BL/10 origin. The growth of these clones has been dependent upon presence of both antigen and interleukin 2. Cytotoxicity and proliferation analysis of AED-specific Kb-restricted CTL clones with target and stimulator cells from Kbm-mutant mice demonstrated two categories of clones (A and B) based on different reactivity patterns against hapten-modified Kbm-mutant cells. AED-modified target cells of bm5, bm6 and bm9 origin were lysed by type A clones but not by type B clones, in contrast to AED-modified bm3, bm8 and bm11 target cells which were lysed by type B but not by type A clones. None of the clones lysed AED-modified bm1 target cells, but all of them lysed AED-modified bm4 and bm10 target cells. The restriction fine specificities for both cytolytic and proliferative activities of the analyzed clones were identical. A panel of monoclonal antibodies (mAb) directed against the Kb molecule was used to inhibit lysis of AED-modified target cells by CTL clones. mAb which recognize a cluster of allodeterminants located in the second external domain (C1 domain) of the Kb molecule [Hammerling, G. J., Rusch, E., Tada, N., Kimura, S. and Hammerling, U., Proc. Natl. Acad. Sci. USA 1982. 79: 4737] only inhibited type B clones, whereas inhibition of both type of clones was observed with mAb specific for allodeterminants clustered in the N-terminal region of Kb. These findings suggest that different regions on the Kb molecule serve as self determinants for H-2-restricted cytotoxicity. We also demonstrate that allodeterminants recognized by mAb and self determinants involved in H-2 restriction need not be identical. Our data also support the notion that covalent AED modification of H-2 molecules is not necessary to generate the self plus X antigen for CTL recognition.

Spleen cells from C57BL/6 mice sensitized to the hapten (4-hydroxy-3-nitrophenyl)acetyl (NP) were hybridized with myeloma cells, and a variety of hybrid cell lines was isolated which secreted homogeneous anti-NP antibodies. The antibodies were purified by affinity chromatography and their chain composition, affinity and fine specificity were determined. All antibodies recovered from the primary immune response carried λ light and μ or γ1 heavy chains. Their variable portions were nonidentical but similar in terms of hapten-binding specificity with a higher affinity for the cross-reacting haptens (4-hydroxy-3,5-dinitro-phenyl)acetyl (NNP) and (4-hydroxy-5-iodo-3-nitro-phenyl)acetyl (NIP) than for the homologous hapten NP. This heteroclitic property as well as the presence of λ, μ and γ1 chains are characteristic for primary anti-NP sera of C57BL/6 mice. In contrast, four families of anti-NP antibodies, each with a characteristic fine specificity pattern, were found among the hybrid cell antibodies derived from the hyperimmune anti-NP response. The antibodies of one of these families were related to the antibodies recovered from the primary immune response in that they were heteroclitic and carried λ light chains. All members of the other groups expressed k chains and were nonheteroclitic. The finding of well-defined antibody families in this system and the isolation of their members enable us to approach the problem of V gene expression and diversification in a new way.

SUMMARY Approximately 50% of fibrosarcomas induced with methylcholanthrene A were found to be defective in H-2 expression. In tumours which lack H-2K antigens, H-2 gene transfection was used to restore H-2K expression. The de novo expression of H-2K reduced tumorigenicity and abolished the formation of metastases in syngeneic mice. Expression of H-2K seems to render the tumour cells immunogenic and leads to effective recognition of the tumour cells by the host immune system.

We and others have previously reported that homologous exchange of alpha 1 or alpha 2 domains between different alleles led to loss of most determinants recognized by cytotoxic T lymphocytes (CTL) raised against the parental H-2 class I antigens. Here we demonstrate that exchange of alpha 1 or alpha 2 domains between the Kk and Kd allele results in the formation of neodeterminants against which allospecific CTL can be generated in responder mice of various related and unrelated H-2 haplotypes. In fact, only CTL against the neodeterminants on the hybrid molecules were found and none against the parental Kk and Kd determinants. In addition our data show that recognition of the neodeterminants is H-2 unrestricted. These findings suggest that in the hybrid molecules the basic structure of a major histocompatibility complex molecule has been preserved although most parental allodeterminants on the alpha 1 and alpha 2 domains have been modified.