Deciphering T Cell Diversity in the Tumor Microenvironment

Evan Newell on decoding immune cell heterogeneity with mass cytometry

Understanding the full complexity of the immune system and its response to infections and diseases, especially in cancer, has eluded researchers for decades. Whether analyzing blood or tissue samples, investigators have struggled to categorize the heterogeneity found in the T cell composition of different tumors.

Compounding the challenge, T cell diversity comes in many forms, such as antigen specificity, differentiation states, functional characteristics and trafficking receptor profiles. Deciphering this diversity is necessary to understand the mechanisms behind cancer progression and immunotherapy response.

In a recently published paper in Nature, Evan Newell, PhD, and colleagues identify a potential biomarker of tumor-antigen specificity and provide a new perspective on the heterogeneity of T cells in the tumor microenvironment. Newell led this research as a principal investigator at the Singapore Immunology Network (SIgN). First author Yannick Simoni, PhD, formally a senior research fellow at Singapore’s Agency for Science, Technology and Research (A*STAR), accumulated data for more than 140 different tumor samples from patients with lung and colorectal cancer to search for correlations between this heterogeneity and various characteristics of the patient samples.

“CyTOF technology has a really unique niche for interrogating the tumor microenvironment,” said Newell. “It has relatively high throughput and high dimensionality, allowing us to open up a lot of doors.”

Newell’s lab developed novel approaches for identifying and characterizing antigen-specific T cells. In this paper, his group used MHC tetramer staining in conjunction with mass cytometry for in-depth analysis of cellular phenotype and function.

His group and others “have been stuck on saying, ‘It’s crazy. There are so many different types of immune cells.’ And you can use all kinds of fancy analyses to show that it’s very diverse,” Newell said. “But we’ve taken it a little bit further by getting to some simple correlates of clinical states.”

Newell and his team used mass cytometry that relies on time-of-flight mass spectrometry (CyTOF® technology) to simultaneously assess antigen specificity and deep phenotypic characteristics of T cells. The high-parameter nature of CyTOF analysis allowed the team to create panels to assess as many as 52 cellular parameters simultaneously. The resulting data shows that T cell populations infiltrating lung and colorectal tumors may be specific for tumor antigens or for a wide range of epitopes unrelated to cancer, such as viral antigens. Moreover, he discovered that these bystander T cells have diverse phenotypes that overlap with tumor-specific cells but lack CD39 expression.

CD39: A more accurate marker of tumor antigen specificity

Newell and Simoni’s discovery of a more accurate marker for tumor antigen specificity relied on a multiplexed approach that allowed them to simultaneously profile T cells within the tumor microenvironment for phenotype and function. Instead of focusing only on tumor-reactive cells, the team looked at all antigen-specific T cells and was able to identify cancer-unrelated antigen-specific T cells in tumors.

“We found a few examples of tumor-specific T cells, and then we also had examples of T cells specific for cancer-unrelated antigens, like the flu and Epstein-Barr virus. This was key in trying to interpret what all this heterogeneity meant,” Newell said.

The next step was to determine what was different about the non-cancer-specific T cells. Simoni observed that many of them expressed what typically have been identified as tumor-infiltrating T cell markers, such as CD69 and PD-1, which may have a role in tumor reactivity.

“The striking thing was that we saw hardly any expression of CD39, which is known as being immunosuppressive,” he said.

Since their findings showed that the tumor-specific T cells expressed CD39, Simoni could build a case for CD39 as a more accurate marker of tumor antigen specificity. Newell now believes CD39 also could be useful as a predictor of response to checkpoint blockade, and as a starting point for the development of novel therapeutics.

Advantage to using CyTOF technology for immune cell profiling

Newell started using mass cytometry for studying human T cell response as a postdoctoral student at Stanford University more than six years ago. He attributes much of his success to the technology’s ability to simultaneously look at many phenotypic markers and antigen specificity, allowing his team to better understand the number of distinct cellular phenotypes and how they are related to each other.

In the Nature paper described here, Newell also used whole transcriptome RNA-seq to validate his mass cytometry findings. He believes the two techniques make a powerful combination for identifying new markers associated with specific cell populations. However, Newell explains that compared to RNA-seq, mass cytometry “can more accurately measure protein and do it on a larger number of cells, which gives a much higher resolution when describing cellular heterogeneity.”

CyTOF applications beyond cancer research

Newell’s ultimate goal is to gain a better understanding of how the human immune system works, and he thinks the best way to accomplish this is by searching for co-variations.

“Mass cytometry is especially great for that because it’s really improving our understanding of immune system variation in humans,” Newell said. “It has also been excellent for mapping out trajectories of cellular development.”

In addition to providing cutting-edge technology platforms to researchers, SIgN aims to support Singapore biotechnology companies such as Newell’s spinoff immunoSCAPE, which provides antigen-specific T cell screening and profiling services. Newell, who established his lab at SIgN in 2012, has moved to the Fred Hutchinson Cancer Research Center in Seattle as part of the Vaccine and Infectious Disease Division. His lab will continue to investigate antigen-specific T cells in cancer and other diseases. He also has been working on developing computational approaches to improve analysis of these large datasets.

Beyond cancer research, Newell and colleagues are using mass cytometry to study a variety of topics from myeloid cell composition in dendritic cell development to T cell antigen specificity in viruses such as hepatitis B and dengue. By taking a multiplexed approach in investigating the phenotype and function of cells from different viral disease stages, Newell’s team can assess the use of T cell phenotypes as future biomarkers for patient outcomes.

“CyTOF technology has a really unique niche for interrogating the tumor microenvironment,” said Newell. “It has relatively high throughput and high dimensionality, allowing us to open up a lot of doors.”

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