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.
Highlights
High-throughput RNA sequencing (RNAseq) is a powerful tool for the quantification and global analysis of the transcriptome of cells, providing valuable insight to cell function and mechanisms of disease
The high salt buffer resulted in a slight improvement in RNA stability and was far more cost-effective when compared to the MARIS protocol, prior research has suggested that higher salt concentrations were expected to impact antibody binding and downstream enzymatic processes [17]
The presence of a 2M NaCl buffer during intracellular staining steps significant reduced the staining of several cytokines (i.e., TNFα, IL-2) when compared to a standard protocol, and appeared to have heterogenous effects on the staining of transcription factors (Supplemental Figures S1C,D)
Summary
High-throughput RNA sequencing (RNAseq) is a powerful tool for the quantification and global analysis of the transcriptome of cells, providing valuable insight to cell function and mechanisms of disease. While high-quality RNA can be obtained from FACS of live cells, investigators are currently limited to isolating cell populations based on surface protein expression. Cell fixation with aldehydes leads to significant RNA-protein crosslinking and fragmentation, while permeabilization with ionic detergents leads to further fragmentation and allows for the introduction of detrimental RNases to the intracellular compartments [2]. This processing can lead to significant degradation of RNA quality, as measured by both the RNA integrity number (RIN) and the DV200 [3, 4]. Maintaining RNA quality is of utmost importance in generating robust, unbiased RNAseq datasets
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