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Immunology and Cell Biology
(2004)
82
, 136–140 doi:10.1046/j.0818-9641.2004.01241.x
© 2004 Australasian Society for Immunology Inc.
Special Feature
Memories of virus-specific CD8
+
T cells
P E T E R C D O H E R T Y
1,2
a n d S T E P H E N J T U R N E R
1
1
Department of Microbiology and Immunology, University of Melbourne, Parkville, VIC, Australia and
2
Department of Immunology, St Jude Children’s Research Hospital, Memphis, TN, USA
Summary
This brief review focuses on the way that our understanding of virus-specific CD8
+
T-cell-mediatedimmunity evolved, giving particular attention to the early impact of the program at the Australian NationalUniversity. The story developed through a sequence of distinct eras, each of which can be defined in the context ofthe technologies available at that time. The progress has been enormous, but there is a great deal still to be learned.A particular challenge is to use what we know for human benefit.
Key words
:
cytotoxic T lymphocyte, ectromelia, influenza, lymphocytic choriomeningitis, transplantation.
Introduction
The story of CD8
+
T-cell-mediated immunity in virus infec-tions has much of its beginnings in the program initiatedduring the late 1960s by R V (Bob) Blanden at the Depart-ment of Microbiology at the John Curtin School of MedicalResearch, Canberra, Australia. This built on local strengthsestablished by the research of FJ Fenner,
1
GL Ada,
2
CAMims,
3
and KJ Lafferty,
4
on the thymus and T lymphocytes inthe laboratory of JFAP Miller in Melbourne,
5
and earlytraining in bacterial pathogenesis
6,7
with GB Mackaness inAdelaide and Saranac Lake, New York. The eve of Bob’sformal retirement from the Australian National Universityseems an appropriate time to reflect on the events thatprogressed our understanding of virus-specific CD8
+
T cellresponses over the subsequent 30 plus years.
Early days
Animal experiments by FJ Fenner
8,9
in Australia, and WPRowe
10
and J Hotchin,
11
in the USA pointed to a key role forthe cellular immune response in both ectromelia (mouse pox)and lymphocytic choriomeningitis (LCM). In particular,Rowe
10
showed that depleting T lymphocytes by neonatalthymectomy prevented the fatal disease that follows theintracerebral (i.c.) injection of LCM virus (LCMV) into adultmice. By the end of the 1960s, there was some understandingthat there are differences between the roles of antibody-mediated and cell-mediated immunity (CMI), but the natureof the divergence was obscure.
12
Most of conceptual focus ofCMI research was, however, concerned with alloreactivity
4,13
rather than the host response to pathogens.
Transfer of immune effector function
The first experiments that identified a specific role for effectorT lymphocytes in virus clearance came from the Canberralaboratory of RV Blanden
14–16
who used antithymocyte serum,then adoptive transfer protocols, to demonstrate that T cellscontrol ectromelia infection. Further experiments
17
with CAMims demonstrated similar effects with LCMV, while theBaltimore group of DH Gilden, GA Cole and N Nathanson
18–20
used a combination of lymphocyte depletion, chemicalimmunosuppression and cell transfer to show that theimmunopathology characteristic of non-cytopathic LCMVinfection in previously unexposed adults is indeed immunecell-mediated.
What we knew by 1972–3 was that T-cell responses werevery powerful and that they could, over a period of 24–48 h,play a key part in the termination of an infectious process. Infact, given that the reduction in virus titres could be measuredas Log
10
, these adoptive transfer models were by far the mostsensitive, quantitative assays then available for measuringT-cell function.
21
The cytotoxic T lymphocyte assay
The problem with the adoptive transfer approach is that,because the analysis is done
in vivo
, the protocol is cumber-some and there are many uncontrolled variables. Thischanged with the development of the simple,
in vitro
51
Crrelease CTL assay, first brought into prominence for the studyof alloreactivity by JC Cerottini and KT Brunner inLausanne.
22,23
Early CTL studies of viral immunity focused onthe LCMV model.
24,25
The approach was then adapted by theBlanden laboratory for the dissection of the ectromelia-specific response
26,27
while the transfer of the LCMV-CTLassay to Canberra led to the discovery of major histocompat-ability complex (MHC)-restricted T-cell function,
28,29
a storythat has been told at length elsewhere and will not be repeatedhere.
30
Further experiments with LCMV and ectromelia led to themapping of CTL activity to the H2K and H2D loci
31–34
thendefined as encoding the strong transplantation antigens (now
Correspondence: Dr Peter Doherty, Department of Microbiologyand Immunology, University of Melbourne, Parkville, Victoria 3010,Australia. Email: pcd@[email protected]
Received 23 December 2003; accepted 5 January 2004.
Memories of virus-specific CD8
+
T cells
137
known as MHC class I glycoproteins). The recognition pro-files that had been established
in vitro
by CTL activity werealso shown to determine T-cell effector function
in vivo,
usinga variety of adoptive transfer approaches with mouse strainsthat were partially matched for MHC phenotype. The initialexperiments, at least, were all done with LCMV
35–38
andectromelia.
39
The CTL analysis then turned to the dissection of viral(rather than MHC) specificity. The obvious approach was touse the influenza A viruses, which vary both as a consequenceof antibody-driven antigenic drift and the reassortment ofgenetic segments (antigenic shift) that occurs when the samecell is infected with two different influenza isolates.
40,41
Thenet consequence is the availability of a spectrum of infectiousprobes that differ for one or other viral component. Unlike thepatterns defined over many years for Ig-mediated recognition,the influenza A virus-specific CTL responses were found tobe highly cross-reactive
42–45
for viruses that show no profile ofreciprocal antibody neutralization. This caused some initialconsternation in the influenza world
46
but it soon becameapparent that, as indicated from the MHC analysis, T cellsand Ig molecules recognize different structures.
What was learned from the CTL experiments and conse-quent cell transfer studies with mice that were partiallymismatched for MHC phenotype was that we were dealingwith a distinct set of lymphocytes that is focused onto thesurface of virus-infected cells by the need to interact with thesame MHC class I (MHCI) glycoproteins that are encoun-tered during the course of T-cell priming. The CTL effectorswere found to discriminate between, for example, MHC-identical targets expressing influenza A or B viruses, but wereapparently unable to tell the difference between cells infectedwith serologically distinct influenza A viruses.
The conclusion reached by the late 1970s was thus that theCTL effectors recognize an entity that is quite distinct fromthat bound by antibody. While the Ig response acts to neutral-ize virions that are free in the blood, at mucosal sites or intissue spaces, the key role of CMI is to terminate theinfectious process by eliminating the cellular ‘factories’ thatproduce new virus progeny. This, in turn, suggested both abiological raison d’être for the extraordinarily polymorphic‘strong transplantation antigens’ (MHCI glycoproteins),and a possible explanation for alloreactivity.
47
What waslacking was any understanding of the underlying molecularmechanisms.
The molecular basis of T-cell recognition
The initial phases of the T-cell effector function and specifi-city story depended essentially on cellular immunologyapproaches, mouse transplantation genetics and the advanced(for the time) state of influenza virology. In fact, the basicbiological role of the MHC
47
was worked out before we hadany DNA sequence information for the MHC genes or even arudimentary understanding of the TCR. As often happens inour very complex subject, what seemed to be a very clearpicture then became confused. The key confounding elementwas evidence that the TCR is, in fact, encoded by the same IgV
H
gene segments that are involved in recognition of whatwas though to be the cognate protein.
48,49
If this were true, thededuction from the biological analysis that T-cell and Ig
recognition profiles are fundamentally different had to bewrong. As a consequence, most immunologists came tobelieve in ‘two receptor’ models of T-cell recognition, withone receptor being specific for the MHC and the other for the‘foreign’ antigen.
The situation was clarified by three separate discoveries.The first major event was the demonstration of the two-chainnature of the TCR.
50–53
The next revelation came when AlainTownsend solved the cross-reactivity problem
42–45
for influ-enza virus-specific CTL
54,55
by defining the viral target as apeptide of the conserved nucleoprotein (NP). The first X-raycrystallographic pictures of the MHC class I molecule
56
thenshowed us how a short viral peptide could be presented to atwo chain, single TCR. The ‘two receptor’ models of T-cellrecognition disappeared without trace, and the course ofT-cell immunology changed with much of the focus shiftingto understanding, particularly, the nature of the cytoplasmicantigen processing pathway.
57
The time lag between the initial statement of the ‘singleTCR-altered self’ hypothesis,
29,47,58
based on the biologicalexperiments and the molecular definition of TCR recognition,was almost exactly 10 years. It then took another 10 yearsto develop tetrameric complexes of peptide +MHC class Iglycoprotein (tetramers) for the direct staining of virus-specificCD8
+
T cells.
59
The former transformed our understanding ofT-cell recognition, the latter has revolutionized our capacityto dissect virus-specific T cell-mediated immunity.
60
Analysis of function and phenotype
The technologies that transformed mammalian biology in thegeneral sense also contributed to great advances in ourunderstanding of T-cell immunity. The effort is enormousand the process continues. The following mentions a fewinstances, largely drawn from our own research program. Theavailability of monoclonal antibodies, combined with increas-ingly powerful flow cytometry instrumentation and comput-ing, allowed us to define lymphocyte subsets and activationstates with much greater precision.
61,62
The monoclonal anti-bodies were also used deplete T cells and cytokines in the
invivo
situation
63–65
complementing analyses based on the use ofgenetically disrupted ‘knockout’ mice.
66,67
The tetramers allowedus to sort single, antigen-specific T cells, then examine theirexpression profiles for both effector molecules
68
and TCRusage.
69
The development of DNA arrays made it possible toscreen for global mRNA expression profiles in differentcategories of, for instance, cytotoxic and memory T cells.
70
Much of the information that has emerged from DNA profil-ing is yet to be analysed, and there are many years of carefulexperimentation ahead.
Beginning the quantitative era
Though we had learned a great deal about the nature of virus-specific CD8
+
T-cell mediated immunity by 1996, our under-standing of the magnitude of these responses was out by afactor of about 10-fold. The realization that much of ourunderstanding was based on numbers that were far too lowcame with the use of short-term peptide stimulationprotocols
71
and the application of the tetramer technology
59,72,73
for demonstrating the prevalence of effector and memory
138
PC Doherty and SJ Turner
T cells. Though the fact that the numbers were so wrong hasnot changed general perceptions of the ways that T cellsfunction, it is still the case that some of our thinking about thenature of T lymphocyte homeostasis and memory is based onmodels that were generated in the prequantitative era.
74
Weare, however, in the process of developing a much betternumerical analysis,
75
though the measurement of lymphocyteturnover and apoptotic elimination rates remains a chal-lenge.
76–78
Anyone entering the CD8
+
T-cell homeostasis fieldshould, however, look very sceptically at ideas and generali-zations that may be based in the prequantitative era.
Application
The major, practical task for immunology is to developinterventions that benefit humanity. Some of the possibilitiesinvolve the manipulation, or understanding, of CMI. Allergyand asthma looks to be increasingly important in our urban-ized societies. Does the answer lie somewhere in the Th1/Th2paradigm?
79,80
Would developing vaccination (or other) strat-egies for reducing the severity of the respiratory virus infec-tions of childhood help to alleviate the later toll of asthma?
81,82
The use of agents that block TNF is proving to be of greatvalue for treating a range of chronic, degenerative diseases.
83,84
Much of the applied interest in T cells is focusing on thepossible use of immunotherapeutic approaches in cancer.
85,86
Recent vaccine strategies to prevent HIV/AIDS have also beenheavily orientated towards priming the CD8
+
T-cell compart-ment, though it is unlikely that this approach will promotesterilizing immunity.
87,88
Are there immunological solutions
89
to chronic, degenerative diseases like multiple sclerosis?A very immediate challenge is to find ways to protect
against pathogens (like tuberculosis and HIV) that normallyestablish, and survive in the presence of an effective immuneresponse. Can we do better than nature? If solutions are to befound, they are likely to come from a mix of further analysisat the most basic level, and initiatives that are activelytargeted to evaluating novel approaches in humans. There isan enormous amount to be done, and great opportunities forinnovative programs, particularly those that interface differ-ent areas of interest and conceptual backgrounds.
Conclusions
A general lesson that can be learned from the T-cell story isthat, by thinking carefully about the interpretation of experi-mental data, we can sometimes reach conclusions that areprofound and substantially true, even though the availabletechnology cannot provide a satisfactory molecular explana-tion.
47
In stark distinction from the early days of the 1970s,what we now face is a plethora of information that sometimestends to obscure what may be solid, underlying principles.The biology of T-cell responses is extremely complex and,even for effector mechanisms that look to be of majorimportance, there can often be compensatory mechanisms.Perhaps immunology has now come to the stage where weneed to think much more in terms of systems approaches.There is also an increasing potential for useful interactionbetween theoreticians and experimentalists. Our greatestpractical challenge is to translate what we know to providereal, human benefit.
Acknowledgements
The work from our laboratory mentioned here was supportedby the block grant to the Australian National University, byfunds from ALSAC at St Jude Children’s Research Hospital,the US Public health Service and a Burnet Fellowship fromthe Australian National Health and medical Research Council.
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