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Team Combines Cell Sorting, NGS to Study Diversity of T Cell Response to Pathogens, Vaccines


NEW YORK (GenomeWeb) – Researchers at the Università della Svizzera Italiana in Switzerland combined cell sorting techniques with next-generation sequencing to elucidate how distinct types of CD4+ T cells respond to pathogens and vaccines.

The study, which was published in Science today, found even more diversity among T cell response than previously known or thought to occur.

"By combining antigenic stimulation with deep sequencing and isolation of T cell clones, we unraveled an unpredicted degree of heterogeneity in the human CD4 T cell response to microbes and vaccines," the authors wrote.

Joseph Blattman, an immunologist at Arizona State University, who was not affiliated with the research, told GenomeWeb that the researchers used "TCRb deep sequencing in a new way to analyze the lineage relationship between different Th subsets." Overall, he added, the study "provides some really interesting clues regarding the recruitment and selection of CD4 T cells during infection and vaccination."

Different types of pathogens evoke different immune responses. Viral and bacterial pathogens trigger T helper 1 cells, while T helper 17 cells are the primary responders to fungi and extracellular bacteria, and T helper 2 cells dominate the response to a vaccine.

In the study, the researchers sought to understand how "priming" the immune system with a specific pathogen or vaccine changes the diversity of CD4+ T cell clones. They also aimed to elucidate the lineage of the T helper cell subsets and whether they arise from different clones or whether the same clone gives rise to the different subsets depending on the stimulus.

To examine this, the group obtained blood samples from healthy donors through the Swiss Blood Donation Center of Basel and Lugano. They did functional studies, examining cytokine secretion in response to different pathogen and vaccine stimuli, as well as sequence analysis of the TCRb repertoire in response to the stimuli.

The group evaluated how the immune cells responded to the microbes Candida albicans and Mycobacterium tuberculosis, as well as the tetanus toxoid vaccine.

The method itself was somewhat novel as it used cell sorting technology to isolate the specific subsets of Th cells, followed by deep sequencing.

Jian Han, the CEO of immune sequencing firm iRepertoire and not affiliated with the study, said that research like this is representative of immune sequencing's growing applications. Until recently, very few researchers were attempting to sequence the immune repertoire because it was thought to be too variable and complex to be amenable to next-gen sequencing technology, said Han, who is also a faculty investigator at HudsonAlpha Institute for Biotechnology.

Now, he said, classical immunologists are "using [NGS technology] in a way to combine old technology with the new," giving them "a different angle to look at things."

In order to analyze the T cell receptor repertoire of antigen-specific memory cells, the researchers first isolated cells from each group — those primed by C. albicans, M. tuberculosis, and the tetanus vaccine — and then enlisted the help of Adaptive Biotechnologies, which used its ImmunoSeq assay to perform multiplex PCR and deep sequencing  to assess the TCRb clonotypes.

Following priming with C. albicans, the researchers found that although specific Th subsets — the Th17 and Th1* subsets — had higher numbers of T cells compared to the Th1 and Th2 subsets, the number of clonotypes in the four subsets from each of five donors were comparable. In addition, the team found that many clonotypes were shared across Th subsets.

Next, they examined the effects of stimulating with M. tuberculosis. They found high numbers of M. tuberculosis–specific T cells in the Th1* subset and lower numbers in the Th17 subset. In addition, compared to the C. albicans-stimulated cells, they found less clonotype sharing.

The findings "indicate that the main patterns of clonotype sharing between different Th subsets are characteristic for different antigens," the authors wrote. They also speculated that the diversity of T cells observed could be attributed in part to the fact that the C. albicans and M. tuberculosis microbes have multiple antigens. To see whether a single antigen induced a less diverse response, they looked at the effect of inducing cells with the tetatnus toxoid vaccine.

Sequencing found a large number of clonotypes in each of the four Th subsets with a "broad distribution of frequencies and a few dominant clonotypes accounting for a large fraction of total reads," the authors wrote. Somewhat surprisingly, they also found a high level of clonotype sharing among the four subsets, indicating that "in contrast to our expectation, the response to [tetanus vaccine] is characterized by a higher TCR diversity and clonotype sharing as compared to the response to complex microbes," the authors wrote.

Blattman agreed that this was a novel finding, and said that it was likely due to the fact that the vaccine was a booster and the donors had previously been exposed to it. He said there is a "prime/boost selection that occurs," which has been suggested in previous studies to "show preferential expansion of higher affinity clones during recall responses."

Overall, the researchers attributed the heterogeneity to both inter- and intra-clonal heterogeneity. "The data suggests that both pathways occur," Blattman said. "Different clones can be stimulated under different conditions leading to clonal Th-bias," he said, but also, "further proliferation and plasticity in the lineage commitment likely results in clones that occur in more than one Th subset."

Han added that the authors' finding of the diversity and flexibility of T cells is somewhat surprising. The classic paradigm of T cells has been that they are not as diverse and mutable as B cells, but "in this case, they showed that T cells can be quite flexible," he said.