The recognition of influenza A virus- infected cells by cytotoxic T lymphocytes

Immunology Today,voL 8, Nos 7andS, ;987

Q

F .VteWS 60 Krensky,A.M., Sanchez-Madrid,F., Robbins,E. etaL (1983)J. Immunol. 131,611-616 61 Fleischer,B., Schendel, D.J.and Steldern, D. (1986) Eur. J. Immu,,ol. 16, 741-746 62 Burns,G.F.,Triglia, T., Werkmeister, J.A., Begley,C.G. and Boyd, A.W. (1985)J. Exp. Med. 161, 1063-1078 63 Shaw, S., Gunther Luce, G.E., Quinones, R. etal. (1986) Nature 323, 262-264 64 Kaufmann, Y., Golstein, P., Pierres, M., Springer,T.A. and Eshhar, Z. (1982) Nature 300, 357-359 65 Carpen, O., Keiser, G. and Saksela,E. Nat. Imm. Cell Growth Reg. (in press) 66 Rothlein, R., Dustin, M.L., Marlin, S.D. and Springer,T.A. (1986)J. ImmunoL 137, 1270-1274 67 Patarroyo,M., Beatty, P.G., Fabre,J.W. and Gahrnberg, C.G. (1985)Stand. J. Immunol. 22, 171-182 68 Santoni, A., Scarpa,S., Testi, R. etaL Nat. Imm. CellGrowth Reg. (in press) 69 Hiseroot, J.C., Chung, A.E. and Reynolds,C. Nat. Imm. Cell Growth Reg. (in press) 70 Klein, E., Kai, C., Sarmay,G., Yefenof, E. and Gergely,J.

The

recognitionof influenzaA virusinfectedcells by cytotoxic T lymphocytes

Cytotoxic T cells (Tc) are important in the immune destruction of virus-infected cells. Recent studies, reviewed here by David Wraith, have disclosed much about the proteins of influenza virus that are recognized by Tc and the nature of the recognition process. These findings have implications for an undervaccines. Influenza viruses are an excellent tool for studying the MHC-restricted recognition of virus-infected cells by cytotoxic T (To) lymphocytes. Their structure is well defined and many recombinant strains are available for antigenic mapping (for review see Ref. !). Human influenza viruses are sub-divided by serological distinction of their internal matrix (M) and nucleoprotein (NP) into three types (A, B and C). Further division of the A types into three groups (H1N1, H2N2 and H3N2) is based on antibody reaction with the surface haemagglutinin (HA) and neuraminidase (NA) glycoproteins. The influenza A virus genome is completed by genes encoding three polymerases (PA, PB1 and PB2) and the non-structural protein (NS) (Table 1). Both the M and NS genes are spliced with the M2 gene encoding a glycoprotein 2. Two types of serological variation have been noted for the A viruses. The first, due to successive mutations in RNA coding for the glycoproteins (HA and NA), is referred to as antigenic 'drift'. The second, most probably due to reassortment between human and other Division of Immunology, National Institute for Medical Research,Mill Hill, London NW7 1AA, UK. Presentaodress: Departmentof Medical Microbiology Stanford University, School of Medicine, Stanford CA 94305, USA (~) 1987, E sevier Publicatio~ Cambridge 0167 - ,!,919/87/50200

Nat. Imm. Cell Growth Reg. (in press) 71 Berger,A.E., Davis, J.E.and Creswell, P. (1981)Human Imm. 3, 231-245 72 Hersey,P., Schibeci,S., Townsend, P., Burns,C. and Cheresh, D.A. (1986) CancerRes. 46, 6083-6090 73 Hersey,P., MacDonald, M., Burns,C. and Cheresh, D.A. (1986) Cancerlmmunol. Immunother. 15, 22-28 74 Clark, E.A.and Ledbetter, J.A. (1986) lmmunol. Today7, 267-270 15 Targan,S.R.and Deem, R.L.Nat. Irnm. Cell Growth Reg. (m press) 76 Burns,G.F.,Werkmeister, J.A. and Triglia, T. (1984) J. Imrnunol. 133. 1391-1396 7/ Werkmeister, J.A., Burns, G.F. and Triglia, T. (1984) J. Immunol. 133, 1385-1390 78 Werkmeister, J.A., Triglia, T. and Burns, G.F. (1985) Cell. Immunol. 92, 123-133 79 Ythier,A., Moingeon, P., Fabbi, M. etal. (1986) Cell. Immunol. 99, 150-159 80 Brenner,M.B., McLean, J., Dialynas,D.P.etal. (1986) Nature 322, 145-149

DavidC. Wraith mammalian or avian viruses, but possibly due to reemergence of a dormant sub-type, leads to a 'shift' from one sub-type to another ~. The recognition patterns of influenza A virus-specific B and T cells are quite distinct. Antibodies directed to HA and NA of influenza A viruses are variant-specific. In contrast, the majority of A virus-specific Tc cells recognize syngeneic cells infected with any virus within the A type 3. For the purpose of this review, such cells will be referred to as being A-virus crossreactive. This, and the fact that cloned influenza A virus-specific Tc cells are important in recovery from infection 4-6, has intensified the search for crossreactive Tc determinants in the hope that once identified they could be incorporated into an effective vaccine for heterotypic immunity to influenza. This review will outline current views on influenza A virus Tc recognition, the Tc antigenic determinants recognized and will consider the implications for both MHC restriction and vaccine development. The infected cell

Because many of the experiments described here concern target cells ~nfected with influenza virus, it seems reasonable to focus briefly on the cellular events involved in infection. A simplified view of influenza virus infection is shown in Fig. 1. It is believed that following endocytosis, influenza viruses fuse with the endosomal membrane, thereby everting the nucleoscapsid (RNA, NP, PA, PBI and PB2) into the cytoplasm. Fusion is effected by a conformational change in HA which is dependent on low pH (Ref. 7). A nucleus-specific stage

239

Immunology Today. vol 8. Nos 7 and 8. 1987

@

F6Vt66 5

~ , 8

L NS1

: 7

'

41

Vl

Y Rg. l. The infec~ cell Influenza viruses bind to the cell surface via HA (1) and are rapid~/ endocytosed (2). Following fusion between viral and endosorne membranes (3), nudeocapsids pass to the cell nucleus where ~nscription into viral (v) and messenger (m) RNA takes pJace (4). Tr~sla~on of viral proteins (5) occurs either on free ribosomes (/xote/mNSI.M. NP.PA.PB1 and 2) or membrane-bound ribosomes (proteins . ~ ..- ~,, ,,zl. ~ - - y,u~ lxnuu~ mrm a[ me c~7 surface oy oUOOlng (b) Cytopia~ic degradation of viral proteins could result in the generation of peptkfes (7) for re~'ognilJonby Tccells It is not dear howsuchpeptides would be ~,~r~l to #,e cetl surface Detaib of the events listed above are described in the text ~

~

JiJ"l|

ILI..~

...~.. . . . .

~-J-

L - -

-=

-,J--

•

,,

• •. . . . .

resulting in transcription of virus RNA into mRNA and regeneration of virus RNA follows, mRNA is translated in the cytoplasm, the glycoproteins being synthesized on membrane=bound polysomes. In productively infected cells, host-cell-dependent, post-translational processing of HA results in cleavage of the nascent chain (HAo) to give a membrane-bound HA2 chain linked by disulphide bonds to the distal HA1 chain. HA-mediated, pHdependent fusion can only occur after cleavage of HAo and, because in most cells used for cytolysis assaysHAo is not cleaved, the virus is limited to a single cycle infection. Strikingly little is known about how influenza viruses assemble at the cell surface before budding and elution.

VirusproteinsrecognizedbyA-virusspecificTc

240

The most notable feature of influenza A-specific Tc recognition is crossreactivity between all A virus subtypes. This became apparent when such cells were first described; their specificity patterns could not be correlated with known serological differences between HA and NA glycoproteins8-1o. Nevertheless, the first virus protein defined as an antigen for Tc was HA itself11.12.

Spleen cells from mice primed with viruses expressing the HA of A/JAP/305/57 (H2N2) proliferated in vitro in response to HA purified from the same virus and generated low levels of Tc cells capable of lysing syngeneic cells. However, the target cells were only lysed if they expressed the homologous HA, indicating that the response to H2 sub-type HA, when presented in this way, was sub-type specific. It was notable that the ablity to induce murine HA-specific Tc cells with purified HA protein did not apply to the H3 sub-type. When spleen cells were restimulated in vitro with purified HA of the H3 sub-type there was no detectable induction of Tc cells (Wraith, Barber and Askonas, unpublished). This showed that, in contrast with results from mice primed with viruses of the H2 sub-type 11.1z, a significant proportion of HA-specific Tc cells could not be selected from mice primed with H3N2 viruses using isolated HA. Moreover, it implied that, while the HA of the H2 sub-type could serve as a target for sub-type-specific To HA was not a significant target for the predominant population of influenza A viruscrossreactive Tc in mice. The proportion of HA-specific Tc cells in a murine response to influenza has more recently been firmly established through the use of recombinant DNA techniques. Tnis approach has confirmed the existence of either H1- or H2- but not H3-sub-type-specific Tc cells in mice primed with the homologous H1 or H2 virus 16.~7. Furthermore, a small but significant population of cells from mice immunized with H2 sub-type HA, but not H3 sub-type, could lyse syngeneic ceils expressing either the homologous or the H1 sub-type HA ~8.~9. To summarize, in primed mice there is a small population of HA-specific Tc capable of recognizing H I and H2 sub-types, but on a population basis, cells which are both HA-specific and crossreactive for all three sub-types are not detectable. As ever, the exception proves the rule and HA-crossreactive Tc ceil ciones have been isolated from both mice ~7 and humans2°. Certainly in mice, following infection with live virus, these cells are extremely rare. Sub-type specific Tc responses have been observed as a primary response soon after immunization 13-1s. However, the detectable proportion of H1 and H3 sub-type HA-specific cells primed by infection is small and it appears that on secondary in-vitro expansion these cells are overwhelmed by crossreactive Tc cells. To reiterate, the analysis of both primary ~3-~s and secondary Tc responses8-1° has shown that the majority of murine anti-influenza A Tc are A virus crossreactive. This was more recently confirmed by limiting dilution analysis of Tc precursors from mice primed by infection. More than 80% of cells generated in vitro from mice primed with A/PR/8/34 (H 1N1) virus lysed syngeneic cells infected with the homologous H 1N 1 or the heterologous H2N2 or H3N2 viruses2~. A similar proportion of 'crossreactive' T¢ could be generated in limiting dilution from mice primed with either X31 or A/PC/l/73 (H3N2) viruses22. However, the latter paper raised questions about the extent and nature of crossreactivity. It was noted that, with X31-primed C57.BL/6 mice, although most limiting dilution clones crossreacted between A/PR/ 8•34 (H 1N 1) and A/X31 (H3N2) infected cells, they would not lyse A/Aichi/2/68 (H3N2) targets, thereby implying that most of the Tc clones were not truly crossreactive for all A viruses. As X31 is a recombinant virus containing

i IrnrnunologyToday, vol. 8, Nos 7andS, 1987

O

I

Table 1. Biochemicalcharacteristicsof humaninfluenzaA virusproteinsa Protein

Abbreviations

Haemagglutinin

HA

Neuraminidase

NA

Nucleoprotein

Sub-:ype distinction

RNAsegment

Polypeptide length

Function

Hlb H2 H3 Nlb N2

4

566

Virusbinding,fusion

6

454

NP

Nonec

5

498

Matrix

M/M2

None

7

252/96

Polymerase Polymerase Polymerase Non-structuralprotein

PA PB1 PB2 NS1

None None Nonec None

3 2 1 8

716 757 759 230

Neuraminidase activity Nascentvirusrelease from cellsurface? Structuralassociation with RNA M: majorprotein, affinityfor lipid, functionuncertain M2: possiblynonstructural glycoprotein, functionunknown Elongationof mRNA Initiation,endonuclease Capbinding Noknownfunction

aFurtherdetailscanbefoundinRef.1 bExamplesof influenzaAvirussub-typesare:A/PR/8/34(H1N1);A/JAPAN/305157(H2N2);AJAICHI/2/68(H3N2) cAlthoughNPandPB2aregroup-specificantigenswhichdistinguishinfluenzaA, BandCvirusestherearesomeaminoacidvariationsbetweendistinctisolates.Such isolateshavebeenusefulinmappingTcspecificityto individualproteins23-25 genes for the internal proteins from A/PR/8/34 and glycoproteins from AIAichi/2/68, these data implied that the internal proteins were the predominant Tcrecognized antigens in these experiments. However, the low level of true A virus crossreactive clones was difficult to explain in the light of previous polyclonal culture analyses. A likely explanation, based on the extraordinarily strong selection in vitro of clonotypes specific for the nucleoprotein gene of the immunizing virus in H-2 b mice, was subsequently revealed through the experiments of Townsend and Skehe123.The data, summarized in Table 2, suggested that for H-2b-restricted To the NP gene encoded a determinant that was different in pre-1943 and post-1946 viruses and a further determinant recognized by crossreactive To. NP genes from either pre-1943 or post-1946 viruses code for proteins with a number of distinct amino acid residues scattered throughout the sequence. In-vitro selection of pre-1943 primed cells with the homologous virus resulted in rapid selection of pre-1943 NP-specific cells. This phenomenon, which is so far peculiar to the H-2 b haplotype for NP recognition, would account for the predominance of such a clonotype in the limiting dilution cultures of Kees and Krammer 22. Further evidence that influenza-specific Tc could recognize antigenic determinants other than the glycoproreins has been provided by Yewdell and Bennink. They used recombinant viruses to map the recognition of two murine Tc clones to the polymerase PB2 gene 25. Taken together, these data provide strong evidence that the conserved A-virus internal proteins could somehow act as antigens for Tc and would be likely candidates as targets for crossreactive To. Influenza A virus NP is indeed an important target antigen for A-virus crossreactive Tc in mice. In one study, L cells transfected with the NP gene were recognized by a high proportion of crossreactive Tc from C3H/He (H-2k)

mice 18. In an independent analysis, appropriate target cells infected with the A virus NP gene incorporated into vaccinia virus (Vac-NP) were lysed by Tc derived from either H-2 k, t-l-2 b or I-I-2 d mice (Ref. 26 and Bennink et al. unpublished) and also by Tc from two of six influenzaprimed human donors 27. Since NP was clearly a major target antigen for crossreactive To experiments were undertaken to test the efficacy of purified NP as a vaccine for influenza A. NP was isolated from purified virus by detergent extraction and following intraperitoneal injection induced high levels of crossreactive memory Tc cells28. Subsequently, both subcutaneous and intramuscular routes of administration have proved effective. Mice receiving two doses of purified NP were substantially protected from a lethal virus infection 29. Recently, using appropriate vaccinia virus recombinants, Bennink and co-workers have defined all three polymerases and NS-1 as further important antigens for crossreactive Tc (Ref. 30), although the response may be less extensive than for NP in mice (see below). As yet, they have not detected activity directed either towards M Table 2. InfluenzaA virusNPgeneis dominantfor in-vitro selectionof

H-2b restrictedTc Virususedfor 2nd in-vitro priming selection

Pre-1943 Post-1946 Post-1946

Pre-1943 Post-1946 Pre-1943

aDatasummarizedfromRefs23and24

Tcrecognitionamappingto the NP genederivedfrom 1934-1943 virus

1946-1979 virus

+ +

÷ + 241

Immunology Today. voL 8, Nos 7and8, 1987

~

~!~ 0 ~C

wide area of the molecule. Class I-restricted recognition is highly sensitive to anti-MHC monoclonal antibody inhibition 39. While the Tc source Restrictingallele NPel0ito~ (aminoacid effect is most likely to be due to steric hindrance 4° the residues) use of such anti-H-2 antibodies has nonetheless revealed C57. BU6 H-.?Db 365-380a both interesting diversity in influenza-specific Tc reperCBA/Ca H-;,;,'k 50_6310 toires between strains of mice 41 and repertoires used for MAN HLA-B37 335-349a different viruses within a strain 42. Vitello and Sherman have provided evidence for the DatafromRefs45aand7(P strong influence of H-2 class I-restricted elements on viral or the M2 glycoprotein. This is not the case for humans specificity and Tc repertoire selection 43. An extensive where class I-restricted (mainly HLA-A2) Tc lyse targets panel of influenza A-specific Tc clones were generated in infected with the vaccinia-M recombinant 62 and M vitro from mice differing at defined class I MHC alleles recognition by a class II-restricted Tc clone was deduced and these were tested for recognition of various virususing recombinant avian/human viruses 2°. It will be infected target cells. The observed repertoire difference interesting to test purified polymerase and M proteins for between C57.BL/6 (KbD b) and BIO.A (SP) (KbD b) may Tc induction in vivo. • again have been due to the strong selection in vitro for Db-restricted NP-specific clones referred to in the preM H C mstdction and 1"=repertoire selection vious section; unlike that of BIO.A (SR) mice, the major Influenza virus-specific Tc cells are MHC restricted. repertoire of C57.BU6 mice, primed with a pre-1943 Most clones and lines so far studied have mapped to virus, was for viruses sharing the pre-1943 NP gene. The class I (K or D in mouse, HLA-A or B in man) restriction D b gene appears to influence the Tc cell repertoire as a elements but there are two recent independent reports result of the type of in-vitro selection shown in Table 2. of class II-restricted Tc in man z°.31 and one in mouse 32. Two recent reports have approached the question of Such class II-restricted cells proliferate, produce IL-2 and class I restriction elements for individual influenza proare T4 or 1.3T4 positive. However, their significance tc~ teins. In the first, Pala and Askonas showed that, within a influenza immunity is somewhat controversial. In mice, particular mouse haplotype, Dd, Dk and Kb were Iowadoptive transfer of influenza immune T cells resulted in responder class I molecules, while Kd, Kk and D b were protective activity only when donor and recipient mice responder alleles for Tc recognition of influenza NP44. shared class I genes33 and significantly, there was no Bennink and co-workert-_ have compared recognition of protective effect after transfer between class I- NS-1 and the polymerases PA, PB1 and PB2 in H-2 k, mismatched/class II -identical mice. In the laboratory, the 14-2b, and H-2 d strains. Following selection in vitro, low class II-restricted cells constitute a minor population (l.J. responders for PA were H-2 d and H-2 k, for PB-1 were Braciale, unpublished) and while one cannot ignore H-2 d and !-1-2b and for PB-2 was H-2 k. They have since them, their contribution to influenza immunity has yet to shown that, as for NP recognition 44, responder haplobe establisht:c,. types have only one set of responder alleles at either the v" ,-~r D 1~,,,,-i ilJ . ~¥ ¥l . vI ~.V. .V.U ~,.a,~u -~.-.,.~ j. R. D,,.....,:.,t, .U.l.l ~. .. /.u u -L, Studies on d_ass__!-m__~_ri~ed, influenza-specific Tc ..have l~. Ul ~;llU ~ II 0111~1 I.~'lllllllr~,, revealed many interesting features of MHC structure and lished). function. For example, some cloned, self-restricted and Table 4. Comparison of the antigenic forms capable of sensitizing influenza-specific Tc can recognize one or more unintarget cellsfor influenza-specificTc recognition fected allogeneic target cells34-36. The mechanism of such apparent "dual specificity' remains a puzzle. HowAntigen and MHC type and antigen expressedby target cell ever, one reasonable explanation is that self-MHC moleinhibitors cules can resemble allogeneic molecules when combined Class I Class I Class II ClassII with antigen such as an influenza protein or peptide. + HA + internals + HA + internals Either a complex may form between antigen and MHC or the normal conformation of the MHC molecule may alter Infectiousvirus + + + + as a result of antigen binding, in both cases, the MHC is Infectiousvirus + IPS + + no longer recognized as 'self' by T cells. Infectiousvirus + LD + + A vast array of MHC recognition patterns have been Inactivatedvirus + + described. Analysis of H-2Kb-mutant recognition showed Inactivatedvirus + + + donal diversity in restriction element, function within an IPS allele and suggested that, to be an effective restriction Inactivatedvirus + . . . . element, the class I molecule depended on the conLD formation of the outer two domains in which the Kb Viral gene 'trans+ + (-) mutations had occurred 37. This was confirmed by confected' cells struction of hybrid H-2 (Kb/D b) molecules 38. Tc restriction Viral gene 'trans(-) (-) (-) (-) sites depending on the conformational interaction of fected' cell + IPS both outer domains and exchange of either domain Viral gene 'trans(+) + (-) abolished recognition. This was an important finding, fected' cells + LD not just because it explained the sensitivity of class I Purifiedprotein +/+ (+) molecules to mutations in either of the domains, but also because it provided structural evidence for a mechanism Thedatashownreferto experimentsdescribedbyvariousauthors(seetextfor by which amino acid substitutions in either of the two details) domains might generate an amplified population of Tc 'Internals':anyoneof thevirusproteins,exceptHA,NAor M2; IP5:inhibitors restriction elements by affecting conformation over a of proteinsynthesis;LD:lysosomotropicdrugs Table

~Z4Z _i

I

~

3, NudeoproteinTcepitopesand their restrictingalleles

Immuno:ogy Today, vol. 8, Nos 7andS, 1987

In conclusion, it is apparent that class I genes can act as immune response genes and can accordingly influence the repertoire of Tc cells responding to a given viral antigen. However, at present it is difficult to see how unequivocal repertoire comparisons can be undertaken when an in-vitro amplification step is required. The strong in-vitro selective effects of particular antigenMHC combinations such as H-2D b and NP are quite striking and should always be considered when repertoire analysis is undertaken.

Is antigen processing required to generate epitopes recognized byte? First, consider certain pieces of evidence: (1) Inactivated virus is ineffective in the induction of Tc in vivo 46-48. (2) Protein synthesis is required for class I-restricted but not class II-restricted Tc recognition of influenzainfected target cells46.49. (3) Lysosomotropic agents inhibit class II-restricted Tc (Refs 20, 49) but not class I-restricted recognition of target cells45.49. (4) Antibodies to influenza proteins do not inhibit Tc recognition of target cells (unpublished observations from many labs). Is infectious virus mandatory for Tcinduction? Ultra-violet light (UV)-inactivated virus induces Tc poorly when injected in suspension. However, both UVinactivated virus and influenza liposomes primed mice for A-virus-crossreactive To but only when adsorbed to spleen cells and injected intravenouslys°. At the clonal level we have recently demonstrated the proliferative response of NP-specific Tc co-cultured with cells treated with UV-inactivated virus5~. The requirement in these experiments was for close association of virions with syngeneic ceiis, followed by uptake and uncoating of virus by these cells but was not dependent on virus infectivity. Is protein synthesis required for Tctarget formation? In contrast with their effect on Tc induction, UVinactivated influenza viruses do not readily form targets for A-virus-crossreactive To. In one report, HA (H2subtype)-specific lysis of inactivated virus-treated target cells was detected when high levels of inoculum were used; such treatment did not sensitize targets for Avirus-crossreactive Tc (Ref. 52). By contrast, Morrison et al. have recently shown that while class II-restricted anti-influenza Tc lysed a B lymphoma cell line treated with UV-inac:i;-ated virus, class I-restricted cells were ineffective, even at 10 O00-fold excess virus49. Furthermore, ementine, an irreversible inhibitor of protein synthesis, blocked class I-restricted but not class IIrestricted lysis. Two clear distinctions can be made from the discussion so far. (1) There is a major difference in the pathway of antigen handling by target cells for lysis by either class I-restricted or class II-restricted cells summarized in Table 4. (2) Class I-restricted anti-influenza Tc respond readily to cells treated with UV-inactivated virus, as measured by primary induction in vivo or secondary proliferation in vitro, but as a rule, these cells will not lyse cells treated with UV-inact!,,ated virus. This observation clearly dis-

tinguishes these cells from paramyxovirus-specit~: To whose fusion-dependent lysis of target cells treated with either inactivated virus or virus-liposomes is well documented (e.g. Ref. 53). It appears that for influenza viruses the requirements for inducing Tc cells to proliferate may differ from those for target formation. Is protein degradatio,~ required for antigen presentation to Tccells? The current belief is that, for recognition by class II-restricted T cells, antigens must first be taken up by antigen-presenting cells and then degraded by ~cid proteases in the 'lysosomal pool' of intracellular vesicles to g~ve linear peptides which can then interact with class II MHC antigens (for review, see Ref. 54). This view is upheld by Morrison et al. 49 and Fleischer et al. 2° who have shown that target cell lysis by class II-restricted anti-influenza Tc can be inhibited by lysosomotropic agents. The same rules do not apply to class I-restricted cells. Targets infected with virus, or 'transfected' with influenza genes, and treated with lysosomotropic drugs such as chloroquine, were nonetheless susceptible to lysis by such Tc ceils4s.49. in this way we can clearly distinguish between class I-restricted and class IIrestricted Tc with regard to both protein synthesis and lysosomal breakdown (Table 4). How absolute is this distinction? It would be conceptually satisfying to believe that class I viral antigen complexes could also be generated by a fragmentation process. Thus we could more readily explain how the internal virus proteins of influenza are recognized in an MHC-restricted fashion and why it has thus far been impossible to inhibit virusspecific target cell lysis with anti-virus antibodies. The strongest evidence that degraded proteins are indeed recognized has come from Townsend and colleagues in a series of experiments dealing with influenza NP18.$5. The fact that cells transfected with the NP gene could be recognized by Tc without detectaDle surface expression of a serological determinant had suggested that some form of antigen processing led to a separation of Tc epitopes from serological epitopes in this case5s. This work culminated with the demonstration that cells incubated with synthetic peptides prepared according to the predicted sequence of NP could be lysed by epitopespecific Tc clones4s. Thus far, three NP epitopes have been described (Table 3) and a further distinct H-2Kd epitope has recently been mapped (P.M. Taylor and B.A. Askonas, unpublished observations). One exciting aspect of this work is that the four non-overlapping peptide epitopes all appear to be exclusively restricted to a particular MHC class I allele. These findings imply that fragmentation of Tc cell antigens such as NP is required for either interaction with class I MHC and/or recognition by the T-cell receptor. The MHC specificity for peptide recognition, although as yet limited to these few alleles, provides molecular evidence in favour of a 'determinant selection' basis for MHC class I allelic variation. The determinant se!ection theory was originally proposed by A.S. Rosenthal as an explanation of MHC-associated immune response gene effects 64. Recent evidence implies that antigen fragmentation may not be an absolute requirement for all class Irestricted responses. Using an in-vitro proliferation assay, we have shown that intact NP can be presented to NP peptide-specific Tc clones without an apparent requirement for fragmentation sl. NP was prepared from purified virus cores by mild detergent treatment 2s'29

243 !

Immunology Today, vol. 8, Nos 7 and 8, 1987

¢ '

i,

;~ i.#

g

which was not denaturing. NP-specific Tc induction was not inhibited either by treatment of antigen-presenting cells with lysosomotropic agents or by prior fixation. These data confirmed similar findings 4s.49 that the class I-restricted Tc epitopes were not generated by lysosomal degradation. Furthermore, it implied that this particular Tc epitope was detectable in or on the folded molecule. To extend these findings, we have recently studied recognit|on of purified NP in a cytotoxicity assay (unpublished data). This was tested by adding various forms of the protein to uninfected stCr-labelled target cells. Intact protein, both before and after reductive carboxymethylation, would not sensitize target cells (H-2 b thymoma cells) at a concentration (0.9 I~M) known to induce proliferation S1. However, after short exposure of the denatured protein to increasing concentrations of trypsin, fragments were generated which were capable of sensitizing target cells at 0.23 I~M. ' , " e interpret this result as showing that a fragmented form of NP, when added in suspension, will interact more readily with the cell surface, or in particular, with MHC molecules. These data do not conflict. -';*~'.~-,.,*~^.,¢obse,wation that intact protein can be presented by paraformaldehyde-~ixed cellss7 if, as suggested above, the signals required to induce proliferation may differ from those required to activate the lytic machinery of particular T¢ cells.

244

There are many questions still to be answered. The major and central problem is antigen association with MHC. Does antigen interact directly with class I MHC, and are MHC gene effects governed by this interaction? Alternatively, is the tripartite interaction between T-cell antigen receptor, MHC and antigen drawn together by the receptor itself and, therefore, do the T-cell-receptor Qenes_ oovem~ T-cell . . . . . .,~e~pnp~_vene~ ... to a niv.~=n~ . . . . antigen? As yet there is no evidence that antigen interacts directly with class I molecules while for class II molecules the evidence is disparate s6.sT. The second question relates to the form of the antigens recognized. AS stated above, peptide recognition by Tc cells is not absolute proof that fragmentation of antigen is required. Indeed, we have demonstrated that To when assayed by proliferation as opposed to cytotoxicity, can recognize both peptide and intact antigen sl. Yet much evidence points to a requirement for antigen fragmentation. How can this question be resolved? The use of peptide-specific antisera should enable the identification of an epitope at the surface of a virus-infected cell and possibly reveal its form. Such reagents could also be used in pulse-chase experiments to follow the fate of a particular epitope from synthesis to the cell surface. If complexes of antigen and class I molecules are generated by fragmentation there are three possible mechanisms: lysosomal degradation; proteolytic attack at the cell surface; and fragmentation by cytoplasmic proteases. Lysosomal degradation seems already exduded 4s.49.sl. Cell surface protease activity has been described (e.g Ref. 58) but as yet little is known about cell and tissue distribution of such activity. As Townsend et ai. have proposed ss, the most plausible mechanism is the cytoplasmic degradation of proteins, which is most likely to occur co-translationally. This mechanism seems most likely when the proteins recognized by crossreacfive, influenza-specific Tc are considered (Fig. 1). They fall

into three groups: NS1 which, as a non-structural protein, would turnover rapidly in infected cells; M, NP, PA, PB1 and PB2, which are translated on membrane-free ribosomes; and HA, NA and M2 which are translated on membrane-bound ribosomes. The evidence so far implies that in infected cells, HA, NA and M2 cannot serve as targets for A-virus crossreadive To. They may be protected from cytoplasmic proteolytic attack by being directly translated into the endoplasmic reticulum. Cytoplasmic fragmentation of protein is well documented $9 even though details of the mechanisms involved, which could include the ubiquitin S9, ATP-stimulated 6° and Ca2+-dependent 61 pathways are limited. Until such pathways are more carefully dissected the mechanism shown as No. 7 in Fig. I must remain purely speculative. There is recent strong evidence in favour of a cotranslational mechanism for fragment generation. Townsend and co-workers have shown that when leader-peptide-free HA was translated on free ribosomes, rather than through the endoplasmic reticulum (see Fig. 1), it was rapidly degraded and cells transfected with leader-free HA genes served as excellent Tc targets 64. Unfortunately the possibility of translation through the endoplasmic reticulum due to a second leader-like sequence could not be excluded. Another series of experiments implies that fragmentation of other antigens, including MHC antigens themselves, can take place and may be a general feature of Tc target formation 65. A synthetic peptide was shown to substitute for antigen in the MHC-restricted recognition of HLA-transfected mouse cells by mouse T¢ cells. This extends the type of protein which can be presented to Tc in a fragmented form. However, as yet, peptide antigens for T¢ cells have all been presented in an MHC-restricted manner. Antigen processing in allo-recognition is less clear. rlJ t,-,cf;rtn ~ l . h ~ ,.I;.lr-~l: . . . . . . ;,~ tig e n rA, ,n / ' t,fvl ~uc ~ r |l..w ~.l~l,,...aw.lvl i r! ~~.l : =, uf ~ e, ~ . ~ ti-,~ ~v ~,,~ u,,,~,~,,~-~ ,,~ an presentation to class I-restricted and class II-restricted To. Why should class II-restricted recognition rely on lysosomal degradation and class I-restricted cytotoxic lysis of cells, at least in influenza, depend on protein synthesis? The answer may lie in both the presenting cell type and the recycling and subcellular distribution of the different MHC glycoproteins. Cresswell has followed the biosynthetic route of class II MHC antigens in antigenpresenting cells and has shown that this pathway intersects with an endocytotic vesicle pathway used by, for example, recycling transferrin receptors66. This intersection could be where endocytosed T-cell antigens interact with class II MHC molecules. Less is known about class I MHC molecules although Pernis has shown that such molecules spontaneously internalize or recycle in T cells but not in most other types67. Germain has proposed that processing of proteins for interaction with class I MHC might occur in a specialized region of the Golgi apparatus 68. This seems unlikely because proteins such as NP, which are synthesized on free ribosomes, would not enter the Golgi apparatus. Furthermore, removal of the leader sequence, which diverts a protein (HA) away from the Gclgi, does not affect the apparent fragmentation 64. However, it is possible that degraded protein fragments are incorporated into spontaneously forming, subcellular vesicles which could subsequently interact with the biosynthetic pathway of class I antigens. As pointed out by Bevan69, it seems unlikely that peptides would simply diffuse out of cells and interact with

Immunology Today, vol. 8, Nos 7 and8, ;987

the surface-associated class I MHC molecules since such peptides would presumably be 'flushed from the system'. Bevan leaves the basis for class I/class II discrimination open but to explain the presentation of exogenously administered antigens, he proposes that a specialized cell type is capable of phagocytosing large molecules but presenting processed antigen only in the context of class I MHC. The existence of such a cell could account for the presentation of intact NP to influenza-specific Tc cells in viv028,29. Finally, what are the implications of these observations for vaccination? First, it is clear that the repertoire of Tc responses to individual influenza proteins in the context of different MHC alleles is only now being delineated. The extent of repertoire variation throughout the population will have to be carefully considered before subunit or peptide vaccines can be designed. Secondly, live virus is more effective than non-infectious preparations in the induction of Tc responses in vivo 46-48, even though injection of an isolated protein can prime for significant Tc memory ~8.29. Further work is required to establish the breadth of response to an individual protein such as NP in outbred populations. If the repertoire turns out to be limited, we will be forced to aim for an effectively attentuated but infectious whole virus particle. I am grateful to Marlene Bertagne and Sharon Contrereas for typing this manuscript; to Ite Askonas and Paul Travers for helpful discussions; and to A. McMichael, J. Bennink, T. Braciale, J. Rothbard, A. Townsend and J. Yewdell for allowing me to review their unpublished results. References

1 Stuart-Harris, C.M., Schild, G.C. and Oxford J.S.Influenza: the Viruses and the Disease (2nd edn), Edward Arnold, 1985 2 Lamb, R.A., Zebedee, S.L. and Richardson, C.D. (1985) Cell 40, 627-633 3 Askonas, B.A., McMichael, A.J. and Webster, R.G. (i982)in Basic and Applied Influenza Research (Beare, A.S. ed.), pp. 159-188, CRC Press 4 Lin, Y.L. and Askonas, B.A. (1981)J. Exp. Med. 154, 225-234 5 Lukacher, A.E., Braciale, V.L. and Braciale, T.J. (1984) J. Exp. Med. 160, 814-826 6 Taylor, P.M. and Askonas, B.A. (1986)Immunology 58, 417-420 7 White, J., Kielion, M. and Helenius, A. (1983) Q. Rev. Biophys. 16, 151-195 8 Zweerink, H.J., Courtneidge, S.A., Skehel, J.J., Crumpton, M. and Askonas, B.A. (1977)Nature 267, 354--356 9 Doherty, P.C., Effros, R.B. and Bennink, J. (1977) Proc. Natl Acad. Sci. USA 74, 1209-1213 10 Braciale, T.J. (1977) Cell. Immunol. 33,423-426 11 Zweerink, H.J., Askonas, B.A., Millican0 D., Courtneidge, S.A. and Skehel, J.J. (I 977)Eur. J. lmmunol. 7, 630-635 12 Braciale, T.J. (1979)J. Exp. Med. 149, 856--869 13 Ennis, F.A., Martin, W.J. and Verbonitz, M.W. (1977) Nature 269, 418-419 14 Ennis, F.A., Martin, W.J., Verbonitz, M.W. and Butchko, G.M. (1977) Proc. Natl Acad. Sci. USA 74, 3006-3010 15 Effros, R.B., Doherty, P.C., Gerhard, W. and Bennink, J. (1977) J. Exp. Med. 145, 557-568 16 Yamada, A., Ziese, M.R., Young, J.F., Yamada, Y.K. and Ennis, F.A. (! 985) J. Exp. Med. 162, 663-674 17 Braciale, l".J., Braciale, V.L., Henkel, T.J., Sambrook, J. and Gething, M.J. (1984)J. Exp. Med. 159, 341-354 18 Townsend, A.R.M., McMichael, A.J., Carter, N.P., Huddleston, J.A. and Brownlee, G.G. (1984) Cell 39, 13-25 19 Bennink, J.R., Yewdell, J.W,, Smith, G.L. and Moss, B. (1986) J. Virol. 57, 786-791

20 Fleischer, B., Becht, H. and Rott, R. (1985)J. Immunol. 135,

2800-2804 21 Owen, J.A., AIIouche, M. and Doherty, P.C. (1982) Cell. Immunol. 67, 49-59 Kees, U. and Krammer, P.H. (1984) J. Exp. Med. 159, 365-377 23 Townsend, A.R.M. and Skehel, J.J.(1984) J. Exp. Med. 160, 552-563 24 Townsend, A.R.M. and Skehel, J.J. (1982)Nature 300, 655-657 25 Bennink, J.R., Yewdell, J.W. and Gerhard, W. (1982) Nature 296, 75-76 26 Yewdell, J.W., Bennink, J.R., Smith, G.L. and Moss, B. (1985) Proc Natl Acad. Sci. USA 82, 1785-1789 27 McMichael, A., Michie, C.A., Gotch, F.M., Sm.th, G.L. and Moss, B. (1986)J. Gen. Vir. 67, 719-726 28 Wraith, D.C. and Askonas B.A. (1985) J. Gen. Virol. 66, 1327-1331 29 Wraith, D.C., Vessey, A.E. and Askonas, B.A. (1987) J. Gen. Virol. 68, 433-440 3~ aennink, J., Yewdell, J.W., Smith, G.L. ~nd Moss, B. (1987) J. Virol. 61, 1098-1102 31 Kaplan, D.R., Griffith, R., Braciale, V.L. and Braciale, T.J. (19U4) Cell. Immunol. 88, 193-206 32 Lukacher, A.E., Morrison, L.A., Braciale, V.L., Malissen, B. and Braciale, T.J. (1985) J. Exp. Med. 162, 171-187 33 Yap, K.L., Ada, G.L. and McKenzie, I.F.C. (1978) Nature 273, 238-239 34 Braciale, T.J., Andrew, M.E. and Braciale, V.L. (1981)J. Exp. Med. 153, 1371-1376 35 Askonas, B.A. and Lin, Y.L. (1982)Immunogenetics 16, 83-87 36 Wraith, D.C. (1984) Immunogenetics 20, 131-139 37 Townsend, A.R.M., Taylor, P.M., Melief, C.J.M. and Askonas, B.A. (1983)Immunogenetics 17, 543-549 38 Allen, H., Wraith, D., Pala, P., Askonas, B. and Flavell, R.A. (1984) Nature 309, 279-281 39 McMichael, A.J., Parham, P., Brodsky, F.M and Pilch, J.R. (1 ~80) J. Exp. Med. 152, 195-203 40 Wraith, D.C., Holtkamp, B. and Askonas, B.A. (1983) Eur. J. Immunol. 13, 762-766 41 Stringfellow, M., Wraith, D.C. and Askonas, B.A. (1983) Immunogenetics 18, 177-181 42 Blanden, R.V., Mullbacher, A. and Ashman, R.B. (1979) J. Exp. Med. 150, 166-173 43 Vitello, A. and Sherman, L.A. (1983)J. Immunol. 131, 1635-1640 44 Pala, P. and Askonas, B.A. (1986) Immunogenetics 23, 379-384 45 Townsend, A.R.M., Rothbard, J., Gotch, F.M. etal. (1985) Cell 44, 959-968 46 Braciale, T.J. and Yap, K.L. (1978)J. Exp. Med. 147, 1236-1252 47 Reiss,C.S. and Schulman, J.L. (1980)J. Immunol. 125, 2182-2188 48 Webster, R.G. and Askonas, B.A. (1980) Eur. J. ImmunoL 10, 396-401 49 Morrison, LA., Lukacher, A.E., Braciale, V.L., Fan, D.P. a;~d Braciale, T.J. (1986)J. Exp. Med. 163,903-921 50 Wraith, D.C. (1986) in Options for the Control of Influenza (Kendal, A.P. and Patriarca, P.A., eds), pp. 461-486, A.R. Liss 51 Wraith, D.C. and Vessey, A.E. (1986) Immunology 59, 173-180 52 Ertl, H. and Ada, G.L. (1981) Immunobiology 158, 239-253 53 Koszinowski, U., Gething, M.J. and Waterfield, M. (1977) Nature 267, 160-163 54 Grey, H.M. and Chesnut, R. (1985)Immunol. Today 6, 101-106 55 Townsend, A.R.M., Gotch, F.M. and Davey, J.(1985} Cell 42,457-467 56 Babbitt,B.P.,Allen,P.M., Matsueda, G., Haber, E. and Unanue, E.R.(1985) Nature 317, 359-361

245

Immunology Today.vol 8. Nos 7 and 8, 1987

$7 Watts, T.H, Gaub, M.E. and McConnell, H.M. (1986) Nature 320, 179-181 Hanski, C., Huhle,T. and Reutter,W. (1985)Biol. Chem. ~Seyler 366, 1169-1176 59 Hershko.A. and Ciechanover.A. (1982)Annu. Rev. Biochem. 51,335-364 68 Tanaka,K., Wakman, L. and Goldberg.A.L. (1983)J. Cell 3iol. 96. 1580-1585 61 Ciesielski-Treska,J., Goetschy,J.E and Aunis, D. (1984) Eur. J. Biochem. 138, 465-471 62 Gotch, F., McMichael,A., Smith, A. and Moss, B. (1987) L................J. Exp. Med. 165,408-416

63 Rosenthal,A.S. (1978) Immunol Rev. 40, 136-152 64 Townsend,A.R.M., Bastin,J., Gould, K. and Brownlee, G.G. (1987) Nature 324, 575-577 65 Maryanski,J.L, Pala,P., Corradin, G., Jordan, B.R.and Cerottini, J-C. (1987) Nature 324, 578-579 66 Cresswell,P. (1985)Proc. NatlAcad. Sci. USA 82, 8188-8192 67 Pernis,B. (1985) lmmunol. Today6, 45-49 68 Germain, R.N.(1986) Nature 322,687-689 68 Bevan,MJ. (1986) Nature 325, 192-194 70 Bastin,J., Rothbard,J., Davey,J., Jones, I. and Townsend,A. (1987) J. Exp. Med. 165, 1508-1523

The immunobiologyof Chlamydia Bacteria of the genus Chlamydia cause a wide variety of disorders in animals and people worldwide. The immune response to chlamydiae is poorly understood and, as Daniel Levitt shows here, there is recent evidence that these organisms induce perturbations in immune function that may assist their own survival in infected hosts and that of co-infecting microbes. Infc~tmation on immune responses towards and caused by the bacterial genus, Chlamydia, is limited despite its identification as a major human and animal pathogen worldwide (Table 1). In humans, Chlarnydia trachomatis, especially serotypes D-K, is the major cause of venereal infections in Western societies I. Conservatively, five million new cases of chlamydial sexual infections occur each year, making it more prevalent than all other sexually transmitted diseases combined. Chlamydiae will invade the cervix in women, occasionally ascending into endometrial epithelium and Fallopian tubes. This Dathway can leacl to salpingitis scarring and ensuing infertility. In fact, chlamydial infections are considered to be a primary cause of female sterility in the West 2-4. Babies that descend down a chlamydia-infected birth canal run a greater than 50% risk of contracting chlamydia conjuncti~,;tis and/or pneumonia, the major bacterial causes of these illnesses in newborns 5. Perhaps the most devastating and overlooked area of chlamydial infections are the 500 000 000 cases of trachoma worldwide, an ocular infection that can lead to conjunctival and corneal scarring, reduction in vision and eventual blindness6.7. This disease, caused by C trachomatis, is found mainly in developing nations, with a clear association between poverty levels and degree of severity of trachoma. Other human diseases associated with C. trachornatis include seronegative arthritis8, endocarditis9 and infer.ions of the male genitourinary tractlO. 1~. Recently, a new strain of Chlarnydia psittaci, designated TWAR, has been described and appears responsible for outbreaks of acute pneumonia in Finland, Nova Scotia and Seattle, Washington12. This strain may account for 10-20% of all radiographically documented pneumonia in adults. Chlamydia, both C trachornatis and C psittaci, can be primary animal pathogens, causing diseases in cattle, fowl and other birds, and contributing to rapidly

1246

1Clinical Oncology Program, Hoffrnann-La Roche Inc, Nutley, NJ 07110, USA; 2and Albert Einstein College of Medicine, Bronx, NY 10461, USA

Daniel Levitt: and JonBard z rising infertility in female Koala bears 13. Thus, the spectrum of chlamydial diseases worldwide is profound both in its scope and magnitude. Creation of rational treatment protocols to prevent some of its devastating chronic effects as well as formulation of protective vaccines for use in high risk populations depend upon an understanding of the biology and immunology of this unique and fascinating microbe. Life cyde of Chlamydia Both C trachomatis and C. psittad share a common life cycle distinct from other bacteria (Fig. 1). An extracellular form, the elementary body (EB, 0.2-0.4 i~m), attaches to certain cells (mainly epithelial cells) and is phagocytosed. Adhesion, at least for the L2 serovar of C. trach_nma.._tis..ma.~y. . . . . . . . . . . ~ . . . . . . . . . . . . ~,o o,,u kDa protease-resistant proteins. Chlamydial uptake into

Table 1. Diseasescausedby chlamydiae C trachoma~is Humans Female:

urethritis;cervicitisand vaginitis;endomc;.ritis; salpingitis;infertility;peritonitis,perihepatitisand periappendicitis;? cervicaldysplasia

Ma;e:

urethritis;epididymitis;prostatitis;anorectalinfections; lymphogranulomavenereum

Infants:

conjunctivitis;pneumoniabronchiolitis;?acuteotitis media

Nongender:ocular-trachoma;arthritis;endocarditis C psittad Humans Nongender:pneumonia- TWARagent; psittacosissyndrome Animals

Meningopneumonitis; encephalitis;inclusion conjundivitis;pneumonia;arthritis;intestinalinfection; infertility;abortion;ornithosissyndrome (~ 1987. ElsevierPublications,Cambridge 0167- 49191871502.00

i