Apr 8, 2015 - LFA-1 in different stages of the cytolytic. The role of the T cell receptor, CD8, and. Vries and H Spits. D Blanchard, C van Els, J Borst, S Carrel, ...
0022-1767/87/1388-2417802.00/0 THEJOURNAL OF IMMUNOLOGY Copyright 0 1987 by The American Aasoclatlonof lmmunolcglsts
VOl. 138.2417-2421. NO. 8. April 15. 1987 Prtnted In U.S.A.
THEROLEOFTHETCELLRECEPTOR,CD8, AND LFA-1 IN DIFFERENT BYALLOREACTIVE T STAGES OF THE CYTOLYTICREACTIONMEDIATED LYMPHOCYTECLONES DOMONIQUE BLANCHARD,' CECILE V A N ELS,~ JANNIE BORST,' STEFAN CARREL,' ART BOYLSTON,' JAN E. DE VRIES," AND HERGEN SPITS' From *Unicet Laboratories. 27 chemin des peupliers.69572Dardilly. France; 'Netherlands Cancer Institute. Amsterdam. The Netherlands;the 'Ludwig Institute for Cancer Research, Lausanne Branch, Epalinges, Switzerland;and 'St. Mary's Hospital Medical School, London. UK
The cytotoxic reaction mediated by cytotoxic T utilizing the T cell receptor (Tcr)' for antigen (1-4). Allymphocytes (CTL) consists of three phases: first, though structural studies on the DNA as well as the the CTL binds to the target cell: next, the CTL is protein level (5-10) have revealed striking similaries betriggered to lyse the target cell: and in the third tween immunoglobulins and theTcr, the latteronly recphase, the CTL detaches from the target cell which ognize cellbound antigen, whereasimmunoglobulins can is lysed in the absence of the CTL. Recently, we bind free antigen as well. The fact that T cells interact obtainedevidence that humanalloreactive CTL only with cellbound antigen may imply that the Tcrclones initially adhere to target cells without the antigen interactionis regulated by interactions involving involvement of the interaction between the T cell other membrane-bound antigens. Indeed, so-called accesreceptor (Tcr) and its specific target antigen.In the sory molecules such as CD4, CD8. CD2, and LFA-1 have present study, we investigated the effect of mono- been shown to be involved in the reactions mediated by clonal antibodies specific for the Tcr on the cyto- T cells (11-17). and it is possible that these antigens are toxic reaction of three CD8+ HLA-A2-specific CTL involved in theregulation of the Tcr-antigen interaction. clones, using a single cell assay in which the binding Cell-cell interactions leading to T cell activity have been event can be distinguished from the post-binding (lytic) phaseof the cytolytic reaction.It was found studied extensively with cytotoxic T lymphocytes (CTL). that monoclonal antibodies directed at a variable The reaction mediated by CTL has been shown to consist the adhere part of the Tcr do not affectthe binding phase but of three phases(18,19).In the first phase, CTL strongly block the lytic phase of the cytotoxic re- to the target cells. After adhesions are established, the lethal hit is delivered. In the third phase, the CTL deaction. An anti-constant region Tcr antibody and an anti- taches from the target cell and recycles, and lysis of the CD3 reagent had asimilar effect onthe two phases target cell proceeds in the absenceof the CTL. Recently, of the reaction as the anti-variable part Tcr anti- we found that CTL bind to target cells in the absenceof bodies. In contrast, antibodies specific for LFA-1 a n interaction between the Tcr and its target antigen strongly blocked the adhesion phase but did not (20). This antigen-nonspecific adhesion was found to be affect the lytic phase. Antibodies specific for CD-8 driven mainly by leukocyte function-associated antigenhad intermediate effects. They could block both the 1 (LFA-1).but it was shownthat CD8 can alsobe involved adhesion as well as the lytic phase. The effect of in antigen-nonspecific adhesions. These findings lndianti-CD8 appeared to be dependent onthe CTL clone cate that the Tcr-antigen interactiondoes not initiate the tested. One clone was found to be inhibited in the formation of conjugates. but occurs after the first adheadhesion phase, but not in the lytic phase, whereas sion between the CTL and the target cell. In this study, anti-CD8 hardly blockedthe adhesion phaseof two we investigated further the role of the Tcr in CTL reacthe lytic step of those other CTL clones. but affected mediated by CD8+ CTLclones by using monoclonal clones. Our data indicate that LFA-1 is a major tions antibodies directed at the variable part of the Tcr. It was adhesion molecule in the CTL reaction, whereas the found that the anti-Tcr antibodies strongly block the lysis Tcr/CD3 complexis implicated ina phase after the initial formation of conjugates. CD8 is associated mediated by CTL but do not affectthe adhesion between with both steps in the cytolytic reaction.In addition the CTL and target cells, supporting the notion that the to its minor role in the adhesion phase, our data Tcr-antigen interaction does not initiate the contact besuggest strongly that CD-8 is involved in the trig- tween the CTL and its target cell. Furthermore, our results demonstrate that CD8. in addition to being involved gering phaseof the cytolytic reaction. in the adhesion phase, also can play a role in the lytic phase.
T cells recognize cellbound antigen ina precise fashion MATERIALS AND METHODS
Recelved for publlcatlon September 26, 1986. Accepted for publication January 5, 1987. The costs of publication of thls artlcle were defrayed In part by the payment of page charges. Thls article must therefore be hereby marked aduertlsement In accordance with 18 U.S.C. Sectfon 1734 solely to In&cate thlsfact.
Monoclonal antlbodles. The monoclonal antibodies 1C1 and 3D6 (both 1 6 1 ) were raised independently against the T cell leukemic cell line HPB-ALL (21-23). These antibodies havebeen shown tobe
' Abbrevlatlons used In thls paper: CTL. cytotoxlc lymphocytes: Tcr.T cell receptor; LFA. leukocyte functlon-assoclatedantigen.
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T CELL ANTIGENS INVOLVED IN CYTOTOXICITY AND ADHESION
specific for the Tcr on HPB-ALL cells. In addition, 3D6 and 1C1 were HPB-ALL and, in addition, react with 2 to 10%of the found to react with the Tcrof 2 to 10%of the peripheral T cells of normal healthy donors (23). The antibody MX-6 was raised against peripheral T cells of healthy donors (23). We have used the T cell leukemic cell line Jurkat and recognizes the Tcr on Jurkat one such antibody (3D6) to raise cytotoxic T cell clones but not the Tcr on HPB-ALL cells. MX-6 reacted with 5% of the expressing the epitope recognized by 3D6. and four other peripheral blood lymphocytes of normal donors (24). The anti-CD8 antibody WT-82 (25) and WT-31. which recognizes a n epitope on independently derived antibodies specific for the Tcr of the constant part of the Tcr a&heterodimer (26-28). werea kind gift HPB-ALL cells (33).Furthermore, we found that theHLAof Dr. W. Tax (University of Nijmegen. The Netherlands). The anti- A2-specific CTL clone JR-2- 16,which has been described body SPV-L7 and SPV-L5 react with the a-chainof LFA-1 (29). The in detail elsewhere (13, 30, 31), reacted with antibody anti-CD3 antibody SPV-T3b has been described (29). CTL clones and other cell lines.The CTL clone JR-2-16 has been MX-6, which recognizes the Tcr of the T leukemic cell described (13, 30, 31).The clone was found to bespecific for HLA- line Jurkat (24). Table I shows that the anti-Tcr antiA2 as determined by panel studies,blocking studies withmonoclonal bodies strongly block the cytolytic activity of the T cell anti-HLA-A2 antibodies [ 13.30).and by its ability to lyse the human fibroblast cell line M1 but only when these cells were transfected clones in the51Cr-releaseassay. In addition, itwas found with HLA-A2 encoding genomic DNA (31).Clone JR-2-16 was found that the LFA-1 antibodies SPV-L5 or SPV-L7 and the to react withthe monoclonal anti-Tcr antibody MX-6. but not with anti-CD8 antibody WT-82 blocked the reactivities of all the anti-Tcr antibodies 3D6 or 1C1. as determined by indirect im- clones (Table I). munofluorescence. The CD8+ CTL clone JS-86 was derived from The eflects of anti-Tcr, anti-CD8, and anti-LFA1 on 3D6+ T cellsof donor JS (HLA-AS; 87: DR1) which were stimulated with the EBV-transformed B cell line J Y (HLA-AS; 87: DR4.6) (32. the different phases of the cytotoxic reaction deter33).The cytotoxic activity of clone JS-86 against JY could be blocked mined with a single cell assay. To determine at which by the anti-HLA-A2 antibody CR-11-351 (a kind gift of Dr. S . Ferrone. New York Medical School, Valhalla. NY). The CTL clone JS-25 phase themonoclonal anti-Tcr, LFA- 1, or anti-CD8 antiwas isolated from 3D6+T cellsof donor JS stimulated withthe EBV- bodies blocked the cytotoxic reactions of the three CD8+ transformed B cell line HEM (HLA-A2. 24: B7, 44; DR3, w6). The CTL clones, a single cell assay was used (20, 37). This 3D6+ 1C1+ clone JS-25 killed M1 cells transfected with HLA-A2 assay allows a distinction between the first adhesion genes. Moreover, its activity could be blocked by the monoclonal anti-HLA-A2 antibody CR-11-351. The clones were expanded as phase and the subsequent lytic phase. Table I1 shows described (34. 35). that the antibodies specific for a variable portion of the Target cells. The EBV-transformed cell lines JY. HEM, and QBL Tcr as well as WT-31, which is specific for a framework (HLA-A26; 818; DR3) and the Burkitt's lymphoma cell line Daudi Tcr a@-heterodimer, do not affectthe (HLA A-, B-. DRw6) were cultured in Yssel's medium with 1%AB+ determinant on the antibodies serum. TheHLA-A2+melanoma cell line 0-MEL I1 (36)was cultured percentage of conjugates, but the anti-Tcr in Dulbecco's modified MEM (GIBCO, Biocult, Glasgow, Scotland) strongly reduced the percentage of conjugates containing supplemented with 10%fetal calf serum. 5'Cr-release assay.The assays to measure cytolytic activity were nonviable targetcells. Similar resultswere obtained with the anti-CD3 reagent SPV-T3b. Furthermore, it is demperformed as described (35). Slngle cell assay. The single cell assay was performed as de- onstrated in Table I1 that theantibodies specific for LFAscribed by Bonavida et al. (37), with a few modifications. Effector 1 block the formation of conjugates. However, the percells were washedtwice in Iscove's medium and were then incubated with 40 ngcarboxy fluorescein diacetate (kindly provided by Dr. H. centage of nonviable cells in the remaining conjugates Bruning, Department of Immunohaematology, University Medical was not affectedby anti-LFA-1 antibodies. The anti-CD8 Center, Leiden. The Netherlands) per milliliter for 10 min at 37°C. antibody WT-82 was found to inhibitthe cytotoxic reacwere washed twice again, and were resuspended a t a concentration of 2 x 10' cells/ml in Iscove's medium supplemented with 0.25% tion in both the adhesion as well as in the lytic phase BSA. The target cells were washed once in Iscove's medium with (Table 11). However, the effect of anti-CD8 was observed 0.25% BSA and were resuspended a t a concentration of 2 x 10' to be dependent on the clone used in the assay.A s was cells/ml. Both cell samples were preincubated at 3OoC for 15 min. found previously (20). the anti-CD8 antibody blocked the At the onset of this preincubation, monoclonal antibodies were added to either the effector or the targetcells. The effector cells were mixed conjugate formation of clone JR-2-16 with J Y cells, but with target cells in a volume of 400 pl in no. 2054 Falcon tubes did not significantly inhibit the lytic phase. In contrast, (Becton Dickinson, Oxnard, CA) and were incubated for 10 min at WT-82 blockedthe lytic phases of the reactions mediated 30°C. followed by centrifugation at 250 X G for 5 min. The supernatant wasremoved and 50 pl Iscove's medium-0.25% BSA (kept a t by the clones JS-86 and JS-25. Theeffect of WT-82 on room temperature) were added to the pellet. Then, 50 pl of medium the conjugate formation of clones JS-25 and JS-86 was containing 1.3%agarose (a blend of Sigma type I and type VI11 not consistent. Although in some experiments WT-82 agarose a t a ratio of 1:5, and kept at 39°C)were addedand the tubes blocked conjugate formation, the average effect ofWTwere vortexed for 10 sec. after which time the agarose was spread 82 on the conjugate formation of clones JS-25 andJS-86 out on a slide (the slide was precoated with 2% agarose and was baked). After gelation of the agarose, the slides were incubated in was not significantly above background. Iscove's medium with 0.25%BSA for 3 hr at 37°C. The slides were The relationship between the presence of specific washed with PBS and were stained with 0.2% trypan blue in PBS followed by a fixation in 0.2% paraformaldehyde. The slides were TABLE I inspected immediately by fluorescence microscopy. The total numThe effectof monoclonal anttbodfes on the cytotoxtc actfulty of three ber of fluorescence-labeled CTL and the numberof CTL conjugated T8+CTL clones as determined with the "Cr-release assay' with viable or nonviable target cells were enumerated. The percentage of conjugates was the numberof conjugated CTL relative to the Inhibition of Lysisb Target Monoclonal total number of CTL. The percentage kill was calculated as: Ant@en JR-2-16JS-86 JS-25 Antibody No. of conjugates containing dead cells x 100 Total no. of conjugates
lClC
3D6 MX-6
Tcr Tcr Tcr
ND 0 60
ND
62
80 0 95
13 0 65
54 CD3 SPV-T3b 89 WT-82 CD8 73 60 40 30 30 1 SPV-L5 LFA50 88 89 4B HLA-A2 The effects of anti-Tcr, anti-CD8, and anti-LFA-1 antibodiesonthecytotoxicreactionsmediated by 'The cytotoxic activity was measured against JY cells. The effector to CD8+ CTL clones as determined with a 51Cr-release target ratio was 5 :1. The control lysis in the absence of monoclonal antibody was: JR-2assay. Recently, monoclonal antibodies have been de- 16.69%: JS-25.65%:and JS-66.71%. e The monoclonalantibodies were usedin a dilution of 1/200 of ascites. scribed which react with the Tcr on the leukemic cell line
RESULTS
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T CELL ANTIGENS INVOLVED IN CYTOTOXICITY AND ADHESION TABLE 11 The effectof monoclonal antibodfes on the conjugateformation and lyslsof JY cells medlated by theCTL clones JR-2-16,JS-25. and JS-86 % lnhibltlonn
Monoclonal Antlbodles formatlon
MX-6' 3D6 1c1 WT-31 SPV-T3b WT-82 SPV-L7
JS-86 CTL clones JS-25
JR-2-16
Conjugate formatlon
0 0 NDE 2 f 0.2 0 88 39 f 38 f 6
LYSIS
43 f 2.16 0
-
60 f 5 39 f 7.5 ? 3.6 0
Conlugate
ND 0 ND ND 0 17 f 9.2 25 f 5
LYSIS
56 f 4 -
47 f 9.2 26 f 4.6 8 f 4
Conjugate formation
ND ND 0 ND 0 13 f 5.1 43 f 1
LYSIS
-
47
* 5.7
-
42 k 8 32 2 0.6 6 4.6
*
"The percentage conjugate formed between the clones and JY. respectively, and the percentages of lysis were JR-2-16 (n = 5).65 1.7 and 57 f 4.9; JS-25 (n = 3), 64 f 4.6 and 33 f 3.4;JS-86 (n = 4). 62 f 4 and 52 f 15. The monoclonal antibodies were used in a 1/50 dilution of ascites. ND. not determined. Standard error ofthe mean.
*
TABLE 111 The numberof conjugates between JR-2-16and allogeneic target cells is not dependent of the presenceor absence of HLA-A2
anti-CD3 antibodies (Table 11). In contrast, LFA-1 antibodies only blocked the formation of conjugates. These findings indicate that the interaction between the Tcr % ConJugate ofCTL Clone JR-2-16 with DifferentTarget Cells and its target antigen, although obligatory for cytolytic QBL (-) JY (+)" M1A2 (+) 0-MEL I1 (+) M1 (-) Daudi (-1 activity, is not required for the formation of conjugates 75 6 f59f ' 1.7 42 3 f0 46f6 72f06+ 4 between CTL and target cells. This notion is supported "(+) or I-)denotes that the target cell expresses HLA-A2 and is killed by observations by us and other investigators (38)that (+) or does not express HLA-A2 and is not killed (-) by JR-2-16. 'Standard error ofthe mean (n = 5). antigen-specific CTL can form conjugates withtarget cells that do not express therelevant target antigen and antigen on the target cell and thenumber of conjugates. are therefore not killed (Table 111) (20). Importantly,LFAThe results presented in Table I1 indicate that CTL can 1 (20) a s well a s CD2 (38)appeared to be involved in form conjugates with target cells in the absence of a n adhesions of CTL with target cells that arekilled a s well interaction between the Tcr and antigen. This finding as with target cells that are not killed, emphasizing the extends the observations published previously that CTL physiologic relevance of the nonspecific conjugate forclones can form conjugates with target cells lacking the mations. Furthermore, it was observed previously that specific antigen but that this conjugate formation does HLA-A2- and -B7-specific CTL clones bind to and lyse not result in lysis of the target cell. To investigate the humanfibroblaststransfectedwith HLA-A2 and -87 relationship betweenthe formation of conjugates and the genes, whereas these clonesfailed to bind and therefore expression of the relevant target antigen, we analyzed to lyse mouse L cells transfected with the same HLA the percentages of conjugates between CTL clone JR-2- genes andexpressing these antigenson their membrane 16 (specific for HLA-A2) and various target cells. Table (20). These observations suggest strongly that nonspe111 shows that the numbers of conjugates formed between cific adhesions can be critical for the recognition of anclone JR-2- 16 and cells of the Class I MHC- negative cell tigen by the Tcr. The finding that theTcr-antigen interline Daudi or of the HLA-AB- cell line QBL (which were action is not involved in the formation of conjugates not killed) were significantly higher than theconjugates between CTL and target cells contrasts with observations obtained with M1A2 or 0-MEL-I1 cells, which express in the mouse, because in murine systems, it has been HLA-A2, and were killed bythis clone. The percentage of found that conjugate formation between CTL and target conjugates of JR-2-16 with M 1 and M1 transfected with cells is mainly antigen-specific (39-41).In most of these HLA-A2-encoding DNA was the same, but only M1A2 studies, however, antigen-independent conjugate formacells were killed (20). The lowest number of conjugates tion was observed, albeit much lower than antigen-dewas observed with the HLA-A2+ melanoma cell line 0- pendent conjugate formation. Therefore. these results do MEL-I1 that was killed by JR-2-16 cells (36).Together, not refute the idea that the Tcr-antigen interaction is these data indicate that the number of conjugates of preceded by a n antigen-independent conjugate formatarget cells with clone JR-2-16 is independent of the tion. It is possible that in these mouse systems, after the presence or absence of HLA-A2. initial nonspecific adhesion, the Tcr-antigen interaction further strengthens theconjugates. The reason why this DISCUSSION does not occur in our system is unclear. Our results and those of other investigators indicate In this paper, we report the effectsof monoclonal antibodies specific for the Tcr, CD8, and LFA-1 on different also that CD8 can play a role in adhesions between CTL phases of the cytolytic reaction mediated by various CD8+ and target cells (42, 43). However, here we show that CTL clones. It wasfound that monoclonal antibodies CD8 is probably also involved in the lytic phase. Antispecific for a variable part on theTcr did not affect the CD8 monoclonal antibodies clearly inhibitedthe lytic formation of conjugates, but strongly inhibited the lytic phase of the cytotoxic reaction of some CD8+ CTL clones phase. These antibodies had the same effect on the cy- (Table 11). The effects of anti-CD8 on the adhesion or on tolytic reaction a s WT 31,which is a monoclonal antibody the lytic phase were variable and were dependent on the specific for a common epitope on the Tcr (26-28) and clone that was used. In this respect, the effects of anti-
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IN CYTOTOXICITYANDADHESION
CD8 antibodies differed from those of the anti-LFA-1 or anti-Tcr antibodies that only blocked the adhesion and the lytic phase, respectively, of each CTL clone. It is unclear at present why anti-CD8 antibodies exert variable effects on the different phases of the reaction. This variable effect is not related to the quantitative differences in CD8,LFA-1, or Tcr expression on the different clones, because allthree clones expressed identical amounts of these antigens as determined with the FACS IV (results not shown). But it is possible that the affinity of the Tcr influences the effects of anti-CD8 antibodies on the cytotoxic reaction. Together, our findings indicate not only that CD8 acts as an adhesion structure, but that it is involved in the triggering of the lethal hit. Because anti-Tcr antibodies also block the lytic phase, it is likely that the Tcr and CD8 act ina coordinate fashion in the induction of lysis. It has been hypothesized that CD8 acts as an associative recognition element whichstrengthens theinteraction of Tcr with low affinity with its antigen, and there is considerablecircumstantial evidence that CD8 interacts with Class I MHC antigens (13, 43-45). CD8 can form homomultimers on the cell surface (46). Therefore, its role could be the mobilization of Class I MHC antigens on the interface between the CTL and the target cells, increasing locally the concentration of the class I MHC antigens which may facilitate the interaction between low affinity Tcr and theClass I MHC antigens. Recently, however, it has been demonstrated that anti-CD8 antibodies can block nonspecific cytotoxicity induced by antiCD3 antibodies in the absence of Class 1 MHC antigens on the targetcells (47.48). To explain this observation in light of the assumption that CD8 interacts with Class 1 MHC antigens, it has been postulated that triggering of CD8 confers a negative signa1 to the CTL, counteracting the positive signal given by the triggering of the Tcr. Our findings are not incompatible with either of these two hypotheses. To determine the exact role of CD8, it will be necessary to characterize precisely the ligand to which the CD8 binds. Moreover, more data are necessary concerning the functional relationship between CD8 and the Tcr.
Acknowledgments. The authors thankDr. S. Ferrone and Dr. W. Tax for their gift of monoclonal antibodies. Dr. H. Yssel, A. Voordouw, and J. Semeyn are gratefully acknowledged for their continuous support during this project, and S. Lorkens for herskillful typing of the manuscript. REFERENCES
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T CELL ANTIGENS
INVOLVED IN CYTOTOXICITY AND ADHESION
30. Spits. H.,M. Breuning. P. Ivanyi, and J. E. de Vries. 1982. In vitro isolated human cytotoxic T-lymphocyte clones detect variations in serologically defined HLA antigens. Immunogenetics 16:503. 40. 31. v. d. Rijn, M.. C. Bernabeu, B. Royer-Pokora, J. Seidman. J. E. de Vries, H. Spits, and C. Terhorst. 1984. Recognition ofHLA-A2by cytotoxic T lymphocytesafter DNA transfer into human and murine 41. cells. Science 226: 1083. 32. Borst. J.. A. W. Boylston, J. E. d e Vries. and H.Spits. 1986. Human T cell lines differing in phenotype and specificity are reactive with 42. the same anti-idiotypic antibody. J. Immunol. 136:601. 33. Borst. J.,H.Spits. A. Voordouw, E. de Vries. A. Boylston, andJ. E.de Vries. 1986. A familyof T cell receptor molecules expressed on 43. T cell clones with different speclficities forallo-major histocompatibility complex antigens. Hum. Immunol. 17:426. 34. Yssel. H..J. E. de Vries. M. Koken, W. van Blitterswijk. and H. Spits. 1984. Serum-freemedium for the generation and propagation of functional human cytotoxic and helper T cell clones. J. Immunol. 44. Methods 72:219. 35. Spits, H.,H.Yssel, C. Terhorst, andJ. E. de Vries. 1982. Establishment of human T lymphocyte clones highly cytotoxic for a n EBVtransformed B cell line in serum-free medium: isolation of clones 45. that differ in phenotype and specificity. J. Immunol. 128:95. 36. De Vries, J. E., and H. Spits. 1984. Cloned human cytotoxic T lymphocyte lines reactive with autologous melanoma cells. I. In vitro generation, isolation. and analysis to phenotype and specificity. J. 46. Immunol. 132:510. 37. B o n a v i d a , B., T. P. Bradley, and E. Grimm. 1983. Frequency determination of killer cells by a single cell cytotoxic assay. In Methods 47. in Enzymology. Vol. 93. J. J. Langone and H. Van Vanukis. eds. Academic Press, New York. P. 270. 38. Shaw. S.. G. E. Ginther-Luce. R. Quinones, R. E. Gress. T. A. Springer, and M.E. Sanders. 1986. Two antigen-independent adhe- 48. sion pathways used by human cytotoxic T cell clones. Nature 323:262. 39. Glasebrook, A. L. 1978. Conjugate formation by primary and sec-
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ondary populations of murine immune T lymphocytes. J. Immunol. 121:1870. Schick, B., and G. Berke. 1978. Is the presence of serologicallydefined target cell antigens sufficient for binding of cytotoxic T lymphocytes. Transplantation 26:14. Whisnant, C. C.. K. H. Sier, and D. B. Amos. 1978. Interaction of cytotoxic T lymphocytes with target cells. I. Specific inhibition by detergent-solubilized partially purified mouse histocompatibility complex antigens. J.Immunol. 121:2253. Landegren, H.,U. Ramsted. I. Axberg, M. Ullberg, and H.Wigzell. 1982. Selective inhibition of human T cell cytotoxicity at levels of target recognition or initiation of lysis. J. Exp. Med. 155:1579. Platsoucas. C. D. 1984. Human T cell antigens involved in cytotoxicity against allogeneic or autologous chemically modified targets. cell binding Association of the Leu 2a/T8 antigen with effector-target and of the T3/Leu 4antigen with triggering. Eur. J. Immunol. 14:566. Meuer, S. C.. S. F. Schlossman. and E. L. Reinhen. 1982. Clonal analysis of human cytotoxic T lymphocytes. T4+ and T8+ effector T cells recognize products of different major histocompatibility complex regions. Proc. Natl. Acad. Sci. USA 79:4395. Spits, H.,H.Yssel, A. Voordouw. and J. E. de Vries. 1985. Therole of T8 in thecytolytic activity of T8* CTL clones specific for Class I and Class I1 MHC antigens. J. Immunol. 134:2294. Snow, P., and C. Terhorst. 1983. The T8 antigen is a multimedc complex of two distinct subunits on human thymocytes but consists of homomultimeric forms on peripheral blood T lymphocytes. J. Biol. 14675. Chem. 258: Fleischer. B.. H.Schrezenmeier, andH.Wagner. 1986. Function of the CD4 and CD8 molecules on human cytotoxic T lymphocytes: regulation of T cell triggering. J. Immunol. 136: 1625. Van Seventer, G.. R.v. Lier, H. Spits, P. Ivanyi, and C.Melief. 1986. Evidence for a regulatory role of the T8 (CD8) antigen in antigenspecific and anti-T3 (CD3)induced lytic activity of allospecific cytotoxic-T-lymphocyte clones. Eur. J. Immunol. 11: 1363.
