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A number of antibodies that efficiently neutralize microbial targets contain long heavy chain complementarity determining region 3 (HCDR3) loops. For HIV, several of the most broad and potently neutralizing antibodies have exceptionally long HCDR3s. Two broad potently neutralizing HIV-specific antibodies, PG9 and PG16, exhibit secondary structure. Two other long HCDR3 antibodies, 2F5 and 4E10, protect against mucosal challenge with SHIV. Induction of such long HCDR3 antibodies may be critical to the design of an effective vaccine strategy for HIV and other pathogens, however it is unclear at present how to induce such antibodies. Here, we present genetic evidence that human peripheral blood antibodies containing long HCDR3s are not primarily generated by insertions introduced during the somatic hypermutation process. Instead, they are typically formed by processes occurring as part of the original recombination event. Thus, the response of B cells encoding antibodies with long HCDR3s results from selection of unusual clones from the naïve repertoire rather than through accumulation of insertions. These antibodies typically use a small subset of D and J gene segments that are particularly suited to encoding long HCDR3s, resulting in the incorporation of highly conserved genetic elements in the majority of antibody sequences encoding long HCDR3s.

The conserved surface of the HIV-1 gp120 envelope glycoprotein that binds to the HIV-1 coreceptor is protected from humoral recognition by multiple layers of camouflage. Here we present sequence and genomic analyses for 12 antibodies that pierce these defenses and determine the crystal structures of 5. The data reveal mechanisms and atomic-level details for three unusual immune features: posttranslational mimicry of coreceptor by tyrosine sulfation of antibody, an alternative molecular mechanism controlling such sulfation, and highly selective VH- gene usage. When confronted by extraordinary viral defenses, the immune system unveils novel adaptive capabilities, with tyrosine sulfation enhancing the vocabulary of antigen recognition.

CD1d-reactive NKT cells are a separate T cell sublineage. Instructive models propose that NKT cells branch off the mainstream developmental pathway because of their T cell antigen receptor specificity, whereas stochastic models would propose that they develop from precursor cells committed to this sublineage before variable-gene rearrangement. We show here that immature double-positive (DP) thymocytes form the canonical rearranged V α gene of NKT cells at nearly equivalent frequencies in the presence or absence of CD1d expression. After interacting with CD1d in the thymus, these cells give rise to expanded populations of NKT cells—including both CD4 + and double-negative lymphocytes in the thymus and periphery—that express this α chain. These results confirm the existence of a DP intermediate for CD1d-reactive NKT cells. They also show that the early developmental stages of these T cells are not governed by a distinct mechanism, which is consistent with the TCR-instructive model of differentiation.

V(D)J recombination creates antibody light chain diversity by joining a Vκ gene segment with one of four Jκ segments. Two Jκ germline-transcript (GT) promoters control Vκ-Jκ joining, but the mechanisms that govern Jκ choice are unclear. Here, we show in gene-targeted mice that the proximal GT promoter helps targeting rearrangements to Jκ1 by preventing premature DNA breaks at Jκ2. Consequently, cells lacking the proximal GT promoter show a biased utilization of downstream Jκ segments, resulting in a diminished potential for receptor editing. Surprisingly, the proximal--in contrast to the distal--GT promoter is transcriptionally inactive prior to Igκ recombination, indicating that its role in Jκ choice is independent of classical promoter function. Removal of the proximal GT promoter increases H3K4me3 levels at Jκ segments, suggesting that this promoter could act as a suppressor of recombination by limiting chromatin accessibility to RAG. Our findings identify the first cis-element critical for Jκ choice and demonstrate that ordered Igκ recombination facilitates receptor editing.

The catfish IGH locus is large (approximately 1 Mb) and complex, having undergone multiple internal duplications and transpositions. To define the structure of the locus that contains the single expressed IGHM gene, two overlapping bacterial-artificial-chromosome (BAC) clones spanning the most 3' end of the channel catfish immunoglobulin heavy (IGH) chain locus have been completely sequenced. The analyses created a contig of 257,153 bp containing 55 VH, 6 D, 12 JH genes and the IGH constant region genes encoding the functional secreted and membrane forms of IgM and the membrane form of IgD. This analysis revealed three major features. First, no C-region genes were found aside from the previously described IGHM1 and IGHD1, with the latter gene being the most 3' C-region gene of the catfish IGH locus. There was no evidence in the region sequenced for genes that could encode an Ig class similar to the IgZ/IgT described in zebrafish, trout and pufferfish. Second, there are a high number of VH pseudogenes, 28 out of 55 (51%). In contrast, the entire zebrafish IGH locus has 40 functional VH genes and eight pseudogenes (17%).

The rearrangement of immunoglobulin (Ig) and T‐cell receptor (TCR) genes in lymphocytes by V(D)J recombinase is essential for immunological diversity in humans. These DNA rearrangements involve cleavage by the RAG1 and RAG2 (RAG1/2) recombinase enzymes at recombination signal sequences (RSS). This reaction generates two products, cleaved signal ends and coding ends. Coding ends are ligated by non‐homologous end‐joining proteins to form a functional Ig or TCR gene product, while the signal ends form a signal joint. In vitro studies have demonstrated that RAG1/2 are capable of mediating the transposition of cleaved signal ends into non‐specific sites of a target DNA molecule. However, to date, in vivo transposition of signal ends has not been demonstrated. We present evidence of in vivo inter‐chromosomal transposition in humans mediated by V(D)J recombinase. T‐cell isolates were shown to contain TCRα signal ends from chromosome 14 inserted into the X‐linked hypo xanthine–guanine phosphoribosyl transferase locus, resulting in gene inactivation. These findings implicate V(D)J recombinase‐mediated transposition as a mutagenic mechanism capable of deleterious genetic rearrangements in humans.

In most vertebrate embryos and neonates studied to date unique antigen receptors (antibodies and T cell receptors) are expressed that possess a limited immune repertoire. We have isolated a subclass of IgM, IgM1gj, from the nurse shark Ginglymostoma cirratum that is preferentially expressed in neonates. The variable (V) region gene encoding the heavy (H) chain underwent V-D-J rearrangement in germ cells (\"germline-joined\"). Such H chain V genes were discovered over 10 years ago in sharks but until now were not shown to be expressed at appreciable levels; we find expression of H1gjin primary and secondary lymphoid tissues early in life, but in adults only in primary lymphoid tissue, which is identified in this work as the epigonal organ. H1gjchain associates covalently with light (L) chains and is most similar in sequence to IgM H chains, but like mammalian IgG has three rather than the four IgM constant domains; deletion of the ancestral IgM C2 domain thus defines both IgG and IgM1gj. Because sharks are the members of the oldest vertebrate class known to possess antibodies, unique or specialized antibodies expressed early in ontogeny in sharks and other vertebrates were likely present at the inception of the adaptive immune system.

Four transgenic Nicotiana tabacum plants were generated that expressed a murine monoclonal antibody kappa chain, a hybrid immunoglobulin A-G heavy chain, a murine joining chain, and a rabbit secretory component, respectively. Successive sexual crosses between these plants and filial recombinants resulted in plants that expressed all four protein chains simultaneously. These chains were assembled into a functional, high molecular weight secretory immunoglobulin that recognized the native streptococcal antigen I/II cell surface adhesion molecule. In plants, single cells are able to assemble secretory antibodies, whereas two different cell types are required in mammals. Transgenic plants may be suitable for large-scale production of recombinant secretory immunoglobulin A for passive mucosal immunotherapy. Plant cells also possess the requisite mechanisms for assembly and expression of other complex recombinant protein molecules

In this study, authors explore the application of modelling and additive layer manufacturing (ALM) for creating and testing materials with interlocking structures aimed to reduce the stress concentration along the edges of a typical lap joint. The effectiveness of this approach is discussed by means of modelling and experimental validation of joints with interlocking structures obtained by ALM. Considering the achieved results, ALM of interlocking structures constitutes an interesting alternative or complement to traditional joining processes, as it may help to minimize stress mismatches in the joining region. It may also prevent the use of adhesive or joining post processes, because the joint is created together with the joined components.

In developing B cells, V(D)J recombination assembles exons encoding IgH and Igκ variable regions from hundreds of gene segments clustered across Igh and Igk loci. V, D and J gene segments are flanked by conserved recombination signal sequences (RSSs) that target RAG endonuclease 1 . RAG orchestrates Igh V(D)J recombination upon capturing a J H -RSS within the J H -RSS-based recombination centre 1 – 3 (RC). J H -RSS orientation programmes RAG to scan upstream D- and V H -containing chromatin that is presented in a linear manner by cohesin-mediated loop extrusion 4 – 7 . During Igh scanning, RAG robustly utilizes only D-RSSs or V H -RSSs in convergent (deletional) orientation with J H -RSSs 4 – 7 . However, for Vκ-to-Jκ joining, RAG utilizes Vκ-RSSs from deletional- and inversional-oriented clusters 8 , inconsistent with linear scanning 2 . Here we characterize the Vκ-to-Jκ joining mechanism. Igk undergoes robust primary and secondary rearrangements 9 , 10 , which confounds scanning assays. We therefore engineered cells to undergo only primary Vκ-to-Jκ rearrangements and found that RAG scanning from the primary Jκ-RC terminates just 8 kb upstream within the CTCF-site-based Sis element 11 . Whereas Sis and the Jκ-RC barely interacted with the Vκ locus, the CTCF-site-based Cer element 12 4 kb upstream of Sis interacted with various loop extrusion impediments across the locus. Similar to V H  locus inversion 7 , DJ H inversion abrogated V H -to-DJ H joining; yet Vκ locus or Jκ inversion allowed robust Vκ-to-Jκ joining. Together, these experiments implicated loop extrusion in bringing Vκ segments near Cer for short-range diffusion-mediated capture by RC-based RAG. To identify key mechanistic elements for diffusional V(D)J recombination in Igk versus Igh , we assayed Vκ-to-J H and D-to-Jκ rearrangements in hybrid Igh–Igk loci generated by targeted chromosomal translocations, and pinpointed remarkably strong Vκ and Jκ RSSs. Indeed, RSS replacements in hybrid or normal Igk and Igh loci confirmed the ability of Igk -RSSs to promote robust diffusional joining compared with Igh- RSSs. We propose that Igk evolved strong RSSs to mediate diffusional Vκ-to-Jκ joining, whereas Igh evolved weaker RSSs requisite for modulating V H joining by RAG-scanning impediments. Experiments in mouse models, and in cell lines that only allow primary Vκ-to-Jκ rearrangements, enable characterization of the mechanisms of V(D)J recombination.