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Jonathan Kagan, Ph.D.

Asst. Professor  |  Harvard Medical School and Division of Gastroenterology, Children’s Hospital Boston

Understanding Cellular Immune Response

While great progress has been made in the last decade to identify the receptors that recognize bacteria and viruses and lead to the initiation of an immune response, a fundamental challenge remains in understanding how these receptors operate. Dr. Jonathan Kagan’s lab at the Harvard Medical School uses a cell biology approach to try to elucidate precisely where in the cell immune signal pathways are activated that lead to protection against infection.

Dr. Kagan’s team recently turned their attention to a family of proteins called RIG-I-like receptors (RLRs), the only receptors that exist in every cell in the body and function to prevent infection. “RLRs represent a wonderful therapeutic target since you can effectively assume that any viral infection could be treated by manipulating the RLR signaling pathway,” said Dr. Kagan.

With this in mind, the researchers were very interested in understanding where in the cell the signal transduction occurs, and hypothesized that “perhaps mitochondria were not the only game in town” in antiviral immunity. For a variety of reasons they decided to focus on peroxisomes - one of the first organelles discovered but only previously known to function in metabolism. “No one had discovered a role for them in immune defense,” said Dr. Kagan.

Through their initial work, Dr. Kagan’s team discovered that peroxisomes contained an essential RLR signaling protein involved in antiviral immunity called MAVS, which was previously only found on mitochondria. This discovery led them to ask: what is it doing there? Is it functioning or acting to induce antiviral gene expression?

“Using the nCounter Analysis System we generated our most informative data because we were able to look at not just a single gene or a couple of genes but the entire repertoire of all the immune signaling pathways,” explained Dr. Kagan. “Effectively we created a diagnostic CodeSet that would report on every known, well-characterized immune signaling pathway.”

Dr. Kagan said the ‘immuno array’ CodeSet they created was much more efficient and sensitive than using a microarray for this experiment. “We had the array data, and simply took the same samples and plugged them into the nCounter and got exactly the same genes induced, but with a much more reliable degree of sensitivity. We also found that the induction was greater and the quantitation was better.”

Because the data produced by the nCounter system is so straightforward they did not need a bioinformaticist, unlike when they used microarrays, Dr. Kagan noted.

“It has been very helpful to have this reliable and robust assay that we can use in a simple plug-and-play workflow,” said Dr. Kagan. “The ability to use the nCounter CodeSets instead of arrays allowed us to do all of our analyses in a couple of weeks, with arrays it would have taken a month to get our data.”

In May, the team published their discoveries in a paper titled “Peroxisomes Are Signaling Platforms for Antiviral Innate Immunity” in the journal Cell.

“Since adopting the nCounter system, my lab has effectively moved entirely away from using microarrays and we exclusively use nCounter CodeSets as our preferred method to interrogate the activity of all the innate immune and inflammatory signaling pathways that we are focused on,” said Dr. Kagan.

Dr. Kagan sees additional application of the nCounter technology for studies that take advantage of the system’s ability to look at mRNA from both the host and pathogen in a single tube.

“One of the great things about the nCounter is the ability to look at mRNAs from several species of organisms at the same time,” added Dr. Kagan. “The ability to lyse your cells and in the same experiment look at both opponents in this battle, the virus and the host, is unique.”

As a next step, Dr. Kagan is working with NanoString to design CodeSets that enable the gene expression analysis of host and viral genes to determine under which conditions the virus is able to replicate at will and under which conditions signal transduction is blocked.

“Using the nCounter system, we can really begin to glean valuable information about at what step exactly the virus’ lifecycle is being blocked, and we can also simultaneously look at which genes the cell is inducing in response to infection. This is a novel approach that is not possible with other types of gene expression technology.”