Transcriptional Profiling of CD8+ CMV-Specific T Cell Functional Subsets Obtained Using a Modified Method for Isolating High-Quality RNA From Fixed and Permeabilized Cells

Abstract

Previous studies suggest that the presence of antigen-specific polyfunctional T cells is correlated with improved pathogen clearance, disease control, and clinical outcomes; however, the molecular mechanisms responsible for the generation, function, and survival of polyfunctional T cells remain unknown. The study of polyfunctional T cells has been, in part, limited by the need for intracellular cytokine staining (ICS), necessitating fixation and cell membrane permeabilization that leads to unacceptable degradation of RNA. Adopting elements from prior research efforts, we developed and optimized a modified protocol for the isolation of high-quality RNA (i.e., RIN > 7) from primary human T cells following aldehyde-fixation, detergent-based permeabilization, intracellular cytokines staining, and sorting. Additionally, this method also demonstrated utility preserving RNA when staining for transcription factors. This modified protocol utilizes an optimized combination of an RNase inhibitor and high-salt buffer that is cost-effective while maintaining the ability to identify and resolve cell populations for sorting. Overall, this protocol resulted in minimal loss of RNA integrity, quality, and quantity during cytoplasmic staining of cytokines and subsequent flourescence-activated cell sorting. Using this technique, we obtained the transcriptional profiles of functional subsets (i.e., non-functional, monofunctional, bifunctional, polyfunctional) of CMV-specific CD8+T cells. Our analyses demonstrated that these functional subsets are molecularly distinct, and that polyfunctional T cells are uniquely enriched for transcripts involved in viral response, inflammation, cell survival, proliferation, and metabolism when compared to monofunctional cells. Polyfunctional T cells demonstrate reduced activation-induced cell death and increased proliferation after antigen re-challenge. Further in silico analysis of transcriptional data suggested a critical role for STAT5 transcriptional activity in polyfunctional cell activation. Pharmacologic inhibition of STAT5 was associated with a significant reduction in polyfunctional cell cytokine expression and proliferation, demonstrating the requirement of STAT5 activity not only for proliferation and cell survival, but also cytokine expression. Finally, we confirmed this association between CMV-specific CD8+ polyfunctionality with STAT5 signaling also exists in immunosuppressed transplant recipients using single cell transcriptomics, indicating that results from this study may translate to this vulnerable patient population. Collectively, these results shed light on the mechanisms governing polyfunctional T cell function and survival and may ultimately inform multiple areas of immunology, including but not limited to the development of new vaccines, CAR-T cell therapies, and adoptive T cell transfer.

Serum and Bronchoalveolar Lavage Fluid Levels of Cytokines in Patients with Lung Cancer and Chronic Lung Disease: A Prospective Comparative Study

“…Thus, the exploration of cytokine production at a cellular level would offer a more comprehensive understanding of the dynamics within the tumor microenvironment. This type of investigation would require more specific techniques, such as cell sorting followed by single-cell RNA sequencing or intracellular cytokine staining followed by flow cytometry [ 43 ], which were beyond the scope of this study.…”

Transcriptomic Approaches in Studies on and Applications of Chimeric Antigen Receptor T Cells

“…This problem is especially pronounced in the work with T cells when it is also necessary to stain intracellular cytokines after permeabilization of cell membrane, exposing the RNA to degradation by enzymes. In this view, development of a protocol for high-quality RNA isolation from human T cells appeared as an especially useful method [ 40 ]. In this protocol, the specific steps consist of fixation with aldehyde, permeabilization with a detergent, staining of intracellular cytokines, and sorting.…”

“…In this protocol, the specific steps consist of fixation with aldehyde, permeabilization with a detergent, staining of intracellular cytokines, and sorting. In addition, an RNase inhibitor and high-salt buffer must be used to prevent activities of nucleases [ 40 ]. It was proved that when using this method of RNA isolation, it is possible to obtain high-quality transcriptional profiles of functional subsets of T cells.…”

“…It was proved that when using this method of RNA isolation, it is possible to obtain high-quality transcriptional profiles of functional subsets of T cells. Therefore, it was suggested that the described protocol can be useful in transcriptomic studies on CAR-T cells [ 40 ].…”

Linking single-cell transcriptomes with secretion using secretion-encoded single-cell sequencing (SEC-seq)

“…In these techniques cells are fixed and permeabilized and then stained with antibodies against the target protein. It is understood that intracellular stores of proteins may not reflect what is secreted, and the process of fixation and permeabilization also is known to degrade mRNA, making downstream transcriptomic measurements noisy 12 . Other techniques, such as ELISpot and the Isolight system (now part of Bruker) enable measurement of proteins secreted from the cell, which are captured on surfaces surrounding cells and detected with fluorophore-or enzyme-labeled antibodies 13 .…”