The Gap in the Immunologist Toolkit
When it comes to developing immunotherapies, having a human cell map can make all the difference in designing and advancing these therapies. However, obtaining the necessary information requires identifying and characterizing functional cells. Existing technologies that scientists use every day to characterize cells (flow cytometry and RNA-Seq) do not provide a complete cellular picture. Because RNA-Seq is used to look at the inside of the cell, and flow cytometry is used to take a look at the cell surface or blocked proteins inside the cell, there is a gap in identifying how each cell is functioning extracellularly. IsoPlexis’ platform, single-cell functional proteomics, fills this gap and is used to characterize the extracellular function of the cell. Now, it is possible to get a complete picture of how each cell is functioning in an immune-correlative manner.
With RNA-Seq or flow cytometry certain cells with unique functions are undetectable, but with IsoPlexis technology, it is possible to detect cell types with an incredible amount of unique function that previously would not have been identified as unique. That unique functional characteristic of each cell is leading to the discovery of the functional mechanisms behind patient response.
The Impact of “Functional Phenotyping”
The purpose of understanding a deeper functional definition of established cell subsets is to reveal the functional drivers which correlate to patient response or disease progression. Cell subsets defined by surface markers are considered phenotypically similar, however, there is functional heterogeneity that is masked by conventional technologies. These differences in cellular function define the biological drivers of response or disease progression. While you might know what the “surface phenotype” of the cell you are working with is, it is the “functional phenotype” that is contributing to the work each immune cell is performing against the tumor. This is the type of information IsoPlexis’ single-cell functional proteomics can provide. This profiling is enabling researchers to accelerate their therapies by uncovering the unique polyfunctional subsets of cells that are secreting a much wider range of proteomic function. We’ve been able to identify heterogeneity in phenotypically identical cells and measure the specific cytokines each single cell secretes with our unique single-cell proteomic technology. This has been proven by our various studies in cancer immunology, cell and gene therapy, inflammation and neurology, infectious diseases, and vaccines with T cells, monocytes, CAR-T, TCR-T, NK cells, and more.
Some of the recent cases highlighting IsoPlexis’ technology in aiding the discovery of new cell types have been published in Blood and Blood Cancer Journal. Interestingly, we’re finding CD4 cell types that are secreting multiple cytokines that are not conventionally seen as simultaneous functions in CD4 defined by surface phenotyping. These rare highly polyfunctional cells have been highly correlated to patient response in various studies. The IsoPlexis single-cell functional proteomics platform is the only technology that is able to uncover this type of information.
Novel Insights into Biological Drivers of Response
Rossi et al. published an article in Blood noting the discovery of polyfunctional subsets in the pre-infusion CAR-T product that significantly correlated with clinical response. CD4+/CD8+ T cells and a relatively similar number of naïve cell phenotypes were defined by flow cytometry, but it was the functional differences in particular that correlated to patient response. Both the CD4+ and CD8+ T cell subsets secreted granzyme B, IFN-γ, IL-5, IL-8, and MIP-1α, and in addition, the highly polyfunctional CD4+ cells also secreted IL-17A. “Strikingly, only 20% to 25% of all product cells were polyfunctional upon stimulation with CD19-expressing target cells.”1 This range of cytokine secretion from the rare polyfunctional CD4+ cells correlated to driving effective and potent immune-mediated responses.
Identifying Unique Cell Subsets Playing Critical Roles in Immune Function
Generally, when researchers are looking at immunotherapies, they are looking at immune cell response:
- how those immune cells are leading to positive outcome in patients
- how the immune cells are doing the action of killing the tumor or attacking the tumor, and
- characterizing how those cells are functioning.
However, this Blood Cancer study, published by Chen et al. at the Mayo Clinic, is a unique case. Here, the researchers looked at how unique cell types are affect the immune responses listed above.
Chen et al. utilized IsoPlexis’ single-cell functional proteomics in their study of the biological and clinical relevance of signal regulatory protein-α (SIRPα) in B-cell non-Hodgkin lymphoma (NHL) to identify cell subsets that correlated with better survival. Three subsets of monocytes/macrophages (Mo/MΦs) were identified and functionally characterized. The researchers discovered monocyte cell types that play a very critical role in immune function, but in a way that is not beneficial to patient outcome. These uniquely functional monocytes suppress T cell function, which leads to poor prognosis in patients.
These subsets possess different characteristics in cytokine profiles, phagocytosis properties, and regulating T cell function. IsoPlexis’ IsoCode technology was then used to functionally profile these identified subsets. It was discovered that the CD14+SIRPαhi subset produced a significantly higher number of polyfunctional cells than the other two subsets. The CD14+SIRPαhi subset also secreted a high percentage of IL-10 (p <0.05), a regulatory, anti-inflammatory cytokine, which plays a vital role in limiting immune response to pathogens and in doing so, prevent damage to the host.2 However, this subset was found to express common Mo/MΦ markers and suppress T cell function, which correlated with poor prognosis in patients with FL. Conversely, the CD14–SIRPαlow subset expressed fewer than typical Mo/MΦ markers and stimulated T cell function instead. Increased numbers of this cell subset correlated with improved patient survival.
This research was published recently in an article titled “SIRPa Expression Delineates Subsets of Intratumoral Monocyte/Macrophages with Different Functional and Prognostic Impact in Follicular Lymphoma” in Blood Cancer Journal.
Speak to an expert today and see how IsoPlexis’ platform can help accelerate your immunotherapy program.
- Rossi J, et al. Preinfusion polyfunctional anti-CD19 chimeric antigen receptor T cells are associated with clinical outcomes in NHL. Blood 132: 804-814, 2018.
- Chen Y-P, et al. SIRPa expression delineates subsets of intratumoral monocyte/macrophages with different functional prognostic impact in follicular lymphoma. Blood Cancer Journal 9: 84, 2019.