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K Hannestad

Publications and source records attributed to K Hannestad.

At least 37 records · Page 2Linked to original sources
PubMed1992

A cytotoxic monoclonal human hybridoma antibody (TrJ3) against HLA-B44(12) and -B45(12).

We have generated a human monoclonal cytotoxic IgM lambda antibody (TrJ3) that reacted specifically with all lymphoblastoid B-cell lines expressing HLA-B44(12) and B45(12). TrJ3 hybridoma supernatant was suitable for HLA-B12 typing of freshly isolated blood mononuclear cells. Analysis of available amino acid sequences of HLA-B molecules indicated that the alpha 1 domain does not contain the TrJ3 serological epitope. Since HLA-B44 is associated with a unique serine residue at position 167 that points towards the peptide binding groove, we propose that S167 of the alpha 2 domain helix is a critical part of the TrJ3 epitope.

Amino Acid Sequence↗
PubMed1991

Igh-1b-specific CD4+CD8- T cell clones of the Th1 subset selectively suppress the Igh-1b allotype in vivo.

The demonstration of major histocompatibility complex (MHC)-restricted T helper (Th) cells specific for peptides from the variable (V) regions of syngeneic immunoglobulin (Ig) (idiopeptides) opens the possibility that Th cells regulate B cell functions via idiopeptide-based cognate T-B interactions. As a model for such interactions we investigated the influence of Ig allotype-specific T cells on the differentiation of H-2-syngeneic B cells expressing that particular Ig allotype. We established a BALB/c (H-2d, Iga) CD4+CD8- T cell line and clones of the Th1 subset (interleukin 2+, interleukin 4-, interferon-gamma+, tumor necrosis factor-alpha+) that recognized Igh-1 (IgG2a) of the b allotype (Igh-1b) together with I-Ad. These T cells specifically suppressed surface Igh-1b+ B cells in vitro and in vivo. In 12 out of 15 6-week-old (BALB/c X B10.D2)F1 mice neonatally injected with Igh-1b-specific T cells, the serum Igh-1b concentrations were less than 5% of the levels in the controls. Thus, allotype suppression can be accomplished solely by adoptive transfer of Igh-1b-specific CD4+ T cells. The in vivo suppression was specific for Igh-1b+ B cells as the recipients' levels of Igh-1a and Igh-4b (IgG1b) were unaffected. The V beta 14-specific anti-T cell receptor (TcR) monoclonal antibody 14-2 inhibited activation of hybridomas derived from two of the clones. Collectively the data indicate that suppression resulted from cognate interactions between allopeptide-specific TcR alpha/beta+ T cells and normal unmanipulated B lymphocytes presenting their endogenous Igh-1b in association with MHC class II molecules. The data support the possibility that normal B cells can be suppressed by idiopeptide-specific T cells in vivo.

Animals↗
PubMed1991

Epitope mapping of human monoclonal antibodies to HLA-B27 by using natural and mutated antigenic variants.

The epitopes defined by three human monoclonal antibodies (mAbs) (Tr3B6, TrCG10, TrBH12) against HLA-B27 have been mapped by flow cytometry. For this purpose we used murine transfected cells expressing at their surface hybrid antigens between HLA-B7 and -B27 and, in addition, Epstein-Barr virus cell lines expressing the six HLA-B27 alleles B*2701 to B*2706. The results indicated that the mAbs are domain specific. TrBH12 recognizes the first external (alpha-1) domain. Residues critical for the TrBH12 epitope are located in the alpha-1 helix and include the polypeptide stretch 63-76 plus a critical amino acid at position 77. Tr3B6 binds the second external (alpha-2) domain, and one mutation (VAL152----GLU152) destroyed its epitope. TrCG10 also binds the alpha-2 domain.

Alleles↗
PubMed1989

Two HLA-DQ-specific human-human hybridoma antibodies (TrG6;TrC5) define epitopes also expressed by a transcomplementing hybrid DQ molecule (DQw7 alpha/DQw4 beta).

We have generated two IgG human-human hybridoma Abs, TrG6 and TrC5, that define subsets of HLA-DQ. TrG6 combined selectively with lymphoblastoid cell lines that expressed DQw1 or DQw4. By sequential immunoprecipitation, competition with other mAbs of defined specificity for binding to antigen, and experiments where HLA antigens from cell lysates crosslinked pairs of mAbs, it was established that TrG6 bound a DQ-molecule. mAb TrC5 specifically recognized DQw2+DR3+ and DQw7+DR5+ cells. The reaction pattern of TrC5 with HLA-loss mutants indicated that TrC5 bound to DQw2 of the DQw2+DR3+ haplotype. Antigens in lysate from DQw7+DR5+ cells crosslinked TrC5 to the murine mAbs Tü22 (anti-DQ monomorphic) and IVD-12 (anti-DQw7 + DQw8 + DQw9), demonstrating that on these cells the TrC5 epitope is located on DQw7 molecules. Lysates from DQw7+DR5+/DQw4+DRw8+ heterozygous cells crosslinked TrG6 and TrC5, and available evidence indicated that the epitopes defined by these two mAbs were expressed by the transcomplementing DQ-molecule DQw7 alpha/DQw4 beta, where the DQw7 alpha chain specifies epitope TrC5 and the DQw4 beta chain specifies epitope TrG6. Taken together with published nucleotide sequences of DQ alpha and beta genes, our data are consistent with the conclusion that the amino acids at positions 69 and/or 75 of the DQ alpha chain of DQw2+DR3+ and DQw7+DR5+ haplotypes are critical for epitope TrC5. The previously reported human-human hybridoma Ab TrB12 reacts with DQw6, DQw8, and DQw9. The specificity of the murine mAb IIB3 is similar to that of TrB12, but, unlike TrB12, IIB3 also binds DQw4+ cells.

Amino Acid Sequence↗
PubMed1989

A supertypic HLA-DP specificity defined by two human-human hybridoma antibodies (TrB50; TrE11).

We here report two human-human hybridoma antibodies: TrB50 (IgG) and TrE11 (IgM), derived from the same donor. They displayed an identical reaction pattern with 76 Epstein-Barr virus-transformed cell lines. Of these, 29 lines were completely HLA-typed and both antibodies recognized all cells expressing DPwl (six lines), DPw3 (five lines), or DPw5 (three lines). In addition, they bound to one out of four DPw2+ cells and three out of four DPblank+ cells. This specificity correlated strikingly with a characteristic DP beta amino acid sequence (DEAV) at positions 84-87 that had been determined by others. Binding of 125I-labeled TrB50 to lymphoblastoid cells was inhibited by unlabeled IVA-12 (anti-DR + DP monomorphic) and by TrE11. Furthermore, antigens in lysates from TrB50+TrE11+ cells cross-linked TrB50 and TrE11 to the monomorphic anti-DP monoclonal antibody B7/21. Collectively the data provide strong evidence that the epitopes reside on DP molecules. TrE11 can be used to type for this DP beta supertypic specificity by microcytotoxicity using isolated blood B lymphocytes as targets or by a rosette assay directly on whole blood.

Antibodies, Monoclonal↗
PubMed1989

Two cytotoxic human-human hybridoma antibodies to HLA: TrAH10 (anti A3.1) and TrAG2 (anti B7, Bw42).

Two cytotoxic human-human hybridoma IgM antibodies to HLA were generated by EBV transformation of PBMC from multiparous women and fusion of EBV transformed cells with the human fusion partners KR4 or KR12. Both mAbs required the sensitive immunomagnetic cytotoxicity method to display killing of freshly prepared PBMC. One mAb (TrAH10) was specific for HLA-A3. Strikingly, TrAH10 reacted much more strongly with lymphoblastoid cell lines of HLA-A3.1 than of the rare variant HLA-A3.2, previously detected by cytotoxic T cells. Thus, in the microcytotoxicity test, the titer of concentrated TrAH10 was approximately 2000 times higher for A3.1 as compared to A3.2, and a clear difference was also observed in radioimmunoassay. Since the two HLA-A3 variants differ by only two amino acids at positions 152 and 156 of the alpha 2-domain's alpha-helix, the epitopes defined by the mAb TrAH10 and HLA-A3.1 specific cytotoxic T cells must be closely related. The observations with TrAH10 suggest that the HLA polymorphism detected by human mAbs may turn out to be as extensive as the T-cell defined HLA polymorphism. The other mAb (TrAG2) bound B7 and Bw42 with equal strength, and in addition bound weakly to some cells that were Bw22 or B39. Magnetic polymerbeads coated with affinity purified human mAbs TrAH10 or TrAG2 formed rosettes with EBV transformed cells carrying relevant HLA antigens; however, rosette formation with freshly isolated PBMC was very weak and unsuitable as a typing assay.

Antibodies, Monoclonal↗
PubMed1989

A cytotoxic human-human hybridoma antibody (TrC7) specific for HLA-A29.

TrC7 is a cytotoxic IgM human-human hybridoma anti-HLA Ab. Its reaction pattern with a panel of 48 HLA-typed EBV-transformed cell lines and PBMC from 348 HLA-typed individuals correlated precisely with expression of HLA-A29. TrC7 did not react with HLA-A30, -31, -32 or -w33, which, like A29, are splits of Aw19.

Antibodies, Monoclonal↗
PubMed1989

Multiple sclerosis patients have a high frequency of an HLA-DQ beta epitope defined by a human-human hybridoma antibody.

Epstein-Barr virus (EBV) transformed B-cells from patients with multiple sclerosis (MS) and healthy controls were analysed for reactivity with the HLA-DQ-specific human-human hybridoma Ab TrB12 (anti-DQw6 + DQw8 + DQw9) by indirect immunofluorescence (IIF). Positive results were obtained with 34 out of 35 MS patients (97.1%) and 79 out of 106 controls (74.5%) (p less than 0.005, RR = 11.6). Thus, DQ molecules that express the TrB12 epitope may contribute to the susceptibility to develop MS.

Antibodies↗
PubMed1989

Direct HLA typing by rosetting with immunomagnetic beads coated with specific antibodies.

Paramagnetic monodisperse polymer beads were coated with purified human hybridoma antibodies specific for polymorphic HLA determinants. The mAb-coated beads bound specifically to cells from individuals with the relevant HLA type. The rosettes formed by beads and cells were isolated with a magnet. Rosette formation was evaluated microscopically and used as criterion for positive typing. By this rosette assay, fast and reliable HLA typing of whole blood was possible.

Antibodies, Monoclonal↗
PubMed1988

Immune responses to an adjuvant-free native syngeneic myeloma protein (M315).

Myeloma protein 315 (M315; isotype IgA, lambda 2) is used in this report as a model to explore the immunogenicity of a syngeneic Ig under nearly physiological conditions. We have previously shown that a synthetic peptide spanning the mutated HV3 loop of the L-315 chain, when emulsified in complete Freund's adjuvant, elicits T helper cells (Th) that respond to a boost with L-315 or M315, indicating that M315 is recognized as a processed protein antigen. We now show that the adjuvant-free 7S monomer of native or of mildly reduced and alkylated M315, given in divided doses totalling 300 or 800 micrograms to BALB/c mice, induced persistent anti-M315 antibodies (Ab), a large part of which was IgG1 directed mainly to idiotypes (Id) associated with M315's hapten-binding site. Polymers of M315 IgA (800 micrograms) failed to induce Ab, due probably to their rapid clearance into bile. Short-term treatment with anti-CD4 monoclonal Ab GK1.5 at the time of priming with 7S M315 inhibited the responses almost completely. The spleens of M315-immune mice contained Th that recognized the L-chain subunit of M315 as a carrier indicating that these Th did not require an assembled (VH-VL) pair of 315 V regions to be activated. We also observed low amounts of Ab specific for epitopes of the C alpha region. This evidence opens the possibility that a distinct autoimmune pathway exists for elicitation of rheumatoid factor (RF; autoAb to Fc gamma) that involves help to RF-producing B cells by Id-specific Th. We suggest that these Th recognize V-region peptides from IgG that have been captured, processed and presented by these B cells.

Adjuvants, Immunologic↗
PubMed1988

Four cytotoxic human-human hybridoma antibodies that react with HLA-B27.

PBMC were isolated from a multiparous woman with HLA-B27 specific Abs in her serum. The HLA type of the donor was A2,9:B7. The PBMC were EBV transformed, and four cell lines making cytotoxic Abs to HLA-B27+ cells prepared. Hybridomas were constructed by fusing the EBV lines with the human fusion partner KR4. All four mAbs were of IgM isotype. One mAb (TrBH12) reacted specifically with B27+, B37+ and Bw47+ lymphoblastoid cell lines and with all B27+ PBMC except for a rare variant so far found only in one Norwegian family. Another mAb (Tr3B6) was cytotoxic for all B27+ cells tested, including the TrBH12- variant; in addition, it showed weaker cross-reactions to Bw42, B49 and a cell line with the probable phenotype B7,38. Supernatant from the Tr3B6 hybridoma was tested in lymphocytotoxicity against a panel of 658 individuals, 141 of whom were B27+. With this panel, Tr3B6 showed perfect correlation with HLA-B27. The two last mAbs (TrCG10 and TrBF1) reacted with all B27+ cells tested, but in addition showed quite extensive cross-reactions.

Antibodies, Monoclonal↗
PubMed1988

A cytotoxic human-human hybridoma antibody (TrH6) specific for HLA-DRw52.

TrH6 is a new human-human hybridoma antibody (Ab) of IgM, lambda isotype. At concentrations of 17-5000 ng IgM/ml, TrH6 killed twelve out of fourteen lymphoblastoid cell lines that expressed DRw52, but none of the nine DRw53 homozygous lines. Both DRw52+ cells (LUY, TAB) not killed by TrH6 were DR8+, suggesting that these lines express a structural variant of DRw52 linked to DR8. The murine cytotoxic DRw52-specific mAb 7.3.19.1 (Koning et al. 1984) exhibited the same pattern of reactivity with DRw52+ cells as TrH6. However, unlike TrH6, 7.3.19.1 cross-reacted weakly with two DR7+DRw53+ homozygous cell lines, suggesting that the TrH6 epitope has a more limited distribution. The reactions with a panel of cells with known deletions in the HLA-region supported the conclusion that TrH6 is specific for DRw52. Further evidence was obtained in a competition RIA, where the murine mAbs L243 (anti-DR monomorphic) and 7.3.19.1 (anti-DRw52) inhibited binding of radiolabeled TrH6 to DRw52 homozygous cells. TrH6-coated magnetic beads provided a simple rosette-assay for typing of "buffy-coat" leukocytes for the TrH6 epitope.

Antibodies, Monoclonal↗
PubMed1988

Are idiotypic peptides from variable domains critical for T-dependent production of rheumatoid factors?

Studies from this laboratory have indicated that isologous myeloma protein 315 (M315, isotype IgA, lambda 2) elicits T helper cells which recognize processed forms (peptides) of its V-domains and that recognition is controlled by H-2 linked immune-response (Ir) genes (J Exp Med 1982; 155:1587-96; Eur J Immunol 1986; 16:889-93). We now report that adjuvant-free soluble M315, particularly the mildly reduced and alkylated form, stimulates a T-dependent antibody response mainly specific for M315's paired V-domains. A small subset of the antibodies appeared to recognize the C-region of IgA, thus being analogous to rheumatoid factors (RF). On the basis of these observations, we propose that one pathway to RF production depends on T helper cells that interact directly with RF-producing B cells across peptide-MHC bridges. These peptides are envisaged to be derived from hypervariable regions of IgG V-domains, and they are therefore called "idiotypic peptides". This hypothesis assumes that the number of different idiotypic peptides is so large that the immune system has not developed tolerance to all of them.

Alkylation↗
PubMed1987

A human-human hybridoma (Tr7E2) producing cytotoxic antibody to HLA-DQw1.

A human-human IgM (lambda) hybridoma antibody (called Tr7E2) was constructed by fusing Epstein-Barr virus-transformed cells from a multiparous woman with the human fusion partner KR12. By eosin exclusion microcytotoxicity the monoclonal antibody killed 12 of 13 human leukocyte antigen DQw1-bearing lymphoblastoid cells. No reaction was seen with any of 19 DQw1-negative cells. The single DQw1+ cell line that was not killed by Tr7E2 was the homozygous cell called 9WS 806 TAB (DR8,8; DQw1,1) of Japanese origin. The radioimmunoassay indicated that this result was probably not because of a decreased expression by this cell of DQ antigens, and these cells were killed by the mouse monoclonal antibody Genox3.53G2a5, reported to be specific for DQw1. Thus, the Tr7E2- cell line TAB probably expresses a novel structural DQw1 variant. Of 213 Norwegians, 107 were Tr7E2+ Genox+; none expressed only one of these epitopes. The putative split is, therefore, probably very rare in this population. Monodisperse magnetic polymer beads coated with Tr7E2 formed rosettes selectively with peripheral blood mononuclear cells from DQw1-positive individuals, suggesting a new approach to typing for class II antigens.

Antibodies, Monoclonal↗
PubMed1987

A human-human hybridoma antibody (TrB12) defining subgroups of HLA-DQw1 and -DQw3.

We have constructed an IgG, kappa human--human hybridoma Ab(TrB12), which precipitates a molecule consisting of two polypeptides of about 33 and 27 kD in size. TrB12 reacted with; (1) 7 out of 10 DQw1-positive cell lines in IIF, and all 10 in a rosette-assay; (2) 5 out of 12 DQw3-positive cells, both in IIF and the rosette-assay; (3) none of 4 DQw2 homozygous cells. Detergent cell lysate of DQw1 homozygous cell lines contained antigens that cross-linked the mouse monoclonal antibody Genox 353 G2a-5 (anti-DQw1) and TrB12. TrB12 competed with the mouse DQw3-specific monoclonal antibody IVD-12 for binding to DQw3 homozygous cells. The data imply that the TrB12 epitope is associated with molecules that carry DQw1 and DQw3 serological specificities. By radioimmunoassay, TrB12 and the mouse monoclonal antibody IIB3 divided both DQw1- and DQw3- bearing cell lines into three phenotypic groups: (1) TrB12+IIB3hi, (2) TrB12-IIB3lo, and (3) TrB12-IIB3-. For DQw1 the results suggest that the first two groups represent structural variants but the third group may reflect low expression of DQw1. For DQw3 the evidence suggests that all three phenotypes represent structural variants. DQw3 has previously been divided into two serologically defined alleles, TA10+IIB3- and TA10-IIB3+. The TrB12+IIB3hi and TrB12-IIB3lo variants of DQw3 described in this study probably represent novel subgroups of the TA10-IIB3+ allele.

Alleles↗
PubMed1987

Two M315 idiotopes defined by isologous monoclonal antibodies: one depends on germline and the other on mutated murine lambda 2 light chain sequences.

We have previously reported that T helper cells of BALB/c mice recognize the unique mutated sequence Phe94, Arg95, Asn96 of the lambda 2 L chain of isologous (BALB/c) myeloma protein 315. Here we study two Id (Id-315.1.4 and Id-315.TH) of the DNP-Lys binding M315 defined by two monoclonal isologous anti-Id Ab (Ab 2-1.4 and Ab 2-TH). Both Id were (1) totally expressed by Fv-315, but not by free unpaired V domains, (2) specifically dependent on VH-315, since lambda 2-315 recombined with four other H chains did not express the Id, (3) related to the hapten-binding site because their expression was blocked by the haptens DNP-Lys and DNP-Gly, and (4) topographically related because Ab 2-1.4 and Ab 2-TH competed with each other for binding to M315. The contribution of lambda chain V regions was studied with the aid of reconstituted Ig molecules of H-315 paired with lambda 1, lambda 2, and lambda 3 L chains. Id-315.TH was expressed equally well by reconstituted Ig containing three different lambda 2 chains (lambda 2-5-7, lambda 2-T952, and lambda 2-315), but its expression was profoundly reduced when H-315 was associated with lambda 3-SAPC15 or lambda 1-J558 L chains; it therefore depended upon amino acids encoded by germline lambda 2 genes. By contrast, Id-315.1.4 was only restored by the lambda 2-315 chain paired with H-315. Since lambda 2-5-7 and lambda 2-T952 differ from lambda 2-315 at positions 38, 94, 95, 96, and 98 or 99, respectively, Id-315.1.4 probably requires the unique mutated amino acids Phe94, Arg95, Asn96 of lambda 2-315. This resembles the effects on Id expression of previously reported unique amino acids of the D region. We failed to confirm that hyperimmunization of BALB/c mice with Ab 2-1.4 cross-linked to KLH induced M315-like Ab. The results are discussed in terms of the contribution of the third hypervariable loop of lambda chains to Id and the immunogenicity of isologous Ig.

Amino Acid Sequence↗
PubMed1987

A human-human hybridoma producing cytotoxic antibody to HLA-B15, cross-reacting with B17, B5, B35 and B18.

Mononuclear blood cells from a multiparous woman were transformed with Epstein Barr virus, and a cell line (Tr2D8) producing anti-HLA antibody was obtained. This cell line was immortalized by hybridization to the human fusion partners KR4 and KR12. While the EBV line died after 7 months, the hybridomas have remained stable for 13 months. The EBV line supernatant (40 micrograms IgM/ml) lysed peripheral blood mononuclear cells (PBMC) bearing B15, B17, B5 and B35. Consistent lysis of B18 bearing cells was only observed with lymphoblastoid cell lines. The supernatant from the Tr2D8 (EBV line X KR4) hybridoma (2.7 micrograms IgM/ml) only lysed B15 bearing PBMC. At a concentration of 13.5 micrograms IgM/ml, the hybridoma antibody lysed lymphoblastoid cell lines bearing B15, B17, B5, B35 and B18.

Antibodies, Monoclonal↗