Two events that were very significant in my life happened in October 1996. The first was an early morning call from Stockhom telling me that Rolf Zinkernagel and I were to share the Nobel Prize for Physiology or Medicine. The second was the publication of the peptide + MHC class I (pMHCI) tetramer staining technology for identifying HIV-specific CD8+ T cells (1). At that time, I was very aware of the former and, being totally distracted, quite ignorant of the latter. That changed in the following year, when Rafi Ahmed at Emory University made contact and suggested we should use the tetramer approach to look at the influenza virus-specific CD8+ T cell response. Kirsten Flynn traveled there to learn the technique from John Altman, who had moved to Atlanta by then, and Kirsten and Gabrielle Belz then did our first experiments (2) using tetramers made at Emory.

For more than a decade before that, part of my research focus had been to put virus-specific CD8+ T cell immunity on a sound, quantitative basis. The original, 1973–1975 definition of lymphocytic choriomeningitis virus (LCMV)-specific MHCI-restricted CD8+ CTL activity, and the subsequent conceptual interpretation that led to the Nobel Prize, depended on the use of the in vitro [51Cr] release assay and in vivo adoptive transfer experiments (3). The latter, although cumbersome and using an approach that was peculiar to the LCMV system, was much more sensitive than the in vitro assay, which gives numbers but is at best semiquantitative.

The subsequent years saw the emergence of the technically demanding limiting dilution analysis (LDA) as the “gold standard” for counting Ag-specific T cells (4, 5). By 1996, our LDA experiments were keeping a 10-channel gamma counter (the Cobra) running almost continuously. If, for example, we wanted to establish the CD62L or CD44 phenotypes of effector or memory T cells, we first had to stain with Ab, separate the cells using the FACS, and then stimulate the sorted population for 6 d in microculture wells before adding the 51Cr-labeled targets (6). It was very hard work and the counts were low. In general, <1:100–1:1000 CD8+ T cells were thought to be responders. Even so, the LDA approach did provide rigorous evidence to support the, at times, disputed (7, 8) view that virus-specific CD8+ T cell responses were characterized by both Ag-driven clonal expansion and the persistence of memory.

This is where the field was (9) when I gave my December 1996 Nobel Lecture in Stockholm (3). Although the overall concepts we were working with at that time remain fundamentally sound, tetramer staining (2, 10) experiments done through 1997–1998 showed that our 1996 understanding of responder CD8+ T cell numbers was at least 10-fold off! Perhaps the inherent inefficiency of the LDA technique reflects that only a small subset of CTL precursors can be expanded under these culture conditions. Although, for example, CD62Lhi and CD62Llo tetramer+CD8+ precursors are found through both the acute and the early memory phases of the influenza-specific response (11), the T cells measured by LDA subsequent to cell sorting all had the more “activated” CD62Llo phenotype, with the CD62Lhi set emerging as late as a year after the initial priming (6).

The first tetramer experiments that provided an accurate picture of response kinetics and the extent of CD8+ T cell proliferation following virus challenge appeared in 1998 (2, 10), along with an IFN-γ ELISPOT analysis from Mike Bevan and Eric Butz (12) that gave much the same results. We retired our Cobra counter, and it sat gathering dust. Then the intracellular cytokine staining (ICS) assay (13), which uses in vitro peptide stimulation in the presence of brefeldin A (to hold protein in the Golgi), came into general use (14) and confirmed both the tetramer and the ELISPOT counts. It is much easier to make peptide than to produce pMHCI tetramers, so ICS has found wide application for the measurement of CD8+ T cell responses (15). In addition to being the “poor man's” counting system, the spectrum of peptide-induced polyfunctional cytokine production has also been used as an estimate of TCR/pMHCI avidity (16). Another way of doing this has been to measure the rate of elution for bound tetramers (17).

Beyond the numbers, though, the tetramers have the advantage over the ICS approach in that they allow us to probe the molecular status of viable, unfixed, Ag-specific CD8+ T cells recovered directly ex vivo from mice, humans (1), nonhuman primates (18, 19), and so forth. Now, if we want to determine the activation phenotype of responding CD8+ T cells, it is simply a matter of staining for cell-surface expression and measuring the numbers of, for instance, tetramer+CD44hiCD62LloIL7RloKLRG1hi cells using the FACS (20). The tetramers have allowed us to make direct measurements of the extent of CD8+ T cell proliferation in “wild-type,” virus-specific CD8+ T cell responses (21). That had been possible for the analysis of adoptively transferred, TCR-transgenic T cells (22), but such experiments gave little insight into, for example, the quantitative basis of the highly reproducible CTL immunodominance hierarchies (21).

Tetramers were soon made to probe the CD4+ T cell (pMHCII), NK cell (pHLA-E), and NKT cell (αGalCer + CD1) responses (2325). Marc Jenkins (26) worked out how to use pMHCII tetramers, in combination with very demanding enrichment procedures, to isolate naive CD4+ T cells from peripheral lymphoid tissue. Application of that approach (21, 27) has allowed us to look at both the size and the spectrum of TCR diversity for naive CD8+ T cell repertoires and to follow how that translates into effector and memory T cell responses following Ag challenge. Our own group has made extensive use of single-cell sorting and RT-PCR to characterize individual TCRβ and, more recently, TCRαβ CDR3 regions (28). Knowing the CDR3αβ sequence at both the amino acid and the nucleotide level means that individual clonotypes can be followed from the initial response through to long-term memory (11). In general, the results confirm the extraordinary stability of the memory T cell compartment, at least for mouse populations held under specific pathogen-free conditions. The same type of approach can be used to characterize the progressive expression and loss of mRNA for various effector molecules, such as the granzymes and perforin, as the CD8+ T cell response progresses and then contracts following the cessation of Ag challenge (29).

Tetramer technology (1, 23) thus transformed T cell immunology by enabling accurate quantitation, facilitating the rigorous definition of activation phenotypes and receptor recognition, and allowing the direct analysis of molecular expression profiles for TCRs and effector molecules in viable lymphocytes taken directly from the spleen of an infected mouse or the arm of a sick human being (2830). The process of technological development continues and, for example, staining with combinations of pMHCI tetramers labeled with different fluorochromes now permits the characterization of multiple T cell specificities within a single, small sample of human blood (31, 32). In general, the tetramers are playing a major part as we seek to translate the insights gained from experimental animal models for application in humans. Beyond that, they also contribute enormously to the process of developing new insights from the direct analysis of cell-mediated immunity in outbred species like us (33).

1
Altman
J. D.
,
Moss
P. A. H.
,
Goulder
P. J. R.
,
Barouch
D. H.
,
McHeyzer-Williams
M. G.
,
Bell
J. I.
,
McMichael
A. J.
,
Davis
M. M.
.
1996
.
Phenotypic analysis of antigen-specific T lymphocytes
.
Science
274
:
94
96
.
2
Flynn
K. J.
,
Belz
G. T.
,
Altman
J. D.
,
Ahmed
R.
,
Woodland
D.L.
,
Doherty
P. C.
.
1998
.
Virus-specific CD8+ T cells in primary and secondary influenza pneumonia
.
Immunity
8
:
683
691
.
3
Doherty
P. C.
1997
.
The Nobel Lectures in Immunology. The Nobel Prize for Physiology or Medicine, 1996. Cell mediated immunity in virus infections
.
Scand. J. Immunol.
46
:
527
540
.
4
Swain
S. L.
,
Panfili
P. R.
,
Dutton
R. W.
,
Lefkovits
I.
.
1979
.
Frequency of allogeneic helper T cells responding to whole H-2 differences and to an H-2K difference alone
.
J. Immunol.
123
:
1062
1067
.
5
Allouche
M.
,
Owen
J. A.
,
Doherty
P. C.
.
1982
.
Limit-dilution analysis of weak influenza-immune T cell responses associated with H-2Kb and H-2Db
.
J. Immunol.
129
:
689
693
.
6
Tripp
R. A.
,
Hou
S.
,
Doherty
P. C.
.
1995
.
Temporal loss of the activated L-selectin-low phenotype for virus-specific CD8+ memory T cells
.
J. Immunol.
154
:
5870
5875
.
7
Eichmann
K.
,
Fey
K.
,
Kuppers
R.
,
Melchers
I.
,
Simon
M. M.
,
Weltzien
H. U.
.
1983
.
Network regulation among T cells; conclusions from limiting dilution experiments
.
Springer Semin. Immunopathol.
6
:
7
32
.
8
Kündig
T. M.
,
Bachmann
M. F.
,
Ohashi
P. S.
,
Pircher
H.
,
Hengartner
H.
,
Zinkernagel
R. M.
.
1996
.
On T cell memory: arguments for antigen dependence
.
Immunol. Rev.
150
:
63
90
.
9
Doherty
P. C.
,
Topham
D. J.
,
Tripp
R. A.
.
1996
.
Establishment and persistence of virus-specific CD4+ and CD8+ T cell memory
.
Immunol. Rev.
150
:
23
44
.
10
Murali-Krishna
K.
,
Altman
J. D.
,
Suresh
M.
,
Sourdive
D.
,
Zajac
A.
,
Ahmed
R.
.
1998
.
In vivo dynamics of anti-viral CD8 T cell responses to different epitopes. An evaluation of bystander activation in primary and secondary responses to viral infection
.
Adv. Exp. Med. Biol.
452
:
123
142
.
11
Kedzierska
K.
,
Stambas
J.
,
Jenkins
M. R.
,
Keating
R.
,
Turner
S. J.
,
Doherty
P. C.
.
2007
.
Location rather than CD62L phenotype is critical in the early establishment of influenza-specific CD8+ T cell memory
.
Proc. Natl. Acad. Sci. USA
104
:
9782
9787
.
12
Butz
E. A.
,
Bevan
M. J.
.
1998
.
Massive expansion of antigen-specific CD8+ T cells during an acute virus infection
.
Immunity
8
:
167
175
.
13
Jung
T.
,
Schauer
U.
,
Heusser
C.
,
Neumann
C.
,
Rieger
C.
.
1993
.
Detection of intracellular cytokines by flow cytometry
.
J. Immunol. Methods
159
:
197
207
.
14
Suni
M. A.
,
Picker
L. J.
,
Maino
V. C.
.
1998
.
Detection of antigen-specific T cell cytokine expression in whole blood by flow cytometry
.
J. Immunol. Methods
212
:
89
98
.
15
Maecker
H. T.
,
Hassler
J.
,
Payne
J. K.
,
Summers
A.
,
Comatas
K.
,
Ghanayem
M.
,
Morse
M. A.
,
Clay
T. M.
,
Lyerly
H. K.
,
Bhatia
S.
, et al
.
2008
.
Precision and linearity targets for validation of an IFNgamma ELISPOT, cytokine flow cytometry, and tetramer assay using CMV peptides
.
BMC Immunol.
9
:
9
.
16
Slifka
M. K.
,
Whitton
J. L.
.
2001
.
Functional avidity maturation of CD8(+) T cells without selection of higher affinity TCR
.
Nat. Immunol.
2
:
711
717
.
17
Kalergis
A. M.
,
Boucheron
N.
,
Doucey
M. A.
,
Palmieri
E.
,
Goyarts
E. C.
,
Vegh
Z.
,
Luescher
I. F.
,
Nathenson
S. G.
.
2001
.
Efficient T cell activation requires an optimal dwell-time of interaction between the TCR and the pMHC complex
.
Nat. Immunol.
2
:
229
234
.
18
Rollman
E.
,
Smith
M. Z.
,
Brooks
A. G.
,
Purcell
D. F.
,
Zuber
B.
,
Ramshaw
I. A.
,
Kent
S. J.
.
2007
.
Killing kinetics of simian immunodeficiency virus-specific CD8+ T cells: implications for HIV vaccine strategies
.
J. Immunol.
179
:
4571
4579
.
19
Kuroda
M. J.
,
Schmitz
J. E.
,
Barouch
D. H.
,
Craiu
A.
,
Allen
T. M.
,
Sette
A.
,
Watkins
D. I.
,
Forman
M. A.
,
Letvin
N. L.
.
1998
.
Analysis of Gag-specific cytotoxic T lymphocytes in simian immunodeficiency virus-infected rhesus monkeys by cell staining with a tetrameric major histocompatibility complex class I-peptide complex
.
J. Exp. Med.
187
:
1373
1381
.
20
Hand
T. W.
,
Morre
M.
,
Kaech
S. M.
.
2007
.
Expression of IL-7 receptor alpha is necessary but not sufficient for the formation of memory CD8 T cells during viral infection
.
Proc. Natl. Acad. Sci. USA
104
:
11730
11735
.
21
La Gruta
N. L.
,
Rothwell
W. T.
,
Cukalac
T.
,
Swan
N. G.
,
Valkenburg
S. A.
,
Kedzierska
K.
,
Thomas
P. G.
,
Doherty
P. C.
,
Turner
S. J.
.
2010
.
Primary CTL response magnitude in mice is determined by the extent of naive T cell recruitment and subsequent clonal expansion
.
J. Clin. Invest.
120
:
1885
1894
.
22
Zinkernagel
R. M.
,
Moskophidis
D.
,
Kündig
T.
,
Oehen
S.
,
Pircher
H.
,
Hengartner
H.
.
1993
.
Effector T-cell induction and T-cell memory versus peripheral deletion of T cells
.
Immunol. Rev.
133
:
199
223
.
23
Crawford
F.
,
Kozono
H.
,
White
J.
,
Marrack
P.
,
Kappler
J.
.
1998
.
Detection of antigen-specific T cells with multivalent soluble class II MHC covalent peptide complexes
.
Immunity
8
:
675
682
.
24
Braud
V. M.
,
Allan
D. S.
,
O'Callaghan
C. A.
,
Söderström
K.
,
D'Andrea
A.
,
Ogg
G. S.
,
Lazetic
S.
,
Young
N. T.
,
Bell
J. I.
,
Phillips
J. H.
, et al
.
1998
.
HLA-E binds to natural killer cell receptors CD94/NKG2A, B and C
.
Nature
391
:
795
799
.
25
Matsuda
J. L.
,
Naidenko
O. V.
,
Gapin
L.
,
Nakayama
T.
,
Taniguchi
M.
,
Wang
C. R.
,
Koezuka
Y.
,
Kronenberg
M.
.
2000
.
Tracking the response of natural killer T cells to a glycolipid antigen using CD1d tetramers
.
J. Exp. Med.
192
:
741
754
.
26
Moon
J. J.
,
Chu
H. H.
,
Pepper
M.
,
McSorley
S. J.
,
Jameson
S. C.
,
Kedl
R. M.
,
Jenkins
M. K.
.
2007
.
Naive CD4(+) T cell frequency varies for different epitopes and predicts repertoire diversity and response magnitude
.
Immunity
27
:
203
213
.
27
Obar
J. J.
,
Khanna
K. M.
,
Lefrançois
L.
.
2008
.
Endogenous naive CD8+ T cell precursor frequency regulates primary and memory responses to infection
.
Immunity
28
:
859
869
.
28
Dash
P.
,
McClaren
J. L.
,
Oguin
T. H.
 III
,
Rothwell
W.
,
Todd
B.
,
Morris
M. Y.
,
Becksfort
J.
,
Reynolds
C.
,
Brown
S. A.
,
Doherty
P. C.
,
Thomas
P. G.
.
2011
.
Paired analysis of TCRα and TCRβ chains at the single-cell level in mice
.
J. Clin. Invest.
121
:
288
295
.
29
Jenkins
M. R.
,
Kedzierska
K.
,
Doherty
P. C.
,
Turner
S. J.
.
2007
.
Heterogeneity of effector phenotype for acute phase and memory influenza A virus-specific CTL
.
J. Immunol.
179
:
64
70
.
30
Gras
S.
,
Kedzierski
L.
,
Valkenburg
S. A.
,
Laurie
K.
,
Liu
Y. C.
,
Denholm
J. T.
,
Richards
M. J.
,
Rimmelzwaan
G. F.
,
Kelso
A.
,
Doherty
P. C.
, et al
.
2010
.
Cross-reactive CD8+ T-cell immunity between the pandemic H1N1-2009 and H1N1-1918 influenza A viruses
.
Proc. Natl. Acad. Sci. USA
107
:
12599
12604
.
31
Newell
E. W.
,
Klein
L. O.
,
Yu
W.
,
Davis
M. M.
.
2009
.
Simultaneous detection of many T-cell specificities using combinatorial tetramer staining
.
Nat. Methods
6
:
497
499
.
32
Hadrup
S. R.
,
Bakker
A. H.
,
Shu
C. J.
,
Andersen
R. S.
,
van Veluw
J.
,
Hombrink
P.
,
Castermans
E.
,
Thor Straten
P.
,
Blank
C.
,
Haanen
J. B.
, et al
.
2009
.
Parallel detection of antigen-specific T-cell responses by multidimensional encoding of MHC multimers
.
Nat. Methods
6
:
520
526
.
33
Davis
M. M.
2008
.
A prescription for human immunology
.
Immunity
29
:
835
838
.

The author has no financial conflicts of interest.