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Biomedical subjects

G Gaudernack

Publications and source records attributed to G Gaudernack.

At least 55 records · Page 3Linked to original sources
PubMed1995

Sialomucin CD34 is the major L-selectin ligand in human tonsil high endothelial venules.

Peripheral node addressin (PNAd) is a complex mixture of glycoproteins with L-selectin ligand activity that functions in lymphocyte homing. We have investigated the contribution of the sialomucin CD34 relative to other components of PNAd in lymphocyte tethering and rolling in in vitro laminar flow assays. PNAd was isolated with MECA-79 mAb-Sepharose from tonsillar stroma, and the CD34 component (PNAd,CD34+) and CD34-negative component (PNAd,CD34-) separated on CD34 mAb-Sepharose. Lymphocytes on the PNAd,CD34- fraction tether less efficiently, roll faster and are less resistant to shear detachment than on PNAd. The PNAd,CD34+ fraction constitutes about half the total functional activity. These studies show that CD34 is a major functional component of PNAd. Ligand activity in both the PNAd,CD34+ and PNAd,CD34- fractions is expressed on mucin-like domains, as shown with O-sialoglycoprotease. The CD34 component of PNAd has about four times higher tethering efficiency than total tonsillar CD34. CD34 from spleen shows no lymphocyte tethering. Although less efficient than the PNAd,CD34+ fraction from tonsil, CD34 from the KG1a hematopoietic cell line is functionally active as an L-selectin ligand despite lack of reactivity with MECA-79 mAb, which binds to a sulfation-dependent epitope. All four forms of CD34 are active in binding to E-selectin. KG1a CD34 but not spleen CD34 are active as L-selectin ligands, yet both lack MECA-79 reactivity and possess E-selectin ligand activity. This suggests that L-selectin ligands and E-selectin ligands differ in more respects than presence of the MECA-79 epitope.

Antigens, CD34↗
PubMed1995

A human melanoma cell line, recognized by both HLA class I and class II restricted T cells, is capable of initiating both primary and secondary immune responses.

We have characterized a melanoma cell line, FM3, established from a metastasis of a 75 year old female patient (HLA-A2, HLA-DQ7) with malignant melanoma. This cell line expresses both HLA class I and class II antigens, as well as several important accessory molecules at high levels. FM3 cells were shown to function as a stimulator of both allogeneic as well as autologous mixed lymphocyte tumour cell culture (MLTC). From these autologous MLTC we were able to generate cytotoxic T cell clones indicating that FM3 is capable of processing and presenting endogenous antigens. We have used this cell line in a model system to investigate whether these cells were able to initiate and support an immune response with specificity for selected peptide antigens. The FM3 cell line was capable of presenting a HLA-DQ7 restricted ras derived peptide (5-21, 13Gly- >Asp) to a previously established T cell clone, RM70. The ability of FM3 to function as an antigen presenting cell (APC) was comparable to that of an autologous Epstein Barr virus (EBV) transformed B cell line. The CD4+ T cell clone RM70 showed a peptide-specific anti-proliferative effect on FM3 cells. This growth inhibition was not due to cytotoxicity as measured in a standard 4 h chromium release assay. The FM3 cell line also presented a HLA-A2 restricted nonapeptide derived from the influenza matrix protein, M1(58-66) to a CD8+ T cell line specific for this peptide. This resulted in an effective killing of the melanoma cells. Together, these data suggest that some melanomas may initiate an immune response by presenting their own specific antigens in an immunogenic context, and subsequently serve as targets for T cells of both the CD4+ and CD8+ phenotype.

Aged↗
PubMed1995

RG1, a new murine monoclonal antibody recognizing a "supertypic" determinant on HLA-A molecules.

Monomorphic and polymorphic anti-HLA monoclonal antibodies (mAb) are valuable reagents for assessment of the structural and functional importance of different class I determinants. We have generated a new mAb, RG1, reacting with an epitope variably expressed on normal and leukemic hematopoietic cells of different lineages. Immunoprecipitation of the RG1 antigen disclosed a bimolecular complex characteristic of class I proteins. The RG1 epitope was expressed on an HLA-A2 transfected cell line but not on cells transfected with HLA-E, -F or -G molecules. MAb reactivity with reference B-lymphoblastoid cell lines and HLA typing of RG1 reactive and unreactive cells demonstrated that the epitope was expressed in conjunction with defined HLA-A molecules. Cells expressing HLA-A2, -A24(9) and -A68(28) proteins were brightly stained with RG1 whereas mAb binding to HLA-A1, -A11 and a split of A3 molecules was significantly lower. In contrast, the RG1 epitope was apparently not expressed on HLA-A23(9), -A25(10), -A26(10), -A29(19), -A30(19), -A31(19), -A32(19), -A33(19) and some HLA-A3 molecules. Based on class I alpha sequence data, these results suggest that the RG1 epitope is localized to a region of the alpha 2 helix accessible to the T cell receptor for antigen on cytotoxic T lymphocytes. Lys in position 144 and His in position 151 are apparently critical for RG1 binding.

Amino Acid Sequence↗
PubMed1995

Characterization and epitope mapping of four HLA class II reactive mouse monoclonal antibodies using transfected L cells and human cells transfected with mutants of DQB1*0302.

To study epitopes of HLA class II molecules, four mouse monoclonal antibodies (mAbs) 13B6, 17F8, 19A1 and 12G6 were made using HLA-DQ8, DP2 and DP4 expressing mouse transfectants for immunization. Three of the mAbs, 13B6, 17F8 and 19A1, bound to all DQ1, 4, 8 or 9 positive B-lymphoblastoid cell lines (B-LCLs) and transfectants tested, i.e. cells carrying the DQB1 genes 0302-3, 0401-2, 0501-3, 0601-4 and 0609 irrespective of the accompanying DQA1 gene. These DQB1 genes code for the shared amino acids (aa) GVY in position 45-47 of the DQ beta chain. DQ1+4+8+9 specific (IIB3) and DQ3 specific (IVD12) reference mAbs inhibited binding of all three mAbs. Testing 13B6, 17F8 and 19A1 with cells made using aa substitutions in various positions of DQ beta 1*0302 indicated involvement of aa 45 in the epitopes of all three mAbs. The last mAb (12G6) bound to all B-LCLs and all DP transfected cells. However, only some DR transfectants and a single DQ transfectant (carrying DQA1*0201 and DQB1*0202) bound mAb 12G6. This reactivity pattern correlates with a shared sequence of aa (RFDSDVGE) in position 39-46 of DR- and DQ- and 37-44 of DP beta chains.

Amino Acid Sequence↗
PubMed1994

A K-ras 13Gly-->Asp mutation is recognized by HLA-DQ7 restricted T cells in a patient with colorectal cancer. Modifying effect of DQ7 on established cancers harbouring this mutation?

We have characterized and described in detail 2 CD4+ T-lymphocyte clones (TLC) from a colonic cancer patient. These TLC specifically recognize a K-ras-derived peptide carrying the 13Asp mutation commonly found in adenocarcinomas of the colon. The TLC were independently derived, as they carried 2 different T-cell receptors. The TLC recognized partly overlapping epitopes within the 13Asp peptide, presented by HLA-DQ7 molecules, suggesting that this molecule might confer some protective immunity against the mutation. On the basis of analysis of 251 colonic carcinomas, the presence of HLA-DQ7 did not seem to protect against the establishment of carcinomas carrying the 13Asp mutation, since the frequency of the DQ7 haplotype was not decreased among patients having this mutation. A modifying effect of DQ7 on the development of carcinomas with a 13Asp mutation was, however, observed, resulting in fewer tumours reaching advanced Dukes stages when DQ7 was present.

Amino Acid Sequence↗
PubMed1994

p21-ras-peptide-specific T-cell responses in a patient with colorectal cancer. CD4+ and CD8+ T cells recognize a peptide corresponding to a common mutation (13Gly-->Asp).

Peptides derived from mutated ras are immunogenic in mice and humans, and represent a group of specific tumor antigens that are potential targets for immunotherapy. T-cell responses against mutant p21 ras can be initiated in vitro by repeated stimulation of peripheral-blood mononuclear cells with mutant ras-derived peptides. Patients with tumors commonly harbouring ras mutations may therefore show evidence of in vivo reactivity against such mutations. Peripheral-blood mononuclear cells from 10 patients with colorectal adenocarcinoma were screened for reactivity against synthetic ras-derived peptides corresponding to the most commonly found mutations in this type of cancer. In one patient, T-cell reactivity against the 1-25,13Gly-->Asp peptide was detected. From this patient, both CD4+ and CD8+ T-cell clones specific for the 1-25,13Gly-->Asp mutation could be raised. We were not, however, able to detect the corresponding mutation in the cancer. The 13Gly-->Asp mutation in the ras oncogene is frequent and constitutes 9 to 27% of all K ras mutations found in biopsies from patients with colorectal carcinomas. Our study demonstrates a mutant ras-specific T-cell response of both the CD4+ and the CD8+ phenotype in a cancer patient. We speculate that in this patient a specific T-cell response resulted in eradication of tumor cells harboring the 13Gly-->Asp mutation.

Adenocarcinoma↗
PubMed1994

T cell epitopes encompassing the mutational hot spot position 61 of p21 ras. Promiscuity in ras peptide binding to HLA.

Activated ras carry a point mutation either in codon 12, 13 or 61 which is tumor specific. Peptides derived from this oncoprotein are therefore potential tumor antigens. Essential for the feasibility of using ras-derived peptides in therapy of cancer is whether p21 ras-derived peptides can be processed, bind to human histocompatibility leukocyte antigen (HLA) and be recognized by T cells. Here we report the fine specificity and HLA restriction of several T lymphocyte clones (TLC) specific for a peptide which is derived from the second mutational hot spot in ras encoding residue 61. These TLC were generated from memory T cells present in the blood of a cancer patient and recognized a ras-derived peptide carrying Leu instead of Gln at residue 61. By sequencing of the T cell receptor (TcR) genes three sets of "sister" TLC carrying highly different TcR were identified. Two of the TLC recognized a peptide carrying the 61 Leu mutation presented by HLA-DQ8 and one recognized the same peptide presented by HLA-DQ4. By using truncated peptides derived from residues 51 to 69 of p21 ras, partially overlapping minimal epitopes could be defined. All three TLC recognized the corresponding recombinant mutant p21 ras oncoprotein carrying Leu at residue 61 presented by autologous B-lymphoblastoid cell lines (B-LCL). This demonstrates that naturally derived ras peptides from this region of p21 ras encompass the three epitopes recognized by the TLC. These results indicate that immunogenic ras-derived peptides may be used in immunotherapy of cancer where transforming ras oncoproteins are involved.

Amino Acid Sequence↗
PubMed1994

Cross-linking of a sequential epitope within the beta-chain of HLA-DR/DP molecules suppressing B lymphocyte growth and inducing homotypic cell aggregation.

A monoclonal antibody (SU2) directed against HLA class II antigens has been produced by immunizing BALB/c mice with a lymphoblastoid cell line, RPMI-8866 cells. The specificity of this mAb has been determined using a panel of HLA class II transfectants. SU2 stained all HLA-DR and HLA-DP transfectants tested, but reacted with only one HLA-DQ (HLA-DQw2). From comparison of the available data sequences of known HLA class II molecules, it appeared that the epitope recognized by the SU2 mAb contains a sequence of 6 amino acids (DSDVGE) at position 41-46 of the beta-chain of HLA-DR/DP. Functional studies indicated that SU2 mAb strongly induced cell aggregation and large clumping in resting tonsillar B cells. SU2 mAb inhibited the spontaneous growth and proliferation of B lymphoblastoid cell lines and drastically inhibited [3H]thymidine uptake by phorbol 12-myristate 13-acetate (PMA)-activated resting B cell. Results are discussed in relation to the dual recognition of HLA-DR/DP by SU2 mAb.

Amino Acid Sequence↗
PubMed1994

Heterogeneity of T cells specific for a particular peptide/HLA-DQ complex.

Whether T cells specific for a particular peptide/HLA-DQ complex are restricted with respect to TCR usage has not been fully established. TCR usage of T cells specific for a peptide presented by a given HLA-DQ molecule has not been studied before. We therefore sequenced the TCR genes of five different TCCs derived from the same donor, which were specific for a p21 ras-derived synthetic peptide presented by the HLA-DQ(alpha 1*0102,beta*0602) (DQ6) molecule. We found that these T cells which recognized the same peptide/HLA-DQ complex used highly diverse TCRs. However, dose-response experiments using various truncations of the p21 ras-derived peptide revealed that the peptide fine specificities of the five TCCs were not completely identical. This may explain the heterogeneity in TCR usage.

Amino Acid Sequence↗
PubMed1994

K-ras mutation in colorectal cancer: relations to patient age, sex and tumour location.

DNA from 251 primary tumours obtained from 123 male and 125 female Norwegian patients with colorectal carcinoma was analysed for the presence of K-ras point mutations at codons 12 and 13. Mutations were found in 99 (39%) of the samples. The frequency of K-ras mutations was significantly related to age and sex of the patients, and to the location of the tumours (overall: P = 0.008). K-ras mutations were much less frequent in colonic tumours from male than female patients at younger ages (< 40 years, odds ratio < 0.014). The low frequency might indicate that a different, ras-independent, pathway to neoplasia is dominating in the colon of younger males. In contrast, older men had more mutations than older women (e.g. 90 years, odds ratio = 5.8). An inverse but less pronounced relationship was seen for rectal tumours. The type of mutation was found to be associated to sex of patient and location of tumour. G-->C transversions accounted for 35% of the mutations in rectal tumours from females, in contrast to only 2.5% in the rest of the material (P = 0.0005). This may indicate that there are specific carcinogens acting in this location.

Adult↗
PubMed1994

Binding of ras oncogene peptides to purified HLA-DQ(alpha 1*0102,beta 1*0602) and -DR(alpha,beta 1*0101) molecules.

Mutated oncogene peptides may be presented to T cells by HLA molecules. To be able to design the optimal peptides for stimulation of T cells in individuals with different HLA molecules, it is important to analyse the binding characteristics of oncogene peptides to HLA. HLA-DQ6 (DQ(alpha 1*0102,beta 1*0602)) and HLA-DR1 (DR(alpha,beta 1*0101)) molecules were purified from lysates of homozygous EBV-transformed cell lines. Purified HLA molecules were then tested for their ability to bind synthetic peptides in gel filtration assays. A p21 ras oncogene peptide (previously found to stimulate DQ6-restricted T-cell clones) and an influenza matrix peptide were labelled with 125I and served as indicator peptides for binding to DQ6 and DR1 respectively. Binding of homologous truncated and mutated p21 ras peptides and unrelated peptides was then evaluated by their capacity to inhibit binding of the indicator peptides. p21 ras-derived peptides were found to bind to both DQ6 and DR1 molecules indicating the existence of a promiscuous binding motif in these peptides. The binding affinities seemed to vary between the different peptides, but the amino acid substitutions resulting from natural mutations were not critical for binding. Notably, the results obtained for DQ6 in the biochemical peptide binding assay correlated well with results obtained in a functional assay using T-cell clones as probes.

Amino Acid Sequence↗
PubMed1994

Growth inhibition of a colonic adenocarcinoma cell line (HT29) by T cells specific for mutant p21 ras.

Mutations at codons 12, 13 or 61 of the ras proto-oncogenes are found in adenocarcinomas of the colon and rectum. Mutated ras encode tumor-specific proteins, and can elicit CD4+ HLA-class-II-restricted T cell responses both in mouse and man. The function of such T cells is, however, unclear. In a model system, we investigated whether HLA-class-II restricted CD4+ T cells, specific for a particular peptide derived from mutant p21 ras (Gln61-->Leu), might inhibit the growth of a colonic cancer cell line, when it was cultured in the presence of the corresponding peptide. We found in this case that the growth of the colonic adenocarcinoma cell line HT29, when also induced to express HLA class II molecules by interferon gamma treatment, was inhibited. The inhibition was peptide-specific and required the presence of HLA-DQ8 molecules on the target cell. However, HLA-DQ8-expressing HT29 cells functioned poorly as antigen-presenting cells and could only induce a weak proliferative T cell response in the presence of interleukin-2. The results suggest that colonic cancer cells expressing peptides derived from mutant p21 ras protein in a complex with HLA class II molecules may be a target for tumor-specific T cells. The results also indicate, however, that an initiation of the immune response will require "professional" antigen-presenting cells.

Adenocarcinoma↗
PubMed1993

T cell clones specific for p21 ras-derived peptides: characterization of their fine specificity and HLA restriction.

Peptides corresponding to the mutated regions of the oncoprotein p21 ras are immunogenic and capable of eliciting HLA class II-restricted T cell responses. Here we report studies on the fine specificity of four T lymphocyte clones (TLC) from a single donor, using various truncated peptides derived from the residues 6-19 of p21 ras and a panel of well-characterized HLA homozygous cells as antigen-presenting cells. Putative minimum peptides of nine or ten amino acids could be defined for each TLC. Two of the TLC recognized peptides presented by DR2, and the two others recognized peptides presented by DQ6. Some notable differences in the requirement for certain amino acids were seen between the DR- and DQ-restricted TLC. Thus, Ser at residue 17 was required for stimulation of the DQ6-but not the DR2-restricted TLC. Val at residue 8 was essential for stimulation of all TLC, whereas one of the DR2-restricted TLC also required Val at residue 7. Some peptides which were nonstimulatory were still capable of binding to DQ6 molecules in peptide competition experiments. The results may be of importance for potential immunotherapy of cancer where transforming ras oncoproteins are involved.

Amino Acid Sequence↗
PubMed1993

Overlapping epitopes encompassing a point mutation (12 Gly-->Arg) in p21 ras can be recognized by HLA-DR, -DP and -DQ restricted T cells.

Mutations in ras genes which result in transforming gene products carrying amino acid substitutions in position 12, 13 or 61 are common in human cancer. Peptides encompassing these mutations in ras are shown to be immunogenic in both mice and humans. The potential usefulness of such peptides in cancer therapy, depends on their ability to bind to HLA molecules. We therefore stimulated T cells from healthy donors with mutated ras-derived peptides. By repeated in vitro stimulation of peripheral blood mononuclear cells, several T cells clones could be generated which recognized a p21 ras derived peptide carrying a position 12 Gly-->Arg substitution. This peptide (1-25,12 Arg) could be specifically recognized by T cells restricted by either HLA-DQ7 or -DP3. Previously, we showed that this peptide is also recognized by a T cell clone restricted by HLA-DR2. The core region of the peptide was determined to span positions 9-16 for all three HLA restriction elements, and accordingly contains the mutational hot spots in position 12 and 13. The observation that the mutant 1-25,12 Arg ras-derived peptide may contain a promiscuous epitope encompassing the Gly-->Arg mutation in position 12 indicates that lack of peptide presentation by given HLA molecules may not be a major constraint in responsiveness against ras mutations.

Amino Acid Sequence↗