It is common knowledge that immunoglobulin (Ig) is produced by B lymphocytes and mainly functions as an antibody. However, it has been shown recently that myeloblasts from acute myeloid leukemia (AML) could also express Ig and that AML-Ig played a role in leukemogenesis and AML progression. The difference between Ig from myeloblasts and B cells has not been explored. Studying the characteristics of the Ig repertoire in myeloblasts and B cells will be helpful to understand the function and significance of AML-Ig. We performed 5\' RACE-related PCR coupled with PacBio sequencing to analyze the Ig repertoire in myeloblasts and B cells from Chinese AML patients. Myeloblasts expressed all five classes of IgH, especially Igγ, with a high expression frequency. Compared with B-Ig in the same patient, AML-Ig showed different biased V(D)J usages and mutation patterns. In addition, the CDR3 length distribution of AML-Ig was significantly different from those of B-Ig. More importantly, mutations of AML-IgH, especially Igμ, Igα, and Igδ, were different from that of B-IgH in each AML patient, and the mutations frequently occurred at the sites of post-translational modification. AML-Ig has distinct characteristics of variable regions and mutations, which may have implications for disease monitoring and personalized therapy.

Immunoglobulin (Ig) has been widely acknowledged to be produced solely by B-lineage cells. However, growing evidence has demonstrated the expression of Ig in an array of cancer cells, as well as normal cells including epithelial cells, epidermal cells, mesangial cells, monocytes, and neutrophils. Ig has even been found to be expressed in non-B cells at immune-privileged sites such as neurons and spermatogenic cells. Despite these non-B cell-derived Igs (non-B-Igs) sharing the same symmetric structures with conventional Igs (B-Igs), further studies have revealed unique characteristics of non-B-Ig, such as restricted variable region and aberrant glycosylation. Moreover, non-B-Ig exhibits properties of promoting malignant behaviours of cancer cells, therefore it could be utilised in the clinic as a potential therapeutic biomarker or target. The elucidation of the generation and regulation of non-B-Ig will certainly broaden our understanding of immunology.

It is widely acknowledged that immunoglobulins (Igs) are produced solely by B-lineage cells. The Ig gene is created by the rearrangement of a group of gene segments [variable (V), diversity (D), and joining (J) segments rearrangement, or V(D)J recombination], which results in the vast diversity of B cell-derived Ig responsible for recognising various antigens. Ig subsequently undergoes somatic hypermutation (SHM) and class switch recombination (CSR) after exposure to antigens, thus converting the low-affinity IgM to IgG, IgA, or IgE antibodies. IgM and IgD are primarily expressed in naïve B cells that have not been exposed to antigens, they do not undergo somatic hypermutation; hence, their variable region sequences remain the same as those in the germline. In contrast, IgG, IgA, and IgE are expressed in antigen-stimulated memory B cells or plasma cells, and thus, they often possess high-frequency mutations in their variable region sequences. Since the discovery that Ig can be produced by non-B cells, Qiu\'s group has investigated and compared the genetic characteristics of B cell-derived Ig and non-B cell-derived Ig. These findings demonstrated that non-B cell-derived Ig shares certain similarities with B cell-derived Ig in that the sequence of its constant region is identical to that of B cell-derived Ig, and its variable region is also strictly dependent on the rearrangement of V, D, and J gene segments. Moreover, akin to B cell-derived Ig, the V regions of IgM and IgD are rarely mutated, while IgG, IgA, and IgE produced by cancer cells are frequently mutated. However, the non-B cell-derived Ig V region sequence displays unique characteristics. (1) Unlike the vast diversity of B cell-derived Igs, non-B cell-derived Igs exhibit restricted diversity; cells from the same lineage always select the same V(D)J recombination patterns; (2) Both mRNA and proteins of RAG1/RAG2 recombinase have been detected in Ig positive cancer cell lines and normal tissues. But Ig recombination could also be found in RAG1-/- and RAG2-/- mice, suggesting that they are not necessary for the rearrangement of non-B cell-derived Igs. These features of non-B cell-derived Igs suggest a potentially undiscovered mechanism of V(D)J recombination, ligation, and SHM in non-B cells, which necessitates further investigation with advanced technology in molecular biology.

Antibody-coding genes accumulate somatic mutations to achieve antibody affinity maturation. Genetic dissection using various mouse models has shown that intrinsic hypermutations occur preferentially and are predisposed in the DNA region encoding antigen-contacting residues. The molecular basis of nonrandom/preferential mutations is a long-sought question in the field. Here, we summarize recent findings on how single-strand (ss)DNA flexibility facilitates activation-induced cytidine deaminase (AID) activity and fine-tunes the mutation rates at a mesoscale within the antibody variable domain exon. We propose that antibody coding sequences are selected based on mutability during the evolution of adaptive immunity and that DNA mechanics play a noncoding role in the genome. The mechanics code may also determine other cellular DNA metabolism processes, which awaits future investigation.

Advances in high-throughput sequencing technologies have facilitated the large-scale characterization of B cell receptor (BCR) repertoires. However, the vast amount and high diversity of the BCR sequences pose challenges for efficient and biologically meaningful analysis. Here, we introduce fastBCR, an efficient computational approach for inferring B cell clonal families from massive BCR heavy chain sequences. We demonstrate that fastBCR substantially reduces the running time while ensuring high accuracy on simulated datasets with diverse numbers of B cell lineages and varying mutation rates. We apply fastBCR to real BCR sequencing data from peripheral blood samples of COVID-19 patients, showing that the inferred clonal families display disease-associated features, as well as corresponding antigen-binding specificity and affinity. Overall, our results demonstrate the advantages of fastBCR for analyzing BCR repertoire data, which will facilitate the identification of disease-associated antibodies and improve our understanding of the B cell immune response.

We have employed mathematical modeling techniques to construct a comprehensive framework for elucidating the intricate response mechanisms of the immune system, facilitating a deeper understanding of B-cell clonal deletion and somatic hypermutation. Our improved model introduces innovative mechanisms that shed light on positive and negative selection processes during T-cell and B-cell development. Notably, clonal deletion is attributed to the attenuated immune stimulation exerted by self-antigens with high binding affinities, rendering them less effective in eliciting subsequent B-cell maturation and differentiation. Secondly, our refined model places particular emphasis on the crucial role played by somatic hypermutation in modulating the immune system\'s functionality. Through extensive investigation, we have determined that somatic hypermutation not only expedites the production of highly specific antibodies pivotal in combating microbial infections but also serves as a regulatory mechanism to dampen autoimmunity and enhance self-tolerance within the organism. Lastly, our model advances the understanding of the implications of antibody in vivo evolution in the overall process of organismal aging. With the progression of time, the age-associated amplification of autoimmune activity becomes apparent. While somatic hypermutation effectively delays this process, mitigating the levels of autoimmune response, it falls short of reversing this trajectory entirely. In conclusion, our advanced mathematical model offers a comprehensive and scholarly approach to comprehend the intricacies of the immune system. By encompassing novel mechanisms for selection, emphasizing the functional role of somatic hypermutation, and illuminating the consequences of in vivo antibody evolution, our model expands the current understanding of immune responses and their implications in aging.

Striking antibody evasion by emerging circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants drives the identification of broadly neutralizing antibodies (bNAbs). However, how a bNAb acquires increased neutralization breadth during antibody evolution is still elusive. Here, we identify a clonally related antibody family from a convalescent individual. One of the members, XG005, exhibits potent and broad neutralizing activities against SARS-CoV-2 variants, while the other members show significant reductions in neutralization breadth and potency, especially against the Omicron sublineages. Structural analysis visualizing the XG005-Omicron spike binding interface reveals how crucial somatic mutations endow XG005 with greater neutralization potency and breadth. A single administration of XG005 with extended half-life, reduced antibody-dependent enhancement (ADE) effect, and increased antibody product quality exhibits a high therapeutic efficacy in BA.2- and BA.5-challenged mice. Our results provide a natural example to show the importance of somatic hypermutation during antibody evolution for SARS-CoV-2 neutralization breadth and potency.

The majority of neutralizing antibodies (NAbs) against SARS-CoV-2 recognize the receptor-binding domain (RBD) of the spike (S) protein. As an escaping strategy, the RBD of the virus is highly variable, evolving mutations to thwart a natural immune response or vaccination. Targeting non-RBD regions of the S protein thus provides a viable alternative to generating potential, robust NAbs. Using a pre-pandemic combinatorial antibody library of 1011, through an alternate negative and positive screening strategy, 11 non-RBD-targeting antibodies are identified. Amongst one NAb that binds specifically to the N-terminal domain of the S protein, SA3, shows mutually non-exclusive binding of the angiotensin-converting enzyme 2 receptor with the S protein. SA3 appears to be insensitive to the conformational change and to interact with both the \"open\" and \"closed\" configurations of the trimeric S protein. SA3 shows compatible neutralization as S-E6, an RBD-targeting NAb, against the wild type and variant of concern (VOC) B.1.351 (Beta) of the SARS-CoV-2 pseudo virus. More importantly, the combination of SA3 with S-E6 is synergistic and recovers from the 10-fold loss in neutralization efficacy against the VOC B.1.351 pseudo virus.

Somatic hypermutation (SHM), initiated by activation-induced cytidine deaminase (AID), generates mutations in the antibody-coding sequence to allow affinity maturation. Why these mutations intrinsically focus on the three nonconsecutive complementarity-determining regions (CDRs) remains enigmatic. Here, we found that predisposition mutagenesis depends on the single-strand (ss) DNA substrate flexibility determined by the mesoscale sequence surrounding AID deaminase motifs. Mesoscale DNA sequences containing flexible pyrimidine-pyrimidine bases bind effectively to the positively charged surface patches of AID, resulting in preferential deamination activities. The CDR hypermutability is mimicable in in vitro deaminase assays and is evolutionarily conserved among species using SHM as a major diversification strategy. We demonstrated that mesoscale sequence alterations tune the in vivo mutability and promote mutations in an otherwise cold region in mice. Our results show a non-coding role of antibody-coding sequence in directing hypermutation, paving the way for the synthetic design of humanized animal models for optimal antibody discovery and explaining the AID mutagenesis pattern in lymphoma.

Despite the initially reported high efficacy of vaccines directed against ancestral SARS-CoV-2, repeated infections in both unvaccinated and vaccinated populations remain a major global health challenge. Because of mutation-mediated immune escape by variants-of-concern (VOC), approved neutralizing antibodies (neutAbs) effective against the original strains have been rendered non-protective. Identification and characterization of mutation-independent pan-neutralizing antibody responses are therefore essential for controlling the pandemic. Here, we characterize and discuss the origins of SARS-CoV-2 neutAbs, arising from either natural infection or following vaccination. In our study, neutAbs in COVID-19 patients were detected using the combination of two lateral flow immunoassay (LFIA) tests, corroborated by plaque reduction neutralization testing (PRNT). A point-of-care neutAb LFIA, NeutraXpress™, was validated using serum samples from historical pre-COVID-19 negative controls, patients infected with other respiratory pathogens, and PCR-confirmed COVID-19 patients. Surprisingly, potent neutAb activity was mainly noted in patients generating both IgM and IgG against the Spike receptor-binding domain (RBD), in contrast to samples possessing anti-RBD IgG alone. We propose that low-affinity, high-avidity, germline-encoded natural IgM and subsequent generation of class-switched IgG may have an underappreciated role in cross-protection, potentially offsetting immune escape by SARS-CoV-2 variants. We suggest Reverse Vaccinology 3.0 to further exploit this innate-like defense mechanism. Our proposition has potential implications for immunogen design, and provides strategies to elicit pan-neutAbs from natural B1-like cells. Refinements in future immunization protocols might further boost long-term cross-protection, even at the mucosal level, against clinical manifestations of COVID-19.