- Email: [email protected]
Cell-mediated processing and presentation of T cell antigenic determinants
SCdL-ZS60 NSSI Prl -ws
PS ?‘QW IWN md ‘/t’la Ya@yWl) UmqPo 30 ‘Jo@1 DgIa8oun~ m 30 uogtmasaJd ag u! pa~agp q~fq~ sauop 3d.v OMI 30 suospedmos LLIOJ~ PIJE (L89-189:6T ‘6861 Iounurw iAW ‘rv la J~uJnyenSqS) aSep!SOl~@-d 30 ~103m2uj aq~~0 sap@4 tpf~ pmnptg qa3 L 30 sas -ptus woJ3 paymaJ aJa~ suo!snl3uo3 ms 2QssamJd ~t-wu 30 SmpoJd pua ay, @wsa~u JOU am s)v -Jalap pans-m ler~, swmpq adm!da u~ 30 suuo3 ~~aq.wis pue passamrd @mm~ uaawaq ibpq&D uog -a!m= Dm uf UO!D~sgl ~.‘uo!m@xa alq!ssod e a 3dv30 naga qnpnmw xp w pm.1~~~8aq 01smu -waiap aq~ 30 bg!qm! 8ur~eununa smp ‘8wssamJd @.Tn -1~ Jayt: adholdzq 3m maJagl~ E 30 3fl Lq pawasaJd aq pIno suo@aJ ~wa2lgm aum aw .swaioJd arm se paXyIoJ1~ a~a~bql uaq~ lou lnq sappdad ~gaqmk SE pappoJd aJa~kaql J!lxaluo3 3m uag.8 t IJ!pazw -80~3~aq pIno s’lmaiap asau [z] sa&oqdtwQ .L palD!JlSaJ-1 Sq3 UEUInq JO3 X3!XdS WloJd x!Jl -eur SIUM muangrr! atp 30 sadol!da Jo3 put? [ 11 sp L 3fXOlOti
palD!JlSaJ-nSqD~~aU&InUI~q~Z!t.I8O~JUIqO~OhII
30 sadol!da Jo3 pamasqo %M sg~_ *SUO$bJ word aums aq 30 sappdad ~paqlu.As pans-@uI!unu ueql ap=louI 3H&VW’ads E VIM ~‘T’os= 01 h!qE J!ayl u! PaFVl aJOUI aq h?u~ S.~E~alap
warm
0
SllnsaJ mp uo~c.wasaJd put? 8wssamJd uai@uE aI_p8~ -zpammqD uo pairiuamoJ aJo3aJatp 5~4 B~~ZIE q3~eas -aH 'pz0~ s! (3~) %aD 8ugUaSaJd-Ua8gw u! sJn33o @XLLIOU PU, 8ufssamJd ua@w aql uo apwoJd ut?3 sa! -pIW
UOQE3glpoW
ID@lUE
1EXfl UO~IX.JJO~
aI_fl ‘JXIMOH
ay, 30 uo!muau&J3 ~~OaioJd iaye 40 @uog.my aJe y3~ sado@ 01 pue ‘aJfumm, hwai agm arF,30 8~ .-plo3un alduns Aq pap~3~ sal!s01 ‘a~ze~our 3m atp 01 Apz3~1ppwq 01 avfotu a4wu arF, u! uropaa~3 @uog -euuo3uo~ luapgns aAt?qletp asotg ~0~3 a2knzJasau 'UO~I?!DOSSE 3m
JO3 aJfIIbaJ bql SUO~ESI3!pouI?qLl01
~u!pJO~~~(GLZ_OLZ:8‘L861 kW%! lounmw ‘mw) paz!J -0%1m 3q UT3 sl mufuuaiap Dwa8gut2na3 1 *uog~!2osse aiap atp ,SSamJd, ~0 _f@xm um swacu 3HbU Jo3 lm -ma pmalxa snow ‘adoitda ~gmds atp uo Z?u~puadag
papaau aJo3aJatp s! JoldaDaJ na3 L ay, pue sa~~otu 3m ql" UO~E~DOSSE JO3 wav aaJ3 ol.,uJs~y3aw v ~sl~lsuo~~~alsJatpoJouogc1O~~u~alu~~~y~3oasn~3 -5q salwalour 3wq qlwAp=Jyp 13araluf01 alqwn & -Jaua8 aJtzsqaloJd aAgzu v sadoltda ~~a2?~ue na3 z .apz3lotu
na3 L passamJd @TJIllriN
‘3dv Lq 2Qpucq ua@m 30 wad -m pams uo uo~muJo3u~ iua3aJ azpmrtuns p 1 ‘aJaH .waioJd aAr$TuUOdn &gl~ JvflaD 30 uofm aI_p~0~3
li:~-,g~:z
‘686~ LBolounLuuy
uog3npor)t.q
LI! uoydo
v_mm-g
118
Antigen recognition
USA 1989,86:331&3320). Cellular processing may result in a less completely degraded product which is able to associate with some, but not all, MHC class 1 or class 11 gene products.
Kinetics of antigen presentation Synthetic peptides added externally sensitize APC for T
cell recognition as a function of time and concentration without a detectable delay interval [3]. In contrast, in the presentation of native proteins, there is a mandatoty lag period before the appearance of dekrminants on APC membranes. Using measurement of calcium influx to assay T cell stimulation rapidly, a lag period of 6Omin at 37°C was needed to obtain T cell recognition of native tetanus toxoid [3]. This delay agrees well with the lag period previously descnbed for other protein antigens (Ziegler and Unanue, J Immunoll981, 127:1869-1875; Chesnut et al, J Immunoll982,129:2382-2385). Sign& cantly, the delay interval was shown to be essentially independent of antigen concentration, suggesting that this interval is close to the time needed to process and present the native antigen rather than simply reflecting the accumulation of a threshold number of determinants on APC surfaces. Ionger times (6-8 h or more) are needed to achieve maximal expression of T cell epitopes [4,5]. Again, this was found to be independent of initial antigen concentration and of whether the antigen was introduced by binding to an AEC surface receptor or by fluid-phase pinocytosis [41. Dissociation of antigens from Ia complexes is known to occur very slowly with isolated components (halflives of around 30 h; Buus et al, Cel1 1986, 47:1071-1077; Nature 1989, 337: 274 SadeghNasseri and MCCOMd, -276). In contrast, antigenic determinants presented by viable APC can functionally turn over either more rapidly or more slowly. The expression of an epitope of pigeon cytochrome c, introduced to functional AEC either as the Intact protein or as a short peptide, had declined to halfmaximal levels only 10 h after the antigen pulse [4]. Individual T cell determinants delivered as part of the same protein can have halflives on AFC surfaces that dilfer greatly~ two regions of the influenza virus hemagglutinin glycoprotein, botb restricted to the same I-Ed molecule, had vety different functional longevities when presented after pulsing of APC with tbe isolated protein. The expression of one determinant (in the region of amino acids 111-119) continuously increased over a 48 h experiment; expression of tbe other determinant (positions 302-313) peaked at 8h post-pulse, but declined sharply thereafter [ 51. An I-Ad-restricted epitope of influenza hemagluttinin (residues 126-138) which, as a syn thetic peptide, binds with high avidity to isolated Ia (Buus et al., Science 1987,235:1353-13581, had a shorter functional half-life when presented from me native protein than an 1-Ed-restricted determinant (residues 111-119) which bound less strongly to its restricting element in vitro. Thus, the lifetime of antigenic determinants on tiable APC cannot necessarily be predicted from the be havior of synthetic peptides with Ia in isolated systems.
Distinct processing pathways Although peptides added externally can associate freely
with either MHC class 1 or class 11 molecules according to their avidities, the MHC presentation of determinants of native proteins is strongly dependent upon the processing pathway. Antigens which enter the cell exogenously by endocytosis are generally used exclusively in MHC class Ikestricted presentation, while class I-restricted determinants are presented from antigens either biosynthesized internally or introduced into the cytoplasm (Germain, Nature 1986,322:687-X%9 Moore et al, Cel1 1988, 54:777-785). These two major pathways, tbe exogenous/endocytic and the biosyntbetic/cytoplasmic, have many distinct properties, such as the sensitivity of the former to lysosomotropic agents (Ziegler and Unanue, Proc Nat1 Acad Sci USA 1982, 793175178) and of the latter to the fungal metabolite brefeldin A [6,7], suggesting that they use many non-overlapping cellular activities. Association with MHC The cellular location of MHC association with proces&
antigen is not clearly established but, in general, it does not appear to occur simply by fluid-phase dilfusion at tbe cell surface as observed with exogenously added syntbetic peptides. For example, maximal T cell stimulation by functional APC can be achieved with much lower concentrations of a determinant delivered as the native antigen man as the synthetic peptide 131. This suggests that there is some form of facilitated association with MHC molecules, possibly in a cellular compartment of antigen-Ia intersection (Creswell, Proc Nat1 Acad Sci USA1985,82:8188) where local concentrations of a processed determinant may be relatively high. Altematively, anchoring structures may stabilize processed antigen at the cell surface (Falo et al, Proc Nat1 Acad Sci USA1987,
84~522-526; lakey et al Proc Nat1 Acad Sci US4 1987, 84:165%X%3), preventing rapid diffusion before it has had an opportunity to interact with MHC molecules. It has been proposed that MHC molecules also fùnction in the intracellular acquisition and transport of processed antigen. As cell surface glycoproteins which are synthesized vectorially and can be recycled into the cell (Tse et al., J Mol Cel1Bio1 1986, 2315329) [8], MHC molecules have properties typical of specific transport proteins. By virtue of their ligand-binding capabilities, MHC molecules may not only acquire antigen within processing compart ments, but may also contribute to the specificity of antigen processing by protecting labile determinants from destruction after binding (IeyvaCobian and Unanue, J
Immunol1988, 141:144-145).
The cellular mechanism of antigen presentation A complex series of events is involved in the processing and presentation of T cell determinants by APC. A conceptual scheme of cellular antigen presentation (outlined
Cell-mediated
processing and presentation of T cell antigenic determinants Hackett
in Fig. 1) involves: antigen acquisition for the different pathways, structural modiikation of antigen to allow association with h4HC molecules, and transport of processed material to the cell surface where antigen/MHC is avail able for interaction witb T cell receptors. As with many cellular processes, antigen presentation appears to be cyclic, with presented determinants having distinct half lives of expression on APC surfaces. The cell biology of antigen handling is not yet well’understood. For example, it is not possible to determine with certainty which events in the cellular handling of a radioWeled molecule are significant for antigen presefitation. At present, the functional assay of T cell recognitick must be used to identiiy whether a given epitope is being expres&. Future research must use multiple approaches; new insights from such studies have recently emerged on the mechanism of antigen presentation by APC. Antigen acquisition
Tbe method by which potential APC acquire antigens for processing bas a critical impact on the outcome of antigen presentation. Entry into a specihc pathway can favour MHC class 1 or class II association (see above).
In the endocytic route, uptake by antigen-specilïc surface immunoglobulin or other membrane receptor results in effective h@IC class Krestricted presentation of antigen present at concentrations lower by several orders of magnitude than those required when uptake is strictly by fluid-phase pinocytosis (Ianzavecchia, Nuture 1985, 314:537-539). The efficient use of endocytosed antigen may also involve the invariant chain of Ia [9]. Murine fibroblasts, which have a low endogenous leve1 of invariant chain expression, were transfected witb MHC class II genes. Some clones were then ‘supertransfected’ with genes encoding invariant chain polypeptides. Fibroblasts transfected only with MHC class II genes were unable to present the antigens of the C5 component of complement or inIluenza virus hemagglutinin to T cells . However, the presence of the invariant chain endowed these fibroblasts with the ability to present these proteins even at’low input concentrations. The leve1 of surface expression of IvlHC class II molecules was not influenced by invariant chain presence. Peptides of C5 or of hemagglutinin, which do not require cellular processing for MHC interaction, were presented equally well by invariant chain-positive or negative cells. The function of the invariant chain in ei%Fig. 1. Conceptual scheme of cellular events in processing and presentation of antigens.
T cel1 determinants
T cel1 determinants
requiring minimal processing
requiring extensive processing
Proteolysis/
association with MHC
further degradatior / loss of labile determinants
Intracellular antigen handling
Uptake
Native protein
119
120
Antigen recognition
cient antigen presentation is not known. The polypeptide itself is not expressed on cell surfaces, and must function in the intemal processing of antigens. Presentation of MHC class 11-restricted measles virus antigens prcduced by biosynthesis in APC has been shown to occur in the absente of the invariant chain (Sekaly et al., Proc Nat2 Acad Sci USA 1988, 85:120+1212). The most important role of this molecule may therefore be in handling of endocytosed antigens. Although degradation appears to be the ultimate fate of endocytosed antigen, some intemalized antigens recycle to the cell surface in a relatively unaltered form. In human B cell clones with tetanus toxoid-specilìc surface immunoglobulin, this antigen was endocytosed and recycled to the surface complexed with immunoglobulin [ 101. This cycling occurred with monovalent antigen and did not depend upon capping of surface immunoglobulin. Indeed, an ultrastructural study [ 111 showed that antigens which were unable to bind to cellular receptors als0 recycled to the cell surface after uptake. Although the relevante of the reappearance of such antigens to T cell recognition is not yet clear, these observations indi cate that there are cellular mechanisms that handle foreign antigens in pathways other than their unidirectional degradation within cells.
Antigen modification While uptake can be assessed at the leve1 of the whole
protein, antigen modifìcation and subsequent steps must be studied on the basis of individual T cell epitopes, which can have processing requirements that diifer greatly. Certain MHC class 1-restricted epitopes of the influenza virus nucleoprotein were not well presented when introduced to APC by vaccinia vectors encoding the nucleoprotein gene [12]. This inhibition of expression of inlluenza epitopes by vaccinia could be at least partially reversed by the introduction of a modiiíed form of nucleoprotein which could be readily degmded in the cell. This was accomplished by making a construct of the ubiquitin gene sequence and the nucleoprotein gene with the codon for the N-terminal methionine changed to one for arginine. This encodes a fusion protein which engages the ubiquitindependent cellular degradative path way (for review, see Dice, FRSEBJ 1987,1:359-357). This observation suggests that intracellular protein breakdown may function in the processing of at least some epitopes for MHC class 1 association, although the specifìc defect caused by vaccinia infection has not yet been established. Epitopespeciiìc perturbation of exogenously supplied antigen was studied using monoclonal antibodies [13]. The antigen j3galactosidase was treated with individual monoclonal antibodies specilïc for different regions of the molecule and then introduced to macrophage APC as an antigekantibody complex. These various APC were then assayed for the ability to be recognized by a panel of distinct class II-restricted T hybridoma clones. Although in most cases antibodies resulted in a more efficient use of antigen by enhancing uptake via FC receptors, some T cells showed greatly reduced responses to antigen treated
with specilïc monoclonal antibodies. Epitope-specifìc inhibition of antigen processing or MHC association was a possibility as antibodies able to reduce recognition by one T cell clone had no effect upon T cells of other speciiïcities. These data not only suggest that surface immunoglobulin has a role in determining T cell epitope availability, but also shows that it is feasible that epitopes are protected in the processing compartment, a possible effect of intracellular MHC binding.
Transport The ability of processed antigen to translocate to the cell surface is a prerequisite for T cell recognition (for review, see Long, Zmmunol7”~ 1989,10:232-234). Transport of MHC molecules to the APC surface may convey antigenie dete rminants associated with the binding site. A pool of intracellular MHC class 11 molecules which may associate internally with uptake antigen was shown to exist [8]. In some APC, intracellular Ia is maintained by recycling from the cell surface, while in ethers, particularly adherent macrophages, new synthesis appears to be the major source of Ia. This could explain the loss of the antigen-presenting function of macrophages, but not of non-adherent B cells, upon treatment with protein synthesis inhibitors (Jensen, J Zmmunol 1988, 141:25452550). Non-MHC structures have also been implicated in the transport of processed antigens. A peptide-binding protein has been demonstrated at the cell surface where antisera to the peptide-binding protein were able to perturb presentation (Lakey, et al, Proc Nat1 Acad Sci USA 1987, 84:165+1663). The antigenic peptide angiotensin 11was shown to associate with a high molecular weight molecule when introduced to various cell types, independent of Ia expression (Betancourt et al, J Zmmunol 1987, 1393725-3729). The efficiency of formation of these complexes varied with the cell line and the peptide used. The role of these peptide-binding molecules in the association of antigen with h4HC molecules is not known. Possibly, they may convey antigen to APC membranes for external association with Ia, or may anchor T cell determinants on cell surfaces, as has been suggested for some antigens by fatty acylation (Falo et aL, Proc Natl Acad Sci USA 1987, 84:522-526). Information has recently become available on the role of intracellular transport in presentation of MHC class Irestricted determinants. The fungal metabolite brefeklin A, which interrupts intracellular transport without af fecting protein synthesis, was used by two independent groups to study recognition of influenza virus antigens by cytotoxic T cells [ 6,7]. Brefeklin A was found to black the transport of newly synthesized antigens from the endoplasmic reticulum and the transGolgi, its presence resulting in failure to present MHC class 1-restricted antigens which were either biosynthesized or introduced into the cytoplasm of APC. Reversal of brefeklin A blocking was readily achieved and could occur in the presence of pro-
Cell-mediated wocessinn and wesentationof T cel1antieenicdeterminantsHackett tein synthesis inhibitors. Externally added synthetic peptides recognized in tbe context of MHC class 1 molecules could sensitize target cells for lysis in tbe presence of brefeldin A, indicatlng that MHC molecules on cell surfaces were not tiected. Presentation of MHC class IK restricted dete rmlnants was only marglnally alkcted by brefeldin A, suggesting that this drug is relatively specik and does not affect endocytosis or cellular tknsport beyond the trans Golgi. Since the presentation pf nonrepllcative, cytoplasmicaJly introduced virus was also inhlbited by brefeldin A, it is clear that the effect:did not rely on disruption of tbe transport of biosynthesized viral antigen. The data suggest that newly synthesized MHC class 1 molecules may be responsible for convhg antigenie determlnan ts proces& in the cytoplasm to the cell surface. While direct proof of this pathway is not available, the sensitization of target cells for cytotoxic T cell lysis paralleled the expression of newly synthesized MHC class 1 molecules, even though large amounts of MHC molecules were already avklable at the cell surface and non-infectieus, cytoplasmically loaded influenza virus, which does not require viral biosynthesis for expression, was used as antigen.
Turnover Once expressed on the surfaces of viable APC, MHC class Krestricted T cell determinants are lost as a function of time. There is llttle information on tbe mechanism of tumover of presented antigen. A cellular activity inhibited by aldehyde fïxation, is required, but it is not known whether the tumover is due to loss of Ia-antigen complexes or to the removal or replacement of antigen at the binding site of stable h4HC molecules. NO treatments short of cell Iïxation have been shown to aker the halfllves of specifïc epitopes [ 51. At present, tumover rates measure net T cell stimulation for a given epitope and the fate of individual antigen-MHC r;omplexes is not known. For example, all cell surface Ia may recycle at tbe same rate, but differences in re-expression of individual T cell epitopes may result in the perceived functional stability or lability of individual determinants. Tumover occurs more quickly tban dissociation of peptides from isolated Ia. However, release of antigen from Ia may be accelerated in an acidic environment (Buus et aC, Cel2 1986, 4731071-1077), which could occur during recycling into tbe cell. If individual determinants have Merential susceptibility to removal from tbe antigen-binding site under stringent conditions, then these determinants may exhibit prolonged su&val on APC surfaces. The longevity of T cell determinan t expression may be a factor in the relative immuncdominance of T cell sites on a protein.
and present many different molecular forms of processed protein antigens. Evidente that heterogeneous, ‘partklly processed’ antigenic forms associate with h4HC molecules on APC surfaces is provided by the recent observations that: (1) relatively unaltered antigen can reappear on the cell surface after endocytosis; (2) some epitopes of a given protein required little or no proteolytic modi&ation for MHC association; (3) end procucts of natural processing need not correspond to minimal peptides delìned by chemical synthesis, and (4) relatively large antigens may functionally associate with MHC molecules. Aldehyde-lïxed APC can be sensitlzed for T cell recognition by natlve protein (Lee et al, J Immunol 1988, 140:1063-10681, conformationally altered hemagglutinin as part of intact tiuenza virus (Eisenlohr et al, J Immunol 1988, 141:187Ck-18761, or synthetic peptide antigen covalently coupled to large protein molecules Qne specu(Casten et al, JEq Med 1988,168:171-180). lation is that partial processing of antigen nïay lead to the expression of a large antigenic molecule bearing both exposed and hidden T cell detemWants. Re-uptake of the antigen may subject it to more extensive processing, resulting both in the expression of determinants requiring more complete fragmentation and in the loss of labile epitopes. In this scheme, antigen presentation may be viewed as a continuous ‘sarnpling’of processing compartments for transport of any molecule exhibiting sufkient avidity for MHC or other transport molecules. The cell biology of T cell epitope expression requires further study. In particular, basic information is needed on the mechanism of transport of proces& forms, the cellular location of antigen-MHC association, and the basis of T cell determination half-lives on APC surfaces.
Annotated
0 ??e
The distinctive requirements for expression of in&vidual T cell epitopes suggest that APC can transport
and recommended
Of interest Of outstanding interest
1.
BREIT SJ, CEASEKB, BERZOWY JA: Inhences of antlgen processing on the expression of the T cell repertoire. Evidence for MHC-specilïc hindering structuren on the products of processing. J Eqb Med 1988, 168:357-374. T cell determlnan ts that could be recognlzed by T cells when introduced to APC as synthetic peptides were not presentexl by processing of the intact myoglobln protein. This fallure to present protein determinants occurred only in certain MHC haplotypes and was related to the MHC molecule needed for T cell recognition, slnce Ft APC pre sented the determlnants to the responder haplotype patent only. End products produced by natural processing seemed to lnteract with MHC molecules dlfferently from the correspondlng synthetic peptldes. The ln!luence of antlgen processing on MHC-llnked T cell responslveness was supported by the dernonstratlon that lmmunlzatlon tith natlve whole proteln vexsus synthetic farms of antigens yielded Werent antlgenlc specisícities of responding T cells. ??e
Bo~hc% HC, GOTCH FM, MCMKXAELAJ: class 1 crossrestrlcted T cells rcveal low respondcr allele due to processing of vlral antigen. Nature 1989, 337:65+654. Human cytotoxic T cell clones restricted to HLA-Aw69were able to rec ognize a synthetic peptlde of influenza virus matrix proteln but not the same detertninant produced in tirwinfected cells. Thus, as in [ 11,class 2.
Conclusion
references
reading
??
121
122
Antigen recognition 1-restricted peptides produced from native proteins can differ iimctionaUy from the minimal dete rminant defìned by chemical synthesis. 3.
ROOSNEK E, DEMOIZ S, CORRADIN G, IANZAVECCHIA A: Kinetics
of MHC-antigen complex fortnation on antigen-presenting cells. J Immund 1988, 14040794Ot32. A Iag pedod of 1 h at 3PC was needed for the functionaI appearance of antigenic determinants on antigen-puIsed AFC. This intenal was essentialiyindependent of initiaIantigen concentmtion, and probabIy reflects the time tequked for antIgen processing and presentation, rather than the accumulation of anngenIc determinants to some thteshold leve1 on APC surfaces. A high concentration of a 12-residue synthetic peptide, which does not need antigen processing, was able to sen.sItIzeAPC for T ceU recognItion without a Iag perk& By using a sensitive, rapid meas ure of T ceU acthation by calcium uptake, the assocIation of the iag period with antIgen processing/presentation was cleady estabUshed. 00
4.
E, PIERCESK: L+XEYEK, CASTENIA, NIEBLINGWL, MARGOIIASH Time dependence of B cell processing and presentation of peptide and native protein antigens. J Immurwl 1983, 140:3309-3314. Antigen deUvered as the native cytochrome c required 6-8 h for APC to be maxima& stimulatory for T cek ThIs time was Independent of .startIngantIgen concentration or of whether the antigen was Introduced by specific or non-specUic uptake. LeveIs of presented antigen decUned after 8-12 h postpuk. The kinetics of the Ioss of determhrants were sImUar to these for determmants Introduced to Uve APC either as the protein or as the synthetic peptide. 0
EISENIQHR LC, GERHARDW, HACKE?TCJ:
Individual class IIrestricted antigenic det enninants of the same protein exhibit distinct kinetics of appearance and persistente on antigen-presenting celk. J Immutwl 1988, 141:2581-2584. After puismg APC with IsoIated hemagglutinin giycoprotein or the nonreplicative infhtenza virus, the time course~ of expression of IndIvidual T ceU dete mrhrants was measured. Expression of 2 detemrinants rec ognbzed In the context of 1-Ed differed greatiy, with one continuousiy Increasmg over 48 h whUe the other peaked at 8 h and decIined there after. Ahhough the proteoIysIis InhIbitor leupeptin altered processing of each dete rmInant, it dId not aikct their haIfUves on APC surfaces. 5. 0
NUCHTE~NJG, BONIFACINO JS, BIDDISONWE, K~AUSNERRD: (Letter to the Fditor) Brefeldin A impiicates egress from endoplasmic reticulum in class 1 restricted antigen presentation. Nature 1989, 339:22%225. BrefeldIn A was able to InhIbit MHC cIass I-mstricted cytotoxk T ceU recognition of InIluenza virus mattix promIn. ThIs was reked to its abII ity to hak transport of proteIns from the endoplasmk reticuium. Protein synthesis was not akted by this drug, nor was the recogmtion of MHC class B-restricted dete nninants processed by the exogenous/endocytic pathway. ThIs Is the first evidente of IntraceUuIar transport as a requhement for presentation of MHC ciass 1-restricted antigens.
6. oo
Brefeldin A specItïcaUy Inhibits presentatkm of protein antigens to cytotoxk T Iymphocytes. Science 1989, 244:1072-1074. Using cytopiasmicaUy processed non-repkative virus as antigen, a correhttion was shown between transpott of newiy synthesked MHC cIass I molecules from the endopiasmk reticulum or tralzs Go@ and T ceU recognition. This is important evidente that MHC molecuIes may convey proces& antigen to the ceU surface.
7. 00
YJ?WDELL JW, BENNINKJR:
8. 0
HARDINGCV, UME ER Antigen processing and Intracellular Ia: possible roles of endocytosIs and protein synthesis In Ia function. J Immund 1988, 142:12-19.
A pool of intracektiar MHC cIass 11molecuIes were identIikd, at kast a propottion of which resukd from recycling of ceU surface-expressed molecuIes. Ia was InternaU& Into Ught endosomes, Into whkh ttansfenin is taken, suggestmg that the recycUng molecule may be avaUable to intetact with antIgen in an IntraceUuIar processmg compartment. STOCKINGER B, PESSARAU, L~NRH, HABICHTJ, GREZ M, KOCH N: A role of Ia-associa ted invariant chains in antigen processing and presentation. Cel1 1989, 566.!33&39. The invariant chain of Ia was introduced to MIX ciass II-expressing fibroblasts by gene transfection. The inwriant chain conferred the abiiity to present the Influenza virus hemagghttinin or complement C5 antigens, even at low concentrations. The invariant chain did not increase MHC ciass II expression on fibrobIasw, its ftmction may be at the leve1 of intraceUuIar antigen handUng. This Unding suggests a physiological role for the Invariant chain in APC iünction.
9. 00
10.
WARS C, DAVIDSONHW: EndocytosIs and recycling of specifîc antigen by human B cell iines. EMBO J 1988, 7~1937-1946. Antigen taken up by surface immunogIobuUn receptors of antigen-spe ciEc B ceils was endocytosed rapidly, but a certain propottion reappeared at the ceU surface in a reiativeIyintact form complexed with im munogiobuUn. Uptake and recycUng did not depend upon crossUnking of the surface immunogIobuUn. Although it is nat known whether such antigen handUng is directly rekatedto MHC-associated presentation to T ceUs, these data indicate the presence of ceUuIar mechanisms to return incompletely degraded material to ceU surfaces. ??
LINJ, BERZOFSKYJA, DE~~VITCHTL Ukrastructural study of IntemaIixation and recycling of antipen by antIgen presentIng ceIIs. J Mol Cell Immund 1988, 3~321-343. The ceUuIar routing of different coUoIdai gold-Iabeled antigens was studied In thIn-section. Both pork insuUn and a tryptic fragment of pork insuiin Iacking receptor-binding abUitywere observed to recycle to the surface after endocytosis. Myogiobin, in contrast, did not readUy reappear on ceU surfaces. Thus, receptor-mediated uptake is not necessary for antigen reyciing, and routing behavior can differ between proteins. 11. ??
TOWNSENDA, BA~TINJ, Gourr, K, BROWNLEEG, ANDREWM, COUPAUB, BOYIE D, CHAN S, Slilrm G: DefectIve presentation to class 1-restrkted cytotoxic T lymphocytes in vaccinia-infected celIs is overcome by enhanced degradation of antigen. J Eq Med 1988, 168:1211-1224. By introducing a form of Iniluenza nucleoprotein able to k readUy degraded by the ImraceUuiar ubiquUin-Unked pathway, the vaccinka MNSinduced black of presentation of certain nucleoprotein T ceU determinants could be avoided. This correiates the processing of some MIK cIass I-restricted T ceU determinants with proteoIytic fmgmenta tion within the ceU. 12. ??
-CA F, FENOGUOD, KUNIUA, CAMBIAGGI C, Qsso M, CEIADA F: Difkrential activation of T cell clones stimulated hy macrophages exposed to antigen complexed with monoclonal antibodies: a possible infhtence of paratope speciEcity on the mode of antigen processing. J Immund 1988, 140:289H898. MonocIonaI antibodIes comphexed to g-gakìctosidase before introduction to macrophage APC could block presentation of determInants to T ceU clones of certain speciEcities. AbUityto black presemation de pended upon the speciEcities both of antibodies and of T ceUs. WhUe neither the precise effect of antibodies upon presentation, nor the positional correspondence between B and T ceU epitopes in effective combinations are known, the data suggest that antibodies may speciEcaUy aifect the intmceUuIarprocessing of tegions of &aIactosidase by sterk inhibition. 13. ??
PS ?‘QW IWN md ‘/t’la Ya@yWl) UmqPo 30 ‘Jo@1 DgIa8oun~ m 30 uogtmasaJd ag u! pa~agp q~fq~ sauop 3d.v OMI 30 suospedmos LLIOJ~ PIJE (L89-189:6T ‘6861 Iounurw iAW ‘rv la J~uJnyenSqS) aSep!SOl~@-d 30 ~103m2uj aq~~0 sap@4 tpf~ pmnptg qa3 L 30 sas -ptus woJ3 paymaJ aJa~ suo!snl3uo3 ms 2QssamJd ~t-wu 30 SmpoJd pua ay, @wsa~u JOU am s)v -Jalap pans-m ler~, swmpq adm!da u~ 30 suuo3 ~~aq.wis pue passamrd @mm~ uaawaq ibpq&D uog -a!m= Dm uf UO!D~sgl ~.‘uo!m@xa alq!ssod e a 3dv30 naga qnpnmw xp w pm.1~~~8aq 01smu -waiap aq~ 30 bg!qm! 8ur~eununa smp ‘8wssamJd @.Tn -1~ Jayt: adholdzq 3m maJagl~ E 30 3fl Lq pawasaJd aq pIno suo@aJ ~wa2lgm aum aw .swaioJd arm se paXyIoJ1~ a~a~bql uaq~ lou lnq sappdad ~gaqmk SE pappoJd aJa~kaql J!lxaluo3 3m uag.8 t IJ!pazw -80~3~aq pIno s’lmaiap asau [z] sa&oqdtwQ .L palD!JlSaJ-1 Sq3 UEUInq JO3 X3!XdS WloJd x!Jl -eur SIUM muangrr! atp 30 sadol!da Jo3 put? [ 11 sp L 3fXOlOti
palD!JlSaJ-nSqD~~aU&InUI~q~Z!t.I8O~JUIqO~OhII
30 sadol!da Jo3 pamasqo %M sg~_ *SUO$bJ word aums aq 30 sappdad ~paqlu.As pans-@uI!unu ueql ap=louI 3H&VW’ads E VIM ~‘T’os= 01 h!qE J!ayl u! PaFVl aJOUI aq h?u~ S.~E~alap
warm
0
SllnsaJ mp uo~c.wasaJd put? 8wssamJd uai@uE aI_p8~ -zpammqD uo pairiuamoJ aJo3aJatp 5~4 B~~ZIE q3~eas -aH 'pz0~ s! (3~) %aD 8ugUaSaJd-Ua8gw u! sJn33o @XLLIOU PU, 8ufssamJd ua@w aql uo apwoJd ut?3 sa! -pIW
UOQE3glpoW
ID@lUE
1EXfl UO~IX.JJO~
aI_fl ‘JXIMOH
ay, 30 uo!muau&J3 ~~OaioJd iaye 40 @uog.my aJe y3~ sado@ 01 pue ‘aJfumm, hwai agm arF,30 8~ .-plo3un alduns Aq pap~3~ sal!s01 ‘a~ze~our 3m atp 01 Apz3~1ppwq 01 avfotu a4wu arF, u! uropaa~3 @uog -euuo3uo~ luapgns aAt?qletp asotg ~0~3 a2knzJasau 'UO~I?!DOSSE 3m
JO3 aJfIIbaJ bql SUO~ESI3!pouI?qLl01
~u!pJO~~~(GLZ_OLZ:8‘L861 kW%! lounmw ‘mw) paz!J -0%1m 3q UT3 sl mufuuaiap Dwa8gut2na3 1 *uog~!2osse aiap atp ,SSamJd, ~0 _f@xm um swacu 3HbU Jo3 lm -ma pmalxa snow ‘adoitda ~gmds atp uo Z?u~puadag
papaau aJo3aJatp s! JoldaDaJ na3 L ay, pue sa~~otu 3m ql" UO~E~DOSSE JO3 wav aaJ3 ol.,uJs~y3aw v ~sl~lsuo~~~alsJatpoJouogc1O~~u~alu~~~y~3oasn~3 -5q salwalour 3wq qlwAp=Jyp 13araluf01 alqwn & -Jaua8 aJtzsqaloJd aAgzu v sadoltda ~~a2?~ue na3 z .apz3lotu
na3 L passamJd @TJIllriN
‘3dv Lq 2Qpucq ua@m 30 wad -m pams uo uo~muJo3u~ iua3aJ azpmrtuns p 1 ‘aJaH .waioJd aAr$TuUOdn &gl~ JvflaD 30 uofm aI_p~0~3
li:~-,g~:z
‘686~ LBolounLuuy
uog3npor)t.q
LI! uoydo
v_mm-g
118
Antigen recognition
USA 1989,86:331&3320). Cellular processing may result in a less completely degraded product which is able to associate with some, but not all, MHC class 1 or class 11 gene products.
Kinetics of antigen presentation Synthetic peptides added externally sensitize APC for T
cell recognition as a function of time and concentration without a detectable delay interval [3]. In contrast, in the presentation of native proteins, there is a mandatoty lag period before the appearance of dekrminants on APC membranes. Using measurement of calcium influx to assay T cell stimulation rapidly, a lag period of 6Omin at 37°C was needed to obtain T cell recognition of native tetanus toxoid [3]. This delay agrees well with the lag period previously descnbed for other protein antigens (Ziegler and Unanue, J Immunoll981, 127:1869-1875; Chesnut et al, J Immunoll982,129:2382-2385). Sign& cantly, the delay interval was shown to be essentially independent of antigen concentration, suggesting that this interval is close to the time needed to process and present the native antigen rather than simply reflecting the accumulation of a threshold number of determinants on APC surfaces. Ionger times (6-8 h or more) are needed to achieve maximal expression of T cell epitopes [4,5]. Again, this was found to be independent of initial antigen concentration and of whether the antigen was introduced by binding to an AEC surface receptor or by fluid-phase pinocytosis [41. Dissociation of antigens from Ia complexes is known to occur very slowly with isolated components (halflives of around 30 h; Buus et al, Cel1 1986, 47:1071-1077; Nature 1989, 337: 274 SadeghNasseri and MCCOMd, -276). In contrast, antigenic determinants presented by viable APC can functionally turn over either more rapidly or more slowly. The expression of an epitope of pigeon cytochrome c, introduced to functional AEC either as the Intact protein or as a short peptide, had declined to halfmaximal levels only 10 h after the antigen pulse [4]. Individual T cell determinants delivered as part of the same protein can have halflives on AFC surfaces that dilfer greatly~ two regions of the influenza virus hemagglutinin glycoprotein, botb restricted to the same I-Ed molecule, had vety different functional longevities when presented after pulsing of APC with tbe isolated protein. The expression of one determinant (in the region of amino acids 111-119) continuously increased over a 48 h experiment; expression of tbe other determinant (positions 302-313) peaked at 8h post-pulse, but declined sharply thereafter [ 51. An I-Ad-restricted epitope of influenza hemagluttinin (residues 126-138) which, as a syn thetic peptide, binds with high avidity to isolated Ia (Buus et al., Science 1987,235:1353-13581, had a shorter functional half-life when presented from me native protein than an 1-Ed-restricted determinant (residues 111-119) which bound less strongly to its restricting element in vitro. Thus, the lifetime of antigenic determinants on tiable APC cannot necessarily be predicted from the be havior of synthetic peptides with Ia in isolated systems.
Distinct processing pathways Although peptides added externally can associate freely
with either MHC class 1 or class 11 molecules according to their avidities, the MHC presentation of determinants of native proteins is strongly dependent upon the processing pathway. Antigens which enter the cell exogenously by endocytosis are generally used exclusively in MHC class Ikestricted presentation, while class I-restricted determinants are presented from antigens either biosynthesized internally or introduced into the cytoplasm (Germain, Nature 1986,322:687-X%9 Moore et al, Cel1 1988, 54:777-785). These two major pathways, tbe exogenous/endocytic and the biosyntbetic/cytoplasmic, have many distinct properties, such as the sensitivity of the former to lysosomotropic agents (Ziegler and Unanue, Proc Nat1 Acad Sci USA 1982, 793175178) and of the latter to the fungal metabolite brefeldin A [6,7], suggesting that they use many non-overlapping cellular activities. Association with MHC The cellular location of MHC association with proces&
antigen is not clearly established but, in general, it does not appear to occur simply by fluid-phase dilfusion at tbe cell surface as observed with exogenously added syntbetic peptides. For example, maximal T cell stimulation by functional APC can be achieved with much lower concentrations of a determinant delivered as the native antigen man as the synthetic peptide 131. This suggests that there is some form of facilitated association with MHC molecules, possibly in a cellular compartment of antigen-Ia intersection (Creswell, Proc Nat1 Acad Sci USA1985,82:8188) where local concentrations of a processed determinant may be relatively high. Altematively, anchoring structures may stabilize processed antigen at the cell surface (Falo et al, Proc Nat1 Acad Sci USA1987,
84~522-526; lakey et al Proc Nat1 Acad Sci US4 1987, 84:165%X%3), preventing rapid diffusion before it has had an opportunity to interact with MHC molecules. It has been proposed that MHC molecules also fùnction in the intracellular acquisition and transport of processed antigen. As cell surface glycoproteins which are synthesized vectorially and can be recycled into the cell (Tse et al., J Mol Cel1Bio1 1986, 2315329) [8], MHC molecules have properties typical of specific transport proteins. By virtue of their ligand-binding capabilities, MHC molecules may not only acquire antigen within processing compart ments, but may also contribute to the specificity of antigen processing by protecting labile determinants from destruction after binding (IeyvaCobian and Unanue, J
Immunol1988, 141:144-145).
The cellular mechanism of antigen presentation A complex series of events is involved in the processing and presentation of T cell determinants by APC. A conceptual scheme of cellular antigen presentation (outlined
Cell-mediated
processing and presentation of T cell antigenic determinants Hackett
in Fig. 1) involves: antigen acquisition for the different pathways, structural modiikation of antigen to allow association with h4HC molecules, and transport of processed material to the cell surface where antigen/MHC is avail able for interaction witb T cell receptors. As with many cellular processes, antigen presentation appears to be cyclic, with presented determinants having distinct half lives of expression on APC surfaces. The cell biology of antigen handling is not yet well’understood. For example, it is not possible to determine with certainty which events in the cellular handling of a radioWeled molecule are significant for antigen presefitation. At present, the functional assay of T cell recognitick must be used to identiiy whether a given epitope is being expres&. Future research must use multiple approaches; new insights from such studies have recently emerged on the mechanism of antigen presentation by APC. Antigen acquisition
Tbe method by which potential APC acquire antigens for processing bas a critical impact on the outcome of antigen presentation. Entry into a specihc pathway can favour MHC class 1 or class II association (see above).
In the endocytic route, uptake by antigen-specilïc surface immunoglobulin or other membrane receptor results in effective h@IC class Krestricted presentation of antigen present at concentrations lower by several orders of magnitude than those required when uptake is strictly by fluid-phase pinocytosis (Ianzavecchia, Nuture 1985, 314:537-539). The efficient use of endocytosed antigen may also involve the invariant chain of Ia [9]. Murine fibroblasts, which have a low endogenous leve1 of invariant chain expression, were transfected witb MHC class II genes. Some clones were then ‘supertransfected’ with genes encoding invariant chain polypeptides. Fibroblasts transfected only with MHC class II genes were unable to present the antigens of the C5 component of complement or inIluenza virus hemagglutinin to T cells . However, the presence of the invariant chain endowed these fibroblasts with the ability to present these proteins even at’low input concentrations. The leve1 of surface expression of IvlHC class II molecules was not influenced by invariant chain presence. Peptides of C5 or of hemagglutinin, which do not require cellular processing for MHC interaction, were presented equally well by invariant chain-positive or negative cells. The function of the invariant chain in ei%Fig. 1. Conceptual scheme of cellular events in processing and presentation of antigens.
T cel1 determinants
T cel1 determinants
requiring minimal processing
requiring extensive processing
Proteolysis/
association with MHC
further degradatior / loss of labile determinants
Intracellular antigen handling
Uptake
Native protein
119
120
Antigen recognition
cient antigen presentation is not known. The polypeptide itself is not expressed on cell surfaces, and must function in the intemal processing of antigens. Presentation of MHC class 11-restricted measles virus antigens prcduced by biosynthesis in APC has been shown to occur in the absente of the invariant chain (Sekaly et al., Proc Nat2 Acad Sci USA 1988, 85:120+1212). The most important role of this molecule may therefore be in handling of endocytosed antigens. Although degradation appears to be the ultimate fate of endocytosed antigen, some intemalized antigens recycle to the cell surface in a relatively unaltered form. In human B cell clones with tetanus toxoid-specilìc surface immunoglobulin, this antigen was endocytosed and recycled to the surface complexed with immunoglobulin [ 101. This cycling occurred with monovalent antigen and did not depend upon capping of surface immunoglobulin. Indeed, an ultrastructural study [ 111 showed that antigens which were unable to bind to cellular receptors als0 recycled to the cell surface after uptake. Although the relevante of the reappearance of such antigens to T cell recognition is not yet clear, these observations indi cate that there are cellular mechanisms that handle foreign antigens in pathways other than their unidirectional degradation within cells.
Antigen modification While uptake can be assessed at the leve1 of the whole
protein, antigen modifìcation and subsequent steps must be studied on the basis of individual T cell epitopes, which can have processing requirements that diifer greatly. Certain MHC class 1-restricted epitopes of the influenza virus nucleoprotein were not well presented when introduced to APC by vaccinia vectors encoding the nucleoprotein gene [12]. This inhibition of expression of inlluenza epitopes by vaccinia could be at least partially reversed by the introduction of a modiiíed form of nucleoprotein which could be readily degmded in the cell. This was accomplished by making a construct of the ubiquitin gene sequence and the nucleoprotein gene with the codon for the N-terminal methionine changed to one for arginine. This encodes a fusion protein which engages the ubiquitindependent cellular degradative path way (for review, see Dice, FRSEBJ 1987,1:359-357). This observation suggests that intracellular protein breakdown may function in the processing of at least some epitopes for MHC class 1 association, although the specifìc defect caused by vaccinia infection has not yet been established. Epitopespeciiìc perturbation of exogenously supplied antigen was studied using monoclonal antibodies [13]. The antigen j3galactosidase was treated with individual monoclonal antibodies specilïc for different regions of the molecule and then introduced to macrophage APC as an antigekantibody complex. These various APC were then assayed for the ability to be recognized by a panel of distinct class II-restricted T hybridoma clones. Although in most cases antibodies resulted in a more efficient use of antigen by enhancing uptake via FC receptors, some T cells showed greatly reduced responses to antigen treated
with specilïc monoclonal antibodies. Epitope-specifìc inhibition of antigen processing or MHC association was a possibility as antibodies able to reduce recognition by one T cell clone had no effect upon T cells of other speciiïcities. These data not only suggest that surface immunoglobulin has a role in determining T cell epitope availability, but also shows that it is feasible that epitopes are protected in the processing compartment, a possible effect of intracellular MHC binding.
Transport The ability of processed antigen to translocate to the cell surface is a prerequisite for T cell recognition (for review, see Long, Zmmunol7”~ 1989,10:232-234). Transport of MHC molecules to the APC surface may convey antigenie dete rminants associated with the binding site. A pool of intracellular MHC class 11 molecules which may associate internally with uptake antigen was shown to exist [8]. In some APC, intracellular Ia is maintained by recycling from the cell surface, while in ethers, particularly adherent macrophages, new synthesis appears to be the major source of Ia. This could explain the loss of the antigen-presenting function of macrophages, but not of non-adherent B cells, upon treatment with protein synthesis inhibitors (Jensen, J Zmmunol 1988, 141:25452550). Non-MHC structures have also been implicated in the transport of processed antigens. A peptide-binding protein has been demonstrated at the cell surface where antisera to the peptide-binding protein were able to perturb presentation (Lakey, et al, Proc Nat1 Acad Sci USA 1987, 84:165+1663). The antigenic peptide angiotensin 11was shown to associate with a high molecular weight molecule when introduced to various cell types, independent of Ia expression (Betancourt et al, J Zmmunol 1987, 1393725-3729). The efficiency of formation of these complexes varied with the cell line and the peptide used. The role of these peptide-binding molecules in the association of antigen with h4HC molecules is not known. Possibly, they may convey antigen to APC membranes for external association with Ia, or may anchor T cell determinants on cell surfaces, as has been suggested for some antigens by fatty acylation (Falo et aL, Proc Natl Acad Sci USA 1987, 84:522-526). Information has recently become available on the role of intracellular transport in presentation of MHC class Irestricted determinants. The fungal metabolite brefeklin A, which interrupts intracellular transport without af fecting protein synthesis, was used by two independent groups to study recognition of influenza virus antigens by cytotoxic T cells [ 6,7]. Brefeklin A was found to black the transport of newly synthesized antigens from the endoplasmic reticulum and the transGolgi, its presence resulting in failure to present MHC class 1-restricted antigens which were either biosynthesized or introduced into the cytoplasm of APC. Reversal of brefeklin A blocking was readily achieved and could occur in the presence of pro-
Cell-mediated wocessinn and wesentationof T cel1antieenicdeterminantsHackett tein synthesis inhibitors. Externally added synthetic peptides recognized in tbe context of MHC class 1 molecules could sensitize target cells for lysis in tbe presence of brefeldin A, indicatlng that MHC molecules on cell surfaces were not tiected. Presentation of MHC class IK restricted dete rmlnants was only marglnally alkcted by brefeldin A, suggesting that this drug is relatively specik and does not affect endocytosis or cellular tknsport beyond the trans Golgi. Since the presentation pf nonrepllcative, cytoplasmicaJly introduced virus was also inhlbited by brefeldin A, it is clear that the effect:did not rely on disruption of tbe transport of biosynthesized viral antigen. The data suggest that newly synthesized MHC class 1 molecules may be responsible for convhg antigenie determlnan ts proces& in the cytoplasm to the cell surface. While direct proof of this pathway is not available, the sensitization of target cells for cytotoxic T cell lysis paralleled the expression of newly synthesized MHC class 1 molecules, even though large amounts of MHC molecules were already avklable at the cell surface and non-infectieus, cytoplasmically loaded influenza virus, which does not require viral biosynthesis for expression, was used as antigen.
Turnover Once expressed on the surfaces of viable APC, MHC class Krestricted T cell determinants are lost as a function of time. There is llttle information on tbe mechanism of tumover of presented antigen. A cellular activity inhibited by aldehyde fïxation, is required, but it is not known whether the tumover is due to loss of Ia-antigen complexes or to the removal or replacement of antigen at the binding site of stable h4HC molecules. NO treatments short of cell Iïxation have been shown to aker the halfllves of specifïc epitopes [ 51. At present, tumover rates measure net T cell stimulation for a given epitope and the fate of individual antigen-MHC r;omplexes is not known. For example, all cell surface Ia may recycle at tbe same rate, but differences in re-expression of individual T cell epitopes may result in the perceived functional stability or lability of individual determinants. Tumover occurs more quickly tban dissociation of peptides from isolated Ia. However, release of antigen from Ia may be accelerated in an acidic environment (Buus et aC, Cel2 1986, 4731071-1077), which could occur during recycling into tbe cell. If individual determinants have Merential susceptibility to removal from tbe antigen-binding site under stringent conditions, then these determinants may exhibit prolonged su&val on APC surfaces. The longevity of T cell determinan t expression may be a factor in the relative immuncdominance of T cell sites on a protein.
and present many different molecular forms of processed protein antigens. Evidente that heterogeneous, ‘partklly processed’ antigenic forms associate with h4HC molecules on APC surfaces is provided by the recent observations that: (1) relatively unaltered antigen can reappear on the cell surface after endocytosis; (2) some epitopes of a given protein required little or no proteolytic modi&ation for MHC association; (3) end procucts of natural processing need not correspond to minimal peptides delìned by chemical synthesis, and (4) relatively large antigens may functionally associate with MHC molecules. Aldehyde-lïxed APC can be sensitlzed for T cell recognition by natlve protein (Lee et al, J Immunol 1988, 140:1063-10681, conformationally altered hemagglutinin as part of intact tiuenza virus (Eisenlohr et al, J Immunol 1988, 141:187Ck-18761, or synthetic peptide antigen covalently coupled to large protein molecules Qne specu(Casten et al, JEq Med 1988,168:171-180). lation is that partial processing of antigen nïay lead to the expression of a large antigenic molecule bearing both exposed and hidden T cell detemWants. Re-uptake of the antigen may subject it to more extensive processing, resulting both in the expression of determinants requiring more complete fragmentation and in the loss of labile epitopes. In this scheme, antigen presentation may be viewed as a continuous ‘sarnpling’of processing compartments for transport of any molecule exhibiting sufkient avidity for MHC or other transport molecules. The cell biology of T cell epitope expression requires further study. In particular, basic information is needed on the mechanism of transport of proces& forms, the cellular location of antigen-MHC association, and the basis of T cell determination half-lives on APC surfaces.
Annotated
0 ??e
The distinctive requirements for expression of in&vidual T cell epitopes suggest that APC can transport
and recommended
Of interest Of outstanding interest
1.
BREIT SJ, CEASEKB, BERZOWY JA: Inhences of antlgen processing on the expression of the T cell repertoire. Evidence for MHC-specilïc hindering structuren on the products of processing. J Eqb Med 1988, 168:357-374. T cell determlnan ts that could be recognlzed by T cells when introduced to APC as synthetic peptides were not presentexl by processing of the intact myoglobln protein. This fallure to present protein determinants occurred only in certain MHC haplotypes and was related to the MHC molecule needed for T cell recognition, slnce Ft APC pre sented the determlnants to the responder haplotype patent only. End products produced by natural processing seemed to lnteract with MHC molecules dlfferently from the correspondlng synthetic peptldes. The ln!luence of antlgen processing on MHC-llnked T cell responslveness was supported by the dernonstratlon that lmmunlzatlon tith natlve whole proteln vexsus synthetic farms of antigens yielded Werent antlgenlc specisícities of responding T cells. ??e
Bo~hc% HC, GOTCH FM, MCMKXAELAJ: class 1 crossrestrlcted T cells rcveal low respondcr allele due to processing of vlral antigen. Nature 1989, 337:65+654. Human cytotoxic T cell clones restricted to HLA-Aw69were able to rec ognize a synthetic peptlde of influenza virus matrix proteln but not the same detertninant produced in tirwinfected cells. Thus, as in [ 11,class 2.
Conclusion
references
reading
??
121
122
Antigen recognition 1-restricted peptides produced from native proteins can differ iimctionaUy from the minimal dete rminant defìned by chemical synthesis. 3.
ROOSNEK E, DEMOIZ S, CORRADIN G, IANZAVECCHIA A: Kinetics
of MHC-antigen complex fortnation on antigen-presenting cells. J Immund 1988, 14040794Ot32. A Iag pedod of 1 h at 3PC was needed for the functionaI appearance of antigenic determinants on antigen-puIsed AFC. This intenal was essentialiyindependent of initiaIantigen concentmtion, and probabIy reflects the time tequked for antIgen processing and presentation, rather than the accumulation of anngenIc determinants to some thteshold leve1 on APC surfaces. A high concentration of a 12-residue synthetic peptide, which does not need antigen processing, was able to sen.sItIzeAPC for T ceU recognItion without a Iag perk& By using a sensitive, rapid meas ure of T ceU acthation by calcium uptake, the assocIation of the iag period with antIgen processing/presentation was cleady estabUshed. 00
4.
E, PIERCESK: L+XEYEK, CASTENIA, NIEBLINGWL, MARGOIIASH Time dependence of B cell processing and presentation of peptide and native protein antigens. J Immurwl 1983, 140:3309-3314. Antigen deUvered as the native cytochrome c required 6-8 h for APC to be maxima& stimulatory for T cek ThIs time was Independent of .startIngantIgen concentration or of whether the antigen was Introduced by specific or non-specUic uptake. LeveIs of presented antigen decUned after 8-12 h postpuk. The kinetics of the Ioss of determhrants were sImUar to these for determmants Introduced to Uve APC either as the protein or as the synthetic peptide. 0
EISENIQHR LC, GERHARDW, HACKE?TCJ:
Individual class IIrestricted antigenic det enninants of the same protein exhibit distinct kinetics of appearance and persistente on antigen-presenting celk. J Immutwl 1988, 141:2581-2584. After puismg APC with IsoIated hemagglutinin giycoprotein or the nonreplicative infhtenza virus, the time course~ of expression of IndIvidual T ceU dete mrhrants was measured. Expression of 2 detemrinants rec ognbzed In the context of 1-Ed differed greatiy, with one continuousiy Increasmg over 48 h whUe the other peaked at 8 h and decIined there after. Ahhough the proteoIysIis InhIbitor leupeptin altered processing of each dete rmInant, it dId not aikct their haIfUves on APC surfaces. 5. 0
NUCHTE~NJG, BONIFACINO JS, BIDDISONWE, K~AUSNERRD: (Letter to the Fditor) Brefeldin A impiicates egress from endoplasmic reticulum in class 1 restricted antigen presentation. Nature 1989, 339:22%225. BrefeldIn A was able to InhIbit MHC cIass I-mstricted cytotoxk T ceU recognition of InIluenza virus mattix promIn. ThIs was reked to its abII ity to hak transport of proteIns from the endoplasmk reticuium. Protein synthesis was not akted by this drug, nor was the recogmtion of MHC class B-restricted dete nninants processed by the exogenous/endocytic pathway. ThIs Is the first evidente of IntraceUuIar transport as a requhement for presentation of MHC ciass 1-restricted antigens.
6. oo
Brefeldin A specItïcaUy Inhibits presentatkm of protein antigens to cytotoxk T Iymphocytes. Science 1989, 244:1072-1074. Using cytopiasmicaUy processed non-repkative virus as antigen, a correhttion was shown between transpott of newiy synthesked MHC cIass I molecules from the endopiasmk reticulum or tralzs Go@ and T ceU recognition. This is important evidente that MHC molecuIes may convey proces& antigen to the ceU surface.
7. 00
YJ?WDELL JW, BENNINKJR:
8. 0
HARDINGCV, UME ER Antigen processing and Intracellular Ia: possible roles of endocytosIs and protein synthesis In Ia function. J Immund 1988, 142:12-19.
A pool of intracektiar MHC cIass 11molecuIes were identIikd, at kast a propottion of which resukd from recycling of ceU surface-expressed molecuIes. Ia was InternaU& Into Ught endosomes, Into whkh ttansfenin is taken, suggestmg that the recycUng molecule may be avaUable to intetact with antIgen in an IntraceUuIar processmg compartment. STOCKINGER B, PESSARAU, L~NRH, HABICHTJ, GREZ M, KOCH N: A role of Ia-associa ted invariant chains in antigen processing and presentation. Cel1 1989, 566.!33&39. The invariant chain of Ia was introduced to MIX ciass II-expressing fibroblasts by gene transfection. The inwriant chain conferred the abiiity to present the Influenza virus hemagghttinin or complement C5 antigens, even at low concentrations. The invariant chain did not increase MHC ciass II expression on fibrobIasw, its ftmction may be at the leve1 of intraceUuIar antigen handUng. This Unding suggests a physiological role for the Invariant chain in APC iünction.
9. 00
10.
WARS C, DAVIDSONHW: EndocytosIs and recycling of specifîc antigen by human B cell iines. EMBO J 1988, 7~1937-1946. Antigen taken up by surface immunogIobuUn receptors of antigen-spe ciEc B ceils was endocytosed rapidly, but a certain propottion reappeared at the ceU surface in a reiativeIyintact form complexed with im munogiobuUn. Uptake and recycUng did not depend upon crossUnking of the surface immunogIobuUn. Although it is nat known whether such antigen handUng is directly rekatedto MHC-associated presentation to T ceUs, these data indicate the presence of ceUuIar mechanisms to return incompletely degraded material to ceU surfaces. ??
LINJ, BERZOFSKYJA, DE~~VITCHTL Ukrastructural study of IntemaIixation and recycling of antipen by antIgen presentIng ceIIs. J Mol Cell Immund 1988, 3~321-343. The ceUuIar routing of different coUoIdai gold-Iabeled antigens was studied In thIn-section. Both pork insuUn and a tryptic fragment of pork insuiin Iacking receptor-binding abUitywere observed to recycle to the surface after endocytosis. Myogiobin, in contrast, did not readUy reappear on ceU surfaces. Thus, receptor-mediated uptake is not necessary for antigen reyciing, and routing behavior can differ between proteins. 11. ??
TOWNSENDA, BA~TINJ, Gourr, K, BROWNLEEG, ANDREWM, COUPAUB, BOYIE D, CHAN S, Slilrm G: DefectIve presentation to class 1-restrkted cytotoxic T lymphocytes in vaccinia-infected celIs is overcome by enhanced degradation of antigen. J Eq Med 1988, 168:1211-1224. By introducing a form of Iniluenza nucleoprotein able to k readUy degraded by the ImraceUuiar ubiquUin-Unked pathway, the vaccinka MNSinduced black of presentation of certain nucleoprotein T ceU determinants could be avoided. This correiates the processing of some MIK cIass I-restricted T ceU determinants with proteoIytic fmgmenta tion within the ceU. 12. ??
-CA F, FENOGUOD, KUNIUA, CAMBIAGGI C, Qsso M, CEIADA F: Difkrential activation of T cell clones stimulated hy macrophages exposed to antigen complexed with monoclonal antibodies: a possible infhtence of paratope speciEcity on the mode of antigen processing. J Immund 1988, 140:289H898. MonocIonaI antibodIes comphexed to g-gakìctosidase before introduction to macrophage APC could block presentation of determInants to T ceU clones of certain speciEcities. AbUityto black presemation de pended upon the speciEcities both of antibodies and of T ceUs. WhUe neither the precise effect of antibodies upon presentation, nor the positional correspondence between B and T ceU epitopes in effective combinations are known, the data suggest that antibodies may speciEcaUy aifect the intmceUuIarprocessing of tegions of &aIactosidase by sterk inhibition. 13. ??