NIAID investigators have developed a microscopy technique capable of identifying, locating, and quantifying many distinct immune cells in whole tissues. This technology could enable researchers to better understand immune cell behavior at rest and in response to a stimulus such as infection, vaccination, or cancer progression. The method appears in a study available online in the August 2nd issue of Immunity.
The immune system is composed of hundreds of different types of immune cells. Scientists traditionally characterize these cells by labeling them with various antibodies tagged with fluorescent dyes. Antibody-labeled cells are then analyzed using a special technique called flow cytometry. Although this approach has helped researchers better understand the diversity of cells participating in immune responses, it only allows scientists to examine single cells extracted from a tissue. Flow cytometry does not permit scientists to learn about the location of the cells in tissues or to understand how different cells interact with one another or their environment.
Confocal microscopy is a technique that allows scientists to view cells in whole tissues. Until now, this approach has been limiting because it was difficult to interpret the data if scientists tried to use more than a few cell-labeling dyes at one time. This restriction has prevented scientists from using the same combinations of labeled antibodies to identify subsets of cells in tissue sections that were used in flow cytometry. One method for accomplishing useful tissue staining with many antibodies was previously reported, but it is very time-consuming and requires specialized instrumentation that is not widely available.
The technique honed by the NIAID team, called histo-cytometry, allows researchers to use as many as eight dyes (currently) to find specific cells in a whole tissue using a combination of confocal microscopy and specialized image analysis. This method is performed with widely available equipment and software, and thus can be employed easily by other research centers.
The histo-cytometry method was developed by Michael Gerner, Ph.D., a postdoctoral fellow in the NIAID Laboratory of Systems Biology, headed by Ronald Germain, M.D., Ph.D. To test the technique, the research team examined many types of immune cells in the lymph nodes of a mouse.
They first identified the major immune cell populations in the lymph nodes (lymphocytes) and then visualized immune activation of one type of lymphocyte, the T cell, in response to vaccination. Next, the team used histo-cytometry to locate specific subsets of immune cells called dendritic cells that also are found in the lymph nodes. They discovered that certain subsets of dendritic cells seem to preferentially reside in specific areas of the lymph nodes, as opposed to being randomly distributed in the tissue. Because subsets of dendritic cells have different roles during an immune response, the team’s findings provide striking evidence suggesting that subregions of the lymph nodes may be specialized to induce particular types of immunity.
Histo-cytometry is an improvement on current microscopy techniques that allows scientists to identify and examine multiple immune cell types in their natural environment. This method could eventually help improve the understanding of how such cells respond to a vaccine, thereby helping manufacturers increase its effectiveness. Histo-cytometry also could be used to examine the immune cells within a tumor biopsy. By understanding which immune cells associate with a tumor, doctors could make better decisions about using therapeutics that inhibit or stimulate specific cell types to fight the cancer.
Because the method builds upon software and microscopy equipment found at research institutions outside NIAID, the team hopes the histo-cytometry technique can be readily adopted by other labs interested in cell localization and quantitation in whole tissues. They have already planned partnerships with other NIH investigators and view this publication as the first step in sharing the method with the greater scientific community.
Gerner MY, Kastenmuller W, Ifrim I, Kabat J, Germain RN. Histo-cytometry: a method for highly multiplex quantitative tissue imaging analysis applied to the dendritic cell subset micro-anatomy in lymph nodes. Immunity. Aug. 2, 2012 (online ahead of print).
Dr. Germain’s Lab
Last Updated August 02, 2012