Immunology Today, vol. 4, No. 1, 1983
) The delineation of antigen receptors on human T lymphocytes Ellis L. Reinherz, Stefan C. Meuer and Smart F. Schlossman Monoclonal antibodies have identified several suoCacemolecules involved in target cell recognition by cytotoxic human T cells. In this article it is proposed that T cells have two recognition units: a complex composed of the T3 molecule and a clonally unique glycoprotein which binds antigen associated with polymorphic M H C gene product," and the T4 or T8 molecule which binds to a constant region of an M H C gene product. T lymphocytes recognize antigen in the context of membrane-bound products of the major histocompatibility complex (MHC) with exquisite specificity '3. While the surface receptors responsible for their cognitive properties are not known, two major theories have been proposed to explain this dual specificity of T lymphocytes4. One, the single-receptor model or interaction-antigen model, suggests that self-MHC molecules interact with antigen ' X ' to form a 'neoantigen' which is presented as one entity to a single T-cell receptor. The second model, a two'receptor model, assumes that one surface component of the T cell sees antigen ' X ' and another views the cellular M H C restricting element on the antigen-presenting ceil. Recently, the development of technologies for cloning antigen-specific human T lymphocytes and producing monoclonal antibodies against their surface structures has identified surface glycoproteins involved in their recognition mechanisms ~-9. This information allows one to construct a unifying hypothesis regarding the roles of polymorphic (clonotypic) and non-polymorphic human T-cell surface structures in this process. Given the known analogies between certain murine and human M H C gene products, immunoglobulin molecules and T-cell markers, it is likely that structures similar to those described below will be defined in murine systems.
T-cell surface structures involved in recognition of allogeneic and autologous target cells In order to present a model for the human T-cell receptor, it is first necessary to review several points of information concerning the various T-cell surface glycoproteins and the effects of antibodies to these structures on the function of clones of human antigen-specific cytotoxic T lymphocytes (CTL). In man, alloreactive cytotoxic effector cells are derived from the two major T-cell subpopulations which have been termed T4 + and T8 + on the basis of their uniquely expressed 62KD (T4) and 76KD (T8) glycoproteins. The target antigens recognized by individual T-cell subsets (i.e. T4 + or T8 + ) have been shown to be products of different gene regions of the major histocompatibility complex 8.~°-'2. Specifically, allosensitized T4 + T cells are directed at class II M H C antigens The Division of Tumor Immunology, Sidney Farber Cancer Institute and the Department of Medicine, Harvard Medical School, Boston, MA 02115, U.S.A.
on target cells whereas T8 + T cells are directed at class I M H C antigens on target cells. The association between surface phenotype of C T L and the class of M H C molecules that is recognized suggests that these subsetrestricted structures might themselves be required to facilitate recognition of and/or binding to different target antigens. This view has recently been supported by the finding that certain monoclonal antibodies to the T4 or T8 glycoproteins selectively inhibit cytolytic effector function o f T 4 + or T8 + C T L clones, respectively 8a,11. In addition to the subset-restricted surface glycoproteins T4 and T8, a 20KD T-cell surface molecule, T3, present on all mature T cells, participates in cell-mediated lysis by both of the major C T L subsets: antibodies to this structure block killing by T4 and T8 C T L 7-9,13.Moreover, anti-T3 antibodies are able to block the induction and effector phase of cell-mediated lympholysis, inhibit T-cell proliferative responses to soluble antigen and be mitogenic for resting T lymphocytes~*-~8.Both the appearance of T3 in late intrathyrnic ontogeny at the time of acquisition of immunological competence and its critical role in T-lymphocyte function suggested that it is closely linked to a recognition structure for antigen ~7,~8. Given that antibodies to the T3, T4 and T8 surface molecules could block C T L activity, it was necessary to define whether these structures serve as recognition elements or alternatively, components of the lytic mechanism itself. To this end, a series oflectin approximation studies was performed. It was reasoned that if antibodies bind to surface structures related to the lytic mechanism, then artificial approximation by lectin would not be capable of reconstituting effective lysis. In contrast, if antibodies blocked structures required for target recognition, lectin approximation would result in effective lysis. The experiments proved the latter alternative to be correct7. Perhaps more importantly, although lectin reversed the inhibitory effects of all three anti-T cell monoclonal antibodies, there was loss of target specificity. Antigen-specific C T L clones recognizing autologous Epstein-Barr virus (EBV)-transformed B lymphoblastoid lines are also governed by a series of recognition elements identical to the series in clones directed to allogeneic targets (Meuer et al., unpublished observations). Specifically, C T L expressing the T8 phenotype recognize the autologous B lymphoblastoidline in the context of class I M H C molecules; anti-HLA antibodies block C T L effector © Elsevier Biomedicnl Pros 1983 0167-4919183/0000-0000/$1.00
Immunology Today, vol. 4, No. 1, 1983
6
TABLE I. Summary of human T-cell surface molecules involved in target recognition by CTL T-cell surface molecule
Mol. wt
Distribution
Functional effect of monoclonal antibodies to the structure
T3
20 000
All mature T lymphocytes and a minority of thymocytes
T4
62 000
T8
76 000a
Tin
49000; 43000 b
Majority of thymocytes and 60% of peripheral T lymphocytes Majority of thymocytes and 30% of peripheral T cells Specific for an individual T-cell clone (clonotypic)
(1) Inhibits antigen-specific T-cell proliferative responses and cytotoxic effector function of all CTL (2) Enhances IL-2 responsiveness (3) Modulates by external shedding Inhibits CTL effectors directed at class II MHC gene products Inhibits CTL effectors directed at class I MHC gene products (1) Identical to anti-T3 effects but inhibits response only of an individual clone with which it reacts (2) Co-modulates by external shedding with T3
a Non-reducedstate; reduces to mol. wt 33 000 and mol. wt 31 000 subunitswith 2-mercaptoethanol.b Reduced state
function at the target level and anti-T8 antibodies abrogate the ability of these class I-specific killers to kill. I n contrast, T 4 + C T L recognize Ia (class II) determinants on the autologous lymphoblastoid cell a n d are inhibited b y a n t i - T 4 b u t not anti-T8 antibodies. As with the allogeneic C T L , anti-T3 antibody preincubation inhibits the killing ability of both T 4 + a n d T 8 + effector T cells. Because these clones do not lyse autologous B cells not infected with E B V or pokeweed mitogen-stimulated B-cell blasts, such T 4 + a n d T8 + effectors seem to recognize vir .ally encoded surface glycoproteins in association with class II or class I molecules, respectively 19. At present, however, these viral proteins have not been identified. The above results indicate that at least several T-cell surface molecules are involved in the lysis of both allogeneic a n d autologous target cells: T3 and T 4 molecules on T 4 + C T L , and T3 a n d T8 molecules on T 8 + C T L . Moreover, all three structures appear to be necessary for target recognition. T h e T 3 surface s t r u c t u r e is r a p i d l y e x t e r n a l i z e d b y shedding
Receptor structures for hormones a n d growth factors u n d e r g o rapid ligand-induced modulation 2°. Given the central role of the T3 molecule in h u m a n T-lymphocyte function, it was not surprising to find that T3 is rapidly induced to modulate by anti-T3 via external shedding from the cell without affecting viability or surface expression of other unrelated structures. M o r e importantly, concomitant functional studies show that antigen-specific recognition and effector function of both T 4 and T 8 h u m a n C T L are linked to surface expression of the T3 molecular complex 9. Thus, after antibody-induced modulation of T3, there is a marked d i m i n u t i o n in antigenspecific recognition by C T L as defined by loss of alloantigen-induced proliferative capacity a n d specific cytotoxicity. Furthermore, return of both antigen-specific functions parallels the kinetics o f T 3 re-expression on the cell surface. Clones modulated by anti-T3 m o u n t a n enhanced proliferative response to interleukin 2 (IL-2), equivalent to that of the u n m o d u l a t e d clones triggered with IL-2 a n d aUoantigen. This observation suggests that naodulation of the T3 structure might activate the cell in a parallel fashion
to antigen itself, at least in so far as becoming more receptive to the second proliferative signal, IL-2. Moreover, it clearly indicates that the effect of anti-T3 modulation is not due to a generalized d i m i n u t i o n in cellular function. of c l o n o t y p i c T-ceU s u r f a c e m o l e c u l e s Because T lymphocytes recognize antigen in a precise fashion, one cannot account for their u n i q u e specificity on the basis ofmonomorphic portions ofT3, T 4 a n d T 8 molecules. Discriminative surface structures restricted to individual T-cellclones ('clonotypic') m u s t exist. Monoclonal antibodies to such structures (Tin) haye now been produced by i m m u n i z i n g mice with C T L clones, screening the resulting antibodies on the i m m u n i z i n g h u m a n C T L clone, and then selecting those which lack reactivity with additional clones from the same donor 21. Such antibodies are u n i q u e in that they inhibit cell-mediated killing a n d antigen-specific proliferation of the individual imm u n i z i n g clone without affecting the function of a n y othei clone. Like anti-T3 antibodies, the anti-clonotypic antibodies enhance IL-2 responsiveness a n d induce modulation of the Ti~ structure which co-modulates with T3. These studies indicate that the T i n structure is closely associated with T3 in the m e m b r a n c e of C T L . Nevertheless, competitive b i n d i n g a n d immunoprecipitation experiments demonstrate that the anti-clonotypic antibodies are distinct from anti-T3 a n d define two glycoprotein chains of 4 9 K D a n d 43KD. I n addition, Tin is unrelated to T 4 or'l"8 surface structures 21. It is likely that a n t i - T in (anti-clonotypic antibodies) define variable regions of the h u m a n T-cell receptor since they recognize clonally u n i q u e structures a n d selectively inhibit the antigen-specific function of individual clones. I n contrast, the wide distribution of the 20KD T3 glycoprotein a n d the ability of anti-T3 antibodies to inhibit antigen-specific function of all clones suggests that T3 comprises a constant region of the antigen receptor complex. A s u m m a r y of those h u m a n C T L surface molecules Definition
*Althoughno structuresanalogousto T3, T4 or Tin have been foundin the mouse,the murineLyt2structureappearsto be homologous to the humanTG molecule both in terms of the ability of antibodiesto that glycoproteinto functionallyinhibitCTL effectorsdirectedat class I MHC geneproductsand its similarmolecularweightunder reduced and non-reducedconditions.
Immunology Today, vol. 4, No. 1, 1983
7
which are involved in target recognition is shown in Table I. These include the 20KD T3 molecules expressed on all GTL, the subset-restricted 62KD T4 and 76KD T8 molecules linked to class II and class I C T L , respectively, and the clonally unique 49-43KD Tin molecules. * A model of the T-ceU receptor Fig. 1 provides a working model of the h u m a n T-cell receptor based upon the above experimental findings. As shown, each T cell displays two major recognition units on its surface. One structure consists of an antigen-binding region which views antigen ' X ' in the context of a polymorphic portion of an M H C molecule, whereas a second serves as an associative recognition structure for a constant region of a class I or class II molecule. The antigen-binding structure is comprised ofa donally unique glycoprotein subunit, Tin, in association with a T3 molecule. While the latter may not be directly involved in antigen binding, its linkage to Ti n could be critical for conformational stability required for the antigen-binding capacity o f T i n itself, thus explaining the major functional effects of anti-T3 antibodies. Since Tin is comprised of two polypeptides, one or both may be involved in antigen binding, analogous to heavy and light chains of the immunoglobulin molecule. The associative recognition element is either T8 or T4, depending on the subset derivation of the individual T lymphocyte. These glycoproteins bind to a constant region of class I or class II molecules, respectively, and are independent of the Tin-T3 complex. This modelcontains features of both the dual and single receptor hypotheses: the clonotype-bearing Tin-T3 molecular complex recognizes antigen ' X ' plus a polymorphic M H C gene product, while T8 or T4 bind to a monomorphic portion of an M H C gene product. In this
scheme, the affinity of two sets of receptors for various ligands would be multiplicative and thus advantageous in facilitating the juxtaposition of the surface target antigen with the effector T cell itself. This might be particularly important for killer-target conjugate formation as well as in the triggering of a primary immune response prior to clonal selection of high-affinity antigen-responsive cells. One prediction from the present model is that after selection of clones with high affinity for' neoantigen', such T cells might no longer require the T4 or T8 associative recognition structures in order to interact with the stimulating target. Indeed, the findings that anti-T4 and antiT8 antibodies block lytic activity of C T L effector clones, but not their ability to proliferate to antigen, suggests a process which demands a less stringent cell-cell interaction than the former. A second prediction from the above is that the Tin-T3 complex from T4 and T8 subsets will be similar: the class II and class I restrictions ofT4 and T8 clones result only as a consequence of the ability of T4 clones to bind class II-antigen ' X ' molecules on the stimulating cell more efficiently than T8 clones (and vice versa for class I- a n t i g e n ' X ' molecules) during the primary immune response. Nevertheless, given the extent ofclonal diversity, one might expect to find an occasional clone with an intrinsically high-affinity Tin-T3 complex viewing class I I - ' X ' or class I - ' X ' directly and in apparent contradiction to the T4-class II, TS-class I 'rule'H.2L As noted above, the T3 structure and its associated clonotype, Tin, are rapidly externalized following modulation by antibodies. In this regard, it may be that the shed Tin-T3 complex serves as an antigen-specific soluble mediator in the fluid phase of the immune response. Although there is no experimental evidence to support this to date, it is likely that a regulatory structure not depicted in Fig. 1 would be linked to the Tin-T3 complex and/or
RECEPTORS FOR ANTIGEN ON HUMAN T LYMPHOCYTES
T8
T4
T Lymphocyte
T Lymphocyte Fig. 1 Model of the human T-cell receptor
Each T lymphocyte displays two recognition units on its surface: the Tin-T3 complex and the associativerecognition structure, T8 or T4. The T8 and T4 glycoproteinsbind to non-polymorphic regions of class I (~t,) and class II (0,) MHC gene products, respectively. In contrast, the Tin-T3 complex recognizes specificantigen in the context of a polymorphic MHC gene product (~li~). Note that the precise domains of class I and class II which the T cells recognize are unknown and assigned above only for purposes of illustration.
8
Immunology Today, vol. 4, No. 1, 1983
associative recognition elements. Given the known inducer function of T4 + cells and suppressor function of T8 + cells, one would suspect that the former would be associated with inducer and the latter with suppressor molecules (reviewed in Ref. 23).
3 4 5 6
Testing the model Now that functional human T-cell clones are available in many laboratories and multiple monoclonal antibodies to their surface molecules exist, it should be possible to obtain more precise biochemical data regarding variability in the functionally critical T-cell surface molecules at both protein and DNA levels. Specifically, two-dimensional gel electrophoresis, peptide maps and nudeotide sequences will be critical for the characterization ofT[,, T3, T4 and T8 structures. In addition, examination of the functional effects of the purified glycoproteins themselves and their binding properties will further test the validity of the proposed model.
8
7
9 10 11 12 13 14 15 16 17
Acknowledgements This work was supported by N I H grants CA 19589 and RO1 NS 17182. Stefan Meuer is a recipient of a fellowship from the Deutsche Forschungsgemeinschaft (DFG; Me 693/1-1).
20 21
References
22
1 Benacerraf, B. and McDevitt, H. O. (1972) &'knee 175, 273 2 Zinkerrmgel, R. M. and Doherty, P. C. (1975)J. F_~. Med. 141, 1427
18 19
Schlossman, S. F. (1972) Tramp/ant. Rev. 10, 97 Zinkernagel, R. M. (1978) lmmunol. Rev. 42, 224 Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495 Meuer, S. C., Schlossman, S. F. and Reinherz, E. L. (1982) Prv¢. Natl Ac.ad. Sa" U.S.A. 79, 4590 Meuer, S. C., Hussey, R. E., Hodgdon, J. C., Hercend, T., Schlossrnan, S. F. and Reinherz, E. L. (1982) S¢/ence218, 471 Reinherz, E. L., Hussey, R. E., Fitzgerald, K. A., Snow, P., Terhorst, C. and Scl'dossman, S. F. (1981) Nature(London) 294, 168 Reinherz, E. L., Meuer, S., Fitzgerald, K. A., Hussey, R. E., Levine, H. and Scl'dossman, S. F. (1982) Cell 30, 735 Krensky, A. M., Reiss, C. S., Mier, J. W., Strominger, J. L. and Burakoff, S.J. (1982) Pr0~. NatlAcad. Sci_ U.S.A. 79, 2365 Biddison, W. E., Rao, P. E., Talle, M. A., Goldstein, G. and Shaw, S. (1982)J. Exp. Meg. 156, 1065 Ball, E. J. and Stasny, P. (1982) lmmunogenetics 16, 157 Chang, T. W., Kung, P. C., Gingras, S. P. and Goldstein, G. (1981) Proc. Natl Acad. Sci. U.S.A. 78, 1805 Reinherz, E. L., Hussey, R. E. and Schlossman, S. F. (1980) Eur. J. lmmunol. 10, 758 Bums, G. F., Boyd, A. W. and Beverley, P. C. (1982)J. lmmunol. 129, 1451 van Wauwe, F. P., DeMay, J. R. and Goossener, J. G. (1980)ft. ImmunoL 124, 2708 Reinherz, E. L., Kung, P. C., Goldstein, G., Levey, R. H. and Schlossman, S. F. (1980)/~'oc. NatlAtad. &'i. U.S.A. 77, 1588 Umiel, T., Daley, J. F., Bhan, A. K., Levey, R. H., Schlossman, S. F. and Reinherz, E. L. (1982)J. Immunol. 129, 1054 Wallace, L. E., Rickinmn, A. B., Rowe, M. and Epstein, M. A. (1982) Nature (London) 297, 413 Pastan, J. H. and Willingham, M. C. (1981) ,.Terence214, 504 Meuer, S. C., Fitzgerald, K. A., Hussey, R. E., Hodgdon, J. C., Schlossman, S. F. and Reinherz, E. L.J. F__~. Med. (in press) Malisnen, B., Rebai, N., Liabeuf, A. and Mawas, C. Eur. J. Immunol. (in
press)
23 Reinherz, E. L. and Schlossman, S. F. (1980) Cell 19, 821
D N A strand breaks and differentiation Cell differentiation is the process by which genetic information is selectively expressed to produce cells with various morphologies and functions. This process is fundamentally important, not least in the immune system where co-ordinated control of progression along several branched developmental pathways is vital for correct functioning. It has been known for some time that different types of differentiated cells contain different populations of messenger R N A which are translated to produce the proteins peculiar to each type. However, many of the processes involved in the integrated changes necessary for selectively expressing genetic information during differentiation are still obscure. Recent work I has produced some intriguing results on early nuclear changes following mitogen stimulation of human peripheral blood lymphocytes, which may be relevant to a generalized mechanism for controlling the expression of genes during differentiation 1-3. Analysis of the rate of sedimentation of 'nucleoids '+ (supercoiled DNA) showed that D N A from mitogenstimulated lymphocytes contained fewer strand breaks than D N A from resting cells a. This implies that (a) circulating quiescent lymphocytes contain DN~k strand breaks; and (b) these breaks are rejoined after stimulation. This D N A ligation occurs rapidly [ 1-8h after addition ofphyto© ElsevierBiomedicalFrel119830167-4919153~$1.{KI
haemaglutinin (PHA)] and represents one of the earliest nuclear responses to stimulation to be detected (the reported acetylation and phosphorylation of nuclear proteins may be attributable to changes in the permeability of the plasma membrane to the added radiochemicalsS). The enzyme ADP-ribosyl transferase (ADPRT) was implicated in the DNA rejoining process by showing that competitive inhibitors of the enzyme slowed the ligation and prevented lymphocyte activation 1. A D P R T is known to regulate D N A repair by enhancing the activity of D N A ligase II (Ref. 6). Inhibitors of A D P R T were only effective early during lymphocyte activation; they did not prevent subsequent cell proliferation if added later'. This suggests that similar chemicals may be of eventual clinical use in suppressing newly initiated immune responses whilst allowing established ones to continue (cf. use of cyclosporin A in transplantation managemend). The inhibitors presently available affect differentiation in general ~a and so may not be specific enough for use as drugs. The presence of breaks in the D N A strands of resting lymphocytes may explain several previously puzzling observations. One example is the extreme susceptibility of circulating lymphocytes to radiation damage, which decreases after P H A stimulation s. The authors of a recent report on the blocking of a mitogenic response by U V ir-
) The delineation of antigen receptors on human T lymphocytes Ellis L. Reinherz, Stefan C. Meuer and Smart F. Schlossman Monoclonal antibodies have identified several suoCacemolecules involved in target cell recognition by cytotoxic human T cells. In this article it is proposed that T cells have two recognition units: a complex composed of the T3 molecule and a clonally unique glycoprotein which binds antigen associated with polymorphic M H C gene product," and the T4 or T8 molecule which binds to a constant region of an M H C gene product. T lymphocytes recognize antigen in the context of membrane-bound products of the major histocompatibility complex (MHC) with exquisite specificity '3. While the surface receptors responsible for their cognitive properties are not known, two major theories have been proposed to explain this dual specificity of T lymphocytes4. One, the single-receptor model or interaction-antigen model, suggests that self-MHC molecules interact with antigen ' X ' to form a 'neoantigen' which is presented as one entity to a single T-cell receptor. The second model, a two'receptor model, assumes that one surface component of the T cell sees antigen ' X ' and another views the cellular M H C restricting element on the antigen-presenting ceil. Recently, the development of technologies for cloning antigen-specific human T lymphocytes and producing monoclonal antibodies against their surface structures has identified surface glycoproteins involved in their recognition mechanisms ~-9. This information allows one to construct a unifying hypothesis regarding the roles of polymorphic (clonotypic) and non-polymorphic human T-cell surface structures in this process. Given the known analogies between certain murine and human M H C gene products, immunoglobulin molecules and T-cell markers, it is likely that structures similar to those described below will be defined in murine systems.
T-cell surface structures involved in recognition of allogeneic and autologous target cells In order to present a model for the human T-cell receptor, it is first necessary to review several points of information concerning the various T-cell surface glycoproteins and the effects of antibodies to these structures on the function of clones of human antigen-specific cytotoxic T lymphocytes (CTL). In man, alloreactive cytotoxic effector cells are derived from the two major T-cell subpopulations which have been termed T4 + and T8 + on the basis of their uniquely expressed 62KD (T4) and 76KD (T8) glycoproteins. The target antigens recognized by individual T-cell subsets (i.e. T4 + or T8 + ) have been shown to be products of different gene regions of the major histocompatibility complex 8.~°-'2. Specifically, allosensitized T4 + T cells are directed at class II M H C antigens The Division of Tumor Immunology, Sidney Farber Cancer Institute and the Department of Medicine, Harvard Medical School, Boston, MA 02115, U.S.A.
on target cells whereas T8 + T cells are directed at class I M H C antigens on target cells. The association between surface phenotype of C T L and the class of M H C molecules that is recognized suggests that these subsetrestricted structures might themselves be required to facilitate recognition of and/or binding to different target antigens. This view has recently been supported by the finding that certain monoclonal antibodies to the T4 or T8 glycoproteins selectively inhibit cytolytic effector function o f T 4 + or T8 + C T L clones, respectively 8a,11. In addition to the subset-restricted surface glycoproteins T4 and T8, a 20KD T-cell surface molecule, T3, present on all mature T cells, participates in cell-mediated lysis by both of the major C T L subsets: antibodies to this structure block killing by T4 and T8 C T L 7-9,13.Moreover, anti-T3 antibodies are able to block the induction and effector phase of cell-mediated lympholysis, inhibit T-cell proliferative responses to soluble antigen and be mitogenic for resting T lymphocytes~*-~8.Both the appearance of T3 in late intrathyrnic ontogeny at the time of acquisition of immunological competence and its critical role in T-lymphocyte function suggested that it is closely linked to a recognition structure for antigen ~7,~8. Given that antibodies to the T3, T4 and T8 surface molecules could block C T L activity, it was necessary to define whether these structures serve as recognition elements or alternatively, components of the lytic mechanism itself. To this end, a series oflectin approximation studies was performed. It was reasoned that if antibodies bind to surface structures related to the lytic mechanism, then artificial approximation by lectin would not be capable of reconstituting effective lysis. In contrast, if antibodies blocked structures required for target recognition, lectin approximation would result in effective lysis. The experiments proved the latter alternative to be correct7. Perhaps more importantly, although lectin reversed the inhibitory effects of all three anti-T cell monoclonal antibodies, there was loss of target specificity. Antigen-specific C T L clones recognizing autologous Epstein-Barr virus (EBV)-transformed B lymphoblastoid lines are also governed by a series of recognition elements identical to the series in clones directed to allogeneic targets (Meuer et al., unpublished observations). Specifically, C T L expressing the T8 phenotype recognize the autologous B lymphoblastoidline in the context of class I M H C molecules; anti-HLA antibodies block C T L effector © Elsevier Biomedicnl Pros 1983 0167-4919183/0000-0000/$1.00
Immunology Today, vol. 4, No. 1, 1983
6
TABLE I. Summary of human T-cell surface molecules involved in target recognition by CTL T-cell surface molecule
Mol. wt
Distribution
Functional effect of monoclonal antibodies to the structure
T3
20 000
All mature T lymphocytes and a minority of thymocytes
T4
62 000
T8
76 000a
Tin
49000; 43000 b
Majority of thymocytes and 60% of peripheral T lymphocytes Majority of thymocytes and 30% of peripheral T cells Specific for an individual T-cell clone (clonotypic)
(1) Inhibits antigen-specific T-cell proliferative responses and cytotoxic effector function of all CTL (2) Enhances IL-2 responsiveness (3) Modulates by external shedding Inhibits CTL effectors directed at class II MHC gene products Inhibits CTL effectors directed at class I MHC gene products (1) Identical to anti-T3 effects but inhibits response only of an individual clone with which it reacts (2) Co-modulates by external shedding with T3
a Non-reducedstate; reduces to mol. wt 33 000 and mol. wt 31 000 subunitswith 2-mercaptoethanol.b Reduced state
function at the target level and anti-T8 antibodies abrogate the ability of these class I-specific killers to kill. I n contrast, T 4 + C T L recognize Ia (class II) determinants on the autologous lymphoblastoid cell a n d are inhibited b y a n t i - T 4 b u t not anti-T8 antibodies. As with the allogeneic C T L , anti-T3 antibody preincubation inhibits the killing ability of both T 4 + a n d T 8 + effector T cells. Because these clones do not lyse autologous B cells not infected with E B V or pokeweed mitogen-stimulated B-cell blasts, such T 4 + a n d T8 + effectors seem to recognize vir .ally encoded surface glycoproteins in association with class II or class I molecules, respectively 19. At present, however, these viral proteins have not been identified. The above results indicate that at least several T-cell surface molecules are involved in the lysis of both allogeneic a n d autologous target cells: T3 and T 4 molecules on T 4 + C T L , and T3 a n d T8 molecules on T 8 + C T L . Moreover, all three structures appear to be necessary for target recognition. T h e T 3 surface s t r u c t u r e is r a p i d l y e x t e r n a l i z e d b y shedding
Receptor structures for hormones a n d growth factors u n d e r g o rapid ligand-induced modulation 2°. Given the central role of the T3 molecule in h u m a n T-lymphocyte function, it was not surprising to find that T3 is rapidly induced to modulate by anti-T3 via external shedding from the cell without affecting viability or surface expression of other unrelated structures. M o r e importantly, concomitant functional studies show that antigen-specific recognition and effector function of both T 4 and T 8 h u m a n C T L are linked to surface expression of the T3 molecular complex 9. Thus, after antibody-induced modulation of T3, there is a marked d i m i n u t i o n in antigenspecific recognition by C T L as defined by loss of alloantigen-induced proliferative capacity a n d specific cytotoxicity. Furthermore, return of both antigen-specific functions parallels the kinetics o f T 3 re-expression on the cell surface. Clones modulated by anti-T3 m o u n t a n enhanced proliferative response to interleukin 2 (IL-2), equivalent to that of the u n m o d u l a t e d clones triggered with IL-2 a n d aUoantigen. This observation suggests that naodulation of the T3 structure might activate the cell in a parallel fashion
to antigen itself, at least in so far as becoming more receptive to the second proliferative signal, IL-2. Moreover, it clearly indicates that the effect of anti-T3 modulation is not due to a generalized d i m i n u t i o n in cellular function. of c l o n o t y p i c T-ceU s u r f a c e m o l e c u l e s Because T lymphocytes recognize antigen in a precise fashion, one cannot account for their u n i q u e specificity on the basis ofmonomorphic portions ofT3, T 4 a n d T 8 molecules. Discriminative surface structures restricted to individual T-cellclones ('clonotypic') m u s t exist. Monoclonal antibodies to such structures (Tin) haye now been produced by i m m u n i z i n g mice with C T L clones, screening the resulting antibodies on the i m m u n i z i n g h u m a n C T L clone, and then selecting those which lack reactivity with additional clones from the same donor 21. Such antibodies are u n i q u e in that they inhibit cell-mediated killing a n d antigen-specific proliferation of the individual imm u n i z i n g clone without affecting the function of a n y othei clone. Like anti-T3 antibodies, the anti-clonotypic antibodies enhance IL-2 responsiveness a n d induce modulation of the Ti~ structure which co-modulates with T3. These studies indicate that the T i n structure is closely associated with T3 in the m e m b r a n c e of C T L . Nevertheless, competitive b i n d i n g a n d immunoprecipitation experiments demonstrate that the anti-clonotypic antibodies are distinct from anti-T3 a n d define two glycoprotein chains of 4 9 K D a n d 43KD. I n addition, Tin is unrelated to T 4 or'l"8 surface structures 21. It is likely that a n t i - T in (anti-clonotypic antibodies) define variable regions of the h u m a n T-cell receptor since they recognize clonally u n i q u e structures a n d selectively inhibit the antigen-specific function of individual clones. I n contrast, the wide distribution of the 20KD T3 glycoprotein a n d the ability of anti-T3 antibodies to inhibit antigen-specific function of all clones suggests that T3 comprises a constant region of the antigen receptor complex. A s u m m a r y of those h u m a n C T L surface molecules Definition
*Althoughno structuresanalogousto T3, T4 or Tin have been foundin the mouse,the murineLyt2structureappearsto be homologous to the humanTG molecule both in terms of the ability of antibodiesto that glycoproteinto functionallyinhibitCTL effectorsdirectedat class I MHC geneproductsand its similarmolecularweightunder reduced and non-reducedconditions.
Immunology Today, vol. 4, No. 1, 1983
7
which are involved in target recognition is shown in Table I. These include the 20KD T3 molecules expressed on all GTL, the subset-restricted 62KD T4 and 76KD T8 molecules linked to class II and class I C T L , respectively, and the clonally unique 49-43KD Tin molecules. * A model of the T-ceU receptor Fig. 1 provides a working model of the h u m a n T-cell receptor based upon the above experimental findings. As shown, each T cell displays two major recognition units on its surface. One structure consists of an antigen-binding region which views antigen ' X ' in the context of a polymorphic portion of an M H C molecule, whereas a second serves as an associative recognition structure for a constant region of a class I or class II molecule. The antigen-binding structure is comprised ofa donally unique glycoprotein subunit, Tin, in association with a T3 molecule. While the latter may not be directly involved in antigen binding, its linkage to Ti n could be critical for conformational stability required for the antigen-binding capacity o f T i n itself, thus explaining the major functional effects of anti-T3 antibodies. Since Tin is comprised of two polypeptides, one or both may be involved in antigen binding, analogous to heavy and light chains of the immunoglobulin molecule. The associative recognition element is either T8 or T4, depending on the subset derivation of the individual T lymphocyte. These glycoproteins bind to a constant region of class I or class II molecules, respectively, and are independent of the Tin-T3 complex. This modelcontains features of both the dual and single receptor hypotheses: the clonotype-bearing Tin-T3 molecular complex recognizes antigen ' X ' plus a polymorphic M H C gene product, while T8 or T4 bind to a monomorphic portion of an M H C gene product. In this
scheme, the affinity of two sets of receptors for various ligands would be multiplicative and thus advantageous in facilitating the juxtaposition of the surface target antigen with the effector T cell itself. This might be particularly important for killer-target conjugate formation as well as in the triggering of a primary immune response prior to clonal selection of high-affinity antigen-responsive cells. One prediction from the present model is that after selection of clones with high affinity for' neoantigen', such T cells might no longer require the T4 or T8 associative recognition structures in order to interact with the stimulating target. Indeed, the findings that anti-T4 and antiT8 antibodies block lytic activity of C T L effector clones, but not their ability to proliferate to antigen, suggests a process which demands a less stringent cell-cell interaction than the former. A second prediction from the above is that the Tin-T3 complex from T4 and T8 subsets will be similar: the class II and class I restrictions ofT4 and T8 clones result only as a consequence of the ability of T4 clones to bind class II-antigen ' X ' molecules on the stimulating cell more efficiently than T8 clones (and vice versa for class I- a n t i g e n ' X ' molecules) during the primary immune response. Nevertheless, given the extent ofclonal diversity, one might expect to find an occasional clone with an intrinsically high-affinity Tin-T3 complex viewing class I I - ' X ' or class I - ' X ' directly and in apparent contradiction to the T4-class II, TS-class I 'rule'H.2L As noted above, the T3 structure and its associated clonotype, Tin, are rapidly externalized following modulation by antibodies. In this regard, it may be that the shed Tin-T3 complex serves as an antigen-specific soluble mediator in the fluid phase of the immune response. Although there is no experimental evidence to support this to date, it is likely that a regulatory structure not depicted in Fig. 1 would be linked to the Tin-T3 complex and/or
RECEPTORS FOR ANTIGEN ON HUMAN T LYMPHOCYTES
T8
T4
T Lymphocyte
T Lymphocyte Fig. 1 Model of the human T-cell receptor
Each T lymphocyte displays two recognition units on its surface: the Tin-T3 complex and the associativerecognition structure, T8 or T4. The T8 and T4 glycoproteinsbind to non-polymorphic regions of class I (~t,) and class II (0,) MHC gene products, respectively. In contrast, the Tin-T3 complex recognizes specificantigen in the context of a polymorphic MHC gene product (~li~). Note that the precise domains of class I and class II which the T cells recognize are unknown and assigned above only for purposes of illustration.
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Immunology Today, vol. 4, No. 1, 1983
associative recognition elements. Given the known inducer function of T4 + cells and suppressor function of T8 + cells, one would suspect that the former would be associated with inducer and the latter with suppressor molecules (reviewed in Ref. 23).
3 4 5 6
Testing the model Now that functional human T-cell clones are available in many laboratories and multiple monoclonal antibodies to their surface molecules exist, it should be possible to obtain more precise biochemical data regarding variability in the functionally critical T-cell surface molecules at both protein and DNA levels. Specifically, two-dimensional gel electrophoresis, peptide maps and nudeotide sequences will be critical for the characterization ofT[,, T3, T4 and T8 structures. In addition, examination of the functional effects of the purified glycoproteins themselves and their binding properties will further test the validity of the proposed model.
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7
9 10 11 12 13 14 15 16 17
Acknowledgements This work was supported by N I H grants CA 19589 and RO1 NS 17182. Stefan Meuer is a recipient of a fellowship from the Deutsche Forschungsgemeinschaft (DFG; Me 693/1-1).
20 21
References
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Schlossman, S. F. (1972) Tramp/ant. Rev. 10, 97 Zinkernagel, R. M. (1978) lmmunol. Rev. 42, 224 Kohler, G. and Milstein, C. (1975) Nature (London) 256, 495 Meuer, S. C., Schlossman, S. F. and Reinherz, E. L. (1982) Prv¢. Natl Ac.ad. Sa" U.S.A. 79, 4590 Meuer, S. C., Hussey, R. E., Hodgdon, J. C., Hercend, T., Schlossrnan, S. F. and Reinherz, E. L. (1982) S¢/ence218, 471 Reinherz, E. L., Hussey, R. E., Fitzgerald, K. A., Snow, P., Terhorst, C. and Scl'dossman, S. F. (1981) Nature(London) 294, 168 Reinherz, E. L., Meuer, S., Fitzgerald, K. A., Hussey, R. E., Levine, H. and Scl'dossman, S. F. (1982) Cell 30, 735 Krensky, A. M., Reiss, C. S., Mier, J. W., Strominger, J. L. and Burakoff, S.J. (1982) Pr0~. NatlAcad. Sci_ U.S.A. 79, 2365 Biddison, W. E., Rao, P. E., Talle, M. A., Goldstein, G. and Shaw, S. (1982)J. Exp. Meg. 156, 1065 Ball, E. J. and Stasny, P. (1982) lmmunogenetics 16, 157 Chang, T. W., Kung, P. C., Gingras, S. P. and Goldstein, G. (1981) Proc. Natl Acad. Sci. U.S.A. 78, 1805 Reinherz, E. L., Hussey, R. E. and Schlossman, S. F. (1980) Eur. J. lmmunol. 10, 758 Bums, G. F., Boyd, A. W. and Beverley, P. C. (1982)J. lmmunol. 129, 1451 van Wauwe, F. P., DeMay, J. R. and Goossener, J. G. (1980)ft. ImmunoL 124, 2708 Reinherz, E. L., Kung, P. C., Goldstein, G., Levey, R. H. and Schlossman, S. F. (1980)/~'oc. NatlAtad. &'i. U.S.A. 77, 1588 Umiel, T., Daley, J. F., Bhan, A. K., Levey, R. H., Schlossman, S. F. and Reinherz, E. L. (1982)J. Immunol. 129, 1054 Wallace, L. E., Rickinmn, A. B., Rowe, M. and Epstein, M. A. (1982) Nature (London) 297, 413 Pastan, J. H. and Willingham, M. C. (1981) ,.Terence214, 504 Meuer, S. C., Fitzgerald, K. A., Hussey, R. E., Hodgdon, J. C., Schlossman, S. F. and Reinherz, E. L.J. F__~. Med. (in press) Malisnen, B., Rebai, N., Liabeuf, A. and Mawas, C. Eur. J. Immunol. (in
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23 Reinherz, E. L. and Schlossman, S. F. (1980) Cell 19, 821
D N A strand breaks and differentiation Cell differentiation is the process by which genetic information is selectively expressed to produce cells with various morphologies and functions. This process is fundamentally important, not least in the immune system where co-ordinated control of progression along several branched developmental pathways is vital for correct functioning. It has been known for some time that different types of differentiated cells contain different populations of messenger R N A which are translated to produce the proteins peculiar to each type. However, many of the processes involved in the integrated changes necessary for selectively expressing genetic information during differentiation are still obscure. Recent work I has produced some intriguing results on early nuclear changes following mitogen stimulation of human peripheral blood lymphocytes, which may be relevant to a generalized mechanism for controlling the expression of genes during differentiation 1-3. Analysis of the rate of sedimentation of 'nucleoids '+ (supercoiled DNA) showed that D N A from mitogenstimulated lymphocytes contained fewer strand breaks than D N A from resting cells a. This implies that (a) circulating quiescent lymphocytes contain DN~k strand breaks; and (b) these breaks are rejoined after stimulation. This D N A ligation occurs rapidly [ 1-8h after addition ofphyto© ElsevierBiomedicalFrel119830167-4919153~$1.{KI
haemaglutinin (PHA)] and represents one of the earliest nuclear responses to stimulation to be detected (the reported acetylation and phosphorylation of nuclear proteins may be attributable to changes in the permeability of the plasma membrane to the added radiochemicalsS). The enzyme ADP-ribosyl transferase (ADPRT) was implicated in the DNA rejoining process by showing that competitive inhibitors of the enzyme slowed the ligation and prevented lymphocyte activation 1. A D P R T is known to regulate D N A repair by enhancing the activity of D N A ligase II (Ref. 6). Inhibitors of A D P R T were only effective early during lymphocyte activation; they did not prevent subsequent cell proliferation if added later'. This suggests that similar chemicals may be of eventual clinical use in suppressing newly initiated immune responses whilst allowing established ones to continue (cf. use of cyclosporin A in transplantation managemend). The inhibitors presently available affect differentiation in general ~a and so may not be specific enough for use as drugs. The presence of breaks in the D N A strands of resting lymphocytes may explain several previously puzzling observations. One example is the extreme susceptibility of circulating lymphocytes to radiation damage, which decreases after P H A stimulation s. The authors of a recent report on the blocking of a mitogenic response by U V ir-
