How Single-Cell Proteomics Adds New Data Layers to Our Understanding of The Adaptive Immune Response

How Harnessing the Immune System Can Lead to Better Therapeutics and Vaccines

The body’s first line of defense against external pathogens is the innate, or non-specific, immune response. This immune response consists of physical barriers, such as the skin, as well as chemical and cellular defenses designed to prevent the spread of pathogens throughout the body. In contrast, the adaptive or specific immune response mounts an attack of cells designed to target a particular substance (i.e., antigen). When the innate immune response fails to eliminate a pathogen, the adaptive immune response acts as a more specific defense. The ability to study the adaptive immune system enables development of effective vaccines and therapeutics.

T cells attacking a tumor

There are different types of cells involved in the adaptive immune response, but here we will focus on T cells, which are involved in attacking the foreign pathogens. If the innate defense system is unable to remove a pathogen, the adaptive immune response kicks in, typically a few days after the innate response begins. A primary role of the adaptive immune system is to identify non-self antigens, or antigens originating outside of the body. After the identification of a foreign pathogen, the adaptive immune system generates a response which mounts an army of immune cells designed to target that specific pathogen. Finally, the adaptive immune system saves immunological memory of the pathogen through the creation of memory cells which can be quickly called upon to provide protection if the pathogen is encountered again in the future.

cancer immunity cycle

When looking at the cancer immunity cycle, depicted above, it’s clear that T cells play a huge role. Once tumor cells release antigens, T cells are able to identify, infiltrate, and kill the tumor cells to begin the process of eliminating the tumor in the body. IsoPlexis’ single-cell proteomics technology has the ability to reveal key mechanisms at each stage of the immunity cycle, providing critical insights to researchers.

T Cells & Cytokines: What are Functional Proteins and Why Does This Matter

T cells do “work” with the proteins they release (cytokines), which we refer to as functional proteins. Functional characterization of these cells is significant to cancer immunology, vaccine development, engineered cell therapies, and other critical research areas. Immune therapies are designed to harness the immune system and direct the patient’s immune system to attack cancer. These therapies harness the patient’s killer T cells to work to attack and destroy the cancer. Some of the T cells will recruit other cells and orchestrate the attack, as well as modulate the immune response, such as helper T cells and regulatory T cells, using cytokines to carry out these functions.

In order for cells to know what their functions are and what cytokines to secrete, they need instructions. Every cell type has the same blueprints (DNA) for these instructions. The cell will take these blueprints and turn them into work instructions (RNA), and then they will have to read those instructions and do the work they specify (cytokines/functional proteins). Even though every cell has these work instructions, it doesn’t mean that each cell always does that work. Some cells are low functioning, and others are very high functioning. Low functioning cells are not very active and therefore do not perform many functions, if at all. Then some cells are a little more active and functional, but the highly functional cells are highly active, perform numerous functions, and act like superheroes. T cells can perform between 20 and 30 separate functions, with the most effective T cells performing all of them, and the least effective cells potentially performing none. Studies have shown that a polyfunctional response is highly correlative of patient outcome.

Immunotherapies are designed to increase the number of highly active, more functional immune cells, which are the cells that secrete multiple cytokines simultaneously. We call these cells polyfunctional because they are “poly” secretors, meaning they do many different types of work (multiple cytokines at once from one immune cell). A critical factor in the development of immune therapies is to determine if the therapy is producing the right type of immune cells.

adaptive immune response

Critical Gaps with Current Technologies & How IsoPlexis’ Single-Cell Proteomics Adds New Data Layers to Provide Previously Inaccessible Insights

Some technologies currently in existence:

  • Predominantly identify cell typees (their phenotype): flow cytometry
  • Identify the blueprints: DNA sequencing
  • Identify the work instructions: RNA sequencing

The information from the above technologies can be valuable a lot of research, however, none of these technologies identify the true function of immmune cells. It is crucial to identify functional proteins at the single-cell level, which has previously proved challenging. When looking at a mixture of immune cells, the individual cell identity is lost. Technologies such as bulk ELISA, genomics, and flow cytometry can see identify the function of the entire group of cells as a whole, like looking at a fruit smoothie, for example. With these methods, and with the cell population “blended together”, it is impossible to know if there are more highly active and polyfunctional cells present. This is what IsoPlexis has solved with single-cell proteomics. IsoPlexis technology can identify the full range of cellular function, from cells that have low function and low potency to the potent and durable super cells. So, in the smoothie example, this would be analogous to having the ability to identify the individual pieces of fruit. Rather than looking at the function of the population of cells as a whole, you now have the ability to look at the function of each individual cell that is contributing to that population.

What only IsoPlexis can do

While flow cytometry tells us what cells look like (and they all may look the same), and RNA-Seq tells us what the cells might do, IsoPlexis tells us what the cells are functionally doing and which are the most active, which is how IsoPlexis has predicted a number of immune responses to therapies.

IsoPlexis’ unique proteomic barcode adds the missing functional data layer that other technologies are unable to provide. We’ve developed our assays to measure the true functionally relevant proteins from live, naïve cells or from serum analysis. This data is truly reflective of in vivo biology, as IsoPlexis’ systems are directly measuring functional proteins rather than measurements based on estimations. Our system runs multiplexed assays of single-cells and bulk cells, using ultra-low sample volumes. This allows researchers to accelerate discoveries with meaningful correlations to in vivo biology.

IsoPlexis' Proprietary Proteomic Barcode

IsoPlexis captures the complexity connected to in vivo biology with walk-away automation to cut down therapeutic timelines by a year or more, while increasing efficacy with:

  • Proteins reflecting and predicting in vivo biology
  • High multiplexing from single cells and bulk to capture complexity
  • Time saved with analytics automation

IsoPlexis’ single-cell proteomics technology has helped accelerate therapy development through providing meaningful data that predicts biology by multiplexing and capturing native protein environments to show greater connectivity to in vivo biology. Liquid “PBMC” biopsies, tissue analysis, and serum/CSF have predicted durable effector response in cancer immunology, infectious disease, and cell and gene therapy, and have predicted early, treatable sources of inflammation in inflammation and targeted therapies.

What is the clinical meaning of this?

  • >Predicting survival: In a study with 38 metastatic melanoma patients, IsoPlexis’ blood-based biomarker predicted response and progression-free survival uniquely in Bristol Myers Squibb & Nektar Therapeutics’ checkpoint inhibitor and IL-2 agonist, presented at SITC 2020
  • Predicting inflammation: In a COVID-19 patient study published in Cell, IsoPlexis’ monocyte cytokine biomarkers identified mechanisms of COVID-19 inflammation uniquely in collaboration with the ISB, Merck, Seattle Consortium
  • Unlocking pre-clinical meaning: cancer metastasis and tumor inhibition strategies revealed from cytokine driven inflammation in small samples, published in Nature Communications

IsoPlexis’ Unique Applications at the Convergence of Single-Cell Biology & Proteomics

IsoPlexis offers panels for human, mouse, and non-human primate to study the adaptive immune system, as well as a variety of panels for other research interests. IsoPlexis’ technology is at the convergence of single-cell biology and proteomics, with fully automated solutions and push-button analysis, provided by the integrated IsoSpeak software, to accelerate the development of novel vaccines and therapeutics. These solutions enable functional biomarker discovery and accelerate development through complete single-cell and bulk population functional characterization. IsoPlexis’ platform is the gold standard technology for functional single-cell proteomics, providing consistent and sensitive detection of up to 30+ cytokines per single immune cell or bulk sample.

See how IsoPlexis’ Single-Cell Intracellular Proteome technology was applied in identifying prognostic biomarkers in COVID-19 in this Cell paper review.

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