In this article, Roederer and his team propose a method for analyzing individual T cells using multiple TaqMan reagents on the BioMark platform. First, BD FACSAriaTM was used to separate the cells according to the label of the cells to obtain single cells. Next, Roederer refines its methodology to ensure that a sufficient amount of RNA is obtained for single-cell expression profiling. Roederer et al. demonstrated a limit of detection (LOD) down to an mRNA transcript. The results indicate that BioMark's high sensitivity and wide dynamic range allow for the detection of single cell equivalent mRNA. The team monitored T cell activation at the single cell level and found a subpopulation of CD4+ cells in the population. In fact, it is rare for CD4+ cells to express genes like CSCR5/CCL5 and DPPR/TIA1, not the common events observed with large amounts of mRNA. The heterogeneity of (cells) was further established by comparing the responses of CD154+ and CD154-T cells to Staphlyococcal enterotoxin B (SEB, Staphlyococcal enterotoxin B). When stimulated, analysis showed a strong association between the activation of a panel of genes and T cells, but only 5-20% of T cells possess receptor Vβ chain variants that bind to SEB. In this study, cellular heterogeneity proved to be highly immune-related. Roederer and his team are continuing to advance systematic, single-cell approaches to better understand the interaction of cells and molecules in the central immune system. references 1. Dominguez, MH, Chattopadhyay, PK, Ma, S., Lamoreaux, L., McDavid, A., Finak, G., Gottardo, R., Koup, R., &Roederer, M. Highly multiplexed quantitation of gene expression On single cells. Journal of Immunological Methods in press 2013 (13 pages). 2. Kern, F., LiPira, G., Gratama, J., Manca, F. & Roederer, M. Measuring antigen specific immune responses: Understanding immunopathogenesis and improving diagnostics in infectious disease, autoimmunity and cancer. Trends in Immunology 26, 477 -484 (2005). 3. Chattopadhyay, PK, Yu, J. & Roederer, M. A live-cell assay to detect antigen-specific CD4 T cells with diverse cytokine profiles. Nature Medicine 11, 1113-1117 (2005). 4. Mattapallil, JM, Douek, DC, Hill, B., Nishimura, Y., Martin, M. & Roederer, M. Massive infection and loss of memory CD4 T cells in multiple tissues during acute SIV infection. Nature 434, 1093- 1097 (2005). 5. Chattopadhyay, PK, Perfetto, SP & Roederer, M. The colorful future of cell analysis by flow cytometry. Discovery Medicine 4, 255-262 (2004). 6. De Rosa, SC, Lu, FX, Perfetto, SP, Yu, J., Moser, S., Miller, CJ, Evans, TG & Roederer, M. Vaccination in humans generating broad T cell cytokine responses. J. Immunol. 173, 5372-5380 (2004). 7. Perfetto, SP, Chattopadhyay, PK & Roederer, M. 17-Color Flow Cytometry: Unraveling the Immune System. Nature Reviews Immunology 4, 648-655 (2004). 8. De Rosa, SC, Andrus, JP, Perfetto, SP, Mantovani, JJ, Herzenberg, LA, Herzenberg, LA & Roederer, M. Ontogeny of gamma-delta T cells in man. J. Immunol. 172, 1637-45 ( 2004). 9. Berhanu, D., Mortari, F. & Roederer, M. Optimized lymphocyte isolation methods for analysis of chemokine receptor expression. J. Immunol. Methods 279, 199-207 (2003). De Rosa, SC, Brenchley, JM & Roederer, M. Beyond six colors: A new era in flow cytometry. Nature Medicine 9, 112-117 (2003). 10. De Rosa, SC, Herzenberg, LA & Roederer, M. 11-color, 13-parameter flow cytometry: identification of human naive T cells by phenotype, function, and T-cell receptor diversity. Nature Medicine 7, 245-8. (2001). 11. Wille-Reece, U., Flynn, BJ, Lore, K., Koup, RA, Kedl, RM, Mattapallil, JM, Weiss, WR, Roederer, M. & Seder, RA HIV Gag protein conjugated to a TLR7/ 8 agonist influences the magnitude and quality of Th1 and CD8+ T cell responses in nonhuman primates. ProcNatlAcadSci (USA) , in press (2005). 12. Nishimura, Y., Brown, CR, Mattapallil, JM, Igarashi, T., Buckler-White, A., Lafont, BAP, Hirsch, VM, Roederer, M. & Martin, M. Non-activated naive CD4 T Cells are the principal source of progeny virus during acute infections of monkeys with highly pathogenic X4-tropic SHIVs. ProcNatlAcadSci (USA) 102, 8000-8005 (2005). 13, Song, K., Rabin, RL, Hill, BJ, De Rosa, S., Perfetto, SP, Zhang, HH, Foley, JF, Reiner, JS, Douek, DC, Roederer, M. & Farber, JM CXCR3 And CCR4 Reveal Polarized Early Central Memory CD4+ T Cells in Humans. ProcNatlAcadSci USA 102, 7916-7921 (2005). 14, Betts, MR, Exley, B., Price, DA, Bansal, A., Camacho, ZT, Teaberry, V., West, SM, Ambrozak, DR, Tomaras, G., Roederer, M., Kilby, JM , Tartaglia, J., Belshe, R., Gao, F., Douek, DC, Weinhold, KJ, Koup, RA, Goepfert, P. & Ferrari, G. Characterization of functional and phenotypic changes in anti-Gag vaccine-induced T cell responses and their role in protection after HIV-1 infection. ProcNatl AcadSci ( USA) 102, 4512-7 (2005). 15. Stuge, T., Holmes, SP, Saharan, S., Rubio, V., Roederer, M., Weber, J. & Lee, PP Complexity of endogenous versus vaccine-elicited T cell responses to cancer. PLoS Medicine 1 , e28 (2004). 16, Betts, MR, Price, DA, Brenchley, JM, Lore, K., Guenaga, F., Smed-Sorensen, A., Ambrozak, DR, Migueles, SA, Connors, M., Roederer, M., Douek , DC &Koup, R. The functional profile of of primary human antiviral CD8(+) T cell effector activity is dictated by cogyl peptide concentration. J. Immunol . 10, 6407-6417 (2004). 17. Sullivan, NJ, Geisbert, TW, Geisbert, JB, Xu, L., Yang, Z.-y., Roederer, M., Koup, RA, Jahrling, PB & Nabel, G. Accelerated vaccination for Ebola virus hemorrhagic fever In nonhuman primates. Nature 424, 681-684 (2004). 18. Rubio, V., Stuge, T., Betts, MR, Weber, J., Roederer, M. & Lee, PP Ex vivo identification, isolation, and analysis of tumor-cytolytic T cells. Nature Medicine 9, 1377- 1382 (2003). 19, Brenchley, JM, Karandikar, NJ, Betts, MR, Ambrozak, DR, Hill, BJ, Crotty, LE, Casazza, JP, Kuruppu, J., Migueles, SA, Connors, M., Roederer, M., Douek , DC &Koup, RA Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T-cells. Blood 101, 2711-2720 (2003). 20, Yang Shih, TA, Meffre, E., Roederer, M. & Nussenzweig, MC Role of BCR affinity in T cell dependent antibody responses in vivo. Nature Immunology 3, 570-5. (2002). 21, Shih, TA, Roederer, M. & Nussenzweig, MC Role of antigen receptor affinity in T cell-independent antibody responses in vivo. Nature Immunology 3, 399-406. (2002). |
