Characterization and humanization of VNARs targeting human serum albumin from the whitespotted bamboo shark (Chiloscyllium plagiosum).

The Shark-derived immunoglobulin new antigen receptors (IgNARs) have gained increasing attention for their high solubility, exceptional thermal stability, and intricate sequence variation. In this study, we immunized whitespotted bamboo shark (Chiloscyllium plagiosum) to create phage display library of variable domains of IgNAR (VNARs) for screening against Human Serum Albumin (HSA), a versatile vehicle in circulation due to its long in vivo half-life. We identified two HSA-binding VNAR clones, 2G5 and 2G6, and enhanced their expression in E. coli with the FKPA chaperone. 2G6 exhibited a strong binding affinity of 13 nM with HSA and an EC50 of 1 nM. In vivo study with a murine model further provided initial validation of 2G6's ability to prolong circulation time by binding to HSA. Additionally, we employed computational molecular docking to predict the binding affinities of both 2G6 and its humanized derivative, H2G6, to HSA. Our analysis unveiled that the complementarity-determining regions (CDR1 and CDR3) are pivotal in the antigen recognition process. Therefore, our study has advanced the understanding of the potential applications of VNARs in biomedical research aimed at extending drug half-life, holding promise for future therapeutic and diagnostic progressions.

Leveraging the biotechnological promise of the hagfish variable lymphocyte receptors: tools for aquatic microbial diseases.

The jawless vertebrates (agnathans/cyclostomes) are ancestral animals comprising lampreys and hagfishes as the only extant representatives. They possess an alternative adaptive immune system (AIS) that uses leucine-rich repeats (LRR)-based variable lymphocyte receptors (VLRs) instead of the immunoglobulin (Ig)-based antigen receptors of jawed vertebrates (gnathostomes). The different VLR types are expressed on agnathan lymphocytes and functionally resemble gnathostome antigen receptors. In particular, VLRB is functionally similar to the B cell receptor and is expressed and secreted by B-like lymphocytes as VLRB antibodies that bind antigens with high affinity and specificity. The potential repertoire scale of VLR-based antigen receptors is believed to be at least comparable to that of Ig-based systems. VLR proteins inherently possess characteristics that render them excellent candidates for biotechnological development, including tractability to recombinant approaches. In recent years, scientists have explored the biotechnological development and utility of VLRB proteins as alternatives to conventional mammalian antibodies. The VLRB antibody platform represents a non-traditional approach to generating a highly diverse repertoire of unique antibodies. In this review, we first describe some aspects of the biology of the AIS of the jawless vertebrates, which recognizes antigens by means of unique receptors. We then summarize reports on the development of VLRB-based antibodies and their applications, particularly those from the inshore hagfish (Eptatretus burgeri) and their potential uses to address microbial diseases in aquaculture. Hagfish VLRB antibodies (we call Ccombodies) are being developed and improved, while obstacles to the advancement of the VLRB platform are being addressed to utilize VLRBs effectively as tools in immunology. VLRB antibodies for novel antigen targets are expected to emerge to provide new opportunities to tackle various scientific questions. We anticipate a greater interest in the agnathan AIS in general and particularly in the hagfish AIS for greater elucidation of the evolution of adaptive immunity and its applications to address microbial pathogens in farmed aquatic animals and beyond.

The genetic spectrum of a Chinese series of patients with 46, XY disorders of the sex development.

PURPOSE: The etiology of 46, XY disorders of sex development (46, XY DSD) is complex, and studies have shown that different series of patients with 46, XY DSD has different genetic spectrum. In this study, we aimed to investigate the underlying genetic etiology in a Chinese series of patients with 46, XY DSD by whole exome sequencing (WES). METHODS: Seventy patients with 46, XY DSD were enrolled from the Peking Union Medical College Hospital (Beijing, China). The detailed clinical characteristics were evaluated, and peripheral blood was collected for WES to find the patients' rare variants (RVs) of genes related to 46, XY DSD. The clinical significance of the RVs was annotated according to American College of Medical Genetics and Genomics (ACMG) guidelines. RESULTS: A total of 57 RVs from nine genes were identified in 56 patients with 46, XY DSD, which include 21 novel RVs and 36 recurrent RVs. Based on the American ACMG guidelines, 43 variants were classified as pathogenic(P) or likely pathogenic (LP) variants and 14 variants were defined as variants of uncertain significance (VUS). P or LP variants were identified in 64.3% (45/70) patients of the series. Thirty-nine, 14, and 4 RVs were involved in the process of androgen synthesis and action, testicular determination and developmental process, and syndromic 46, XY DSD, respectively. The top three genes most frequently affected to cause 46, XY DSD were AR, SRD5A2, and NR5A1. Seven patients were found harboring RVs of the 46, XY DSD pathogenic genes identified in recent years, namely DHX37 in four patients, MYRF in two patients, and PPP2R3C in one patient. CONCLUSION: We identified 21 novel RVs of nine genes, which extended the genetic spectrum of 46, XY DSD pathogenic variants. Our study showed that 60% of the patients were caused by AR, SRD5A2 or NR5A1 P/LP variants. Therefore, polymerase chain reaction (PCR) amplification and Sanger sequencing of these three genes could be performed first to identify the pathogeny of the patients. For those patients whose pathogenic variants had not been found, whole-exome sequencing could be helpful in determining the etiology.

Evolution of two distinct variable lymphocyte receptors in lampreys: VLRD and VLRE.

Jawless vertebrates possess an alternative adaptive immune system in which antigens are recognized by variable lymphocyte receptors (VLRs) generated by combinatorial assembly of leucine-rich repeat (LRR) cassettes. Three types of receptors, VLRA, VLRB, and VLRC, have been previously identified. VLRA- and VLRC-expressing cells are T cell-like, whereas VLRB-expressing cells are B cell-like. Here, we report two types of VLRs in lampreys, VLRD and VLRE, phylogenetically related to VLRA and VLRC. The germline VLRD and VLRE genes are flanked by 39 LRR cassettes used in the assembly of mature VLRD and VLRE, with cassettes from chromosomes containing the VLRA and VLRC genes also contributing to VLRD and VLRE assemblies. VLRD and VLRE transcription is highest in the triple-negative (VLRA-/VLRB-/VLRC-) population of lymphocytes, albeit also detectable in VLRA+ and VLRC+ populations. Tissue distribution studies suggest that lamprey VLRD+ and VLRE+ lymphocytes comprise T-like sublineages of cells.

Phage Display of Bovine Ultralong CDRH3.

Phage display is an in vitro technique used in the discovery of monoclonal antibodies that has been used successfully in the discovery of both camelid VHH and shark variable new antigen receptor domains (VNAR). Bovines also contain a unique "ultralong CDRH3" with a conserved structural motif, comprising a knob domain and ß-stalk. When removed from the antibody scaffold, either the entire ultralong CDRH3 or the knob domain alone, is typically capable of binding an antigen, to produce antibody fragments that are smaller than both VHH and VNAR. By extracting immune material from bovine animals and specifically amplifying knob domain DNA sequences by PCR, knob domain sequences can be cloned into a phagemid vector producing knob domain phage libraries. Target-specific knob domains can be enriched by panning the libraries against an antigen of interest. Phage display of knob domains exploits the link between phage genotype and phenotype and could prove to be a high throughput method to discover target-specific knob domains, helping to explore the pharmacological properties of this unique antibody fragment.

Genetic Lesions in Russian CLL Patients with the Most Common Stereotyped Antigen Receptors.

Chronic lymphocytic leukemia (CLL) is one of the most common B-cell malignancies in Western countries. IGHV mutational status is the most important prognostic factor for this disease. CLL is characterized by an extreme narrowing of the IGHV genes repertoire and the existence of subgroups of quasi-identical stereotyped antigenic receptors (SAR). Some of these subgroups have already been identified as independent prognostic factors for CLL. Here, we report the frequencies of TP53, NOTCH1, and SF3B1 gene mutations and chromosomal aberrations assessed by NGS and FISH in 152 CLL patients with the most common SAR in Russia. We noted these lesions to be much more common in patients with certain SAR than average in CLL. The profile of these aberrations differs between the subgroups of SAR, despite the similarity of their structure. For most of these subgroups mutations prevailed in a single gene, except for CLL#5 with all three genes affected by mutations. It should be noted that our data concerning the mutation frequency in some SAR groups differ from that obtained previously, which could be due to the population differences between patient cohorts. The research in this area should be important for better understanding the pathogenesis of CLL and therapy optimization.

An updated definition of V(D)J recombination signal sequences revealed by high-throughput recombination assays.

In the adaptive immune system, V(D)J recombination initiates the production of a diverse antigen receptor repertoire in developing B and T cells. Recombination activating proteins, RAG1 and RAG2 (RAG1/2), catalyze V(D)J recombination by cleaving adjacent to recombination signal sequences (RSSs) that flank antigen receptor gene segments. Previous studies defined the consensus RSS as containing conserved heptamer and nonamer sequences separated by a less conserved 12 or 23 base-pair spacer sequence. However, many RSSs deviate from the consensus sequence. Here, we developed a cell-based, massively parallel assay to evaluate V(D)J recombination activity on thousands of RSSs where the 12-RSS heptamer and adjoining spacer region contained randomized sequences. While the consensus heptamer sequence (CACAGTG) was marginally preferred, V(D)J recombination was highly active on a wide range of non-consensus sequences. Select purine/pyrimidine motifs that may accommodate heptamer unwinding in the RAG1/2 active site were generally preferred. In addition, while different coding flanks and nonamer sequences affected recombination efficiency, the relative dependency on the purine/pyrimidine motifs in the RSS heptamer remained unchanged. Our results suggest RAG1/2 specificity for RSS heptamers is primarily dictated by DNA structural features dependent on purine/pyrimidine pattern, and to a lesser extent, RAG:RSS base-specific interactions.

The influence of antibody humanization on shark variable domain (VNAR) binding site ensembles.

Sharks and other cartilaginous fish produce new antigen receptor (IgNAR) antibodies, as key part of their humoral immune response and are the phylogenetically oldest living organisms that possess an immunoglobulin (Ig)-based adaptive immune system. IgNAR antibodies are naturally occurring heavy-chain-only antibodies, that recognize antigens with their single domain variable regions (VNARs). In this study, we structurally and biophysically elucidate the effect of antibody humanization of a previously published spiny dogfish VNAR (parent E06), which binds with high affinity to the human serum albumin (HSA). We analyze different humanization variants together with the parental E06 VNAR and the human Vκ1 light chain germline DPK9 antibody to characterize the influence of point mutations in the framework and the antigen binding site on the specificity of VNARs as reported by Kovalenko et al. We find substantially higher flexibility in the humanized variants, reflected in a broader conformational space and a higher conformational entropy, as well as population shifts of the dominant binding site ensembles in solution. A further variant, in which some mutations are reverted, largely restores the conformational stability and the dominant binding minimum of the parent E06. We also identify differences in surface hydrophobicity between the human Vκ1 light chain germline DPK9 antibody, the parent VNAR E06 and the humanized variants. Additional simulations of VNAR-HSA complexes of the parent E06 VNAR and a humanized variant reveal that the parent VNAR features a substantially stronger network of stabilizing interactions. Thus, we conclude that a structural and dynamic understanding of the VNAR binding site upon humanization is a key aspect in antibody humanization.

Monogenic TCRß Assembly and Expression Are Paramount for Uniform Antigen Receptor Specificity of Individual αß T Lymphocytes.

The ability of individual T and B cells to display Ag receptors of unique uniform specificity is the molecular basis of adaptive immunity. Most αß T cells achieve uniform specificity by assembling in-frame genes on only one allelic copy of TCRß and TCRα loci, while others prevent incorporation of TCRα protein from both alleles into TCRs. Analysis of mice expressing TCR proteins from a restricted combination of transgenes showed that TCR protein pairing restrictions achieve uniform specificity of cells expressing two types of TCRß protein. However, whether this mechanism operates in the physiological context where each dual-TCRß cell expresses one set of a vast number of different TCRß proteins remains an open question, largely because there is a low, but significant, portion of cells carrying two in-frame TCRß genes. To resolve this issue, we inactivated one allelic copy of the TCRα locus in a new mouse strain that assembles two in-frame TCRß genes in an elevated fraction of cells. This genetic manipulation has no effect on the frequency of cells that display multiple types of αß TCR, yet increases the representation of cells displaying TCRß proteins that generate more highly expressed TCRs. Our data demonstrate that some TCRß proteins exhibit differential functional pairing with TCRα proteins, but these restrictions have negligible contribution for ensuring uniform specificity of cells that express two types of TCRß protein. Therefore, we conclude that mechanisms governing monogenic assembly and expression of TCRß genes in individual cells are paramount for uniform specificity of αß T lymphocytes.

Generation of Redirected Engineered Human Chimeric Antigen Receptor (CAR) T Cells.

Chimeric antigen receptor (CAR) T cell therapy that involves genetic engineering a patient's own immune cells with antigen-specific receptors has shown remarkable efficacy in blood cancer treatment. Numerous clinical studies with CAR T cells targeting the blood cell surface protein CD19 led to the FDA 's first approval of a genetically engineered cell therapy. The process of generating potent CAR T cells involves several carefully performed manufacturing steps. Here, we describe the generation of redirected engineered human CAR T cells for preclinical studies starting with the CAR design, retroviral gene transfer, detection of CAR expression, and expansion of transduced T cells.

Cas9-Based Local Enrichment and Genomics Sequence Revision of Megabase-Sized Shark IgNAR Loci.

The 0.8-Mb Ig new Ag receptor (IgNAR) region of the whitespotted bamboo shark (Chiloscyllium plagiosum) is incompletely assembled in Chr_44 of the reference genome. Here we used Cas9-assisted targeting of chromosome segments (CATCH) to enrich the 2 Mb region of the Chr_44 IgNAR loci and sequenced it by PacBio and next-generation sequencing. A fragment >3.13 Mb was isolated intact from the RBCs of sharks. The target was enriched 245.531-fold, and sequences had up to 94% coverage with a 255× mean depth. Compared with the previously published sequences, 20 holes were filled, with a total length of 3508 bp. In addition, we report five potential germline V alleles of IgNAR1 from six sharks that may belong to two clusters of the IgNAR. Our results provide a new method to research the germline of large Ig gene segments, as well as provide the enhanced bamboo shark IgNAR gene loci with fewer gaps.

Evolution of thymopoietic microenvironments.

In vertebrates, the development of lymphocytes from undifferentiated haematopoietic precursors takes place in so-called primary lymphoid organs, such as the thymus. Therein, lymphocytes undergo a complex differentiation and selection process that culminates in the generation of a pool of mature T cells that collectively express a self-tolerant repertoire of somatically diversified antigen receptors. Throughout this entire process, the microenvironment of the thymus in large parts dictates the sequence and outcome of the lymphopoietic activity. In vertebrates, direct genetic evidence in some species and circumstantial evidence in others suggest that the formation of a functional thymic microenvironment is controlled by members of the Foxn1/4 family of transcription factors. In teleost fishes, both Foxn1 and Foxn4 contribute to thymopoietic activity, whereas Foxn1 is both necessary and sufficient in the mammalian thymus. The evolutionary history of Foxn1/4 genes suggests that an ancient Foxn4 gene lineage gave rise to the Foxn1 genes in early vertebrates, raising the question of the thymopoietic capacity of the ancestor common to all vertebrates. Recent attempts to reconstruct the early events in the evolution of thymopoietic tissues by replacement of the mouse Foxn1 gene by Foxn1-like genes isolated from various chordate species suggest a plausible scenario. It appears that the primordial thymus was a bi-potent lymphoid organ, supporting both B cell and T cell development; however, during the course of vertebrate, evolution B cell development was gradually diminished converting the thymus into a site specialized in T cell development.

Strategies and Tactics for the Development of Selective Glycan-Binding Proteins.

The influences of glycans impact all biological processes, disease states, and pathogenic interactions. Glycan-binding proteins (GBPs), such as lectins, are decisive tools for interrogating glycan structure and function because of their ease of use and ability to selectively bind defined carbohydrate epitopes and glycosidic linkages. GBP reagents are prominent tools for basic research, clinical diagnostics, therapeutics, and biotechnological applications. However, the study of glycans is hindered by the lack of specific and selective protein reagents to cover the massive diversity of carbohydrate structures that exist in nature. In addition, existing GBP reagents often suffer from low affinity or broad specificity, complicating data interpretation. There have been numerous efforts to expand the GBP toolkit beyond those identified from natural sources through protein engineering, to improve the properties of existing GBPs or to engineer novel specificities and potential applications. This review details the current scope of proteins that bind carbohydrates and the engineering methods that have been applied to enhance the affinity, selectivity, and specificity of binders.

Lost structural and functional inter-relationships between Ig and TCR loci in mammals revealed in sharks.

Immunoglobulins and T cell receptors (TCR) have obvious structural similarities as well as similar immunogenetic diversification and selection mechanisms. Nevertheless, the two receptor systems and the loci that encode them are distinct in humans and classical murine models, and the gene segments comprising each repertoire are mutually exclusive. Additionally, while both B and T cells employ recombination-activating genes (RAG) for primary diversification, immunoglobulins are afforded a supplementary set of activation-induced cytidine deaminase (AID)-mediated diversification tools. As the oldest-emerging vertebrates sharing the same adaptive B and T cell receptor systems as humans, extant cartilaginous fishes allow a potential view of the ancestral immune system. In this review, we discuss breakthroughs we have made in studies of nurse shark (Ginglymostoma cirratum) T cell receptors demonstrating substantial integration of loci and diversification mechanisms in primordial B and T cell repertoires. We survey these findings in this shark model where they were first described, while noting corroborating examples in other vertebrate groups. We also consider other examples where the gnathostome common ancestry of the B and T cell receptor systems have allowed dovetailing of genomic elements and AID-based diversification approaches for the TCR. The cartilaginous fish seem to have retained this T/B cell plasticity to a greater extent than more derived vertebrate groups, but representatives in all vertebrate taxa except bony fish and placental mammals show such plasticity.

Changes of Taste, Smell and Eating Behavior in Patients Undergoing Bariatric Surgery: Associations with PROP Phenotypes and Polymorphisms in the Odorant-Binding Protein OBPIIa and CD36 Receptor Genes.

Bariatric surgery is the most effective long-term treatment for severe obesity and related comorbidities. Although patients who underwent bariatric surgery report changes of taste and smell perception, results from sensory studies are discrepant and limited. Here, we assessed taste and smell functions in 51 patients before, one month, and six months after undergoing bariatric surgery. We used taste strip tests to assess gustatory function (including sweetness, saltiness, sourness, umaminess, bitterness and oleic acid, a fatty stimulus), the "Sniffin' Sticks" test to assess olfactory identification and the 3-Factor Eating Questionnaire to assess eating behavior. We also explored associations between these phenotypes and flavor-related genes. Results showed an overall improvement in taste function (including increased sensitivity to oleic acid and the bitterness of 6-n-propylthiouracil (PROP)) and in olfactory function (which could be related to the increase in PROP and oleic acid sensitivity), an increase in cognitive restraint, and a decrease in disinhibition and hunger after bariatric surgery. These findings indicate that bariatric surgery can have a positive impact on olfactory and gustatory functions and eating behavior (with an important role of genetic factors, such PROP tasting), which in turn might contribute to the success of the intervention.

Reconsidering phosphorylation in the control of inducible CARD11 scaffold activity during antigen receptor signaling.

Protein phosphorylation is a commonly used regulatory step that controls signal transduction pathways in a wide array of biological contexts. The finding that a residue is phosphorylated, coupled with the observation that mutation of that residue impacts signaling, often forms the basis for concluding that the phosphorylation of that residue is a key signaling step. However, in certain cases, the situation is more complicated and warrants further study to obtain a clear mechanistic understanding of whether and how the kinase-mediated modification in question is important. CARD11 is a multi-domain signaling scaffold that functions as a hub in lymphocytes to transmit the engagement of antigen receptors into the activation of NF-κB, JNK and mTOR. The phosphorylation of the CARD11 autoinhibitory Inhibitory Domain in response to antigen receptor triggering has been proposed to control the signal-induced conversion of CARD11 from an inactive to an active scaffold in a step required for lymphocyte activation. In this review, I discuss recent data that suggests that this model should be reconsidered for certain phosphorylation events in CARD11 and propose possible experimental avenues for resolution of raised issues.

The immune system of jawless vertebrates: insights into the prototype of the adaptive immune system.

Jawless vertebrates diverged from an ancestor of jawed vertebrates approximately 550 million years ago. They mount adaptive immune responses to repetitive antigenic challenges, despite lacking major histocompatibility complex molecules, immunoglobulins, T cell receptors, and recombination-activating genes. Instead of B cell and T cell receptors, agnathan lymphocytes express unique antigen receptors named variable lymphocyte receptors (VLRs), which generate diversity through a gene conversion-like mechanism. Although gnathostome antigen receptors and VLRs are structurally unrelated, jawed and jawless vertebrates share essential features of lymphocyte-based adaptive immunity, including the expression of a single type of receptor on each lymphocyte, clonal expansion of antigen-stimulated lymphocytes, and the dichotomy of cellular and humoral immunity, indicating that the backbone of the adaptive immune system was established in a common ancestor of all vertebrates. Furthermore, recent evidence indicates that, unlike previously thought, agnathans have a unique classical pathway of complement activation where VLRB molecules act as antibodies instead of immunoglobulins. It seems likely that the last common ancestor of all vertebrates had an adaptive immune system resembling that of jawless vertebrates, suggesting that, as opposed to jawed vertebrates, agnathans have retained the prototype of vertebrate adaptive immunity.

Biological characterisation, expression and functional analysis of non-specific cytotoxic cell receptor protein 1 in Nile tilapia (Oreochromis niloticus).

Non-specific cytotoxic cell receptor protein 1 (NCCRP-1) plays a role in recognition of target cell and activation of non-specific cytotoxic cell (NCC). In this study, the full length of Nile tilapia NCCRP-1 (On-NCCRP-1) was cloned. cDNA is composed of 1045 bp with a 90 bp of 5'-Untranslated Regions (UTR), 702 bp open reading frame (ORF) and 253 bp 3'-UTR, encoding 233 amino acids (GenBank accession no: MF162296). The On-NCCRP-1 genomic sequence is 4471 bp in length and contains six exons and five introns. On-NCCRP-1 possesses some inherent conservative domains, such as proline-rich motifs, antigen recognition site, and F-box-related domain. Subcellular localisation and Western blot analysis indicated that On-NCCRP-1 is located in the cell membrane. The transcript of On-NCCRP-1 was detected in all the examined tissues of healthy Nile tilapia by using qRT-PCR, with the highest expression levels in the liver. Following Streptococcus agalactiae challenged in vivo, the On-NCCRP-1 expression was up-regulated significantly in brain, intestines, head kidney and spleen. In the in vitro analysis, the On-NCCRP-1 expression in NCCs was up-regulated significantly from 8 h to 12 h after LPS challenge, and up-regulated significantly at 12 h after challenged with polyI:C. After NCCs were challenged with inactivated S. agalactiae, the On-NCCRP-1 expression was down-regulated significantly after 24 h. NF-кB pathway was strongly activated by the over-expression of On-NCCRP-1 in HEK-293T cells. These results indicate that On-NCCRP-1, as a membrane surface receptor of NCCs, may play an important role in immune response to pathogenic infection in Nile tilapia.

Sequence-dependent dynamics of synthetic and endogenous RSSs in V(D)J recombination.

Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigen-receptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG-RSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAG-12RSS-23RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with Ca2+ leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAG-RSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination.

Genome Topology Control of Antigen Receptor Gene Assembly.

The past decade has increased our understanding of how genome topology controls RAG endonuclease-mediated assembly of lymphocyte AgR genes. New technologies have illuminated how the large IgH, Igκ, TCRα/δ, and TCRß loci fold into compact structures that place their numerous V gene segments in similar three-dimensional proximity to their distal recombination center composed of RAG-bound (D)J gene segments. Many studies have shown that CTCF and cohesin protein-mediated chromosome looping have fundamental roles in lymphocyte lineage- and developmental stage-specific locus compaction as well as broad usage of V segments. CTCF/cohesin-dependent loops have also been shown to direct and restrict RAG activity within chromosome domains. We summarize recent work in elucidating molecular mechanisms that govern three-dimensional chromosome organization and in investigating how these dynamic mechanisms control V(D)J recombination. We also introduce remaining questions for how CTCF/cohesin-dependent and -independent genome architectural mechanisms might regulate compaction and recombination of AgR loci.