(T0930-06-36) scRNA-seq - A Potential ELISpot Alternative?

Purpose: Monitoring patients’ immune responses to gene therapies is critical to understanding product safety and efficacy. The ELISpot, a cell-based immunoassay, is often used to monitor cellular immune responses by measuring one or two cytokines produced in response to gene therapy product antigens. However, cellular immune responses require coordinated interactions between multiple cell types and monitoring a single analyte may not adequately capture the full response. In this study, single-cell RNA sequencing was evaluated as an alternative to ELISpot for measuring cellular immune responses to gene therapies.

Methods: Human PBMCs from two healthy donors were cultured with and without CEFX and pp65 peptide pools to model antigens produced by gene therapy products following administration. T cells expressing the CD137 activation marker were enriched by magnetic-activated cell sorting (MACS) or by fluorescence-activated cell sorting (FACS). Single cells were then isolated using droplet generation and cDNA libraries were constructed using the Chromium Next GEM Single Cell 5’ Reagent Kit (10X Genomics). Libraries were sequenced using either the NovaSeq 6000 or NextSeq 550. Raw reads were aligned to a reference genome and assigned to cells using the Cell Ranger pipeline. Seurat was used to normalize the resulting UMI count matrices, remove doublets and cells with high mitochondrial gene expression, and integrate cells expressing CD137 transcripts for further analysis, including pathway enrichment analysis using the pathfindR tool.

Results: The proportion of cells expressing CD137 transcripts was 46.5% for FACS-enriched cells, compared to only 7.9% for MACS-enriched cells. When sequencing quality and cell viability were evaluated to determine which cell sorting strategy was best suited for studying cellular immune responses, the results were comparable between cell sorting approaches. The regulatory T cells, cytotoxic T cells, gamma delta (γδ) T cells, and type 1 helper (Th1) T cells found expressing CD137 each increased in frequency with pp65 or CEFX peptide pool stimulation, suggesting antigen-specific T cell responses. When pathway enrichment analysis was performed on their differentially expressed genes, the most common statistically enriched biological process was “response to a virus”. Many of the genes involved in this process were either interferon response factors or interferon inducible genes. To compare the potential anti-viral response between ELISpot and scRNA-seq, ELISpot was used to evaluate responses to CEF and pp65 in the same two donors. In comparison to the negative control (mock), IFN-gamma spot forming units increased ~100 fold in donor 1 and ~40 fold in donor 2 following either pp65 or CEF peptide pool stimulation. Similar trends were observed by scRNA-seq, with the number of T cells expressing IFN-gamma increasing ~130 fold in donor 1 and ~20 fold in donor 2 following either pp65 or CEFX peptide pool stimulation. IFN-gamma gene expression by scRNA-seq was primarily observed in cytotoxic T cells, but was also found in some γδ T cells, Th1 T cells, and a subpopulation of regulatory T cells. Differential gene expression analysis revealed eight additional cytokines were significantly upregulated in these cell types. Cytotoxic T cells showed increased XCL1, XCL2, CCL3, CCL4, and CCL4L2 levels while γδ T cells showed increased CCL5 levels. Both TNF-α and lymphotoxin-α upregulation distinguished Th1 T cells.

Conclusion: By sorting for activated T cells using the CD137 marker, it was possible to study the cellular immune response in antigen-specific cells to pp65 or CEFX. This strategy may also allow for the study of antigen-specific T cells responding to a peptide pool of gene therapy capsid or transgene antigens. CD137 has been reported to induce CD4+ and CD8+ T cell proliferation, survival, and cytokine production. Multiple cytokines were found elevated in CD137+ cytotoxic T cells. Differential gene expression suggested the chemokines XCL1 and XCL2 may be more specific to cytotoxic T lymphocyte (CTL) responses than IFN-gamma. In contrast to the analysis of a single analyte by ELISpot, scRNA-seq revealed γδ T cells, Th1 T cells, and even some regulatory T cells expressed IFN-gamma following stimulation. Regulatory T cells are thought to play an immunosuppressive role by modulating the duration and size of CTL responses. However, some research has shown in inflammatory environments peripheral-derived murine regulatory T cells may adopt cytotoxic functions. Based on the results from this experiment, single-cell RNA sequencing may offer a deeper analysis of the cellular immune response to gene therapies. Observed responses were complex and are likely shaped by multiple cell types varying in frequency and function. Some of these cell responses were unexpected, suggesting this technology has the potential to improve our understanding and ability to monitor gene therapy safety and efficacy.

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UMAP visualization of integrated MACS and FACS datasets containing cells expressing CD137 transcripts. Cell type annotations included to distinguish clusters from each other.

Bar plots depicting IFN-gamma ELISpot and scRNA-seq detection after culturing human PBMCs from two donors with and without peptide pools.

Violin plots depicting the distribution of each marker’s gene expression for clusters identified in both the MACS and FACS datasets.