The Tetramer Transformation

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, CD62L^hi and CD62L^lo 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” CD62L^lo phenotype, with the CD62L^hi 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⁺CD44^hiCD62L^loIL7R^loKLRG1^hi 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 (23–25). 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 (28–30). 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).