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A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells

Abstract

Cellular immunity has an inherent high level of functional heterogeneity. Capturing the full spectrum of these functions requires analysis of large numbers of effector molecules from single cells. We report a microfluidic platform designed for highly multiplexed (more than ten proteins), reliable, sample-efficient (1 × 104 cells) and quantitative measurements of secreted proteins from single cells. We validated the platform by assessment of multiple inflammatory cytokines from lipopolysaccharide (LPS)-stimulated human macrophages and comparison to standard immunotechnologies. We applied the platform toward the ex vivo quantification of T cell polyfunctional diversity via the simultaneous measurement of a dozen effector molecules secreted from tumor antigen–specific cytotoxic T lymphocytes (CTLs) that were actively responding to tumor and compared against a cohort of healthy donor controls. We observed profound, yet focused, functional heterogeneity in active tumor antigen–specific CTLs, with the major functional phenotypes quantitatively identified. The platform represents a new and informative tool for immune monitoring and clinical assessment.

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Figure 1: Design of the SCBC for single-cell protein secretome analysis.
Figure 2: On chip secretion measurements of macrophage differentiated from THP-1 monocyte cell.
Figure 3: Single-cell secretion measurements of CTLs from individuals with melanoma and healthy donors.
Figure 4: Polyfunctional diversity analysis for CTLs from a subject with metastatic melanoma and from healthy donors.

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Acknowledgements

We thank B. Marzolf at the Institute for Systems Biology for printing DNA-spotted arrays and the UCLA nanolab for photomask fabrication. We thank L. Yang, S. Wang, R. Diamond and H. Wu for valuable discussion. C.M. acknowledges the support of the Benjamin M. Rosen Fellowship. R.F. is supported by the US National Institutes of Health K99 Pathway to Independence Award (No. 1 K99 CA136759-01). This work was funded by the US National Cancer Institute Grant No. 5U54 CA119347 (J.R.H.), by the Ivy Foundation and the Jean Perkins Foundation (J.R.H.), by the California Institute for Regenerative Medicine New Faculty Award RN2-00902-1 (A.R.), by the Caltech/UCLA Joint Center for Translational Medicine (A.R. and J.R.H.) and the Melanoma Research Alliance (A.R. and J.R.H.). The UCLA Flow Cytometry Core Facility is supported by the US National Institutes of Health awards CA-16042 and AI-28697.

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Authors and Affiliations

Authors

Contributions

C.M. conducted T cell experiments and analyzed data. R.F. conducted macrophage experiments. C.M. and R.F. performed validation experiments and designed the chip. H.A. wrote Excel macros. Q.S., C.-C.L. and G.A.K. helped with experiments. B.C.-A., T.C. and R.C.K. collected T cell samples and conducted flow cytometry phenotyping experiments. C.M., R.F. and J.R.H. conceived of the experiments. C.M., R.F., C.G.R., A.R. and J.R.H. wrote the manuscript.

Corresponding author

Correspondence to James R Heath.

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The authors declare no competing financial interests.

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Supplementary Figures 1–9, Supplementary Table 1–3 and Supplementary Methods (PDF 1568 kb)

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Ma, C., Fan, R., Ahmad, H. et al. A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells. Nat Med 17, 738–743 (2011). https://doi.org/10.1038/nm.2375

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