When your immune system turns on you, it attacks healthy cells. These autoimmune diseases—roughly 80 of them, from Addison disease to rheumatoid arthritis—can be deadly and different from one patient to the next. “The clinical course of systemic autoimmune diseases (SADs) varies greatly, even between individuals with the same disease,” wrote immunologist Yves Renaudineau of the Université de Brest in France. “Understanding of the immune actors is informative and could lead to significant improvements in diagnosis, monitoring, initial treatment decisions, and/or follow-up.” To study the features of these diseases—and much of biology in general—scientists can turn to immunophenotyping.
To study the features of diseases—and much of biology in general—scientists can turn to immunophenotyping.
This technology identifies cells based on surface molecules. Antibodies bind to antigens on those molecules, and adding markers to the antibodies allows cells to be identified and sorted. So, using immunological features—the antigens—this technology categorizes cells based on their surface molecules—the phenotype. Those cell phenotypes can be used to study, diagnose, and improve the treatment of a range of diseases, including leukemia and HIV/AIDS.
As Bob Balderas, vice president biological sciences at BD, explains, “moving ahead in immunophenotyping depends on three things.” First, cellular surface molecules must be identified. Second, scientists require markers, such as fluorochromes, that attach to antibodies that bind the antigens on the receptors. Third, instruments analyze and manipulate the cells. It takes all three to advance immunophenotyping.
Defining diseases
Clinical scientists use immunophenotyping to study various diseases. “The most well-known use of immunophenotyping in infectious disease research, diagnosis, and response to therapy is quantitation of CD4+ T cells in patients with HIV/AIDS,” says Joy Bickle, product manager, flow cytometry, life science solutions at Thermo Fisher Scientific. “However, immunophenotyping is also being used to study immune responses to other diseases, such as cytomegalovirus, Epstein-Barr virus, and Mycobacterium tuberculosis.”
The range of diseases that can be studied with immunophenotyping is very broad, as are the potential approaches to treatment. As Bickle points out, for example, “Experimental models are being used to screen vaccine candidates for T-cell responses by combining intracellular cytokine or transcription factor staining with surface phenotyping to detect functional populations and the quality of the immune response.”
So, these techniques help clinical scientists delve deeply into the mechanisms of disease, diagnosis, and treatments. For instance, Bickle says, “Standardized panels of target markers can provide consistency in cross-site studies for disease diagnosis.”
Cancer makes up a particularly heterogeneous collection of diseases. Moreover, the same kind of cancer varies between people and even over time in one person. One of the most promising treatments for cancer is immunotherapy, which engineers a person’s immune cells to fight the cancer. “Cancer immunotherapy presents a new realm of clinical immunophenotyping needs, which will require new markers, improved reagents, and expanded panels in order to detect rare events and gain more information from a limited sample,” Bickle explains.
Pushing for purity
To study the cellular basis of disease more carefully, scientists need ways to identify and collect the kinds of cells that make up the disease. Similar techniques can also reveal how the cells change with a specific treatment. This can all be done with flow cytometry (FC) and cell markers.
To identify cells via surface molecules, scientists use FC analysis. “This gives you a representation of the heterogeneity of cells in, say, blood or a tumor,” says Balderas.
The various subsets of cells in a sample can also be sorted with FC. “This gives us an opportunity to isolate the cells we want based on a phenotype,” notes Balderas. The antibodies define a cellular phenotype, and then cells can be sorted based on that. As Marcello Stein, global product manager T cell research at Miltenyi Biotec, says, “When applying flow cytometry methods, the key benefits of immunophenotyping are the fast, convenient, and reliable determination of immune cell subsets.”
The more surface molecules that can be marked, the more finely a sample can be divided, focusing on more and more specific versions of cells. With two markers, for example, cells could be divided into four groups: ones that bind both antibodies, bind just one antibody, bind just the other antibody, or bind no antibodies. Some of today’s FC platforms can handle as many as 40 different markers, and that creates about one billion possibilities.
Biocompare’s Flow Cytometry Antibodies Search Tool
Find, compare and review flow cytometry
antibodies from different suppliers Search
The increasing level of selectivity leads to ever more homogenous samples of cells. The immunophenotyping reveals information about the surface of the cells. Then, the sorted samples can be analyzed with next-generation sequencing (NGS) to tell scientists about the genetic differences in the cells.
Endless opportunities
Improvements in immunophenotyping come from many different areas. For one thing, Stein mentions the use of recombinantly engineered antibodies. This allows the creation of limitless forms of antibodies for binding surface molecules.
Beyond making more antibodies, I wondered how many colors could be used at once. When I ask Balderas, he says, “That’s a good question!” Then he adds, “We have not seen where a top level of antibodies and colors on them max out to where you can’t do more.” Only more time and ongoing advances in technology will tell scientists where the limit lies. Looking ahead, the number of markers that can be used promises to keep rising.
With immunophenotyping, the amount of markers is not the only number that matters. It is also possible to use immunophenotyping to study single cells. As Irene Roberts, professor of pediatric hematology at the UK’s University of Oxford, says, “Knowing what the cells are by index sorting clearly provides the reassurance that the gene expression being measured in that cell does at least derive from the cell type you are trying to study.” That way, scientists can truly analyze and attack diseases cell by cell.
So, from autoimmune diseases to cancer and more, new knowledge and technology make immunophenotyping faster and increasingly able to subdivide samples into more homogeneous groups.
Image: Shutterstock Images