Ahuman cell map is vital information for researchers designing immunotherapies. To create these maps, scientists use techniques such as flow cytometry and RNA sequencing to characterize immune cells based on their physical properties. However, researchers have identified numerous cells with identical surface marker phenotypes that show vast functional diversity, highlighting a gap between the information provided by these traditional techniques and the ability to characterize cell function.

Immune Therapy Advancement with IsoPlexis "Functional Phenotyping"

IsoPlexis’ functional extracellular phenotyping is addressing urgent challenges in cancer immunology.IsoPlexis’ single-cell functional proteomics platform fills this gap by providing a more complete picture of how individual cell functionality provides critical information missing from genotypes and marker expression through “functional phenotyping.” The platform can identify functional differences in phenotypically identical cells and characterize and measure cytokine secretion in single cells. This allows scientists to uncover unique polyfunctional cellular subsets that can produce multiple cytokines simultaneously. This technology has advanced cancer immunology research and development of...

Insights into Biological Drivers of Anti-Tumor Response 

Recently, IsoPlexis technology helped uncover highly polyfunctional CD4+ cell subsets that conventional surface marker-based phenotyping techniques had missed. In 2018, Rossi et al. published an article in the journal Blood1 identifying single-cell polyfunctionality as a potential predictor of patient response to CAR-T cell therapy. By examining the CAR-T products from 20 lymphoma patients, the researchers identified separate CD4+ and CD8+ subsets that secreted granzyme B, IFN-γ, IL-5, IL-8, and MIP-1α in a polyfunctional manner. In addition to these, the highly polyfunctional CD4+ cellular subset also secreted IL-17A. Rossi et al. found that only 20% to 25% of all CAR-T product cells were polyfunctional upon stimulation with CD19+ target cells. This range of cytokine secretion from the rare polyfunctional CD4+ cells correlated to driving effective and potent immune-mediated responses.

Further experiments revealed that cellular polyfunctionality associated with clinical outcomes, including adverse effects such as cytokine release syndrome and neurologic toxicity. This study outlined how functional phenotyping could be a useful tool for discovering improved biomarkers of response to treatment.

Identifying Unique Cell Subsets with Critical Roles in Immune Suppression

Immunotherapy research typically focuses on direct immune cell responses. However, a study in Blood Cancer2 from Chen et al. at the Mayo Clinic explored how other cell types modulate immune cell responses. This study used IsoPlexis' single-cell functional proteomics technology to study the biological and clinical relevance of signal regulatory protein-α (SIRPα) expression in B-cell non-Hodgkin lymphoma (NHL). They found and functionally characterized three monocyte/macrophage (Mo/MΦ) subsets based on differences in CD14 and SIRPα expression levels. These three subsets differed functionally in terms of cytokine secretion profiles, phagocytosis properties, and how they regulated T cell activity. IsoPlexis’ single-cell proteomics technology revealed that the CD14+SIRPαhi subset contained a higher number of polyfunctional cells than the other subsets.

The CD14+SIRPαhi subset expressed common Mo/MΦ markers, suppressed T cell function, and was correlated with poor prognosis in follicular lymphoma patients. Conversely, the CD14-SIRPαlow subset expressed fewer typical Mo/MΦ markers, stimulated T cell function, and correlated with improved patient survival. This study identified unique Mo/MΦ subsets that modulated cellular function and affected the immune response as a whole based on their functional phenotypes.

Powering Immune Medicine

Unable to be detected by conventional technologies, cellular functional heterogeneity leads to differences in patient response and disease progression. Filling this gap, IsoPlexis’ single-cell functional proteomics is allowing researchers to characterize the range of extracellular functions (32+ cytokines) per single cell. By exploring, understanding, and characterizing cells based on function, researchers can accelerate therapeutic development by revealing new phenotypes that specifically correlate to individual disease or response states.


  1. J. Rossi et al., “Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL,” Blood, 132(8):804-814, 2018.
  2. Y. Chen et al., “SIRPα expression delineates subsets of intratumoral monocyte/macrophages with different functional and prognostic impact in follicular lymphoma,” Blood Cancer J, 9:84, 2019.

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