The human humoral immune system has evolved to recognize a vast array of pathogenic threats. This ability is primarily driven by the immense diversity of antibodies generated by gene rearrangement during B cell development. However, different people often produce strikingly similar antibodies when exposed to the same antigen-known as public antibodies. Public antibodies not only reflect the immune system's ability to consistently select for optimal B cells but can also serve as signatures of the humoral responses triggered by infection and vaccination. In this Minireview, we examine and compare public antibody identification methods, including the identification criteria used based on V(D)J gene usage and similarity in the complementarity-determining region three sequences, and explore the molecular features of public antibodies elicited against common pathogens, including viruses, protozoa, and bacteria. Finally, we discuss the evolutionary significance and potential applications of public antibodies in informing the design of germline-targeting vaccines, predicting escape mutations in emerging viruses, and providing insights into the process of affinity maturation. The ongoing discovery of public antibodies in response to emerging pathogens holds the potential to improve pandemic preparedness, accelerate vaccine design efforts, and deepen our understanding of human B cell biology.

The assembly of Tcrb and Tcra genes require double negative (DN) thymocytes to undergo multiple rounds of programmed DNA double-strand breaks (DSBs), followed by their efficient repair. However, mechanisms governing cell cycle checkpoints and specific survival pathways during the repair process remain unclear. Here, we report high-resolution scRNA-seq analyses of individually sorted mouse DN3 and DN4 thymocytes, which reveals a G2M cell cycle checkpoint, in addition to the known G1 checkpoint, during Tcrb and Tcra recombination. We also show that inactivation of GSK3β by phosphorylation on Ser389 is essential for DN3/DN4 thymocytes to survive while being stalled at the G1 and G2/M checkpoints. GSK3β promotes death by necroptosis, but not by apoptosis, of DN3/DN4 thymocytes during V(D)J recombination. Failure to inactivate GSK3β in DN3 thymocytes alters the Tcrb gene repertoire primarily through Trbv segment utilization. In addition, preferential recombination of proximal V segments in Tcra depends on GSK3β inactivation. Our study identifies a unique thymocyte survival pathway, enabling them to undergo cell cycle checkpoints for DNA repair during V(D)J recombination of Tcrb and Tcra genes. Thymocyte survival during cell cycle checkpoints for V(D)J recombination DNA repair determines TCRα/β repertoire.

Effective vaccines are urgently needed for the control of tuberculosis (TB). Here, we report that an mRNA TB vaccine is highly effective and exhibits both prophylactic and therapeutic activity in the zebrafish model of TB. Adult zebrafish immunized with the mRNA vaccine survived significantly longer after Mycobacterium marinum challenge compared to those immunized with the DNA vaccine. Furthermore, post-infection treatment with the mRNA vaccine drastically reduced the bacterial burden. The mRNA vaccine activated multiple DNA break repair systems that are essential for the normal development and function of adaptive immunity, but did not activate the canonical DNA damage responses that promote cell death. This highlights a profound connection between DNA damage repair and the activation of immune responses under physiological processes of immunization. Remarkably, the mRNA vaccine induced autophagy in granulomas, coinciding with bacterial killing and cell survival. Collectively, these findings demonstrate that the mRNA vaccine elicits potent innate and adaptive immunity, providing effective host protection against mycobacterial challenge.

Human monoclonal antibodies (mAbs) are an important segment in precision therapeutics. Various methodologies are available for generating them. Recombinant human mAbs expression from sorted single B cells is preferred for its rapid expression using mammalian vectors while maintaining in vivo immunoglobulin (Ig) pairing. The success rate of generating recombinant mAbs from single sorted human B cells directly relies on Ig heavy (IgH) and light (IgL) gene coverage of the PCR primers. Existing primer sets fail to cover all functional human Ig gene rearrangements, exhibit high degeneracy leading to non-specific amplifications and mutations arising from primer mismatch/degeneracy, and require high amplification cycles. Some existing primer sets have high coverage but are not designed for expression as recombinant mAbs. Here, we have designed a primer set to amplify all functional V(D)J transcripts in human B cell repertoire using a nested RT-PCR approach. The resultant amplicons can be cloned into mammalian vectors for expression of recombinant mAb. Non-specific amplifications were minimized using isotype-specific primers for cDNA synthesis and limiting primer degeneracy. We validated the designed primers on single sorted B cells, bulk sorted B cells and peripheral blood mononuclear cells. We were successfully able to amplify paired heavy and light chain transcripts in 38.46 % (80/208) from naive, memory and B1 B cell subsets sorted as single B cells. Paired Ig transcripts from five single B cells were cloned into expression vectors and purified from mammalian cells as recombinant mAbs. Thus, our new primer set offers significant advantages over existing primers as it allows amplification of all functional V(D)J rearrangements, facilitating rapid generation of antigen-specific recombinant antibodies from diverse human B cell repertoires following vaccinations and infections previously inaccessible due to primer limitations.

Immune responses require complex, spatially coordinated interactions between immune cells and their tissue environment. For decades, we have imaged tissue sections to visualize a limited number of immune-related macromolecules in situ, functioning as surrogates for cell types or processes of interest. However, this inevitably provides a limited snapshot of the tissue's immune landscape. Recent developments in high-throughput spatial -omics technologies, particularly spatial transcriptomics, and its application to human samples has facilitated a more comprehensive understanding of tissue immunity by mapping fine-grained immune cell states to their precise tissue location while providing contextual information about their immediate cellular and tissue environment. These data provide opportunities to investigate mechanisms underlying the spatial distribution of immune cells and its functional implications, including the identification of immune niches, although the criteria used to define this term have been inconsistent. Here, we review recent technological and analytic advances in multiparameter spatial profiling, focusing on how these methods have generated new insights in translational immunology. We propose a 3-step framework for the definition and characterization of immune niches, which is powerfully facilitated by new spatial profiling methodologies. Finally, we summarize current approaches to analyze adaptive immune repertoires and lymphocyte clonal expansion in a spatially resolved manner.

T-cell receptor (TCR) sequencing provides a novel platform for insight into and characterization of intricate T-cell profiles, advancing the understanding of tumor immune heterogeneity. Recently, transarterial chemoembolization (TACE) combined with systemic therapy has become the recommended regimen for advanced hepatocellular carcinoma. The regulation of the immune microenvironment after TACE and its impact on tumor progression and recurrence has been a focus of research. By examining and tracking fluctuations in the TCR repertoire following combination treatment, novel perspectives on the modulation of the tumor microenvironment post-TACE and the underlying mechanisms governing tumor progression and recurrence can be gained. Clarifying the distinctive metrics and dynamic alterations of the TCR repertoire within the context of combination therapy is imperative for understanding the mechanisms of anti-tumor immunity, assessing efficacy, exploiting novel treatments, and further advancing precision oncology in the treatment of hepatocellular carcinoma. In this review, we initially summarized the fundamental characteristics of TCR repertoire and depicted immune microenvironment remodeling after TACE. Ultimately, we illustrated the prospective applications of TCR repertoires in TACE combined with systemic therapy.

This study investigates the immunological factors underlying the differential susceptibility of two chicken strains, E- and M-lines, to avian leukosis virus subgroup J (ALV-J). During the eradication of avian leukosis at a chicken breeder farm in Guangdong, we observed strain-specific differences in susceptibility to ALV-J. Moreover, E-line chickens exhibited a slower antibody response to ALV-J compared to M-line chickens. As the T cell receptor (TCR) and B cell receptor (BCR) are critical for antigen recognition, their activation triggers specific immune responses, including antibody production. Using high-throughput sequencing, we characterized the T cell receptor beta (TCRβ) and immunoglobulin heavy chain (IGH) repertoires in spleen tissues from both chicken strains. The M-line demonstrated higher clonal diversity in both TCRβ and IGH repertoires under normal conditions compared to the E-line, suggesting a broader baseline antigen recognition capacity. Following ALV-J infection, the TCRβ repertoire diversity remained unchanged, while the IGH repertoire displayed distinct clonal expansion patterns and complementarity-determining region 3 (CDR3) length distributions between the two lines, potentially affecting their ability to recognize ALV-J antigens. Our study provides the first comprehensive comparison of TCRβ and IGH repertoire dynamics in chickens with different ALV-J susceptibilities, offering new insights into the molecular and immunological mechanisms underlying resistance to ALV-J.

The efficacy of antibody responses is inherently linked to paratope diversity, as generated through V(D)J recombination and somatic hypermutation. Despite this, it is unclear how genetic diversification mechanisms evolved alongside codon optimality and affect antibody expression. Here, we analyze germline immunoglobulin (IG) genes, natural V(D)J repertoires, serum IgG, and monoclonal antibody (mAb) expression through the lens of codon optimality. Germline variable genes (IGVs) exhibit diverse optimality that is inversely related to mutability. Hypermutation deoptimizes heavy-chain (IGH) VDJ repertoires within human tonsils, bone marrow, lymph nodes (including SARS-CoV-2-specific clones), blood (HIV-1-specific clones), mice, and zebrafish. Analyses of mutation-affected codons show that targeting to complementarity-determining regions constrains deoptimization. Germline IGHV optimality correlates with serum variable fragment (VH) usage after influenza vaccination, while synonymous deoptimization attenuated mAb yield. These findings provide unanticipated insights into an antagonistic relationship between diversification mechanisms and codon optimality. Ultimately, the need for diversity takes precedence over that for the most optimal codon usage.

REV7, also known as MAD2B, MAD2L2, and FANCV, is a HORMA-domain family protein crucial to multiple genome stability pathways. REV7's canonical role is as a member of polymerase ζ, a specialized translesion synthesis polymerase essential for DNA damage tolerance. REV7 also ensures accurate cell cycle progression and prevents premature mitotic progression by sequestering an anaphase-promoting complex/cyclosome activator. Additionally, REV7 supports genome integrity by directing double-strand break repair pathway choice as part of the recently characterized mammalian shieldin complex. Given that genome instability is a hallmark of cancer, it is unsurprising that REV7, with its numerous genome maintenance roles, is implicated in multiple malignancies, including ovarian cancer, glioma, breast cancer, malignant melanoma, and small-cell lung cancer. Moreover, high REV7 expression is associated with poor prognoses and treatment resistance in these and other cancers. Promisingly, early studies indicate that REV7 suppression enhances sensitivity to chemotherapeutics, including cisplatin. This review aims to provide a comprehensive overview of REV7's myriad roles in genome maintenance and other functions as well as offer an updated summary of its connections to cancer and treatment resistance.

Gene expression is regulated by chromatin architecture and epigenetic remodeling in cell homeostasis and pathologies. Histone modifications act as the key factors to modulate the chromatin accessibility. Different histone modifications are strongly associated with the localization of chromatin. Heterochromatin primarily localizes at the nuclear periphery, where it interacts with lamina proteins to suppress gene expression. In this review, we summarize the potential bridges that have regulatory functions of histone modifications in chromatin organization and transcriptional regulation at the nuclear periphery. We use lamina-associated domains (LADs) as examples to elucidate the biological roles of the interactions between histone modifications and nuclear lamina in cell differentiation and development. In the end, we highlight the technologies that are currently used to identify and visualize histone modifications and LADs, which could provide spatiotemporal information for understanding their regulatory functions in gene expression and discovering new targets for diseases.

V(D)J recombination secures the production of functional immunoglobulin (Ig) genes and antibody diversity during the early stages of B-cell development through long-distance interactions mediated by cis-regulatory elements and trans-acting factors. O-GlcNAcylation is a dynamic and reversible posttranslational modification of nuclear and cytoplasmic proteins that regulates various protein functions, including DNA-binding affinity and protein-protein interactions. However, the effects of O-GlcNAcylation on proteins involved in V(D)J recombination remain largely unknown. To elucidate this relationship, we downregulated O-GlcNAcylation in a mouse model by administering an O-GlcNAc inhibitor or restricting the consumption of a regular diet. Interestingly, the inhibition of O-GlcNAcylation in mice severely impaired Ig heavy-chain (IgH) gene rearrangement. We identified several factors crucial for V(D)J recombination, including YY1, CTCF, SMC1, and SMC3, as direct targets of O-GlcNAc modification. Importantly, O-GlcNAcylation regulates the physical interaction between SMC1 and SMC3 and the DNA-binding patterns of YY1 at the IgH gene locus. Moreover, O-GlcNAc inhibition downregulated DDX5 protein expression, affecting the functional association of CTCF with its DNA-binding sites at the IgH locus. Our results showed that locus contraction and long-range interactions throughout the IgH locus are disrupted in a manner dependent on the cellular O-GlcNAc level. In this study, we established that V(D)J recombination relies on the O-GlcNAc status of stage-specific proteins during early B-cell development and identified O-GlcNAc-dependent mechanisms as new regulatory components for the development of a diverse antibody repertoire.

In people living with HIV (PLWH), the susceptibility to malignancies is notably augmented, with lymphoma emerging as a predominant malignancy. Even in the antiretroviral therapy (ART) era, aggressive B-cell lymphoma stands out as a paramount concern. Yet, the pathogenesis of HIV related lymphoma (HRL) largely remains an enigma. Recent insights underscore the pivotal role of the dysregulated B cell receptor (BCR) signaling cascade, evidencing its oncogenic potential across a spectrum of lymphomas. Intricate interplays between HIV and BCR structural-functional integrity have been identified in PLWH. In this review, we elucidated the mechanism by which the BCR signaling pathway is involved in HRL, mainly including the following aspects: HIV can reshape BCR structure by modulating of activation-induced cytidine deaminase (AID) and recombination-activating gene (RAG) dynamics; HIV can act as a chronic antigen to activate the BCR signaling pathway, such as upregulating PI3K and MAPK signaling pathway and reducing the expression of CD300a; HIV co-infection with other oncogenic viruses may also influence tumor formation mediated by the BCR signaling pathway. This review aims to elucidate the intricate regulation of the BCR signaling pathway by HIV in B cell lymphoma, providing a novel perspective on the pathogenesis of lymphoma in HIV-affected environments.

Atherosclerosis (AS) is a chronic inflammatory disease characterized by the formation of atherosclerotic plaques that consist of numerous cells including smooth muscle cells, endothelial cells, immune cells, and foam cells. The most abundant innate and adaptive immune cells, including neutrophils, monocytes, macrophages, B cells, and T cells, play a pivotal role in the inflammatory response, lipoprotein metabolism, and foam cell formation to accelerate atherosclerotic plaque formation. In this review, we have discussed the underlying mechanisms of activated immune cells in promoting AS and reviewed published clinical trials for the treatment of AS by suppressing immune cell activation. We have also presented some crucial shortcomings of current clinical trials. Lastly, we have discussed the therapeutic potential of novel compounds, including herbal medicine and dietary food, in alleviating AS in animals. Despite these limitations, further clinical trials and experimental studies will enhance our understanding of the mechanisms modulated by immune cells and promote widespread drug use to treat AS by suppressing immune system-induced inflammation.

Adaptive Immune Receptor Repertoire sequencing (AIRR-seq) is critical for our understanding of the adaptive immune system's dynamics in health and disease. Reliable analysis of AIRR-seq data depends on accurate rearranged immunoglobulin (Ig) sequence alignment. Various Ig sequence aligners exist, but there is no unified benchmarking standard representing the complexities of AIRR-seq data, obscuring objective comparisons of aligners across tasks. Here, we introduce GenAIRR, a modular simulation framework for generating Ig sequences alongside their ground truths. GenAIRR realistically simulates the intricacies of V(D)J recombination, somatic hypermutation, and an array of sequence corruptions. We comprehensively assessed prominent Ig sequence aligners across various metrics, unveiling unique performance characteristics for each aligner. The GenAIRR-produced datasets, combined with the proposed rigorous evaluation criteria, establish a solid basis for unbiased benchmarking of immunogenetics computational tools. It sets up the ground for further improving the crucial task of Ig sequence alignment, ultimately enhancing our understanding of adaptive immunity.

T cells play an important role in adaptive immunity. Mature T cells specifically recognize antigens on major histocompatibility complex molecules through T-cell receptors (TCRs). As the TCR repertoire is highly diverse, its analysis is vital in the assessment of T cells. Advances in sequencing technology have provided convenient methods for further investigation of the TCR repertoire. In the present review, the TCR structure and the mechanisms by which TCRs function in tumor recognition are described. In addition, the potential value of the TCR repertoire in tumor diagnosis is reviewed. Furthermore, the role of the TCR repertoire in tumor immunotherapy is introduced, and the relationships between the TCR repertoire and the effects of different tumor immunotherapies are discussed. Based on the reviewed literature, it may be concluded that the TCR repertoire has the potential to serve as a biomarker for tumor prognosis. However, a wider range of cancer types and more diverse subjects require evaluation in future research to establish the TCR repertoire as a biomarker of tumor immunity.

T cells, the "superstar" of the immune system, play a crucial role in antitumor immunity. T-cell receptors (TCR) are crucial molecules that enable T cells to identify antigens and start immunological responses. The body has evolved a unique method for rearrangement, resulting in a vast diversity of TCR repertoires. A healthy TCR repertoire is essential for the particular identification of antigens by T cells.

In this article, we systematically summarized the TCR creation mechanisms and analysis methodologies, particularly focusing on the application of next-generation sequencing (NGS) technology. We explore the TCR repertoire in health and cancer, and discuss the implications of TCR repertoire analysis in understanding carcinogenesis, cancer progression, and treatment.

The TCR repertoire analysis has enormous potential for monitoring the emergence and progression of malignancies, as well as assessing therapy response and prognosis. The application of NGS has dramatically accelerated our comprehension of TCR diversity and its role in cancer immunity.

To substantiate the significance of TCR repertoires as biomarkers, more thorough and exhaustive research should be conducted. The TCR repertoire analysis, enabled by advanced sequencing technologies, is poised to become a crucial tool in the future of cancer diagnosis, monitoring, and therapy evaluation.

Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the human and pig parasitic nematode Ascaris to characterize the DSBs. Using END-seq, we demonstrate that DSBs are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3'-overhangs before the addition of neotelomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends may be due to the sequestration of the eliminated DNA into micronuclei, preventing neotelomere formation at their ends. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for PDE. Overall, our data indicate that telomere healing of DSBs is specific to the break sites responsible for nematode PDE.

G0-G1 phase alternative end joining (A-EJ) is a recently defined mutagenic pathway characterized by resected deletion and translocation joints that are predominantly direct and are distinguished from A-EJ in cycling cells that rely much more on microhomology-mediated end joining (MMEJ). Using chemical and genetic approaches, we systematically evaluate potential A-EJ factors and DNA damage response (DDR) genes to support this mechanism by mapping the repair fates of RAG1/2-initiated double-strand breaks in the context of Igκ locus V-J recombination and chromosome translocation. Our findings highlight a polymerase theta-independent Parp1-XRCC1/LigIII axis as central A-EJ components, supported by 53BP1 in the context of an Ataxia-telangiectasia mutated (ATM)-activated DDR. Mechanistically, we demonstrate varied changes in short-range resection, MMEJ, and translocation, imposed by compromising specific DDR activities, which include polymerase alpha, Ataxia-telangiectasia and Rad3-related (ATR), DNA2, and Mre11. This study advances our understanding of DNA damage repair within the 53BP1 regulatory domain and the RAG1/2 postcleavage complex.

The evolutionary conservation of non-core RAG regions suggests significant roles that might involve quantitative or qualitative alterations in RAG activity. Off-target V(D)J recombination contributes to lymphomagenesis and is exacerbated by RAG2' C-terminus absence in Tp53-/- mice thymic lymphomas. However, the genomic stability effects of non-core regions from both Rag1c/c and Rag2c/c in BCR-ABL1+ B-lymphoblastic leukemia (BCR-ABL1+ B-ALL), the characteristics, and mechanisms of non-core regions in suppressing off-target V(D)J recombination remain unclear. Here, we established three mouse models of BCR-ABL1+ B-ALL in mice expressing full-length RAG (Ragf/f), core RAG1 (Rag1c/c), and core RAG2 (Rag2c/c). The Ragc/c (Rag1c/c and Rag2c/c) leukemia cells exhibited greater malignant tumor characteristics compared to Ragf/f cells. Additionally, Ragc/c cells showed higher frequency of off-target V(D)J recombination and oncogenic mutations than Ragf/f. We also revealed decreased RAG cleavage accuracy in Ragc/c cells and a smaller recombinant size in Rag1c/c cells, which could potentially exacerbate off-target V(D)J recombination in Ragc/c cells. In conclusion, these findings indicate that the non-core RAG regions, particularly the non-core region of RAG1, play a significant role in preserving V(D)J recombination precision and genomic stability in BCR-ABL1+ B-ALL.

The rearrangement and expression of the immunoglobulin μ heavy chain (Igh) gene require communication of the intragenic Eμ and 3' regulatory region (RR) enhancers with the variable (VH) gene promoter. Eμ binding of the transcription factor YY1 has been implicated in enhancer-promoter communication, but the YY1 protein network remains obscure. By analyzing the comprehensive proteome of the 1-kb Eμ wild-type enhancer and that of Eμ lacking the YY1 binding site, we identified the male-specific lethal (MSL)/MOF complex as a component of the YY1 protein network. We found that MSL2 recruitment depends on YY1 and that gene knockout of Msl2 in primary pre-B cells reduces μ gene expression and chromatin looping of Eμ to the 3' RR enhancer and VH promoter. Moreover, Mof heterozygosity in mice impaired μ expression and early B cell differentiation. Together, these data suggest that the MSL/MOF complex regulates Igh gene expression by augmenting YY1-mediated enhancer-promoter communication.

The canonical role of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in repairing DNA double-strand breaks combined with its reported dysregulation in several malignancies has driven the development of DNA-PKcs inhibitors as therapeutics. However, until recently the relationship between DNA-PKcs and tumorigenesis has been primarily investigated with regard to its role in non-homologous end joining (NHEJ) repair. Emerging research has uncovered non-canonical DNA-PKcs functions involved with transcriptional regulation, telomere maintenance, metabolic regulation, and immune signaling all of which may also impinge on tumorigenesis. This review mainly discusses these non-canonical roles of DNA-PKcs in cellular biology and their potential contribution to tumorigenesis, as well as evaluating the implications of targeting DNA-PKcs for cancer therapy.

The ATP-dependent RNA helicase UPF1 plays a crucial role in various mRNA degradation pathways, most importantly in nonsense-mediated mRNA decay (NMD). Here, we show that UPF1 is upregulated during the early stages of B cell development and is important for early B cell development in the bone marrow. B-cell-specific Upf1 deletion in mice severely impedes the early to late LPre-B cell transition, in which VH-DHJH recombination occurs at the Igh gene. Furthermore, UPF1 is indispensable for VH-DHJH recombination, without affecting DH-JH recombination. Intriguingly, the genetic pre-arrangement of the Igh gene rescues the differentiation defect in early LPre-B cells under Upf1 deficient conditions. However, differentiation is blocked again following Ig light chain recombination, leading to a failure in development into immature B cells. Notably, UPF1 interacts with and regulates the expression of genes involved in immune responses, cell cycle control, NMD, and the unfolded protein response in B cells. Collectively, our findings underscore the critical roles of UPF1 during the early LPre-B cell stage and beyond, thus orchestrating B cell development.

Mitochondria carry their own genetic information encoding for a subset of protein-coding genes and translational machinery essential for cellular respiration and metabolism. Despite its small size, the mitochondrial genome, its natural genetic variation and molecular phenotypes have been challenging to study using bulk sequencing approaches, due to its variation in cellular copy number, non-Mendelian modes of inheritance and propensity for mutations. Here we highlight emerging strategies designed to capture mitochondrial genetic variation across individual cells for lineage tracing and studying mitochondrial genetics in primary human cells and clinical specimens. We review recent advances surrounding single-cell mitochondrial genome sequencing and its integration with functional genomic readouts, including leveraging somatic mitochondrial DNA mutations as clonal markers that can resolve cellular population dynamics in complex human tissues. Finally, we discuss how single-cell whole mitochondrial genome sequencing approaches can be utilized to investigate mitochondrial genetics and its contribution to cellular heterogeneity and disease.

Transcripts longer than 200 nucleotides that are not translated into proteins are known as long non-coding RNAs, or lncRNAs. Now, they are becoming more significant as important regulators of gene expression, and as a result, of many biological processes in both healthy and pathological circumstances, such as blood malignancies. Through controlling alternative splicing, transcription, and translation at the post-transcriptional level, lncRNAs have an impact on the expression of genes. In multiple myeloma (MM), the majority of lncRNAs is elevated and promotes the proliferation, adhesion, drug resistance and invasion of MM cells by blocking apoptosis and altering the tumor microenvironment (TME). To control mRNA splicing, stability, and translation, they either directly attach to the target mRNA or transfer RNA-binding proteins (RBPs). By expressing certain miRNA-binding sites that function as competitive endogenous RNAs (ceRNAs), most lncRNAs mimic the actions of miRNAs. Here, we highlight lncRNAs role in the MM pathogenesis with emphasize on their capacity to control the molecular mechanisms known as "hallmarks of cancer," which permit earlier tumor initiation and progression and malignant cell transformation.

The nervous and immune systems are highly developed, and each performs specialized physiological functions. However, they work together, and their dysfunction is associated with various diseases. Specialized molecules, such as neurotransmitters, cytokines, and more general metabolites, are essential for the appropriate regulation of both systems. Tryptophan, an essential amino acid, is converted into functional molecules such as serotonin and kynurenine, both of which play important roles in the nervous and immune systems. The role of kynurenine metabolites in neurodegenerative and psychiatric diseases has recently received particular attention. Recently, we found that hyperactivity of the kynurenine pathway is a critical risk factor for septic shock. In this review, we first outline neuroimmune interactions and tryptophan derivatives and then summarized the changes in tryptophan metabolism in neurological disorders. Finally, we discuss the potential of tryptophan derivatives as therapeutic targets for neuroimmune disorders.

Ten-eleven translocation (TET) proteins are DNA dioxygenases that mediate active DNA demethylation. TET3 is the most highly expressed TET protein in thymic developing T cells. TET3, either independently or in cooperation with TET1 or TET2, has been implicated in T cell lineage specification by regulating DNA demethylation. However, TET-deficient mice exhibit complex phenotypes, suggesting that TET3 exerts multifaceted roles, potentially by interacting with other proteins. We performed liquid chromatography with tandem mass spectrometry in primary developing T cells to identify TET3 interacting partners in endogenous, in vivo conditions. We discover TET3 interacting partners. Our data establish that TET3 participates in a plethora of fundamental biological processes, such as transcriptional regulation, RNA polymerase elongation, splicing, DNA repair, and DNA replication. This resource brings in the spotlight emerging functions of TET3 and sets the stage for systematic studies to dissect the precise mechanistic contributions of TET3 in shaping T cell biology.

Class switch recombination (CSR) plays an important role in adaptive immune response by enabling mature B cells to replace the initial IgM by another antibody class (IgG, IgE or IgA). CSR is preceded by transcription of the IgH constant genes and is controlled by the super-enhancer 3' regulatory region (3'RR) in an activation-specific manner. The 3'RR is composed of four enhancers (hs3a, hs1-2, hs3b and hs4). In mature B cells, 3'RR activity correlates with transcription of its enhancers. CSR can also occur in primary developing B cells though at low frequency, but in contrast to mature B cells, the transcriptional elements that regulate the process in developing B cells are ill-known. In particular, the role of the 3'RR in the control of constant genes' transcription and CSR has not been addressed. Here, by using a mouse line devoid of the 3'RR and a culture system that highly enriches in pro-B cells, we show that the 3'RR activity is indeed required for switch transcription and CSR, though its effect varies in an isotype-specific manner and correlates with transcription of hs4 enhancer only.

Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well-characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the nematode Ascaris to study the timing of PDE breaks and examine the DSBs and their end processing. Using END-seq, we characterize the DSB ends and demonstrate that DNA breaks are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3' overhangs before the addition of telomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends in Ascaris may be due to the sequestration of the eliminated DNA into micronuclei, preventing their ends from telomere healing. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for nematode PDE.

T cell receptor (TCR) repertoire sequencing has emerged as a powerful tool for understanding the diversity and functionality of T cells within the host immune system. Yet, the chicken TCR repertoire remains poorly understood due to incomplete genome annotation of the TCR loci, despite the importance of chickens in agriculture and as an immunological model. Here, we addressed this critical issue by employing 5' rapid amplification of complementary DNA ends (5'RACE) TCR repertoire sequencing with molecular barcoding of complementary DNA (cDNA) molecules. Simultaneously, we enhanced the genome annotation of TCR Variable (V), Diversity (D, only present in β and δ loci) and Joining (J) genes in the chicken genome. To enhance the efficiency of TCR annotations, we developed VJ-gene-finder, an algorithm designed to extract VJ gene candidates from deoxyribonucleic acid (DNA) sequences. Using this tool, we achieved a comprehensive annotation of all known chicken TCR loci, including the α/δ locus on chromosome 27. Evolutionary analysis revealed that each locus evolved separately by duplication of long homology units. To define the baseline TCR diversity in healthy chickens and to demonstrate the feasibility of the approach, we characterized the splenic α/β/γ/δ TCR repertoire. Analysis of the repertoires revealed preferential usage of specific V and J combinations in all chains, while the overall features were characteristic of unbiased repertoires. We observed moderate levels of shared complementarity-determining region 3 (CDR3) clonotypes among individual birds within the α and γ chain repertoires, including the most frequently occurring clonotypes. However, the β and δ repertoires were predominantly unique to each bird. Taken together, our TCR repertoire analysis allowed us to decipher the composition, diversity, and functionality of T cells in chickens. This work not only represents a significant step towards understanding avian T cell biology, but will also shed light on host-pathogen interactions, vaccine development, and the evolutionary history of avian immunology.

In rheumatoid arthritis (RA), the activation of T and B cell clones specific for self-antigens leads to the chronic inflammation of the synovium. Here, we perform an in-depth quantitative analysis of the seven chains that comprise the adaptive immune receptor repertoire (AIRR) in RA.

In comparison to controls, we show that RA patients have multiple and strong differences in the B cell receptor repertoire including reduced diversity as well as altered isotype, chain, and segment frequencies. We demonstrate that therapeutic tumor necrosis factor inhibition partially restores this alteration but find a profound difference in the underlying biochemical reactivities between responders and non-responders. Combining the AIRR with HLA typing, we identify the specific T cell receptor repertoire associated with disease risk variants. Integrating these features, we further develop a molecular classifier that shows the utility of the AIRR as a diagnostic tool.

Simultaneous sequencing of the seven chains of the human AIRR reveals novel features associated with the disease and clinically relevant phenotypes, including response to therapy. These findings show the unique potential of AIRR to address precision medicine in immune-related diseases.

The vast diversity of mammalian adaptive antigen receptors allows for robust and efficient immune responses against a wide number of pathogens. The antigen receptor repertoire is built during the recombination of B and T cell receptor (BCR, TCR) loci and hypermutation of BCR loci. V(D)J recombination rearranges these antigen receptor loci, which are organized as an array of separate V, (D), and J gene segments. Transcription activation at the recombining locus leads to changes in the local three-dimensional architecture, which subsequently contributes to which gene segments are utilized for recombination. The endogenous retrovirus (ERV) mouse mammary tumor provirus 8 (Mtv8) resides on mouse chromosome 6 interposed within the large array of light chain kappa V gene segments. As ERVs contribute to changes in genomic architecture by driving high levels of transcription of neighboring genes, it was suggested that Mtv8 could influence the BCR repertoire. We generated Mtv8-deficient mice to determine if the ERV influences V(D)J recombination to test this possibility. We find that Mtv8 does not influence the BCR repertoire.

Deep learning models have been shown to accurately predict protein structure from sequence, allowing researchers to explore protein space from the structural viewpoint. In this paper we explore whether "novel" features, such as distinct loop conformations can arise from these predictions despite not being present in the training data. Here we have used ABodyBuilder2, a deep learning antibody structure predictor, to predict the structures of ~1.5M paired antibody sequences. We examined the predicted structures of the canonical CDR loops and found that most of these predictions fall into the already described CDR canonical form structural space. We also found a small number of "new" canonical clusters composed of heterogeneous sequences united by a common sequence motif and loop conformation. Analysis of these novel clusters showed their origins to be either shapes seen in the training data at very low frequency or shapes seen at high frequency but at a shorter sequence length. To evaluate explicitly the ability of ABodyBuilder2 to extrapolate, we retrained several models whilst withholding all antibody structures of a specific CDR loop length or canonical form. These "starved" models showed evidence of generalisation across CDRs of different lengths, but they did not extrapolate to loop conformations which were highly distinct from those present in the training data. However, the models were able to accurately predict a canonical form even if only a very small number of examples of that shape were in the training data. Our results suggest that deep learning protein structure prediction methods are unable to make completely out-of-domain predictions for CDR loops. However, in our analysis we also found that even minimal amounts of data of a structural shape allow the method to recover its original predictive abilities. We have made the ~1.5 M predicted structures used in this study available to download at https://doi.org/10.5281/zenodo.10280181.

The breadth of pathogens to which T cells can respond is determined by the T cell receptors (TCRs) present in an individual's repertoire. Although more than 90% of the sequence diversity among TCRs is generated by terminal deoxynucleotidyl transferase (TdT)-mediated N-nucleotide addition during V(D)J recombination, the benefit of TdT-altered TCRs remains unclear. Here, we computationally and experimentally investigated whether TCRs with higher N-nucleotide diversity via TdT make distinct contributions to acute or chronic pathogen control specifically through the inclusion of TCRs with lower antigen binding strengths (i.e., lower reactivity to peptide-major histocompatibility complex (pMHC)). When T cells with high pMHC reactivity have a greater propensity to become functionally exhausted than those of low pMHC reactivity, our computational model predicts a shift toward T cells with low pMHC reactivity over time during chronic, but not acute, infections. This TCR-affinity shift is critical, as the elimination of T cells with lower pMHC reactivity in silico substantially increased the time to clear a chronic infection, while acute infection control remained largely unchanged. Corroborating an affinity-centric benefit for TCR diversification via TdT, we found evidence that TdT-deficient TCR repertoires possess fewer T cells with weaker pMHC binding strengths in vivo and showed that TdT-deficient mice infected with a chronic, but not an acute, viral pathogen led to protracted viral clearance. In contrast, in the case of a chronic fungal pathogen where T cells fail to clear the infection, both our computational model and experimental data showed that TdT-diversified TCR repertoires conferred no additional protection to the hosts. Taken together, our in silico and in vivo data suggest that TdT-mediated TCR diversity is of particular benefit for the eventual resolution of prolonged pathogen replication through the inclusion of TCRs with lower foreign antigen binding strengths.

Helix-loop-helix (HLH) transcription factors (TFs) play a key role in various cellular differentiation and function through the regulation of enhancer activity. E2A, a member of the mammalian E-protein family (class I HLH protein), is well known to play an important role in hematopoiesis, especially in adaptive lymphocyte development. E2A instructs B- and T-cell lineage development through the regulation of enhancer activity for B- or T-cell signature gene expression, including Rag1 and Rag2 (Rag1/2) genes. In this chapter, we mainly focus on the function of E2A in B-cell development and on the roles of E2A in establishing the enhancer landscape through the recruitment of EP300/KAT3B, chromatin remodeling complex, mediator, cohesion, and TET proteins. Finally, we demonstrate how E2A orchestrates the assembly of the Rag1/2 gene super-enhancer (SE) formation by changing the chromatin conformation across the Rag gene locus.

SUMMARYCiliated protozoa undergo large-scale developmental rearrangement of their somatic genomes when forming a new transcriptionally active macronucleus during conjugation. This process includes the fragmentation of chromosomes derived from the germline, coupled with the efficient healing of the broken ends by de novo telomere addition. Here, we review what is known of developmental chromosome fragmentation in ciliates that have been well-studied at the molecular level (Tetrahymena, Paramecium, Euplotes, Stylonychia, and Oxytricha). These organisms differ substantially in the fidelity and precision of their fragmentation systems, as well as in the presence or absence of well-defined sequence elements that direct excision, suggesting that chromosome fragmentation systems have evolved multiple times and/or have been significantly altered during ciliate evolution. We propose a two-stage model for the evolution of the current ciliate systems, with both stages involving repetitive or transposable elements in the genome. The ancestral form of chromosome fragmentation is proposed to have been derived from the ciliate small RNA/chromatin modification process that removes transposons and other repetitive elements from the macronuclear genome during development. The evolution of this ancestral system is suggested to have potentiated its replacement in some ciliate lineages by subsequent fragmentation systems derived from mobile genetic elements.

The stringent regulation of RAGs (Recombination activating genes), the site-specific endonuclease responsible for V(D)J recombination, is important to prevent genomic rearrangements and chromosomal translocations in lymphoid cells. In the present study, we identify a microRNA, miR-501, which can regulate the expression of RAG1 in lymphoid cells. Overexpression of the pre-miRNA construct led to the generation of mature miRNAs and a concomitant reduction in RAG1 expression, whereas inhibition using anti-miRs resulted in its enhanced expression. The direct interaction of the 3'UTR of miR-501 with RAG1 was confirmed by the reporter assay. Importantly, overexpression of miRNAs led to inhibition of V(D)J recombination in B cells, revealing their impact on the physiological function of RAGs. Of interest is the inverse correlation observed for miR-501 with RAG1 in various leukemia patients and lymphoid cell lines, suggesting its possible use in cancer therapy. Thus, our results reveal the regulation of RAG1 by miR-501-3p in B cells and thus V(D)J recombination and its possible implications on immunoglobulin leukemogenesis.

The T cell receptor (TCR) determines specificity and affinity for both foreign and self-peptides presented by the major histocompatibility complex (MHC). Although the strength of TCR interactions with self-pMHC impacts T cell function, it has been challenging to identify TCR sequence features that predict T cell fate. To discern patterns distinguishing TCRs from naive CD4+ T cells with low versus high self-reactivity, we used data from 42 mice to train a machine learning (ML) algorithm that identifies population-level differences between TCRβ sequence sets. This approach revealed that weakly self-reactive T cell populations were enriched for longer CDR3β regions and acidic amino acids. We tested our ML predictions of self-reactivity using retrogenic mice with fixed TCRβ sequences. Extrapolating our analyses to independent datasets, we predicted high self-reactivity for regulatory T cells and slightly reduced self-reactivity for T cells responding to chronic infections. Our analyses suggest a potential trade-off between TCR repertoire diversity and self-reactivity. A record of this paper's transparent peer review process is included in the supplemental information.

Somatic hypermutation (SHM) drives affinity maturation and continues over months in SARS-CoV-2-neutralizing antibodies (nAbs). However, several potent SARS-CoV-2 antibodies carry no or only a few mutations, leaving the question of how ongoing SHM affects neutralization unclear. Here, we reverted variable region mutations of 92 antibodies and tested their impact on SARS-CoV-2 binding and neutralization. Reverting higher numbers of mutations correlated with decreasing antibody functionality. However, for some antibodies, including antibodies of the public clonotype VH1-58, neutralization of Wu01 remained unaffected. Although mutations were dispensable for Wu01-induced VH1-58 antibodies to neutralize Alpha, Beta, and Delta variants, they were critical for Omicron BA.1/BA.2 neutralization. We exploited this knowledge to convert the clinical antibody tixagevimab into a BA.1/BA.2 neutralizer. These findings broaden our understanding of SHM as a mechanism that not only improves antibody responses during affinity maturation but also contributes to antibody diversification, thus increasing the chances of neutralizing viral escape variants.

The spatial distribution of lymphocyte clones within tissues is critical to their development, selection, and expansion. We have developed spatial transcriptomics of variable, diversity, and joining (VDJ) sequences (Spatial VDJ), a method that maps B cell and T cell receptor sequences in human tissue sections. Spatial VDJ captures lymphocyte clones that match canonical B and T cell distributions and amplifies clonal sequences confirmed by orthogonal methods. We found spatial congruency between paired receptor chains, developed a computational framework to predict receptor pairs, and linked the expansion of distinct B cell clones to different tumor-associated gene expression programs. Spatial VDJ delineates B cell clonal diversity and lineage trajectories within their anatomical niche. Thus, Spatial VDJ captures lymphocyte spatial clonal architecture across tissues, providing a platform to harness clonal sequences for therapy.