CIPHER-seq bridges the RNA-protein divide to reveal real-time cytokine dynamics in single immune cells
A novel single-cell multiomics platform developed at the Sylvester Comprehensive Cancer Center simultaneously quantifies intracellular proteins and transcriptomes, overcoming a longstanding limitation of RNA-based immune profiling. By capturing RNA, surface proteins, intracellular cytokines, and multiplexing data in one workflow, CIPHER-seq delivers a five-layer picture of immune activation with markedly reduced cellular stress artefacts – offering new precision for cancer immunotherapy research and patient stratification.
Single-cell RNA sequencing (scRNA-seq) has transformed immune cell biology, yet carries a fundamental limitation: transcript abundance correlates poorly with protein levels, with RNA-protein correlations typically ranging from r = 0.1–0.4, and cytokines consistently ranking among the least concordant gene classes. To close this gap, researchers at the University of Miami’s Sylvester Comprehensive Cancer Center, with collaborators at the University of California, San Francisco, have developed CIPHER-seq – Cytokine Intracellular Protein High-throughput Expression with RNA-sequencing. The findings were published in Scientific Reports on 8 April 2026.
The RNA-protein gap and why it matters
To establish the scale of the problem, the team reanalysed two external proteotranscriptomic datasets – a breast cancer cohort and the SPARC single-cell dataset – finding Spearman correlation coefficients distributed near zero across more than 3,300 shared genes, with cytokines and immune activation markers identified as the least concordant features in both cohorts.
The discordance is not technical noise but a biological reality. As co-senior author Emiliano Cocco, Ph.D., assistant professor of biochemistry and molecular biology at the Miller School of Medicine, explained: “In immune cells, RNA and protein don’t always rise and fall together. RNA can appear quickly and disappear just as fast, while proteins take time to build up and may linger longer. Studying RNA alone can miss this timing.”
Developing and benchmarking CIPHER-seq
CIPHER-seq integrates optimised fixation chemistry, controlled perme-abilisation, abbreviated oligonucleotide-conjugated antibody incubation, and RNase-protective conditions to capture five simultaneous information layers from a single cell: whole-transcriptome scRNA-seq, cell-surface protein profiles, intracellular cytokine measurements, broader intracellular protein detection, and sample multiplexing via oligo-tagged barcodes.
The platform was benchmarked against commercially available intracellular protocols from Proteintech, BioLegend, and BD using resting and PMA/ionomycin-stimulated peripheral blood mononuclear cells (PBMCs). Flow cytometry screening for tubulin accessibility showed that BD and BioLegend protocols failed to provide adequate intracellular antigen access, leaving only CIPHER-seq and Proteintech to advance to single-cell sequencing.
Despite comparable immune subset recovery across both methods, RNA quality metrics diverged markedly. Proteintech-processed samples exhibited substantially elevated mitochondrial transcript percentages, indicative of fixation-induced cellular stress, whereas CIPHER-seq samples maintained low mitochondrial content. The authors note that CIPHER-seq “retains high-quality transcriptomes and preserves biologically meaningful RNA-protein relationships while matching Proteintech in immune cell recovery.”
“We wanted a method that lets cells stay as close as possible to their natural state,” said co-senior author Justin Taylor, M.D., a Sylvester physician-scientist and The Pap Corps Endowed Professor in Leukemia.
Capturing cytokine kinetics during immune activation
Applied to stimulated PBMCs, CIPHER-seq resolved robust upregulation of interferon-gamma (IFNG) and tumour necrosis factor (TNF) at both RNA and protein levels across multiple immune lineages, with cytokines ranking among the top effect-size responders. Polyfunctionality analysis demonstrated stimulation-dependent expansion of IFNG⁺, TNF⁺, and double-positive cells.
Pseudotime trajectory analysis revealed that IFNG RNA induction preceded peak protein accumulation with a Δpeak pseudotime of 0.027 – a short but consistent temporal offset supporting the expected biological ordering of transcription before protein accumulation. First author Avni Bhalgat, Ph.D., described the significance: “It’s like seeing the plan before the action. Cytokines help determine whether immune cells attack cancer, ignore it or even help tumours grow. Understanding how and when immune cells produce these signals is critical.”
Translational outlook
Taylor highlighted the clinical potential: “RNA gives us clues about where a cell is headed. Proteins show us where it actually arrives, and this clearer picture could help scientists design better immunotherapies and help clinicians predict which patients are most likely to benefit from them.”
The authors conclude that “by minimising fixation-induced stress while enabling sensitive detection of intracellular cytokines, CIPHER-seq generates high-quality, interpretable data that capture both the magnitude and timing of immune responses.” Data are publicly available via the Gene Expression Omnibus (accession GSE314400).
Journal reference: Bhalgat, A., Micin, K., Affer, M., et al. (2026). CIPHER-seq enables intracellular multimodal profiling of cytokine responses in single immune cells. Scientific Reports, 16, 9693. https://doi.org/10.1038/s41598-026-44946-y
Co-senior author Justin Taylor, M.D., a Sylvester physician-scientist and The Pap Corps Endowed Professor in Leukemia at Sylvester Comprehensive Cancer Centre.
