April 5-10, 2024 at the San Diego Convention Center in San Diego, California
Stop by and say hello at Booth 1110!
Exhibit Hall Hours
Sunday, April 7, 1:00 - 5:00 PM
Monday, April 8, 9:00 AM - 5:00 PM
Tuesday, April 9, 9:00 AM - 5:00 PM
Wednesday, April 10, 9:00 AM - 12:30 PM
I. Exhibitor Spotlight Theatre
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Tuesday, April 9, 2024 - 3:00 - 4:00 PM Exhibitor Spotlight Theatre B (Refreshments will be provided.)
Applications of TCR & BCR Repertoire Profiling:
Insights into the Adaptive Immune Response in T-cell Lymphomas and more
Introductory Remarks: Paul Diehl, PhD, COO, Cellecta
Speaker 1: Alex Chenchik, PhD, President, Cellecta
Talk: TCR & BCR Repertoire Analysis and Other Approaches for the Discovery of Drug Targets, Resistance Mechanisms and Biomarkers
T-cell receptor (TCR) and B-cell receptor (BCR) repertoire profiling holds great potential for understanding disease mechanisms. We introduce a novel technology for profiling of all human TCR and BCR variable regions together with phenotypic characterization of immune cells in bulk in PBMCs and immune cell fractions. Preliminary data shows that TCR/BCR clonotype analysis combined with targeted expression profiling of immune cells can be applied for large-scale discovery in several immune-responsive model systems.
Speaker 2: Utpal Davé, MD, Associate Professor of Medicine, Associate Professor of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN.
Talk: Dissecting the role of T-cell receptors in T-cell large granular lymphocytic leukemias
The etiology of T-LGL (T-cell large granular lymphocytic leukemias) and its accompanying autoimmune disease manifestations are mostly unknown. With the help of Cellecta’s sequencing technologies, we investigated the most abundant TCR clonotypes in a T-LGLL patient with severe neutropenia. Further analysis of the CDR3 region (VDJdb server) identified putative antigens recognized by the expanded clonotypes, allowing us to devise a model for the onset and development of T-LGL.
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II. Poster Presentations
Sunday, April 7, 1:30 - 5:00 PM
The DriverMap™ Adaptive Immune Receptor (AIR) repertoire profiling assay employs multiplex RT-PCR for comprehensive profiling of CDR3 or full-length receptor profiling of all variable heavy and light chains of T-cell receptors (TCR) and B-cell receptors (BCR) from either RNA or DNA. In this study, initiated by a U.S. Food and Drug Administration (FDA) consortium to compare performance of different BCR repertoire profiling technologies, we tested spike-ins in peripheral blood mononuclear cell (PBMC) RNA and DNA control mixtures prepared from nine different B-cell lines. We measured the sensitivity, linearity and accuracy of AIR RNA and DNA BCR profiling for the IGH, IGK and IGL chains of the spike-in controls. Our comprehensive analysis, encompassing CDR3 profiling and full-length receptor profiling (CDR1, CDR2, and CDR3) sequencing, revealed a 100X increase in sensitivity of detection of spike-in controls in RNA compared to the DNA assay. The high sensitivity of the full-length DriverMap AIR RNA assay allowed the detection of controls from all nine cell lines, with 8 out of 9 cell lines detected in the DNA assay, which demonstrates the robustness of the DriverMap AIR profiling technology. This study highlights the remarkable sensitivity of the AIR RNA assay in quantifying transcripts. This approach enables the detection of low-frequency BCR clonotypes, which is particularly advantageous when working with samples containing low numbers of B cells. Furthermore, our AIR DNA assay in combination with spike-in controls offers a quantitative tool for minimal residual disease (MRD) applications, providing accurate insight into cell numbers, which then facilitates tracking clonal expansion of immune cells.
Adaptive immune receptor (AIR) repertoire diversity assays are susceptible to biases arising from variations in conditions in the RT-PCR and next-generation sequencing (NGS) steps. We designed synthetic TCR and BCR spike-in controls to mitigate these biases and to serve as universal standards for any PCR-based immune receptor profiling assay. We synthesized 48 BCR constructs representing different IGH, IGK, and IGL genes, and 39 TCR constructs for TRB, TRA, TRG, and TRD genes. The spike-in controls were tested as (16x3) BCR constructs and (13x3) TCR Triplex isoform pools, added to multiple samples in the same batch to detect cross-contamination across samples. We successfully discriminated between controls and background sequences by combining a unique molecular identifier (UMI)-based correction strategy with spike-in controls at the data analysis step. We also tested 48 BCR and 39 TCR Premixed Controls by spiking into peripheral blood mononuclear cells (PBMC) RNA samples before reverse transcription using Cellecta’s DriverMap™ Adaptive Immune Receptor Profiling Assay that uses a multiplex RT-PCR approach with gene-specific primers and UMIs. We successfully used it to evaluate assay performance by adding premixed controls at different concentrations. Results showed a linear trend as the number of spike-in molecules increased. Our analysis revealed an average sequencing error rate of 0.4%-0.8% per base, aligning with the reported error rate range of Illumina sequencing. This suggests the reliability of our spike-in controls, which can be used to rectify biases in the AIR protocol and accurately estimate error and mutation rates for the DriverMap AIR assay or any other sequencing-based immune receptor profiling assay. This innovative approach enhances the robustness of immune receptor profiling technology, facilitating more accurate assessments of repertoire diversity.
Monday, April 8, 1:30 - 5:00 PM
This study presents a novel approach for performing 10X platform-based targeted single-cell RNA-seq analysis in combination with CRISPR perturbation screens. For the analysis, we constructed a custom sgRNA library focused on a small subset of genes associated with the TNFα response (i.e., NFκB pathway). We evaluated the specific trade offs with regard to the size of the CRISPR library, the size of the panel of genes targeted for expression analysis, and NGS sequencing depth. This flexible, targeted approach not only refines the precision of RNA-seq but also provides a more cost-effective solution for comprehensive genomic analyses.
This study explores the utilization of orthogonal CRISPR-based gene editing/modulation systems for combinatorial genetic screens using CRISPRknockout (CRISPR-KO), CRISPR activation (CRISPRa), and CRISPR interference (CRISPRi) functionalities. For this purpose, a complete set of tools for CRISPR/saCas9 gene editing and gene modulation was developed, compatible with CRISPR/spCas9 co-expression. Specifically, we developed and validated optimized saCas9 and sg(sa)RNA lentiviral and AAV vectors, dual expression (sp)/(sa) sgRNA lentiviral library vectors, as well as (sa)CRISPR-KO, (sa)CRISPRi, (sa)CRISPRa fluorescence-based activity kits for the functional validation of saCas9expressing cell lines. Results will be presented, with different combinations of CRISPR-KO, CRISPRa, and CRISPRi systems in multiple cell lines.
Tuesday, April 9, 9:00 AM - 12:30 PM
Single-cell immune receptor profiling is a revolutionary approach that allows investigators to combine clonotype repertoire identification with paired-chain information and the phenotype of cells (e.g., cell subtype). Single-cell immune receptor profiling can be performed using a medium-throughput approach (1,000-5,000 cells) using microwell arrays or droplet microfluidics technologies. However, these assays are more complicated to run and require expensive reagents and limited sequencing throughput when compared to bulk immune receptor profiling methods. Here, we describe a low-throughput, single-cell immune profiling strategy using sorted cells in 96-well plates. The plate is pre-aliquoted with either T-cell receptor (TCR) 𝝰/𝛃 or TCR γ/δ primers along with 30 crucial T-cell markers. We perform multiplex RT-PCR amplification and sequencing of the CDR3 regions. The resulting data provides the abundant clonotype counts along with the chain pairing information for these 𝝰/𝛃 and γ/δ chains, together with the T-cell subtype information using gene-expression profiles. By analyzing the TCR gene rearrangement at the single-cell level, researchers can better understand T-cell development, proliferation, and clonality, which are crucial for studying diseases such as cancer, immunodeficiency, and autoimmunity. Furthermore, single-cell TCR sequencing when combined with RNA sequencing datasets, facilitates the identification of γδ T cells. This method provides a standardized tool for identifying potential γδ T cell-based cancer immunotherapies. The technology's cost-effectiveness and ability to analyze clonotypes and immunophenotypes of cells in a single assay make it a valuable tool for unraveling the immune dynamics in various diseases.
Tuesday, April 9, 1:00 - 5:00 PM
Microsampling lancet-induced blood drops enables frequent and comprehensive analysis of various metabolites, lipids, cytokines, and proteins. This approach holds promise for monitoring immunotherapy patients using RNA biomarkers, but a suitable method for processing RNA has been lacking. In this study, we employed a targeted RNA-sequencing protocol, the DriverMap™ EXP assay, to process 30 ul of dried blood. We compared the gene expression profile in traditionally collected blood samples with that of blood absorbed onto a Mitra® microsampling device containing an RNA-stabilization reagent. Following endotoxin incubation, RNA was extracted from stimulated and unstimulated blood samples. Targeted PCR amplification of 274 immune/inflammatory genes using the DriverMap targeted RNA-Seq protocol demonstrated robust detection and high correlation (r = 0.94) between the two methods in both unstimulated and endotoxin-stimulated blood. Moreover, differentially expressed genes (DEGs) identified in standard and microsampling methods exhibited substantial overlap with publicly available datasets from similar experiments. Furthermore, we compared whole blood extracted from Tempus™ blood RNA tubes to Mitra microsamples pre- and post-immunization with the Pneumovax® vaccine using the DriverMap EXP genome-wide 19K panel for targeted RNA-sequencing. We observed approximately 90% overlap in the top 10K genes between Tempus and Mitra microsamples. Notably, microsamples stored at 4ºC for over a year exhibited similar expression profiles to more recently drawn whole blood samples.
