Biomedical subjects

K Hannestad

Publications and source records attributed to K Hannestad.

At least 19 recordsLinked to original sources

PubMed2001

The primary IgM antibody repertoire: a source of potent idiotype immunogens.

A widely held view is that, to elicit adaptive immune responses, most protein antigens must be given with adjuvants that activate the innate immune system. It has also been proposed that the immune system is tolerant to idiotypes (Id) of the syngeneic primary antibody (Ab) repertoire. We now show that among 73 purified noncomplexed secretory IgM monoclonal antibodies (mAb), 4 (5.5%) elicited high levels of IgG Ab against the Id even though no adjuvant was added. The responses were controlled by H2-linked immune response genes. IgG1, but no IgG2a or IgG2b, anti-Id Ab were detected, indicating involvement of T helper type 2 (Th2) cells. All 4 IgM mAb are likely germ-line gene-encoded, and 1 was shown to represent a recurrent Id. After endotoxin depletion the most potent immunogen of the 4 still provoked robust humoral anti-Id responses. The results suggest that a natural protein of the primary IgM Ab repertoire can be immunogenic without an adjuvant.

Animals↗

PubMed2000

Genes predisposing to autoimmunity augment constitutive major histocompatibility complex class II-associated presentation of the self-antigen IgG2a in vivo.

The self-antigen IgG2ab is poorly presented to a gamma2ab 435-451-reactive I-Ad-restricted T-cell hybridoma unless available in high concentrations or targeted to Fcgamma- or complement receptors. Environmental factors, probably the extent of microbial challenge, profoundly influence the constitutive gamma2ab/I-Ad presentation in IgCHb, H-2d mice. Here we report also a strong genetic impact. Constitutive presentation was highly efficient in spleen and thymus of (NZB x BXSB)F1 mice, which inherit a predisposition to develop lupus. Presentation correlated with disease progression and the serum levels of IgG2ab and IgG2ab complement factor 3 complexes. The finding that constitutive presentation was by far most efficient in males indicated that it was augmented by the Y chromosome-linked autoimmune acceleration Yaa gene. In line with previous data for healthy mice, constitutive gamma2ab/I-Ad presentation was most pronounced in the adherent spleen cell fraction and improved by further enrichment for dendritic cells. Notably, however, whereas in normal mice the gamma2ab determinant was undetectable on B cells lacking surface IgG2ab, such B cells contributed considerably to constitutive presentation in (NZB x BXSB)F1 hybrids. Presumably this resulted from complement receptor-mediated internalization of IgG2ab-containing immune complexes formed in lupus. These data add to the evidence that B cells with self-reactive receptors, known to exist in the mature repertoire, may present non-cognate foreign antigen to anti-foreign helper T lymphocytes and thus differentiate into autoantibody-secreting cells, and might likewise account for the polyclonal B-cell activation characteristic of several autoimmune syndromes.

Animals↗

PubMed2000

Native IgG2a(b) is barely antigenic to major histocompatibility complex class II-restricted T cells owing to inefficient internalization by professional antigen-presenting cells.

Peptide epitopes derived from immunoglobulin variable regions represent tumour-specific antigens on B-cell neoplasms and can be recognized by syngeneic, major histocompatibility complex (MHC) class II-restricted T cells. Immunoglobulin peptide/MHC class II complexes may also be involved in autoimmunity and CD4+ T-cell-mediated B-cell regulation. Thus, the IgG2a(b) H-chain allopeptide gamma2a(b) 435-451 presented on I-Ad mimics the epitope implicated in herpes simplex virus-induced autoimmune stromal keratitis and is the target of T helper 1 (Th1) clones that suppress IgG2a(b) production in vivo. We here report that spleen and thymus cells constitutively present the autologous gamma2a(b) epitope to a gamma2a(b) 435-451/I-A(d) reactive T-cell hybridoma as a function of the animal housing conditions (specific pathogen-free or not) and the serum levels of IgG2a(b). Constitutive presentation in the spleen was predominantly performed by dendritic cells. Whereas spleen cells poorly presented native IgG2a(b) to a gamma2a(b) 435-451/I-A(d) reactive T-cell hybridoma, IgG2a(b) in the form of immune complexes were presented > 200-fold more efficiently owing to internalization via low-affinity FcgammaR on macrophages. The antigenicity could also be improved by homotypic aggregation and by targeting IgG2a(b) to complement receptors on the A20 B-cell lymphoma. Mice without detectable IgG2a(b)-containing immune complexes typically exhibited minimal constitutive presentation. Nevertheless, native IgG2a(b) can sensitize antigen-presenting cells in vivo, as mice that were devoid of immune complexes and carried an IgG2a(b)-producing tumour did present constitutively, even at physiological IgG2a(b) serum levels. Whereas the amounts of IgG released from most B-cell lymphomas may be too low to allow spontaneous priming of tumour-specific MHC class II-restricted T cells, administration of tumour immunoglobulin in aggregated form might improve the efficacy of idiotype vaccination.

Animals↗

PubMed1999

The P9 peptide sidechain specificity of I-Ad.

The murine MHC class II variant I-Ad confers susceptibility to herpes simplex virus (HSV)-induced keratitis and relative protection against type 1 diabetes mellitus. The association to these autoimmune diseases appears to be largely determined by the peptide sidechain specificity of the P9 pocket, which we therefore have analyzed in detail. Assessment of T-cell responses and I-Ad binding capacity of position 446-substituted analogs of an IgG2a allotype b (IgG2a(b)) heavy chain peptide demonstrates that engagement of the P9 pocket is crucial for effective peptide presentation. Sidechain size rather than charge decides the capacity to engage the P9 pocket. Thus, small, uncharged sidechains are accepted, whereas acidic and aromatic amino acids as well as lysine and arginine are disfavored. The specificity of the P9 pocket of I-Ad (serine beta57) is distinct from that of the diabetes-associated I-Ag7 (aspartic acid beta57), supporting the contention that the polymorphism at residue beta57 influences diabetes susceptibility via P9-specific effects on the repertoires of self peptides presented to T cells. Furthermore, the data rationalize the susceptibility to HSV-induced keratitis conferred by the a and the protection conferred by the b allotypes of the IgG2a heavy chain. Keratitogenic T cells, which cross-react with the viral UL6 protein and a corneal antigen, are silenced in IgG2a(b) mice because of antigenic mimicry with gamma2a(b) 435-451. Our finding that the lysine P9 residue of the corresponding gamma2a(a) allopeptide precludes high-affinity binding to I-Ad indicates that the susceptibility of IgG2a(a) mice reflects inefficient thymic presentation of autologous IgG2a and thus failure to purge the T-cell repertoire of the pathogenic clones.

Animals↗

PubMed1999

N-terminal elongation of a peptide determinant beyond the first primary anchor improves binding to H-2 I-Ad and HLA-DR1 by backbone-dependent and aromatic side chain-dependent interactions, respectively.

The IgG2a(b) heavy chain allopeptide determinant gamma2a(b) 436-451 (Kabat numbering) presented by the major histocompatibility complex (MHC) class II molecule I-Ad is recognized by T cells which cross-react with a corneal self antigen and with the UL6 protein of the herpes simplex virus which induce autoimmune keratitis, and is the target of Th1 clones that suppress IgG2a(b) production in vivo. In the gamma2a(b) peptide/l-Ad complex, tyrosine438 is the first primary anchor (P1) and residues 440-445 encompass the T cell receptor contact residues. Amino-terminal elongation of gamma2a(b) 437-451 by a single residue (P-2) augmented the I-Ad binding capacity 10-fold and the antigenicity 55-195-fold. This was a function of the peptide main chain, since non-conservative substitutions were accepted. The gamma2a(b) peptide also bound HLA-DR1, and amino-terminal extension by a single aromatic amino acid at P-3 augmented binding 15-fold. The interaction between HLA-DR1 and P-3 specifically required an aromatic peptide side chain, and computer simulations indicated that the aromatic ring at P-3 engaged conserved HLA-DR1 phenylalanine residues at the edge of the peptide binding groove. Thus, these data demonstrate that residues amino terminal to P1 may substantially increase peptide affinity for MHC class II by main chain-dependent as well as side chain-dependent interactions, and imply that the HLA-DR1 motif should be extended to include an aromatic amino acid at P-3.

Animals↗

PubMed1997

Engagement of the B lymphocyte antigen receptor induces presentation of intrinsic immunoglobulin peptides on major histocompatibility complex class II molecules.

By means of the clonotypic variable region, the immunoglobulin (Ig) is a tumor-specific antigen on B cell neoplasms. We report that engagement of the B cell antigen receptor (BcR) promotes presentation of peptides derived from the B cell's intrinsic Ig to major histocompatibility complex (MHC) class II-restricted T cells. Thus, anti-Ig endowed normal, ex vivo B lymphocytes from H-2d, Ig constant heavy chain allotype b (IgCHb) mice with the capacity to stimulate an I-Ad-restricted T cell clone which recognizes the gamma 2ab 435-451 allopeptide. The corresponding self gamma 2aa peptide is cryptic and 6000-fold less antigenic than the gamma 2ab allopeptide. Even so, the syngeneic B cell lymphoma A20 which expresses surface(s) IgG2aa, was also recognized by the T cells after BcR ligation. Thus, anti-Ig triggered the disclosure of a cryptic tumor antigen determinant. We propose that autoantigens, by engaging the BcR of self-reactive B cells, induce presentation of intrinsic Ig peptides to which the T helper cell (Th) repertoire is not tolerant. In this way, B cells with anti-self potential may be activated without Th recognition of nominal autoantigen.

Animals↗

PubMed1997

A retro-inverso analog mimics the cognate peptide epitope of a CD4+ T cell clone.

Synthetic analogs of peptide epitopes may activate specific T helper cells, antagonize their antigen receptors, or block recognition by competing for major histocompatibility complex (MHC) class II binding sites. Rationally designed peptides may therefore prove useful as vaccines and for treatment of autoimmune diseases and allergies mediated by CD4+ T cells. However, their susceptibility to proteolytic degradation limits the applicability of conventional peptides in vivo. By contrast, retro-inverso analogs, in which a native sequence is substituted with D-amino acids linked with a reversed backbone, resist proteolysis and still maintain the side chain topology of the corresponding natural peptide. We report here that an end group-modified retro-inverso analog of the IgG2ab heavy chain allopeptide determinant gamma 2ab 435-447 was recognized by an I-Ad-restricted, gamma 2ab 435-447-reactive T cell clone. The pseudopeptide elicited near-maximal interleukin-2 responses, although 300-fold higher concentrations were needed than the native determinant. The weaker antigenicity of the retro-inverso analog could be fully accounted for by an impaired I-Ad binding capacity, which might reflect reduced ability of the distorted main chain to form hydrogen bonds with I-Ad. Glycine substitution at the residue corresponding to the first primary anchor (P1) of the native peptide abrogated I-Ad binding and antigenicity of the retro-inverso analog. Thus, the pseudopeptide resembled the native determinant with respect to orientation in the class II binding site, configuration of the epitopic side chains, and the constraints that governed the interactions between a major anchoring side chain and I-Ad. In conclusion, proteolytically resistant compounds with predefined capacity to interact with MHC class II allelic products and T cell antigen receptors may be designed by retro-inverso modification of native determinants.

Animals↗

PubMed1997

A novel first primary anchor extends the MHC class II I-Ad binding motif to encompass nine amino acids.

The MHC class II molecule I-Ad has been reported to bind peptides containing a motif of six consecutive amino acids. We demonstrate that binding of the murine IgG2ab heavy chain allopeptide gamma 2ab 435-451 (Kabat numbering) to I-Ad is strongly enhanced by a novel first primary anchor (P1) three residues N-terminal to this hexamer. This is based on flow cytometric assessment of the I-Ad binding capacity of gamma 2ab peptide analogues, their antigenicity for I-Ad-restricted T cell clones and molecular modelling. The P1 pocket is broadly specific since allphatic, aromatic, acidic, the basic histidine and small polar side chains all allowed good binding. By contrast, asparagine, arginine and glycine reduced the binding capacity 10-, 16- and > 100-fold respectively. Truncation or glycine substitution at P1 decreased antigenicity by a factor > 1000. Nevertheless, I-Ad-restricted T cells are not completely dependent on this anchor since high concentrations of a peptide with glycine-substituted P1 elicited maximal responses. Additional anchoring side chains are found at P4, P6 and P9. The autologous IgG2aa heavy chain shares prominent epitopic residues with gamma 2ab 435-451 at P3, P5 and P8. However, the lysine of gamma 2aa at P9 impairs binding to I-Ad, which may explain why the gamma 2ab allopeptide-reactive T cells escaped negative selection. The data rationalize our observation (Bartnes, K. and Hannestad, K. 1997. Eur. J. Immunol. 27:1124) that these T cells recognize a syngeneic B cell lymphoma, provided its presentation of intrinsic gamma 2aa is enhanced by surface IgG2aa ligation.

Animals↗

PubMed1995

A syngeneic idiotype is immunogenic when borne by IgM but tolerogenic when joined to IgG.

Some syngeneic monoclonal antibodies (mAb) elicit immune responses like conventional T-dependent antigens. To find out whether the heavy chain class (isotype) plays a role for the immunogenicity of an idiotype (Id), we isolated rare subclones of an IgM mAb (termed Id3) in which the variable region of the heavy chain (VH) is associated with a new constant region (CH). The VH-Id3 gene is a member of the murine 36-60 family and probably has three replacement mutations. The light chain V gene is germ-line V lambda 2. IgM, IgG1, IgG2a and IgG2b variants of Id3 were purified from protein-free medium and injected without adjuvant into BALB/c mice. The parental 19S IgM mAb given subcutaneously (s.c.) elicited a vigorous humoral response against Id3; in comparison, monomeric 8S IgM was a much weaker immunogen. Unlike IgM, multiple challenges with the IgG switch variants failed to induce anti-Id3 Ab. IgG variants gained immunogenicity if they were purified from medium containing fetal calf serum, mixed with complete Freund's adjuvant or injected into mice primed with IgM-Id3. Pretreatment with 100 micrograms s.c. + 50 micrograms of the IgG2a variant extinguished the Ab response to parental IgM, but the response to adjuvant-free bovine serum albumin was intact. Therefore, the tolerance induced by the IgG2a switch variant is antigen-specific and not due to toxicity. Significant inhibition of the Ab response to parental IgM was observed after treatment with 4 micrograms of the IgG2a switch variant. Administration of the IgG1 and IgG2b switch variants also inhibited this response significantly. Thus, the outcome of an encounter with Id3 is strongly influenced by the CH isotype to which the Id is joined. This suggests novel ways to minimize unwanted Ab responses against Id of human therapeutic mAb. In the context of the theory of Id networks, we suggest that dominant B cell clones can preempt anti-Id Ab responses against themselves by early switching from IgM to IgG secretion, before immunogenic IgM Ab have had time to activate anti-Id B cells.

Amino Acid Sequence↗

PubMed1995

A human hybridoma monoclonal antibody (TrJ11) recognizing a new HLA-DR epitope shared by DR4, DR8, DR11, and DRB1*1303.

The cytotoxic IgM lambda human hybridoma mAb TrJ11 reacts with lymphoblastoid B-cell lines expressing DR4, DR8, DR11, and DRB1*1303. However, TrJ11 was monospecific when normal B cells freshly isolated from blood served as targets in that it only killed HLA-DR4-positive cells. Thus, of 235 HLA-typed persons TrJ11 was strongly cytotoxic for normal B cells of all 90 DR4-positive individuals, but it did not react with B cells from any of the 145 DR4-negative donors. Hence, mAb TrJ11 proved to be suitable for routine DR4 typing. The specific binding of TrJ11 to a DR4-positive cell line was profoundly blocked by the mouse HLA-DR beta chain-specific monomorphic mAb TAL 14.1, indicating that the epitope recognized by TrJ11 is located in the DR beta chain. The possibility that amino acids located in the floor of the peptide-binding site are critical for the TrJ11 epitope is discussed.

Alleles↗

PubMed1994

Identification of the gene encoding a novel HLA-B39 subtype. Two amino acid substitutions on the beta-sheet out of the peptide-binding floor form a novel serological epitope.

Serological analysis suggests the existence of a novel HLA-B39 subtype (HLA-B39N) in the Japanese population. To identify this novel allele, a gene encoding HLA-B39N was cloned and the exons were sequenced. A gene encoding HLA-B39N (B*3904) and B*39011 differs by two nucleotide substitutions at codons 11 and 12 whereas B*3904 and B*39013 differ by three nucleotide substitutions at codons 11, 12, and 312. One nucleotide difference at codon 11 produces a change from serine in B*3901 to alanine in B*3904 whereas another difference at codon 12 changes valine in B*3901 to methionine in B*3904. The residues 11 and 12 are located on the beta-sheet out of the peptide-binding floor and are completely buried in the molecule. These results suggest that the substitutions at these residues alter the conformation of other residues forming epitopes of alloantibodies. Analysis of HLA-B*3901 genes in the Japanese population showed that both B*39011 and B*39013 were observed in the Japanese population. The present study suggests that B*3904 may have evolved from B*39011 rather than B*39013.

Amino Acid Sequence↗

PubMed1994

A cytotoxic human hybridoma monoclonal antibody (TrJ6) defining an epitope expressed by HLA-DQ4 and -DQ5.

We have generated a cytotoxic human hybridoma monoclonal IgM lambda antibody, designated TrJ6, that is specific for a new epitope shared by HLA-DQ4 and -DQ5. TrJ6 strongly killed all ten DQ4-bearing cells and weakly killed all four DQ5-bearing cell lines. In contrast, none of the 36 cell lines lacking DQ4 and DQ5 antigens was recognized by TrJ6. This was confirmed by fluorescence cytometry. The specific binding of TrJ6 to a DQ4-bearing line was efficiently blocked by IIB3 (murine anti-DQ8+9+4+5+6 mAb) and TrG6 (human IgG mAb against DQ4+5+6), confirming that TrJ6 is specific for a polymorphic DQ epitope. TrJ6 can be used to distinguish DQ5+ from DQ6+ B-lymphoblastoid cells. DQ4 beta and DQ5 beta chains share one unique residue (Ser-74) and one relatively unique residue (Val-75), which may therefore need to be coexpressed in order for the TrJ6 epitope to be formed. Alternatively, Ser-74 alone contributes critically to the allospecificity of this epitope. In addition, one or more of three residues unique for DQ4 (Leu-56, Glu-70, and Asp-71 on the DQ4 beta chain) could also contribute to the TrJ6 epitope because TrJ6 reacted stronger with DQ4- than with DQ5-bearing cell lines.

Amino Acid Sequence↗

PubMed1994

A novel HLA-A determinant recognized by a cytotoxic human hybridoma IgG1 monoclonal antibody (TrJ14).

TrJ14 is a cytotoxic human IgG1 lambda hybridoma mAb that recognized a novel HLA-A epitope expressed by lymphoblastoid B cells that are homo- or heterozygous for A2, A3, A11, A30, A31, A33, A68 and A69. Based on these results, the HLA type of cell line TEM (10w9057) was retyped as A66. When peripheral blood T cells isolated freshly from 265 HLA-typed normal individuals served as targets, TrJ14 killed cells expressing two TrJ14-positive HLA-A alleles, as well as the majority of cells having one TrJ14-positive and one TrJ14-negative HLA-A antigen. However, TrJ14 failed to recognize or reacted weakly with most HLA-A2 and -A3 heterozygous normal T cells when A2 or A3 was coexpressed together with a TrJ14-negative antigen. The serological reactivity of TrJ14 correlated with the amino acid valine and aspartic acid at positions 76 and 77 of the alpha 1-domain helix. These amino acids were shared exclusively by all the identified TrJ14+ alleles.

Amino Acid Sequence↗

PubMed1993

Th1 clones that suppress IgG2ab specifically recognize an allopeptide determinant comprising residues 435-451 of gamma 2ab.

We have previously reported that gamma 2ab/I-A(d)-specific Th1 clones from BALB/c mice (gamma 2aa, H-2d) mediated a long-lasting, selective suppression of serum IgG2ab levels when transferred to newborn (BALB/c x B10.D2)F1 (gamma 2a/b, H-2d) mice (Bartnes, K. and Hannestad, K. Eur. J. Immunol. 1991. 21: 2365). We here analyze the peptide specificity of hybridomas derived from two suppressive T cell clones. The shortest synthetic peptide with optimal antigenicity comprises gamma 2ab residues 435-451 (Kabat numbering). The determinant core encompasses the gamma 2ab 440-446 (KLRVQKS) sequence which contains an I-A(d) allele-specific motif. Challenge with single amino acid-substituted gamma 2ab 435-447 analogs revealed that residues K440, R442 and K445 which are shared by the autologous and allogeneic gamma 2a, as well as residues Q444 and S446 which represent allogeneic differences, are critical for recognition. We obtained evidence that K440, R442 and Q444 are epitope residues, while K445 and S446 contribute to anchoring of the peptide to I-A(d). Amino acids located outside of the core also influence antigenicity, the most striking effect being a 340-870-fold augmentation of potency when gamma 2ab 437-451 is extended by F436. IgG2ab required processing in order to stimulate the hybridomas. The data support the contention that the Th1 clones specific for Fc of gamma 2ab mediated IgG2ab suppression by cognate interaction with sIgG2ab+ B cells that presented a C gamma 2ab peptide(s) derived from their endogenous Ig on major histocompatibility complex class II. The T cells cross-reacted weakly with peptide 435-451 of the autologous gamma 2aa allotype. This opens the possibility that self-peptides from Ig C regions can target B cells for regulatory interactions with autologous Th cells.

Amino Acid Sequence↗

PubMed1993

A cytotoxic human hybridoma monoclonal antibody (TrJ5) specific for HLA-B38(16) and -B39(16).

We have generated a cytotoxic IgM lambda human hybridoma mAb (TrJ5), that is specific for HLA-B38(16) and -B39(16). Among a panel of 42 HLA-defined cell lines, TrJ5 killed all five B38-positive and both B16-positive cell lines, as well as the single B39-positive cell line, but not any cell lines lacking these antigens. It could be ruled out that the TrJ5 epitope is located on the alpha 1 domain because TrJ5 did not react with cells bearing HLA-B14, the alpha 1 domain of which is identical to alpha 1 of B39. In the alpha 2 domain of B38 and B39, a unique threonine residue at position 158 (Thr-158) is not shared by any of 29 other HLA-B alleles. This suggests that Thr-158 of the alpha 2-domain helix is critical for the TrJ5 epitope.

Antibodies, Monoclonal↗

PubMed1993

A human monoclonal hybridoma antibody (TrJ1) specific for HLA-DQ2.

TrJ1 is a cytotoxic human hybridoma mAb (IgM lambda). Its reaction pattern with a panel of 42 HLA-defined lymphoblastoid B-cell lines correlated precisely with expression of DQ2. By flow cytometry it was shown that the binding of TrJ1 to DQ2 was efficiently blocked by the murine anti-DQ2 mAb 358.4, indicating that the TrJ1 and 358.4 epitopes overlap. TrJ1 reacted much better with EBV-transformed B cells than with B cells freshly isolated from blood. TrJ1 seemed suitable for typing freshly isolated B cells provided the incubation with complement lasted for 115 min in Terasaki plates. One or more of the DQ2-specific polymorphic amino acids E46, F47, L52, L55, K71 or A74, situated on the alpha-helix of the DQ2 beta chain, are probably critical for the TrJ1 epitope.

Antibodies, Monoclonal↗

PubMed1992

Anti-idiotypic immune responses against adjuvant-free isologous IgM monoclonal antibodies and their augmentation by complex formation between IgM and albumin in bovine serum.

In previous studies we demonstrated that the hypermutated isologous myeloma protein MOPC 315 (isotype IgA; lambda 2) is recognized by T helper cells like an ordinary foreign protein antigen. To what extent can an immune system recognize and respond to V domains from the primary (pre-immune) repertoire? To study this question we made 21 BALB/c hybridoma anti-2,4,6 trinitrophenyl monoclonal antibodies (mAb) of IgM; lambda isotype. All mAb purified from supernatants containing fetal bovine serum had formed spontaneous complexes with bovine serum albumin possibly by way of disulfide interchange. Twenty of twenty-one mAb from this source elicited IgG1 anti-idiotypic (Id) Ab when given as a single adjuvant-free dose of 200 micrograms. For 12 of them even 10 micrograms was sufficient. This indicated that BSA augmented the anti-Id responses by a carrier effect. Three of the mAb were therefore purified from ascites fluid and from serum-free medium. Only one of them then induced humoral anti-Id responses in BALB/c mice when given as a single adjuvant-free dose of 100 micrograms. The other two became immunogenic when emulsified in Freund's complete adjuvant. The results indicate that some IgM mAb exist whose Id determinants alone can elicit substantial anti-Id responses because they are recognized like ordinary foreign protein antigens. Since albumin in fetal bovine serum forms complexes with IgM and greatly augments its immunogenicity, serum-free medium should be used for production of human or humanized therapeutic IgM mAb. A possible role for Id for IgM Ab as cardinal autoantigens is discussed.

Adjuvants, Immunologic↗

PubMed1992

Mapping of an epitope defined by a human hybridoma antibody (TrD3): a new HLA-B supertype associated with a subset of HLA-Bw6.

The new human-human hybridoma TrD3 secretes a cytotoxic IgM mAb, which reacted with 28 of a panel of 56 HLA-typed lymphoblastoid cells. All 28 TrD3+ cells expressed the HLA-B supertype Bw6, whereas 10 Bw6+ cells were not recognized by the mAb. None of the 17 Bw4 homozygous cells were positive with TrD3. Thus, TrD3 divided the Bw6+ HLA-B specificities of the cell lines into two subgroups, namely, Bw6+TrD3+ and Bw6+TrD3-, and therefore defines a new HLA-B supertype. TrD3 reacted strongly with some B8+ cell lines and weakly or not at all with others, suggesting a new split of HLA-B8. Compared with cell lines, TrD3 reacted more weakly with freshly isolated T cells from blood. The Bw6-specific rat mAb SFR8-B6 partially blocked the binding of 125I-labeled TrD3 to a Bw6+ cell line. By using cell lines transfected with hybrid genes between HLA-B7 (Bw6+) and HLA-B27 (Bw6-) as targets in flow cytometry, critical residues for the TrD3 epitope could be mapped to the amino acid region 24-62 of the HLA class-I alpha 1 domain. Comparison of deduced amino acid sequences of TrD3-positive and -negative cells indicated that a tryptophane residue at position 95 destroyed the TrD3 epitope, and that one or more of the residues in positions 24, 45, and 46 may be critical, suggesting that it is a discontinuous epitope. It is notable that none of these residues are located on alpha-helixes.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗