Due to the low frequency of antigen-specific B-cells in the normal repertoire, we have developed methods to enrich for these rare cells among all the other cells in blood and tissues to enable phenotypic and functional studies using high dimensional spectral flow cytometry, mass cytometry (CyTOF), and single cell RNA sequencing (10X Genomics).
Current Projects:
Determine differences in the gene expression and clonal repertoire of islet-reactive (e.g. insulin-reactive) B cells in healthy and autoimmune prone individuals
Determine better phenotypic markers for anergic autoreactive B cells
Elucidate the B cell endotype in young onset, rapid progressing T1D patients compared to older onset, slow progressing T1D patients
Investigate the mechanism by which certain T1D risk alleles, e.g. PTPN2, affect maintenance of B cell anergy
Techniques
Breakthroughs in the ability to analyze and visualize high dimensional datasets have provided immunologists the ability to decipher the phenotype and actions of immune cells with a high level of detail. Below are some of the techniques and technologies we employ most frequently to achieve our research goals.
spectral Flow Cytometry
Spectral flow cytometry uses spectral data to separate cell populations based on size, complexity, and fluorescent antibody markers. Using markers specific to our cells of interest, we are able to identify specific B cell populations and phenotypically characterize them, including their activation and functional status.
Mass Cytometry
Mass cytometry (CyTOF) is similar to flow cytometry but uses antibodies labeled to rare earth metals instead of fluorochromes. Mass cytometry can yield higher dimensionality than flow cytometry with minimal need for compensation, allowing us to discover new, more complex, cell subtypes.
cite-seq (10X-genomics)
Since its conception DNA sequencing has become a cornerstone of biological research. Cellular Indexing of Transcriptomes and Epitopes (CITE-Seq) allows one to combine transcriptional and protein data to better understand your cells of interest. In addition, we have recently generated antigen-oligo probes that bind specifically to the BCR to allow identification of rare antigen-specific B cells. This enables multiplexing of gene expression, V(D)J sequencing, CITE-Seq, and antigen-specificity to better understand how autoreactive B cells contribute to autoimmunity.
