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Immunology Today, vol. 6, No. 3, 1985 28 Gearhart, P. J. and Bogenhagen, D. F. (1983) Proc. NatlAcad. Sci. USA 80, 3439-3443 29 Tonegawa, S. (1983) Nature (London) 302, 575-581 30 Siekevitz, M., Huang, S. Y. and Gefter, M. L. (1983) Eur. J. Immunol. 13, 123-132 31 Oudin, J. and Cazenave, P. A. (1971) Proc. Natl Acad. Sci. USA 68, 2616-2660 32 Kohno, Y., Berkower, I., Minna, J. and Berzofsky, J. A. (1982)dr. ImmunoL 128, 1742 1748 33 Krieger, N . J . , Pesce, A . J . and Michael, J. G. (1983)Ann. NYAcad. Sci. 418, 305-312 34 Wysoeki, L. J. and Sato, V. L. (1981) Eur. J. lmmunol. 11, 832-839 35 Smith, J. A. and Margolies, M. N. (1984) Biochem. 23, 4726-4732 36 Rothstein, T. L. and Gefter, M. L. (1983) MoL lmmunoL 20, 161-168 37 Rodwetl, J. D., Gearhart, P . J . and Karush, F. (1983)`]. ImmunoL 130, 313-316 38 Kaartinen, M., Griffiths, G., Hamlyn, P., Markam, A. F., Karjalainen, K., Pelhonen, J. L. T., Miikelii, O. and Milstein, C. (1983)J. ImmunoL 130, 937 944 39 Ehrlich, P. (1910) in Studies in Immunity Wiley, New York
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
Jerne, N. K. (1955) Proc. Natl Amd. Sci. USA 41,849-857 Tatmage, D. W. (1957)Annu. Rev. Med. 8, 239-250 Lederberg, J. (1959) Science 129, 1649-1653 Burnet, F. M. (1957) Aust. J. Sci. 20, 67-68 Cunningham, A. J. (1976) Cold Spring Harbor Symp. Quant. Biol. 41, 761-770 Jerne, N. K. (1974)Ann. Immunol. (Paris) 125C, 373-389 Kuettner, M. G., Wang, A. and Nisonoff, A. (1972)d" Exp. Med 135, 579-597 Dzierzak, E. A., Rosenstein, R. W. andJaneway, C. A. (1981)J Exp Med. 154, 1432-1441 Jerne, N. K. (1984) Immunol. Rev. 79, 5-24 Bottomly, K. (1984) Immunol. Rev. 79, 45 62 Manser, T., Huang, S-Y. and Gefter, M. L. (1984) Science 226, 1283-1288 Griffiths, G. M., Berek, C., Kaartinen, M. and Milstein, C. (1984) Nature 312, 271-275 Clarke, S. and Weigert, M. (1985)`]. Exp. Med., in press Rajewsky, K. and Takemori, T. (1983) Ann. Rev. Immunol. 1,569 608 Near, R. I., Manser, T. and Gefter, M. L. (1985),]. Immunol, in press
Antigen processing and presentation to T cells Howard M. Grey and Robert Chesnut In this articleHoward Grey and Robert Chesnut describe recentinsights into the mechanism of antigen presentation and discuss the needfor antigen processing in the stimulation of T cells. In the past few years our understanding of the role of antigen presenting accessory cells in the induction of an immune response has greatly increased. In addition, it has become evident that an increasing number of diverse cell types besides macrophages can express Ia molecules and have antigen presenting capabilities. Although workers for several decades had realized that accessory cells were somehow involved in the activation o f T cells by both mitogens as well as antigen it was not until Rosenthal and Shevach began to examine the function of immune response (Ir) genes at the accessory cell level that the critical role of accessory cells in the activation of helper T cells (Th) began to unfold. In their now classical studies these workers examined a group of synthetic co-polymers composed of L-glutamic acid plus L-lysine (GL) and Lglutamic acid plus L-tyrosine (GT), the immune response to which in the guinea-pig is under Ir gene control i . They found that (2 x 13)F~ T cells proliferated to G T only on pulsed F 1 or strain 13 macrophages and not on strain 2 macrophages; whereas they proliferated to D N P - G L on F 1 or strain 2 macrophages and not on strain 13 macrophages. Thus, although the FI T cells were capable of responding to either G T or GL, they did so only when the appropriate Ia bearing macrophage presented the antigen. Additional results from studies in the guinea-pig along with similar findings using mouse 2'3 and h u m a n 4'5 cells showed that the T-cell response requires accessory cells and that the interaction between accessory cells and the responding T cells is Ia restricted.
Cell types other than macrophages can present antigen Until recently attention has been focused almost excluDivision of Basic Immunology, Department of Medicine, National Jewish Hospital and Research Center, and the Departments of Pathology, Microbiology and Immunology, and Medicine, University of Colorado Health Sciences Center, Denver, CO 80206, USA.
sively on macrophages as the critical accessory cell in the immune response.. However, an increasing number of studies have documented the capacity of other cell types to act as accessory cells, some of which because of their unique tissue or organ distribution, may be more important than macrophages in certain in-vivo situations. O u r own studies, as well as those of others, have demonstrated the capacity of both mouse and human B cells, to present soluble protein antigens to primed T h cells in an antigen-specific, MHC-restricted fashion 6 9. In addition we have found that B cells can present Ia to alloreactive T cells for stimulation of a 'secondary' mixed lymphocyte reaction ( M L R ) response but appear to lack the capacity to elaborate the soluble cytokine, interleukin 1, required for stimulation of a primary M L R (Yanover, M., Kubo, R., Grey, H., Augustin, A., Sire, G. and Chesnut, R., unpublished observations). The functional capacity of B cells to serve as antigen presenting cells appears to play a pivotal role by focusing antigen-specific T a cells to the surface of antigen-specific B cells. This results in the T - B interaction that leads to B-cell differentiation and antibody production. Other cell types with demonstrated antigen presenting capacity include skin Langerhans' cells, liver Kupffer cells as well as spleen dendritic cells 1°-'2. These three cell types have been shown to present soluble protein antigens to primed T cells and to act as stimulator cells in the allogeneic M L R . Langerhans' cells are likely to be the primary antigen presenting cell for contact sensitivity while the exact role of Kupffer cells and spleen dendritic cells in the immune response remains to be elucidated. The major non-lymphoid cell which has been studied for its antigen presenting capacity is the endothelial cell. Several groups have independendy demonstrated the capacity of human umbilical vein endothelial cells to stimulate an M L R and to present soluble antigens to T cells~3. Recent evidence suggests that endothelial cells do not constitutively express Ia ( H L A - D R ) antigens but are © 1985,
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102 induced to do so by y-interferon ~4. How endothelial cells might function in the immune response is not yet resolved either. There are in addition several other cell types which because of their anatomic location and expression of Ia molecules m a y have an antigen presenting capacity although to date evidence is insufficient to allow a clear definition of this functional capacity. A m o n g these cells are veiled cells and interdigitating cells in the skin and lymph node, both of which express Ia molecules 15'~6. T cells, in the human and guinea-pig, which have been activated by mitogen or allo-antigen synthesize and express Ia antigen on their surfacC 7'18and can stimulate an M L R . There are some data which suggest that Ia expressing T cells when presenting soluble protein antigens may not be capable of activating the responding T cells but on the contrary may deliver 'tolerogenic' signals 19. Various kinds of epithelial cells such as the 'nurse cells' of the thymus which appear to express Ia constitutively2° and m a m m a r y epithelial cells which, following stimulation by lactotropic hormones, express Ia antigens appear likely, at least under certain conditions, to have functional roles. For example, the nurse cells of the thymus have been proposed to play a major role in the ' education' of thymocytes with regard to self-MHC antigens. Recent studies have also shown that skin keratinocytes which are normally Ia negative are capable of expressing Ia following 'activation TM and have been shown to produce a soluble cytokine (ETAF) with properties very similar to those of IL-1. The apparent multiplicity of Ia-expressing cell types that can present antigen raises the question of whether any cell type can perform this function and that the only special property required for antigen presentation is the expression of Ia. Two experimental approaches are now available to answer this question. First, it has been shown that mouse L cells transfected with the genes encoding the a and /3 chains of I-A are capable of processing and presenting keyhole limpet hemocyanin ( K L H ) to K L H specific T-cell hybrids 22. Secondly purified I-A antigen can be transferred to Ia-negative L cells or B-cell tumors by fusion of I-A containing liposomes to these cells. Such cells to which I-A has been transferred have been shown to be capable of presenting ovalbumin peptides to ovalbumin-specific-MHC restricted T-cell hybrids (Coeshott, C. and Grey, H. M., unpublished observations). Experiments of this nature with a variety of other recipient cell types should answer the question posed above as well as provide an interesting tool for the further dissection of the mechanisms of antigen processing and presentation. Finally, there are two cell types found in the spleen and lymph nodes which do not apparently express Ia but because of their intimate association with lymphocytes may play a role in antigen presentation. These are the marginal zone macrophages and the follicular dendritic cells 23. While these cells may not present antigen leading to the activation of T h cells, their ability to retain antigen bound to their surface for extended periods of time may provide a persistent source of antigen for recogpition by lymphocytes such as B cells which, in contrast to T cells, recognize antigens independent of Ia molecules.
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In spite of these and many additional results from invitro experiments showing the functional capacity of various cell types to present antigen, because of the m a n y difficulties in carrying out similar experiments in vivo we know very little about which cells that actually carry out the antigen presenting function within the animal.
The processing requirement for antigen recognition by T ceRs O u r ideas about the handling of antigen prior to presentation have changed dramatically over the last decade. It was first thought that antigens bound to the surface of accessory cells, where at least in part they remained for recognition by T cells. In the case of B cells it was the role of surface Ig to bind and display antigen to T cells allowing antigen to form a 'bridge' between the carrier-specific T-cell receptor and the hapten-specific Igreceptor on B cells 24. Continued research by several groups has produced a growing body of evidence indicating that at least for most soluble protein antigens as well as for bacterial antigens 'processing' of antigen prior to presentation involves, in addition to binding, endocytosis followed by active .catabolic steps 2527. The binding of antigen to the accessory cell surface occurs either by specific receptors such as those for Ig, Fc or C3, or by binding via nonspecific, non-covalent interactions to undefined structures on the plasma membrane of accessory cells. Most protein antigens and all particulate antigens studied can be demonstrated to bind to the surface of accessory cells to greater or lesser extents. In the case of soluble protein antigens, the extent of binding appears to be directly related to the size of the protein; i.e. larger molecules such as K L H bind in significantly greater quantities than smaller molecules such as ovalbumin. Such binding can be demonstrated by incubation of accessory cells with antigen at 4 ° C, or in the presence of metabolic inhibitors that block the endocytic pathway. In the case of nonreceptor mediated binding, the extent of binding is directly related to antigen concentration and it is not a readily saturable process 2~'29.The uptake of antigen by B cells via membrane Ig is highly efficient compared to antigen binding by non-specific interaction to B cells so that approximately 10 4 fold lower concentration of antigen is required to effect antigen presentation by Ig mediated uptake than by non-specific means 3°. Virtually no information is available concerning the elements involved in the non-specific binding of antigen to accessory cells and whether there may be specialized structures expressed on accessory cell membranes that participate in the binding of antigen that will eventually be presented to T cells is not known. Another potential pathway of antigen uptake which does not involve antigen binding, i.e. fluid phase pinocytosis, may also serve as a means for the endocytosis of antigen that results in appropriate antigen processing and presentation; however, this mechanism has not yet been clearly established. The second step in antigen processing appears to involve active metabolic events that are required prior to T cells being capable of recognizing antigen. Studies suggesting this first came from kinetic experiments that
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showed that there was a lag period between the time antigen was bound to the accessory cell surface and the time when antigen recognition by T cells could be detected. The requirement for such metabolic events following antigen binding is well illustrated by experiments in which antigen was allowed to bind to the surface of accessory cells in the cold, the cells were subsequently washed, then incubated at 37°C for varying periods of time and finally fixed with paraformaldehyde or glutaraldehyde to prevent any further active metabolic event from taking place. These antigen-pulsed and fixed cells were then examined for their capacity to present antigen to T cells. O u r own results using antigen-specific M H C restricted T-cell hybridomas have shown that when antigen was bound to an antigen-presenting B cell lymphoma or to macrophages at 40°C, a lag period of 45 to 60 min at 37°C was evident before antigen presentation could be detected 26. The kinetics we observed with a soluble protein antigen were essentially identical to those reported by Ziegler and Unanue for the processing of a particulate antigen Listeria monocytogenes 31. In another series of experiments, where the aim was to obtain information about the fate of immunologically relevant antigen following binding to accessory cells, it has been shown that antigen is not entirely exposed o/1 the cell surface but is rather, to a significant extent, sequestered within the accessory cell. The initial observations were made by Ellner and associates employing guinea-pig macrophages and subsequently by ourselves using both murine B-cell tumors as well as macrophages as antigen presenting cells. In these experiments the effects of removing surface-bound antigen from accessory cells, on the capacity of accessory cells to present antigen was tested 32. The results suggest that at least one pool of immunologically relevant antigen is sequestered in a cellular compartment not accessible to trypsin while a second pool of antigen is exposed on the cell surface where it is accessible to trypsin and recognized by the T h cell. While the kinetic data reviewed above suggested that antigen processing required active metabolic events that took approximately one hour to complete, the exact nature of the metabolic events remained obscure. The first evidence that proteolysis might be important came from studies carried out in U n a n u e ' s laboratory and our own using the lysosomotropic agents, ammonia and chloroquine, that inhibit degradation of proteins by lysosomal acid hydrolases by raising the lysosomal p H from about p H 4.5 to p H 6-7. W e found that chloroquine blocked the presentation of a soluble antigen such as K L H by a B-cell tumor or macrophages when it was present during the time of antigen processing. The presence of chloroquine at >/4/~M during the one hour antigen pulse period completely abrogated the capacity of both the B-cell tumor and macrophages to present antigen while the presentation of antigen which was allowed to undergo processing prior to exposing accessory cells to chloroquine was not inhibited. Similarly, Unanue found that chloroquine or ammonium chloride inhibited presentation of L. monocytogenes to T cells and that the inhibitory effect of varying doses of these lysosomotropic agents was closely correlated with the capacity of these agents to inhibit the degradation of bacterial proteins into trichloroacetic acid soluble materiaP 3.
103 Taken collectively these results suggest that the antigen sequestration observed in macrophages or B-cell tumors involves the trafficking of the antigen to lysosomes where antigen processing in the form of partial proteolysis takes place followed by recycling of the processed antigen to the plasma membrane. Recent studies from our laboratory have more directly demonstrated the role of proteolysis in antigen processing 34. The system studied employed a series of ovalbumin specific H-2d-restricted T-cell hybridomas. Utilizing fkxation of the accessory cells as a means of preventing active metabolic processing events from taking place we showed that T-cell hybridomas would respond to ovalbumin pulsed accessory cells that were fixed by a brief exposure to glutaraldehyde after a 1-4 h pulse period; however when the cells were fixed prior to ovalbumin being added to the accessory cells, as would be expected, no effective presentation occurred. The striking observation that was made, however, was that in 8 out of 11 hybrids studied prefixed accessory cells could trigger T-cell proliferation if, instead of intact ovalbumin, various peptides of ovalbumin were used. Thus, for this set ofovalbumin specific T-cell hybridomas in-vitro fragmentation of ovalbumin into peptides substituted for the active metabolic processing events that were necessary when intact ovalbumin was used as an antigen; i.e. fragmentation of ovalbumin was not only necessary but sufficient to account for antigen processing. W e have been able to demonstrate the same phenomenon with normal T cells derived from ovalbumin-primed mice in that prefixed antigen-presenting cells could stimulate significant T-cell proliferation if a tryptic digest of ovalbumin was used as the antigen, whereas no proliferation was observed when intact ovalbumin was used together with prefixed cells. D i s p l a y of processed antigen to T h cells Despite efforts by many investigators, it remains unclear how accessory cells display processed antigen to T h cells for recognition in the context o f l a molecules. As corollaries to the single receptor and two receptor models for T-cell recognition of antigen, there are two major possibilities for how processed antigen is displayed on the surface of accessory cells 35. If the one receptor, 'altered self' theory is correct then the simplest (but not exclusive) explanation for how a single T-cell receptor recognizes both Ia and antigen is that a physical complex is formed between Ia and antigen, creating a single macromolecular ligand. Alternatively, if the T cell has two receptors, one for Ia and one for antigen, then no such physical interaction between antigen and Ia would be required for T-cell recognition of antigen. Is there evidence for an Ia-antigen complex? There are three sets of studies, one conducted by Erb and Feldman 36, a second carried out by Purl and Lonai 37, and more recently a third performed by Freedman et al. 38, that claim to have demonstrated the existence of such a complex in the supernatant fluid of macrophages cultured in the presence of antigen. These experiments, if repeatable, may give important information about how antigen is displayed to T h cells and clearly warrant extensive further investigation. More indirect evidence for a bimolecular complex has also been reported. Based on the idea that a part of the Ir
104 gene control ofT-cell responses was imposed by the accessory cells' capacity to present a particular antigen determinant 39Werdelin found that two non-cross-reacting copolymers containing poly-L-lysine could compete with one another, apparently at the level of antigenic presentation 4°. Thus, exposure of guinea-pig responder strain accessory cells to the co-polymer of L-glutamic acid and Llysine (GL) inhibited the subsequent presentation of dinitrophenyl poly-L-lysine (DNP-PLL) to D N P - P L L primed T cells. Other antigens, not under the same Ir gene control, had no such inhibitory effect. Werdelin concluded that the competitive inhibition observed between G L and D N P - P L L may reflect a competition for the Ir gene product (i. e. the Ia antigens) produced by the antigen presenting cell. These findings were confirmed and extended by Rock and BenacerraP 1 using different synthetic polymers as antigens in mice. However, the results of the antigen competition studies only demonstrate that closely related antigens appear to compete at some level of antigen processing and presentation by accessory cells; they do not prove or disprove that an antigen-Ia complex is recognized by T cells as a unit. Using yet another approach, Schwartz and associates found that T-cell hybridomas as well as primed T cells which responded to pigeon cytochrome peptide 81-104 when presented by B10.A accessory cells did not respond to the same peptide when presented by B10.A (5R) accessory cells42. In contrast, these T cells responded to moth cytochrome C peptide 81-103 equally well when the moth peptide was presented by either B10.A or B10.A (5R) accessory cells. Since these results were obtainable with cloned T cells the following conclusions could be drawn: (1) The T-cell receptor must be capable of recognizing the restriction element on 5R-antigen presenting cells (IE] E~) since they were stimulated by moth cytochrome presented by 5R-antigen presenting cells. (2) These same T cells must be capable of recognizing pigeon cytochrome since they were stimulated by pigeon cytochrome presented by B10.A-antigen presenting cells. (3) The failure of these T cells to respond to pigeon cytochrome when presented by 5R-antigen presenting cells cannot therefore be explained by a failure of the T cell to recognize either the restriction element or the antigen but must be explained by a defect in the interaction between the restricting element and the antigen. Based on such data, Schwartz and associates have proposed a model depicting a trimolecular complex between Ia molecules, antigen and a T-cell receptor, each component of which has some positive affinity for the other two components. Although the data thus far appears to support the model they propose, the results are also interpretable in other ways, as described below. Using our previously described system in which fixed antigen presenting cells could be shown to present a 'preprocessed' ovalbumin peptide to ovalbumin-specific T-cell hybrids we have conducted experiments to examine the capacity of this immunogenic peptide to react with the peptide-specific T-cell hybrids and with the antigen presenting cells 3~. The data we have obtained thus far can be summarized as follows: the extent of binding of peptide to accessory cells is the same whether the accessory cell possesses the requisite restricting element (I-Ad) or not I-Ad + cells and cells lacking I-~;d bound
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similar amounts ofpeptide. Furthermore, anti-I-Ad antibodies failed to inhibit the binding of peptide to I-Ad+ accessory cells. Thus, there is no evidence that I-Ad represents a significant binding structure for the immunogenic peptide on the surface of the antigen presenting cells. Finally , we attempted to inhibit the stimulation of a n OVA-specific T-cell hybridoma, DO-11.10 by the immunogenic O V A peptide 323-339 using a truncated form of the peptide, 323-336, to which DO-11.10 did not respond. If there was a significant ~[a-peptide interaction site it would be predicted that the truncated peptide would have this site since it was capable of being presented by I-Ad antigen presenting cells to three other OVA-specific T-cell hybridomas. The results of these inhibition experiments showed that a preincubation of fixed I-Ad presenting cells with a large excess of the truncated peptide had no effect on their ability to present peptide 323-339 to DO-11.10 (Ref. 43). The most straightforward interpretation of this experiment is that there is no high affinity site on the I-A d molecule for the binding of the peptide. W e cannot at present formally rule out the alternative possibility that even for such a small 17 residue peptide there exist two distinct sites of interaction with I-Ad and that the 14 residue peptide lacked one of these interaction sites. However, we consider this latter possibility rather remote, thus leading us to conclude that the interaction of the peptide with I-A d is either of an extremely low affinity or that an interaction does not occur at all prior to the engagement of the T-cell receptor. A lack of any significant binding between Ia and antigen is in keeping with the conceptual problems posed when postulating significant specific bimolecular interactions between proteins of limited structural variation such as Ia molecules, and the myriad of diverse antigenic structures that they would presumably have to interact with to account for the large number of antigen specificities to which T cells must respond. A n alternative view, which to us seems more reasonable, to explain the relationship between antigenspecificity and MHC-restriction is that interaction between antigen and Ia occurs only after each of these components is independently engaged by a T-cell receptor. Then, due to the close proximity of the T-cell receptor binding sites for antigen and Ia, these two elements are juxtaposed, thereby imposing interactions between Ia and antigen. The nature of these interactions could be positive and result in greater stabilization of the interaction between Ia, antigen and the T-cell receptor or negative and result in a destabilization and dissociation of the complex. A model similar to this has recently been proposed by Parham for the recognition of antigens such as viral proteins in the context of c l a s s I M H C molecules 44. With respect to how such a trimolecular complex initially is formed, it is thermodynamically improbable that the three components would interact with one another simultaneously. Therefore it would appear reasonable to postulate that the reaction between T-cell receptor, antigen and Ia proceeds by two independent bi-molecular events. Since there is considerable evidence for the interaction between self-Ia and T-cell receptors as exemplified by autologous mixed lymphocyte reactivity, an interaction between T-cell receptor and Ia molecules would appear to be the most
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°
2.
3,
Syngeneic 'permissive' la
AIIogeneic 'non-permissive' la
'Related' antigen
Fig. 1. A model of antlgen-specific, MHC-restrlcted recognition by T cells 1. Processed antigen is expressed on the surface of an Ia-positive accessory cell. Ia and antigen have no inherent affinity for one another and both are freely diffusible within the plasma membrane of the accessory cell. 2. T cells with receptors that have distinct subsites capable of recognizing processed antigen and Ia separately, interact initially with Ia. 3. Antigen diffuses into the site of I a - T cell interaction. In the case of the syngeneic 'permissive' interaction (upper panel), antigen can bind to the antigen-specific subsite on the T-cell receptor, leading to a stable trimolecular complex which initiates T-ceU triggering. In the case of allogeneic Ia (middle panel) or certain related antigens (lower panel) the stereochemistry of the Ia or antigen are such that the antigen cannot bind to the T-cell receptor in the presence of the Ia-receptor interaction. This failure to bind antigen results in dissociation of the low affmity I a - T cell receptor complex and failure to stimulate the T cell.
likely first bimolecular event. This would be followed by the diffusion of antigen into the region of interaction between T-cell receptor and Ia and subsequent binding of the antigen to a subsite on the T-ceU receptor. If an inappropriate Ia molecule is bound or a structurally related but different antigen is used, although the epitopes recognized by the T cell may be identical, the stereochemistry or non-covalent interactions may be such that the antigen cannot be engaged by the T-cell receptor in the presence of the Ia-receptor interaction; an illustration of this model for T-cell-Ia-antigen interaction is shown in Fig. 1. W h y is antigen processing required? Having defined for at least one globular protein antigen, OVA, that processing consists of fragmentation of the antigen into peptides, the question of why such frag-
mentation would be necessary remains to be answered. The enigma is accentuated by the fact that the recognition of antigens by class I restricted cytotoxic T lymphocytes does not appear to require an antigen processing event since within minutes of integration of viral capsular proteins into the surface of target cells, such cells can be recognized by C T L (Ref. 44). Current knowledge of the structure of the T-cell receptors for C T L and T-helper cells suggests that they are very similar to one another and furthermore that they both possess a common polypeptide chain, the//-chain. This high degree of homology between the structure of receptors on C T L and T~, makes it unlikely that the requirement for antigen processing is due to differences in the nature of the T-cell receptor. Therefore the need for processing must either be a characteristic of the restricting elements, i.e. class I vs. class II, or due to the nature of the antigens being recognized. It is possible, for
106
instance, that the portions of the class I and class II molecules recognized by T cells are such that they either permit (class I) or sterically interfere with (class II) the engagement of another macromolecule by the T-cell receptor. Thus processing of antigen to small peptides would minimize the problem of steric hindrance in the case of class II restricted responses but would not be necessary in the case of class I restricted responses. An alternative explanation centers around the vast difference in the types of antigens that are usually employed in studying C T L and T-helper cell induction, i.e. viral glycoproteins or haptenated cell surfaces being the usual antigens for C T L studies and soluble protein antigens being used for T-helper cell studies. The most striking difference between these two types of molecules of course is how they are represented on the surface of a presenting cell. Viral glycoproteins are integral membrane proteins that exist in a stable form on the surface of an antigen presenting cell for long periods of time, as are hapten molecules that are conjugated directly to integral membrane proteins on the surface of antigen presenting cells. O n the other hand, soluble protein antigens bind very poorly and transiently to the surface of accessory cells. Thus, it could be postulated that the reason why processing is required is to convert a soluble protein antigen that binds poorly to the surface of an accessory cell to a peptide that for one reason or another can bind with more stability to the cell surface. Some data to support this latter point of view have come from studies examining the requirement for processing of Sendai viral glycoproteins for recognition by Ia-restricted T cells (Wegmann, D., unpublished observations). These studies suggest that, unlike soluble protein antigens, Sendai glycoproteins may not require a processing event for T-cell recognition. If these results can be confirmed and extended to the recognition of other integral membrane proteins by Ia restricted T h cells, then it would strongly suggest that it is the nature of the antigen that dictates the requirement for processing. []
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2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39
Acknowledgement This work was supported in part by grants from the National Institutes of Health, U S P H S number AI-09758, AI-17510, AI-18634.
References 1 Shevach, E. and Rosenthal, A. (1973)~ Exp. Med. 138, 1213
40 41 42 43 44
Schwartz, R. and Paul, W. (1976).]. Exp. Med. 143, 529 Schwartz, R., Yano, A. and Paul, W. (1978) ImmunoL Rev. 40, 153 Rosenwasser, L. and Rosenthal, A. (1978).]i ImmunoL 121, 2479 Rosenwasser, L. and Rosenthal, A. (1979)J. ImmunoL 123, 1141 Chesnut, R. and Grey, H. (1981),f Immunol. 126, 1075 Walker, E., Warner, N., Chesnut, R., Kappler, J. and Marrack, P. (1982)J. Immunol. 128, 2164 Bergholtz, B. and Thorsby, E. (1979) Stand. J. Immunol. 10, 267 Abramson, S., Puck, J. and Rich, R. (1981).]. Exp. Med. 154, 1005 Stingl, G., Gazze-Stingl, L., Aberer, W. and Wolff, K. (1981)J. Immunol. 127, 1707 Nadler, P., Klingenstein, J., Richman, L. and Ahman,~G. (1980).}i ImmunoL 125, 2521 Sunshine, G., Katz, D. and Feldmann, M. (1980).]i. Exp. Med. 152, 1817 Burger, D., Ford, D., Vetto, R., Hamblin, A., Goldstein, A., Hubbard, M. and Dumonde, D. (1981) Hum. Immunol. 3, 209 Pober, J., Grimbrone, M., Cotran, R:, Reiss, C., Burakoff, S., Friers, W. and Ault, K. (1983).]. Exp. Med. 157, 1339 Spry, C., Pflug, A., Janossy, G. and Humphrey, J. (1980) Clin. Exp. ImmunoL 39, 750 Lampert, I., Pizzolo, G., Thomas, J. andJanossy, G. (1980).]. Pathol. 131, 145 Hercend, T., Ritz, J., Scholossman, S., Reinherz, E. (1981) Hum. Immunol. 3, 247 Lyons, C., Lipscomb, M., Schlossman, S., Tucker, T. and Uhr, J. (1981).]. Immunol. 127, 1879 Lamb, J., Skidmore, B., Green, N., Chiller, J. and Feldmann, M. (1983)J. Exp. Med. 157, 1434 Wekerle, H. and Ketelsen, U. (1980) Nature (London) 283, 402 Daynes, R., Emam, M., Krueger, G. and Roberts, L. (1983)3~ ImmunoL 130, 1536 Malissen, B., Price, M. P., C-overman, M. et al. (1984) Cell 36, 319 Humphrey, J. and Grennan, D. (1981) gur. J. ImmunoL 11,221 Mitchison, N. (1971) Eur. J. Immunol. 1, 18 Unanue, E. (1981)Adv. Immunol. 31, 1 Chesnut, R., Colon, S. and Grey, H. (1982).}i ImmunoL 129, 2382 Grey, H., Colon, S. and Chesnut, R. (1982),]i Immunol. 129, 2389 Unanue, E. and Cerottini, J. (1970)j. Exp. Med. 131, 711 Steinman, R. and Cohn, Z. (1972)J. Cell. Biol. 55, 186 Kakiuehi, T., Chesnut, R. and Grey, H. (1983).]. Immunol. 131, 109 Ziegler, K. and Unanue, E. (1981)J. ImmunoL 127, 1869 Ellner, J., Lipsky, P. and Rosenthal, A. (1977)J. ImmunoL 118, 2053 Ziegler, K. and Unanue, E. R. (1982) Pr0e. NatlAcad. Sei. USA 79, 175 Shimonkevitz, R., Kappler, J., Marrack, P. and Grey, H. (1983)J. Exp. Med. 158, 303 Zinkernagel, R. and Doherty, P. (1974) Nature (London) 251,547 Erb, P. and Feldmann, M. (1975) Eur. jr. Immunol. 5, 759 Puri, J. and Lonai, P. (1980) Eur..jr. ImmunoL 10, 273 Friedman, A., Zerubavel, R., Gitler, C. and Cohen, I. (1983) Immunogenetics (NY) 18, 291 Rosenthal, A., Barcinski, M. and Blake, J. (1977) Nature (London) 267, 156 Werdlen, O. (1982)J. Immunol. 129, 1883 Rock, K. and Benacerraf, B. (1983).]. Exp. Med. 157, 1618 Heber-Katz, E., Hansburg, D. and Schwartz, R. (1983)J. Mol. Celt. Immunol. 1, 3 Shimonkevitz, R., Colon, S. C., Kappler, J., Marraek, P. and Grey, H. M. (1984).]i ImmunoL 133, 2067 Parham, P. (1984) Immunol. Today 5, 89
Immunology Today, vol. 6, No. 3, 1985 28 Gearhart, P. J. and Bogenhagen, D. F. (1983) Proc. NatlAcad. Sci. USA 80, 3439-3443 29 Tonegawa, S. (1983) Nature (London) 302, 575-581 30 Siekevitz, M., Huang, S. Y. and Gefter, M. L. (1983) Eur. J. Immunol. 13, 123-132 31 Oudin, J. and Cazenave, P. A. (1971) Proc. Natl Acad. Sci. USA 68, 2616-2660 32 Kohno, Y., Berkower, I., Minna, J. and Berzofsky, J. A. (1982)dr. ImmunoL 128, 1742 1748 33 Krieger, N . J . , Pesce, A . J . and Michael, J. G. (1983)Ann. NYAcad. Sci. 418, 305-312 34 Wysoeki, L. J. and Sato, V. L. (1981) Eur. J. lmmunol. 11, 832-839 35 Smith, J. A. and Margolies, M. N. (1984) Biochem. 23, 4726-4732 36 Rothstein, T. L. and Gefter, M. L. (1983) MoL lmmunoL 20, 161-168 37 Rodwetl, J. D., Gearhart, P . J . and Karush, F. (1983)`]. ImmunoL 130, 313-316 38 Kaartinen, M., Griffiths, G., Hamlyn, P., Markam, A. F., Karjalainen, K., Pelhonen, J. L. T., Miikelii, O. and Milstein, C. (1983)J. ImmunoL 130, 937 944 39 Ehrlich, P. (1910) in Studies in Immunity Wiley, New York
40 41 42 43 44 45 46 47 48 49 50 51 52 53 54
Jerne, N. K. (1955) Proc. Natl Amd. Sci. USA 41,849-857 Tatmage, D. W. (1957)Annu. Rev. Med. 8, 239-250 Lederberg, J. (1959) Science 129, 1649-1653 Burnet, F. M. (1957) Aust. J. Sci. 20, 67-68 Cunningham, A. J. (1976) Cold Spring Harbor Symp. Quant. Biol. 41, 761-770 Jerne, N. K. (1974)Ann. Immunol. (Paris) 125C, 373-389 Kuettner, M. G., Wang, A. and Nisonoff, A. (1972)d" Exp. Med 135, 579-597 Dzierzak, E. A., Rosenstein, R. W. andJaneway, C. A. (1981)J Exp Med. 154, 1432-1441 Jerne, N. K. (1984) Immunol. Rev. 79, 5-24 Bottomly, K. (1984) Immunol. Rev. 79, 45 62 Manser, T., Huang, S-Y. and Gefter, M. L. (1984) Science 226, 1283-1288 Griffiths, G. M., Berek, C., Kaartinen, M. and Milstein, C. (1984) Nature 312, 271-275 Clarke, S. and Weigert, M. (1985)`]. Exp. Med., in press Rajewsky, K. and Takemori, T. (1983) Ann. Rev. Immunol. 1,569 608 Near, R. I., Manser, T. and Gefter, M. L. (1985),]. Immunol, in press
Antigen processing and presentation to T cells Howard M. Grey and Robert Chesnut In this articleHoward Grey and Robert Chesnut describe recentinsights into the mechanism of antigen presentation and discuss the needfor antigen processing in the stimulation of T cells. In the past few years our understanding of the role of antigen presenting accessory cells in the induction of an immune response has greatly increased. In addition, it has become evident that an increasing number of diverse cell types besides macrophages can express Ia molecules and have antigen presenting capabilities. Although workers for several decades had realized that accessory cells were somehow involved in the activation o f T cells by both mitogens as well as antigen it was not until Rosenthal and Shevach began to examine the function of immune response (Ir) genes at the accessory cell level that the critical role of accessory cells in the activation of helper T cells (Th) began to unfold. In their now classical studies these workers examined a group of synthetic co-polymers composed of L-glutamic acid plus L-lysine (GL) and Lglutamic acid plus L-tyrosine (GT), the immune response to which in the guinea-pig is under Ir gene control i . They found that (2 x 13)F~ T cells proliferated to G T only on pulsed F 1 or strain 13 macrophages and not on strain 2 macrophages; whereas they proliferated to D N P - G L on F 1 or strain 2 macrophages and not on strain 13 macrophages. Thus, although the FI T cells were capable of responding to either G T or GL, they did so only when the appropriate Ia bearing macrophage presented the antigen. Additional results from studies in the guinea-pig along with similar findings using mouse 2'3 and h u m a n 4'5 cells showed that the T-cell response requires accessory cells and that the interaction between accessory cells and the responding T cells is Ia restricted.
Cell types other than macrophages can present antigen Until recently attention has been focused almost excluDivision of Basic Immunology, Department of Medicine, National Jewish Hospital and Research Center, and the Departments of Pathology, Microbiology and Immunology, and Medicine, University of Colorado Health Sciences Center, Denver, CO 80206, USA.
sively on macrophages as the critical accessory cell in the immune response.. However, an increasing number of studies have documented the capacity of other cell types to act as accessory cells, some of which because of their unique tissue or organ distribution, may be more important than macrophages in certain in-vivo situations. O u r own studies, as well as those of others, have demonstrated the capacity of both mouse and human B cells, to present soluble protein antigens to primed T h cells in an antigen-specific, MHC-restricted fashion 6 9. In addition we have found that B cells can present Ia to alloreactive T cells for stimulation of a 'secondary' mixed lymphocyte reaction ( M L R ) response but appear to lack the capacity to elaborate the soluble cytokine, interleukin 1, required for stimulation of a primary M L R (Yanover, M., Kubo, R., Grey, H., Augustin, A., Sire, G. and Chesnut, R., unpublished observations). The functional capacity of B cells to serve as antigen presenting cells appears to play a pivotal role by focusing antigen-specific T a cells to the surface of antigen-specific B cells. This results in the T - B interaction that leads to B-cell differentiation and antibody production. Other cell types with demonstrated antigen presenting capacity include skin Langerhans' cells, liver Kupffer cells as well as spleen dendritic cells 1°-'2. These three cell types have been shown to present soluble protein antigens to primed T cells and to act as stimulator cells in the allogeneic M L R . Langerhans' cells are likely to be the primary antigen presenting cell for contact sensitivity while the exact role of Kupffer cells and spleen dendritic cells in the immune response remains to be elucidated. The major non-lymphoid cell which has been studied for its antigen presenting capacity is the endothelial cell. Several groups have independendy demonstrated the capacity of human umbilical vein endothelial cells to stimulate an M L R and to present soluble antigens to T cells~3. Recent evidence suggests that endothelial cells do not constitutively express Ia ( H L A - D R ) antigens but are © 1985,
Elsevier Science
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102 induced to do so by y-interferon ~4. How endothelial cells might function in the immune response is not yet resolved either. There are in addition several other cell types which because of their anatomic location and expression of Ia molecules m a y have an antigen presenting capacity although to date evidence is insufficient to allow a clear definition of this functional capacity. A m o n g these cells are veiled cells and interdigitating cells in the skin and lymph node, both of which express Ia molecules 15'~6. T cells, in the human and guinea-pig, which have been activated by mitogen or allo-antigen synthesize and express Ia antigen on their surfacC 7'18and can stimulate an M L R . There are some data which suggest that Ia expressing T cells when presenting soluble protein antigens may not be capable of activating the responding T cells but on the contrary may deliver 'tolerogenic' signals 19. Various kinds of epithelial cells such as the 'nurse cells' of the thymus which appear to express Ia constitutively2° and m a m m a r y epithelial cells which, following stimulation by lactotropic hormones, express Ia antigens appear likely, at least under certain conditions, to have functional roles. For example, the nurse cells of the thymus have been proposed to play a major role in the ' education' of thymocytes with regard to self-MHC antigens. Recent studies have also shown that skin keratinocytes which are normally Ia negative are capable of expressing Ia following 'activation TM and have been shown to produce a soluble cytokine (ETAF) with properties very similar to those of IL-1. The apparent multiplicity of Ia-expressing cell types that can present antigen raises the question of whether any cell type can perform this function and that the only special property required for antigen presentation is the expression of Ia. Two experimental approaches are now available to answer this question. First, it has been shown that mouse L cells transfected with the genes encoding the a and /3 chains of I-A are capable of processing and presenting keyhole limpet hemocyanin ( K L H ) to K L H specific T-cell hybrids 22. Secondly purified I-A antigen can be transferred to Ia-negative L cells or B-cell tumors by fusion of I-A containing liposomes to these cells. Such cells to which I-A has been transferred have been shown to be capable of presenting ovalbumin peptides to ovalbumin-specific-MHC restricted T-cell hybrids (Coeshott, C. and Grey, H. M., unpublished observations). Experiments of this nature with a variety of other recipient cell types should answer the question posed above as well as provide an interesting tool for the further dissection of the mechanisms of antigen processing and presentation. Finally, there are two cell types found in the spleen and lymph nodes which do not apparently express Ia but because of their intimate association with lymphocytes may play a role in antigen presentation. These are the marginal zone macrophages and the follicular dendritic cells 23. While these cells may not present antigen leading to the activation of T h cells, their ability to retain antigen bound to their surface for extended periods of time may provide a persistent source of antigen for recogpition by lymphocytes such as B cells which, in contrast to T cells, recognize antigens independent of Ia molecules.
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In spite of these and many additional results from invitro experiments showing the functional capacity of various cell types to present antigen, because of the m a n y difficulties in carrying out similar experiments in vivo we know very little about which cells that actually carry out the antigen presenting function within the animal.
The processing requirement for antigen recognition by T ceRs O u r ideas about the handling of antigen prior to presentation have changed dramatically over the last decade. It was first thought that antigens bound to the surface of accessory cells, where at least in part they remained for recognition by T cells. In the case of B cells it was the role of surface Ig to bind and display antigen to T cells allowing antigen to form a 'bridge' between the carrier-specific T-cell receptor and the hapten-specific Igreceptor on B cells 24. Continued research by several groups has produced a growing body of evidence indicating that at least for most soluble protein antigens as well as for bacterial antigens 'processing' of antigen prior to presentation involves, in addition to binding, endocytosis followed by active .catabolic steps 2527. The binding of antigen to the accessory cell surface occurs either by specific receptors such as those for Ig, Fc or C3, or by binding via nonspecific, non-covalent interactions to undefined structures on the plasma membrane of accessory cells. Most protein antigens and all particulate antigens studied can be demonstrated to bind to the surface of accessory cells to greater or lesser extents. In the case of soluble protein antigens, the extent of binding appears to be directly related to the size of the protein; i.e. larger molecules such as K L H bind in significantly greater quantities than smaller molecules such as ovalbumin. Such binding can be demonstrated by incubation of accessory cells with antigen at 4 ° C, or in the presence of metabolic inhibitors that block the endocytic pathway. In the case of nonreceptor mediated binding, the extent of binding is directly related to antigen concentration and it is not a readily saturable process 2~'29.The uptake of antigen by B cells via membrane Ig is highly efficient compared to antigen binding by non-specific interaction to B cells so that approximately 10 4 fold lower concentration of antigen is required to effect antigen presentation by Ig mediated uptake than by non-specific means 3°. Virtually no information is available concerning the elements involved in the non-specific binding of antigen to accessory cells and whether there may be specialized structures expressed on accessory cell membranes that participate in the binding of antigen that will eventually be presented to T cells is not known. Another potential pathway of antigen uptake which does not involve antigen binding, i.e. fluid phase pinocytosis, may also serve as a means for the endocytosis of antigen that results in appropriate antigen processing and presentation; however, this mechanism has not yet been clearly established. The second step in antigen processing appears to involve active metabolic events that are required prior to T cells being capable of recognizing antigen. Studies suggesting this first came from kinetic experiments that
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showed that there was a lag period between the time antigen was bound to the accessory cell surface and the time when antigen recognition by T cells could be detected. The requirement for such metabolic events following antigen binding is well illustrated by experiments in which antigen was allowed to bind to the surface of accessory cells in the cold, the cells were subsequently washed, then incubated at 37°C for varying periods of time and finally fixed with paraformaldehyde or glutaraldehyde to prevent any further active metabolic event from taking place. These antigen-pulsed and fixed cells were then examined for their capacity to present antigen to T cells. O u r own results using antigen-specific M H C restricted T-cell hybridomas have shown that when antigen was bound to an antigen-presenting B cell lymphoma or to macrophages at 40°C, a lag period of 45 to 60 min at 37°C was evident before antigen presentation could be detected 26. The kinetics we observed with a soluble protein antigen were essentially identical to those reported by Ziegler and Unanue for the processing of a particulate antigen Listeria monocytogenes 31. In another series of experiments, where the aim was to obtain information about the fate of immunologically relevant antigen following binding to accessory cells, it has been shown that antigen is not entirely exposed o/1 the cell surface but is rather, to a significant extent, sequestered within the accessory cell. The initial observations were made by Ellner and associates employing guinea-pig macrophages and subsequently by ourselves using both murine B-cell tumors as well as macrophages as antigen presenting cells. In these experiments the effects of removing surface-bound antigen from accessory cells, on the capacity of accessory cells to present antigen was tested 32. The results suggest that at least one pool of immunologically relevant antigen is sequestered in a cellular compartment not accessible to trypsin while a second pool of antigen is exposed on the cell surface where it is accessible to trypsin and recognized by the T h cell. While the kinetic data reviewed above suggested that antigen processing required active metabolic events that took approximately one hour to complete, the exact nature of the metabolic events remained obscure. The first evidence that proteolysis might be important came from studies carried out in U n a n u e ' s laboratory and our own using the lysosomotropic agents, ammonia and chloroquine, that inhibit degradation of proteins by lysosomal acid hydrolases by raising the lysosomal p H from about p H 4.5 to p H 6-7. W e found that chloroquine blocked the presentation of a soluble antigen such as K L H by a B-cell tumor or macrophages when it was present during the time of antigen processing. The presence of chloroquine at >/4/~M during the one hour antigen pulse period completely abrogated the capacity of both the B-cell tumor and macrophages to present antigen while the presentation of antigen which was allowed to undergo processing prior to exposing accessory cells to chloroquine was not inhibited. Similarly, Unanue found that chloroquine or ammonium chloride inhibited presentation of L. monocytogenes to T cells and that the inhibitory effect of varying doses of these lysosomotropic agents was closely correlated with the capacity of these agents to inhibit the degradation of bacterial proteins into trichloroacetic acid soluble materiaP 3.
103 Taken collectively these results suggest that the antigen sequestration observed in macrophages or B-cell tumors involves the trafficking of the antigen to lysosomes where antigen processing in the form of partial proteolysis takes place followed by recycling of the processed antigen to the plasma membrane. Recent studies from our laboratory have more directly demonstrated the role of proteolysis in antigen processing 34. The system studied employed a series of ovalbumin specific H-2d-restricted T-cell hybridomas. Utilizing fkxation of the accessory cells as a means of preventing active metabolic processing events from taking place we showed that T-cell hybridomas would respond to ovalbumin pulsed accessory cells that were fixed by a brief exposure to glutaraldehyde after a 1-4 h pulse period; however when the cells were fixed prior to ovalbumin being added to the accessory cells, as would be expected, no effective presentation occurred. The striking observation that was made, however, was that in 8 out of 11 hybrids studied prefixed accessory cells could trigger T-cell proliferation if, instead of intact ovalbumin, various peptides of ovalbumin were used. Thus, for this set ofovalbumin specific T-cell hybridomas in-vitro fragmentation of ovalbumin into peptides substituted for the active metabolic processing events that were necessary when intact ovalbumin was used as an antigen; i.e. fragmentation of ovalbumin was not only necessary but sufficient to account for antigen processing. W e have been able to demonstrate the same phenomenon with normal T cells derived from ovalbumin-primed mice in that prefixed antigen-presenting cells could stimulate significant T-cell proliferation if a tryptic digest of ovalbumin was used as the antigen, whereas no proliferation was observed when intact ovalbumin was used together with prefixed cells. D i s p l a y of processed antigen to T h cells Despite efforts by many investigators, it remains unclear how accessory cells display processed antigen to T h cells for recognition in the context o f l a molecules. As corollaries to the single receptor and two receptor models for T-cell recognition of antigen, there are two major possibilities for how processed antigen is displayed on the surface of accessory cells 35. If the one receptor, 'altered self' theory is correct then the simplest (but not exclusive) explanation for how a single T-cell receptor recognizes both Ia and antigen is that a physical complex is formed between Ia and antigen, creating a single macromolecular ligand. Alternatively, if the T cell has two receptors, one for Ia and one for antigen, then no such physical interaction between antigen and Ia would be required for T-cell recognition of antigen. Is there evidence for an Ia-antigen complex? There are three sets of studies, one conducted by Erb and Feldman 36, a second carried out by Purl and Lonai 37, and more recently a third performed by Freedman et al. 38, that claim to have demonstrated the existence of such a complex in the supernatant fluid of macrophages cultured in the presence of antigen. These experiments, if repeatable, may give important information about how antigen is displayed to T h cells and clearly warrant extensive further investigation. More indirect evidence for a bimolecular complex has also been reported. Based on the idea that a part of the Ir
104 gene control ofT-cell responses was imposed by the accessory cells' capacity to present a particular antigen determinant 39Werdelin found that two non-cross-reacting copolymers containing poly-L-lysine could compete with one another, apparently at the level of antigenic presentation 4°. Thus, exposure of guinea-pig responder strain accessory cells to the co-polymer of L-glutamic acid and Llysine (GL) inhibited the subsequent presentation of dinitrophenyl poly-L-lysine (DNP-PLL) to D N P - P L L primed T cells. Other antigens, not under the same Ir gene control, had no such inhibitory effect. Werdelin concluded that the competitive inhibition observed between G L and D N P - P L L may reflect a competition for the Ir gene product (i. e. the Ia antigens) produced by the antigen presenting cell. These findings were confirmed and extended by Rock and BenacerraP 1 using different synthetic polymers as antigens in mice. However, the results of the antigen competition studies only demonstrate that closely related antigens appear to compete at some level of antigen processing and presentation by accessory cells; they do not prove or disprove that an antigen-Ia complex is recognized by T cells as a unit. Using yet another approach, Schwartz and associates found that T-cell hybridomas as well as primed T cells which responded to pigeon cytochrome peptide 81-104 when presented by B10.A accessory cells did not respond to the same peptide when presented by B10.A (5R) accessory cells42. In contrast, these T cells responded to moth cytochrome C peptide 81-103 equally well when the moth peptide was presented by either B10.A or B10.A (5R) accessory cells. Since these results were obtainable with cloned T cells the following conclusions could be drawn: (1) The T-cell receptor must be capable of recognizing the restriction element on 5R-antigen presenting cells (IE] E~) since they were stimulated by moth cytochrome presented by 5R-antigen presenting cells. (2) These same T cells must be capable of recognizing pigeon cytochrome since they were stimulated by pigeon cytochrome presented by B10.A-antigen presenting cells. (3) The failure of these T cells to respond to pigeon cytochrome when presented by 5R-antigen presenting cells cannot therefore be explained by a failure of the T cell to recognize either the restriction element or the antigen but must be explained by a defect in the interaction between the restricting element and the antigen. Based on such data, Schwartz and associates have proposed a model depicting a trimolecular complex between Ia molecules, antigen and a T-cell receptor, each component of which has some positive affinity for the other two components. Although the data thus far appears to support the model they propose, the results are also interpretable in other ways, as described below. Using our previously described system in which fixed antigen presenting cells could be shown to present a 'preprocessed' ovalbumin peptide to ovalbumin-specific T-cell hybrids we have conducted experiments to examine the capacity of this immunogenic peptide to react with the peptide-specific T-cell hybrids and with the antigen presenting cells 3~. The data we have obtained thus far can be summarized as follows: the extent of binding of peptide to accessory cells is the same whether the accessory cell possesses the requisite restricting element (I-Ad) or not I-Ad + cells and cells lacking I-~;d bound
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similar amounts ofpeptide. Furthermore, anti-I-Ad antibodies failed to inhibit the binding of peptide to I-Ad+ accessory cells. Thus, there is no evidence that I-Ad represents a significant binding structure for the immunogenic peptide on the surface of the antigen presenting cells. Finally , we attempted to inhibit the stimulation of a n OVA-specific T-cell hybridoma, DO-11.10 by the immunogenic O V A peptide 323-339 using a truncated form of the peptide, 323-336, to which DO-11.10 did not respond. If there was a significant ~[a-peptide interaction site it would be predicted that the truncated peptide would have this site since it was capable of being presented by I-Ad antigen presenting cells to three other OVA-specific T-cell hybridomas. The results of these inhibition experiments showed that a preincubation of fixed I-Ad presenting cells with a large excess of the truncated peptide had no effect on their ability to present peptide 323-339 to DO-11.10 (Ref. 43). The most straightforward interpretation of this experiment is that there is no high affinity site on the I-A d molecule for the binding of the peptide. W e cannot at present formally rule out the alternative possibility that even for such a small 17 residue peptide there exist two distinct sites of interaction with I-Ad and that the 14 residue peptide lacked one of these interaction sites. However, we consider this latter possibility rather remote, thus leading us to conclude that the interaction of the peptide with I-A d is either of an extremely low affinity or that an interaction does not occur at all prior to the engagement of the T-cell receptor. A lack of any significant binding between Ia and antigen is in keeping with the conceptual problems posed when postulating significant specific bimolecular interactions between proteins of limited structural variation such as Ia molecules, and the myriad of diverse antigenic structures that they would presumably have to interact with to account for the large number of antigen specificities to which T cells must respond. A n alternative view, which to us seems more reasonable, to explain the relationship between antigenspecificity and MHC-restriction is that interaction between antigen and Ia occurs only after each of these components is independently engaged by a T-cell receptor. Then, due to the close proximity of the T-cell receptor binding sites for antigen and Ia, these two elements are juxtaposed, thereby imposing interactions between Ia and antigen. The nature of these interactions could be positive and result in greater stabilization of the interaction between Ia, antigen and the T-cell receptor or negative and result in a destabilization and dissociation of the complex. A model similar to this has recently been proposed by Parham for the recognition of antigens such as viral proteins in the context of c l a s s I M H C molecules 44. With respect to how such a trimolecular complex initially is formed, it is thermodynamically improbable that the three components would interact with one another simultaneously. Therefore it would appear reasonable to postulate that the reaction between T-cell receptor, antigen and Ia proceeds by two independent bi-molecular events. Since there is considerable evidence for the interaction between self-Ia and T-cell receptors as exemplified by autologous mixed lymphocyte reactivity, an interaction between T-cell receptor and Ia molecules would appear to be the most
105
Immunology Today, voL 6, No. 3, 1985
°
2.
3,
Syngeneic 'permissive' la
AIIogeneic 'non-permissive' la
'Related' antigen
Fig. 1. A model of antlgen-specific, MHC-restrlcted recognition by T cells 1. Processed antigen is expressed on the surface of an Ia-positive accessory cell. Ia and antigen have no inherent affinity for one another and both are freely diffusible within the plasma membrane of the accessory cell. 2. T cells with receptors that have distinct subsites capable of recognizing processed antigen and Ia separately, interact initially with Ia. 3. Antigen diffuses into the site of I a - T cell interaction. In the case of the syngeneic 'permissive' interaction (upper panel), antigen can bind to the antigen-specific subsite on the T-cell receptor, leading to a stable trimolecular complex which initiates T-ceU triggering. In the case of allogeneic Ia (middle panel) or certain related antigens (lower panel) the stereochemistry of the Ia or antigen are such that the antigen cannot bind to the T-cell receptor in the presence of the Ia-receptor interaction. This failure to bind antigen results in dissociation of the low affmity I a - T cell receptor complex and failure to stimulate the T cell.
likely first bimolecular event. This would be followed by the diffusion of antigen into the region of interaction between T-cell receptor and Ia and subsequent binding of the antigen to a subsite on the T-ceU receptor. If an inappropriate Ia molecule is bound or a structurally related but different antigen is used, although the epitopes recognized by the T cell may be identical, the stereochemistry or non-covalent interactions may be such that the antigen cannot be engaged by the T-cell receptor in the presence of the Ia-receptor interaction; an illustration of this model for T-cell-Ia-antigen interaction is shown in Fig. 1. W h y is antigen processing required? Having defined for at least one globular protein antigen, OVA, that processing consists of fragmentation of the antigen into peptides, the question of why such frag-
mentation would be necessary remains to be answered. The enigma is accentuated by the fact that the recognition of antigens by class I restricted cytotoxic T lymphocytes does not appear to require an antigen processing event since within minutes of integration of viral capsular proteins into the surface of target cells, such cells can be recognized by C T L (Ref. 44). Current knowledge of the structure of the T-cell receptors for C T L and T-helper cells suggests that they are very similar to one another and furthermore that they both possess a common polypeptide chain, the//-chain. This high degree of homology between the structure of receptors on C T L and T~, makes it unlikely that the requirement for antigen processing is due to differences in the nature of the T-cell receptor. Therefore the need for processing must either be a characteristic of the restricting elements, i.e. class I vs. class II, or due to the nature of the antigens being recognized. It is possible, for
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instance, that the portions of the class I and class II molecules recognized by T cells are such that they either permit (class I) or sterically interfere with (class II) the engagement of another macromolecule by the T-cell receptor. Thus processing of antigen to small peptides would minimize the problem of steric hindrance in the case of class II restricted responses but would not be necessary in the case of class I restricted responses. An alternative explanation centers around the vast difference in the types of antigens that are usually employed in studying C T L and T-helper cell induction, i.e. viral glycoproteins or haptenated cell surfaces being the usual antigens for C T L studies and soluble protein antigens being used for T-helper cell studies. The most striking difference between these two types of molecules of course is how they are represented on the surface of a presenting cell. Viral glycoproteins are integral membrane proteins that exist in a stable form on the surface of an antigen presenting cell for long periods of time, as are hapten molecules that are conjugated directly to integral membrane proteins on the surface of antigen presenting cells. O n the other hand, soluble protein antigens bind very poorly and transiently to the surface of accessory cells. Thus, it could be postulated that the reason why processing is required is to convert a soluble protein antigen that binds poorly to the surface of an accessory cell to a peptide that for one reason or another can bind with more stability to the cell surface. Some data to support this latter point of view have come from studies examining the requirement for processing of Sendai viral glycoproteins for recognition by Ia-restricted T cells (Wegmann, D., unpublished observations). These studies suggest that, unlike soluble protein antigens, Sendai glycoproteins may not require a processing event for T-cell recognition. If these results can be confirmed and extended to the recognition of other integral membrane proteins by Ia restricted T h cells, then it would strongly suggest that it is the nature of the antigen that dictates the requirement for processing. []
Immunology Today, voL 6, No. 3, 1985
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Acknowledgement This work was supported in part by grants from the National Institutes of Health, U S P H S number AI-09758, AI-17510, AI-18634.
References 1 Shevach, E. and Rosenthal, A. (1973)~ Exp. Med. 138, 1213
40 41 42 43 44
Schwartz, R. and Paul, W. (1976).]. Exp. Med. 143, 529 Schwartz, R., Yano, A. and Paul, W. (1978) ImmunoL Rev. 40, 153 Rosenwasser, L. and Rosenthal, A. (1978).]i ImmunoL 121, 2479 Rosenwasser, L. and Rosenthal, A. (1979)J. ImmunoL 123, 1141 Chesnut, R. and Grey, H. (1981),f Immunol. 126, 1075 Walker, E., Warner, N., Chesnut, R., Kappler, J. and Marrack, P. (1982)J. Immunol. 128, 2164 Bergholtz, B. and Thorsby, E. (1979) Stand. J. Immunol. 10, 267 Abramson, S., Puck, J. and Rich, R. (1981).]. Exp. Med. 154, 1005 Stingl, G., Gazze-Stingl, L., Aberer, W. and Wolff, K. (1981)J. Immunol. 127, 1707 Nadler, P., Klingenstein, J., Richman, L. and Ahman,~G. (1980).}i ImmunoL 125, 2521 Sunshine, G., Katz, D. and Feldmann, M. (1980).]i. Exp. Med. 152, 1817 Burger, D., Ford, D., Vetto, R., Hamblin, A., Goldstein, A., Hubbard, M. and Dumonde, D. (1981) Hum. Immunol. 3, 209 Pober, J., Grimbrone, M., Cotran, R:, Reiss, C., Burakoff, S., Friers, W. and Ault, K. (1983).]. Exp. Med. 157, 1339 Spry, C., Pflug, A., Janossy, G. and Humphrey, J. (1980) Clin. Exp. ImmunoL 39, 750 Lampert, I., Pizzolo, G., Thomas, J. andJanossy, G. (1980).]. Pathol. 131, 145 Hercend, T., Ritz, J., Scholossman, S., Reinherz, E. (1981) Hum. Immunol. 3, 247 Lyons, C., Lipscomb, M., Schlossman, S., Tucker, T. and Uhr, J. (1981).]. Immunol. 127, 1879 Lamb, J., Skidmore, B., Green, N., Chiller, J. and Feldmann, M. (1983)J. Exp. Med. 157, 1434 Wekerle, H. and Ketelsen, U. (1980) Nature (London) 283, 402 Daynes, R., Emam, M., Krueger, G. and Roberts, L. (1983)3~ ImmunoL 130, 1536 Malissen, B., Price, M. P., C-overman, M. et al. (1984) Cell 36, 319 Humphrey, J. and Grennan, D. (1981) gur. J. ImmunoL 11,221 Mitchison, N. (1971) Eur. J. Immunol. 1, 18 Unanue, E. (1981)Adv. Immunol. 31, 1 Chesnut, R., Colon, S. and Grey, H. (1982).}i ImmunoL 129, 2382 Grey, H., Colon, S. and Chesnut, R. (1982),]i Immunol. 129, 2389 Unanue, E. and Cerottini, J. (1970)j. Exp. Med. 131, 711 Steinman, R. and Cohn, Z. (1972)J. Cell. Biol. 55, 186 Kakiuehi, T., Chesnut, R. and Grey, H. (1983).]. Immunol. 131, 109 Ziegler, K. and Unanue, E. (1981)J. ImmunoL 127, 1869 Ellner, J., Lipsky, P. and Rosenthal, A. (1977)J. ImmunoL 118, 2053 Ziegler, K. and Unanue, E. R. (1982) Pr0e. NatlAcad. Sei. USA 79, 175 Shimonkevitz, R., Kappler, J., Marrack, P. and Grey, H. (1983)J. Exp. Med. 158, 303 Zinkernagel, R. and Doherty, P. (1974) Nature (London) 251,547 Erb, P. and Feldmann, M. (1975) Eur. jr. Immunol. 5, 759 Puri, J. and Lonai, P. (1980) Eur..jr. ImmunoL 10, 273 Friedman, A., Zerubavel, R., Gitler, C. and Cohen, I. (1983) Immunogenetics (NY) 18, 291 Rosenthal, A., Barcinski, M. and Blake, J. (1977) Nature (London) 267, 156 Werdlen, O. (1982)J. Immunol. 129, 1883 Rock, K. and Benacerraf, B. (1983).]. Exp. Med. 157, 1618 Heber-Katz, E., Hansburg, D. and Schwartz, R. (1983)J. Mol. Celt. Immunol. 1, 3 Shimonkevitz, R., Colon, S. C., Kappler, J., Marraek, P. and Grey, H. M. (1984).]i ImmunoL 133, 2067 Parham, P. (1984) Immunol. Today 5, 89
