Overcoming Challenges in Neuroinflammation with Highly-Multiplexed Functional Proteomics (Part 2)

In Part 1 of our blog series, we highlighted the importance of understanding neuroinflammation in neurodegenerative diseases. Part 2 examines common challenges and applications that our Single-Cell Secretome can help address.

To recap, neuroinflammation is a complex biological response to nervous tissue injury and plays a key role in neurodegenerative diseases. Nevertheless, it can be tough to assess due to heterogeneity in cytokine production and aberrant cytokine signatures.

For this reason, IsoPlexis has developed a platform to accelerate workflows by functionally defining every cell type, helping to inform more effective therapies. Traditional technologies average serum protein information for all cells in a sample. With IsoPlexis’ cellular functional phenotyping, cellular differences are uncovered to identify functional mechanisms in neuroinflammation and neurotoxicity. Data has shown that which specific cytokines are produced by each heterogeneous immune cell is crucial. Our single-cell functional proteomic helps to complete neuroinflammatory characterization.

Through analysis of cellular RNA or surface phenotypes alone, functional extracellular and intracellular phenotypic differences reveal the biological drivers of patient response may be missed. Isoplexis’ IsoLight system allows researchers to obtain highly multiplexed cytokine data without advanced training and interaction with samples.

Below we highlight three applications/challenges in which our technology was used and found to be beneficial.1

Challenges & Applications

Application 1 – Understanding Mechanism of Disease Progression in Neuroinflammation

Functional Phenotyping Reveals Differences between Circulating Monocytes in Patients with Frontotemporal Degeneration (FTLD)

Identifying predictive biomarkers that have the capacity to show a correlation between efficacy and adverse effects of immunotherapies can be challenging thanks to the heterogeneous functionality and complex immune response of immune cells. Using Isoplexis’ single-cell IsoCode chip with a fluorescent ELISA-based assay, researchers simultaneously identified up to 40+ proteins from live single immune cells, allowing for accurate assessment of polyfunctional heterogeneity of each immune cell type.

Increased inflammatory polyfunctional monocyte subsets with unique cytokine signatures can potentially provide a basis for biomarkers for peripheral immune pathology unique to FTLD.2

By applying IsoPlexis’ Single-Cell Secretome to FTLD the following were achieved: 2

  • Single-cell polyfunctional heatmap revealed a more diverse secretion profile of both CD14+CD16- and CD14+CD16+ monocytes within diseased patients.
  • Single-Cell Secretome analysis revealed increased polyfunctional profiles of monocyte subsets in FTLD patients compared to healthy control.
  • Increased inflammatory polyfunctional monocyte subsets with unique cytokine signatures provided a potential basis for biomarkers for peripheral immune pathology unique to FTLD.

Application 2 – The Role of Innate Immunity in Multiple Sclerosis (MS) Pathogenesis

Assessing Dysfunctional Innate Immune Cell Subsets in MS with Functional Phenotyping

The role the microbiome and innate immunity play in the progression of multiple sclerosis (MS) remains unclear. Very low levels of a microbiome-derived Toll-like receptor (TLF)2-stimulating bacterial lipid in the blood of MS patients has been previously documented and suggests that a deficiency in innate immune regulating function of the microbiome in MS. The authors believe that the resulting enhanced TLR2 responsiveness play a role in the progressions of MS.3

The objective of this study was to characterize TLR2 responses for MS patients using a variety of methods. The team found that 50% of MS patients in the study showed enhanced responsiveness to TLR2 stimulation. What’s more, the enhanced TLR2 responders include a significant percentage of those with progressive MS, a subset of patients who were considered unresponsive to adaptive immune system-targeting therapies. These results could help further the understanding of the role of innate immunity in MS.3

Highlights from the study: 3

  • The IsoPlexis Single-Cell Secretome identified a pathologically and therapeutically relevant Toll-like receptor (TLR) previously uncharacterized in MS.
  • Data suggests that the highest frequency of enhanced TLR2 responders within the MS cohort may be found among the patients with progressive forms of the disease.
  • The ability to stratify MS patients based on monocyte functionality may be especially powerful given the observed differences in therapeutic response for those with the different forms of the disease.

Application 3 – Biomarkers of Neurotoxicity and Immune Related Adverse Events (IRAEs)

Preinfusion CAR-T Cell Product Metrics Uniquely Correlate with Grade 3+ Cytokine Release Syndrome (CRS)

Following treatment with CAR T cells, interleukin-15 (IL-15) elevation and CAR T cell expansion have been found to be associated with non-Hodgkin lymphoma (NHL) outcomes. Yet, the association of preinfusion CAR product T cell functionality with clinical outcomes has not been reported.4

In this study, a single-cell analysis of a preinfusion CD19 CAR product from patients with NHL showed that CAR products have polyfunctional T cell subsets that can cause multiple immune responses.

A predetermined T cell polyfunctionality strength index (PSI) applied to the preinfusion CAR product was found to be significantly associated with clinical response. In addition, PSI combined with CAR T cell expansion or pretreatment serum IL-15 elevation also showed significance.

The team’s discoveries indicate that a preinfusion CAR product T-cell subset with a definable polyfunctional profile has a major association with clinical outcomes of CAR T-cell therapy.4

Highlights of IsoPlexis’ Functional Proteomics and Correlation to CRS: 4

  • The IsoPlexis Single-Cell Secretome revealed association between inflammatory functional phenotype in conjunction with either CAR peak or pretreatment in vivo IL-15 levels in blood, and grade 3+ NT.
  • IL-17A functional phenotype combined with IL-15 levels at day 0 had a statistically significant association with grade ≥3 NT, suggesting a critical role for IL-17A producing polyfunctional cell subsets in neurologic toxicities.
  • Secretion profiles from single peripheral immune cells fundamentally impact the understanding of the underlying mechanism of adverse events correlated with immunotherapies.

A Fully Automated Solution for Single-cell Functional Phenotyping of Immune Cells

Our Single-Cell Secretome solution enables the discovery of better biomarkers and accelerated development through functional immune landscaping of each immune cell, allowing for complete single-cell functional characterization. Detect rare subsets of “super cells” to reveal functional biological drivers of persistence, potency, durability and more.

Be sure to look for Part 3 of our series in which we address neurological manifestations of COVID-19 in patients and how our highly multiplexed CodePlex Secretome Solution can help.

 

References

  1. https://isoplexis.com/literature/isoplexis-proteomic-product-suite-for-neuroinflammation/
  2. Liu D, Paczkowski P, Mackay S, Ng Colin, and Zhou J., Single-Cell Multiplexed Proteomics on the IsoLight Resolves Cellular Functional Heterogeneity to Reveal Clinical Responses of Cancer Patients to Immunotherapies. Biomarkers for Immunotherapy of Cancer – Springer, 2019.
  3. Fujiwara M, Anstadt EJ, Flynn B, Morse K, Ng C, Paczkowski P, Zhou J, Mackay S, Wasko N, Nichols F, Clark RB., Enhanced TLR2 Responses in Multiple Sclerosis. Clinical and Experimental Immunology, 2018.
  4. Rossi J, Paczkowski P, Shen Y, Morse K, Flynn B, Kaiser A, Ng C, Gallatin K, Cain T, Fan R, Mackay S, Heath JR, Rosenberg SA, Kochenderfer JN, Zhou J, and Bot A., Preinfusion Polyfunctional Anti-CD19 Chimeric Antigen Receptor T Cells Associate with Clinical Outcomes in NHL. Blood, 2018.
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