Abstract
Existing approaches that quantify cytotoxic T cell responses rely on bulk or surrogate measurements which impede the direct identification of single activated T cells of interest. Single cell microscopy or flow cytometry methodologies typically rely on fluorescent labeling, which limits applicability to primary cells such as human derived T lymphocytes. Here, we introduce a quantitative method to track single T lymphocyte mediated cytotoxic events within a mixed population of cells using live cell interferometry (LCI), a label-free microscopy technique that maintains cell viability. LCI quantifies the mass distribution within individual cells by measuring the phase shift caused by the interaction of light with intracellular biomass. Using LCI, we imaged cytotoxic T cells killing cognate target cells. In addition to a characteristic target cell mass decrease of 20–60% over 1–4 h following attack by a T cell, there was a significant 4-fold increase in T cell mass accumulation rate at the start of the cytotoxic event and a 2–3 fold increase in T cell mass relative to the mass of unresponsive T cells. Direct, label-free measurement of CD8+ T and target cell mass changes provides a kinetic, quantitative assessment of T cell activation and a relatively rapid approach to identify specific, activated patient-derived T cells for applications in cancer immunotherapy.
Highlights
CD8+ T lymphocyte mediated cytotoxicity is a critical component of the adaptive immune response against viruses and cancers, and is implicated in autoimmunity [1,2]
As a proof of concept, we demonstrate tracking of up to 2,000 individual CTLs with specificity toward Melanocytic Antigen Recognized by T lymphocytes (MART1) responding against human leukocyte antigen (HLA) matched MART1+ target cells [15]
We developed a model system for analyzing cytotoxicity events by establishing the antigen specificity of healthy human donor CD8+ enriched lymphocytes against HLA matched target cell lines
Summary
CD8+ T lymphocyte mediated cytotoxicity is a critical component of the adaptive immune response against viruses and cancers, and is implicated in autoimmunity [1,2]. The introduction of peptide-MHC tetramers and microfluidic platforms has allowed for surrogate measures of cytotoxicity through analysis of T cell antigen specificity and cytokine secretion [3,5,6]. Tracking T lymphocyte mediated cytotoxicity at the single cell level is advantageous for analyzing cytotoxic T cells (CTLs) within a mixed population, which is of particular relevance in assessing T cell recognition against cancer cells. Viable CTLs can potentially be cultured and expanded further, or the corresponding T cell receptors (TCRs) bearing optimal specificity toward immunogenic peptides can be molecularly cloned for utilization in a clinical setting [7]
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