Xiangang Zou

Dominant expression of a 1.3 Mb human Ig kappa locus replacing mouse light chain production.

Expression studies of multigene families, such as the immunoglobulin (Ig) loci, are difficult because of their large size and the necessity to introduce germline configured regions into an animal. Antibody diversity from Ig gene miniloci is limited by the number of variable (V) region genes and the need for distal regulatory elements to control expression. Here, we show germline transfer into mice of a 1300 kb human Igκ light chain locus on a yeast artificial chromosome that resulted in early DNA rearrangement and highly efficient human light chain expression. The human locus was assembled from a 300 kb authentic region using contig extension by addition of cosmid multimers to supplement the variable gene cluster. This resulted in the addition of about 100 V region genes in germline configuration from different families. In transgenic animals with Igκ disruption, this large human κ locus replaced the endogenous locus, and subsequent down‐regulation of Igλ light chain contribution led to a dominant expression of the rearranged human genes. Contrary to expectation, rather than providing a solely selective advantage for ensuring repertoire formation controlled by the sheer number of introduced genes, the λ/κ ratio in serum appears to be the result of competition for early surface Ig expression maintained in the developing B cell.—Zou, X., Xian, J., Davies, N. P., Popov, A. V., Brüggemann, M. Dominant expression of a 1.3 Mb human Igκ locus replacing mouse light chain production. FASEB J. 10, 1227‐1232 (1996)

Heavy-chain only antibodies derived from dromedary are secreted and displayed by mouse B cells

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 × 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.

Block in Development at the Pre-B-II to Immature B Cell Stage in Mice Without Igκ and Igλ Light Chain

Silencing individual C (constant region) λ genes in a κ−/− background reduces mature B cell levels, and L chain-deficient (λ−/−κ−/−) mice attain a complete block in B cell development at the stage when L chain rearrangement, resulting in surface IgM expression, should be completed. L chain deficiency prevents B cell receptor association, and L chain function cannot be substituted (e.g., by surrogate L chain). Nevertheless, precursor cell levels, controlled by developmental progression and checkpoint apoptosis, are maintained, and B cell development in the bone marrow is fully retained up to the immature stage. L chain deficiency allows H chain retention in the cytoplasm, but prevents H chain release from the cell, and as a result secondary lymphoid organs are B cell depleted while T cell levels remain normal.

Expression of a Dromedary Heavy Chain-Only Antibody and B Cell Development in the Mouse

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 μ-H chain in the absence of surrogate and conventional L chain has been identified. Despite this, Ag-specific single H chain Ig repertoires, using μ-, γ-, ε-, or α-H chains found in conventional Abs, are not produced. Moreover, removal of H chain or, separately, L chain (κ/λ) 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 CH1 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 γ2a 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 μ- or δ-H chain, at early developmental stages.

Heavy chain-only antibodies are spontaneously produced in light chain-deficient mice

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−/− (κ−/−λ−/−-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) γ exon 1, which is accomplished by genomic alterations in CH1-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 CH1 (VHDJH-hinge-CH2-CH3) are selected for and lead to the formation of fully functional and diverse H chain–only antibodies in L−/− animals.

Rapid induction of B-cell lymphomas in mice carrying a human IgH/c-mycYAC.

Activation of the c-myc proto-oncogene by one of the immunoglobulin (Ig) loci after chromosomal translocation is a consistent feature of Burkitts lymphoma. Different subtypes of this tumor vary in the molecular architecture of the translocation region. In most cases there are no known regulatory elements of the Ig locus neighboring the oncogene and this considerably obscures the mechanism of its deregulation. In order to assess possible oncogene activation signals, we produced an experimental translocation region by insertion of a c-myc gene about 50 kb from the IgH intron enhancer in a yeast artificial chromosome (YAC) containing a 220 kb region of the human Ig heavy chain (IgH) locus. Single copy integration of this YAC into the genome of mouse embryonic stem (ES) cells was achieved by spheroplast fusion. Chimeric mice derived from these ES cells developed monoclonal B-cell lymphomas expressing surface IgM by 8 – 16 weeks of age. The IgH/c-myc translocus showed different VHDJH rearrangement in almost all tumors without any alterations of the distance between c-myc and the IgH intron enhancer. This mouse model can be used for the in vivo analysis of c-myc deregulation and the tumor formation capacity of the IgH locus in aberrant rearrangements.

Novel Control Motif Cluster in the IgH δ-γ3 Interval Exhibits B Cell-Specific Enhancer Function in Early Development

The majority of the human Ig heavy chain (IgH) constant (C) region locus has been cloned and mapped. An exception is the region between Cδ and Cγ3, which is unstable and may be a recombination hot spot. We isolated a pBAC clone (pHuIgH3′δ-γ3) that established a 52-kb distance between Cδ and Cγ3. Sequence analysis identified a high number of repeat elements, explaining the instability of the region, and an unusually large accumulation of transcription factor-binding motifs, for both lymphocyte-specific and ubiquitous transcription activators (IKAROS, E47, Oct-1, USF, Myc/Max), and for factors that may repress transcription (ΔEF1, Gfi-1, E4BP4, C/EBPβ). Functional analysis in reporter gene assays revealed the importance of the Cδ-Cγ3 interval in lymphocyte differentiation and identified independent regions capable of either enhancement or silencing of reporter gene expression and interaction with the IgH intron enhancer Eμ. In transgenic mice, carrying a construct that links the β-globin reporter to the novel δ-γ3 intron enhancer (Eδ-γ3), transgene transcription is exclusively found in bone marrow B cells from the early stage when IgH rearrangement is initiated up to the successful completion of H and L locus recombination, resulting in Ab expression. These findings suggest that the Cδ-Cγ3 interval exerts regulatory control on Ig gene activation and expression during early lymphoid development.

Immunoglobulin aggregation leading to Russell body formation is prevented by the antibody light chain.

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.

Removal of the BiP-retention domain in Cμ permits surface deposition and developmental progression without L-chain

Nascent, full length, immunoglobulin (Ig) heavy (H)-chains are post-translationally associated with H-chain-binding protein (BiP or GRP78) in the endoplasmic reticulum (ER). The first constant (C) domain, CH1 of a C gene (Cmu, Cgamma, Calpha), is important for this interaction. The contact is released upon BiP replacement by conventional Ig light (L)-chain (kappa or lambda). Incomplete or mutated H-chains with removed variable (VH) and/or C(H)1 domain, as found in H-chain disease (HCD), can preclude stable BiP interaction. Progression in development after the preB cell stage is dependent on surface expression of IgM when association of a micro H-chain with a L-chain overcomes the retention by BiP. We show that IgM lacking the BiP-binding domain is displayed on the cell surface and elicits a signal that allows developmental progression even without the presence of L-chain. The results are reminiscent of single chain Ig secretion in camelids where developmental processes leading to the generation of fully functional H-chain-only antibodies are not understood. Furthermore, in the mouse the largest secondary lymphoid organ, the spleen, is not required for H-chain-only Ig expression and the CD5 survival signal may be obsolete for cells expressing truncated IgM.

Establishment of mouse expanded potential stem cells

Mouse embryonic stem cells derived from the epiblast contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.