Cell, Vol. 44, 959-968, March 28, 1986, Copyright 0 1986 by Cell Press The Epitopes of Influenza Nucleoprotein Recognized by Cytotoxic T Lymphocytes Can Be Defined with Short Synthetic Peptides A. R. M. Townsend,’ J. Rothbard,? F. M. Gotch, G. Bahadur,* D. Wraith,* and A. J. Mcklichael’ * Nuffield Department of Clinical Medicine John Radcliffe Hospital Headington Oxford OX3 9DU, England tlmperial Cancer Research Fund Lincoln’s Inn Fields London WC2, England t National Institute for Medical Research The Ridgeway, Mill Hill London NW7 lAA, England Summary A proportion of cytotoxic T lymphocytes (CTL) re- sponding to infection by influenza recognize target cells that express the viral nucleoprotein. Recent work showed that CTL can recognize short overlapping regions of large nucleoprotein fragments expressed in transfected L cells. This led to the suggestion that CTL recognize segmental epitopes of denatured or degraded proteins in a similar way to helper T cells. One corollary of this idea is that CTL should recognize appropriate short peptides on the target cell surface. We demonstrate that the epitopes of nucleoprotein recognized by CTL in association with class I mole- cules of the major histocompatibility complex in both mouse and man can be defined with short synthetic peptides derived from the nucleoprotein sequence. Introduction Mice and humans retaliate with a vigorous cytotoxic T lym- phocyte response to infection by influenza A viruses (reviewed by Askonas et al., 1982; Townsend and Mc- Michael, 1985). Cytotoxic T lymphocytes (CTL) gener- ally express the Lyt2 positive (CD8 in the human) surface phenotype and recognize and kill infected target cells that share class I molecules of the major histocompatibility complex (MHC) with the host in which the CTL developed (reviewed by Swain and Dutton, 1980; Dialynas et al., 1983; Blanden et al., 1975; Zinkernagel and Doherty, 1979). CTL may play an important role in limiting the spread of virus in vivo (reviewed by McMichael et al., 1983). Little is known about the nature of the viral epitopes rec- ognized by CTL on the surface of an infected target cell. Recently a major population of CTL from influenza in- fected mice have been shown to recognize murine L cells (a fibroblast line) that express the viral nucleoprotein (NP) in isolation after DNA mediated gene transfer (Townsend et al., 1984a). Recombinant vaccinia engineered to ex- press influenza NP also has been used to demonstrate human (McMichael et al., 1986) and murine (Yewdell et al., 1985) CTL that recognize the NP molecule. Recent evi- dence has shown that other nonglycosylated proteins of the virus can also be responsible for inducing cytotoxic T cells (Yewdell, personal communication). These findings raised the question of how viral compo- nents that are not transmembrane proteins and do not have recognizable amino-terminal leader sequences are transported to the plasma membrane of the target cell, where CTL recognition is assumed to occur. To investigate this, L cells were transfected with a series of deletion mu- tants of the influenza NP gene in an attempt to identify se- quences required either as epitopes recognized by CTL or as signals for membrane transport (Townsend et al., 1985). Epitopes were localized by comparing L cells express- ing large fragments of NP that overlapped over short regions. In this way two regions of sequence recognized by CTL were identified in a continuous segment of 59 amino acids between residues 328 and 386 of the mole- cule. No leader-like sequence could be located because nonoverlapping amino-terminal and carboxy-terminal frag- ments of NP were recognized equally well by appro- priate CTL. This implied that the two ends of the molecule could be transported to the plasma membrane indepen- dently of each other. In addition, although all of the frag- ments of NP expressed in transfected cells could be rec- ognized by specific CTL, some were no longer detected with antibodies that bound the complete folded molecule. One possible explanation for these results is that non- membrane viral proteins are degraded in the cytoplasm of the infected cell, producing short denatured peptides that are exported from the cell by some unknown mechanism. Such degraded viral proteins may then become available for recognition by CTL in association with class I MHC molecules in a way similar to that in which helper T cells recognize denatured or degraded proteins with class II molecules (reviewed by Grey and Chesnut, 1985; Unanue et al., 1984). One of the main predictions of this proposal is that CTL should be able to recognize appropriate short synthetic peptides corresponding to linear regions of the NP se- quence when they are added in vitro to the target cell sur- face. We describe experiments that define the epitopes of influenza nucleoprotein recognized by class I restricted CTL from both man and mouse using short synthetic pep- tides. Both the minimum length and the concentration re- quired are similar to those required for recognition of pro- tein antigens by class II restricted helper T cells. The results are consistent with the view that all somatic cells bearing class I molecules may be capable of degrading and presenting newly synthesized viral proteins to CTL. Results Murine and human CTL were tested for their ability to lyse SICr-labeled target cells exposed to a variety of peptides derived from the nucleoprotein sequence (see Tables 1 and 3). Cloned murine CTL also were tested for their