PATHOGENESIS ESSAY

From influenza to HIV—and back?

Andrew J McMichael

Andrew McMichael recounts the seminal data he and colleagues produced demonstrating that cytotoxic T lymphocytes kill cells expressing antigenic peptides presented on major histocompatibility complex class I molecules.

At the end of 1976 I returned to the UK from A puzzling question in those early days was Hugh McDevitt’s laboratory at Stanford, how the influenza virus–specific CTL could excited by the phenomenon of major his- be so fastidious about tiny differences in HLA tocompatibility complex (MHC) restric- type while at the same time appearing to cross- http://www.nature.com/natureimmunology tion, newly discovered by Zinkernagel and react broadly with the influenza virus subtypes. Doherty1, and planning to examine its rele- This was not solved until Alain Townsend came vance to humans. I was particularly interested onto the scene. He first visited me in 1980, sent in asking whether the restricting role of HLA by Stan Peart, who was Professor of Medicine class I molecules on T cell function might help at St. Mary’s Hospital Medical School, to ask for explain HLA genetic polymorphism. My first advice on taking temporary leave from his clini- idea was to work on herpes simplex virus, but cal career to work for a PhD. Stan and I intro- my former PhD supervisor and mentor, Ita duced him to Ita Askonas, who took him on Askonas, persuaded me to switch to influ- for the first two years of a PhD. In year three he enza virus. She had just started to work on joined my small group in Oxford. Our original

Nature Publishing Group Group Nature Publishing T cell immunity in influenza virus infections intention was that he would start working on 7 in mice, with help from John Skehel at the human cells, but this idea was shelved because

200 UK National Institute of Medical Research he had come up with a really exciting finding

© and helped me adapt the mouse protocols in Ita’s laboratory. She had been one of the first to humans. I was soon able to show that rec- to generate virus-specific CTL clones in mice, ognition of influenza virus–infected cells nearly all of which appeared to cross-react to all by cytotoxic T lymphocytes (CTLs) is HLA influenza virus subtypes. However, Alain had Alain Townsend in 1984. class I restricted2. Then I started to search for sought and found a CTL clone, F5, that distin- responder and nonresponder (to influenza guished between certain influenza A viruses. length influenza virus RNA segment5 and had virus) HLA alleles. This search was compli- In collaboration with John Skehel, he used a made cDNA clones for all the virus segments. cated by the subtle variability in the amino set of reassorted influenza viruses in which the He generously gave us genes for the nucleo- acid sequences of HLA molecules such as eight RNA segments of a 1934 and a 1968 virus protein and, for good measure, the hemag- HLA-A2, not helped by the fact that my main had been shuffled, to identify the gene segment glutinin (H3). Alain soon transfected them blood donor (myself) turned out to have a that encoded the antigen his cloned T cells rec- into mouse L cells, cotransfecting the MHC rare A2 subtype, subsequently identified as ognized. Surprisingly, this turned out to the allele H-2 Db to match the MHC restrict- HLA-A*0205. These small differences in HLA nucleoprotein3. A couple of years before, Jack ing element for F5. Although the hemag- molecules affected T cell recognition of virus- Bennink, in a similar experiment, had identi- glutinin expressed well, there were problems infected cells. Fortunately, Bill Biddison and fied another internal virus protein, one of the with the nucleoprotein; it could barely be Steve Shaw at the US National Institutes of viral polymerases, as a target for CTLs4. These detected after transfection by immunostain- Health, also studying influenza-specific CTLs, were surprising results, because until that time ing. After several weeks trying and failing to pioneered identification of the HLA-A2 sub- almost everyone believed that CTL must rec- increase surface expression, Alain performed types, which were then gradually sorted out. ognize the viral glycoproteins, hemagglutinin the critical experiment anyway and found and neuraminidase, which are present as intact that the F5 clone killed the nucleoprotein- Andrew J. McMichael is at the MRC Human proteins on the surfaces of infected cells. transfected L cells beautifully. The manuscript Immunology Unit, Weatherall Institute of Molecular Soon after Alain came to Oxford in 1982, describing this result was rejected by Nature Medicine, John Radcliffe Hospital, Oxford, OX3 we went to see George Brownlee in the Sir (with one referee’s comment that it might only 9DS, UK. William Dunn School of Pathology. George appeal to those with a ‘modicum of afficion’ e-mail: [email protected] had been the first to sequence cDNA for a full for T cells), but was readily accepted by Cell 6.

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The failure of antibodies to detect nucleo- Identification of peptides as antigens for protein on the surface of infected or trans- CTLs transformed human T cell immunology. fected cells, which were readily recognized by By adding epitope peptides instead of live virus F5 T cells (and also by polyclonal influenza to T cell cultures (together with IL-2) it became virus–specific CTLs) provoked much discus- possible to grow T cell lines and then clone sion in the lab. Over many beers, we wondered them very readily. This facilitated precise char- whether the antigens presented in association acterization of virus-specific T cell responses with class I MHC might be peptides, similar and, subsequently, T cell responses to more to the peptides presented in association with complex pathogens and tumors. Around this class II MHC, as shown elegantly by Emil time, 1987, HIV had emerged as a major health Unanue7. To test the idea, Alain worked with threat. Using recombinant vaccinia viruses Frances Gotch to determine whether fragments expressing HIV gene products and synthetic of nucleoprotein might be recognized by his peptides, Doug Nixon, Frances Gotch and I clone F5. John Davey in Warwick had broken were able to identify the first epitope in HIV, the influenza nucleoprotein gene into three now usually called KK10, presented by HLA- cDNA fragments, which he transfected into B27 (ref. 13). Many other epitopes followed, cells to investigate the mechanisms of nuclear Andrew McMichael in 1984. and there are now more than 1,100 mapped targeting. Using the same cDNA, Alain and and optimized HIV epitopes in the Los Alamos Frances showed that F5 recognized a nucleo- to be sent around so that the experiments could database! Cloning of virus-specific T cells led protein fragment8. be repeated in other laboratories, but the data us into studies of the fine specificity of T cell At that time peptides were hard to make, were soon confirmed and accepted by all. Then receptors, including structural studies of HLA- but John Skehel had made some based on John Skehel introduced us to Don Wiley, who peptide complexes with CD8 and T cell recep- the nucleoprotein sequence. He gave these to with Pam Bjorkman and Jack Strominger was tors with Yvonne Jones and David Stuart in http://www.nature.com/natureimmunology Alain and over several more beers we discussed working on the crystal structure of HLA-A2. Oxford. how to give these to target cells, assuming they They had been puzzled by unidentified elec- Having learned how to refold HLA class I would have to be taken into the cells before F5 tron density that did not fit the HLA-A2 amino molecules with epitope peptides in vitro, we T cells could recognize them. The discussion acid sequence; our results implied that the den- were excited when Mark Davis proposed a led not very far, but again Alain just did the sity was a mixture of peptides. The paper that collaboration with John Bell and ourselves to experiment that most of us thought would not followed, describing the structure of HLA-A2 test a method that he had developed with John work. He added the peptides to the wells con- with its peptide binding groove, is one of the Altman of staining human virus-specific T cells taining F5 T cells and 51chromium-labeled H-2 most influential in immunology in the twen- with tetramers of MHC molecules. Together, Db-positive L cells. That afternoon he looked at tieth century10. we made HLA-A2–HIV gag tetramers and the L cells under the inverted microscope and After these heady days, much followed. showed not only that they stained HIV-specific

Nature Publishing Group Group Nature Publishing was very excited to see the L cells coming off Alain started to question how internal viral T cells, but that there were a lot more virus-spe- 7 the plastic bottom of the wells. By early evening proteins such as nucleoproteins that are made cific cells present than previously suspected14.

200 the assay had been going long enough to mea- in the cytosol are degraded to peptides, and HLA-peptide tetramers are now widely used

© sure chromium release, and sure enough, the how those crossed a membrane to get into the and have transformed our ability to detect, target cells had been killed. It is still a puzzle endoplasmic reticulum to join the MHC class I quantify and characterize antigen-specific why the experiment (and thousands more like molecules with their empty peptide binding T cells. We also used them to identify the NK it since) works, because one would think the grooves. He proposed that there must be a cell ligand for HLA-E (ref. 15). T cells would also kill one another, but they peptide transporter and that newly synthesized In HIV infection, identification of the precise do not. Thus, the principle that CTLs see short MHC class I might require peptide binding for epitopes recognized by CTLs facilitated very peptide fragments of foreign antigen was estab- its stability and export to the cell surface. He, detailed examination of these T cell responses lished. together with Enzo Cerundolo and Tim Elliott and their role in controlling the virus. In 1990, Frances and I had some human-specific in his lab, sought out mutant cell lines that in questions following a talk I gave at a small T cells in culture generated from the blood of lacked surface expression of class I MHC even meeting, Simon Wain-Hobson asked me Frances’s husband, Mike, who made a really though the genes were present, arguing that whether HIV might be able to escape from strong influenza nucleoprotein–specific HLA- they might lack the transporter. This led to a CTL responses by mutation. Rodney Phillips, B37–restricted T cell response. We knew the collaboration with Klas Karre, who had such a Sarah Rowland-Jones, Doug Nixon and I then viral antigen specificity because his CTLs lysed cell line, and Alain and colleagues showed that looked at this question in a small group of HLA-B37–matched target cells infected with adding peptide to the cells increased expression patients, sequencing their provirus and asking recombinant vaccinia expressing influenza of the class I molecules (stabilizing empty class I whether the variations found in the epitopes nucleoprotein, generously given to us by Bernie molecules on the cell surface), making the cells undermined the T cell responses. This turned Moss and Geoff Smith. The next day, we tested a target for CTLs11. Even better, Bob De Mars out to be the case, and we proposed that HIV the same peptides in the same way as Alain had had previously given us a mutant human B cell was capable of escaping from CTL responses by done and one worked. Thus the first mouse line, 721.174, that failed to express HLA class I mutation of epitopes16. For a long time this idea and human peptide epitopes restricted by class on its surface, and it behaved in the same way12. was not generally accepted, but in 1997 papers I MHC were found. This cell had a gene deletion in the HLA class II by Philip Goulder in my group17, by David We rapidly wrote a paper and sent it off to region, prompting a successful search for the Price in Rodney Philips’ group in Oxford18 9 Cell . When it was published, there was early peptide transporter in that region, in collabo- and by Persephone Borrow and George Shaw skepticism in certain quarters and reagents had ration with John Trowsdale. in the United States19 showed convincing evi-

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Having helped to acquire evidence that CTLs subtype specific and cross-reactive cytotoxic T cells. are important in controlling HIV infection, we J. Exp. Med. 160, 552–563 (1984). 4. Bennink, J.R., Yewdell, J.W. & Gerhard, W. A viral have worked on vaccines that stimulate HIV- polymerase involved in recognition of influenza virus– specific CTLs. This approach is likely to be less infected cells by a cytotoxic T-cell clone. Nature 296, 75–76 (1982). effective than a vaccine that stimulates broadly 5. Sleigh, M.J., Both, G.W. & Brownlee, G.G. The influenza neutralizing antibodies, but may be less elu- virus haemagglutinin gene: cloning and characterisa- sive. This type of translational research is slow, tion of a double-stranded DNA copy. Nucleic Acids Res. 7, 879–893 (1979). expensive and highly regulated, but the need is 6. Townsend, A.R., McMichael, A.J., Carter, N.P., great, and we are working with the Center for Huddleston, J.A. & Brownlee, G.G. Cytotoxic T cell HIV/AIDS Vaccine Immunology (CHAVI) and recognition of the influenza nucleoprotein and hemag- glutinin expressed in transfected mouse L cells. Cell the International AIDS Vaccine Initiative as part 39, 13–25 (1984). of a Global Enterprise for HIV Vaccines. Two of 7. Ziegler, K. & Unanue, E.R. Identification of a macro- phage antigen-processing event required for I-region- the most advanced CTL-stimulating vaccines restricted antigen presentation to T lymphocytes. (from Merck and the NIH Vaccine Research J. Immunol. 127, 1869–1875 (1981). Center) are now in clinical trials to test efficacy. 8. Townsend, A.R., Gotch, F.M. & Davey, J. Cytotoxic T cells recognize fragments of the influenza nucleopro- Our early studies of CTL immunity to influ- tein. Cell 42, 457–467 (1985). enza sparked off many scientific adventures. By 9. Townsend, A.R. et al. The epitopes of influenza nucleo- a curious twist of fate, pandemic influenza is protein recognized by cytotoxic T lymphocytes can be defined with short synthetic peptides. Cell 44, 959– Frances Gotch and Andrew McMichael circa 1990. now a looming threat. A paper that Frances 968 (1986). Gotch and I wrote in 1983 on CTL-mediated 10. Bjorkman, P.J. et al. Structure of the human class I histocompatibility antigen, HLA-A2. Nature 329, 506– dence for selection by CTLs of escape mutants. protection against experimental influenza virus 512 (1987). This was followed by a very clear and detailed infection in volunteers22 has suddenly been 11. Townsend, A. et al. Association of class I major histo- demonstration of escape from CTLs in acute noticed again and is being used as an argument compatibility heavy and light chains induced by viral peptides. Nature 340, 443–448 (1989). http://www.nature.com/natureimmunology 20 SIV infection in macaques by David Watkins for developing ‘cross-reactive’ T cell vaccines 12. Cerundolo, V. et al. Presentation of viral antigen con- and then by many excellent papers, in particular against avian influenza. Using the quantitative trolled by a gene in the major histocompatibility com- plex. Nature 345, 449–452 (1990). from Bruce Walker’s group, with sophisticated assays that we and others have developed for 13. Nixon, D.F. et al. HIV-1 gag-specific cytotoxic T lym- data analysis from Bette Korber, Simon Mallal studies of T cell immunity to HIV, we can ask phocytes defined with recombinant vaccinia virus and and collaborators, that have built up extensive whether preinfection influenza-specific CTL synthetic peptides. Nature 336, 484–487 (1988). 14. Altman, J.D. et al. Phenotypic analysis of antigen- evidence that HIV mutants are selected to evade responses protect against natural infection; if specific T lymphocytes. Science 274, 94–96 (1996). CTL responses. Now it is recognized that one of so a CTL vaccine aimed at conserved internal 15. Braud, V.M. et al. HLA-E binds to natural killer cell the most serious obstacles to the development influenza viral proteins might be realistic as a receptors CD94/NKG2A, B and C. Nature 391, 795– 799 (1998). of an effective HIV vaccine is virus variability protection against pandemic influenza. In a 16. Phillips, R.E. et al. Human immunodeficiency virus and its ability to escape from both humoral and sense I have come full circle. genetic variation that can escape cytotoxic T cell rec- ognition. Nature 354, 453–459 (1991).

Nature Publishing Group Group Nature Publishing T cell immune responses. 17. Goulder, P.J. et al. Late escape from an immunodomi-

7 ACKNOWLEDGMENTS The different types of escape mutation nant cytotoxic T-lymphocyte response associated with I am very grateful for all the generous contributions progression to AIDS. Nat. Med. 3, 212–217 (1997). 200 found in HIV, dependent on different HLA of all those mentioned above as well as many more 18. Price, D.A. et al. Positive selection of HIV-1 cytotoxic © types, show one way that HLA type might with whom I have worked but could not mention T lymphocyte escape variants during primary infection. affect the outcome of a serious virus infec- because of space constraints. In this type of science, Proc. Natl. Acad. Sci. USA 94, 1890–1895 (1997). tion, something that had first interested me rapid advance depends on good collaborations and 19. Borrow, P. et al. Antiviral pressure exerted by HIV-1- friendly rivalries, and above all on willing patients and specific cytotoxic T lymphocytes (CTLs) during pri- in 1977. HLA-B27 and HLA-B57 are associ- blood donors. mary infection demonstrated by rapid selection of CTL ated with better prognosis in untreated HIV escape virus. Nat. Med. 3, 205–211 (1997). 1. Zinkernagel, R.M. & Doherty, P.C. Restriction of in vitro 20. Evans, D.T. et al. Virus-specific cytotoxic T-lymphocyte infection; data has been accumulating over T cell–mediated cytotoxicity in lymphocytic choriomen- responses select for amino-acid variation in simian several years and was recently confirmed and ingitis within a syngeneic or semiallogeneic system. immunodeficiency virus Env and Nef. Nat. Med. 5, extended in a whole-genome scan for single- Nature 248, 701–702 (1974). 1270–1276 (1999). 2. McMichael, A.J., Ting, A., Zweerink, H.J. & Askonas, 21. Fellay, J. et al. A whole-genome association study of 21 nucleotide polymorphisms . The protec- B.A. HLA restriction of cell-mediated lysis of influenza major determinants for host control of HIV-1. Science tive effect of HLA-B57 is one of the three virus–infected human cells. Nature 270, 524–526 317, 944–947 (2007). (1977). 22. McMichael, A.J., Gotch, F.M., Noble, G.R. & Beare, strongest disease associations with any class 3. Townsend, A.R. & Skehel, J.J. The influenza A virus P.A. Cytotoxic T-cell immunity to influenza. N. Engl. J. I MHC allele. nucleoprotein gene controls the induction of both Med. 309, 13–17 (1983).

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