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Generation of effector cytotoxic T cells from cytotoxic T cell precursors: Role of soluble factors
$24
Journal of the Neurological Sciences, 115 (Suppl.) (1993) $24---$28 © 1993 Elsevier Science Publishers B.V. All rights reservecd 0022-510X/93/$06.00
JNS 04012
Generation of effector cytotoxic T cells from cytotoxic T cell precursors: role of soluble factors G. P a l l a d i n o The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104-4268, USA
Key words: Cytotoxic T lymphocytes; Lymphokines;Proliferation; Differentiation;Antigen presenting cells
Summary Cytotoxic T lymphocytes (CTL) have long been recognized as playing a major role in the immune response to alloantigens and viral antigens as well as tumor antigens. The progress of the last decade in the identification and characterizationof soluble factors involved in the regulation of the immune response has greatly improved our knowledge of the mechanisms of CTL activation and regulation, This review wilt summarize the data available in the literature regarding different lymphokines and their specific activity on CTL. In addition it will point out a few of the elornents of the systems that hamper its full understanding and it will suggest possible directions for future research.
Introduction Cytotoxic activity, the ability of cells to recognize other cells as target and kill them by direct lysis is a feature common to different cell types: natural killer cells, lymphokine activated killer cells and cytotoxic T lymphocytes (CTL). While the mechanisms oflysis appear to be common to all of them, the mechanisms of target recognition and activation are very different and CTL are the ones best characterized in this regard. Not all T lymphocytes express lytic activity: cytotoxic activity can be generated from CD4+ peripheral lymphocytes as well as thymocytes, but the majority of CTL are found in the CD8+ compartment of mature T lymphocytes and it is on this specific population, that is phenotypically characterized by the expression of T cell receptor (TcR) and CD8 antigen and the lack of Fc receptor, that the attention of this review will be focused. These cells are effectors of cytotoxic activity against alloantigens and viral antigens and are also involved in antineoplastic responses as well as various immonopathogenetic mechanisms. Cytotoxic activity is not always expressed by CTL: on the contrary, it is highly regulated. Resting CTL (precursor CTL = pCTL), either naive or memory, are unable to lyse specific target cells unless they undergo a complex process of activation and differentiation that are under the control of many different soluble factors. After antigen stimulation Correspondence to: Dr. G. Palladino, The Wistar Institute of Anatomy and Biology, 360I Spruce Street, Philadelphia, PA 19104-4268, USA.
the cytotoxic activity of CTL increase progressively, at day 5-7 and rapidly declines to reach background level usually by day 10. In the period between contact with the antigen and the peak of eytotoxic activity three different phases can be recognized that are usually indicated as (i) activation, (ii) proliferation, and (iii) differentiation (Bach et al. 1989).
Activation The activation phase is characterized by the binding of the T cell receptor ofpCTL to the antigen in the context of class I MHC molecule on the surface of accessory cells (antigen presenting cells or APC). The activation through TcR/Ag-MHC interaction, as well as through other mechanisms that mimic this interaction, i.e. mitogen or anti-TcR antibodies, induces modifications in the pCTL that make them sensitive to factors stimulating proliferation and/or differentiation. The increase sensitivity to these factors appears to be mediated, in some cases, by the induction or upregulation of the expression of specific receptors. Upregulation of interleukin-2 receptors (IL-2R) has been reported on CTL clones after antigen stimulation (Andrew et al. 1985) and upregnlation of intedeukin-4 receptors (IL4R) has been shown in murine splenic T cells after stimulation with ConA (Ohara and Paul 1987), Conversely, mitogenic stimuli do not appear to have any effect on the expression of receptors for other lympholdnes, i.e. interleukin(IL)-5 and IL-6 (Taga et al. 1987; Mita et al. 1988).
$25 No lymphokines are directly involved in the activation phase, but there are indications that the strength of the activation signal may influence the factor requirements of CTL during the following proliferation and differentiation. De Jong et al. (1990) reported that CTL activity can be generated in human PBL in the absence of exogenous lymphokines after stimulation with optimal concentration of antibodies anti-TcR, while the generation of CTL activity after stimulation with suboptimal concentration of the same antibodies requires the addition of exogenous factors. Proliferation
Many different lymphokines can regulate the proliferation of activated CD8+ T cells, some of them do it through a direct effect, others can modulate the expression of IL-2R and significantly affect CTL proliferation in the presence of IL-2. IL-2 can directly stimulate proliferation of activated pCTL from murine peripheral lymphocytes (Raulet and Bevan 1982; Wagner et al. 1982) as well as IL-6 (Uyttenhove et al. 1988) and IL-4 (Pfeifer et al. 1987; Widmer and Grabstein 1987), even if the ability of the latter to do it through an IL-2 independent mechanism is still controversial (Miller et al. 1990; Selvan et al. 1990). IL-7 and IL-12 also induce proliferation of activated CDS+ human PBL, and the mechanism of stimulation has been shown to be 1L-2-independent for the latter (Gately et al. 1991) and partially so for the former (Annitage et al. 1990; Grabstein et al. 1990). IL-7 can also upregulate the expression of IL-2R on activated CD8+ human PBL as well as murine splenocytes (Armitage et al. 1990; Grabstein et al. 1990), while TNF-ct synergize with IL-2 to increase the expression of IL-2R on activated human PBL (Robinet et al. 1990). The data relative to the effect of IL-4 on the expression of 1L-2R are controversial: Martinez and al. (1990) reported downregulation of IL-2R by IL-4 in CD8+ activated human PBL while Bertagnolli et al. (1991) described upregulation of the same receptor by IL-4 in activated murine thymocytes. An inhibitory effect of TGFq3 on the generation of alloreactive murine CTL supported by TNFmt has been also described (Ranges et al. 1987). The inhibition is at least partially due to the ability of TGF-13 to downregulate the expression of 1L-2R on peripheral T lymphocytes (Kehrl et al. 1986), in so doing counteracting the effect of T N F ~ . Differentiation
To acquire significant cytotoxic activity, CD8+ T cells need to undergo a process of differentiation which is characterized by the accumulation in the cytoplasm of granules containing an array of different molecules that are the me-
diators of cytolytic activity (Tschopp and Nabholz 1990). Among them the best characterized are perforin, a protein with complement-like lytic activity, and a group of closely related proteolytic enzymes called granzymes and sometime referred to also as serine esterases or BLT esterases. The increase of intracellular concentration of these enzymes or their corresponding messenger RNAs has been used in recent years as markers for CTL differentiation. The differentiation of CD8+ to cytolytic cells, measured by the induction of these markers, can be modulated by different factors. IL-2, in relatively high concentration, can induce mRNA synthesis of perforin and BLT esterase in resting CD8+ human PBL (Liu et al. 1989; Smyth et al. 1990). IL-4 has been shown to increase intracellular concentration of BLT esterase in activated CD8+ human PBL and murine peripheral T cells (Horohov et al. 1988; Trerm et al. 1988). IL-6 alone does not appear to have a significant differentiative effect but it can synergize with low concentration of 1L-2 to increase the intracellular concentration of perforin mRNA in resting human PBL (Smyth et al. 1990). Finally, high concentration of IL-7 induces increase in intracellular perforin mRNA in CD8+ resting human PBL and can also synergize with low concentration of IL-2 to generate a larger and faster accumulation of this messenger (Smyth et al. 1991). Factors with undefined function
Other factors have been involved in the generation of CTL but their specific mechanism of activity is still undetermined: ¥-IFN has been shown to be required in addition to IL-2 during the early phase of CTL generation from activated CD8+ murine T cells (Maraskovsky et al. 1989; Rogers et al. 1991) and it appears to modulate IL-2-dependent CTL proliferation (Simon et al. 1986). IL-5 has also been involved in CTL generation: it has been shown to increase cytotoxic activity of human CD8+ PBL and mouse peripheral T cells, but the evidence suggesting that it has an activity on differentiation and not proliferation of CTL is only circumstantial (Ramos 1989; Nagasawa et al. 1991). IL-1 has been suggested earlier to play a role in the generation of alloreactive CTL (Farrar et al. 1980), however, later evidence indicates that it is required to support IL-2 secretion and not cytotoxic activity of CD8+ cells (Renauld et al. 1989). Discussion
Despite this large amount of available data a clear and comprehensive understanding of the activity of lymphokines on CTL generation is still elusive. The numerous attempts to analyze the involvement and the role of different factors during in vitro or in vivo generation of CTL activity and the experiments directed to identify the minimal factor
$26 requirements for the same activities have generated different and very otten contradictory results. Three elements of the system, most of all, concur to hamper its explanation: (i) the variability in the source of antigen stimulation, (ii) the variability in the source of factors, and (iii) the variability within the CD8+ population of responder cells. The vast majority of cells express class I MHC antigens and can thus present antigen to CD8+ lymphocytes, but not all class I+ cells can stimulate CTL activity equally well (Sprent and Schaefer 1990). Among others, dendritic cells appear to be particularly efficient and, as opposed to other cell types, they can induce primary in vitro CTL activity against allo-antigens or viral antigens in the absence of exogenous help (Young and Steinman 1990; Mercadal et al. 1991). In addition the state of maturation and/or activation of other APC, i.e. macrophages and T lymphocytes can affect their ability to stimulate CD8+ cells and may change CTL requirement for exogenous factors (Sprent and Schaefer 1990). CD4+ lymphocytes able to produce IL-2, IL-4, IL-5, IL-6, IL-7, y-IFN, TNF-~ and TGF-13 are the best known and characterized source of helper factors relevant to CTL activation, but different accessory cells are also able to produce many of the same factors. Macrophages secrete IL-6, TNF-ct and TGF-I3 (Van Snick 1990) while B cells can produce IL-2, IL-6 and IL-12 (Taira et al. 1987; Stem et al. 1990; Van Snick 1990). Even though experimental systems can be envisaged, and have been actually tested (De Jong et al. 1990; Rogers et al. 1991), that are free of both CD4+ and accessory ceils, an uncomroiied source of t'actors would still be left in the form of the CD8+ cells themselves. These cells, or a population thereof, have been shown to produce a variety of factors relevant for their own stimulation: alloreactive murine CD8+ clones have been described that produce IL-2, y-IFN and TNF-et (Klein et al. 1982; Kelso and Glasebrook 1984; Fong and Mosmann 1990; Gajewski and Fitch 1990) and more recently mRNA encoding for IL-4 and IL-5 has been demonstrated in CD8+ murine splenocytes after activation by antigen or mitogen (Cardell et al. 1991; Horvat et al. 1991). Human CD8+ clones have also been generated producing in various combinations IL2, IL-4, IL-5, IL-6, y-IFN and TNF-et (Salgame et at. 1991). The finding of both cytotoxic activity and factor secretion within the CD8+ compartment suggested the possibility of the existence of two functionally distinct subpopulations within this cell type. This hypothesis has been supported by the results of studies on alloreactive clones indicating that the majority of IL-2 secreting clones were not cytolytic and vice versa the majority of cytolytic clones did not produce IL-2 (Kelso and Glasebrook 1984; Heeg et al. 1987). These two subpopulations have later been found to have also different factor requirements: IL-2 secreting CD8+ cells but not cytolytic ones express IL-1 receptors and require this
lymphokine for their activation (Mizuochi et al. 1988; Klamet et al. 1989). More differences have been shown within CD8+ cells with regard to their previous contact with antigen. Human as well as murine peripheral CD8+ T lymphocytes include naive and memory cells that can be distinguished by the expression of different isoforms of the surface antigen CD45R (Merkenschlager and Beverly 1989; Marvel et al. 1991). The two groups may have different factor requirements as it is suggested by experiment showing that CD8+ cells require CD4+ help to generate primary CTL response to tumor or viral antigens but are able to generate a CD4-independant secondary response against the same antigens (Horvat et al. 1991; Jennings et al. 1991). While the possibility to distinguish phenotypically between naive and memory cells will allow further characterization of their differences the inability to date to distinguish phe: notypically cytolytic from factor-secreting cells strongly hampers the possibility of any further purification of pCTL.
Conclusions
While the vast majority of the data that have been collected so far have been helpful to the identification of the various elements involved in CTL activation, much work remains to be done to understand the way all these different elements work together during the normal immune response, and this direction appears to be the most promising for future studies. The use of new approaches and new technologies will be helpful to allow further development in the field. One example or'these new tools are the transgenic mice deficient in various factors that have been recently generated (Kuhn et al. 1991; Schorle et al. 1991). They may offer useful information regarding the need for a given lymphokine during in vivo response as well as help to clarify the mechanisms by which the immune system compensates for the loss of one single factor. Another possible
Fig. I. Lymphokinesinvolvedin CTL generationand their sources.
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approach may involve the use of molecular probes for perforin and granzimes that have been already used to detect cytotoxic activity in vivo (Griffiths and Mueller 1991) and that, combined with probes specific for lymphokines, may allow the study of pathological processes during their normal in vivo development (Young et al. 1989). References Andrew, M.E., A.M. Churilla, T.R. Malek, V.L. Braciale and T.J. Braciale (1985) Activation of virus specific CTL clones: antigen-dependent regulation of interleukin 2 receptor expression. J. Immunol., 134: 9 2 ~ 925. Amfitage, R.J., A.E. Namen, H.M. Sasseufeld and K.H. Grabstein (1990) Regulation of human T cell proliferation by IL-7. J. Immunol., 144: 938 941. Bach, F.H., R.L. Geller, P.J. Nelson, et al. (1989) A "'Minimal Signal-Stepwise Activation" analysis of ftmctional maturation of T lymphocytes. Immunol. Rev.. 111 : 35 57. Bertaguolli, M.M., Y. Takai and S.H. Herrmam~ (1991) IL-4-supported induction of cytolytic lymphocytes-T requires 1L-2 and 1L-6. Cell. lmmunol., 133:327 341. Cardell, S., B. Sander and G. Moiler (1991) Helper interleukhls are produced by both CD4 and CD8 splenic T-cells after mitogen stimulation. Enr. J. lmmtmol., 21:2495 2500. De Jong, R., M. Brouwer, V.I. Rebel, G.A. Van Seventer, F. Miedema and R.A.W. Van Lier (1990) Generation of alloreactive cytolytic T lymphocytes by immobilized anti-CD3 mouoclonal antibodies. Analysis of requirements for human cytotoxic T-lymphocyte differentiation, hnmunology, 70:357 364. Farrar, W.L., S.B. Mizel and J.J. Farrar (1980) Participation of lymphocyte activating factor (interleukin 1) in the induction of cytotoxic T cell responses. J. lmmunol., 124:1371 1377. Fong, Y.A.'I'. and T.R. Mosmann (1990) Alloreactive murine CD8+ T cell clones secrete the Thl pattern of cytokines. J. Immunol., 144: 17441752. Gajewski, T.H. and F.W. Fitch (1990) Anti-proliferative effect of ifi~-y in immune regulation. IV. Murine CTL clones produce IL-3 and GM-CSF, the activity of which is masked by the inhibitory action of secreted IFN-'y. J. Immunol., 144: 548- 556. Gately, M.K., B.B. Desai, A.G. Wolitzky et al. (1991) Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotox ic lymphocyte maturation factor). J. lnmaunoL, 147: 874-882. Grabstein, K.H., A.E. Namen, K. Shanebeck, R.F. Voice, S.G. Reed and M.B. Widmer (1990) Regulation of T cell proliferation by IL-7. J. hnmunol., 144:3015 3020. Griffiths, G.M. and C. Mueller (1991) Expression of perforin and granzymes invivo potential diagnostic markers for activated cytotoxic cells. Immuuol. Today, 12: 415-419. Heeg, K., C. Steeg, C. Hardt and H. Wagner (1987) Identification of interleukin 2 prodncmg T helper cells within murine Lyt-2+ T lymphocytes: frequency, specificity and clonal segregation from Lyt-2+ precursor ofcytotoxic T lymphocytes. Eur. J. Inununol., 17:229 2336. Horohov, D.W, J.A. Crim, EL. Smith and J.P. Siegel (1988) IL-4 (B cell-stimulatory factor 1) regulates multiple aspects of influenza viresspecific cell-mediated immunity. J. lrmnunol., 141:4217 23. Horvat, B., J.A. Loukides, L. Anandan, E. Brewer and P.M. Flood (1991) Production of interleukin-2 and iuterleukio-4 by immune CD4-CD8+ and their role in the generation of antigen-specific cytotoxic Y-cells. Eur. J. hnmunol., 21:1863 1871. Jennings, S.R., R.H. Bonneau, P.M. Smith, R.M. Wolcott and R. Chervenak (1991) CD4-positive lymphocytes-T are required for the generation of the primary but not the secondary CDS-positive cytolytic lymphocyteT response to herpes simplex virus in C57BL/6 mice. Cell. Immunol., 133:234 252.
Kehrl, J.H., L.M. Wakefield, A.B. Roberts et al. (1986) Production of transforming growth factor ]3 by human T lymphocytes aqd its potential role in the regulation o f T cell growth. J. Exper. Med., 163:1037 1050. Kelso, A. and A.L. Gtasebrook (1984) Secretion of interleukin 2, macrophage-activating factor, interferon, and colony-stimulating factor by alloreactive T lymphocyte clones. J. linmtmol., 132:2924-2931. Klarnet, J.E, D.E. Kern, S.K. Dower, L.A. Matis, M.A. Cheever and ED. Greenberg (1989) Helper-independent CDS+ cytotoxic T lymphocytes express IL-I receptors and require IL-1 for secretion oflL-2. J. Immunol., 142: 2187-2191. Klein, J.R., D.H. Raulet, M.S. Pasternack and M.J. Bevan (1982) Cytotoxic T lymphocytes produce immune interferon in response to antigen or mytogen. J. Exp Med., 155:1198 1203. Kuhn, R., K. Rajewsky and W. Muller (1991) Generation and analysis of interleukin-4 deficient mice. Science, 254:707 710. Liu, C., Rafii, A. Granelli-Pipemo, J.A. Yrapani and J.D. Young (1989) Perforin and serine esterase gene expression in stimulated human T cells. J. Exp. Med., 170: 2105-2118. Maraskovsky, E., W.F. Chen and K. Shortman (1989) 1L-2 and IFN-gamma are ~vo necessary lymphokines in the development ofcytolytic T cells. J. lmmunoh, 143:1210 1214. Martinez, O.M., R.S. Gibbons, M.R. Garovoy and F.R. Aronson (1990) IL-4 inhibits 1L-2 receptor expression and IL-2-dependent proliferation of human T ceils. J. lmmunol., 144:2211 -2215. Marvel, J., E. Lightstone, N.L. Samberg, D. Ettinghausen and H.J. Stauss (1991) The CD45RA molecule is expressed in naive murine CTL precursors but absent in memory and effector CTL. Int. hnmunol., 3: 21 28. Mercadal, C.M., S. Martin and B.T. Rouse (1991) Apparent requirement for CD4+T cells in primary auti-herpes simplex virus cytotoxic T-lymphocyte induction can be overcome by optimal antigen presentation. Viral lmmunol., 4: 177-186. Merkenschlager, M. and P.C. Beverly (1989) Evidence for differential expression of CD45 isofovm by precursors for memory-dependent and independent cyototoxic responses: human CD8 memory CTLp selectively express CD45R0 (UCHL1). hat. Immunol., 1: 450M59. Miller, C.L., J.W. Hooton, S. Gillis and V. Paetkau (1990) IL-4 potentiates the lL-2-dependent proliferation of mouse cytotoxic T cells. J. Immunol., 144: 1331-7. Mita, S., N. Harada, S. Naomi, et al. (1988) Receptors fi)r T cell-replacing factor/interleukin 5. Specificity, quantitation, and its implication J. Exp. Med., 168:863 78. Mizuochi, T., D.J. McKean and A. Singer (1988) IL-1 as a co-factor for lymphokine-secreting CD8+ routine T cells. J. lmmunol., 141:1571 1575. Nagasawa, M., A. Ohshiba and J. Yata (1991) Effect of recombinant interleukin-5 on the generation of cytotoxic T-cells (CTL). Cell. Immuuol., 133:317 326. Ohara, J. and W.E. Paul (1987) Receptor for B-cell stimulatory factor-I expressed on cells of haematopoietic lineage. Nature, 325: 537-540. Pfeifer, J.D., D.T. McKenzie, S.L. Swain and R.W. Duttou (1987) B cell stimulatory factor 1 (interleukin 4) is sufficient for the proliferation and differentiation of lectin-stimulated cytolytic T lymphocyte precursors. J. Exp. Med., 166:1464 70. Ramos, T. (1989) Interleukin 5 is a differentiation factor for cytotoxic T lymphocytes, hnmunol. Lett., 21:277 -83. Ranges, G.E., I.S. Figari, "12 Espevik and M.A. Palladino (1987) Inhibition of cytotoxic T cell development by transforming growth factor [3 and reversal by recombinant tumor necrosis factor or. J. Exp. Med., 166: 991- 998. Raulet, D.H. and M.J. Bevan (1982) A differentiation factor required for the expression of cytotoxic T-cell function. Nature, 296: 754-756. Renauld, J.C., A. Vink and J. Vau Snick (1989) Accessory signals in murine cytolytic T cell responses. Dual requirement for [L-1 and IL-6. J. lmmunol., 143:1894 1898. Robinet, E., D. Branellec, A.M. Termijtelen, J.Y. Blay, f=. Gay and S. Chouaib (1990) Evidence for tttmor necrosis factor-~t involvement in
$28 the optimal induction of class I allospecific cytotoxic T cells. J. Immunol., 144: 45554561. Rogers, L.A., A. Zlotnik, F. Lee and K. Shortman (1991) Lymphokine requirements for the development of specific cytotoxic T-cells from single precursors. Eur. J. Immunol., 21: 1069-1072. Salgame, P., J.S. Abrams, C. Clayberger et al. (t991) Differing Lymphokine profiles of functional subsets of human CD4 and CD8 T cell clones. Science, 254: 279-282. Schorle, H., T. Holtschke, T. Hiinig, A. Schimpl and I. Horak (1991) Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature, 352: 621~524. Selvan, R.S., P.S. Nagarkatti and M. Nagarkatti (1990) Role of IL-2, IL-4 and IL-6 in the growth and differentiation of tumor-specific CD4+ T helper and CD8+ T cytotoxic ceils. Int. J. Cancer, 45: 1096-1104. Simon, M.M., U. Hochgeschwender, U. Brugger and S. Landolfo (1986) Monoclonal antibodies to interferon-,{ inhibit interleukin 2-dependent induction of growth and maturation in lectin/antigen-reactive cytotoxic T lymphocyte precursors. J. Immunol., 136: 2755-2761. Smyth, M.J., Y. Norihisa, J.R. Gerard, H.A. Young and J.R. Ortaldo (1991) IL-7 regulation of cytotoxic lymphocytes - pore-forming protein gene expression, interferon-gamma production, and cytotoxicity of human peripheral blood lymphocyte subsets. Cell. Immunol., 138: 390-403. Smyth, M.J., J.R. Ortaldo, W. Bere, et al. (1990) IL-2 and IL-6 synergize to augment the pore-forming protein gene expression and cytotoxic potential of human peripheral blood T cells. J. I mmunol., 145: ! 1591166. Smyth, M.J., J.R. Ortaldo, Y. Shinkai et al. (1990) Interleukin 2 induction of pore-forming protein gene expression in human peripheral blood CDS+ T cells. J. Exp. Med., 171: 1269-1281. Sprent, J. and M. Schaefer (1990) Antigen-presenting cells for CD8+ T cells. Immunol. Rev., 117: 213-234. Stem, A.S., F.J. Podlaski, J.D. Hulmes, et al. (1990) Purification to homogeneity and partial characterization of cytotoxic lymphocyte matura-
tion factor from human B-lymphoblastoid cells. Immunology, 87: 6808~5812. Taga, T., Y. Kawanishi, R.R. Hardy, T. Hirano and T. Kishimoto (1987) Receptors for B cell stimulatory factor 2. Quantitation, specificity, distribution, and regulation of their expression. J. Exp Med., 166: 967-981. Taira, S., M. Matsui, K. Hayakawa, Y. Yokoyama and H. Nariuchi (1987) Interleukin 2 secretion by B cell tines and splenic B ceils stimulated with calcium ionophore and phorbol ester. J. Immunol., 139: 2957. Trenn, G., H. Takayama, L.J. Hu, W.E. Paul and M.V. Sitkovsky (1988) B cell stimulatory factor 1 (IL-4) enhances the development of cytotoxic T cells from Lyt-2+ resting murine T lymphocytes. J. hrununol., I40: 1101-1106. Tschopp, J. and M. Nabholz (1990) Perforin-mediated target cell lysis by cytolytic T lymphocytes. Annu. Rev. Immunol., 8! 279--302. Uyttenhove, C., P.G. Coulie and J. Van Snick (1988) T cell growth and differentiation induced by interleukin-HPlflL-6, the murine hybridoma/plasma cytoma growth factor. J. Exp. Med., 167: 1417-1427. Van Snick, J. (1990) lnterleukin-6: an overview. Annu. Rev. lmmtmol., 8: 253--278. Wagner, H., C. Hardt, B.Y. Rouse, M. Rrllinghoff, P. Scheurich and K. Pfizenmaier (1982) Dissection of the proliferative and differentiative signals controlling murine cytotoxic T lymphocyte responses. J. Exp. Med., 155: 1876-1881. Widmer, M.B. and K.H. Grabstein (1987) Regulation of cytolytic T-lymphocyte generation by B-cell stimulatory factor. Nature, 326: 795-8. Young, J.W. and R.M. Steinman (1990) Dendritic cells stimulate primary human cytolitic lymphocyte responses in the absence of CD4+ helper T cells. J. Exp. Med., 171: 1315-1332. Young, L.H.Y., L.S. Klavinskis, M:B. Oldstone and J.D.-E. Young (1989) In vivo expression of perforin by CD8+ lymphocytes during an acute viral infection. J. Exp. Med., 169: 2159-2171.
Journal of the Neurological Sciences, 115 (Suppl.) (1993) $24---$28 © 1993 Elsevier Science Publishers B.V. All rights reservecd 0022-510X/93/$06.00
JNS 04012
Generation of effector cytotoxic T cells from cytotoxic T cell precursors: role of soluble factors G. P a l l a d i n o The Wistar Institute of Anatomy and Biology, Philadelphia, PA 19104-4268, USA
Key words: Cytotoxic T lymphocytes; Lymphokines;Proliferation; Differentiation;Antigen presenting cells
Summary Cytotoxic T lymphocytes (CTL) have long been recognized as playing a major role in the immune response to alloantigens and viral antigens as well as tumor antigens. The progress of the last decade in the identification and characterizationof soluble factors involved in the regulation of the immune response has greatly improved our knowledge of the mechanisms of CTL activation and regulation, This review wilt summarize the data available in the literature regarding different lymphokines and their specific activity on CTL. In addition it will point out a few of the elornents of the systems that hamper its full understanding and it will suggest possible directions for future research.
Introduction Cytotoxic activity, the ability of cells to recognize other cells as target and kill them by direct lysis is a feature common to different cell types: natural killer cells, lymphokine activated killer cells and cytotoxic T lymphocytes (CTL). While the mechanisms oflysis appear to be common to all of them, the mechanisms of target recognition and activation are very different and CTL are the ones best characterized in this regard. Not all T lymphocytes express lytic activity: cytotoxic activity can be generated from CD4+ peripheral lymphocytes as well as thymocytes, but the majority of CTL are found in the CD8+ compartment of mature T lymphocytes and it is on this specific population, that is phenotypically characterized by the expression of T cell receptor (TcR) and CD8 antigen and the lack of Fc receptor, that the attention of this review will be focused. These cells are effectors of cytotoxic activity against alloantigens and viral antigens and are also involved in antineoplastic responses as well as various immonopathogenetic mechanisms. Cytotoxic activity is not always expressed by CTL: on the contrary, it is highly regulated. Resting CTL (precursor CTL = pCTL), either naive or memory, are unable to lyse specific target cells unless they undergo a complex process of activation and differentiation that are under the control of many different soluble factors. After antigen stimulation Correspondence to: Dr. G. Palladino, The Wistar Institute of Anatomy and Biology, 360I Spruce Street, Philadelphia, PA 19104-4268, USA.
the cytotoxic activity of CTL increase progressively, at day 5-7 and rapidly declines to reach background level usually by day 10. In the period between contact with the antigen and the peak of eytotoxic activity three different phases can be recognized that are usually indicated as (i) activation, (ii) proliferation, and (iii) differentiation (Bach et al. 1989).
Activation The activation phase is characterized by the binding of the T cell receptor ofpCTL to the antigen in the context of class I MHC molecule on the surface of accessory cells (antigen presenting cells or APC). The activation through TcR/Ag-MHC interaction, as well as through other mechanisms that mimic this interaction, i.e. mitogen or anti-TcR antibodies, induces modifications in the pCTL that make them sensitive to factors stimulating proliferation and/or differentiation. The increase sensitivity to these factors appears to be mediated, in some cases, by the induction or upregulation of the expression of specific receptors. Upregulation of interleukin-2 receptors (IL-2R) has been reported on CTL clones after antigen stimulation (Andrew et al. 1985) and upregnlation of intedeukin-4 receptors (IL4R) has been shown in murine splenic T cells after stimulation with ConA (Ohara and Paul 1987), Conversely, mitogenic stimuli do not appear to have any effect on the expression of receptors for other lympholdnes, i.e. interleukin(IL)-5 and IL-6 (Taga et al. 1987; Mita et al. 1988).
$25 No lymphokines are directly involved in the activation phase, but there are indications that the strength of the activation signal may influence the factor requirements of CTL during the following proliferation and differentiation. De Jong et al. (1990) reported that CTL activity can be generated in human PBL in the absence of exogenous lymphokines after stimulation with optimal concentration of antibodies anti-TcR, while the generation of CTL activity after stimulation with suboptimal concentration of the same antibodies requires the addition of exogenous factors. Proliferation
Many different lymphokines can regulate the proliferation of activated CD8+ T cells, some of them do it through a direct effect, others can modulate the expression of IL-2R and significantly affect CTL proliferation in the presence of IL-2. IL-2 can directly stimulate proliferation of activated pCTL from murine peripheral lymphocytes (Raulet and Bevan 1982; Wagner et al. 1982) as well as IL-6 (Uyttenhove et al. 1988) and IL-4 (Pfeifer et al. 1987; Widmer and Grabstein 1987), even if the ability of the latter to do it through an IL-2 independent mechanism is still controversial (Miller et al. 1990; Selvan et al. 1990). IL-7 and IL-12 also induce proliferation of activated CDS+ human PBL, and the mechanism of stimulation has been shown to be 1L-2-independent for the latter (Gately et al. 1991) and partially so for the former (Annitage et al. 1990; Grabstein et al. 1990). IL-7 can also upregulate the expression of IL-2R on activated CD8+ human PBL as well as murine splenocytes (Armitage et al. 1990; Grabstein et al. 1990), while TNF-ct synergize with IL-2 to increase the expression of IL-2R on activated human PBL (Robinet et al. 1990). The data relative to the effect of IL-4 on the expression of 1L-2R are controversial: Martinez and al. (1990) reported downregulation of IL-2R by IL-4 in CD8+ activated human PBL while Bertagnolli et al. (1991) described upregulation of the same receptor by IL-4 in activated murine thymocytes. An inhibitory effect of TGFq3 on the generation of alloreactive murine CTL supported by TNFmt has been also described (Ranges et al. 1987). The inhibition is at least partially due to the ability of TGF-13 to downregulate the expression of 1L-2R on peripheral T lymphocytes (Kehrl et al. 1986), in so doing counteracting the effect of T N F ~ . Differentiation
To acquire significant cytotoxic activity, CD8+ T cells need to undergo a process of differentiation which is characterized by the accumulation in the cytoplasm of granules containing an array of different molecules that are the me-
diators of cytolytic activity (Tschopp and Nabholz 1990). Among them the best characterized are perforin, a protein with complement-like lytic activity, and a group of closely related proteolytic enzymes called granzymes and sometime referred to also as serine esterases or BLT esterases. The increase of intracellular concentration of these enzymes or their corresponding messenger RNAs has been used in recent years as markers for CTL differentiation. The differentiation of CD8+ to cytolytic cells, measured by the induction of these markers, can be modulated by different factors. IL-2, in relatively high concentration, can induce mRNA synthesis of perforin and BLT esterase in resting CD8+ human PBL (Liu et al. 1989; Smyth et al. 1990). IL-4 has been shown to increase intracellular concentration of BLT esterase in activated CD8+ human PBL and murine peripheral T cells (Horohov et al. 1988; Trerm et al. 1988). IL-6 alone does not appear to have a significant differentiative effect but it can synergize with low concentration of 1L-2 to increase the intracellular concentration of perforin mRNA in resting human PBL (Smyth et al. 1990). Finally, high concentration of IL-7 induces increase in intracellular perforin mRNA in CD8+ resting human PBL and can also synergize with low concentration of IL-2 to generate a larger and faster accumulation of this messenger (Smyth et al. 1991). Factors with undefined function
Other factors have been involved in the generation of CTL but their specific mechanism of activity is still undetermined: ¥-IFN has been shown to be required in addition to IL-2 during the early phase of CTL generation from activated CD8+ murine T cells (Maraskovsky et al. 1989; Rogers et al. 1991) and it appears to modulate IL-2-dependent CTL proliferation (Simon et al. 1986). IL-5 has also been involved in CTL generation: it has been shown to increase cytotoxic activity of human CD8+ PBL and mouse peripheral T cells, but the evidence suggesting that it has an activity on differentiation and not proliferation of CTL is only circumstantial (Ramos 1989; Nagasawa et al. 1991). IL-1 has been suggested earlier to play a role in the generation of alloreactive CTL (Farrar et al. 1980), however, later evidence indicates that it is required to support IL-2 secretion and not cytotoxic activity of CD8+ cells (Renauld et al. 1989). Discussion
Despite this large amount of available data a clear and comprehensive understanding of the activity of lymphokines on CTL generation is still elusive. The numerous attempts to analyze the involvement and the role of different factors during in vitro or in vivo generation of CTL activity and the experiments directed to identify the minimal factor
$26 requirements for the same activities have generated different and very otten contradictory results. Three elements of the system, most of all, concur to hamper its explanation: (i) the variability in the source of antigen stimulation, (ii) the variability in the source of factors, and (iii) the variability within the CD8+ population of responder cells. The vast majority of cells express class I MHC antigens and can thus present antigen to CD8+ lymphocytes, but not all class I+ cells can stimulate CTL activity equally well (Sprent and Schaefer 1990). Among others, dendritic cells appear to be particularly efficient and, as opposed to other cell types, they can induce primary in vitro CTL activity against allo-antigens or viral antigens in the absence of exogenous help (Young and Steinman 1990; Mercadal et al. 1991). In addition the state of maturation and/or activation of other APC, i.e. macrophages and T lymphocytes can affect their ability to stimulate CD8+ cells and may change CTL requirement for exogenous factors (Sprent and Schaefer 1990). CD4+ lymphocytes able to produce IL-2, IL-4, IL-5, IL-6, IL-7, y-IFN, TNF-~ and TGF-13 are the best known and characterized source of helper factors relevant to CTL activation, but different accessory cells are also able to produce many of the same factors. Macrophages secrete IL-6, TNF-ct and TGF-I3 (Van Snick 1990) while B cells can produce IL-2, IL-6 and IL-12 (Taira et al. 1987; Stem et al. 1990; Van Snick 1990). Even though experimental systems can be envisaged, and have been actually tested (De Jong et al. 1990; Rogers et al. 1991), that are free of both CD4+ and accessory ceils, an uncomroiied source of t'actors would still be left in the form of the CD8+ cells themselves. These cells, or a population thereof, have been shown to produce a variety of factors relevant for their own stimulation: alloreactive murine CD8+ clones have been described that produce IL-2, y-IFN and TNF-et (Klein et al. 1982; Kelso and Glasebrook 1984; Fong and Mosmann 1990; Gajewski and Fitch 1990) and more recently mRNA encoding for IL-4 and IL-5 has been demonstrated in CD8+ murine splenocytes after activation by antigen or mitogen (Cardell et al. 1991; Horvat et al. 1991). Human CD8+ clones have also been generated producing in various combinations IL2, IL-4, IL-5, IL-6, y-IFN and TNF-et (Salgame et at. 1991). The finding of both cytotoxic activity and factor secretion within the CD8+ compartment suggested the possibility of the existence of two functionally distinct subpopulations within this cell type. This hypothesis has been supported by the results of studies on alloreactive clones indicating that the majority of IL-2 secreting clones were not cytolytic and vice versa the majority of cytolytic clones did not produce IL-2 (Kelso and Glasebrook 1984; Heeg et al. 1987). These two subpopulations have later been found to have also different factor requirements: IL-2 secreting CD8+ cells but not cytolytic ones express IL-1 receptors and require this
lymphokine for their activation (Mizuochi et al. 1988; Klamet et al. 1989). More differences have been shown within CD8+ cells with regard to their previous contact with antigen. Human as well as murine peripheral CD8+ T lymphocytes include naive and memory cells that can be distinguished by the expression of different isoforms of the surface antigen CD45R (Merkenschlager and Beverly 1989; Marvel et al. 1991). The two groups may have different factor requirements as it is suggested by experiment showing that CD8+ cells require CD4+ help to generate primary CTL response to tumor or viral antigens but are able to generate a CD4-independant secondary response against the same antigens (Horvat et al. 1991; Jennings et al. 1991). While the possibility to distinguish phenotypically between naive and memory cells will allow further characterization of their differences the inability to date to distinguish phe: notypically cytolytic from factor-secreting cells strongly hampers the possibility of any further purification of pCTL.
Conclusions
While the vast majority of the data that have been collected so far have been helpful to the identification of the various elements involved in CTL activation, much work remains to be done to understand the way all these different elements work together during the normal immune response, and this direction appears to be the most promising for future studies. The use of new approaches and new technologies will be helpful to allow further development in the field. One example or'these new tools are the transgenic mice deficient in various factors that have been recently generated (Kuhn et al. 1991; Schorle et al. 1991). They may offer useful information regarding the need for a given lymphokine during in vivo response as well as help to clarify the mechanisms by which the immune system compensates for the loss of one single factor. Another possible
Fig. I. Lymphokinesinvolvedin CTL generationand their sources.
$27
approach may involve the use of molecular probes for perforin and granzimes that have been already used to detect cytotoxic activity in vivo (Griffiths and Mueller 1991) and that, combined with probes specific for lymphokines, may allow the study of pathological processes during their normal in vivo development (Young et al. 1989). References Andrew, M.E., A.M. Churilla, T.R. Malek, V.L. Braciale and T.J. Braciale (1985) Activation of virus specific CTL clones: antigen-dependent regulation of interleukin 2 receptor expression. J. Immunol., 134: 9 2 ~ 925. Amfitage, R.J., A.E. Namen, H.M. Sasseufeld and K.H. Grabstein (1990) Regulation of human T cell proliferation by IL-7. J. Immunol., 144: 938 941. Bach, F.H., R.L. Geller, P.J. Nelson, et al. (1989) A "'Minimal Signal-Stepwise Activation" analysis of ftmctional maturation of T lymphocytes. Immunol. Rev.. 111 : 35 57. Bertaguolli, M.M., Y. Takai and S.H. Herrmam~ (1991) IL-4-supported induction of cytolytic lymphocytes-T requires 1L-2 and 1L-6. Cell. lmmunol., 133:327 341. Cardell, S., B. Sander and G. Moiler (1991) Helper interleukhls are produced by both CD4 and CD8 splenic T-cells after mitogen stimulation. Enr. J. lmmtmol., 21:2495 2500. De Jong, R., M. Brouwer, V.I. Rebel, G.A. Van Seventer, F. Miedema and R.A.W. Van Lier (1990) Generation of alloreactive cytolytic T lymphocytes by immobilized anti-CD3 mouoclonal antibodies. Analysis of requirements for human cytotoxic T-lymphocyte differentiation, hnmunology, 70:357 364. Farrar, W.L., S.B. Mizel and J.J. Farrar (1980) Participation of lymphocyte activating factor (interleukin 1) in the induction of cytotoxic T cell responses. J. lmmunol., 124:1371 1377. Fong, Y.A.'I'. and T.R. Mosmann (1990) Alloreactive murine CD8+ T cell clones secrete the Thl pattern of cytokines. J. Immunol., 144: 17441752. Gajewski, T.H. and F.W. Fitch (1990) Anti-proliferative effect of ifi~-y in immune regulation. IV. Murine CTL clones produce IL-3 and GM-CSF, the activity of which is masked by the inhibitory action of secreted IFN-'y. J. Immunol., 144: 548- 556. Gately, M.K., B.B. Desai, A.G. Wolitzky et al. (1991) Regulation of human lymphocyte proliferation by a heterodimeric cytokine, IL-12 (cytotox ic lymphocyte maturation factor). J. lnmaunoL, 147: 874-882. Grabstein, K.H., A.E. Namen, K. Shanebeck, R.F. Voice, S.G. Reed and M.B. Widmer (1990) Regulation of T cell proliferation by IL-7. J. hnmunol., 144:3015 3020. Griffiths, G.M. and C. Mueller (1991) Expression of perforin and granzymes invivo potential diagnostic markers for activated cytotoxic cells. Immuuol. Today, 12: 415-419. Heeg, K., C. Steeg, C. Hardt and H. Wagner (1987) Identification of interleukin 2 prodncmg T helper cells within murine Lyt-2+ T lymphocytes: frequency, specificity and clonal segregation from Lyt-2+ precursor ofcytotoxic T lymphocytes. Eur. J. Inununol., 17:229 2336. Horohov, D.W, J.A. Crim, EL. Smith and J.P. Siegel (1988) IL-4 (B cell-stimulatory factor 1) regulates multiple aspects of influenza viresspecific cell-mediated immunity. J. lrmnunol., 141:4217 23. Horvat, B., J.A. Loukides, L. Anandan, E. Brewer and P.M. Flood (1991) Production of interleukin-2 and iuterleukio-4 by immune CD4-CD8+ and their role in the generation of antigen-specific cytotoxic Y-cells. Eur. J. hnmunol., 21:1863 1871. Jennings, S.R., R.H. Bonneau, P.M. Smith, R.M. Wolcott and R. Chervenak (1991) CD4-positive lymphocytes-T are required for the generation of the primary but not the secondary CDS-positive cytolytic lymphocyteT response to herpes simplex virus in C57BL/6 mice. Cell. Immunol., 133:234 252.
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