Genetic and mutational analysis of the T-cell antigen receptor.

Annu. Rev. Immunol. 1990.8:139-67

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GENETIC AND MUTATIONAL

Annu. Rev. Immunol. 1990.8:139-167. Downloaded from www.annualreviews.org by University of Sherbrooke on 06/10/14. For personal use only.

ANALYSIS OF THE T-CELL ANTIGEN RECEPTORl Jonathan D. Ashwell

Biological Response Modifiers Program, Division of Cancer Treatment, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892 Richard D. Klausner

Cellular Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892 KEY

WORDS:

T-cell receptor, T-cell activation, T-cell mutants

INTRODUCTION The T-cell antigen-specific receptor (TCR) has a number of remarkable properties. Unlike most cell-surface receptors, which bind non polymorphic and soluble ligands, the TCR recognizes a complex ligand composed of antigen and a major histocompatibility complex (MHC)� encoded glycoprotein (antigen/MHC) present on the surface of a second cell ( I). Second, this receptor (in slightly modified forms) recognizes a large number of chemically and structurally distinct antigen and MHC-encoded molecules. Finally, regardless of the fine structure of its ligand-binding components, occupancy of the TCR initiates an extensive array of tempo rally ordered biological responses common to all T cells of a given I

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functional phenotype. The basis for these properties lies in the complex genetic organization and protein structure of the TCR. Many different forms of this receptor (each unique to a given clone ofT cells) are generated during the course of T-cell maturation, largely by the pseudo-random rearrangement of the genomic elements encoding the ligand-binding chains of the receptor (the so-called Ti chains, for T-cell idiotype) (2). In this way, each clone of T cells possesses a molecule that, while having a great deal of structural similarity to other TCRs, has critical amino acid differences that confer unique ligand-binding specificity. In addition to the two poly morphic chains that form the ligand-binding heterodimer (a/3 chains in most peripheral T lymphocytes), the cell-surface form of the TCR has at least five additional nonpolymorphic chains. Three of these chains, 'l', b, and 8, comprise the CD3 complex (3-5). More recently it has become clear that at least two other chains are present in the cell surface TCR: ( and 1] (6, 7). The variable chain-associated invariant polypeptides play little or no role in determining antigen-specificity. They are, however, responsible for coupling TCR occupancy to intracellular signal transduction pathways that result in the events comprising T-cell activation. The intricate genetic organization and protein structure of the TCR has in many ways been a barrier to understanding its biology. At the same time, these properties can be exploited using genetic techniques to create partially formed TCR complexes; this allows an examination of how the different components interact with themselves and with other molecules. In this review, we present an overview of the structural and functional properties of the TCR, emphasizing recent approaches that involve the analyses of genetically manipulated or structurally aberrant receptors. Such work has involved the generation of mutant T ceIIs that lack one or more TCR chains, the transfection of individual chains into TCR - ceIIs, the introduction into cells of other molecules involved in T-cell activation, or a combination of these methods. These approaches have yielded insights into the assembly, expression, and function of the TCR, and they form the basis for new and provocative speculations about the biological properties of this molecule. CURRENT VIEW OF THE TCR STRUCTURE Components of the TCR

Although the structure of the TCR has been the subject of several recent reviews (8- 1 0), an understanding ofthis molecular complex is still evolving. In this section we update our current view of the receptor structure. As with many other integral membrane proteins, the TCR is a multichain, hetero-oligomeric transmembrane assembly. A tremendous amount of


MUTATIONAL ANALYSIS OF THE TCR

141

work has been directed toward defining the subunits o f the receptor, clearly an essential step in understanding how this complex is actually configured. However, we have only the sketchiest information on how these multiple subunits interact. Figure 1 shows a cartoon based upon the primary sequence of six of the gene products that comprise the receptors on the surface of a T cell. Two types of subunits comprise this receptor. The variable chain com ponents are highly polymorphic, being structurally unique for each clone of T cells; the rest of the subunits are identical in all T cells. Two structural features are common to all of the known subunits. First, they are trans membrane proteins possessing a single membrane-spanning and pre sumably helical domain. Second, all of the components of the TCR possess the unusual feature of having charged amino acids within the predicted transmembrane domain. All of the invariant chains have single negative

Variable Chains

CD3 &

or

,

798 - 9 27aa

4S-55u

0, @ •

,

9aa

{

{

21 aa

113aa

Charged amino acid(s) in the transmembrane region

(®

indicates unknown)

Homology to a consensus nucleotide-binding sequence

Figure 1

Schematic diagram of the cell surface TCR.


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ASHWELL

&

KLAUSNER

charges that are conserved between mouse and human, and all of the clonotypic chains possess one or two positive charges in that domain. Based upon structural and genetic considerations, we have divided the TCR into three groups of genes/gene products. The first genetic group of receptor subunits are the variable chain components. These are im munoglobulin-like structures possessing constant and variable immuno globulin domains and very short (4- 1 2 amino acids) cytoplasmic tails. All receptors containing rxf3 chains have these two subunits linked by a single disulfide bond. The next genetic group of subunits is the CD3 complex (4-6, 11). These consist of three proteins referred to as y, 6, and 2. Gamma and 6 are glycoproteins whose core protein sizes ate approxi mately 1 5 kd. As a result of glycosylation, the apparent molecular weight of 6 is 25 kd in the mouse "}nd 21 kd in the human. Likewise, the mature form of the y chain due to N-linked glycosylation has an apparent molec ular weight of 2 1 kd in mouse and 25-28 kd in human. The e chain is not glycosylated. All of these chains are likely to contain intrachain d isulfide bonds, a point demonstrated by mobility differences on SDS-PAGE as a function of their state of reduction. This is most marked for the e chain. Like the variable chains, the CD3-complex components can all be con sidered members of the immunoglobulin gene superfamily because of predicted structural homology to immunoglobulin domains found in their extracellular portions ( 1 2) . The CD3 components are highly homologous to each other both in structure and sequence; this is most marked when one compares the sequence of y to (5. These three chains likely arose by gene duplication. In fact, the genes encoding these three chains are all closely linked. They are found within 50-300 kb on human chromosome l lq23 and mouse chromosome 9 ( 1 3, 1 4); the CD3-y and -6 genes lie within 1 . 5 kb of each other. In contrast to the variable chains, each of the CD3 components possess significant fractions of their primary structure as cytoplasmic domains. The third genetic grouping of subunits of the TCR is composed of the ( and 1] chains (7, 1 5). The ( chain is a 1 6-kd nonglycosylated protein with no sequence or structural homology to either of the other genetic groupings of the receptor complex. In the majority of receptors, ( exists as a disulfide-linked homodimer. In contrast to the other chains, it has a very short 6-9 amino acid extracellular domain, with the vast majority of the ( protein ( 1 1 3 amino acids) existing as the cytoplasmic domain. The ( chain is encoded by a gene found on human and mouse chromosome 1 within a linkage group conserved between these two species ( 1 6). An unusual observation was made concerning the structure of (. While the majority of ( was found to be part of a 32-kd homodimer, 5-20% of ( appears to be disulfide linked to a 22-kd protein (7). Thus, under



143

nonreducing conditions ( could be detected either as part of a 32-kd (homodimeric) structure or as part of a 38-kd (heterodimeric) structure. In most cells, the ratio of homodimer to heterodimer is reproducible and fixed. Single-cell cloning of T-cell hybridomas does not change this ratio, and we believe that all cells contain, at the single cell level, both homo dimers of ( and heterodimers. In most cells the ratio of homodimer to heterodimer ranges from 5 to 1 0: 1 . Although steady-state metabolic labeling data suggest that the relative stoichiometry is as shown in Figure I , no definitive data indicate how many of each subunit exist per complex. Although we currently favor the model of two different heptameric complexes, one containing the (-( «(2) homodimer and the other containing the (-1] «(1]) heterodimer, we do not have any direct evidence that complexes containing the heterodimer do not also contain the homodimer. The potential functional implications of the existence of two classes of TCRs will be addressed later in this chapter. Subunit Interactions Within the TCR

Two approaches have been taken to define the structural relationships between the subunits of the TCR. One is covalent cross-linking studies; the other is the identification and characterization of partial complexes. The only covalent cross-linking study in the literature demonstrated that the {3 chain could be cross-linked to the CD3-y chain in human T cells ( 1 7). Partial complexes have been examined to infer nearest neighbor relationships within the full TCR complex ( 1 8). There are three general ways in which partial complexes have been observed. First, the TCR can be partially dissociated by altering the detergent conditions. Second, spontaneously occurring variants ofT cells or specifically derived mutants of T cells containing only a subset of the receptor chains have been examined. Third, groups of chains have been transfected into non-T cells, and the assembly of these incomplete sets has been studied ( 1 9). The TCR can be isolated in its full heptameric state by solubilizing the cells in detergents such as Triton X-lOO or digitonin. If ionic detergents are utilized, the complex dissociates. However, if limiting amounts of ionic detergents are added, one can observe partial dissociation. Several partial complexes observed by this sort of procedure include CD3-y&, rx{3b, and partial CD3 complexes, including ye and be. The examination of a wide variety of T cells that only express a limited number of the chains of the receptor complex has been a rich source for the observation of partial complexes. Finally, transfection experiments demonstrate that rx and {3 can pair in the absence of any other chains (20). The efficiency of this pairing

may, however, vary depending upon the particular rx-{3 chains examined. CD3 chains can assemble independently of the presence of any of the other


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chains, and partial CD3 complexes such as By can be observed when only those chains are co-transfected into fibroblasts ( 19, 2 1). The ( chain readily dimerizes when expressed in fibroblasts. A clear conclusion to be drawn from all of these observations is that the interaction of subunits within the TCR complex does not require the presence of all of the subunits. A very tentative proposal based upon these partial interactions for the nearest neighbor relations within the TCR complex can be made. Accordingly, all members of the CD3 group mutually interact. The (J chain directly interacts with the y chain of CD3. Because /3 and CD3-y are structurally so similar to a and CD3-b, respectively, it is tempting to speculate that CD3-b interacts with the a chain. Finally, the ( chain primarily interacts with a /3. However, data suggest that this last interaction is quite unstable in the absence of CD3 (18). THE STRUCTURE OF THE COMPLEX DETERMINES ITS INTRACELLULAR FATE The lives of all of the components of the TCR complex begin in the endoplasmic reticulum. From studies on a variety of integral membrane proteins, it is becoming clear that many, if not the majority, of these proteins assemble into oligomeric structures (22). Two important obser vations about oligomerization of newly synthesized membrane proteins have emerged. First, oligomerization virtually always takes place and is completed within the endoplasmic reticulum. Second, full oligomerization appears to be, in general, required to leave the endoplasmic reticulum (ER) and follow the secretory pathway through the Golgi and to the plasma membrane. The TCR is no exception. Assembly of the TCR Complex

Work from several laboratories has led to agreement on two conclusions about the assembly of the TCR complex. First, assembly takes place within the ER and begins soon after biosynthesis (19, 23, 24). Second, within the endoplasmic reticulum there is a transient, noncovalent association with a 26-kd nonglycosylated protein (21, 25). This protein has been called CD3-w or TRAP (T-cell receptor-associated protein). TRAP can be found associated with newly synthesized components of the TCR from the earliest times after synthesis. It remains associated with the complex for 1 0-20 minutes within the ER before it disappears. Studies in human T cells have suggested that TRAP binds both to CD3 components and to unassembled a or (J chains. These studies have suggested that TRAP disappears upon the association of CD3 with a-p. In mouse cells we have been unable



1 45

to identify a direct association between TRAP and the variable chain components. Although TRAP may well be involved in the assembly of the newly synthesized complex, its dissociation is clearly not a prerequisite for complete assembly because TRAP dissociation can be inhibited by a va riety of manipulations, including low temperature and the drug Brefeldin A. Despite these manipulations, no inhibition of the complete assembly of the entire TCR occurs within the endoplasmic reticulum. The two most rapid events in subunit assembly are the dimerization of , and the formation of partial and complete CD3 complexes. In a number of human T cells that lack either the rt. or the f3 chain, the remaining variable chain has been observed to assemble with the CD3 complex. This is not always the case, however, and in the 2B4. 1 1 murine T-cell hybridoma the failure to synthesize f3 results in no detectable assembly of rt. with the remaining chains (26). Regardless of these discrepancies, studies in all systems suggest that rt. and f3 chains are capable of stably interacting with CD3 before they are disulfide linked. It is extremely difficult to define an exact order of assembly within the CD3 complex. At very early points we observe what appear to be partial complexes containing either ')'e or ')'b. The ( chain homodimerizes or heterodimerizes with IJ within minutes after synthesis. Even if one observes an order of assembly in a T cell, one cannot be sure that it represents a unique or requisite pathway. For example, rt. and f3 chains, when expressed in fibroblasts in the absence of any other TCR subunits, rapidly and efficiently dimerize within the endoplasmic reticulum (ER) (20). In addition, although the ( chain dimer may assemble quite early with other TCR components, the pentameric complex in the absence of any ( is formed with no apparent kinetic or structural differ ences. Likewise, in T-cell hybridomas lacking the CD3-b chain, there is a rapid and efficient assembly of the remaining TCR components. Inter estingly, in this situation, there is some suggestion that in c)-deficient cells more than one')' chain is now assembled, perhaps replacing the missing b chain (18). Thus, despite a variety of observations about the details of the assembly of the TCR within the ER, we are still far from a clear consensus on this process. The Fate of Newly Assembled Complexes

Initial studies on the fate of newly synthesized components of the TCR provided a surprising finding. In the 2B4. 1 1 T-cell hybridoma the vast majority of newly assembled receptor subunits are rapidly degraded after synthesis (24). This observation presented a puzzle whose solution is found in the recognition of a set of cellular processes aimed at ensuring that only correctly assembled multicomponent complexes are transported to the cell surface. We have chosen to refer to these processes as architectural editing.


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Closer examination of the 2B4. 1 1 hybridoma revealed that, in fact, not all newly synthesized chains were rapidly degraded. In particular, virtually all of the ( chain made in these cells assembled into full receptor heptamers within the endoplasmic reticulum and survived long term (greater than 1 0-20 hr half-life), consistent with the measured half-life of the mature fully assembled cell surface receptor. In contrast, from 80 to 95% of the other TCR chains in these cells were degraded within 2 to 4 hours of their synthesis. The small fraction of these chains that survived long-term were those that assembled into full heptamers within the ER. The fact that, in these cells, the ( chain protein is made at approximately one tenth the rate of any of the other subunits explains the disparity in subunit survival. The complete survival of ( occurred because essentially 1 00% of the newly synthesized ( chain was rapidly assembled into full TCR complexes. In contrast, the remaining chains formed partial complexes (the majority were able to form pentamers lacking O. The incomplete but pentameric complexes containing r:t., {J, 'Y, b, and c emerged efficiently from the endo plasmic reticulum and were transported through the entire Golgi system. This could be monitored by the processing of N-linked carbohydrate side chains. During this transit through the Golgi, no degradation was observed. However, after Golgi processing was complete the vast majority (greater than 99%) of these chains were transported for complete degra dation to lysosomes (as demonstrated by the fact that their degradation was readily inhibited by a number of pharmacologic agents known to inhibit lysosomal degradation, and that we were able to observe mor phologically a large trafficking of receptor components to lysosomes; 20, 26). These data allowed the initial proposal of a sorting model for the transport of newly synthesized and assembled TCR components through the secretory organellar system. Such a model proposes a unique role for the assembly of the ( chain onto the pentameric complex-either that it provides a specific targeting and/or transport signal to the plasma membrane, or it specifically blocks a targeting signal present in the pen tameric complex that directed that complex to lysosomes. Support for this role of ( was obtained by the isolation of mutant and/or variant 2B4. 1 1 hybridoma cells that expressed no ( chain despite normal levels of synthesis of the remaining five subunits of the receptor (27). In these cells only pentameric complexes could be formed. As predicted, pentamers efficiently assembled within the endoplasmic reticulum and then moved out through the Goigi complex. However, considerably less than 1 % of these complexes arrived at the plasma membrane; the rest were directed from the Goigi to lysosomes where the complex was completely degraded. What is the fate of TCR complexes or chains that fail to assemble, at



1 47

least into a minimal pentameric complex? All current data suggest that none of these subpentameric complexes reach the Golgi. This could be demonstrated both morphologically, using immunoelectron microscopy, and by biochemical studies that confirmed the absence of any Golgi associ ated-posttranslational modifications. This is true of all of the partial complexes described in the previous section, including those arising from transfection of individual or groups of genes encoding TCR chains, nat urally occurring variants of T-cell lines, and mutant T cells. TCR subunits can have either of two fates when they cannot reach the Golgi because of incomplete assembly. Either subunits are rapidly degraded or they are retained for long periods of time within the ER. We have referred to the former as ER degradation for a number of reasons (20). First, it can be pharmacologically distinguished from lysosomal degradation because none of the drugs that readily inhibit lysosomal degradation affect ER degradation. Second, this pathway of degradation is seen only when these chains are not capable of moving beyond the ER system. Third, chains undergoing this degradation do not leave the ER, as assessed either mor phologically or biochemically. This degradation appears to be selective in that only certain components of the TCR complex are subject to ER degradation. Among the TCR components susceptible to ER degradation are all of the glycosylated subunits (ct, [3, (j, and y); the' chain and the I:: chain apparently are not targets for ER degradation. In this respect the y chain is of some interest. If one examines the BW51 47 lymphoma cell invariant chains, only I:: and y chains are synthesized. These assemble efficiently but remain within the ER. However, neither y nor I:: appear to dissociate from each other, and y is as long-lived as 1::. In contrast, if one transfects the y chain cDNA into fibroblasts, the y chain protein is rapidly degraded in an ER system. As in the T cell, the I:: chain, when transfected into these fibroblasts, demonstrates no sensitivity to ER degradation. When the cDNAs encoding both chains are cotransfected, all of the assembled y chain is not degraded. A similar situation holds for the (j chain. In a variant of 2B4. l l that synthesizes no [3 chain, the CD3 triplet readily assembles within the ER. However, in the absence of the [3 chain, no transfer to the Golgi is seen. Interestingly, with time the (j chain is lost from the complex and degraded. The disassembly of the CD3 trimer can be blocked by minimally reducing the temperature (to 30°C). As one blocks disassembly of c5 within the ER, one also blocks c5 ER degradation. This has led us to propose that it is the state of assembly of the complex that, at least in part, can explain the selectivity of subunit degradation (28). It also raises the very intriguing observation that in this organelle, apparently built to allow the assembly of this multi subunit complex, the failure to leave the ER will result in the gradual disassembly of the components.

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The various intracellular fates of partial TCR complexes illustrate that the cell has an elaborate set of mechanisms that ensure that only fully assembled TCR complexes are efficiently transported to and expressed on the cell surface. Underlying this phenomenon is a whole array of intriguing cellular processes that are illuminating quality control pathways clearly central to normal cell physiology. A cartoon i llustrating the relationship between the nature of the TCR complex formed and its fate within the cell is shown in Figure 2.


GENETIC RECONSTITUTION STUDIES OF THE TCR The fact that the receptor, as a functional unit, is composed of at least seven chains derived from at least six distinct genes poses a formidable challenge to the reconstitution of the entire complex in non-T cells. In fact, even if all seven chains of the majority TCR, as described above, were transfected into fibroblasts we do not know that those chains would correctly assemble. It appears that fibroblasts do not make TRAP, and since the gene for TRAP is not yet available, we do not know what the consequences would be of the expression of the remaining chains in the

TRAP dissociation

a�y3€t;2"""""'--+"""""'�---""'�"'�/ a�y& Y3� �"'''''' a�

J

retention

B

a a�

degradation Figure 2

Intracellular fate of the TCR and its partial complexes. A schematic diagram of

the fate of partial TCR complexes: cis, cis-Golgi, med, medial-Golgi; trans, t rans-Golgi; TGN, Trans-Golgi network; PM, plasma membrane. See text for discussion.



1 49

absence of this protein. The isolation of mutant or variant T cells that fail to express one or more TCR chains may provide the best cells in which to study the function of individual components of the TCR. In the absence of most of the molecularly characterized chains (a, /3, y, b, or 8) the TCR is effectively nonfunctional because, as a manifestation of architectural editing, the incomplete receptor is simply not expressed efficiently on the cell surface. In fact, some of the earliest studies that suggested the critical requirements for all components of the receptor for the expression of any of the components on the cell surface, came from examining mutants or variants of human T -cell lines that failed to express either the variable or the CD3 chains on the surface. Whenever one of these two components was lost, the other failed to be expressed on the cell surface or was expressed at substantially reduced levels (29). Of course, T cells that lack the expression of individual chains provide us with a superb setting in which to reintroduce the missing chain and thus define the function of that chain. In T-cell tumor lines lacking expression of either rx or /3, the reintroduction of the missing chain by either cDNA or genomic transfection reconstituted surface expression of the full receptor complex and a functional receptor (30-32). Recently, a similar approach has been used to reconstitute the surface expression of the TCR in a murine hybridoma that failed to transcribe the gene for ( (33). When the ( cDNA was expressed in these cells, surface receptor expression was restored. Such reconstitution studies can shed light on the structural requirements for both assembly and correct intracellular transport, as well as for the function of the reconstituted surface receptor. One study reported the use of site-directed mutagenesis to alter one of the conserved, positively charged residues in the trans membrane region of the f3 chain (34). Mutated or wild-type f3 chains were transfccted into a fJ-ncgative human T cell, and thc ability to express surface receptor complexes was examined. When the wild-type protein containing a lysine at position 290 was introduced into the cell, surface expression was regained. However, if the amino acid at position 290 was changed to a negatively charged glutamic acid, no surface expression was seen. The replacement with a polar (serine) or a hydrophobic (leucine) amino acid similarly failed to restore surface expression. When the posi tively charged lysine residue was replaced by arginine, again no surface expression was seen. One would conclude from this study that this par ticular lysine is critical to the assembly of the f3 chain with the complex and thus essential for surface expression. However, at this time no detailed experiments have been reported on the effect of these or any changes on the intracellular assembly and fate of the mutated complex. Another approach that can be used for genetic reconstitution studies of the TCR is the ability to form genetic chimeras between human and mouse


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receptor subunits. Because of the availability of species-specific antibody reagents, the structural and functional consequences of mutations in an introduced heterologous TCR gene can be assessed. Such heterologous reconstitution between variable and invariant chains was demonstrated when the ex and {3 chains derived from the murine T-cell hybridoma of known antigen/MHC specificity were introduced into a human T-cell tumor line (3 1). The successful surface expression of the murine ri-{3 heterodimer demonstrated successful heterologous assembly of the TCR complex. Fur thermore, this heterologous receptor displayed antigen/MHC specificity identical to that possessed by the murine hybrid oma from which the IX and {3 chains were derived. Recently in another study the human CD3-B chain was introduced into murine T cells (c. Transy et ai, 1 989. PNAS 86: 71081 2). Again, surface expression of the G chain was addressed, utilizing a monoclonal antibody that recognizes only the human G chain. The finding of the human G chain on the surface again supported the ability to obtain heterologous assembly. When the entire cytoplasmic tail of the human G chain was truncated by genetic manipulation of the human G eDNA, this truncated protein was still expressed on the cell surface. Complexes containing this truncated human G chain reportedly were still capable of functioning in receptor-mediated signal transduction. This study provided no evidence that receptor complexes containing the human s either also contained mouse G or stably interacted with complexes containing mouse s. The conclusions from such studies that the cytoplasmic tail of the G chain is irrelevant for signal transduction must be taken as only tentative. This type of heterologous reconstitution provides an intriguing approach that obviates the need to have a mutant T cell lacking the expression of any given endogenous chain. However, the presence of a normal endogenous counterpart to the mutated heterologous introduced gene will always con found the certainty with which one can interpret the results of such experi ments. FUNCTIONAL PROPERTIES OF THE TCR Occupancy of the TCR by antigen/MHC is the major physiological means of activating T cells. The role of the heterodimeric variable chains in ligand recognition was conclusively proved by experiments in which DNAs encoding the IX and {3 chains of the TCR from antigen-specific T-cell clones were transfected into T cells with different antigen/MHC reactivities (3 1 , 3 5). The transfectants responded to the same antigen/MHC combination as the 1X{3 chain donor, proving that the heterodimeric variable chains are entirely responsible for the T-cell's exquisite ligand specificity. In addition to antigen/MHC, T cells may be activated with antibodies



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that cross-link the TCR complex, antibodies that cross-link an ever-grow ing list of cell surface molecules not known to be part of the TCR itself (such as CD2, Thy-I, and Ly-6), lectins, and some bacterial products such as the staphylococcal enterotoxins (36, 37) . Table 1 contains a partial list of some of the early and late phenomena that comprise T-cell activation. One of the central issues of modern cell biology is how information about actions occurring at the plasma membrane is rendered into intracellular events. For T cells in particular, what are the TCR-dependent biochemical and cellular events that mediate intracellular signaling and result in cellular activation? The analysis of TCR mutants and transfectants is providing some of the answers to these fundamental questions. TCR-Mediated Signal Transduction

Two simple and fundamentally different models concern the way in which the TCR transduces multiple signals. One is that TCR occupancy or perturbation generates a single event (such as the GTP-binding protein coupled activation of phospholipase C) that, via a cascade of enzymatic activity or intracellular ligand/receptor interactions, yields multiple bio logical outcomes. A second possibility is that the TCR generates multiple Table 1 A.

Some consequences of TCR-mediated T-cell activation

Early Events (Seconds to minutes) 1 Hydrolysis of phosphatidylinositol, generating 1 ,2-diacyglycerol and inositol trisphosphate

2 3 4 5

Increase in protein kinase C activity Redistribution of Ca2+ from intracellular organelles to the cytosol 2 Influx of Ca + from the extracellular medium Increased ph·osphorylation of proteins on tyrosine residues (due to increased activity of tyrosine kinase(s)?)

6 Redistribution of spectrin from near the Golgi to the plasma membrane 7 Intracellular redistribution of CD45

B.

Late Events (Minutes to hours)

I Nuclear gene activation, including increases in mRNA for • cellular proto-oncogenes, e.g. c-myc and c-fos • lymphokines, e.g. IL-2, IL-4, and l'-IFN • receptors for growth-promoting molecules, e.g. IL-2 2 Reorientation of the Golgi(microtubule organizing center 3 Increase in protein synthesis 4 Cell size enlargement (blast transformation)

5 Lymphokine secretion 6 Entry into the cell cycle 7 G ,/S cell cycle block (transformed T cells)

8 Cell lysis(DNA fragmentation (T-cell hybridomas, thymocytes)

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concurrent and independent signals. In this case, the TCR's extreme struc tural complexity may permit individual chains (or chain combinations) to act as functional domains that couple to distinct intracellular pathways. This model implies that it might be possible for the cell to regulate its array of activation responses (e.g. the lymphokines produced, whether to enter into the cell cycle, etc) at the level of the TCR. Recent analyses of structural and functional variants of the TCR support the latter model. STRUCTURAL VARIANTS OF THE TCR A series of T cells with aberrant TCRs, all derived from the 2B4. 1 1 T-celI hybridoma, were established independently and by a variety of approaches. As for most T cells, stimu lation of the 2B4. 1 1 wild type cell with either antigen or antibodies against CD3-e causes a rapid (seconds to minutes) increase in the hydrolysis of phosphoinositides (PI). When the I]-deficient subclones of 2B4. l l were analyzed, a correlation was found between (I] heterodimer content and the generation of inositol phosphates (IP) (38). TrxPl .2 celIs, for example, express 3 5--40% as much (11 as do 2B4. 1 1 cells, and the maximal level of IP they generate in response to antigen or anti-CD3 antibodies was comparably decreased (39). Even more striking were subclones whose (I] levels were lower than in the 2B4. 1 1 parental cell by 85-95%; in these cells activation induced IP generation was decreased to almost undetectable levels (38). Activation-induced PKC activity was also diminished in the (IJ-deficient cells, as assessed by the phosphorylation of the CD3-,), chain of the TCR . In contrast, the tyrosine kinase-mediated phosphorylation of the ( chain (40) was relatively preserved. Further studies with subclones from two additional and independent sets of 2B4. 1 1 -derived T cells that express absolutely no detectable (1J confirmed these initial observations. Although the response of all 2B4. l 1 -derived T-cell hybridomas to AIF;;- (a direct activator of G protein-dependent PI hydrolysis in 2B4. 1 1 cells; 4 1 ) was comparable, concentrations of antigen that generated maximal amounts of PI hydrolysis in 2B4.11 cells yielded little if any IP production in the (I]-negative subclones (42). These TCR variants have provided an impetus for the examination of signal transduction pathways leading to IL-2 production. One hypothesis is that two early biochemical events, elevation of [Ca2+]i and activation of PKC, are necessary and (probably) sufficient for TCR-mediated late cellu lar activation responses. A large body of circumstantial evidence supports this notion. Perhaps the best argument in favor of this model is that occupancy of the TCR results in the rapid cleavage of phosphatidylinositol bisphosphate to 1 ,2-diaclyglycerol and inositol trisphosphate, which in turn cause an increase in [Ca2+]; and PKC activity, and that the com bination of PKC-activating phorbol esters and Ca 2+ ionophores induces



1 53

IL-2 secretion in almost all T cells. However, as yet there is no direct and definitive proof that these biochemical events represent the only, or indeed the physiologic, second messengers for TCR-mediated activation. It has been possible to dissociate IL-2 production from these second messengers in the case of the ('I-deficient 2B4. 1 1-derived cells. Whereas anti-Thy- l stimulated virtually undetectable amounts of PI turnover or increases in [Ca 2+]j from Texf3 1 .2 cells (39), IL-2 production was normal. An important observation was that the Tocf31.2 cells could respond to PMA and a calcium ionophore with a dose-response curve similar to that of2B4. 1 1 cells, ruling out the possibility that Tocf3 1 .2 cells were simply sensitive to very small (unmeasurable) increases in [Ca 2+]j and PKC activity. Furthermore, to determine if the fluorescence assay used to measure [Ca 2+] was sensitive enough to detect biologically meaningful changes, the response of these cells to low concentrations of ionomycin was measured. The lowest con centration of ionomycin that could synergize with PMA to yield any detectable IL-2 secretion caused a large and sustained increase in [Ca2+]j (28), an increase in fact much greater than that seen with the stimulatory anti-Thy- l monoclonal antibody (mAb). These results have been con firmed with the ('I-negative 2B4. 1 1 variants; although antigen induced little PI hydrolysis in these cells, IL-2 production was preserved (42). These data, obtained in one extensively studied model system, obviously do not refute the model that IP 3, PKC activation, and CA 2+ flux are the initiators of IL-2 production in many, or even most, T cells. However, they do suggest that the question of what signaling pathways are used under physiological conditions is worthy of further experimentation. For example, what is the role of other second messengers? One clue may be in the finding that unlike PI hydrolysis, activation-induced increases in tyro sine phosphorylation were relatively normal in the absence of ('I-containing TCRs. To ask directly if phosphorylation of proteins on tyrosyl residues could initiate IL-2 production, a retroviral construct containing a func tional v-src gene was introduced into 2B4. l l cells (43). Numerous isolates with spontaneously increased tyrosine phosphorylation were studied. Cells with active v-src, but not cells that expressed only low levels of v-src or that had received the retroviral construct alone, constitutively produced small but easily detectable amounts of IL-2. Importantly, treatment with sodium butyrate (which enhanced transcription of the v-src gene) increased the production of IL-2 to levels that approached those induced by a mitogenic anti-Thy- l mAb. No spontaneous increase appeared in PKC activity in the cells expressing v-src, as judged by the normal levels of spontaneous phosphorylation of a PKC substrate, c-raf As with acti vation-induced IL-2 production, cyc1osporin A completely inhibited the secretion of IL-2 by 2B4. l l cells expressing v-src (unpublished obser-

1 54

ASHWELL & KLAUSNER


vations). These results indicate that an intracellular pathway that appears to be largely independent of the PlICa 2+ IPKC signals is capable of inducing IL-2 production. Whether TCR-mediated IL-2 production in normal T cells predominantly uses a Ca 2+ jPKC or a tyrosine kinase signaling pathway, whether these two pathways synergize, or whether an as yet unidentified series of second messengers are critical are matters for further study. FUNCTIONAL T-CELL VARIANTS In the lurkat T-cell model system, several T-cell variants have been obtained that are likely to represent mutations in components critical for TCR signaling pathways. Goldsmith & Weiss used lectin to kill a population of chemically mutagenized lurkat cells. The surviving cells were subjected to selection by alternate rounds of cell sorting, first for cell surface TCR expression, and then for the inability to mobilize [Ca2+ li in response to an anti-clonotypic mAb. One subclone, termed lCAM . l , that was obtained after limiting dilution cloning of cells with this phenotype was analyzed in detail (44). In addition to its failure to exhibit increases in [Ca 2+L JCAM . I did not manifest increases in PI hydrolysis in response to most anti-TCR antibodies, although both were observed in response to one particular anti-CD3 mAb (named 235) or to a combination of anti-CD-3 and anti-Ti mAbs, each unable to activate when used individually. JCAM. l cells were able to produce IL-2 when stimulated with PMA and a Ca 2+ ionophore, but the cells did not do so when PMA and anti-TCR mAbs were used, even when the mAbs used were those capable of inducing substantial increases in [Ca2+li (45). A reason for this surprising result may be that the 235 mAb induced markedly blunted PI hydrolysis. Furthermore, although this mAb induced rapid and large increases in [ea 2 + 1 in lCAM. l cells, the elevation was short-lived (returning to baseline levels by 90 min) compared to that induced in the parental Jurkat cells (remaining on a plateau after 2 h). These data were interpreted as indicating that unsustained elevations of [Ca 2+li, even in the presence of PMA, are insufficient to result in IL-2 production and are reminiscent of an independent study in which TCR - Jurkat cells trans fected with the murine Thy- l gene demonstrated increases in [Ca 2+li but no production of IL-2 when Thy- l was cross-linked by antibody (46). A second Jurkat signaling variant, JCAM.2, was isolated using a strat egy similar to that used to obtain JCAM . l (47). Unlike JCAM . I, this cell did not alter its levels of IP or [Ca 2+l in response to any of the anti-TCR mAbs tested, indicating different lesions in the signaling pathway. Initial biochemical analyses of both JCAM . l cells and JCAM.2 cells failed to reveal any gross structural defects in the TCR chains. Furthermore, when the 2B4. 1 1 murine T-cell ex chain was transfected into JCAM. I , mAb



155

binding o f the hybrid TCRs failed t o mobilize Ca 2+, making it unlikely that alterations in the Jurkat IX chain were responsible for the abnormal signal transduction. To facilitate the analysis of the signaling variants, an acute heterokaryon assay was devised in which one cell loaded with the indo- l Ca 2+ -sensitive fluorescent dye and another cell surface labeled with specific fluorescein-conjugated antibody were fused with polyethylene glycol. After 1 hr the resulting population, containing both fused and unfused cells, was exposed to a nonstimulatory (for the variants) anti TCR mAb and the "double positive" cells (heterokaryons) were analyzed by flow cytometry for the ability to mobilize Ca2 + . It was found that fusing JCAM . l or JCAM.2 to the parental Jurkat cell line, or to a series of T cells that lacked either IX or f3 chains, restored Ca 2+ responses. Interestingly, fusing of JCAM. l to JCAM .2 produced cells that exhibited an increase in [Ca 2+]i when stimulated with the anti-TCR mAb, allowing one to assign these variants to separate complementation groups. The structural bases for the signaling defects in JCAM . l and JCAM.2 are unknown. Since PI hydrolysis and changes in [Ca 2+1 occur so rapidly after stimulation, it is tempting to speculate that they reside in the TCR itself (particularly in the invariant chains) or in closely related molecules such as GTP-binding proteins. Given the correlation between (11 levels and PI hydrolysis/Ca 2+ signaling and that ( 2-containing receptors transduce signals for IL-2 production in 2B4. 1 1 cells, it may be speculated that a mutation in ( could account for the observations. The detailed char acterization of these variants may require the cloning of all their TCR chains and/or the determination of which molecules physically couple the TCR to the different signaling pathways. The TCR and Growth Regulation of Transformed T Cells

Although most assays of cellular responses after TCR perturbation have focused on IL-2 production, there are, of course, biological responses other than IL-2 production that are regulated via the TCR. One phenomenon observed in spontaneously transformed T cells is the inhibition of growth. Activation with antigen, antibodies to the TCR, antibodies to Thy-l or Ly-6, or lectins, causes a GtiS cell cycle block in such cells (48, 49). In normal cells as well, activation with large amounts of antigen or immobilized anti TCR antibody inhibits IL-2--driven proliferation, perhaps because of a block in late G, (50, 5 1 ). Some tumor cells such as 2B4. l 1 die after stimulation, an event manifested by the specific release of 5'Cr and DNA fragmentation (49, 52, 53). Recently, the phenomena of the cell cycle block and the cell lysis have been mechanistically differentiated (54). Growth inhibition (a decrease in [3H]thymidine incorporation due to the cell cycle block) occurs within minutes, does not require extracellular Ca 2+, and is


1 56

ASHWELL & KLAUSNER

not inhibited by cyclosporin A (CsA). Cell lysis occurs after 4-6 hours, requires extracellular Ca 2+, and is prevented by CsA. These results dem onstrate that the cell cycle block does not require Ca2+ flux across the plasma membrane as a signal. Further, its resistance to Ca 2+ depletion clearly differentiates it from IL-2 production. Cell lysis, however, obeys the same "rules" as IL-2 production. Analysis of the (I}-deficient variants has distinguished cell lysis from IL2 secretion and the G,/S cell cycle block. In contrast to 2B4. 1 1 , the (1} deficient subclones lysed either poorly or not at all by 1 2 hr after stimu lation with antigen. One 2B4. 1 1 subclone that contains more (I} than the wild type cell actually lysed better than 2B4. 1 1 , while another that contains 20% less (I} than 2B4. 1 1 lysed less well. Interestingly, Taf3 l .2, which contains 60% less (1] than 2B4. l l cells failed to lyse at all after antigen activation. These results suggest that for 2B4. 1 1 cells there may be a (I} level, or (I} : ( 2 ratio, below which lysis does not occur. Of course, there may be other independently varying cellular mechanisms that differentiate these cells and that account for some of the differences between them. Nonetheless, when the lytic response of these hybrid om as is viewed as an �

�

all-or-none

phenomenon,

there

is

an

excellent

correlation

with

''1

expression. Table 2 contains a partial summary of (I}-deficient 2B4. 1 1 derivatives and their responses to antigen. Table 2

TCR (I)-deficient variants of the 2B4. 1 1 T cell hybridomaa Antigen-Induced:

Cell

Derivation

2B4. 1 1

Parent cell

TlXfJ 1 .2

Transfection of a spon-

Percentage

PI

IL-2

(I)

Hydrolysis

Production

Cell Death

1 00

++++ +

++++ ++++

++++

35

15

±

8

±

0

±

+++

0

±

+++

taneous IXfJ-loss variant with 2B4 2M.44

IX

and fJ

Growth in mice treated with an anti-CD3 mAb

EY.3

Growth in mice treated with an anti-CD3 mAb

2A7

Transfection of a (loss variant with ( eDNA (protoplast fusion) Transfeetion of a' loss

1 .2

variant with ( eDNA (eleetroporation) a

Data summarized from references 33, 38, 39, and 42

h Also produced little or no IL-2 when stimulated with PMA plus ionomycin

'Less than

10%

specific release of "Cr after activation with antigen.



157

These observations are especially intriguing in view of the similarities between the antigen-induced death of T-cell hybridomas and the death of developing thymocytes during the process of negative selection. Both phenomena are antigen-specific, accompanied by nuclear DNA frag mentation, and sensitive to cyclosporine A (48, 52-56). Therefore, it is tempting to speculate that the pathway(s) to which (1]-containing TCRs are coupled may be involved in negative selection in the thymus. If so, the positive (proliferation) or negative (death) response of an immature T cell might be regulated by the level of (IJ-versus (2-containing TCR expression. Alternatively, the (1] signaling pathway could be regulated in these cells at a level distal to the TCR. The validity of these speculations can be deter mined experimentally when the appropriate reagents (mAbs and eDNA probes for the IJ chain) become available. Activation via TCR-Independent Molecules

Cell surface molecules other than the TCR, such as CD2, CD28, Thy-I, and Ly-6, can transduce some or all of the signals necessary for IL-2 production or T-cell proliferation when cross-linked by antibody (57). With the exception of CD28, antibody-mediated perturbation of these molecules leads to the PI hydrolysis and Ca 2+ mobilization that are charac teristic of TCR occupancy. Due largely to studies with TCR mutants, it has become apparent that these molecules utilize the TCR, directly or indirectly, in their signaling pathways. CD2 is a 50-kd transmembrane glycoprotein present on the surface of human thymocytes, peripheral T cells, and natural killer cells (58, 59). CD2 serves as a cell-cell adhesion-promoting molecule by binding to LFA3, a molecule with a wide tissue distribution (60-62). In addition to its role in cellular adhesion, CD2 can participate in the activation of T cells; certain combinations of non-cross-blocking anti-CD2 antibodies initiate most of the cellular responses that comprise T-cell activation (63). The judgement of whether activation via CD2 depends upon concomitant TCR expression depends upon the model system studied. Arguing against such a dependency is the observation that both CD3 + and CD3� thymocytes manifest increases in IL-2 receptor expression and [Ca2+]j levels when stimulated by the appropriate anti-CD2 mAb combination (64, 65). Fur thermore, NK cells that express cell surface CD2 but not CD3 manifest increases in [Ca2+ 1 (65, 66) or enhanced cytotoxicity (67, 68) when stimulated via CD2. Finally, Moretta et al (69) generated variants of the lurkat human leukemic T-cell line that were TCR � /CD2+. Whereas these variants failed to respond to PMA plus anti-CD3, they all produced normal levels of IL2 when stimulated with PMA plus the appropriate combination of anti CD2 mAbs. CO2


1 58

ASHWELL

&

KLAUSNER

Arguing in favor of a functional relationship between CD2 and the TCR, anti-CD3-mediated modulation of CD3 from the cell surface has been reported to make T cells refractory to anti-CD2-induced Ca2+ mobi lization, PI hydrolysis, and proliferation (63, 70, 71). The interpretation of these results is ambiguous because the anti-CD3 mAbs used to modulate the TCR might also have initiated transmembrane signals interfering with independent activation pathways. This potential problem was circum vented by others with the use of TCR + T-cell tumors and their TCR variants. Wild type (TCR +) REX human T lymphoblastoid cells (71 ) and Jurkat cells (72, 73) responded to a mitogenic combination of anti-CD2 antibodies, or to a single anti-CD2 mAb plus soluble LF A-3, with elev ations of rCa 2 + ] j, IP production, and IL-2 secretion. Neither of the TCR mutants of these cells, nor a Jurkat variant thought to possess a proximal defect in TCR-mediated signaling, responded to the stimuli. Restoration of TCR expression in the mutant Jurkat T cells by DNA-mediated gene transfer of the f3 chain yielded cells that once again manifested these activation-dependent events upon stimulation with anti-CD2, making it unlikely that the failure of the variants to respond was due to TCR independent mutations. Furthermore, fusion of the TCR - Jurkat cell with the TCR + Jurkat signaling variant yielded cells that increased their rCa 2 + ] j after stimulation via CD2, suggesting that at least two (complementing) defects accounted for the failure of either cell alone to respond. A different inference about the relationship between CD2 and the TCR can be drawn from recent studies in which some isolates of TCR - Jurkat T cells were capable of responding to anti-CD2 mAbs (plus PMA) by producing IL-2 (T. Saito and R . Germain, personal communication). Close inspection of these subclones revealed that they consistently expressed 3to lO-fold more CD2 than did the parental TCR - cell. In another series of experiments, the TCR - Jurkat T cells were supertransfected with human CD2, resulting in very high surface expression of CD2; these cells also produced IL-2 when stimulated with anti-CD2 mAbs, despite the lack of a cell-surface TCR. These data argue that the requirement for TCR co expression in CD2-mediated IL-2 production may be quantitative rather than qualitative. The studies outlined here do not present a simple picture of the func tional relationship between CD2 and the TCR. Some CD2-mediated acti vation responses, such as IL-2 receptor expression in immature thymocytes or Ca 2+ elevation and cytotoxicity in NK cells, may likely be initiated in the absence of a TCR. However, it seems clear that in some cases stimulation of mature T cells via the CD2 molecule to produce IL-2 requires, or is at least amplified by, co-expression of a normally functional TCR. Consistent with the hypothesis that the TCR, or at least tissue-specific auxiliary



1 59

molecules, are required for CD2-mediated signal transduction is that per turbation of CD2 molecules that are transfected into TCR - transformed fibroblasts (L cells) (74, 75) or insect cells (76) fails to alter [Ca 2+]j. Possible evidence for a TCR/CD2 physical interaction has recently been provided by the observation that, with certain detergents, antibodies to the TCR co-precipitate CD2 from cell lysates (77). Data obtained with transfected CD2 suggest that its critical interactions with the TCR occur in the cyto plasm. Rat CD2 cDNA was transfected into lurkat cells in forms that were normal (with an intracellular domain of 1 1 6 amino acids) or truncated (containing 6 or 40 intracytoplasmic amino acids) (75). The appropriate combination of anti-rat CD2 mAbs caused [Ca 2 + ] j increases in trans fectants expressing the full length CD2 molecule, but little or no increase in those expressing the truncated forms. Increases of [Ca 2+]j were comparable between the cell lines when anti-CD3 or PHA was used to stimulate. Similarly, anti-CD2 mAbs, or liposomes containing LF A-3 and HLA-DR, were able to elicit IL-2 production from an HLA-DR-specific murine T cell hybridoma that was transfected with full-length human CD2, but not from one transfected with CD2 from which the C-terminal 1 00 amino acids had been truncated (7S). Thy-l is a 25-30 kd molecule attached to the plasma membrane of murine T cells via a phosphatidylinositol linkage (79). The finding that antibody-mediated cross-linking of Thy- l is mitogenic presents a problem: How can a cell-surface molecule transduce signals without a trans membrane or intracytoplasmic portion? At least part of the answer appears to lie in a functional relationship with the TCR. Schmitt-Verhulst and colleagues generated a TCR-IX chain loss variant of a normal murine cytolytic-T-cell clone (SO). As with the IX or f3 chain-negative lurkat T cells, this variant failed to express any TCR components on the cell surface, although it did express unchanged levels of the Thy-l molecule. Stimu lation of the wild-type cell with Con A or antibodies to Thy-l caused a rapid increase in [Ca 2+]j and the secretion of lymphokine. The TCR variant manifested neither response. One objection to the conclusion that signaling via Thy- l requires co-expression of the TCR was that no TCR revertant (or transfectant) was analyzed, so it was possible that the vari ant's phenotype was due to some independent mutation. This was directly addressed by studies in two different model systems. In one, the human T leukemia cell lurkat and a TCR-f3- variant of lurkat were transfected with the murine Thy- l gene (46). External cross-linking of the Thy- l molecule on the Jurkat transfectant caused mobilization of [Ca 2 + J and, in the presence of PM A, secretion of IL-2. In contrast, the Thy- l transfected TCR - lurkat did not respond. Restoration ofTCR expression in the latter

THY·]


1 60

ASHWELL & KLAUSNER

cell by transfection of the TCR-f3 chain generated a cell that once more could respond to cross-linked anti-Thy- l plus PMA. In another study that used a strictly murine system in which no chemical co-stimulators were necessary, a T-cell TCR mutant was created that expressed normal Thy I levels in the absence of cell-surface TCR (27). Transfection of this T-cell hybridoma with the original antigen-specific rx and f3 chains restored cell surface TCR expression. Activation of these cells with an anti-Thy- l mAb that does not require external cross-linking to be mitogenic, G7, caused the wild-type cell and the transfectant to secrete IL-2; the TCR - mutant was unresponsive. The spontaneous growth of transformed 2B4. 1 1 cells is inhibited by activation. In multiple analyses, stimulation of the TCR + clones with anti-Thy- l decreased their incorporation of [3H]thymidine 7 5� 80% 24 hr after stimulation. In contrast, high concentrations of the mAb decreased [3H]thymidine incorporation by the TCR - mutant by only 1 5 % . Therefore, although activation via Thy- l in the absence of the TCR had a small effect upon cell growth, optimal inhibition appeared to require the presence of the intact TCR. Transmembrane signaling via Thy- l does not depend upon TCR expression in all cases. Thy- l cDNA has been transfected into murine B cell tumors, where it is expressed on the plasma membrane (8 1 ). Antibody mediated cross-linking causes [Ca2+]j mobilization in these transfectants just as it does in T cells, proving that the TCR is not always required to couple Thy- l to intracellular pathways (although it is possible that B cells have a structure that is analogous to CD3 and that serves a similar function in this regard). Analysis of TCR mutants has also demonstrated that transmembrane activation of some biological responses can occur. In one case, the Thy- I -mediated induction of tyrosine phosphorylation has been studied (8 1 a). The 2B4. 1 1 T-cell hybridoma and its TCR - variant mani fested grossly similar patterns of phosphorylation after incubation with the anti-Thy- l antibody G 7. In another example, anti-Thy-I -mediated DNA fragmentation has been used as a measure of activation-induced cell death (D. Ucker, J. Ashwell, G. Nickas, in preparation). These studies have shown that the TCR - variant of 2B4. 1 1 manifests the fragmentation response, although these cells do so only after at least a 1 5 hr delay, compared to the 4-6 hours required for the TCR + parental cell. '"

Ly-6 is a complex family of cell-surface proteins ( 1 4- 1 7 kd) found on murine T and B lymphocytes (82-84). As with Thy- I , these molecules are linked to the plasma membrane by a phosphatidylinositol anchor (85� 87), and many externally cross-linked anti-Ly-6 antibodies are mitogenic (88-90) . The relationship between Ly-6 and the TCR has been directly tested in two studies employing T-cell lines with absent cell-surface TCR. We have measured IL-2 production and inhibition of transformed growth

LY-6



161

i n the 2B4. 1 1 murine T-cell hybridoma, a TCR-[) chain mutant, and the Ta[) 1 .2 cell in which cell-surface TCR expression has been restored by gene transfection (32). Whereas the wild type hybridoma responded to anti-Ly-6 in both assays, the TCR - mutant did not. Importantly, the [) chain transfectant responded as well as the wild-type cell. In another study (9 1 ), two different T-cell hybridomas were mutagenized and selected for loss of cell-surface TCR expression. Whereas all cells produced IL-2 when stimulated with PMA plus a Ca 2 + ionophore, the parental cells produced IL-2 in response to anti-Ly-6 while the TCR - subclones did not. One revertant (from TCR - to TCR + ) once again secreted IL-2 in response to anti-Ly-6. Together, these results strongly implicate the TCR as a par ticipant in transmembrane signaling pathways leading to activation induced IL-2 production and growth inhibition. A recent report has raised the unexpected possibility that the relationship between Ly-6 and the TCR may be bidirectional; that is, signaling through the TCR may be affected by co-expression of Ly-6 (92). In this study, mutants of a T-cell hybridoma were isolated that either transcribed and synthesized low levels of Ly-6 (class-I mutants) or that were unable to form the membrane phosphatidylinositol linkage required for the expression of molecules such as Ly-6 or Thy- l (class-II mutants). Both types of mutant cells produced lower levels of IL-2 than the wild type when stimulated with antigen/MHC, anti-TCR mAbs, or lectin, although a quantitative correlation was clearly seen only with the class-I mutants. This is consistent with the finding that enzymatic removal 'Of phosphatidylinositol-linked membrane proteins from normal lymphocytes impairs their responses to lectin (87, 92). The significance of these observations is unknown at this time, and proof that TCR-mediated signaling is modified by Ly-6 co expression will require that transfection of the DNA encoding Ly-6 into the c1ass-1 mutants restores their response to transmembrane stimuli. These data provide good evidence that the transfer of information from the T-cell surface to responsive intracellular compartments via these "activatioQ molecules" usually requires the expression of a functional TCR; in the absence of the TCR, however, a subset of the usual signals and biological responses can be generated. The most straightforward model to account for these findings is that the TCR physically associates with these activating molecules, either before or after they are bound by ligand or antibody, or that it shares with them intermediary signaling molecules, such as GTP-binding proteins. Alternatively, the TCR may exert tonic effects upon the cell that modify transmembrane signaling. Support for such a possibility comes from recent work with TCR - T-cell mutants. A cytoskeletal protein, spectrin, was found localized near the Golgi in an unactivated T-cell hybridoma (93). After activation with anti-CD3 a rapid

1 62

ASHWELL & KLAUSNER

dispersal of the spectrin occurred. Interestingly, a TCR - mutant of this hybridoma had a spectrin pattern resembling that of the activated T cell (i.e. diffuse distribution). Analogous results have been obtained with CD45, a molecule recently shown to have tyrosine phosphatase activity (94). When the 2B4. l l T-cell hybridoma was homogenized, the majority of the tyrosine phosphatase activity was found in the postnuclear membrane preparation (Y. Minami, J. Lippincott-Schwartz, and R. D. Klausner, manuscript in preparation). However, this activity segregated with the cytoskeletal/nuclear fraction when fractionation was performed after acti vating the cells with anti-Thy- I . In a TCR 2B4. 1 1 mutant cell line the tyrosine phosphatase activity was always found in the nuclear fraction, even in the absence of activation. When these cells had TCR expression restored by eDNA transfection of the variable chain genes, the normal tyrosine phosphatase distribution of unactivated cells was restored. The enzymatic activity could be completely attributed to CD45, since anti bodies to this molecule specifically removed the tyrosine phosphatase activity. Although the mechanism underlying these phenomena is unknown, it is possible that the unengaged (by ligand) TCR generates


-

signals that influence the cellular organization of other molecules. This

would allow the TCR to act at a distance and could explain why the presence of the TCR has a pivotal role in transmembrane signal trans duction. SUMMARY

The studies reviewed here exploit the fact that the TCR is a multisubunit complex whose perturbation initiates an assortment of biochemical path ways and diverse biological responses. The creation and analysis ofT cells bearing aberrant TCRs has led to a number of important conclusions and provided a framework for some educated speculation about T-cell biology. The assembly of the TCR is a highly regulated process in which the majority of the synthesized material is rapidly degraded. Partial complexes, which potentially might interfere with ligand binding by, or the function of, complete receptor molecules, are not tolerated; this "architectural editing" is performed in a compartment(s) associated with the ER or, in some cases, lysosomes. The individual chains of the TCR can be separated into subgroups that are, pcrhaps, functionally autonomous. The disulfide linked variable chains bind ligand. The (I]. heterodimer appears to be largely responsible for coupling receptor occupancy to PI hydrolysis, the (2 heterodimer may couple to tyrosine kinase activation and/or other signaling pathways. The (rcontaining receptors are fully capable of trans ducing signals leading to IL-2 production and growth inhibition, while the


163


presence of the 'Yf heterodimer is associated with the autolytic response of T-cell hybridomas to activation. Finally, an intact and functional TCR must be present for optimal expression of some, although not all, mani festations of activation that are initiated via independent cell-surface mol ecules such as Thy- l , Ly-6, and CD2. Future experiments in which TCR chains that incorporate site-directed mutations are transfected into T and non-T cells are certain to enhance our appreciation of how the structure of this receptor determines its many biological attributes. ACKNOWLEDGMENTS We are grateful to Drs. Ronald Germain and Ethan Shevach for critical review of this manuscript , and to Drs. T. Saito , R. Germain, and E. Reinherz for sharing unpublished data. We would also like to acknowledge the members of our laboratories who have contributed to our current view of the complex biology of the T-cell antigen receptor.

Literature Cited I . Schwartz, R. H. 1 985. T-1ymphocyte rec ognition of antigen in association with gene products of the major histocom patibility complex. Annu. Rev. Immunol. 3: 237-6 1 2. Kronenberg, M . , Siu, G., Good, L. E., Shastri, N . 1 986. The molecular genetics of the T-cell antigen receptor and T-cell antigen recognition. Annu. Rev. Immu nol. 4: 529-9 1 3. Van Wauwe, J. P., De May, J. R., Goosens, J. G. 1 980. OKT3: a mono clonal anti-human T lymphocyte anti body with potent mitogenic properties. J. lmmunol. 1 24: 2708- 1 3 4. Borst, J . , Alexander, S. Elder, J . , Terhorst, C . 1 9 8 3 . The T 3 complex o n human T lymphocytes involves four structurally distinct glycoproteins. 1. Bioi. Chern. 258: 5 1 35-4 1 5 . Borst, J., Prendiville, M . A., Terhorst, C. 1983. Thc T3 complcx on human thy mus-derived lymphocytes contains two different subunits of 20 kDa Eur. J. Immunol. 1 3: 576-80 6. Samelson, L. E., Harford, J. B., Klausner, R. D . 1 98 5 . Identification of the components of the murine T cell anti gen receptor complex. Cell 43: 223-3 1 7. Baniyash, M . , Garcia-Morales, P., Bon ifacino, J . S., Samelson, L. E., Klausner, R. D. 1 988. Disulfide linkage of the zeta and eta chains of the T cell receptor.

8.

9.

10.

II.

12.

1 3.

1 4.

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