Peter Doherty is Chair of the Department of Immunology at St. Jude Children's Research Hospital, Memphis, Tenn.

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Viral Studies Illuminate the Nature of Immunity

The AIDS epidemic reversed the notion among immunologists that studying viruses was too complex and therefore unfashionable

Peter C. Doherty

The vertebrate immune system has evolved to protect what is essentially a complex of interdependent, multicellular organ systems from the consequences of parasitism by much simpler life forms. Despite this, the study of immunityto infectious agents became, for a time, very unfashionable in the world of basic immunology.

Part of the reason was that pathogenic mechanisms are very complicated, and it was thought that analysis of specific host response to simple chemical entities would greatly facilitate progress. This belief turned out to be only partially true. Many of the major paradigm shifts in immunology have, in fact, been triggered by dissecting infectious diseases. An example from protozoology is the recognition of the differential effects of the Th1 and Th2 cytokine spectra in both the control and the induction of pathology in leishmaniasis.

This brief account looks at some of the ways that studies with the simplest of all parasites, the viruses, have advanced our understanding of T cell-mediated immunity.

T. M. Rivers and Autoimmunity

The German scientist Paul Ehrlich pointed out the possibility of autoreactivity in the early 1900s, coining the term "horror autotoxicus." The first demonstration that such effects could occur with the as-yet-unrecognized T cell system came when Thomas Rivers analyzed the perivascular pathology and demyelination that are associated with the delivery of a brain-derived rabies vaccine.

Rivers and his colleagues showed conclusively that rabbit brain emulsified in Freund's complete adjuvant alone caused disseminated inflammatory pathology and demyelination in monkeys. This discovery developed into the experimental allergic encephalomyelitis (EAE) model that is still the system of choice for modeling human multiple sclerosis.

Experiments with EAE and other autoimmune systems, using a variety of "self" antigens, indicate that immunological tolerance to antigens that are not expressed in the thymus is far from complete. A recent evolution of this line of thinking is the "danger" hypothesis, which puts infectious agents at the center of the induction of autoimmunity, an idea for which there is surprisingly little formal proof.

F. M. Burnet, Immunological Tolerance, and Clonal Selection

During the 1930s, the German virologist E. Traube isolated an unusual virus from mice. The virus was named lymphocytic choriomeningitis virus (LCMV) because it induces inflammatory pathology following its intracerebral inoculation into previously unexposed adults. He recognized that LCMV is maintained as a lifelong, inapparent infection contracted in utero.

Knowledge of Traub's work with LCMV and Ray Owen's studies with consanguineous cattle twins led the Australian virologist, then immunologist, F. M. Burnet to formulate the theory of immunological tolerance. His conceptual analysis of self-nonself discrimination has been central to the immunology debate for 40 years and led to his sharing the 1960 Nobel Prize with P. B. Medawar, who had been studying the protected nature of xenografts implanted without lymphatic drainage. We celebrate the 100th anniversary of Burnet's birth this year, and much will no doubt be said about him during the course of the International Virology Congress in Sydney during August 1999.

Burnet himself considered that his most important contribution to immunology was his formulation of the clonal selection hypothesis, which evolved from ideas proposed by Nils Jerne and David Talmage. It seems likely that his thinking in terms of clonal lineages for specifically committed lymphocytes arose, in fact, from his earlier work with viruses. He was the first to recognize the power of both plaque counting and the selection of variants for studying bacteriophages, then mammalian viruses.

Much of the latter work was done with the chick chorioallantoic membrane (CAM) model that he developed for this purpose. Burnet was also to use CAM to quantitate the graft-versus-host effect and alloreactivity in some of the last experiments that were published from his laboratory.

J. F. A. P. Miller, the Thymus, and Cell-Mediated Immunity

By the end of the 1950s, the British immunologist J. L. Gowans determined that lymphocytes recirculate from blood to lymph and had begun to establish the role of organized lymphoid tissue in these processes. The realization that separate categories of T and B lymphocytes fulfilled distinct functional roles came in the early 1960s.

Working at an out-station of the Chester Beatty Institute for Cancer Research in London, Jacques Miller developed the technique of neonatal thymectomy to analyze the leukemia caused by a murine retrovirus, Gross virus. He discovered that the absence of the thymus from birth led to severe immunosuppression, particularly in the capacity to deal with experimentally induced tumors and allografts.

Though cell-mediated immunity (CMI) had been studied for many years in viral and bacterial infections, there was no understanding that such effects were due to a separate category of thymus-derived lymphocytes. The first experiments applying the techniques and ideas developed by Miller, N. A. Mitchison, and others to the study of infectious diseases were done by G. B. Mackaness and R. V. Blanden at the Trudeau Institute in Saranac Lake, New York. Blanden later extended these initial studies with bacterial models to ectromelia (mouse pox) at the Australian National University (ANU) in Canberra, where he had been recruited by F. J. Fenner. Fenner, who was a coauthor of Burnet's book on immunological tolerance, had published important studies on CMI to ectromelia in the late 1940s.

The experiments of Miller, Blanden, and Fenner were all done when they were Ph.D. students, in the British tradition that focuses on the production of a substantial research thesis but does not require course work. Are the mindsets required to absorb a diverse spectrum of established knowledge on the one hand, or to do creative science on the other, fundamentally different?

T Cell Recognition and the MHC

The major histocompatibility complex

In the early part of the 1970s, research efforts led by Baruj Benacerraf at Harvard and Hugh McDevitt at Stanford showed that genes mapping to the major histocompatibility complex (MHC) were in some way controlling T cell responses to linear peptides. This immune response (Ir), or I region, is now known to encode the class II MHC glycoproteins that determine the characteristics of CD4+ T helper cells, though the early emphasis was that the Ir genes specified the then-elusive T cell receptor.

Prior to the work of McDevitt and Benacerraf, the MHC had been studied largely by geneticists and immunologists interested in transplantation and alloreactivity. Though T cell responses to the strong transplantation antigens, now called the MHC class I glycoproteins, were among the most powerful known to immunology, there was no understanding of the biological function of these molecules.

Zinkernagel and Doherty experiment

This condition changed in 1973 when Rolf Zinkernagel and I stumbled onto the phenomenon of MHC restriction. We were very junior scientists in the excellent virology program that had been founded at the ANU by Frank Fenner and was at that time led by G. L. Ada, a viral biochemist, then immunologist, who had grown up in a research environment dominated by Burnet. Using mice infected with LCMV, we discovered that virus-specific CD8+ cytotoxic T lymphocytes (CTLs) are lytic only for virally modified cells expressing the spectrum of MHC class I glycoproteins present throughout the course of the host response. The phenomenon was quickly confirmed in Blanden's laboratory with the ectromelia model.

Rolf and I thought initially that our results might be explained by the types of ideas then favored by Benacerraf, McDevitt, and colleagues. However, we also argued (and generated evidence for) an alternative proposal, that the effector T cells were using a single T cell receptor to recognize some complex of "self" MHC class I glycoproteins and virus. It is worth noting that similar experiments with almost any other virus would not have given such a clear-cut result, perhaps explaining why the initial finding was not made with ectromelia. Priming with LCMV induces a very powerful response, while the background in the CTL assay is minimal due to the low level of cytopathology caused by this virus.

Viruses, Antigen Processing via Endogenous Pathway

Having found that CD8+ T cells are in some sense specific for self MHC class I glycoproteins, I next questioned the nature of the viral entity recognized by the uncharacterized T cell receptor. The influenza A viruses offered what seemed to be an ideal experimental system. These viruses have a broad species distribution with related, though serologically distinct, surface hemagglutinin (H) and neuraminidase (N) molecules defining a spectrum of subtypes. Reassortants are readily generated, as the viral genome consists of eight segments that will simply "repackage" when the same cell is infected with two different variants.

Collaborative studies during 1976-1977 with Walter Gerhard and two graduate students, Rita Effros and Jack Bennink, at the Wistar Institute in Philadelphia led, however, to the surprising finding that the virus-specific CTL response is highly cross-reactive for the H1N1 and H3N2 influenza A viruses. This lack of identity with antibody-defined specificity profiles was further confirmed by experiments with reassortant viruses. We learned soon after doing these studies of similar findings by B. A. Askonas at the National Institute of Medical Research, Mill Hill.

These observations, and similar evidence of cross-reactivities with other viruses that had no relationship to serologically defined specificity profiles, remained unexplained for almost a decade. Most of us working in this area felt more than a little obtuse when Alain Townsend and Andrew McMichael (both trained by Ita Askonas) showed that the immunodominant influenza-specific CD8+ T cell response was specific for peptides derived from the influenza virus nucleoprotein, a conserved component that is internal to the virion and is not subject to antibody-mediated selection.

Their observations formed the basis for the field of antigen processing via the endogenous pathway, though it had been known for some years that CD4+ T cells recognize peptides derived from the degradation of proteins in the "exogenous" lysosome/endosome compartment. Townsend's findings can also be considered to have triggered the modern era of tumor immunology.

Coevolution and Viruses That Steal from the Immune System

Advances in molecular technology are driving the linked processes of discovery and innovation in contemporary immunology. This generalization is probably more true for viral immunology than for any other field, as we are dealing with the interaction between complex (though accessible) genomes.

Some of the most exciting work illuminating how immune modulators operate is coming from analysis of the large DNA viruses. The herpesviruses, in particular, can be considered to have evolved to induce a state of stable, but persistent, parasitism in their mammalian hosts. It is also intriguing to speculate how these pathogens, some of which (e.g., smallpox) have had a profound effect on the shape of human populations, have influenced the evolution of the vertebrate immune system.

Such studies have focused largely on the detection and biological analysis of genes homologous to known elements of the mammalian defense system, particularly cytokine and cytokine receptor homologs. More recently, however, investigators have been using unassigned open reading frames in the viruses to probe back into the mammalian genome. Apart from enriching our understanding of immunity, there seems a good possibility that therapeutically useful molecules may emerge from this analysis.

The AIDS Epidemic: a Harsh Tutor

Human immunodeficiency virus (HIV) particles inside a cell. The emergence of HIV and the AIDS epidemic put the focus of immunology back on infectious disease. (©Hans Gelderblom/Visuals Unlimited.)

The mindset among the immunology community in the 1960s and early 1970s was that the study of immunity to pathogens was simply too complicated and did not merit the attention of "serious" immunologists. Part of this strange snobbism reflected the rejection of an earlier era, when much of immunology was the province of people like Rivers who were essentially interested in infectious processes.

By the late 1960s, Burnet wrote confidently that the era of infectious disease was over, having switched the Walter and Eliza Hall Institute (WEHI) that he led for more than 20 years from virology to investigations of basic and applied immunology. Burnet was clearly not attuned to the problems posed by diseases such as malaria and schistosomiasis in the developing world, though this crusade was taken up in the late 1970s by Gustav Nossal, his successor as head of the WEHI.

The prejudice against working with pathogens was to some extent changed by the discovery of MHC restriction with LCMV, which induced, for a time, a few members of the T cell immunology community to do experiments with viral models. However, this interlude soon passed after the revelation from Townsend's discovery that "peptide" could be substituted for "virus."

Any move away from viral immunology as a substantial focus has, however, been totally reversed by the AIDS epidemic. The pressing need to deal with the human immunodeficiency viruses has already told us an immense amount about the functional characteristics and limitations of the vertebrate immune system. Both the inherent danger of this disease, and the enormous levels of research funding that have been provided to combat it, have drawn some of the best minds in immunology towards the problem of dealing with viruses. There is little doubt that much of what is discovered about the fundamental nature of immunity over the next 10 to 20 years will continue to come from studies with viruses and other parasites.