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HIT: a versatile proteomics platform for multianalyte phenotyping of cytokines, intracellular proteins and surface molecules

Nature Medicine volume 14, pages 1284–1289 (2008)Cite this article

Abstract

We have developed a multianalyte fluid-phase protein array technology termed high-throughput immunophenotyping using transcription (HIT). This method employs a panel of monoclonal antibodies, each tagged with a unique oligonucleotide sequence that serves as a molecular bar code. After staining a sample, T7 polymerase amplifies the tags, which are then hybridized to a DNA microarray for indirect measurement of each analyte. Although there are many potential applications for this technology, here we report its suitability for profiling cytokines, intracellular molecules and cell surface markers. Using HIT, we profiled 90 surface markers on human naive T helper cells activated in vitro. The markers identified in this screen are consistent with previously described activation markers and were validated by flow cytometry. Additionally, a HIT screen of surface markers expressed on T helper cells activated in the presence of transforming growth factor-β identified downregulation of CD26 in these cells. HIT arrays are an ideal platform for rapidly identifying markers for further characterization and therapeutic intervention.

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Figure 1: Schematic depicting the general format of HIT.
Figure 2: ELISA format HIT.
Figure 3: Surface marker profiling format of HIT.
Figure 4: Surface marker phenotype of activated versus resting primary human naive T helper cells.
Figure 5: HIT identifies downregulation of CD26 on TGF-β–treated human T helper cells and a subset of circulating FOXP3+ cells.

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References

  1. Fathman, C.G., Soares, L., Chan, S.M. & Utz, P.J. An array of possibilities for the study of autoimmunity. Nature 435, 605–611 (2005).

    Article  CAS  Google Scholar 

  2. Chan, S.M. & Utz, P.J. The challenge of analyzing the proteome in humans with autoimmune diseases. Ann. NY Acad. Sci. 1062, 61–68 (2005).

    Article  Google Scholar 

  3. Chan, S.M., Ermann, J., Su, L., Fathman, C.G. & Utz, P.J. Protein microarrays for multiplex analysis of signal transduction pathways. Nat. Med. 10, 1390–1396 (2004).

    Article  CAS  Google Scholar 

  4. Robinson, W.H. et al. Autoantigen microarrays for multiplex characterization of autoantibody responses. Nat. Med. 8, 295–301 (2002).

    Article  CAS  Google Scholar 

  5. Balboni, I. et al. Multiplexed protein array platforms for analysis of autoimmune diseases. Annu. Rev. Immunol. 24, 391–418 (2006).

    Article  CAS  Google Scholar 

  6. Schweitzer, B. et al. Multiplexed protein profiling on microarrays by rolling-circle amplification. Nat. Biotechnol. 20, 359–365 (2002).

    Article  CAS  Google Scholar 

  7. Nielsen, U.B., Cardone, M.H., Sinskey, A.J., MacBeath, G. & Sorger, P.K. Profiling receptor tyrosine kinase activation by using Ab microarrays. Proc. Natl. Acad. Sci. USA 100, 9330–9335 (2003).

    Article  Google Scholar 

  8. Belov, L. et al. Analysis of human leukaemias and lymphomas using extensive immunophenotypes from an antibody microarray. Br. J. Haematol. 135, 184–197 (2006).

    Article  CAS  Google Scholar 

  9. Hendrickson, E.R., Truby, T.M., Joerger, R.D., Majarian, W.R. & Ebersole, R.C. High sensitivity multianalyte immunoassay using covalent DNA-labeled antibodies and polymerase chain reaction. Nucleic Acids Res. 23, 522–529 (1995).

    Article  CAS  Google Scholar 

  10. Niemeyer, C.M., Adler, M. & Wacker, R. Immuno-PCR: high sensitivity detection of proteins by nucleic acid amplification. Trends Biotechnol. 23, 208–216 (2005).

    Article  CAS  Google Scholar 

  11. Sano, T., Smith, C.L. & Cantor, C.R. Immuno-PCR: very sensitive antigen detection by means of specific antibody-DNA conjugates. Science 258, 120–122 (1992).

    Article  CAS  Google Scholar 

  12. Zhou, H. et al. Two-color, rolling-circle amplification on antibody microarrays for sensitive, multiplexed serum-protein measurements. Genome Biol. 5, R28 (2004).

    Article  Google Scholar 

  13. Zhang, H.T., Kacharmina, J.E., Miyashiro, K., Greene, M.I. & Eberwine, J. Protein quantification from complex protein mixtures using a proteomics methodology with single-cell resolution. Proc. Natl. Acad. Sci. USA 98, 5497–5502 (2001).

    Article  CAS  Google Scholar 

  14. Zhang, H., Cheng, X., Richter, M. & Greene, M.I. A sensitive and high-throughput assay to detect low-abundance proteins in serum. Nat. Med. 12, 473–477 (2006).

    Article  CAS  Google Scholar 

  15. Fredriksson, S. et al. Multiplexed protein detection by proximity ligation for cancer biomarker validation. Nat. Methods 4, 327–329 (2007).

    Article  CAS  Google Scholar 

  16. Gullberg, M. et al. Cytokine detection by antibody-based proximity ligation. Proc. Natl. Acad. Sci. USA 101, 8420–8424 (2004).

    Article  CAS  Google Scholar 

  17. Nam, J.M., Thaxton, C.S. & Mirkin, C.A. Nanoparticle-based bio–bar codes for the ultrasensitive detection of proteins. Science 301, 1884–1886 (2003).

    Article  CAS  Google Scholar 

  18. Howarth, M. et al. A monovalent streptavidin with a single femtomolar biotin binding site. Nat. Methods 3, 267–273 (2006).

    Article  CAS  Google Scholar 

  19. Kattah, M.G., Alemi, G.R., Thibault, D.L., Balboni, I. & Utz, P.J. A new two-color Fab labeling method for autoantigen protein microarrays. Nat. Methods 3, 745–751 (2006).

    Article  CAS  Google Scholar 

  20. Brown, J.K., Pemberton, A.D., Wright, S.H. & Miller, H.R. Primary antibody-Fab fragment complexes: a flexible alternative to traditional direct and indirect immunolabeling techniques. J. Histochem. Cytochem. 52, 1219–1230 (2004).

    Article  CAS  Google Scholar 

  21. Kozlov, I.A. et al. Efficient strategies for the conjugation of oligonucleotides to antibodies enabling highly sensitive protein detection. Biopolymers 73, 621–630 (2004).

    Article  CAS  Google Scholar 

  22. Tweeddale, M.E. et al. The presence of clonogenic cells in high-grade malignant lymphoma: a prognostic factor. Blood 69, 1307–1314 (1987).

    CAS  PubMed  Google Scholar 

  23. Fantini, M.C. et al. Cutting edge: TGF-β induces a regulatory phenotype in CD4+CD25− T cells through Foxp3 induction and down-regulation of Smad7. J. Immunol. 172, 5149–5153 (2004).

    Article  CAS  Google Scholar 

  24. Bettelli, E., Oukka, M. & Kuchroo, V.K. TH17 cells in the circle of immunity and autoimmunity. Nat. Immunol. 8, 345–350 (2007).

    Article  CAS  Google Scholar 

  25. Wong, J., Mathis, D. & Benoist, C. TCR-based lineage tracing: no evidence for conversion of conventional into regulatory T cells in response to a natural self-antigen in pancreatic islets. J. Exp. Med. 204, 2039–2045 (2007).

    Article  CAS  Google Scholar 

  26. Morimoto, C. & Schlossman, S.F. The structure and function of CD26 in the T-cell immune response. Immunol. Rev. 161, 55–70 (1998).

    Article  CAS  Google Scholar 

  27. Reinhold, D. et al. Inhibitors of dipeptidyl peptidase IV/CD26 suppress activation of human MBP-specific CD4+ T cell clones. J. Neuroimmunol. 87, 203–209 (1998).

    Article  CAS  Google Scholar 

  28. Krakauer, M., Sorensen, P.S. & Sellebjerg, F. CD4+ memory T cells with high CD26 surface expression are enriched for TH1 markers and correlate with clinical severity of multiple sclerosis. J. Neuroimmunol. 181, 157–164 (2006).

    Article  CAS  Google Scholar 

  29. Love, J.C., Ronan, J.L., Grotenbreg, G.M., van der Veen, A.G. & Ploegh, H.L. A microengraving method for rapid selection of single cells producing antigen-specific antibodies. Nat. Biotechnol. 24, 703–707 (2006).

    Article  CAS  Google Scholar 

  30. Michaud, G.A. et al. Analyzing antibody specificity with whole proteome microarrays. Nat. Biotechnol. 21, 1509–1512 (2003).

    Article  CAS  Google Scholar 

  31. Tusher, V.G., Tibshirani, R. & Chu, G. Significance analysis of microarrays applied to the ionizing radiation response. Proc. Natl. Acad. Sci. USA 98, 5116–5121 (2001).

    Article  CAS  Google Scholar 

  32. Saeed, A.I. et al. TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34, 374–378 (2003).

    Article  CAS  Google Scholar 

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Acknowledgements

We thank S. Chan, D. Thibault, J. Mollick, P. Teo, A. Venteicher, S. Wrenn, R. Tibshirani, M. Schaner, J. Chi and other members of our laboratory for helpful discussion and technical assistance. We thank A. Ting (Massachusetts Institute of Technology) for kindly providing mSA plasmids. We also thank the Stanford University Human Immune Monitoring Center for assistance with Luminex cytokine data acquisition. M.G.K. was funded by the Stanford Medical Scientist Training Program and the Floren Family Trust. N.O. was funded by the Center for Clinical Immunology at Stanford Summer Research Program and the Northern California Chapter of the Arthritis Foundation. R.K.C. was funded by the Stanford Program in Immunology training grant 5 T32 AI07290. P.J.U. is the recipient of a Donald E. and Delia B. Baxter Foundation Career Development Award and was supported by the Dana Foundation, the Floren Family Trust, the Northern California Chapter of the Arthritis Foundation, US National Institutes of Health Grants DK61934, AI50854, AI50865 and AR49328 and National Heart, Lung, and Blood Institute Proteomics Contract N01-HV-28183.

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  1. Department of Medicine, Division of Immunology and Rheumatology, Stanford University School of Medicine, Stanford, 94305, California

    Michael G Kattah, Regina K Cheung, Neekaan Oshidary & Paul J Utz

  2. Stanford Functional Genomics Facility, Stanford University School of Medicine, Stanford, 94305, California

    John Coller

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Correspondence to Paul J Utz.

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Competing interests

In the past three years, P.J.U. has served as a consultant to Centocor, Biogen Idec, Genentech, Johnson & Johnson, AstraZeneca, Avanir, Gilead Sciences, Amgen, and UCB. P.J.U. is also a member of the Scientific Advisory Boards of Monogram Biosciences, Argos and xDx and is a cofounder and consultant of Bayhill Therapeutics.

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Kattah, M., Coller, J., Cheung, R. et al. HIT: a versatile proteomics platform for multianalyte phenotyping of cytokines, intracellular proteins and surface molecules. Nat Med 14, 1284–1289 (2008). https://doi.org/10.1038/nm.1755

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