|
在這篇文章里，Roederer和他的團隊提出了一種在平臺使用多重TaqMan試劑對單個T細胞進行分析的方法。首先，使用BD FACSAria™，根據(jù)細胞的標記將其分離以得到單細胞。接下來，Roederer細化其方法學，確保獲得足量的RNA以進行單細胞的表達譜分析。Roederer等展示了低至一個mRNA轉錄本的檢測極限（limit of detection, LOD），結果表明的高靈敏度和寬的動態(tài)范圍，可以檢測單細胞當量的mRNA。
該團隊在單細胞水平監(jiān)測了T細胞的激活，發(fā)現(xiàn)了群體中CD4+細胞的亞群。實際上，CD4+細胞表達類似CSCR5/CCL5和DPPR/TIA1這樣的基因是稀有事件，并非之前使用大量mRNA觀察到的是普遍事件
通過比較CD154+和CD154- T細胞對葡萄球菌腸毒素B（SEB, Staphlyococcal enterotoxin B）的反應，（細胞）的異質性被進一步確立。當被刺激時，分析顯示有一組基因和T細胞的激活強烈相關，但是，只有5-20%的T細胞擁有可以結合SEB的受體Vβ鏈變體。
在這項研究中，細胞異質性被證明有很強的免疫相關性。Roederer和他的團隊正繼續(xù)推進系統(tǒng)的、單細胞的方法以更好地理解中樞免疫系統(tǒng)中細胞和分子的相互作用。
參考文獻
1、Dominguez, M.H., Chattopadhyay, P.K., 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, P. K., 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, J. M., Douek, D. C., 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, P. K., Perfetto, S. P. &Roederer, M. The colorful future of cell analysis by flow cytometry. Discovery Medicine 4, 255-262 (2004).
6、De Rosa, S. C., Lu, F. X., Perfetto, S. P., Yu, J., Moser, S., Miller, C. J., Evans, T. G. &Roederer, M. Vaccination in humans generates broad T cell cytokine responses. J. Immunol. 173, 5372-5380 (2004).
7、Perfetto, S. P., Chattopadhyay, P. K. &Roederer, M. 17-Color Flow Cytometry: Unraveling the Immune System. Nature Reviews Immunology 4, 648-655 (2004).
8、De Rosa, S. C., Andrus, J. P., Perfetto, S. P., Mantovani, J. J., Herzenberg, L. A., Herzenberg, L. A. &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, S. C., Brenchley, J. M. &Roederer, M. Beyond six colors: A new era in flow cytometry. Nature Medicine 9, 112-117 (2003).
10、De Rosa, S. C., Herzenberg, L. A. &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, B. J., Lore, K., Koup, R. A., Kedl, R. M., Mattapallil, J. M., Weiss, W. R., Roederer, M. & Seder, R. A. 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, C. R., Mattapallil, J. M., Igarashi, T., Buckler-White, A., Lafont, B. A. P., Hirsch, V. M., 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, R. L., Hill, B. J., De Rosa, S., Perfetto, S. P., Zhang, H. H., Foley, J. F., Reiner, J. S., Douek, D. C., Roederer, M. & Farber, J. M. CXCR3 and CCR4 Reveal Polarized Early Central Memory CD4+ T Cells in Humans. ProcNatlAcadSci USA 102, 7916-7921 (2005).
14、Betts, M. R., Exley, B., Price, D. A., Bansal, A., Camacho, Z. T., Teaberry, V., West, S. M., Ambrozak, D. R., Tomaras, G., Roederer, M., Kilby, J. M., Tartaglia, J., Belshe, R., Gao, F., Douek, D. C., Weinhold, K. J., Koup, R. A., 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. ProcNatlAcadSci (USA) 102, 4512-7 (2005).
15、Stuge, T., Holmes, S. P., Saharan, S., Rubio, V., Roederer, M., Weber, J. & Lee, P. P. Complexity of endogenous versus vaccine-elicited T cell responses to cancer.PLoS Medicine 1, e28 (2004).
16、Betts, M. R., Price, D. A., Brenchley, J. M., Lore, K., Guenaga, F., Smed-Sorensen, A., Ambrozak, D. R., Migueles, S. A., Connors, M., Roederer, M., Douek, D. C. &Koup, R. The functional profile of of primary human antiviral CD8(+) T cell effector activity is dictated by cognate peptide concentration. J. Immunol. 10, 6407-6417 (2004).
17、Sullivan, N. J., Geisbert, T. W., Geisbert, J. B., Xu, L., Yang, Z.-y., Roederer, M., Koup, R. A., Jahrling, P. B. &Nabel, G. Accelerated vaccination for Ebola virus hemorrhagic fever in nonhuman primates. Nature 424, 681-684 (2004).
18、Rubio, V., Stuge, T., Betts, M. R., Weber, J., Roederer, M. & Lee, P. P. Ex vivo identification, isolation, and analysis of tumor-cytolytic T cells. Nature Medicine 9, 1377-1382 (2003).
19、Brenchley, J. M., Karandikar, N. J., Betts, M. R., Ambrozak, D. R., Hill, B. J., Crotty, L. E., Casazza, J. P., Kuruppu, J., Migueles, S. A., Connors, M., Roederer, M., Douek, D. C. &Koup, R. A. Expression of CD57 defines replicative senescence and antigen-induced apoptotic death of CD8+ T-cells. Blood 101, 2711-2720 (2003).
20、Yang Shih, T. A., Meffre, E., Roederer, M. &Nussenzweig, M. C. Role of BCR affinity in T cell dependent antibody responses in vivo. Nature Immunology 3, 570-5. (2002).
21、Shih, T. A., Roederer, M. &Nussenzweig, M. C. Role of antigen receptor affinity in T cell-independent antibody responses in vivo. Nature Immunology 3, 399-406. (2002). |
