Kristian Hannestad

Acid Elution of Blood Group Antibodies from Intact Erythrocytes

Abstract. Human IgM and IgG antibodies against blood group antigens (A, B, D, C, c, E, e, Fya, K), autoantibodies and mouse IgM and IgG antibodies against sheep erythrocytes have been eluted from intact human and sheep red cells by glycine‐HC1 buffer, pH 3.0. The yield of human antibodies was higher with acid than with heat and ether elution, and the contamination of hemoglobin in the eluate was negligible. The acid elution method is very simple and rapid and, therefore, highly suitable for experimental as well as routine immuno‐hematological work.

Immunoglobulin heavy chain constant regions regulate immunity and tolerance to idiotypes of antibody variable regions

Particular syngeneic adjuvant-free monoclonal antibodies are immunogenic and elicit antibody responses against the variable region idiotypes (Ids). We here study how heavy-chain constant regions (CH) regulate immune responses to Ids of free, uncomplexed monoclonal antibodies. To this end, we selected two hybridomas, called Id3 and IdA.01, that produce immunogenic IgMλ2 directed toward 2,4,6-trinitrophenyl, and subcloned rare IgG1, IgG3, IgE, or IgA class switch variants. The purified switch variants, which possessed the Ids of their IgM progenitors, were injected repeatedly without added adjuvant into BALB/c mice, and anti-Id IgG responses were determined. These repeated injections revealed that the immunogenicity of Ids was lost by switching to IgG1 and IgG3, restored when the Fc piece of IgG1 was removed, maintained by switching to IgE and monomeric IgA, and lost in polymeric IgA. Loss of immunogenicity was associated with acquisition of Id-specific tolerogenicity, as determined by immunization challenge with Id borne by IgM. An Id borne by IgG induced tolerance when injected at least 90 days before or 3–21 days after immunization with IgM Id was begun. Ids of IgG were also tolerogenic in mice deficient in FcγRIIB or FcγRI + III. The results suggest that Ids that have switched to IgG and pIgA negatively control immune responses to shared Ids, including the Ids of their IgM progenitors.

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.

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.

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 IgG2ab heavy chain allopeptide determinant γ2ab 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 IgG2ab production in vivo. In the γ2ab peptide/I‐Ad complex, tyrosine438 is the first primary anchor (P1) and residues 440 – 445 encompass the T cell receptor contact residues. Amino‐terminal elongation of γ2ab 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 γ2ab 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.

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.

H-2-linked Ir genes have a striking influence on the immunogenicity of idiotopes of myeloma protein 315 for T Helper cells

H‐2‐linked immune response (Ir) genes control T helper cells (Th) that recognize idiotopes of the V domains of myeloma protein 315 as carriers; Th recognition was detected by augmentation of antibody responses of hapten (4‐hydroxy‐3‐iodo‐5‐nitrophenylacetyl (NIP))‐primed B cells boosted with NIP conjugated to Fab315. The present study indicates that the responder k allele of the Ir VH315 gene maps to the I‐A subregion of H‐2. A responder s allele of the Ir Vλ2315 gene on an A‐strain background was identified, which also most likely maps to I‐A. Although the d allele of the Ir Vλ2315 gene is a responder allele on DBA/2 background, the D2.GD strain (with I‐region haplotype AdBbJbEbCb) was non‐responder to Vλ2315, suggesting either that the responder d allele maps to I‐E or that the b allele of a second Ir Vλ2315 gene located to the right of I‐A exerts a strong suppressive influence. The H‐2b haplotype conferred non‐responsiveness to VH315, Vλ2315, and Fv315.

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.

Two M315 Idiotopes Defined by Isologous Monoclonal Antibodies: One Depends on Germline and the Other on Mutated Murine λ Light Chain Sequences

We have previously reported that T helper cells of BALB/c mice recognize the unique mutated sequence Phe91, Arg95, Asn96 of the λ2 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, since λ2‐315 recombined with four other H chains did not express the Id, (3) related to the haptenbinding 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 λ chain V regions was studied with the aid of reconstituted Ig molecules of H‐315 paired with λ1, λ2, and #LD3 L chains. Id‐315. TH was expressed equally well by reconstituted Ig containing three different λ2 chains (λ2‐5‐7, λ‐T952, and λ2‐315), but its expression was profoundly reduced when H‐315 was associated with #LD‐SAPC15 or #LD2‐T952 differ from λ2‐315 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 λ2‐315. 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 bypervariable loop of λ chains to Id and the immunogenicity of isologous Ig.

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.