Radically altered T cell receptor signaling in glycopeptide-specific T cell hybridoma induced by antigen with minimal differences in the glycan group.

A T cell hybridoma raised against the synthetic glycopeptide T(72)(Tn) was used to study whether the initial TCR signaling events are markedly different when the hybridoma is stimulated with glycopeptides closely related to the cognate glycopeptide antigen. T(72)(Tn) has an alpha-D-GalNAc group O-linked to the central threonine in the decapeptide VITAFTEGLK, and the hybridoma is known to be highly specific for this carbohydrate group. T(72)(Tn)-pulsed APC induced tyrosine phosphorylation of the TCR-zeta 21- and 23-kDa proteins and the downstream p42/44 MAP kinase and strong IL-2 secretion. APC pulsed with T(72)(alpha-D-GlcNAc), which differs from T(72)(Tn) solely by the orientation of a hydroxy group in the carbohydrate structure, completely failed to induce detectable tyrosine phosphorylation and IL-2 secretion. APC pulsed with S(72)(Tn), which differs from T(72)(Tn) by not having a methyl group in the serine amino acid side chain to which the glycan is attached, induced partial tyrosine phosphorylation of the TCR-zeta 21-kDa protein, no tyrosine phosphorylation of the MAP kinases and no IL-2 production. Molecular modeling of the MHC/glycopeptide complex revealed that the dramatic difference between the stimulatory power of T(72)(Tn) and T(72)(alpha-D-GlcNAc) is mainly due to very small differences in the TCR exposed carbohydrate structure.

CD8+ T cells are crucial for the ability of congenic normal mice to reject highly immunogenic sarcomas induced in nude mice with 3-methylcholanthrene.

An attempt was made to identify the selection pressures put upon a growing tumour by CD8+ T cells. To this end tumours induced with 3-methylcholanthrene in T cell-deficient nude mice and in congenic T cell-competent nu/+ mice were transplanted to nu/+ recipients. The rejection rate of the sarcomas from nude mice was almost twice that of the sarcomas from nu/+ mice. Depletion of CD8+ T cells from nu/+ recipients prior to transplantation made them accept nude tumours that were consistently rejected by untreated nu/+ recipients. These findings suggest that a methylcholanthrene sarcoma during its growth in a T cell-competent host adapts to the T cell system through a selective elimination of highly immunogenic tumour cells that are susceptible to CD8+ T cell-mediated lysis.

Synthesis of tumor associated sialyl-T-glycopeptides and their immunogenicity.

Sialyl-T-glycopeptides were synthesized by solid-phase techniques, using a PEGA resin as the solid support. An appropriately protected building block containing alpha-Neu5Ac-(2 --> 3)-beta-Gal-(1 --> 3)-alpha-GalN3-(1-->) attached to Fmoc-Thr/Ser-OPfp was employed in a solid phase glycopeptide assembly of a 10-mer glycopeptide, using a general Fmoc/OPfp-ester strategy. Reduction of the azido group of the GalN3 residue was effected on solid-phase, using DTT and DBU. After acidolytic cleavage from the resin, the methyl ester of the sialic acid residue and acetyl groups were removed with 30% NaOMe/MeOH in MeOH and water pH 14, at -30 degrees C for 2 h. At this low temperature, the highly basic conditions did not result in any detectable beta-elimination. However, one O-acetyl group, located at the 2-position of the Gal was resistant to hydrolysis. To remove this remaining acetyl group, reaction with hydrazine hydrate in CHCl3 and MeOH at room temperature for 2.5 h was successful. The two target sequences of sialyl-T-glycopeptides were obtained in good yield. In contrast to the the analogs carrying the T-antigen, the Sial-T-glycopeptides were nonimmunogenic, supporting the idea that the sialylation is a method of circumventing the recognition by the immune system.

Shared structural motifs in TCR of glycopeptide-recognizing T cell hybridomas.

The TCR structure of T cell hybridomas recognizing a tumor glycan-defined epitope has been studied using reverse transcriptase-PCR and gene sequencing. The hybridomas had been raised against a glycopeptide, T72(Tn), consisting of the mouse hemoglobin-derived decapeptide Hb(67 - 76), O-glycoslated in position 72 with alpha-D-GalNAc. The glycan-specific hybridomas varied widely in their use of Valpha genes although Valpha4 was predominant, being present in one third of them. The Vbeta gene usage was more restricted and dominated by Vbeta1 and Vbeta15. There was no correlation between Valpha and Vbeta usage and antigen fine specificity of the hybridomas. The overall amino acid composition of the complementarity-determining region (CDR) 3 of the hybridomas was dominated by small polar residues such as Gly, Asn, Ser, Glu and Ala, amino acids reported in the literature to be frequent in glycan-recognizing proteins. Furthermore, the CDR3 of most hybridomas also contained an aromatic residue with preference for Tyr. A few of the hybridomas raised against the T72(Tn) glycopeptide were peptide specific, i. e. they responded to the unglycosylated peptide only. The amino acid usage of their CDR3 regions was not radically different from that of the glycopeptide specific hybridomas. They also preferentially used Valpha4. However, Vbeta4 and Vbeta8 were the dominating beta chains.

Aberrant expression of tissue-specific proteins in the thymus: a hypothesis for the development of central tolerance.

Herein we present the case for the existence of a thymic cortical epithelial cell that possesses an unusual gene transcription. It produces tissue-specific proteins that have their usual physiological functions outside the thymus and presents them, as well as household proteins, to the differentiating thymocytes. We suggest that this specialized cell enforces tolerance to most self-proteins by causing release of a signal for programmed cell death to thymocytes that express receptors for these self-antigens.

High MHC class I expression correlates with slow growth in UV-induced skin carcinomas in hairless mice.

An experiment was set up to investigate the relationship, if any, between cell surface MHC class I expression and the growth rate for skin tumors induced by two different UV radiation regimens in hairless mice. Two groups of 20 hairless mice were each irradiated with either a UVA radiation source (2 SED per session) or broad-spectrum UV radiation (UVB) (8.1 SED per session) 5 days a week during the entire experiment. In the UVA group, 17 out of 20 animals developed tumors, and 10 of these grew to a diameter of > or = 5 mm. In the UVB group, 19 out of 20 animals developed tumors, and 15 of these grew to a diameter of > or = 5 mm. The tumor induction time, i.e. the time from the start of UV treatment to tumor appearance, was found to be significantly longer (p<0.01) in the UVA than in the UVB group. This is in accordance with previous findings. Of the 25 tumors growing to a diameter of > or = 5 mm, 11 were established as cultured cell lines (4 UVA and 7 UVB tumors). These uncloned cell lines were analyzed for surface expression of major histocompatibility complex class I by FACS analysis. There was a clear correlation between high MHC class I expression and slow growth of the individual tumors (p<0.05). This suggests a role for the MHC class I governed, i.e. cytotoxic T-cell-mediated, reactions in deciding the fate of UV-induced skin cancers. No correlation was found between MHC class I expression and tumor induction time.

Murine thymic nurse cells express ICAM-1 on caveolar and vacuolar membranes.

The thymic nurse cell is a unique type of epithelial cell in the thymic cortex. It is in intimate contact with the developing thymocytes by harbouring up to 200 thymocytes in distinct vacuoles, called caveoles. This investigation is concerned with the nurse cell expression of the intercellular adhesion molecule ICAM-1, the ligand for thymocyte LFA-1. Nurse cells from young Balb/c mice were isolated in a density gradient. ICAM-1 expression was studied by using two different immunotechniques: alkaline phosphatase labelled cryosections, and immunogold electron microscopy. The specific antibody was a monoclonal rat anti-mouse ICAM-1. Immunostaining of cryosections demonstrated that ICAM-1 is expressed on the surface membrane and in the internal caveolar membranes of thymic nurse cells. Electron microscopy of immunogold labelled sections revealed ICAM-1 on the surface membrane of thymic nurse cells and on the membranes of the caveoles, the small cytoplasmic vesicles, as well as on the Golgi apparatus.

Interferon-gamma-induced MHC class I expression and defects in Jak/Stat signalling in methylcholanthrene-induced sarcomas.

Seventy-eight uncloned tumour cell lines, each established from a primary sarcoma induced with methyl-cholanthrene in immunocompetent nu/+ BALB/c and C.B.-17 mice or in immunodeficient nu/nu BALB/c and severe combined immunodeficient (SCID) mice, were examined for sensitivity to interferon-gamma (IFN-gamma) as measured by tumour cell augmentation of major histocompatibility complex (MHC) class I expression. The tumour cells were cultured with IFN-gamma and their expression of Kd, Dd and Ld was measured by fluorescence-activated cell sorter analysis. All but three of the 78 tumour lines up-regulated Kd, Dd and Ld to a variable degree in response to IFN-gamma, indicating that IFN-gamma resistance is not a common property of these sarcomas. The tumour cell lines varied greatly in their MHC class I expression before as well as after IFN-gamma stimulation. There was a tendency towards a higher MHC expression after IFN-gamma stimulation in tumour lines from immunocompetent mice compared to immunodeficient mice, but no common maximum MHC class I expression level was found for the 78 tumour cell lines. Three of the tumour lines, all from immunodeficient mice, completely failed to respond to IFN-gamma by up-regulating MHC class I expression. The same three also displayed absence of IFN-gamma-induced Stat1 beta tyrosine phosphorylation and low Stat1 alpha tyrosine phosphorylation, indicating a defect in the signal transduction pathway affecting phosphorylation of Stat1. These findings strongly suggest a link between defects in Stat1 phosphorylation and the failure to up-regulate MHC class I. In all tumour lines tested, the Stat1 Western blotting revealed a 78 kDa protein (p78) not previously described.

T-cell recognition of tumor-associated carbohydrates: the nature of the glycan moiety plays a decisive role in determining glycopeptide immunogenicity.

Aberrant glycosylation is one of the most constant traits of the malignant cell phenotype. To study T-cell responses to tumor-associated glycans, the mouse hemoglobin-derived decapeptide Hb(67-76), which binds well to the MHC class II molecule E(k) and is nonimmunogenic in CBA/J mice, was either O- or N-glycosylated at its primary T-cell receptor contact residue, position 72, with different glycans attached to either threonine, serine, or asparagine. The carbohydrate moieties included tumor-associated mucins, i.e., the Tn and T antigens, mucin-related glycans, and mucin-unrelated glycans. The side chain of the amino acid in position 72 points away from the MHC binding site when the Hb(67-76) peptide is bound to E(k), so the assumption was that this was also the case for glycans attached to this position. The glycosylated Hb(67-76) peptide analogues were then studied for binding to E(k) and for immunogenicity in CBA/J mice. All 16 glycopeptides bound well to E(k), although those with the more complex carbohydrates bound more weakly than those with monosaccharides. Six of 12 O-glycosylated and 0 of 4 N-glycosylated glycopeptides were able to induce a T-cell proliferative response with a stimulation index above 3.0. Some glycopeptides were not immunogenic, suggesting that there may be holes in the T-cell repertoire due to a lack of T-cell receptor regions accommodating certain glycan structures. The four strongest immunogenic glycopeptides were all O-glycosylated, and interestingly, three of them carried the tumor-associated Tn or T antigen. On the other hand, the Hb(67-76) peptide analogue with the natural mucin Core2 structure attached did not elicit any T-cell response. T cells primed to a glycopeptide with a simple glycan structure such as Tn did not cross-respond significantly to other glycopeptides, indicating a high degree of carbohydrate specificity in T-cell recognition. T cells primed to a glycopeptide carrying the more complex T antigen showed a complicated pattern of cross-responses to glycopeptides with simpler glycan moieties. The fact that it is possible to raise MHC class II-restricted T-cell responses against tumor-associated carbohydrate structures opens new perspectives for the designing of cancer vaccines.

Molecular aberrations in the MHC class I-restricted pathway for antigen presentation in methylcholanthrene sarcomas from nude mice: discrepancies between MHC mRNA and surface protein.

In a previous study, we demonstrated that eight sarcomas induced by chemical carcinogenesis in nude mice were rejected by syngeneic immunocompetent recipients at a much higher rate than eight sarcomas induced with the same method in syngeneic immmunocompetent mice. In the present study, we investigated these 16 sarcomas for structural and quantitative aberrations in components of the MHC class I-restricted antigen-processing and -presentation pathway. Considerable discrepancies between mRNA levels and cell surface protein expression of MHC class I (Kd, Dd and Ld) molecules were observed almost exclusively in the tumours derived from nude mice. Several of the nude mouse-derived tumours also displayed incongruent levels of heavy chain mRNA and beta2-microglobulin mRNA. These findings are taken as indications of abnormal regulation of gene transcription in nude mouse tumours, and if this abnormal regulation extends to the entire genome, it may explain the pronounced immunogenicity of these tumours. Proteasome composition, heat shock protein expression, TAP-molecule inducibility and intercellular adhesion molecule-1 expression were investigated in the same tumours. We found no indications of structural defects or quantitative differences in these molecules between the two groups of tumours.

MCA sarcomas induced in scid mice are more immunogenic than MCA sarcomas induced in congenic, immunocompetent mice.

With the aim of studying possible T-cell mediated selection of the cells in growing tumours, 108 mice of the C.B-17 strain, either immunocompetent C.B-17 mice or histocompatible immunodeficient C.B-17 severe combined immune deficiency (scid) were treated with 3-methylcholanthrene (MCA) in two different dosages. A total of 51 tumours were obtained, 44 of which were established as uncloned tumour cell lines, and used for further study. Tumour incidence correlated with carcinogen-dosage in that more tumours developed in groups treated with a high MCA dose than in groups treated with a low MCA dose, but not with immune status of the tumour host. No significant difference in the level of MHC class I molecule expression was found between the two groups of tumours. The rate of rejection after transplantation to syngeneic immunocompetent hosts was significantly higher for the scid tumours than for the non-scid tumours. The authors suggest that this reflects an immunoselection performed by T cells in the immunocompetent host in which the tumour originated, which has eliminated highly immunogenic tumour cells, leaving non-immunogenic tumour cells to grow.

Carbohydrate and peptide specificity of MHC class II-restricted T cell hybridomas raised against an O-glycosylated self peptide.

MHC class II E(k)-restricted, IL-2 secreting T cell hybridomas were raised against the synthetic glycopeptide Hb(67-76)-alpha-GalNAc, (T72(Tn)), in CBA/J mice (H-2(k)). The fine specificity of the hybridomas against the glycan moiety was investigated by testing their response against a panel of Hb(67-76)-derived glycopeptides, all with a glycan attached to serine or threonine at the position 72 in the peptide, but with different glycans. The hybridomas showed a high degree of specificity for the alpha-GalNAc moiety with few and faint cross-responses to the glycopeptides having other glycans attached even though some of these were structurally very similar to alpha-GalNAc. The fine specificity of the hybridomas for the peptide moiety was investigated by testing their responses to a panel of Hb(67-76)-alpha-GalNAc glycopeptides with alanine substitutions at all positions except at the two MHC binding anchor positions, I68 and K76, and the T72 to which the alpha-GalNAc was attached. Glycopeptides substituted with alanine at positions where the amino acid side chain pointed toward the TCR did not stimulate the hybridomas, whereas glycopeptides substituted with alanine at positions orientated down into the MHC binding groove stimulated many of the hybridomas. These results indicate that the glycan attached to the peptide as well as solvent-accessible parts of the peptide are recognized with a high degree of specificity by the T cells, whereas the parts of the peptide buried in the MHC binding site are less important or totally ignored by the T cells.

The susceptibility to cytotoxic T lymphocyte mediated lysis of chemically induced sarcomas from immunodeficient and normal mice.

A panel of sarcomas induced with 3-methylcholanthrene in normal and immunodeficient mice was studied for their capacity to present antigen by the endogenous, MHC class I restricted pathway. Lymphocytic choriomeningitis virus was used to infect cultured tumour cells, and the infected cells were tested for susceptibility to cytolysis by virus specific cytotoxic T cells. Tumour cells originating from tumours induced in immunocompetent C.B.-17 mice presented virus antigen more efficiently than tumour cells from immunodeficient SCID mice. No significant difference in virus antigen presentation was found between tumours from nude and nu/+ BALB/c mice. The sensitivity of target cells from the individual tumours to cytotoxic T lymphocyte (CTL) mediated lysis correlated negatively with their sensitivity to natural killer (NK) cell mediated lysis. There was a positive correlation between the sensitivity to CTL mediated lysis and surface expression of the MHC class I molecule Ld of the tumour cells. Tumour cells incapable of in vitro presentation of viral antigen to specific cytotoxic T cells originated from tumours known from previous experiments to be readily accepted after transplantation to immunocompetent, histocompatible recipients.

Sertoli cells, but not tumor cells, of seminoma in situ express ICAM-1.

Tumor and surrounding testicular tissue from six seminomas and one combined seminoma/embryonal carcinoma were examined for the expression of ICAM-1, VCAM-1 and ELAM-1. This was done by immunohistochemical staining of frozen samples using monoclonal antibodies and the avidin-biotin/ peroxidase or alkaline phosphatase staining method. ICAM-1 was expressed by Sertoli cells of intratubular germ cell neoplasia, but not by any of the cells in normal seminiferous epithelium, or by neoplastic germ cells whether invading or not. In addition inflammatory cells and endothelium expressed ICAM-1. VCAM-1, and also occasionally ELAM-1, was expressed only on endothelial cells in and outside the tumors. These results are discussed in relation to lymphocytic infiltration and immune surveillance of seminomas and T-cell tolerance to the antigens of the immunologically privileged seminiferous epithelium.

Methylcholanthrene-induced sarcomas in nude mice have short induction times and relatively low levels of surface MHC class I expression.

In order to study the role of the T-cell-mediated immune defense in tumor development, a total of 93 sarcomas were induced using different doses (8 micrograms (0.1%), 40 micrograms (0.5%) and 400 micrograms (5%)) of 3-methylcholanthrene in athymic nude Balb/c mice and phenotypically normal immunocompetent Balb/c mice. A shorter tumor induction time and a higher tumor incidence after treatment with low doses of methylcholanthrene were seen in nude mice than in immunocompetent mice, indicating that they have a lower resistance to the carcinogen. Contrary to expectations we found that the MHC class I expression of tumors from nude mice was lower than that of tumors from normal mice. Higher surface expression of MHC class I was demonstrated on high dose tumors from normal mice than on low dose tumors from normal mice. The cellular composition of the individual tumors raised in nude mice was more heterogeneous with respect to MHC class I expression. Since the mice differ genetically only with respect to the nu gene, these results indicate that a lack of T-cell-mediated defense mechanisms may confer upon the bearer a lower resistance to 3-methylcholanthrene and a different MHC profile of the ensuing tumor.

Chemically induced sarcomas from nude mice are more immunogenic than similar sarcomas from congenic normal mice.

To detect possible differences in immunogenicity between tumors induced in T cell-deficient mice and phenotypically normal congenic mice, 16 sarcomas, 8 having developed in nude BALB/c mice and 8 having developed in congenic normal (nu/+) mice, were transplanted to normal BALB/c recipients and the rates of rejection or acceptance were registered. The 16 tumors were chosen randomly from a panel of 39 sarcomas induced with 0.5% or 0.1% 3-methylcholanthrene and maintained as cell lines in culture. Out of the tumors originating from nude mice, 66% were rejected by the normal BALB/c recipients, while only 30% of the tumors originating from normal mice were rejected. Tumors with short induction times from normal mice were more readily accepted than tumors with long induction times. Tumors originating from nude mice had significantly longer mean latency times after transplantation to both normal and nude recipients than tumors originating from normal mice. Contrary to what has been reported by others, there was no correlation between the rejection rates of the individual tumors and their Kd, Dd or Ld major histocompatibility complex (MHC) class I surface expression as measured by flow cytometric analysis of cultured tumor cells. The Kd, Dd and Ld proteins of the transplanted tumor lines were analyzed by isoelectric focusing for the occurrence of mutations resulting in altered charge of the MHC protein. No such mutations were found, ruling out MHC mutations of that kind as the source of immunogenicity in the cell lines used in these experiments. Our results suggest the existence of a T cell-mediated selection in the original tumor cell mass of tumors induced in normal mice, adapting the tumor to growth in a host with a functional T cell system, but apparently there is no connection between this loss of immunogenicity and loss of MHC class I expression.

Multiple column synthesis of a library of T-cell stimulating Tn-antigenic glycopeptide analogues for the molecular characterization of T-cell-glycan specificity.

A series of peptides and glycopeptides derived by amino acid and glycosyl amino acid scans through the self peptide from CBA/J mouse haemoglobin Hb (67-76). VITAFNEGLK, was synthesized by multiple column peptide synthesis (MCPS). Investigation of glycopeptide binding to the mouse major histocompatibility class II molecule Ek showed that glycans in position 72 did not interfere with the binding to Ek. Immunization experiments revealed that glycopeptides with the glycan in position 72 were immunogenic. Therefore a series of N-linked and O-linked glycopeptides with the glycan attached in the position 72 either to serine, threonine or asparagine was synthesized by MCPS. The glycan structure was furthermore varied with respect to monosaccharide component, size of oligosaccharide, anomer configuration and stereochemistry of essential hydroxyl groups in order to investigate the specificity of the interaction with the T-cell receptor. Easy synthesis of ready to use Ser and Thr building blocks corresponding to mucin core 1, the Tn-antigen and its beta-anomer were developed using trichloroacetimidates as glycosyl donors and reduction with in situ acetylation of the azide containing glycosylation products. Synthesis of an alpha-linked GlcNAc-Thr building block was achieved by glycosylation of Fmoc-Thr-OPip with 2-azido-2-deoxy-3,4,6-tri-O-acetyl-D-glycopyranosyl trichloroacetimidate as a glycosyl donor. Other building blocks were obtained by previously described procedures.

T cell recognition of Tn-glycosylated peptide antigens.

The mouse hemoglobin-derived decapeptide Hb (67-76), VITAFNEGLK, which binds well to Ek and is non-immunogenic in CBA/J mice, was O-glycosylated with the tumor-associated carbohydrate Tn (alpha-D-N-acetylgalactosamine, or alpha-D-GalNAc). Each of the ten positions in the peptide was substituted with serine or threonine having the Tn antigen attached. The complete set of Tn-glycosylated peptides were then studied for binding to Ek and for immunogenicity in CBA/J mice. All of those glycopeptides which had the Tn attached to serine or threonine at a position in the peptide where, according to the crystal structure determinations, the amino acid side chain was oriented downwards into the binding site of the major histocompatibility complex (MHC) molecule, completely lost their capacity for binding to Ek. This was the case for the glycopeptides with Tn attached at position 68 and 76, which are the major anchor residues and for those with Tn attached at position 71 and 73, which function as secondary anchor residues. Those glycopeptides which had Tn attached to serine or threonine at positions where the side chain pointed away from the binding site maintained their capacity for binding to Ek, except for those with Tn attached at position 70 and 74. Furthermore, some of the MHC-binding glycopeptides were immunogenic. In particular, this was the case for the glycopeptide with Tn attached to the central position 72 in the decapeptide. From previous studies, this is known to be the dominant T cell receptor contact residue of Hb (67-76). The results suggest that T cells may be capable of recognizing epitopes which are partially defined by a small glycan group.

Sertoli cells of intratubular germ cell neoplasia express beta 2 microglobulin.

The cells in intratubular germ cell neoplasia in the vicinity of 38 germ cell tumors of the testis, including 20 pure seminomas, were studied for the expression of beta 2-microglobulin (beta 2m), the constant component of all HLA class I molecules. Immunohistochemistry using antibodies towards beta 2m, vimentin, placental alkaline phosphatase, and ferritin was employed. Whereas the intratubular cells in normal testis are beta 2m negative, beta 2m positive cells were identified in intratubular germ cell neoplasia tubules in 55 per cent of all tumors and in 60 per cent of the seminomas. The tubules with beta 2m positive cells were located in areas with invasive tumor or in the vicinity of such areas. The beta 2m positive cells were identified as Sertoli cells by morphology and by their staining with anti-vimentin. Neoplastic germ cells, identified by morphology and staining with anti-placental alkaline phosphatase and anti-ferritin were beta 2-microglobulin negative. The most intensely beta 2m-stained Sertoli cells were found in tubules with high concentrations of neoplastic germ cells. Intensely stained Sertoli cells were also found in 'Sertoli cell only' tubules inside invasive tumors and in areas without lymphocytic infiltration. The cells in adjacent normal tubules were beta 2m negative.