Graphical Abstract Highlights d Latent polyomavirus infections generate a potent, neutralizing antibody response d The anti-BKV repertoire is clonally diverse and harbors crossreactive antibodies d Memory IgM B cell receptors are affinity matured and isotype restricted d A monoclonal cross-neutralizing IgG binds a conserved quaternary viral epitope SUMMARY Human polyomaviruses cause a common childhood infection worldwide and typically elicit a neutralizing antibody and cellular immune response, while establishing a dormant infection in the kidney with minimal clinical manifestations. However, viral reactivation can cause severe pathology in immunocompromised individuals. We developed a high-throughput, functional antibody screen to examine the humoral response to BK polyomavirus. This approach enabled the isolation of antibodies from all peripheral B cell subsets and revealed the anti-BK virus antibody repertoire as clonally complex with respect to immunoglobulin sequences and isotypes (both IgM and IgG), including a high frequency of monoclonal antibodies that broadly neutralize BK virus subtypes and the related JC polyomavirus. Cryo-electron microscopy of a broadly neutralizing IgG single-chain variable fragment complexed with BK virus-like particles revealed the quaternary nature of a conserved viral epitope at the junction between capsid pentamers. These features unravel a potent modality for inhibiting polyomavirus infection in kidney transplant recipients and other immunocompromised patients.

Type 1 diabetes (T1D) is characterized by a chronic, progressive autoimmune attack against pancreas-specific antigens, effecting the destruction of insulin-producing β-cells. Here we show interleukin-2 (IL-2) is a non-pancreatic autoimmune target in T1D. Anti-IL-2 autoantibodies, as well as T cells specific for a single orthologous epitope of IL-2, are present in the peripheral blood of non-obese diabetic (NOD) mice and patients with T1D. In NOD mice, the generation of anti-IL-2 autoantibodies is genetically determined and their titre increases with age and disease onset. In T1D patients, circulating IgG memory B cells specific for IL-2 or insulin are present at similar frequencies. Anti-IL-2 autoantibodies cloned from T1D patients demonstrate clonality, a high degree of somatic hypermutation and nanomolar affinities, indicating a germinal centre origin and underscoring the synergy between cognate autoreactive T and B cells leading to defective immune tolerance.

High-throughput sequencing of immunoglobulin (Ig) repertoires (Ig-seq) is a powerful method for quantitatively interrogating B cell receptor sequence diversity. When applied to human repertoires, Ig-seq provides insight into fundamental immunological questions, and can be implemented in diagnostic and drug discovery projects. However, a major challenge in Ig-seq is ensuring accuracy, as library preparation protocols and sequencing platforms can introduce substantial errors and bias that compromise immunological interpretation. Here, we have established an approach for performing highly accurate human Ig-seq by combining synthetic standards with a comprehensive error and bias correction pipeline. First, we designed a set of 85 synthetic antibody heavy-chain standards (in vitro transcribed RNA) to assess correction workflow fidelity. Next, we adapted a library preparation protocol that incorporates unique molecular identifiers (UIDs) for error and bias correction which, when applied to the synthetic standards, resulted in highly accurate data. Finally, we performed Ig-seq on purified human circulating B cell subsets (naïve and memory), combined with a cellular replicate sampling strategy. This strategy enabled robust and reliable estimation of key repertoire features such as clonotype diversity, germline segment, and isotype subclass usage, and somatic hypermutation. We anticipate that our standards and error and bias correction pipeline will become a valuable tool for researchers to validate and improve accuracy in human Ig-seq studies, thus leading to potentially new insights and applications in human antibody repertoire profiling.

Determining antigen specificity is vital for understanding B cell biology and for producing human monoclonal antibodies. We describe here a powerful method for identifying B cells that recognize membrane antigens expressed on cells. The technique depends on two characteristics of the interaction between a B cell and an antigen-expressing cell: antigen-receptor-mediated extraction of antigen from the membrane of the target cell, and B cell activation. We developed the method using influenza hemagglutinin as a model viral membrane antigen, and tested it using acetylcholine receptor (AChR) as a model membrane autoantigen. The technique involves co-culturing B cells with adherent, bioorthogonally labeled cells expressing GFP-tagged antigen, and sorting GFP-capturing, newly activated B cells. Hemagglutinin-specific B cells isolated this way from vaccinated human donors expressed elevated CD20, CD27, CD71, and CD11c, and reduced CD21, and their secreted antibodies blocked hemagglutination and neutralized viral infection. Antibodies cloned from AChR-capturing B cells derived from patients with myasthenia gravis bound specifically to the receptor on cell membrane. The approach is sensitive enough to detect antigen-specific B cells at steady state, and can be adapted for any membrane antigen.

Mice lacking the lymphocyte-specific transcription factor Bob1 (also called OBF-1 or OCA-B) fail to generate germinal centers and a robust Ig response. We show that peripheral B cells in Bob1−/− mice bear characteristics of chronically activated or anergic-like B cells and identify the immunosuppressive microRNA-146a, together with other microRNAs, as novel transcriptional targets of Bob1. The inability to restrict B cell signaling could contribute to the immunodeficient phenotype of these mice and is consistent with an important role for Bob1 in suppressing B cell activation in vivo.

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15High-throughput sequencing of immunoglobulin repertoires (Ig-seq) is a powerful method for 16 quantitatively interrogating B cell receptor sequence diversity. When applied to human 17 repertoires, Ig-seq provides insight into fundamental immunological questions, and can be 18 implemented in diagnostic and drug discovery projects. However, a major challenge in Ig-seq 19 is ensuring accuracy, as library preparation protocols and sequencing platforms can 20 introduce substantial errors and bias that compromise immunological interpretation. Here, 21 we have established an approach for performing highly accurate human Ig-seq by combining 22 synthetic standards with a comprehensive error and bias correction pipeline. First, we 23 designed a set of 85 synthetic antibody heavy chain standards (in vitro transcribed RNA) to 24 assess correction workflow fidelity. Next, we adapted a library preparation protocol that 25 incorporates unique molecular identifiers (UIDs) for error and bias correction which, when 26 applied to the synthetic standards, resulted in highly accurate data. Finally, we performed Ig-27 seq on purified human circulating B cell subsets (naïve and memory), combined with a 28 cellular replicate sampling strategy. This strategy enabled robust and reliable estimation of 29 key repertoire features such as clonotype diversity, germline segment and isotype subclass 30 usage, and somatic hypermutation (SHM). We anticipate that our standards and error and 31 bias correction pipeline will become a valuable tool for researchers to validate and improve 32 accuracy in human Ig-seq studies, thus leading to potentially new insights and applications in 33 human antibody repertoire profiling. 34 35 36 42 of the germline-encoded variable (V), diversity (D, heavy chain only), and joining (J) gene 43 segments. V(D)J recombination in B cells creates a highly complex receptor population (generally 44interchangeably referred to as BCR, antibody, or immunoglobulin (Ig) repertoires), which matures 45 upon antigen experience to produce the more targeted, high-affinity memory BCR network. In-46 depth and accurate characterization of these repertoires provides valuable insight into the generation 47 synthetic standards to quantify the impact of errors and bias introduced during multiplex-PCR 92 library preparation, and the robustness with which our previously developed method for UID 93 addition by MAF could correct these artifact sequences. Finally, we implemented MAF-based error 94 and bias correction on human B cell subsets (naïve and memory), which enabled us to make 95 accurate clonal diversity estimates and quantify divergent repertoire features across B cell 96 compartments. 97 98 RESULTS 99 Design of a comprehensive set of human synthetic standards 100 Our previously established set of murine synthetic antibody standards contained 16 unique clones 101 (CDR3s) covering 7 IGHV gene segments (out of more than 140 annotated murine IGHV gene 102 segments) (18). For our human standards, we developed a more comprehensive set ...

SUMMARY In order to investigate the influence of thyroid-stimulating hormone (TSH) on the ratio of newly synthesized thyroxine( T4):tri-iodothyronine (T3), hemithyroidectomy was performed on rats maintained on an iodinerich diet. One and two weeks after the operation the concentration of TSH increased in the plasma. As a result, the weight of the residual lobes and their thyroidal uptake of 131I/mg increased and significant histological signs of increased function in the remaining lobes were observed. The most prominent effect was a significant alteration of the ratio of newly synthesized T4:T3 in favour of T3, both in the thyroid and plasma. Four weeks after the operation, when the residual lobes weighed 57% more than those in the controls, the alterations decreased or returned to normal. The total hormone concentration in the plasma (measured as protein-bound 127I) and the oxygen consumption of the animals remained unchanged during the entire study. These findings support the hypothesis that alteration of the production and secretion ratio of T4:T3 induced by TSH might act as another regulatory factor, if a hormone deficiency originates in the peripheral cells. The results show also that changes of the T4:T3 ratio induced by TSH occur in animals on an iodine-rich diet.