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Pioneering Immune Therapy Research

In the Field: Benaroya Research Institute’s pioneering quest into autoimmune disease

The first commercially available Fluidigm® C1™ system was purchased in 2012 by the Benaroya Research Institute at Virginia Mason (BRI) in Seattle, known worldwide for its autoimmune disease research. To mark the event, Fluidigm employees including the R&D team that brought the technology to life signed the shipping box containing the instrument. You can still find that box with the signatures at BRI—a stamp in time representing Fluidigm and BRI's shared belief that the C1 would revolutionize translational research through single-cell analysis.

BRI is committed to eliminating not just one but the more than 80 known autoimmune diseases—including type 1 diabetes, multiple sclerosis, Crohn's disease and rheumatoid arthritis—that arise when the immune system launches an attack on itself. As a world leader in scientific innovation, BRI continues to make a significant impact in the field by helping predict disease, curbing disease progression and enhancing the safety and efficacy of available therapies. BRI's close integration of laboratory, translational and clinical research accelerates discovery and allows it to take scientific breakthroughs from the laboratory to the clinic.

That first C1 was purchased by BRI's Systems Immunology Division, now directed by Peter Linsley, PhD. Using a multidisciplinary approach including medicine, immunology, genomics and bioinformatics, the team strives to advance personalized medicine with state-of-the-art technologies that enable high-throughput profiling of the immune system for early disease detection or response to treatment. The focus on personalized medicine holds tremendous promise for improving patient outcomes and cutting the cost of care.

Linsley has used the C1 extensively to identify responders for immune therapies, exploring why patients can sometimes produce vastly different responses to the same treatment. For example, tumor necrosis factor blockers (etanercept, infliximab, etc.), a T cell co-stimulation blocker (abatacept) and a B-cell blocker (rituximab) approved for treating arthritis are effective in only a fraction of patients and can cause adverse effects in others. By understanding what causes these differences at the molecular level, Linsley hopes to move closer to personalized immunotherapies.

“Monitoring phenotypes of pathogenic T cells will enable researchers to monitor disease and optimize treatment effects for autoimmune and allergic diseases with greater precision than previously possible,” Linsley said.

As the person initially in charge of fully integrating the C1 into the genomics platform at BRI, Linsley uses the technology to understand how single-cell profiles fit into responder and non-responder observations, and to determine whether drug specificity is governed by T cell receptors. Single-cell mRNA expression analysis using the C1 workflow is ideal for this study as it helps address questions regarding T cell ancestry.

The C1 system reinvents single-cell genomics with an innovative microfluidics technology that allows scientists and clinicians to seamlessly isolate, process and profile individual cells. As the first commercially available, automated single-cell isolation and preparation system, C1 reproducibly prepares 96 individual cells with less than one hour of hands-on time.

“You bypass the headache of doing sub-purification and all the handling that goes on that is really affecting the cells before you can get any sort of readout,” explains Dr. Damien Chaussabel, former director of BRI’s Systems Immunology Division. Using this complete workflow, researchers can capture and verify single cells, lyse cells, reverse transcribe and preamplify targets and harvest cells for quantitative PCR or library preparation.

Single-cell gene expression analysis is critical to understanding cell-fate specification during an immune response at the molecular level. The standard protocols of fluorescence-activated cell sorting (FACS) and microdissection for isolating single cells are labor-intensive, require a high level of skill and cannot ensure a viable individual cell for downstream analysis.

Linsley's interest in immunology dates to the roughly 30 years he spent in the biotechnology industry. While working at the Seattle startup Oncogen (later acquired by Bristol-Myers Squibb), he investigated the communication networks between T cells and other cells, and co-discovered the CD28/CTLA-4/B7 co-stimulation axis and the immunosuppressive drugs abatacept and belatacept (Orencia® and Nulojix®). Eventually he shifted gears from drug discovery to helping clinicians use current immunotherapies more effectively. The ultimate goal of his drug responder studies at BRI is to allow clinicians to match each patient with the most effective treatment.

“We know that pathogenic effects of T cells are determined by their specificities—antigen receptor sequences, or clonotypes—and by their functional capacity, or phenotypes,” Linsley explains. “However, even among T cells recognizing identical antigens, there may exist a variety of clonotypes and phenotypes, not all of which are equally pathogenic. As a consequence, relationships between the ancestry and phenotypic properties of individual pathogenic T cells are difficult to discern in mixed populations. Single-cell analysis on the C1 enables us to elucidate how T cells recognizing autoantigens expand and contract during disease flares and during therapy.”

Currently, the analysis begins with single-cell mRNA sequencing, which is then downsampled to a targeted gene expression panel using the C1, Biomark™ HD and Dynamic Array™ IFC from Fluidigm. That panel is then used to predict which genes are overexpressed.

Although proteomics is not a routine component of this regimen, BRI plans to include functional assays for single-cell protein analysis in the future using instrumentation such as the CyTOF® 2 mass cytometer.

Innovations in single-cell genomics and proteomics technologies will continue to advance immunology research by allowing scientists to ask broader scientific questions. Through its ongoing relationship with BRI, Fluidigm looks forward to the use of single-cell tools in clinical research applications so rare cells from hard-to-reach tissues can be analyzed in a noninvasive manner.

As one of the handwritten notes on that shipping box reads, “C you on the other side.”