Polyfunctionality Correlates with In Vivo Response to Immune Checkpoint Inhibitor Therapies

Studies indicate IsoPlexis’ Polyfunctional Strength Index (PSI) has the potential to be a clinically relevant biomarker of early immune checkpoint inhibitor response


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Immune checkpoint inhibitors (CPIs) are effective immunotherapies for treating multiple cancer types in some patients. As more CPIs are investigated for potential clinical use, predictive biomarkers that can be used as stratification tools for clinical trials and treatment
decisions are needed.

The IsoPlexis system addresses a limitation of legacy technologies, such as flow cytometry and bulk serum ELISA, in its ability to detect underlying T cell functional heterogeneity through measuring true releases of the full range of functional cytokines. Additional results from several leading medical centers demonstrate how IsoPlexis’ new resolution into the cytokine signatures of single cells points to a novel ability to stratify complex patient responses in combination CPI therapy and offers a glimpse into the future of understanding the mechanisms of these complex immunotherapies, from pre-clinical to clinical applications.

The Polyfunctional Strength Index (PSI)

The IsoPlexis single-cell cytokine-detection system measures, from each single immune cell, true releases of the full range of functional cytokines that orchestrate the immune system. The Polyfunctional Strength Index (PSI™) is defined as the percentage of polyfunctional single cells in a sample that secrete two or more cytokines, multiplied by the average signal intensity of the secreted proteins from each cell. Each cell’s strength, across one thousand or more cells, is then aggregated and simplified to provide a comprehensible visualization of the potent cell subsets and the cytokine types that drive them.

Using the IsoCode Chip, the IsoPlexis platform isolates single cells into individual microchambers, containing a full range of multiplexed cytokine detection. ELISA-based detection determines which combinations of proteins are being secreted by each individual cell, providing the full spectrum characterization of a single cell’s polyfunctional cytokine profile.

Cell detection and cytokine signal detection are performed automatically. With an interactive user-interface, IsoPlexis’ IsoSpeak bioinformatics software rapidly provides advanced visualization of raw data in an easy and automated manner.

Acute Myeloid Leukemia (AML)

Blockade of the PD-1/PD-L1 pathway in T cell subsets can exert anti-leukemia immune responses. The response rates for the azacitidine/nivolumab therapy were almost double the results of either drug alone, and the overall survival rate was better than historical rates with azacitidine alone. To help identify which patients would benefit most from this combination therapy, bone marrow infiltrating T cells, before therapy, were tested for polyfunctionality on IsoPlexis’ platform.1

Patients with highly polyfunctional T cells (i.e. PSI>10) had a response to therapy, while patients with low polyfunctionality (i.e. PSI<10) did not respond. The study demonstrated that pre-therapy PSI was potentially a strong predictor of response and overall survival in relapsed patients treated with azacitidine/nivolumab, which could be used to select patients in prospective clinical trials.

Additionally, single-cell multiplexed-proteomics precision profiling from IsoPlexis identified a polyfunctional downregulation in BM T cells in patients with relapsed AML compared to newly diagnosed patients, and an elevated polyfunctionality compared to those with the TP53 mutation. There was also a significant difference in PSI detected between newly diagnosed AML and relapsed, refractory AML.

BM T cells from relapsed patients with TP53 mutations showed significantly impaired polyfunctional profiles compared to patients with newly diagnosed AML without the mutation.

Non-Small-Cell Lung Cancer (NSCLC)

In an additional study,2 current predictive biomarkers of CPI response in NSCLC include PD-L1 expression, Tumor Mutational Burden-High (TMB-Hi), and Microsatellite Instability-High (MSI-Hi). However, these biomarkers have had mixed results in providing a correlative value in a variety of clinical studies. Moreover, emerging clinical evidence suggests that oncogene-addicted NSCLCs do not respond well to CPIs, regardless of levels of PD-L1 expression status.

In this study, patients were treated with pembrolizumab, now approved for first-line metastatic NSCLC. Using the 32-plex IsoCode Chip from IsoPlexis, an increase in polyfunctionality was discovered in the post-treatment samples compared to the pre-treatment samples; this increase in polyfunctionality was heterogeneous among patients and cell types.

The researchers concluded that the study demonstrated the feasibility of using a single-cell proteomics platform to longitudinally profile, in multiplexed fashion, cytokines released by live, single T cells from NSCLC patients. In addition, the data presented PSI as a potential predictive biomarker of CPI response, linking single-cell cytokine profiling to clinical outcome.

Metastatic Melanoma

Tumor-infiltrating T lymphocytes (TILs) are lymphocytes that infiltrate and disrupt tumor cells: several clinical studies have suggested that their presence is positively associated with patient survival. However, an in-depth dissection of their functionality is needed in order to determine if TILs are a new way to predict clinical outcome in cancer patients receiving immunotherapy.3

Researchers isolated TILs from primary tumor tissues of patients with metastatic melanoma who received anti-PD-1/CTLA-4 CPI immunotherapy. IsoPlexis’ single-cell cytokine-detection system was used for a comprehensive evaluation of the secretion diversity of the TILs.

The analysis of CD8+ TIL samples revealed upregulation of polyfunctional cytokine production in patients who responded to anti-PD-1/CTLA-4 immunotherapy compared to non-responding patients. Bulk ELISA or histology failed to detect these functional differences in TILs and tumor tissues between the two groups.

Responders vs Non-RespondersResponders had 9x more polyfunctional cells than non-responders and 40x higher polyfunctional strength. The contributions to enhanced polyfunctional strength were dominated by effector and stimulatory cytokines—both associated with anti-tumor immunity.

CD8+ TILs from responding patients exhibited highly polyfunctional cell subsets co-secreting Granzyme B, IFN-γ, MIP-1a, Perforin, and TNF-a, which are the major anti-tumor effector molecules, suggesting that PSI can be used as a potential biomarker to predict clinical outcome of melanoma patients treated by CPI immunotherapy.

The PSI revealed previously unappreciated deep functional heterogeneity and dissected the full spectrum of immune functions of TILs across patients. More importantly, these findings suggested that highly polyfunctional TILs, producing multiple cytokines per single cell as measured by IsoPlexis’ single-cell multiplexed cytokine profiling, could be a potential predictive biomarker for melanoma patients treated by checkpoint therapies.

Future Applications

Researchers are using IsoPlexis’ next-generation platform to analyze the true functional proteomic single-cell biology of patients and stratify responders from non-responders to therapy. The IsoPlexis single-cell cytokine-detection technology and PSI address the limitation of traditional methods and deeply decipher the heterogeneous polyfunctional profile of single cells. The ability to determine cellular fitness and reveal how single cells are truly functioning can help move a new generation of immunotherapy treatments forward as well as offer a deeper understanding of mechanism and personalized medicine in these complex immunotherapies. IsoPlexis’ technology is helping to accelerate the development of these complex therapies, so that more novel therapies can be brought to patients.


1.Polyfunctionality determined by single-cell proteomics of bone marrow-derived CD4 T Cells from patients with acute myeloid leukemia Identifies patients responding to anti-PD-1-based therapy and discovers profound T cell defect in mutant TP53 Disease, Daver, N. et al., American Association for Cancer Research Annual Meeting 2019

2. Translational Single Cell Proteomics Profiling of Live T-Lymphocytes in Oncogene-Addictive NSCLC under Immune Checkpoint Inhibitor (CPI) Treatment, Lim Z. et al., Translational Research Cancer Centers Consortium 22nd Annual Meeting 2019
3. Single-cell PSI of CD8+ TILs in melanoma shows uniquely sensitive correlates with response to anti-PD-1/CTLA4 therapy, where histology and serum cytokines were unable to detect significant associations, Mackay S. et al., American Society of Clinical Oncology Annual Meeting 2019


Learn more about how PSI can be used as a potential biomarker to predict patient response in checkpoint therapies in our App Note, “Assessing Functional Quality of Single TILs for Correlative Immunotherapy Biomarkers.”

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