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Human γδ T lymphocyte subsets: Activation by superantigens?
658
33 'a F O R U M
IN IMMUNOLOGY
HUMAN V~ T LYMPHOCYTE SUBSETS: ACTIVATION BY SUPERANTIGENS? R.L.H. Bolhuis (1), E. Sturm (i), p. Fisch (2), P.M. Sondel (2) and E. Braakman (I) (i) Department o f Immunology, Dr. Daniel den Hoed Cancer Center, Rotterdam (The Netherlands), and (2) Departments o f Human Oncology, Pediatrics and Genetics, University o f Wisconsin, Madison, WI (USA)
Introduction. Since the discovery of "r~ T lymphocytes three years ago (Borst et aL, 1987; Brenner et al., 1987), y~ T lymphocytes have become more than popular and are now extensively studied. Despite the large amount of information on genetic, biochemical and functional properties of 78 T lymphocytes in both mice and man, their biological role remains enigmatic. In this contribution, we will summarize and comment on the data presently available in our laboratories on human y~ T lymphocytes and its subsets in peripheral blood and thymus. We provide data and speculation on their origin, functional properties in vitro and their putative physiological role in vivo. "t'~ T lymphocytes in peripheral blood can be dissected into two major nonoverlapping subsets on the basis of their Vy and V~ gene segment usage (Borst et al., 1989; Casorati et al., 1989; Faure et al., 1988; Sturm et al., 1989). The major subset of circulating T8 T lymphocytes expresses the Vy9/V~2-encoded T-cell receptor (TCR), whereas the minor subset expresses a ~ chain encoded by the VS1 gene segment in combination with a 7 chain encoded by a Vy gene segment belonging to the VTI subgroup. Expression of Vy9/V~2 or V~l-encoded T-cell receptors was founa to be associated with distinct molecular forms of the receptor. Whereas all V79/V82-encoded TCR are disulphide-linked heterodimers, both disulphide and nondisulphide-linked heterodimers can be detected within the V~l-encoded TCR
(Lanier et ai., 1988; Seki et ai., 1989; Sturm et al., 1990 in press). Thymic or extrathymic maturation of 7~ T-lymphocyte subsets. The actual limited combinatorial diversity of ~,~ T lymphocytes in peripheral blood, in spite of the large potential combinatorial diversity, suggests that selection must occur either at the genomic level, i.e. through a coordinated rearrangement of 7 and ~ chain genes, or through selective expansion of particular subsets of-r~ T lymphocytes. Evidence in favour of the selective expansion of y8 T lymphocytes was provided by studies documenting agerelated changes in the y~ T lymphocyte subset composition in peripheral blood in combination with studies analysing the expression of the " n a i v e " (CD45RA) and "primed" (CD45RO) markers by "r~ T lymphocyte subsets in the periphery. During the first years after birth, the frequency of y~ T lymphocytes in the periphery increases (Parker et al., 1990; Yachie et al.. 1989). This increase can be completely accounted for by an age-related expansion of the Vy9/V~2+ T-lymphocyte subset (Parker et al., 1990). We and others showed that virtually all V79/V82÷ T lymphocytes in peripheral blood have the CD45RO+ CD45RA- or low phenotype (Miyawaki et al., 1990; Braakman et aL, submitted). In contrast, the minor V~I T-lymphocyte subset in peripheral blood expresses CD45RA and lacks the expression of CD45RO. In vitro activa-
7~ T C E L L S
tion of V~I T lymphocytes with PHA leads to acquisition of CD45RO expression and decreased CD45RA expression. Moreover, all V31 T-lymphocyte clones have a CD45RA-,CD45RO+ phenotype. Therefore, it can be cor,cluded that CD45 isoforms are also genuine markers for naive and primed 78 T lymphocytes, respectively. Thus it appears that virtually all V79/V82+ T lymphocytes in peripheral blood are already primed, whereas the entire V~I ÷ T lymphocyte subset is naive. In contrast to peripheral blood, the majority of 78 T lymphocytes in the thymus express V~I, whereas V79/V~2 thymocytes are rare (Lanier et al., 1988). We found that V~I thymocytes can be subdivided into two subsets based on the expression of either CD45RA or CD45RO (Braakman et ai., submitted). For a[] thymocytes, it is believed that the CD45RA-,CD45RO+ subset is destined to intrathymic death because the generative potential is almost exclusively f o u n d in the CD45RA + ,CD45RO- thymocyte subset (Egerton et aL, 1990). This probably also holds true for 7~ thymocytes, since self-reactive 7~ thymocytes are eliminated in the thymus (Dent et al., 1990). Taken together, these observa*;-'"~ are ~vualaL~nL . . . . :o'~ • --':'~" the concept WI[II that V~I lymphocytes in the periphery descend from V~I ,CD45RA +, CD45RO- thymic precursors. The relatively low frequency of V79/V~2+ T lymphocytes in the thymus, together with the age-related selective expansion of primed V79/V~2 + T lymphocytes in peripheral blood, suggest that V79/V~2+ T lymphocytes are subject to extrathymic selection (Parker et al., 1990; Braakman et al., submitted). This notion is further substantiated by the presence of V-c9/V~2+ T lymphocytes in peripheral blood of athymic individuals (DiGeorge syndrome patients) (Van Dongen et al., submitted). LI%.PlIO
Activation of Vy9/V~2 T lymphocytes in vivo.
The expression of CD45RO on the entire V82+ T-lymphocyte subset in peripheral blood is difficult to reconcile
659
with stimulation of these lymphocytes by an extensive repertoire ol i~munogenic antigens, matching the Oolymorphism of the V79/V82 T ly,'~aplmcy~e population basedon the ~r,tclLive junctional diversity at the V.ii!gr-Jjoints of functionally rearranged YCR-~ genes (Hata et al., 1988). Rath~ r, it favours an activation by a single ,~atigen or by a closely related family of antigens. In analogy to subsets of 0t[3T lymphocytes which share a particular V[3 region and which can be activated by so-called superantigens, we suggest that V79/V~2 T lymphocytes in peripheral blood are in vivo activated by superantigens (see below). V81 T lymphocytes in peripheral blood have the naive CD45RA+ phenotype and consequently these lymphocytes have not been activated in vivo or, alternatively, once activated do not remigrate to peripheral blood. If the hypothesis is correct that V~2 but not VSI T lymphocytes are in vivo activated, it would imply that this superantigen is ubiquitous in man. Attractive candidates for such superantigens are autologous and mycobacterial heat shock (stress) proteins which are ubiquitous in man and to which human and mouse 7~ T lymphocytes reportedly respond (Born et al., 1990; Haregewoin et aL, 1989). The putative superantigen activation in vivo may occur by mature V82 T lymphocytes in the periphery or may be an integral part of the positive selection during extrathymic differentiation of V82 T iymphocytes.
V79/V~2 T lymphocytes specifically recognize Daudi cells. Proliferation of purified a[~ and 7~ T-lymphocyte populations can be sustained by stimulation with B-iymphoblastoid ceil lines (B-LCL) (Bosnes et al., 1989; Van de Griend et al., 1984). We observed that stimulation of fresh unfractionated PBL with irradiated Daudi cells, a Burkitt lymphomaderived cell line, resulted in the selective expansion of 7~ T lymphocytes within the CD3 ÷ population. This selective expansion was strictly confined to the V79/V82 T lymphocytes. Stimulation with several other B-LCL lines, e.g.
660
33 ~a F O R U M I N I M M U N O L O G
APD, BSM or Raji did not result in such selective expansion of V~,9/V~2 T lymphocytes, although the total lymphocyte yield was similar. These results suggest an antigen-specific stimulation of V~.9/V~2 T lymphocytcs by Daudi cells, and involving the V~.9/V~2 T C R It appeared that none of the V~I T-lymphocyte clones, but all V~,9/V82 T-lymphocyte -lones lysed Daudi cells, e ~en when tne-~~were generated in an aut , l o g o u s or non-Daudi B-LCLcomprising culture system (Sturm et al., 1990, in press), i.e. when they had never ';seen" Daudi cells before. The question is now whether the lysis of Daudi cells, cell line known to be susceptible to ".'~X ,t~tivity, is due to: a)MHCunrestricted lytic activity not involving the TCR (the hallmark of T C R / C D 3 - NK lymphocytes, but also exerted by activated "r~ T lymphocytes, and to a lesser extent, by a[3 T !ymphocytes) (Borst et aL, 1988; Christmas: 1989, Dastot et al., 1990; Jitsukawa e~ aL, 1988), or b) whether it involves the V'r9/V~2 TCR as the recognition structure, i The distinct patterns of lysis bet',:-een all these subsets of~'rCR- or TCR ÷ effector lymphocyt~s, and in particular the V-I'9/V~2 versus the V~ 1 T lymphoc.ytes, might reflect differential expresstun and/or involvement of multiple receptors, e.g. acee~or~ molecules in targe~ .~ell reccgnition (Bolhuis and Braakman, 1988). Overall, the disulphide-linked TCR V~.9/V~2 lym~&.-_~:~,';~exert higher MHC-unrestricted cytolyt,.'c activity than either the disulphide or non-disulphide-linked TCR V~I lymphocytes (Christmas, 1989; Dastot et aL, 1990; Jitsukawa et aL, 1988; Sturm et aL, 1989). Although both subsets of -¢~ T lymphocytes express equal levels of adhesion molecules such as CD2, ICAM-1, LFA-I and LFA-3 (Fisch et al., 1990), they have been reported to differ in their cell matrix composition, which is reflected by differences in their respective adherence and motility capacities. These differences may result in different target cell repertoires (Grossi et al., 1989). However, the VS1 cloned T lymphocytes formed conjugates with Daudi ceils as
Y
well as the V~,9/V~2 cloned T lymphocytes did with Daudi cells. Together with our observation that all cloned V~.9/V82 T lymphocytes lyse and specifically proliferate (Fisch et aL, 1990, in press) to Daudi cells even when generated in the absence of Daudi cells, these data support the idea of direct TCR~'~ involvement in Daudi cell recognition. The specific proliferation to and cytolysis of Daudi cells by entire subpopulation of V'r9/V~2 T lymphocytes is reminiscent of a superantigen reponse as described for e.g. V[38 (Marrack and Kappler, 1988) and V't9 (Rust et al., 1990) T lymphocytes to staphylococcal enterotoxins. In superantigen stimulation, the V138- or V'r9-encoded TCR chain in itself is sufficient to impose superantigen specificity, i.e. independent of V0t or V~ usage in the associated TCR0c or TCR~ chain. In the case of this proposed analogy, the V'l'9 chain is expected to dictate Daudi cell specificity. However, we found that two V~.9/V~l clones of thymic origin did not lyse Daudi cells, implying that the V~,9-encoded TCR~, chain in itself is not sufficient to dictate Daudi cell specificity. Therefore, either the expression of a V82-encoded TCR8 chain alone is sufficient to dictate Daudi cell specificity or, alternatively, the combination of a V'r9- and V82-encoded TCR chain is required. Also, the possibility remains that thymic V'r9/V~ 1 clones do not !yse D.audi cells because of their thymic origin. Another feature of "classical" superantigen recognition is restriction of presentation by MHC class II molecules. Dat~di cell specificity of V'r9/V~2 T lymphocytes could not be blocked t~y anti-MHC class II mAb. We therefore suggest that any stimulus that activates an entire subset of lymphocytes with a particular TCR gene rearrangement represents a superantigen. The presentation of such superantigens to ~,~ T lymphocytes may not necessarily be restricted by MHC molecules. Interestingly, the V~,9/V82, but not the VS1, T lymphocytes ~pecifically proliferate to mycobacterial antigens (Fisch et al., 1990, in press). These mycobacterial antigens can be presented by MHC class I negative B-LCL, including Daudi cells. The proliferative
~
T CELLS
response to Daudi cells can be blocked by polyclonal anti-heat shock protein (HSP-58) antibodies. More importantly, preliminary data suggest that heat shock proteins can be isolated from the surface of Daudi target cells (Fisch et al., 1990, in pre~s).
The physiological role of V-f9/V82 T iymphocytes ? Heat shock (stres~ proteins are constitutatively expresseca mtracellularly to sustain essential normal cell growth functions (Welch, 1990, in press). T lymphocytes from a healthy individual can specifically react to a self heat shock protein. Under physiological conditions, such self proteins may not be functionally present on the membrane of normal cells, due to either lack of intracellular processing or too low a surface expression. In contrast, stressed cells may express heat shock proteins in an immunogenic form at their surface. This may result in the reactivation of the C D 4 5 R A - , C D 4 5 R O ÷ V-f9/V~2 T lymphocytes; their physiologic function may be to produce cytokines to "calm" the stressed cells or to eliminate them by cytolysis. I~,..Jll',r~g~#l Tnrlo~-
"~snf~,Lr,"~t,~hl~ ,-~,~,nA;+; . . . . . . .~ U t . , l l I-, I.,~III[,I. YIL]II~ILI.JI~" I.,~JIIIdlLIW~.PlI~
as episodes of infection, V~,9/V82 T lymphocytes can be activated by the evolutionary highly conserved heat shock proteins present on many organ-
66!
isms, such as bacteria (Fisch et aL, 1990, in press). Because of the above mentioned high sequence homology of stress proteins from bacteria to man, this activation of V'r9/V82 T lymphocytes can induce an a u t o r e a c t i v e i m m u n e response against stressed cells at the site of infection. This may depend on whether the conserved or the speciesspecific determinants of the heat shock proteins are immunodominant. One would expect tolerance to the conserved, i.e. self-like, epitopes. Such tolerance may be actively maintained through the immune network. A functional relationship between immune responses against stress proteins and autoimmunity is demonstrated by the following observations, immune responses to stress proteins have been reported during autoimmune diseases. Moreover, in vitro responses of normal lymphocytes against stress proteins have been reported to contain an autoreactive component (Lamb et aL, 1989). Thus, the mechanism of such tolerance for self-like proteins may not be absolute. Dysregulation of actively maintained self tolerance may result from viral and/or bacterial infections and mount a V-f9/V~2 T lymphocyte response against self antigens, i.e. the conserved sequences of the stress proteins shared between bacteria and man. Genotyping and functional analysis of lymphocytes present at the inflammatory sites in patients with autoimmune disease may provide the experimental evidence.
References. BOLHUIS, R.L.H. & BRAAKMAN,E. (1988), Are cognitive receptors involved in nonspecific MHC-unrestricted lytic activity?, in "23th Forum In Immunology". Res. lmmunoL, 139, 460-465. BORN, W., HAPP, M.P., DALLAS,A., REARDON,C., KUBO,R., SHINNICK,T., BRENNAN,P. & O'BRIEN, R. (1990), Recognition of heat shock proteins and ~'~ cell function, hnmunol. Today, 2, 40-43. BORST, J., VANDE GRIEND, R.J., VANOOSFVEEN~J.W., ANG, S.-L., MELIEF,C.J.M., SEIDMAN, J.G. & BoLmnS,R.L.H. (1987), A T-cell receptor ~'/CD3 complex found on cloned functional lymphocytes. Nature (Lond.), 325, 683-688. BORST, J., VANDONGEN,J.J.M., BOLHUIS,R.L.H., PETERS,P.J., HAFLER,D.A., DE VRIES,E. & VANDE GRIEND, R.J. (1988), Distinct molecular forms of the T-cell receptor ~'8 detected on viable cells by a monoclonal antibody. J. exp. Med., 167, 1625-1644. BORST, J., WICHERINK, A., VAN DONGEN, J.J.M., DE VRIES, E., COMANZ-BITTER,W.H., WASSENAAR,F. & VANDENELSEN,P. (1989), Non-random expression of T-cell receptor "r and 8 variable gene segments in functional T lymphocyte clones from human peripheral blood. Europ. J. lmmunol., 19, 1559-1568.
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33 rd FOR U M IN I M M U N O L O G Y
BOSNES,V., HALVORENSEN,R., SKAFTADOTTIR,I., GAUDERNACK,G. & THORSBY,E. (1989), Specificity of -f~ receptor cytotoxic lymphocytes isolated from human peripheral blood. Scand. .L Immunoi., 29, 723-731. BRENNER,M.B., MCLEAN,J., SCHEFT,H., RIBERDY,J., ANG, S.-L., SEIDMAN,J.G., DEVLIN,P. t~ KRANGEL,M.S. (1987), Two forms of the T-cell receptor -f protein found on peripheral blood cytotoxic lymphocytes. Nature (Lond.), 325, 689-694. CASORAT~,G., DE LIBERO,G., LANZAVECCHIA,A. • MIGONE,N. (1989), Molecular analysis of human -f/B÷ clones from thymus and peripheral blood. J. exp. Med., 1"/0, 1521-1535. CHRISTMAS~S.E. (1989), Most human CD3 + WT31- clones with T-cell receptor C'rl rearrangements show strong non-MHC-restricted cytotoxic activity in contrast to those with C3,2 rearrangements. Europ. J. Immunoi., 19, 741-746. DASTOT, H., SCHMID, M., GONTIER, C., AMIOT, M., MATHIEU-MAHuL,D., BENSUSSAN,A. & BOUMSELL,L. (1990), Correlation between T-cell receptor ~ isotypic forms and cytotoxic activity: analysis with human T-cell clones and hnes. Ceil. Immunol., 25, 315-325. DEN1, A.L., MATIS, L.A., HOOSHMAND,F., WIDACKI,S.M., BLUESTONE,J.A. & HEDRICK,S.M. (1990), Self-reactive T~ T cells are eliminated in the thymus. Nature (Lond.), 343, 714-719. EGERTON,M., PRUSKI,E. &; PIEARSKI,L.M. (1990), Cell generation within human thymic subsets defined by selective expression of CD45 (T200) isoforms. Human Immunol., 27, 333-347. FAURE, F., JITSUKAWA,S., TRIEBEL, F. &. HERCEND,T. (1988), Characterization of human peripheral lymphocytes expressing the CD3-T/~ complex with anti-receptor monoc!onat antibodies. J. Immunol., 141, 3357-3360. FlscH, P., MALK~VSKY,M., BRAAKMAN,E., STURM,E., BOLHUIS,R.L.H., PRIEVE,A., SOSMAN, J.A., LAM, V.A. & SONDEL,P. (1990), Gamma/delta T-cell clones and natural killer cell clones mediate distinct patterns of non-MHC-restricted cytolysis. J. exp. Med., 171, 1567-1579. GROSSI,C.E., CICCONE,E., M!GONE,N., BOTTINO,C., ZARCONE,D., MINGARI,M.C., FERRINI,S., TAMBUSI, G., VIALE, O., CASORATI,G., MILLO, R., MORETTA, L. & MORETTA, A. (1989), Human T cells expressing the -~'/Breceptor (TCR-1) : CTI- and C~'2~encoded forms of the receptor correlate with distinctive morphology, cytoskeletal organization, and growth characteristics. Proc. nat. Acad. Sci. (Wash.), 86, 1619-1623. HAREGEWOIN,A., SOMAN,G., HUM, R.C. & FINBERG,R.W. (1989), Human TB+ T cells respond to mycobacterial heat-shock protein. Nature (Lond.), 340, 309-312. HATA, S., SATYANARAYANA,K., DEVEIN, P., BAND, H., MCLEAN, J., STROMINGER, J.L., RRI~NNI:R_M R ,~Z l~RA~r.~t M" ~ (IQRR~ l~vt~nclv~, i n n ~ t l r t r t u l rll,zarclt,tr atF ra~r_ ranged human T-cell receptor ~ genes. Science, 240, 1541-1544. JITSUKAWA,S., TRIEBEL, F., FAURE, F., MIOSSEC,C. & HERCEND,T. (1988), Cloned CD3 ÷ TCRa/[3 TiyA- peripheral blood lymphocytes compared to the TiTA + counterparts : structural differences of the ~'/~ receptor and functional heterogeneity. Europ. J. Immunoi., 18, 1671-1679. LAMB, J.R., BAL, V., MENDEz-SAMPERIO,P., MEHLERT,A., So, A., ROTHBAND,J., JINDAL,S., YOUNG, R.A. & YOUNG,R.B. (1989), Stress proteins may provide a link between the immune response to infection and autoimmunity. Int. lmmunol., l, 191-196. LANIER, L.L., RUITENBERG,J., BOLHUIS,R.L.H., BURST,J., PHILLIPS,J.H. & TESTI, R. (1988), Structural and serological heterogeneity of ~,/~ T-cell antigen receptor expression in thymus and peripheral blood. Europ. J. lmmunol., 18, 1985-1992. MARRACK,P. & KAPPLER,J. (1988), The T-cell receptor repertoire for antigen and MHC. Immunol. Today, 9, 308-315. MIYAWAKI,T., KASAHARA,Y., TAGA,K., YACHIE,A. & TANIGUCHI,N. (1990), Differential expression of CD45RO (UCHLI) and its functional relevance in two subpopulations ' ' of circulating TCR--f/~ + lymphocytes. J. exp. Med., 171, 1833-1838. PARt~.ER, C.M., GROH, V., BAND, H., PORCELL], S.A., MORITA, C., FABBI, M., GLASS, D., STROMINGER,J.L. & BRENNER,M.B. (1990), Evidence for extrathymic changes in the T-cell receptor y/~ repertoire. J. exp. Med., 171, 1597-1612. RUST, C.J.J., VERRECK, F., VIETOR, H. & KONING, F. (1990), Specific recognition of staphylococcal enterotoxin A by TCR Vy9- bearing human T cells. Nature (Lond.), 346, 572-574. SEKI, H., NANNO,M., CHEN, P.-F., ITOH, K., IOANNIDES,C., GOOD, R.A. & PLATSOUCAS,C.D. (1989), Molecular heterogeneity of T~ T-cell antigen receptors expressed by CD4-CD8- T-cell clones from normal donors: both disulphide- and non-disulphide-linked receptors are 8TCSI +. Proc. nat. Acad. Sci. (Wash.), 86, 2326-2330.
"f8 T C E L L S
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STURIvl,E., BRAAKMAN,E., BONTROP,R.E., CHUCHANA,P., VANDEGRIEND,R.J., KONING,F., LEFRANC,M.-P. & BOLHUIS,R.L.H. (1989), Coordinated Vy and V~ gene segment rearrangements in human T-cell receptor ~,/8÷ lymphocytes. Europ. J. ImmunoL, 19, 1261-1265. STURM,E., BRAAKMAN,E., F1SCH,P., VREUGDENHIL,R.J., SONDEL,P. & BOLHUIS,R.L.H. (1990), Human V~'9-V82TCR~ lymphocytes show specificity to Daudi Burkitt's lymphoma cells. J. ImmunoL (in press). VANDEGRIEND,R.J., VANKRIMPEN,B.A., BOL,S.J., THOMPSON,A. & BOLHUIS,R.L.H. (1984), Rapid expansion of human cytotoxic T-cell clones. Growth promotion by heat-labile serum components and by various types of feeder cells. J. lmmunoL Methods, 66, 285-298. WELSCH,W.J. (1990), The mammalian stress response: physiology and biochemistry of stress protein, in "Stress protein in biology and medicine" (R. Morimoto, C. Georgopoulos & A. Tissieres) (pp. 223-278). Cold Spring Harbor Lalaoratory, New York. YACHIE, A., UENO, Y., TAKANO,N., MIYAWAKI,T. & TANIGUCHI,N. (1989), Developmental changes of double-negative (CD3 + 4- 8-) T cells in human peripheral blood. Clin. exp. lmmunoi., 76, 258-261.
REPERTOIRE SELECTION OF HUMAN T~ T CELLS
J. Borst (l) and J.J.M. van Dongen (2)
(i) Division o f Immunology, The Netherlands Cancer Institute, Amsterdam, and (2) Department of Immunology, Erasmus University, Rotterdam (The Netherlands)
Introduction. Research on ~'8 T cells has been very rewarding in the past few years, particularly for molecular biologists and biochemists, since the stage had already been set by previous investigations on 0~13T cells and the experiments to be done were obvious and straightforward. However, now that the molecular basis of -¢~T-cell receptor (TCR) diversity has been elucidated, we are left with the question as to how this diversity is employed in the immune response. It seems fair to say that we are all in the dark on this issue. Only thorough investigations in well defined in vivo systems will provide satisfactory answers. This second stage of ~8 T-cell research is still in its infancy. In this paper, therefore, we would like to focus on what we have learned about the ~ T-cell repertoire in human beings. In most healthy individuals, there is an extreme bias for expression
of the V~f9 and V~2 gene segments in peripheral blood (PB) "r,~T cells. Based on our studies in patients suffering from thyrnic hypoplasia and on analysis of changes in the ~ T-cell repertoire in healthy individuals with ageing, it would appear that this repertoire bias comes about by positive selection in the peripheral compartment. There are suggestions from in vitro experiments of other investigators that superantigens may drive this peripheral expansion. We would like to propose that the predominance of V~.9/V~2 expressing cells in PB of most healthy individuals arises fortuitously and does not provide insight into the inherent potential of peripheral "r~ T cells to recognize conversional antigenic peptides.
Biochemical evidence for TCR T~ repertoire bias. We first encountered human ~ T cells in two types of cellular materl-
33 'a F O R U M
IN IMMUNOLOGY
HUMAN V~ T LYMPHOCYTE SUBSETS: ACTIVATION BY SUPERANTIGENS? R.L.H. Bolhuis (1), E. Sturm (i), p. Fisch (2), P.M. Sondel (2) and E. Braakman (I) (i) Department o f Immunology, Dr. Daniel den Hoed Cancer Center, Rotterdam (The Netherlands), and (2) Departments o f Human Oncology, Pediatrics and Genetics, University o f Wisconsin, Madison, WI (USA)
Introduction. Since the discovery of "r~ T lymphocytes three years ago (Borst et aL, 1987; Brenner et al., 1987), y~ T lymphocytes have become more than popular and are now extensively studied. Despite the large amount of information on genetic, biochemical and functional properties of 78 T lymphocytes in both mice and man, their biological role remains enigmatic. In this contribution, we will summarize and comment on the data presently available in our laboratories on human y~ T lymphocytes and its subsets in peripheral blood and thymus. We provide data and speculation on their origin, functional properties in vitro and their putative physiological role in vivo. "t'~ T lymphocytes in peripheral blood can be dissected into two major nonoverlapping subsets on the basis of their Vy and V~ gene segment usage (Borst et al., 1989; Casorati et al., 1989; Faure et al., 1988; Sturm et al., 1989). The major subset of circulating T8 T lymphocytes expresses the Vy9/V~2-encoded T-cell receptor (TCR), whereas the minor subset expresses a ~ chain encoded by the VS1 gene segment in combination with a 7 chain encoded by a Vy gene segment belonging to the VTI subgroup. Expression of Vy9/V~2 or V~l-encoded T-cell receptors was founa to be associated with distinct molecular forms of the receptor. Whereas all V79/V82-encoded TCR are disulphide-linked heterodimers, both disulphide and nondisulphide-linked heterodimers can be detected within the V~l-encoded TCR
(Lanier et ai., 1988; Seki et ai., 1989; Sturm et al., 1990 in press). Thymic or extrathymic maturation of 7~ T-lymphocyte subsets. The actual limited combinatorial diversity of ~,~ T lymphocytes in peripheral blood, in spite of the large potential combinatorial diversity, suggests that selection must occur either at the genomic level, i.e. through a coordinated rearrangement of 7 and ~ chain genes, or through selective expansion of particular subsets of-r~ T lymphocytes. Evidence in favour of the selective expansion of y8 T lymphocytes was provided by studies documenting agerelated changes in the y~ T lymphocyte subset composition in peripheral blood in combination with studies analysing the expression of the " n a i v e " (CD45RA) and "primed" (CD45RO) markers by "r~ T lymphocyte subsets in the periphery. During the first years after birth, the frequency of y~ T lymphocytes in the periphery increases (Parker et al., 1990; Yachie et al.. 1989). This increase can be completely accounted for by an age-related expansion of the Vy9/V~2+ T-lymphocyte subset (Parker et al., 1990). We and others showed that virtually all V79/V82÷ T lymphocytes in peripheral blood have the CD45RO+ CD45RA- or low phenotype (Miyawaki et al., 1990; Braakman et aL, submitted). In contrast, the minor V~I T-lymphocyte subset in peripheral blood expresses CD45RA and lacks the expression of CD45RO. In vitro activa-
7~ T C E L L S
tion of V~I T lymphocytes with PHA leads to acquisition of CD45RO expression and decreased CD45RA expression. Moreover, all V31 T-lymphocyte clones have a CD45RA-,CD45RO+ phenotype. Therefore, it can be cor,cluded that CD45 isoforms are also genuine markers for naive and primed 78 T lymphocytes, respectively. Thus it appears that virtually all V79/V82+ T lymphocytes in peripheral blood are already primed, whereas the entire V~I ÷ T lymphocyte subset is naive. In contrast to peripheral blood, the majority of 78 T lymphocytes in the thymus express V~I, whereas V79/V~2 thymocytes are rare (Lanier et al., 1988). We found that V~I thymocytes can be subdivided into two subsets based on the expression of either CD45RA or CD45RO (Braakman et ai., submitted). For a[] thymocytes, it is believed that the CD45RA-,CD45RO+ subset is destined to intrathymic death because the generative potential is almost exclusively f o u n d in the CD45RA + ,CD45RO- thymocyte subset (Egerton et aL, 1990). This probably also holds true for 7~ thymocytes, since self-reactive 7~ thymocytes are eliminated in the thymus (Dent et al., 1990). Taken together, these observa*;-'"~ are ~vualaL~nL . . . . :o'~ • --':'~" the concept WI[II that V~I lymphocytes in the periphery descend from V~I ,CD45RA +, CD45RO- thymic precursors. The relatively low frequency of V79/V~2+ T lymphocytes in the thymus, together with the age-related selective expansion of primed V79/V~2 + T lymphocytes in peripheral blood, suggest that V79/V~2+ T lymphocytes are subject to extrathymic selection (Parker et al., 1990; Braakman et al., submitted). This notion is further substantiated by the presence of V-c9/V~2+ T lymphocytes in peripheral blood of athymic individuals (DiGeorge syndrome patients) (Van Dongen et al., submitted). LI%.PlIO
Activation of Vy9/V~2 T lymphocytes in vivo.
The expression of CD45RO on the entire V82+ T-lymphocyte subset in peripheral blood is difficult to reconcile
659
with stimulation of these lymphocytes by an extensive repertoire ol i~munogenic antigens, matching the Oolymorphism of the V79/V82 T ly,'~aplmcy~e population basedon the ~r,tclLive junctional diversity at the V.ii!gr-Jjoints of functionally rearranged YCR-~ genes (Hata et al., 1988). Rath~ r, it favours an activation by a single ,~atigen or by a closely related family of antigens. In analogy to subsets of 0t[3T lymphocytes which share a particular V[3 region and which can be activated by so-called superantigens, we suggest that V79/V~2 T lymphocytes in peripheral blood are in vivo activated by superantigens (see below). V81 T lymphocytes in peripheral blood have the naive CD45RA+ phenotype and consequently these lymphocytes have not been activated in vivo or, alternatively, once activated do not remigrate to peripheral blood. If the hypothesis is correct that V~2 but not VSI T lymphocytes are in vivo activated, it would imply that this superantigen is ubiquitous in man. Attractive candidates for such superantigens are autologous and mycobacterial heat shock (stress) proteins which are ubiquitous in man and to which human and mouse 7~ T lymphocytes reportedly respond (Born et al., 1990; Haregewoin et aL, 1989). The putative superantigen activation in vivo may occur by mature V82 T lymphocytes in the periphery or may be an integral part of the positive selection during extrathymic differentiation of V82 T iymphocytes.
V79/V~2 T lymphocytes specifically recognize Daudi cells. Proliferation of purified a[~ and 7~ T-lymphocyte populations can be sustained by stimulation with B-iymphoblastoid ceil lines (B-LCL) (Bosnes et al., 1989; Van de Griend et al., 1984). We observed that stimulation of fresh unfractionated PBL with irradiated Daudi cells, a Burkitt lymphomaderived cell line, resulted in the selective expansion of 7~ T lymphocytes within the CD3 ÷ population. This selective expansion was strictly confined to the V79/V82 T lymphocytes. Stimulation with several other B-LCL lines, e.g.
660
33 ~a F O R U M I N I M M U N O L O G
APD, BSM or Raji did not result in such selective expansion of V~,9/V~2 T lymphocytes, although the total lymphocyte yield was similar. These results suggest an antigen-specific stimulation of V~.9/V~2 T lymphocytcs by Daudi cells, and involving the V~.9/V~2 T C R It appeared that none of the V~I T-lymphocyte clones, but all V~,9/V82 T-lymphocyte -lones lysed Daudi cells, e ~en when tne-~~were generated in an aut , l o g o u s or non-Daudi B-LCLcomprising culture system (Sturm et al., 1990, in press), i.e. when they had never ';seen" Daudi cells before. The question is now whether the lysis of Daudi cells, cell line known to be susceptible to ".'~X ,t~tivity, is due to: a)MHCunrestricted lytic activity not involving the TCR (the hallmark of T C R / C D 3 - NK lymphocytes, but also exerted by activated "r~ T lymphocytes, and to a lesser extent, by a[3 T !ymphocytes) (Borst et aL, 1988; Christmas: 1989, Dastot et al., 1990; Jitsukawa e~ aL, 1988), or b) whether it involves the V'r9/V~2 TCR as the recognition structure, i The distinct patterns of lysis bet',:-een all these subsets of~'rCR- or TCR ÷ effector lymphocyt~s, and in particular the V-I'9/V~2 versus the V~ 1 T lymphoc.ytes, might reflect differential expresstun and/or involvement of multiple receptors, e.g. acee~or~ molecules in targe~ .~ell reccgnition (Bolhuis and Braakman, 1988). Overall, the disulphide-linked TCR V~.9/V~2 lym~&.-_~:~,';~exert higher MHC-unrestricted cytolyt,.'c activity than either the disulphide or non-disulphide-linked TCR V~I lymphocytes (Christmas, 1989; Dastot et aL, 1990; Jitsukawa et aL, 1988; Sturm et aL, 1989). Although both subsets of -¢~ T lymphocytes express equal levels of adhesion molecules such as CD2, ICAM-1, LFA-I and LFA-3 (Fisch et al., 1990), they have been reported to differ in their cell matrix composition, which is reflected by differences in their respective adherence and motility capacities. These differences may result in different target cell repertoires (Grossi et al., 1989). However, the VS1 cloned T lymphocytes formed conjugates with Daudi ceils as
Y
well as the V~,9/V~2 cloned T lymphocytes did with Daudi cells. Together with our observation that all cloned V~.9/V82 T lymphocytes lyse and specifically proliferate (Fisch et aL, 1990, in press) to Daudi cells even when generated in the absence of Daudi cells, these data support the idea of direct TCR~'~ involvement in Daudi cell recognition. The specific proliferation to and cytolysis of Daudi cells by entire subpopulation of V'r9/V~2 T lymphocytes is reminiscent of a superantigen reponse as described for e.g. V[38 (Marrack and Kappler, 1988) and V't9 (Rust et al., 1990) T lymphocytes to staphylococcal enterotoxins. In superantigen stimulation, the V138- or V'r9-encoded TCR chain in itself is sufficient to impose superantigen specificity, i.e. independent of V0t or V~ usage in the associated TCR0c or TCR~ chain. In the case of this proposed analogy, the V'l'9 chain is expected to dictate Daudi cell specificity. However, we found that two V~.9/V~l clones of thymic origin did not lyse Daudi cells, implying that the V~,9-encoded TCR~, chain in itself is not sufficient to dictate Daudi cell specificity. Therefore, either the expression of a V82-encoded TCR8 chain alone is sufficient to dictate Daudi cell specificity or, alternatively, the combination of a V'r9- and V82-encoded TCR chain is required. Also, the possibility remains that thymic V'r9/V~ 1 clones do not !yse D.audi cells because of their thymic origin. Another feature of "classical" superantigen recognition is restriction of presentation by MHC class II molecules. Dat~di cell specificity of V'r9/V~2 T lymphocytes could not be blocked t~y anti-MHC class II mAb. We therefore suggest that any stimulus that activates an entire subset of lymphocytes with a particular TCR gene rearrangement represents a superantigen. The presentation of such superantigens to ~,~ T lymphocytes may not necessarily be restricted by MHC molecules. Interestingly, the V~,9/V82, but not the VS1, T lymphocytes ~pecifically proliferate to mycobacterial antigens (Fisch et al., 1990, in press). These mycobacterial antigens can be presented by MHC class I negative B-LCL, including Daudi cells. The proliferative
~
T CELLS
response to Daudi cells can be blocked by polyclonal anti-heat shock protein (HSP-58) antibodies. More importantly, preliminary data suggest that heat shock proteins can be isolated from the surface of Daudi target cells (Fisch et al., 1990, in pre~s).
The physiological role of V-f9/V82 T iymphocytes ? Heat shock (stres~ proteins are constitutatively expresseca mtracellularly to sustain essential normal cell growth functions (Welch, 1990, in press). T lymphocytes from a healthy individual can specifically react to a self heat shock protein. Under physiological conditions, such self proteins may not be functionally present on the membrane of normal cells, due to either lack of intracellular processing or too low a surface expression. In contrast, stressed cells may express heat shock proteins in an immunogenic form at their surface. This may result in the reactivation of the C D 4 5 R A - , C D 4 5 R O ÷ V-f9/V~2 T lymphocytes; their physiologic function may be to produce cytokines to "calm" the stressed cells or to eliminate them by cytolysis. I~,..Jll',r~g~#l Tnrlo~-
"~snf~,Lr,"~t,~hl~ ,-~,~,nA;+; . . . . . . .~ U t . , l l I-, I.,~III[,I. YIL]II~ILI.JI~" I.,~JIIIdlLIW~.PlI~
as episodes of infection, V~,9/V82 T lymphocytes can be activated by the evolutionary highly conserved heat shock proteins present on many organ-
66!
isms, such as bacteria (Fisch et aL, 1990, in press). Because of the above mentioned high sequence homology of stress proteins from bacteria to man, this activation of V'r9/V82 T lymphocytes can induce an a u t o r e a c t i v e i m m u n e response against stressed cells at the site of infection. This may depend on whether the conserved or the speciesspecific determinants of the heat shock proteins are immunodominant. One would expect tolerance to the conserved, i.e. self-like, epitopes. Such tolerance may be actively maintained through the immune network. A functional relationship between immune responses against stress proteins and autoimmunity is demonstrated by the following observations, immune responses to stress proteins have been reported during autoimmune diseases. Moreover, in vitro responses of normal lymphocytes against stress proteins have been reported to contain an autoreactive component (Lamb et aL, 1989). Thus, the mechanism of such tolerance for self-like proteins may not be absolute. Dysregulation of actively maintained self tolerance may result from viral and/or bacterial infections and mount a V-f9/V~2 T lymphocyte response against self antigens, i.e. the conserved sequences of the stress proteins shared between bacteria and man. Genotyping and functional analysis of lymphocytes present at the inflammatory sites in patients with autoimmune disease may provide the experimental evidence.
References. BOLHUIS, R.L.H. & BRAAKMAN,E. (1988), Are cognitive receptors involved in nonspecific MHC-unrestricted lytic activity?, in "23th Forum In Immunology". Res. lmmunoL, 139, 460-465. BORN, W., HAPP, M.P., DALLAS,A., REARDON,C., KUBO,R., SHINNICK,T., BRENNAN,P. & O'BRIEN, R. (1990), Recognition of heat shock proteins and ~'~ cell function, hnmunol. Today, 2, 40-43. BORST, J., VANDE GRIEND, R.J., VANOOSFVEEN~J.W., ANG, S.-L., MELIEF,C.J.M., SEIDMAN, J.G. & BoLmnS,R.L.H. (1987), A T-cell receptor ~'/CD3 complex found on cloned functional lymphocytes. Nature (Lond.), 325, 683-688. BORST, J., VANDONGEN,J.J.M., BOLHUIS,R.L.H., PETERS,P.J., HAFLER,D.A., DE VRIES,E. & VANDE GRIEND, R.J. (1988), Distinct molecular forms of the T-cell receptor ~'8 detected on viable cells by a monoclonal antibody. J. exp. Med., 167, 1625-1644. BORST, J., WICHERINK, A., VAN DONGEN, J.J.M., DE VRIES, E., COMANZ-BITTER,W.H., WASSENAAR,F. & VANDENELSEN,P. (1989), Non-random expression of T-cell receptor "r and 8 variable gene segments in functional T lymphocyte clones from human peripheral blood. Europ. J. lmmunol., 19, 1559-1568.
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33 rd FOR U M IN I M M U N O L O G Y
BOSNES,V., HALVORENSEN,R., SKAFTADOTTIR,I., GAUDERNACK,G. & THORSBY,E. (1989), Specificity of -f~ receptor cytotoxic lymphocytes isolated from human peripheral blood. Scand. .L Immunoi., 29, 723-731. BRENNER,M.B., MCLEAN,J., SCHEFT,H., RIBERDY,J., ANG, S.-L., SEIDMAN,J.G., DEVLIN,P. t~ KRANGEL,M.S. (1987), Two forms of the T-cell receptor -f protein found on peripheral blood cytotoxic lymphocytes. Nature (Lond.), 325, 689-694. CASORAT~,G., DE LIBERO,G., LANZAVECCHIA,A. • MIGONE,N. (1989), Molecular analysis of human -f/B÷ clones from thymus and peripheral blood. J. exp. Med., 1"/0, 1521-1535. CHRISTMAS~S.E. (1989), Most human CD3 + WT31- clones with T-cell receptor C'rl rearrangements show strong non-MHC-restricted cytotoxic activity in contrast to those with C3,2 rearrangements. Europ. J. Immunoi., 19, 741-746. DASTOT, H., SCHMID, M., GONTIER, C., AMIOT, M., MATHIEU-MAHuL,D., BENSUSSAN,A. & BOUMSELL,L. (1990), Correlation between T-cell receptor ~ isotypic forms and cytotoxic activity: analysis with human T-cell clones and hnes. Ceil. Immunol., 25, 315-325. DEN1, A.L., MATIS, L.A., HOOSHMAND,F., WIDACKI,S.M., BLUESTONE,J.A. & HEDRICK,S.M. (1990), Self-reactive T~ T cells are eliminated in the thymus. Nature (Lond.), 343, 714-719. EGERTON,M., PRUSKI,E. &; PIEARSKI,L.M. (1990), Cell generation within human thymic subsets defined by selective expression of CD45 (T200) isoforms. Human Immunol., 27, 333-347. FAURE, F., JITSUKAWA,S., TRIEBEL, F. &. HERCEND,T. (1988), Characterization of human peripheral lymphocytes expressing the CD3-T/~ complex with anti-receptor monoc!onat antibodies. J. Immunol., 141, 3357-3360. FlscH, P., MALK~VSKY,M., BRAAKMAN,E., STURM,E., BOLHUIS,R.L.H., PRIEVE,A., SOSMAN, J.A., LAM, V.A. & SONDEL,P. (1990), Gamma/delta T-cell clones and natural killer cell clones mediate distinct patterns of non-MHC-restricted cytolysis. J. exp. Med., 171, 1567-1579. GROSSI,C.E., CICCONE,E., M!GONE,N., BOTTINO,C., ZARCONE,D., MINGARI,M.C., FERRINI,S., TAMBUSI, G., VIALE, O., CASORATI,G., MILLO, R., MORETTA, L. & MORETTA, A. (1989), Human T cells expressing the -~'/Breceptor (TCR-1) : CTI- and C~'2~encoded forms of the receptor correlate with distinctive morphology, cytoskeletal organization, and growth characteristics. Proc. nat. Acad. Sci. (Wash.), 86, 1619-1623. HAREGEWOIN,A., SOMAN,G., HUM, R.C. & FINBERG,R.W. (1989), Human TB+ T cells respond to mycobacterial heat-shock protein. Nature (Lond.), 340, 309-312. HATA, S., SATYANARAYANA,K., DEVEIN, P., BAND, H., MCLEAN, J., STROMINGER, J.L., RRI~NNI:R_M R ,~Z l~RA~r.~t M" ~ (IQRR~ l~vt~nclv~, i n n ~ t l r t r t u l rll,zarclt,tr atF ra~r_ ranged human T-cell receptor ~ genes. Science, 240, 1541-1544. JITSUKAWA,S., TRIEBEL, F., FAURE, F., MIOSSEC,C. & HERCEND,T. (1988), Cloned CD3 ÷ TCRa/[3 TiyA- peripheral blood lymphocytes compared to the TiTA + counterparts : structural differences of the ~'/~ receptor and functional heterogeneity. Europ. J. Immunoi., 18, 1671-1679. LAMB, J.R., BAL, V., MENDEz-SAMPERIO,P., MEHLERT,A., So, A., ROTHBAND,J., JINDAL,S., YOUNG, R.A. & YOUNG,R.B. (1989), Stress proteins may provide a link between the immune response to infection and autoimmunity. Int. lmmunol., l, 191-196. LANIER, L.L., RUITENBERG,J., BOLHUIS,R.L.H., BURST,J., PHILLIPS,J.H. & TESTI, R. (1988), Structural and serological heterogeneity of ~,/~ T-cell antigen receptor expression in thymus and peripheral blood. Europ. J. lmmunol., 18, 1985-1992. MARRACK,P. & KAPPLER,J. (1988), The T-cell receptor repertoire for antigen and MHC. Immunol. Today, 9, 308-315. MIYAWAKI,T., KASAHARA,Y., TAGA,K., YACHIE,A. & TANIGUCHI,N. (1990), Differential expression of CD45RO (UCHLI) and its functional relevance in two subpopulations ' ' of circulating TCR--f/~ + lymphocytes. J. exp. Med., 171, 1833-1838. PARt~.ER, C.M., GROH, V., BAND, H., PORCELL], S.A., MORITA, C., FABBI, M., GLASS, D., STROMINGER,J.L. & BRENNER,M.B. (1990), Evidence for extrathymic changes in the T-cell receptor y/~ repertoire. J. exp. Med., 171, 1597-1612. RUST, C.J.J., VERRECK, F., VIETOR, H. & KONING, F. (1990), Specific recognition of staphylococcal enterotoxin A by TCR Vy9- bearing human T cells. Nature (Lond.), 346, 572-574. SEKI, H., NANNO,M., CHEN, P.-F., ITOH, K., IOANNIDES,C., GOOD, R.A. & PLATSOUCAS,C.D. (1989), Molecular heterogeneity of T~ T-cell antigen receptors expressed by CD4-CD8- T-cell clones from normal donors: both disulphide- and non-disulphide-linked receptors are 8TCSI +. Proc. nat. Acad. Sci. (Wash.), 86, 2326-2330.
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STURIvl,E., BRAAKMAN,E., BONTROP,R.E., CHUCHANA,P., VANDEGRIEND,R.J., KONING,F., LEFRANC,M.-P. & BOLHUIS,R.L.H. (1989), Coordinated Vy and V~ gene segment rearrangements in human T-cell receptor ~,/8÷ lymphocytes. Europ. J. ImmunoL, 19, 1261-1265. STURM,E., BRAAKMAN,E., F1SCH,P., VREUGDENHIL,R.J., SONDEL,P. & BOLHUIS,R.L.H. (1990), Human V~'9-V82TCR~ lymphocytes show specificity to Daudi Burkitt's lymphoma cells. J. ImmunoL (in press). VANDEGRIEND,R.J., VANKRIMPEN,B.A., BOL,S.J., THOMPSON,A. & BOLHUIS,R.L.H. (1984), Rapid expansion of human cytotoxic T-cell clones. Growth promotion by heat-labile serum components and by various types of feeder cells. J. lmmunoL Methods, 66, 285-298. WELSCH,W.J. (1990), The mammalian stress response: physiology and biochemistry of stress protein, in "Stress protein in biology and medicine" (R. Morimoto, C. Georgopoulos & A. Tissieres) (pp. 223-278). Cold Spring Harbor Lalaoratory, New York. YACHIE, A., UENO, Y., TAKANO,N., MIYAWAKI,T. & TANIGUCHI,N. (1989), Developmental changes of double-negative (CD3 + 4- 8-) T cells in human peripheral blood. Clin. exp. lmmunoi., 76, 258-261.
REPERTOIRE SELECTION OF HUMAN T~ T CELLS
J. Borst (l) and J.J.M. van Dongen (2)
(i) Division o f Immunology, The Netherlands Cancer Institute, Amsterdam, and (2) Department of Immunology, Erasmus University, Rotterdam (The Netherlands)
Introduction. Research on ~'8 T cells has been very rewarding in the past few years, particularly for molecular biologists and biochemists, since the stage had already been set by previous investigations on 0~13T cells and the experiments to be done were obvious and straightforward. However, now that the molecular basis of -¢~T-cell receptor (TCR) diversity has been elucidated, we are left with the question as to how this diversity is employed in the immune response. It seems fair to say that we are all in the dark on this issue. Only thorough investigations in well defined in vivo systems will provide satisfactory answers. This second stage of ~8 T-cell research is still in its infancy. In this paper, therefore, we would like to focus on what we have learned about the ~ T-cell repertoire in human beings. In most healthy individuals, there is an extreme bias for expression
of the V~f9 and V~2 gene segments in peripheral blood (PB) "r,~T cells. Based on our studies in patients suffering from thyrnic hypoplasia and on analysis of changes in the ~ T-cell repertoire in healthy individuals with ageing, it would appear that this repertoire bias comes about by positive selection in the peripheral compartment. There are suggestions from in vitro experiments of other investigators that superantigens may drive this peripheral expansion. We would like to propose that the predominance of V~.9/V~2 expressing cells in PB of most healthy individuals arises fortuitously and does not provide insight into the inherent potential of peripheral "r~ T cells to recognize conversional antigenic peptides.
Biochemical evidence for TCR T~ repertoire bias. We first encountered human ~ T cells in two types of cellular materl-