The Tetramer Transformation Peter C

The Tetramer Transformation Peter C. Doherty J Immunol 2011; 187:5-6; ; This information is current as doi: 10.4049/jimmunol.1101297 of September 24, 2021. http://www.jimmunol.org/content/187/1/5 Downloaded from References This article cites 33 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/187/1/5.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2011 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Tetramer Transformation Peter C. Doherty

wo events that were very significant in my life hap- This is where the field was (9) when I gave my December pened in October 1996. The first was an early 1996 Nobel Lecture in Stockholm (3). Although the overall T morning call from Stockhom telling me that Rolf concepts we were working with at that time remain funda- Zinkernagel and I were to share the Nobel Prize for Physiology mentally sound, tetramer staining (2, 10) experiments done or Medicine. The second was the publication of the peptide 1 through 1997–1998 showed that our 1996 understanding of 1 MHC class I (pMHCI) tetramer staining technology for responder CD8 T cell numbers was at least 10-fold off! 1 identifying HIV-specific CD8 T cells (1). At that time, I was Perhaps the inherent inefficiency of the LDA technique very aware of the former and, being totally distracted, quite reflects that only a small subset of CTL precursors can be ignorant of the latter. That changed in the following year, expanded under these culture conditions. Although, for ex- Downloaded from 1 1 when Rafi Ahmed at Emory University made contact and sug- ample, CD62Lhi and CD62Llo tetramer CD8 precursors gested we should use the tetramer approach to look at the in- are found through both the acute and the early memory 1 fluenza virus-specific CD8 T cell response. Kirsten Flynn phases of the influenza-specific response (11), the T cells mea- traveled there to learn the technique from John Altman, who sured by LDA subsequent to cell sorting all had the more had moved to Atlanta by then, and Kirsten and Gabrielle Belz “activated” CD62Llo phenotype, with the CD62Lhi set then did our first experiments (2) using tetramers made at Emory. emerging as late as a year after the initial priming (6). http://www.jimmunol.org/ For more than a decade before that, part of my research The first tetramer experiments that provided an accurate 1 1 focus had been to put virus-specific CD8 T cell immunity on picture of response kinetics and the extent of CD8 T cell a sound, quantitative basis. The original, 1973–1975 defini- proliferation following virus challenge appeared in 1998 (2, tion of lymphocytic choriomeningitis virus (LCMV)-specific 10), along with an IFN-g ELISPOT analysis from Mike Bevan 1 MHCI-restricted CD8 CTL activity, and the subsequent and Eric Butz (12) that gave much the same results. We retired conceptual interpretation that led to the Nobel Prize, de- our Cobra counter, and it sat gathering dust. Then the intra- pended on the use of the in vitro [51Cr] release assay and in cellular cytokine staining (ICS) assay (13), which uses in vitro vivo adoptive transfer experiments (3). The latter, although peptide stimulation in the presence of brefeldin A (to hold by guest on September 24, 2021 cumbersome and using an approach that was peculiar to the protein in the Golgi), came into general use (14) and confirmed LCMV system, was much more sensitive than the in vitro both the tetramer and the ELISPOT counts. It is much easier assay, which gives numbers but is at best semiquantitative. to make peptide than to produce pMHCI tetramers, so ICS 1 The subsequent years saw the emergence of the techni- has found wide application for the measurement of CD8 cally demanding limiting dilution analysis (LDA) as the “gold T cell responses (15). In addition to being the “poor man’s” standard” for counting Ag-specific T cells (4, 5). By 1996, our counting system, the spectrum of peptide-induced polyfunc- LDA experiments were keeping a 10-channel gamma counter tional cytokine production has also been used as an estimate (the Cobra) running almost continuously. If, for example, of TCR/pMHCI avidity (16). Another way of doing this has we wanted to establish the CD62L or CD44 phenotypes of been to measure the rate of elution for bound tetramers (17). effector or memory T cells, we first had to stain with Ab, Beyond the numbers, though, the tetramers have the ad- separate the cells using the FACS, and then stimulate the vantage over the ICS approach in that they allow us to probe 1 sorted population for 6 d in microculture wells before adding the molecular status of viable, unfixed, Ag-specific CD8 the 51Cr-labeled targets (6). It was very hard work and the T cells recovered directly ex vivo from mice, humans (1), 1 counts were low. In general, ,1:100–1:1000 CD8 T cells nonhuman primates (18, 19), and so forth. Now, if we want 1 were thought to be responders. Even so, the LDA approach to determine the activation phenotype of responding CD8 did provide rigorous evidence to support the, at times, dis- T cells, it is simply a matter of staining for cell-surface ex- 1 puted (7, 8) view that virus-specific CD8 T cell responses pression and measuring the numbers of, for instance, tetra- 1 were characterized by both Ag-driven clonal expansion and mer CD44hiCD62LloIL7RloKLRG1hi cells using the FACS the persistence of memory. (20). The tetramers have allowed us to make direct measure- 1 ments of the extent of CD8 T cell proliferation in “wild- 1 type,” virus-specific CD8 T cell responses (21). That had Department of Microbiology and Immunology, University of Melbourne, Mel- bourne, Australia 3010; and Department of Immunology, St. Jude Children’s been possible for the analysis of adoptively transferred, TCR- Research Hospital, Memphis, TN 38105 transgenic T cells (22), but such experiments gave little insight Address correspondence and reprint requests to Dr. Peter C. Doherty, Department into, for example, the quantitative basis of the highly repro- of Microbiology and Immunology, University of Melbourne, Melbourne, Australia ducible CTL immunodominance hierarchies (21). 3010. E-mail address: [email protected] 1 Tetramers were soon made to probe the CD4 Tcell Copyright Ó 2011 by The American Association of Immunologists, Inc. 0022-1767/11/$16.00 (pMHCII), NK cell (pHLA-E), and NKT cell (aGalCer 1 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1101297 6 PILLARS OF IMMUNOLOGY

CD1) responses (23–25). Marc Jenkins (26) worked out how to 8. Ku¨ndig, T. M., M. F. Bachmann, P. S. Ohashi, H. Pircher, H. Hengartner, and use pMHCII tetramers, in combination with very demanding R. M. Zinkernagel. 1996. On T cell memory: arguments for antigen dependence. 1 Immunol. Rev. 150: 63–90. enrichment procedures, to isolate naive CD4 T cells from 9. Doherty, P. C., D. J. Topham, and R. A. Tripp. 1996. Establishment and persistence of virus-specific CD41 and CD81 T cell memory. Immunol. Rev. 150: 23–44. peripheral lymphoid tissue. Application of that approach (21, 10. Murali-Krishna, K., J. D. Altman, M. Suresh, D. Sourdive, A. Zajac, and 27) has allowed us to look at both the size and the spectrum of R. Ahmed. 1998. In vivo dynamics of anti-viral CD8 T cell responses to different 1 TCR diversity for naive CD8 T cell repertoires and to follow epitopes. An evaluation of bystander activation in primary and secondary responses to viral infection. Adv. Exp. Med. 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Detection of antigen-specific be followed from the initial response through to long-term T cell cytokine expression in whole blood by flow cytometry. J. Immunol. Methods memory (11). In general, the results confirm the extraordinary 212: 89–98. stability of the memory T cell compartment, at least for mouse 15. Maecker, H. T., J. Hassler, J. K. Payne, A. Summers, K. Comatas, M. Ghanayem, M. A. Morse, T. M. Clay, H. K. Lyerly, S. Bhatia, et al. 2008. Precision and populations held under specific pathogen-free conditions. The linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytom- same type of approach can be used to characterize the pro- etry, and tetramer assay using CMV peptides. BMC Immunol. 9: 9. 16. Slifka, M. K., and J. L. Whitton. 2001. Functional avidity maturation of CD8(1) gressive expression and loss of mRNA for various effector mol- Downloaded from 1 T cells without selection of higher affinity TCR. Nat. 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