H3K27M neoepitope vaccination in diffuse midline glioma induces B and T cell responses across diverse HLA loci of a recovered patient.

H3K27M, a driver mutation with T and B cell neoepitope characteristics, defines an aggressive subtype of diffuse glioma with poor survival. We functionally dissect the immune response of one patient treated with an H3K27M peptide vaccine who subsequently entered complete remission. The vaccine robustly expanded class II human leukocyte antigen (HLA)-restricted peripheral H3K27M-specific T cells. Using functional assays, we characterized 34 clonally unique H3K27M-reactive T cell receptors and identified critical, conserved motifs in their complementarity-determining region 3 regions. Using detailed HLA mapping, we further demonstrate that diverse HLA-DQ and HLA-DR alleles present immunogenic H3K27M epitopes. Furthermore, we identified and profiled H3K27M-reactive B cell receptors from activated B cells in the cerebrospinal fluid. Our results uncover the breadth of the adaptive immune response against a shared clonal neoantigen across multiple HLA allelotypes and support the use of class II-restricted peptide vaccines to stimulate tumor-specific T and B cells harboring receptors with therapeutic potential.

T-FINDER: A highly sensitive, pan-HLA platform for functional T cell receptor and ligand discovery.

Effective, unbiased, high-throughput methods to functionally identify both class II and class I HLA-presented T cell epitopes and their cognate T cell receptors (TCRs) are essential for and prerequisite to diagnostic and therapeutic applications, yet remain underdeveloped. Here, we present T-FINDER [T cell Functional Identification and (Neo)-antigen Discovery of Epitopes and Receptors], a system to rapidly deconvolute CD4 and CD8 TCRs and targets physiologically processed and presented by an individual's unmanipulated, complete human leukocyte antigen (HLA) haplotype. Combining a highly sensitive TCR signaling reporter with an antigen processing system to overcome previously undescribed limitations to target expression, T-FINDER both robustly identifies unknown peptide:HLA ligands from antigen libraries and rapidly screens and functionally validates the specificity of large TCR libraries against known or predicted targets. To demonstrate its capabilities, we apply the platform to multiple TCR-based applications, including diffuse midline glioma, celiac disease, and rheumatoid arthritis, providing unique biological insights and showcasing T-FINDER's potency and versatility.

Antigen Extraction and B Cell Activation Enable Identification of Rare Membrane Antigen Specific Human B Cells.

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.

Human Memory B Cells Harbor Diverse Cross-Neutralizing Antibodies against BK and JC Polyomaviruses.

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.

Synthetic Standards Combined With Error and Bias Correction Improve the Accuracy and Quantitative Resolution of Antibody Repertoire Sequencing in Human Naïve and Memory B Cells.

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.

Loss of immune tolerance to IL-2 in type 1 diabetes.

Type 1 diabetes (T1D) is characterized by a chronic, progressive autoimmune attack against pancreas-specific antigens, effecting the destruction of insulin-producing ß-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.

Cutting edge: The transcription factor Bob1 counteracts B cell activation and regulates miR-146a in B cells.

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.

A C-terminal acidic domain regulates degradation of the transcriptional coactivator Bob1.

Bob1 (Obf-1 or OCA-B) is a 34-kDa transcriptional coactivator encoded by the Pou2af1 gene that is essential for normal B-cell development and immune responses in mice. During lymphocyte activation, Bob1 protein levels dramatically increase independently of mRNA levels, suggesting that the stability of Bob1 is regulated. We used a fluorescent protein-based reporter system to analyze protein stability in response to genetic and physiological perturbations and show that, while Bob1 degradation is proteasome mediated, it does not require ubiquitination of Bob1. Furthermore, degradation of Bob1 in B cells appears to be largely independent of the E3 ubiquitin ligase Siah. We propose a novel mechanism of Bob1 turnover in B cells, whereby an acidic region in the C terminus of Bob1 regulates the activity of degron signals elsewhere in the protein. Changes that make the C terminus more acidic, including tyrosine phosphorylation-mimetic mutations, stabilize the instable murine Bob1 protein, indicating that B cells may regulate Bob1 stability and activity via signaling pathways. Finally, we show that expressing a stable Bob1 mutant in B cells suppresses cell proliferation and induces changes in surface marker expression commonly seen during B-cell differentiation.