Project description:Recently, a mass spectrometry-based approach was introduced to directly assess the IgG1 immunoglobulin clonal repertoires in plasma. Here we expanded upon this approach by describing a mass spectrometry-based technique to assess specifically the clonal repertoire of another important class of immunoglobulin molecules, IgA1, and show it is efficiently and robustly applicable to either milk or plasma samples. Focusing on two individual healthy donors, whose milk was sampled longitudinally during the first 16 weeks of lactation, we demonstrate that the total repertoire of milk sIgA1 is dominated by only 50-500 clones, even though the human body theoretically can generate several orders of magnitude more clones. We show that in each donor the sIgA1 repertoire only changes marginally and quite gradually over the monitored 16-week period of lactation. Furthermore, the observed overlap in clonal repertoires between the two individual donors is close to non-existent. Mothers provide protection to their newborn infants directly by the transfer of antibodies via breastfeeding. The approach introduced here, can be used to visualize the clonal repertoire transferred from mother to infant and to detect changes in-time in that repertoire adapting to changes in maternal physiology.
Project description:To study longitudinal dynamics of IGH BCR repertoires and clonal lineages evolution of memory B-cells, plasmablasts and plasma cells from peripheral blood of healthy donors, which were sampled three times within a year
Project description:Developing B lymphocytes undergo V(D)J recombination to assemble germline V, D, and J gene segments into exons that encode the antigen-binding variable region of immunoglobulin (Ig) heavy (H) and light (L) chains. IgH and IgL chains associate to form the B cell receptor (BCR), which upon antigen binding activates B cells to secrete BCR as an antibody. Each of the huge number of clonally independent B cells expresses a unique set of IgH and IgL variable regions. Ability of V(D)J recombination to generate vast primary B cell repertoires results from combinatorial assortment of large numbers of different V, D, and J segments, coupled with diversification of the junctions between them to generate the complementary determining region 3 (CDR3) for antigen contact. Approaches to evaluate in depth the content of primary antibody repertoires and, ultimately, to study how they are further molded by secondary mutation and affinity maturation processes are of great importance to the B cell development, vaccine, and antibody fields. We now describe an unbiased, sensitive, and readily accessible assay, referred to as HTGTS repertoire sequencing (HTGTS-Rep-seq), to quantify antibody repertoires. HTGTS-Rep-seq quantitatively identifies the vast majority of IgH and IgL V(D)J exons, including their unique CDR3 sequences, from progenitor and mature mouse B lineage cells via the use of specific J primers. HTGTS-Rep-seq also accurately quantifies DJH intermediates and V(D)J exons in either productive or non-productive configurations. HTGTS-Rep-seq should be useful for studies of human samples, including clonal B-cell expansions and also for following antibody affinity maturation processes. We employed high-throughput genome-wide translocation sequencing adapted repertoire sequencing (HTGTS-Rep-seq) to study antibody repertoires. For HTGTS-Rep-seq libraries, we utilize bait coding ends of J segments to identify, in unbiased fashion, mouse IgH DJH repertoires [processed tlx files] along with both productive and non-productive IgH V(D)J repertoires from both pro-B and peripheral B cells [processed xls files of samples 1-18, 21-51]. Similarly, we also identify mouse productive and non-productive Igk repertoires from peripheral B cells [processed xls files of samples 19,20,52-57].
Project description:Human antibodies are heterogeneous molecules, primarily due to clonal sequence variations. Analytical techniques to assess antibody levels quantitatively, like ELISA assays, lack the power to determine abundances at the clonal level. Recently, we introduced an LC-MS-based approach that can distinguish and quantify antibody clones using the mass and retention time of their corresponding Fab-fragments. We used a specific hinge-cleaving protease IgdE (FabALACTICA) to release the Fab-fragments from the glycosylated constant Fc region of the antibody. Here, we explore an alternative protease, the recently described IgG1-specific hinge-cleaving protease BdpK (FabDELLO) and compare it directly to IgdE for its applicability in IgG1 repertoire profiling. We used IgdE and BdpK in parallel to digest the IgG1s from the same set of plasma samples. Both proteases cleave IgG1 in the hinge region, albeit at two distinct cleavage sites. The LC-MS based analyses of the Fab-fragments generated by IgdE or BdpK produced qualitatively and quantitatively very similar clonal repertoires. However, IgdE required ~16 hours to digest the polyclonal plasma IgG1s, while BdpK required just ~2 hours. We validated the similarity of the clones by top-down proteomics, using Electron Transfer Dissociation (ETD). We conclude that BdpK performs well in digesting polyclonal plasma IgG1 samples, and that neither BdpK nor IgdE displays detectable biases in cleaving IgG1s. We anticipate that BdpK may emerge as the preferred protease for IgG1 hinge- digestion because it offers a shorter digestion time compared to IgdE, an equally specific digestion site, and no bias against any IgG1 present in the plasma repertoires.
Project description:The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the MHC (pMHC) on cells is an essential parameter for efficient T cell-mediated immunity. Yet, whether the TCR-ligand avidity can drive the clonal evolution of virus antigen-specific CD8 T cells, and how this process is determined in latent Cytomegalovirus (CMV)- against Epstein-Barr virus (EBV)-mediated infection remains largely unknown. Here, we quantified monomeric TCR-pMHC dissociation rates on CMV- and EBV-specific individual TCR-alpha-beta clonotypes and polyclonal CD8 T cell populations in healthy donors over a follow-up time of 15-18 years. Within CMV/pp65-specific T cell repertoires, a progressive contraction of clonotypes with high TCR-pMHC avidity and low CD8 binding dependency was observed, leading to an overall avidity decline during long-term antigen exposure. We identified a unique transcriptional signature preferentially expressed by high-avidity CMV/pp65-specific T cell clonotypes, including the inhibitory receptor LILRB1. Interestingly, T cell clonotypes of high-avidity showed higher LILRB1 expression than the low-avidity ones and LILRB1 blockade moderately increased T cell proliferation. Similar findings were made for CD8 T cell repertoires specific for the CMV/IE-1 epitope. There was a gradual in vivo loss of high-avidity T cells with time for both CMV specificities, corresponding to virus-specific CD8 T cells expressing enhanced LILRB1 levels. In sharp contrast, the EBV/BMFL1-specific T cell clonal composition and distribution, once established, displayed an exceptional stability, unrelated to TCR-pMHC binding avidity or LILRB1 expression. Together, these findings reveal an overall long-term avidity decline of CMV- but not EBV-specific T cell clonal repertoires, highlighting the differing role played by TCR-ligand avidity over the course of these two latent herpesvirus infections. Our data suggest that the inhibitor receptor LILRB1 potentially restricts the clonal expansion of high-avidity CMV-specific T cell clonotypes during latent infection. We propose that the mechanisms regulating the long-term outcome of CMV- and EBV-specific memory CD8 T cell clonotypes in humans are distinct.
Project description:Regulatory T (Treg) are present in lymphoid and non-lymphoid tissues and restrict immune activation, prevent autoimmunity and regulate inflammation. While Treg in non-lymphoid tissues are typically resident, Treg in lymph nodes (LNs) predominantly recirculate and residency of Treg in LNs is poorly studied. Here, we show that 10-20% of all Treg in LNs are memory-like resident cells. Both thymic and peripheral Treg contributed to the pool of resident Treg and their residency did not require continuous TCR signaling. Yet, resident and circulating Treg had distinct TCR repertoires and different LNs contained distinct clonal subpopulations of resident Treg. We speculate that stochastic selection and variation in antigen availability governs commitment of individual T cell clones to the resident pool in each LN. Our results describe a framework for local fine-tuning of immune responses in LNs based on previous adaptive immune responses.
Project description:This SuperSeries is composed of the following subset Series: GSE32348: Methylation specifies distinct estrogen-induced binding site repertoires of CBP to chromatin (mRNA) GSE32349: Methylation specifies distinct estrogen-induced binding site repertoires of CBP to chromatin (ChIP-Seq) Refer to individual Series