Search

Russell bodies (RBs) are intracellular inclusions filled with protein aggregates. In diverse lymphoid disorders these occur as immunoglobulin (Ig) deposits, accumulating in abnormal plasma or Mott cells. In heavy-chain deposition disease truncated antibody heavy-chains (HCs) are found, which bear a resemblance to diverse polypeptides produced in Ig light-chain (LC)-deficient (L(-/-)) mice. In L(-/-) animals, the known functions of LC, providing part of the antigen-binding site of an antibody and securing progression of B-cell development, may not be required. Here, we show a novel function of LC in preventing antibody aggregation. L(-/-) mice produce truncated HC naturally, constant region (C)gamma and Calpha lack C(H)1, and Cmicro is without C(H)1 or C(H)1 and C(H)2. Most plasma cells found in these mice are CD138(+) Mott cells, filled with RBs, formed by aggregation of HCs of different isotypes. The importance of LC in preventing HC aggregation is evident in knock-in mice, expressing Cmicro without C(H)1 and C(H)2, which only develop an abundance of RBs when LC is absent. These results reveal that preventing antibody aggregation is a major function of LC, important for understanding the physiology of heavy-chain deposition disease, and in general recognizing the mechanisms, which initiate protein conformational diseases.

In healthy mammals, maturation of B cells expressing heavy (H) chain immunoglobulin (Ig) without light (L) chain is prevented by chaperone association of the H chain in the endoplasmic reticulum. Camelids are an exception, expressing homodimeric IgGs, an antibody type that to date has not been found in mice or humans. In camelids, immunization with viral epitopes generates high affinity H chain-only antibodies, which, because of their smaller size, recognize clefts and protrusions not readily distinguished by typical antibodies. Developmental processes leading to H chain antibody expression are unknown. We show that L(-/-) (kappa(-/-)lambda(-/-)-deficient) mice, in which conventional B cell development is blocked at the immature B cell stage, produce diverse H chain-only antibodies in serum. The generation of H chain-only IgG is caused by the loss of constant (C) gamma exon 1, which is accomplished by genomic alterations in C(H)1-circumventing chaperone association. These mutations can be attributed to errors in class switch recombination, which facilitate the generation of H chain-only Ig-secreting plasma cells. Surprisingly, transcripts with a similar deletion can be found in normal mice. Thus, naturally occurring H chain transcripts without C(H)1 (V(H)DJ(H)-hinge-C(H)2-C(H)3) are selected for and lead to the formation of fully functional and diverse H chain-only antibodies in L(-/-) animals.

Antibodies expressed in mice, humans, and most mammals consist of paired heavy (H) and light (L) chains. Cellular release of single H-chains without L-chains is prevented by chaperone association and retention in the endoplasmic reticulum. Consequently, H-chain-only antibodies are absent, except in pathological conditions known as Heavy Chain Disease, where they occur in mutated form. An exception to this is found in the serum of Camelidae. Homodimeric H-chain antibodies in camelids lack the first constant region domain (CH1) of the H-chain but retain an otherwise intact H-chain polypeptide, thus maintaining antigen specificity and effector functions. In this review, we summarize how H-chain antibodies can be expressed in mice, and whether normal developmental processes leading to antibody expression without L-chain are retained.

In mature B cells of mice and most mammals, cellular release of single H chain Abs without L chains is prevented by H chain association with Ig-specific chaperons in the endoplasmic reticulum. In precursor B cells, however, surface expression of mu-H chain in the absence of surrogate and conventional L chain has been identified. Despite this, Ag-specific single H chain Ig repertoires, using mu-, gamma-, epsilon-, or alpha-H chains found in conventional Abs, are not produced. Moreover, removal of H chain or, separately, L chain (kappa/lambda) locus core sequences by gene targeting has prevented B cell development. In contrast, H chain-only Abs are produced abundantly in Camelidae as H2 IgG without the C(H)1 domain. To test whether H chain Abs can be produced in mice, and to investigate how their expression affects B cell development, we introduced a rearranged dromedary gamma2a H chain into the mouse germline. The dromedary transgene was expressed as a naturally occurring Ag-specific disulphide-linked homodimer, which showed that B cell development can be instigated by expression of single H chains without L chains. Lymphocyte development and B cell proliferation was accomplished despite the absence of L chain from the BCR complex. Endogenous Ig could not be detected, although V(D)J recombination and IgH/L transcription was unaltered. Furthermore, crossing the dromedary H chain mice with mice devoid of all C genes demonstrated without a doubt that a H chain-only Ab can facilitate B cell development independent of endogenous Ig expression, such as mu- or delta-H chain, at early developmental stages.

Silencing or removal of individual C (constant)-region genes and/or adjacent control sequences did not generate fully deficient Ig (immunoglobulin)- mice. A reason is that different C genes share many functional tasks and most importantly are individually capable of ensuring lymphocyte differentiation. Nevertheless, incomplete arrests in B-cell development were found, most pronounced at the onset of H-chain expression. Here we show that removal of 200 kb accommodating all C genes, Cmu-Cdelta-Cgamma3-Cgamma1-Cgamma2b-Cgamma2a-Cepsilon-Calpha, stops antibody production. For this two loxP targeting constructs were introduced into the most 5' C gene and the distal alpha 3' enhancer. Cre-loxP-mediated in vivo deletion was accompanied by extensive germ-line mosaicism, which could be separated by breeding. Homozygous C-gene deletion mice did not express Ig H or L chains and flow cytometry revealed a complete block in B-cell development. However, C-gene removal did not affect DNA rearrangement processes following locus activation, as recombination efficacy appears to be similar to what is found in normal mice., (Copyright 2004 Elsevier Inc.)

Whereas functional heavy (H)-chain antibodies devoid of light (L)- chains account for about half of the circulating immunoglobulins in Camelidae, H-chain only antibodies (HCAbs) are not produced in other healthy mammals including rodents and humans. To test the feasibility of expressing single chain antibodies in the mouse, which on account of their small size and antigen-recognition properties would have a major impact on antibody engineering strategies, we constructed a rearranged dromedary H-chain gene encoding the immunoglobulin G2a (IgG2a) isotype with specificity for hen-egg lysozyme (HEL). This IgG2a H-chain gene was introduced into mouse myeloma cells not expressing endogenous immunoglobulin H- or L-chains. Unexpectedly the mouse cells processed and expressed the introduced H-chain as naturally occurring dromedary antibody. For this the first constant (C) region exon was proficiently removed from the recombinant H-chain transcript. This resulted in specific H-chain antibodies of the correct molecular weight (2 x 50 000 MW) secreted as disulfide-linked homodimers and displayed on the mouse cell surface as glycosyl-phosphatidyl-inositol-linked B-cell receptor. The results indicate that antibody expression and maturation without immunoglobulin L-chain is feasible and paves the way for the generation of transgenic single chain antibody repertoires.