Polyfunctionality in T Cells Correlates Vaccine-Induced Durable Protective Immunity, Revealed with Single-Cell Proteomics

How IsoPlexis’ Single-Cell Functional Phenotyping Identifies Quality T Cell Response and Overcomes Limitations in Traditional Technologies in Vaccine Development

There are limitations to the protein assay technologies, such as ELISpot and flow cytometry, that most companies have been using for decades in infectious disease research and vaccine development. Due to a lengthy and expensive development and approval process, it’s important to utilize the most efficient and effective tools to help accelerate this development pipeline. These decades-old technologies are not providing a clear insight into the direct function of immune cells and quality T cell response, and this lack of clarity leads to delays in the development timeline. For the first time, IsoPlexis’ single-cell proteomics provides true functional phenotyping for a full range (30+) of cytokines for the identification of quality T cell response and accelerated insight into vaccine development. Now, researchers can narrow down their vaccine candidates to the most efficacious and durable products before entering into pre-clinical and clinical trials, saving precious time and money.

ELISpot and flow cytometry are limited to measuring only a few cytokines per cell, albeit the cells must be fixed and permeabilized because cytokines are not surface markers and the sample must be blocked due to a high level of non-specific binding. While these data provide basic information on function, they do not provide full functional insight and quality T cell response. Because of these limitations, researchers using these methods are not able to get information on potent T cell response and whether a potential vaccine is providing some protective value until entering in vivo studies. IsoPlexis is able to provide information on quality T cell response with unique single-cell functional phenotyping, detecting 30+ cytokines per single cell in a completely automated fashion.

Limitations of Traditional Technologies & How IsoPlexis’ Single-Cell Proteomics Provides True Cellular Function for Quality T Cell Response

Human peripheral blood contains a vast heterogeneity of functionally different cell subsets, and distinguishing between these is vital to learning more about pathogenesis and cellular immunity. Subsets of cells, such as T cells, have demonstrated distinct functions that create a change in the immune system, even if it’s in a small number of cells.1 Future studies will need technology, such as IsoPlexis’ single-cell proteomics, that can measure a full range of functional cytokines, while conventionally three or four are measured with traditional flow cytometry from cells that are blocked and fixed.1

Dr. Mario Roederer spoke on the limitations of flow cytometry:

“Even with such tools, proper data analysis, interpretation, and presentation on the part of flow cytometry users will require a high level understanding of the intricacies of compensation and the inherent limitations of compensated data. Without a proper understanding, there will continue to be surfeit of improper conclusions based on assumptions about the rectilinearity of cytometry data.”2

Traditional technologies are not able to provide researchers with a complete picture of a cell’s function because they are limited in the number of cellular functions they can measure simultaneously. With IsoPlexis’ single-cell proteomics and functional phenotyping, you can detect the true range of cellular function, over 30 cytokines per single cell. This information is imperative in all sorts of therapy studies and vaccine research as this information helps identify potent cell subsets and quality T cell response.

The failure of an early HIV vaccine trial demonstrated the different approaches that are needed in vaccine development.

The disappointing results of the recent vaccine trial suggest that a more thorough assessment of vaccine-induced immune responses is urgently needed.”

“As we are still far from understanding the ingredients required for effective vaccines, attempts to better understand the components of the immune response to licensed, efficacious vaccines should be a research priority. This research will help define the elusive correlates of T cell immunity to vaccines in humans.”3

It’s clear that a better way to measure function will be immensely valuable in identifying the quality of these vaccines. For the first time, IsoPlexis’ single-cell proteomics provides the full range of functional phenotyping needed for identifying quality T cell response.

IsoPlexis’ Functional Phenotyping Identifies Durable and Persistent Immune Response Potency, Quality, Characterizes Function, and More

Accelerating Vaccine Development with IsoPlexis’ Single-Cell Proteomics

Traditional analyses involve numerous manual processes and multiple instruments, taking up precious time. However, with the IsoPlexis platform, the entire workflow is completely automated and hands-off. When running single-cell analysis, the samples are loaded onto the chip and inserted into the IsoLight, which automates the incubation, ELISA, data analysis, etc. The same IsoLight system can also run highly multiplexed serum analysis with only five minutes of hands-on sample loading time. The automation for both single-cell and bulk serum analyses allows the user to walk away and save time.

Another important aspect of speed in the vaccine development workflow is being able to predict how a vaccine candidate will perform. “Current immune-monitoring strategies are focused on measuring effector T cell responses. They do not, however, measure memory and its renewal or persistence, despite the fact that absolute numbers of memory T cells and, in particular, central memory T cells have been associated with protection in primate models. Nor do the current strategies assess other immune parameters, such as Th2 cytokines and innate immune responses.”3 IsoPlexis’ platform detects a full range of cytokines (30+) and has predicted durability and quality T cell response, as well as other cell types in various studies.

IsoPlexis’ Single-Cell Proteomics Enables Insights into Polyfunctionality & Quality T Cell Response

Immune cell subsets which are highly polyfunctional act as “superhero” cells in their ability to perform a more diverse range of function via cytokines. These highly polyfunctional “superhero” cells lead to protective immunity, durable responses, and quality t cells response. Conversely, when these “superhero” cells have an aberrant or dysfunctional cytokine signature, they can become “super-villains”,  and lead to autoimmune inflammation or cause immune related adverse effects. It is critical to be able to detect these “superhero” types of polyfunctional cells to identify mechanisms of protective immunity within vaccine development.

Traditionally, polyfunctionality is regarded as anything secreting two or more cytokines. “Polyfunctional T helper (Th)1 cells that make higher levels of cytokines on an individual cell basis have been associated with protection in vaccine trials against other microbes as well as HIV-infected elite controllers.”3 Technologies like flow cytometry and ELISpot are only able to detect a few cytokines per cell, while IsoPlexis’ platform is able to look at more than just IL-2, TNF, and IFN-γ—a range of 30+ cytokines can be detected for full characterization of true functional phenotype for each immune cell. Highly polyfunctional cell subsets have been correlated with more persistent, durable responses in multiple published studies. Using IsoPlexis’ technology, polyfunctionality becomes a predictor of vaccine candidate success in pre-clinical and clinical trials.

Durablity and Quality T Cell Response

Researchers Zhou, et al. talk about the limitations of ELISpot in “CD8+ T-cell mediated anti-malaria protection induced by malaria vaccines; assessment of hepatic CD8+ T cells by SCBC assay”: “ELISpot assay measures true cytokine secretion by a single T cell, but does not identify T cell polyfunctionality due to limitations on multiplexing (<4), which is associated with the functional performance.”4

Dr. Moriya Tsuji states in an interview how IsoPlexis’ single cell proteomics are overcoming limitations in the current technologies being used such as ELISpot and flow cytometry:

“There’s an unlimited number of cytokines to identify, but there are no good tools to show the more than 20-30 cytokines or chemokines at the single-cell level with a high throughput assay. This has not been done until IsoPlexis has established this very novel high throughput cutting edge technology that can measure the amount or level of cytokines and chemokines from more than 30-40 cytokines at the single-cell level at once. This technology will really be able to advance our knowledge for which T cells [contribute to] protective immunity against malaria.”

In identifying quality T cell response, not only is effectiveness important, but so is potency. To understand this, function needs to be identified and studied. What specific cytokines are being produced by each single cell? When you identify this, you can see how the cell is functioning and orchestrating immune response.

For measuring the full range of cytokines per cell, flow cytometry and ELISpot are not sufficient; measuring a few cytokines is not enough, and the direct function of cells needs to be measured. With IsoPlexis’ platform, these challenges presented by traditional technologies can be overcome. IsoPlexis’ single-cell proteomics and functional phenotyping provide insight relevant to specific diseases and patient populations. IsoPlexis’ system can identify the specific cytokines each individual cell is truly secreting, not one or two of the cytokines it might be secreting. It is known that cells are heterogeneous, so the ability to detect specific subsets of cells with unique function is essential. This information provides insight into how a subset of cells are orchestrating the immune system’s function and how that can be utilized in creating therapies and vaccines.

Download our Infectious Disease eBook to learn more about breakthroughs in developing vaccines, protective immune monitoring, and measuring toxicity with functional proteomics.


  1. Perfetto SP, et al. Seventeen-colour flow cytometry: unravelling the immune system. Nature Reviews Immunology 4: 648-655, 2004.
  2. Roederer M. Spectral compensation for flow cytometry: Visualization artifacts, limitations, and caveats. Cytometry 45: 194-205, 2001.
  3. Rafick-Pierre S. The failed HIV Merck vaccine study: a step back or a launching point for future vaccine development? Journal of Experimental Medicine 205: 7-12, 2008.
  4. Zhou J, et al. CD8+ T-cell mediated anti-malaria protection induced by malaria vaccines; assessment of hepatic CD8+ T cells by SCBC assay. Human Vaccines & Immunotherapeutics 13: 1625-1629, 2017.
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