High-Throughput Detection of Autoantigen-Specific B Cells Among Distinct Functional Subsets in Autoimmune Donors

Abstract

Antigen-specific B cells (ASBCs) can drive autoimmune disease by presenting autoantigen to cognate T cells to drive their activation, proliferation, and effector cell differentiation and/or by differentiating into autoantibody-secreting cells. Autoantibodies are frequently used to predict risk and diagnose several autoimmune diseases. ASBCs can drive type 1 diabetes even when immune tolerance mechanisms block their differentiation into antibody-secreting cells. Furthermore, anti-histidyl tRNA synthetase syndrome patients have expanded IgM+ Jo-1-binding B cells, which clinically diagnostic IgG Jo-1 autoantibodies may not fully reflect. Given the potential disconnect between the pathologic function of ASBCs and autoantibody secretion, direct study of ASBCs is a necessary step towards developing better therapies for autoimmune diseases, which often have no available cure. We therefore developed a high-throughput screening pipeline to 1) phenotypically identify specific B cell subsets, 2) expand them in vitro, 3) drive them to secrete BCRs as antibody, and 4) identify wells enriched for ASBCs through ELISA detection of antibody. We tested the capacity of several B cell subset(s) to differentiate into antibody-secreting cells following this robust stimulation. IgM+ and/or IgD+, CD27- memory, memory, switched memory, and BND B cells secreted B cell receptor (BCR) as antibody following in vitro stimulation, whereas few plasmablasts responded. Bimodal responses were observed across autoimmune donors for IgM+ CD21lo and IgM- CD21lo B cells, consistent with documented heterogeneity within the CD21lo subset. Using this approach, we detected insulin-binding B cell bias towards CD27- memory and CD27+ memory subsets in pre-symptomatic type 1 diabetes donors. We took advantage of routine detection of Jo-1-binding B cells in Jo-1+ anti-histidyl tRNA synthetase syndrome patients to show that Jo-1-binding B cells and total B cells expanded 20-30-fold using this culture system. Overall, these studies highlight technology that is amenable to small numbers of cryopreserved peripheral blood mononuclear cells that enables interrogation of phenotypic and repertoire attributes of ASBCs derived from autoimmune patients.

Mass cytometry analysis reveals attrition of naïve and anergized self-reactive non-malignant B cells in chronic lymphocytic leukemia patients

“…B ND cells are IgM lo/- naïve B cells that express an autoreactive BCR and have been functionally censored after recognition of self Ag during early development ( 25 , 26 ). It is also documented that the B ND pool serves as a reservoir for autoreactive B cells in several autoimmune diseases ( 35 ). At face value, it may seem counterintuitive to associate decreased abundance of B ND cells with an increased risk of autoreactivity.…”

TCR-like antibodies targeting autoantigen-mhc complexes: a mini-review

“…Increased frequency of circulating DCs was implicated in regulation of antigen presentation by islet cells and activation of autoreactive CD4+ T cells ( 80 , 81 ). Recently, a novel approach was developed using PBMC to identify autoantigen-specific memory B cells, which are potent MHC-II+ APC ( 82 ). Therapeutic strategies focusing broadly on APC function have been developed, such as B cell depletion in SLE ( 83 ) and tolerogenic DC adoptive therapy ( 84 ).…”

The Application of High‐Throughput Approaches in Identifying Novel Therapeutic Targets and Agents to Treat Diabetes

“…PPARs function as transcription factors that regulate the expression of genes involved in glucose and lipid metabolism, and thiazolidinediones (TZDs), such as pioglitazone and rosiglitazone, bind to PPARγ in adipose tissue to enhance insulin sensitivity, leading to reduced circulating FFA, increased lipid storage capacity of adipocytes and enhanced adiponectin secretion. [210] However, TZDs are associated with side effects, such as weight gain, fluid retention and congestive heart failure, which has largely limited widespread use of these Restore functional β-cell mass [139] The number of β-cells a) Restore functional β-cell mass [140,141] Urocortin 3 (UCN3) a) Reduce immune or metabolic stressesinduced β-cell dedifferentiation; restore functional β-cell mass [144] Antigen-specific B cells Discover novel T1D-associated autoantigens [171] Variable number tandem repeat alleles…”

“…This goal might be achieved by HTS campaigns that identify antigen‐specific B cells (ASBCs) from peripheral blood samples. ASBCs play important roles in autoantigen presentation and T1DM initiation, [ 171 ] and it is now possible to isolate these specific B cell subsets from peripheral blood mononuclear cells of patients with T1DM. After expanding these B cells in vitro and stimulating them to secrete B‐cell receptors as antibodies, ELISA‐bases HTS can then be used to determine the specificity of these ASBCs, which may lead to the discovery of potential autoantigens.…”

“…After expanding these B cells in vitro and stimulating them to secrete B‐cell receptors as antibodies, ELISA‐bases HTS can then be used to determine the specificity of these ASBCs, which may lead to the discovery of potential autoantigens. [ 171 ] Also, phage display libraries have also been used in T1DM to find autoantigens. By displaying random fragments of islet cell antigens on the surface of phages, the aforementioned affinity‐based assays can identify autoantigenic epitopes that could be recognized by autoantibodies.…”