Immunology Letters, 29 (1991) 51-54

Elsevier IMLET 01594

Mechanisms of T lymphocyte activation J o h n E. Kay School of Biological Sciences, University of Sussex, Brighton, U.K.

(Accepted for publication 31 January 1991)

1. Summary The proliferation of T lymphocytes in response to an antigenic stimulus requires the successive ligation of a range of T lymphocyte receptors by MHCpresented antigen, molecules expressed on the surfaces of accessory cells and lymphokines. A range of intracellular signalling systems are involved, with the possibility of alternative activation pathways utilising different intracellular signalling systems, though protein kinase C activation appears to play a key role. Appreciation of the complexity of the response may allow more selective clinical modulation of T lymphocyte-dependent immunological responses.

assessment. However, such systems have contributed little to our understanding of the mechanisms mediating T cell activation, due to the difficulty of studying changes occurring only in the very low proportion of responding cells in the initial cell population. More recently T lymphocyte clones responsive to specific antigen have become available and have been used to confirm that some of the mechanisms identified as mediating non-specific T cell activation also occur during the response to specific antigen. However, it is difficult to be sure how closely the behaviour of such cloned cells in the absence of antigen or lymphokines resembles that of resting lymphocytes in vivo.

2. Specific T lymphocyte activation systems

3. Non-specific T lymphocyte activation systems

The ability of some T lymphocytes present in peripheral blood and lymphoid organs to respond to the presence of allogeneic cells by proliferation and the differentiation of some of the progeny into specific cytotoxic effector cells has been established for over 25 years [1]. In the presence of appropriate antigen presenting cells, T lymphocytes will also proliferate in response to soluble antigen [2]. Such experimental systems have been used to evaluate potential donor-recipient compatibility in transplantation and to assess the effectiveness of potential immunosuppressive drugs. The commonly used [3H]thymidine assay allows extremely sensitive detection of T cell responses, although both theoretical and practical problems limit their quantitative

The study of T cell activation has been facilitated by the development of artificial systems in which T cells of many different antigenic specificities can be activated by mechanisms that appear to resemble those normally induced by specific antigen. The first non-antigen-specific mitogens identified were plant lectins such as phytohaemagglutinin (PHA) and concanavalin A (Con A), which bind strongly to many cell membrane glycoproteins, including the T cell receptor-CD3 (TCR-CD3) complex [3], and induce an activation response in most if not all T lymphocytes which in many cells progresses to proliferation. Following the characterisation of the T cell antigen receptor, monoclonal antibodies specific for either CD3 or the invariant or idiotype-specific regions of the TCR have been used to induce T lymphocyte proliferation for experimental study (though these monoclonals are considerably less potent than lectins such as P H A and Con A). T cell clones that retain TCR-CD3-mediated responses,

Key words: T lymphocyte;Activation Correspondence to: John E. Kay,Schoolof BiologicalSciences, University of Sussex, Brighton, BN1 9QG, U.K.

0165 2478 / 91 / $ 3.50 © 1991 ElsevierSciencePublishers B.V.

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such as the Jurkat cell line, have also been widely used to study the mechanism of these responses, with the advantage that a single cell population can be studied uncomplicated by interactions between the different cell subpopulations present in vivo balancing the disadvantage of studying cells that are never truly resting, referred to above. Strong activation of T lymphocytes or cloned T cells, comparable to that obtained with the most effective lectins, can also be induced by the combination of a protein kinase C activator such as a phorbol ester with a Ca 2+ ionophore [4, 5]. B lymphocytes may also be activated by the same reagents. These agents bypass the requirement for the TCR-CD3 receptor, and are presumed to act, at least in part, by mimicking the intracellular signal generated in response to TCR-CD3 ligation. There have also been many reports of alternative mechanisms by which T lymphocytes can be activated. In particular monoclonal antibodies to a considerable number of different membrane glycoproteins have been found to promote T lymphocyte proliferation. These can in general be divided into two broad categories. Some responses, while induced by monoclonals that recognise membrane receptors distinct from TCR-CD3, are only effective if this complex is present in a functional form and appear to generate identical intracellular signals and responses. Examples include CD2 [6] and the group of receptors anchored to the membrane by a cleavable glycosyl-phosphatidylinositol link [7]. The second category of receptors may assist T lymphocyte proliferation by the delivery of quite different intracellular signals. However, monoclonals to the membrane receptors in this second category do not activate resting T lymphocytes on their own, but are effective only if protein kinase C has already been activated by some other mechanism. In some cases at least these receptors are not expressed in a functional form by unstimulated lymphocytes. Both categories may be indicative of accessory mechanisms important in vivo for the induction of T lymphocyte proliferation. Non-specific T lymphocyte activation systems such as these have been most valuable in establishing the basic features of the process necessary for the induction of proliferation in resting T lymphocytes. Beginning within a few minutes of the cells receiving a strong mitogenic signal, there are complex changes 52

in membrane transport and in the activities of some enzymes. Existing membrane receptors (including TCR-CD3) may be down-regulated, new receptors are expressed and others undergo more subtle changes in their specificity. An equally complex pattern of changes in the expression of specific genes begins during the first few hours, with new genes expressed including genes commonly associated with the induction of cell proliferation (e.g., c-myc) as well as those of specific importance for T lymphocytes (e.g., IL-2 and its receptor). Many of the early changes can be induced, at least in experimental systems, by protein kinase C activation alone, though the synthesis of IL-2 and other key lymphokines requires a second signal, usually provided by the elevation of the cytoplasmic Ca 2÷ concentration. After about 24 h enzymes necessary for DNA synthesis (e.g., DNA polymerase) are synthesised and DNA replication begins.

4. Accessory signals for T lymphocyte activation The commonly accepted model for T lymphocyte activation is that when the TCR component of the TCR-CD3 complex identifies a complementary antigen-MHC structure on the membrane of an antigen-presenting cell, the CD3 component of the TCR-CD3 complex, probably acting via a mediating G protein or tyrosine kinase, activates a phosphatidyl inositol-specific phospholipase C to hydrolyse a minor phospholipid present in the lymphocyte membrane, phosphatidylinositol bisphosphate (PIP2). This generates two intracellular mediators, diacyl glycerol (which activates protein kinase C) and inositol trisphosphate (which causes a transient release into the cytoplasm of Ca 2÷ from the intracellular stores) and these two signals in concert then initiate the chain of events leading to proliferation [5, 8]. Key events are usually identified as the induction of high-affinity receptors for the lymphokine IL-2 and, in the T helper cell subpopulation, the synthesis of IL-2 and other lymphokines. Progression to proliferation usually requires a signal from IL-2 mediated via the newly induced receptors, though in some experimental systems this may be replaced by other lymphokinereceptor interactions (e.g., IL-4). Superficially, there appears to be strong support for this model. PIP2 hydrolysis, diacylglycerol

generation, protein kinase C activation and elevation of the cytoplasmic Ca 2+ concentration have been repeatedly demonstrated to follow ligation of the TCR-CD3 complex, while as noted above activation can be induced more effectively by phorbol ester plus Ca 2+ ionophore than by anti-CD3 [5, 8]. However, the model is far less satisfactory than it may at first appear. The TCR-CD3 receptor is rapidly down-regulated after ligation, and the rate of PIP2 hydrolysis and the cytoplasmic Ca 2+ concentration both fall to close to control values within a few minutes. However, in vitro studies show that for T cell activation to progress to proliferation a Ca 2+ signal must be maintained (though now from extracellular Ca 2+) for some hours [9] while protein kinase C activation appears to be needed throughout the GI phase of the cell cycle at least [10]. The imbalance between the two is itself of interest - PIP2 hydrolysis would provide the intracellular mediators for both signals, but in theory at least diacylglycerol could also arise from the hydrolysis of other phospholipids. Accelerated hydrolysis of phosphatidylcholine during T lymphocyte activation has been reported [11]. While there is no reason to doubt that in vivo MHC-TCR interaction normally supplies the initial trigger for T cell proliferation, it seems clear that accessory signals must be provided from the very early stages of the response to maintain its progression. The synthesis of IL-2 and its receptor, again very probably of real importance in T cell responses, nevertheless occurs only after several hours, much too late to provide more than a partial explanation. There is, however, no shortage of other T lymphocyte receptors through which the response may be maintained [12]. These include CD4 and CD8 (which also interact with the class II and class I M H C on the antigen-presenting cell); CD2 (which interacts with LFA-3); LFA-1 (which interacts with ICAM-1 or -2); many other receptors through which a co-mitogenic signal may be delivered, such as CD5, CD28, CD45 and CD69; receptors for lymphokines which may facilitate T cell proliferation such as IL-1, IL-2, IL-4 and IL-6; and the transferrin receptor. Some of these receptors (e.g., CD8, CD2 and LFA-1) are present on unstimulated T lymphocytes but bind their physiological iigands only after subtle changes induced by protein kinase C activation. One of the earliest effects of T lymphocyte activa-

tion noted during in vitro studies was the increased adhesiveness of activated T cells, to each other and to accessory cells present in the cultures, which becomes established within minutes of the initial triggering via TCR-CD3 (or protein kinase C activation by phorbol esters) [12]. Inhibition of specific interactions known to play a role in this process has been shown to reduce activation, but analysis is complicated by the number of different interactions that appear to be involved and the likelihood that these may vary with the particular T lymphocyte subpopulations, antigen presenting cells and other accessory cells present. Other accessory signals, such as those mediated by lymphokines, may only play their part much later in the response, as the lymphokines and/or their receptors must first be synthesised de novo. Some receptors only apparent after activation may be fully expressed within an hour or two (e.g., CD69), while others (e.g. the transferrin receptor) are not expressed until the end of G1.

5. Signal transmission mediating accessory activation systems In many cases the nature of the intracellular signal(s) contributing to T cell activation consequent on the ligation of these accessory receptors has not been established. However, the few cases in which reliable information is available make it clear that a variety of signals may be involved. For example, it appears that ligation of CD2 results in the hydrolysis of PIP2 and the generation of the same intracellular signals as with TCR-CD3 [13]; CD4 and CD8 are coupled to the cytoplasmic tyrosine kinase p56-1ck [14]; CD69 appears not to release Ca 2+ from the intracellular stores, but a Ca 2+-mediated mechanism, probably dependent on extraceUular Ca 2+, is involved [15]; while CD28 strongly induces IL-2 expression, but by a quite different mechanism that is Ca 2+-independent and thus resistant to immunosuppressive drugs such as cyclosporin A and FK-506 [16, 17]. Thus it seems likely that a wide variety of intracellular signals may be generated, and that specific components of the T lymphocyte response, such as IL-2 induction, may occur in response to alternative signals. Understanding the complexity of T cell activation may be of importance for its successful clinical modulation for transplantation and other purposes. 53

T h u s t h e n a t u r a l l i g a n d for C D 2 8 has r e c e n t l y b e e n i d e n t i f i e d as the B l y m p h o c y t e a c t i v a t i o n a n t i g e n B7 [18], s u g g e s t i n g a n e x p l a n a t i o n for t h e o b s e r v a t i o n t h a t i n i n t a c t a n i m a l s c y c l o s p o r i n a n d F K - 5 0 6 have relatively little effect o n s e c o n d a r y r e s p o n s e s to B cell p r e s e n t e d a n t i g e n , even t h o u g h these are d e p e n d e n t o n T h e l p e r cells w h o s e a c t i o n is n o r m a l l y c o n s i d e r e d sensitive to these drugs. T h e a c c e s s o r y T cell receptors a n d i n t r a c e l l u l a r s i g n a l l i n g systems inv o l v e d m a y well reflect the n a t u r e o f t h e a n t i g e n , the cells i n v o l v e d in its p r e s e n t a t i o n a n d the s u b p o p u l a t i o n s a n d p a s t h i s t o r y o f t h e r e s p o n d i n g T cells. A p p r e c i a t i o n o f these factors m a y e n a b l e t h e r a t i o n a l d e s i g n o f i m m u n o s u p p r e s s i v e regimes t h a t will be m u c h m o r e selective for the specific aspects o f the i m m u n e r e s p o n s e w h o s e m o d u l a t i o n is r e q u i r e d t h a n t h o s e p r e s e n t l y available.

Acknowledgements I t h a n k the S.E.R.C. a n d the Royal Society for financial support.

References [11 Bain, B., Vas, M. R. and Lowenstein, L. (1964) Blood 23, 108.

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[2] Pearmain, G., Lycette, R. R. and Fitzgerald, P. H. (1963) Lancet i, 637. [3] Chilson, O. P. and Kelly-Chilson, A. E. (1989) Eur. J. lmmunol. 19, 389. [4] Truneh, A., Albert, F., Golstein, P. and Schmitt-Verhulst, AM. (1985) Nature 313, 318. [5] Isakov, N., Mally, M. I., Scholz, W. and Altman, A. (1987) lmmunol. Rev. 95, 89. [6] Alcover, A., Alberini, C., Acuto, O., Clayton, L. K., Transy, C., Spagnoli, G. C., Moingeon, P., Lopez, P. and Reinherz, E. L. (1988) EMBO J. 7, 1973. [7] Bamezai, A., Reiser, H. and Rock, K. L. (1988) J. lmmunol. 141, 1423. [8] King, S. (1988) Immunology 65, 1. [9] Gelfand, E. W., Cheung, R. K., Mills, G. B. and Grinstein, S. (1988) Eur. J. Immunol. 18, 917. [101 Davis, L. S. and Lipsky, R E. (1989) Cell. lmmunol. 118, 208. Ill] Rosoff, P. M., Savage, N. and Dinarello, C. A. (1988) Ceil 54, 73. [121 Springer, T. A. (1990) Nature 346, 425. [13] Pantaleo, G., Olive, D., Poggi, A., Kozumbo, W. J., Moretta, L. and Moretta, A. (1987) Eur. J. Immunol. 17, 55. [141 Bolen, J. B. and Veillette, A. (1989) Trends Biochem. Sci. 14, 404. [15] Testi, A., Phillips, J. H. and Lanier, L. L. (1989) J. lmmunol. 143, 1123. [16] June, C. H., Ledbetter, J. A., Gillespie, M. M., Lindsten, T. and Thompson, C. T. (1987) Mol. Cell. Biol. 7, 4472. [17] Kay, J. E. and Benzie, C. R. (1989) Immunol. Lett. 23, 155. [18] Linsley, P. S., Clark, E. A. and Ledbetter, J. A. (1990) Proc. Natl. Acad. Sci. USA 87, 5031.

Mechanisms of T lymphocyte activation.

The proliferation of T lymphocytes in response to an antigenic stimulus requires the successive ligation of a range of T lymphocyte receptors by MHC-p...
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