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Expression of MHC class II and Tac antigens on IL2-activated human T cell clones that can stimulate in MLR, AMLR, PLT and can present antigen
Expression of MHC Class II and Tac Antigens on IL2-Activated Human T Cell Clones That Can Stimulate in MLR, AMLR, PLT and Can Present Antigen Frederic Triebel, Sabine De Roquefeuil, Catherine Blanc, Dominique J. Charron, and Patrice Debre
ABSTRACT: The expression of interleukin 2 (IL2) receptor and HLA-DR, DQ, and DP antigens on the surface of four diphtheria toxoid (DT)-specific T lymphocyte clones (TLC) and two TLC specific for an allogeneic EBV-transformed cell line was investigated with the use of monoclonal antibodies (MoAbs) that recognize defined molecules or epitopes. Incubation of a resting TLC with IL2 resulted in a 1O- to 30-fold increase in the level of DR, DQ, and Tac antigen expression. On the other hand, incubation of an activated TLC with IL2 decreasedfor i to 6 hr the level of expression of these three antigens. Anti-FA MoAbs did not react with any of the TLC tested suggesting that the expression of DR, DQ, and DP antigens is dissociated on activated TLC. Surface-marker analysis with anti-DR MoAbs indicated that DR epitopes were differently expressed at some activation stages of the TLC. Functional studies showed that activated TLC can stimulate in MLR, AMLR, and PLT. These proliferative responses were inhibited by preincubating the TLC with anti-DR MoAbs suggesting that the stimulatory determinants were predominantly DR molecules. In addition, some TLC can act as antigen presenting cells in DTspecific proliferative responses. These results indicate that MHC class II molecules on activated T lymphocytes may be relevant for the control of specific immunologic responses in vivo.
ABBREVIATIONS DT diphtheria toxoid TLC T lymphocyte clone IL2 interleukin 2 MoAb monoclonal antibody MLR mixed lymphocyte reaction
PLT PBM Var
primed lymphocyte testing peripheral blood mononuclear cells varidase
INTRODUCTION The surface membrane of activated lymphocytes expresses a number of gene products that differ from those found on resting T cells. Among them, Tac and Ia antigens represent common markers of T cell activation. These surface molecules have been detected on T cells activated in vitro by antigen or lectin stimulation [1,2]. From the Laboratoire d'Immunog~n~tique, D~partement d'H~matologie, C.H.U. Pitie-Salpetriere, 91, Boulevard de l'H$pital, 75013 Paris, France. Address reprint requests to Docteur Frederic Triebel, Laboratoire D'Immogenetique, Dept. d'Hematologie, CHU PITIE SALPETRIERE 91, Blvd. de l'H~pital, 75013 Paris, France. Received April 18, 1985; acceptedJuly 13, 1985.
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H u m a n Immunology 15, 3 0 2 - 3 1 5 (1986) © Elsevier Science Publishing Co., Inc., 1986 52 Vanderbilt Ave., N e w York, N Y 10017
Activated TLC Can Stimulate in MLR and Present Antigen
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Class II MHC antigens appear to regulate cell-cell interactions that are necessary for the initiation of immune responses including interactions between T cells and antigen presenting cells (APC). Recent progress in the understanding of class II antigens has provided evidence for the existence of at least three types of Ia-like molecules, namely DR, DQ, and DP antigens [3,4]. Class II antigens are expressed primarily on B lymphocytes, monocytes, macrophages, and dendritic cells. None of these antigens were expressed on resting T cells, whereas T cells activated by soluble antigens or alloantigens were found to express DR, DQ, and DP determinants [5]. In addition, the biosynthesis of DR antigens by T cells during the course of their activation has been demonstrated [6]. However, many questions concerning the physiological role of class II molecules on activated T cells have not been resolved. We established different T lymphocyte clones (TLC) that proliferate either after stimulation with diphtheria toxoid (DT) or with an allogeneic EBV-transformed cell line. These TLC required repetitive antigenic stimulation to resume growth. Studying the regulation of cell growth of these activated T cells, we observed that the cell surface density of Tac and class II antigens was affected by the addition every 3 to 4 days of an interleukin 2 (IL2) supplemented medium used for their growth. In the present report we describe experiments designed to monitor the kinetics of DR, DP, DQ antigens appearance and disappearance after stimulation of different TLC by IL2 supplemented medium. Furthermore, in order to relate the expression of class II molecules to their functional properties, we studied their capacity to stimulate in mixed lymphocyte reaction (MLR) autologous MLR (AMLR) primed lymphocyte testing (PLT) and for their capacity to present a soluble antigen. MATERIALS AND METHODS
Cell culture. TLC 24, 28, 29, and 30 are specific for DT and are restricted by the DR7 haplotype in their proliferative response to DT in the presence of APC [7]. They are derived from a DR 6/7 individual and were tested 2 to 3 weeks after the last stimulation with antigen and autologous filler cells. Allogeneic TLC P10 (T4 ÷, T 8 - ) and P16 (T4-, T8 ÷) were derived from a MLR between PBL of a DR2/5 individual and a DR 6/6 EBV-transformed cell line. The cloning procedure, the long-term culture, and the preparation of the growth factor, were described previously [7,8].
Monoclonal antibodies (moAbs). The first group of anti-DR moAbs, CA 141, D112, and VI-15C immunoprecipitates only DR molecules on DR 1-8 and detects very similar epitopic specificities [7]. The second group, CA 206 and CA 135, reacts with DR molecules on DR 1-6, whereas in DR7 individuals DR but also DQ molecules are recognized [7]. CA 206 and CA 135 detect very similar epitopic specificities using reciprocal binding inhibition to target cells in radioimmunoassay. SG 465 (a gift of Dr. Silver) detects DQ molecules on DR 1-7 and DR molecules on DR2 haplotype. Anti-FA MoAb (a gift of DR F.H. Bach) recognizes a determinant present only on DP molecules [9]. Anti-Tac MoAbs was kindly provided by Dr. Waldmann (NIH, Bethesda, MA).
Immunofluorescence analysis. All antibodies were used in antibody excess. D1-12, 141, 206, 135, and SG 465 were used as ascites fluid at a 1:100 dilution and anti-Tac at a 1 : 1000 dilution. The TLC were suspended for 1 hr at room temperature with pure AB serum to reduce back ground fluorescence levels and 3
304
F. Triebel et al. to 5 x 104 cells were then incubated in 50 /zl of MoAb or control ascite of irrelevant specificity. After incubation for 15 min at room temperature, the cells were washed twice in PBS-BSA containing 0.02% sodium azide and fluorescein labeled goat anti-mouse IgG (Cappel laboratories, Cochranville, PA) was added for 15 min at room temperature. The cells were then washed twice and analyzed on a Epics C cytofluorograph (Coulter Electronics, Hialeah, FL). All samples from a single kinetic were analyzed on the same day and back-ground fluorescence in the cytofluorograph was determined with a control antibody by using the same settings as those used for the relevant antibodies. Emission of fluorescence was logarithmically amplified and stored into histograms with a resolution of 256 channels. A minimum of 5000 cells were analyzed.
Proliferative assays. Autologous and allogeneic peripheral blood mononuclear cells (PBM) were tested in MLR (1 × 105 cells) with decreasing numbers of 2500 rad irradiated TLC. Cultures were incubated for 6 days in RPMI 1640 supplemented with 10% pooled human serum and glutamine. During the last 18 hr of culture, cells were pulsed with 3H thymidine (3HTdR) (1 /~Ci -- 37 kBq/well). The cells were harvested onto glass fiber filter paper by using an automatic cell harvester and the incorporated radioactivity was determined by liquid scintillation counting. The results are expressed as the mean counts/min of triplicate determinations. The standard error of the mean was less than 10%. The effect of anti-DR moAbs on these proliferative responses was tested by adding the respective antibody at tenfold dilution steps to appropriate wells at the beginning of cell culture. Only the data obtained with the first dilution (1 : 150) are expressed in the result section. Secondary MLR was performed by testing 10-day cultured lymphocytes (5 x 104 cells) primed against the autologous PBM with different irradiated TLC (1 × 104 cells) from the same individual. The secondary MLR were cultured for 48 hr and 3HTdR was added during the last 8 hr of the culture. TLC were also tested for their capacity to present a soluble antigen. 2500 rad irradiated TLC cells (1 × 104) were used as APC in the presence of the relevant antigen and these cells were incubated with DT and TLC responder cells (2 x 104) in a 3-day culture. Varidase (Streptokinase-Streptodornase) was used as a control antigen [8].
RESULTS Fluorescence Analysis of the Expression of DR, DP, DQ, and Tac Antigens on the ~urface of IL2-Stimulated TLC These studies utilized 4 DT-specific TLC (clones 24, 28, 29, and 30) and two allogeneic TLC (clones P10 and P16) which were maintained by serial stimulation with the antigen every 3 weeks alternating every 3 to 4 days with subculture in IL2-supplemented medium. The kinetics were started at least 15 days after the last antigenic stimulation and 5 days after the addition of IL2. The growth factor preparation was added 1, 6, 24, 48, 72, or 96 hr before the fluorescence analysis as indicated in Figures 1, 2, and 3. All the clones expressed variable amounts of DR and DQ antigens depending upon their activation stage whereas no DP antigen can be detected by the anti-FA MoAbs (see below). Without addition of IL2, these activation antigens were poorly expressed and the TLC cells finally died 3-5 days after the beginning of the kinetic. After addition of IL2, the same kinetic of activation was reproducibly obtained for a given TLC when the kinetics were started at least 15 days after the antigenic stimulation. Each clone exhibited
Activated TLC Can Stimulate in MLR and Present Antigen
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FIGURE 1 Analysis of the time-dependent expression of IL2 receptor, DR, and DQ antigens after stimulation of TLC 24 and 28 by IL2. For each time point the cells were labeled with anti-DR MoAb (D1-12, 141,206, 135), anti-DQ MoAb (465), anti-DP MoAb (anti-FA), and anti-IL2 receptor (anti-Tac).
a different time course o f acquisition or loss of these activation antigens. The peak o f activation was observed either at 1, 2, or 3 days and even at day 4 for P10. Clones 24 and P16 were poorly activated. Clone 29 expressed a high density o f activation antigens before the start of the kinetic and the addition of IL2 diminished the density of its activation antigens by at least a factor of 10 at time points 1 or 6 hr. On the whole, the kinetics of expression o f DR, DQ, and Tac antigens were similar except for TLC, P10, and P16 for which a dissociation between the level o f Tac and D R and D Q antigens was observed. The different mean fluorescence intensities observed on some time points may be accounted for by: (1) the fact that 206 and 135 recognize D R but also D Q antigens on DR7 cells (TLC 24, 28, 29, and 30) and that 206, 135, 141, and D1-12 belong to two different groups o f epitopic specificities on Ia molecules; (2) or alternatively, for the differential expression o f epitopic determinants on Ia molecules which are gradually expressed or lossed at the surface o f the TLC. D i f f e r e n t i a l D e t e c t i o n o f D R Epitopes R e c o g n i z e d by 141 and D1-12 m o A b s The differential expression of two closely related epitopes detected by 141 and D1-12 MoAbs was suggested by the mean fluorescence values obtained at 72 hr with TLC 28 and 30 and also at 24, 48, 72, and 96 hr with TLC 24. It was important to verify that these two MoAbs reacted in the same conditions with a EBV-transformed cell line and a PHA-stimulated T cell line maintained 1 month in culture, both cell lines from the same individual P H (DR 6/7) from which the clones were derived. The same mean fluorescence intensities were obtained with these two cell lines (Table 1). Kinetic studies on the time course of the appearance
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FIGURE 3 Analysis of the time-dependent expression of IL2 receptor, DR, and DQ antigens after stimulation of TLC P10 and P16 by IL2. For each time point the cells were labeled with anti-DR MoAb (D1-12,141,206, 135), anti-DQ MoAb (465), anti-DP MoAb (anti-FA), and anti-IL2 receptor (anti-Tac). ,,,
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TABLE
1
M e a n f l u o r e s c e n c e i n t e n s i t y o f d i f f e r e n t cell l i n e s d e r i v e d f r o m i n d i v i d u a l P H w i t h D 1 - 1 2 , 141, a n d a n t i - F A M o A b s Mean fluorescence intensity ascites
Cell linesa
Control
141
D 1-12
anti-FA
PHA-PH EBV-PH TLC 24 (96 hr) TLC 28 (72 hr) TLC 30 (96 hr)
0.78 1.12 0.54 0.97 0.87
7.74 12.42 2.29 1.34 4.56
7.96 12.36 4.16 6.25 7.56
4.77 11.04 0.61 1.11 0.92
~A EBV-transformed cell line and a PHA-stimulated T cell line cultured with IL2 for 1 month were tested with an optimal dilution of MoAb (1:100) 141, D1-12, and FA. These two lines were used as controls to demonstrate the differential expression of some DR epitopes and the absence of FA-related epitope on TLC 24, 28 and 30 (derived from the same individual PH).
F I G U R E 4 Differential d e t e c t i o n o f D R e p i t o p e s recognized by 141 and D 1-12 M o A b on TLC 28. S h o w n are the fluorescence-activated cell sorter histograms o f reactivity o f 141 and D 1 - 1 2 anti-Dr M o A b with TLC 28 cells 0, 24, 48, and 72 hr after stimulation by IL2.
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308
F. Triebel et al. and loss o f the epitopes recognized by 141 and D1-12 are illustrated in Figure 4 where cytofluorograph histograms o f TLC cells incubated from 0 to 72 hr in IL2-supplemented medium are shown. A significant increase of D R molecules was detected after 24 hr o f incubation. The level of D R antigen continued to increase until 72 hr as detected by moAb D1-12, whereas at this time point 141 did not stain TLC 28.
A n t i - F A m o A b s D o N o t R e a c t w i t h DT-Specific T L C or A l l o g e n e i c T L C This moAb did not recognize any surface antigen during the course o f activation o f the four DT-specific TLC or of P10 (Figures 1, 2, and 3). It is important to note that this moAb reacted with either a EBV-transformed cell line or a P H A stimulated T cell line derived from the same individual D R 6/7. The mean fluorescence intensity was much lower with the PHA-stimulated T cell line than with the EBV-transformed B cell line (Table 1). T L C S t i m u l a t e P r i m a r y L y m p h o p r o l i f e r a t i v e Response o f A u t o l o g o u s and A l l o g e n e i c P B M Forty-eight-hour IL2-activated TLC possessing high number of D R and D Q molecules at their surface (Figures 1 and 2) were irradiated (2500 rad) and titrated against a fixed number of normal PBM to test their stimulating ability in primary MLR. Figure 5 shows results obtained when using TLC 24, 28, and 29 to stimulate autologous PBM (individual A) or allogeneic PBM (individuals B, C, D). These results were compared to a classical MLR directed against irradiated PBM from individual A. Responses caused by irradiated TLC were equally high either with
FIGURE 5 TLC stimulate primary lymphoproliferative response of autologous and allogeneic PBM. 105-103 2500 tad irradiated PBM (MLR) or 104-102 irradiated TLC (clone 24, 28, and 30) were titrated against a fixed number of autologous PBM (A) or three allogeneic PBM (B, C, D) in a 5-day assay. o
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37.8 ( - 2 2 % ) 26.4 ( - 10%) 12.6 (8%)
141 29.5 (5%) 26.4 ( - 10%) 11.6 (15%)
135 9.2 (70%) 2.7 (89%) 3.1 (77%)
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M o A b s on the MLR a
7.5 (75%) 6.3 (74%) 3.7 (73%)
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al05 PBM of individual D (DR 4/6) and A (DR 6/7) were cultured for 5 days with 105 irradiated (2500 rads) PBM from individual A or with 104 irradiated cells from TLC 28 which is derived from individual A.
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TABLE 2
27.0 (13%) 17.0 (29%) 17.2 ( - 2 5 % )
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F. Triebel et al. autologous or allogeneic responder cells. Strong proliferative responses were observed even at a ratio of 1 : 100. Kinetic study indicates a peak proliferative response at 120 hr, consistent with the proliferative time course in classical MLR (data not shown). Very similar results were obtained using eight other TLC either allogeneic or specific for DT and derived from three other individuals (data not shown), indicating that this stimulatory ability was not restricted to the DR 6/7 haplotypes of A (individual PH).
Blocking with MoAb Indicates T h a t DR Antigens Are Involved in Primary Lymphoproliferative Stimulation by TLC The ability of TLC 28 to stimulate autologous or allogeneic PBM in the presence of different class II specific MoAbs was compared to the ability of PBM from the clone donor to stimulate allogeneic PBM. D 1-12 and VI- 15 C that recognized only DR molecules, strongly inhibited the proliferative responses of individual D against A, of D against TLC 28, and of A against TLC 28, at a dilution of 1:150 (Table 2), and 1:1500 (data not shown). 206 and 135, which recognize DR and DQ antigens on TLC 28, did not block the response. Kinetic experiments indicated that 1-hr incubation of the cells with either D1-12 or VI-15 C, followed by three washings with medium, was sufficient to inhibit the response (data not shown). These experiments suggest that early activation events are linked to the stimulatory signals in the MLR. In order to discriminate the functional epitopes of the DR molecule, we incubated either the stimulatory cells (TLC) or the responder cells with one of these moAb. Table 3 shows that maximum inhibition effect was obtained by the incubation of TLC 28 with D1-12 or VI-15 C for 1 hr at 37°C. On this basis, the stimulatory antigens present on activated TLC would appear to be DR determinants. The mechanisms of the stimulation in the MLR and AMLR is under study in our laboratory. TABLE 3
Stimulatory determinants in the MLR are primarily DR molecules on the target cells a 3HTdR incorporation in A cpm (% inhibition) Ascites
MLR D vs. TLC 28 b D vs. TLC 28 (5 days)
D1-12 6258 (0%) 564 (91%)
VI-15C 6258 (0%) 1840 (70%)
D vs. TLC 28 (1 hr)
922 (86%)
D vs. TLC 28 (1 hr)
2561 (60%)
D vs. TLC 28 (1 hr)
518 (92%)
1127 (82%)
6850 (0%) 802 (89%)
6850 (0%) 423 (94%)
A vs. TLC 28 A vs. TLC 28 (5 days)
1183 (81%) 7472 ( - 19%)
A vs. TLC 28 (1 hr)
196 (97%)
655 (90%)
A vs. TLC 28 (1 hr)
5314 (22%)
7048 ( - 2 % )
A vs. TLC 28 (1 hr)
1030 (85%)
999 (96%)
a105 PBM of individual D (Dr 4/6) and A (autologous) were cultured for 5 days with 104 irradiated cells from TLC 28. t~Fhe underlined responder or stimulatory cells were incubated for the indicated time with either Dl-12 or VI-15C.
Activated TLC Can Stimulate in MLR and Present Antigen
311
A c t i v ated TLC Stimulate Secondary Responses Directed Against H L A - D Antigens All TLC were found to stimulate primary lymphoproliferative responses of autologous as well as allogeneic PBM (see above). Moreover, blocking experiments indicated that the stimulatory molecules were predominantly DR antigens on the irradiated TLC. Our results (Table 4) clearly show that different TLC activated for 48 hr with IL2 can induce secondary proliferative responses. TLC 24, 28, and 29 stimulated cells primed against DR determinants present on the autologous PBM (as shown by blocking experiments in Table 3). An I r r a d i a t e d TLC Can Present D T A n t i g e n to A n o t h e r Autologous DT-Specific TLC To test the capacity of activated TLC to present antigen, we incubated the presenting irradiated TLC with antigen and the responder TLC in a 3-day culture. These studies were performed after 3 weeks of clonal expansion with the addition of 5000 rad irradiated feeder cells and antigen at the beginning of the culture. Therefore we tested T-T interactions in the absence of contaminating macrophages or dendritic cells. Table 5 shows an example of stimulation of DT-specific TLC 29 by 2500 rad irradiated TLC 28, acting as APC. TLC 28 was unable to present antigen to resting TLC including itself (TLC 28) or TLC 24, even when the stimulating clone was activated for 1 or 2 days with IL2. Among a series of 20 DT-specific TLC derived from three different individuals, we found two other examples of antigen presentation by irradiated TLC. Surface expression of class II antigens on these TLC was not carefully studied and these results are not presented here. TLC 28 when used as on APC could reproducibly present antigen to TLC 29 and not vice versa. Because TLC 28 reacts with the B fragment of the toxin and TLC 29 reacts with the A fragment [8], it is possible that the epitope recognized by TLC 28 on the B chain is not accessible to TLC 28 when the intact DT molecule is presented by TLC 29.
DISCUSSION In this report, we examined the expression and some of the functional properties of MHC class II molecules on the surface of DT-specific TLC and of TLC derived from a MLR against an EBV-transformed cell line. Incubation of a resting TLC that expressed a low level of activation antigens with a IL2-supplemented medium resulted in a 10-30-fold increase in the level of DR, DQ, and IL2 receptor expression, 24-48 hr after stimulation. The kinetics
TABLE 4
Activated TLC can induce secondary proliferative responses a Irradiated stimulatory cells 3HTdR incorporation (mean ÷ SD)
PLT
A
D
TLC 24
TLC 28
TLC 29
TLC N 4
Dvs. A
55679 ± 3248
1034 ± 201
5913 -+ 6038
20158 ± 4192
32388 ± 6623
892 ± 524
~5 × l04 PI3M of individual D (DR 4/6), primed during 10 days with cells of individual A were cultured for 48 hr with 105 irradiated (2500 rad) PBM or with 104 irradiated TLC cells from A (TLC 24, 28, and 29) or from a different donor (TLC N4) as a control of specificity.
312
F. Triebel et al. TABLE 5
DT-specific proliferative responses of TLC 24, 28, and 29 in response to antigen and 2500 rad irradiated TLC acting as APC a Mean 3HTdR incorporation (± SD) APC
TLC
PBM
TLC 24
TLC 28
TLC 29
TLC 24 + DT TLC 24 + Var TLC 24
28382 (2962) 1002 (76) 348 (136)
188 (33) 190 (90) 146 (45)
322 (65) 264 (57) 185 (35)
141 (27) 191 (94) 123 (23)
TLC 28 + DT TLC 28 + Var TLC 28
47943 (5149) 887 (154) 377 (227)
324 (164) 301 (78) 256 (78)
413 (197) 278 (73) 509 (226)
208 (84) 260 (126) 276 (66)
TLC 29 + DT TLC 29 + Var TLC 29
49264 (4210) 1620 (334) 350 (167)
1901 (139) 475 (378) 582 (412)
17847 (2635) 347 (66) 374 (69)
757 (289) 134 (15) 202 (15)
~2 × 104 TLC cells were cultured for 3 days with 105 irradiated PBM or 104 irradiated TLC to study the antigen presentation capacity of irradiated TLC.
of expression of these antigens on the surface of a single TLC were generally similar except in two occasions where high numbers of DR and DQ molecules were found on cells expressing very low levels of IL2 receptors. The coordinate expression of these three antigens on activated TLC suggests that the regulation of IL2-receptor expression is not readily distinguishable from other antigens that appear on activated T cells. In addition, our results support the concept that IL2 receptor expression is regulatable in antigen-specific TLC and depend not only on the periodic stimulation by antigen [10-12], but also on the addition of exogeneous IL2. This observation with TLC is consistent with previous results, which showed that the expression of Tac antigen on peripheral blood T lymphocytes activated by low, nonmitogenic concentrations of OKT 3 moAb, is dependent upon exogenous IL2, suggesting that IL2 might induce its own receptor [13]. Another example of the modulation of these surface antigens is the decrease of Tac, DR, and DQ antigens 1-6 hr after stimulation by IL2 (Figure 2). The rapid modulation of Tac antigens by their ligand was surprising considering that a large portion of the Tac proteins on activated T cells possess a low affinity for IL2 that would exclude any interactions between these proteins and their ligand [14]. Our results would be explanable if human T-cell clones express a relative prevalence of high-affinity IL2 binding sites. Indeed, the proportion of high and low-affinity binding sites for the different cell types was found to vary [14]. The transient decrease of DR and DQ antigens at the surface of activated TLC could not be related to any changes in the expression of T3, T4, or T l l molecules as determined by immunofluorescence analysis (data not shown). Of interest is that FA moAb do not detect any DP determinant on the surface of the activated TLC. Dissociation in the expression of the three class II antigens has been reported for normal monocytes [5,15], endothelial cells [16], and hairy cell leukemia [ 17]. The question of the absence of DP molecules or more simply of its FA associated epitope on the surface of activated TLC has not been resolved in the absence of other known monospecific DP reagents. Many questions concerning the transport across the cell membrane and the maturation of class II molecules on the surface of the cell have not been resolved.
Activated TLC Can Stimulate in MLR and Present Antigen
313
In order to follow the expression of two closely related epitopes detected on a single molecule, we chose two anti-DR moAbs 141 and D1-12, which recognized very similar epitopes on D R molecules as determined by reciprocal binding inhibition to target cells in radioimmunoassay [7]. Our results suggest a differential expression o f the epitopic determinants recognized by these two moAbs on the surface o f some TLC at the end of the kinetic of activation. This study underlines that differential cell surface detection in selected cell populations is a tool more sensitive than competitive radioimmunoassay to dissect the fine topology of Ia determinants. Relatively little is known about the function of class II antigens on T cells. In particular, functional studies are lacking in mice, simply because activated murine T cells do not express Ia antigens. It is probable that this study with TLC may be relevant to the dissection of the functional properties of class II molecules on normal activated T cells. Several laboratories have reported that activated human T cells can stimulate in MLR and even present antigen [6,18,19]. However, in these experiments the T cell populations were arguably contaminated with monocytic or dendritic cells which could have accounted for the observed stimulation. Experiments with long-term cultured T cells were reported by Pawelec et al. [20] who observed that some but not all allogeneic TLC could stimulate rapid primary but not secondary lymphoproliferative responses. These TLC were found to be strongly suppressive when titrated directly into MLR and this suppression could account for the diverse effects reported in this study. All TLC tested were found to stimulate primary lymphoproliferative responses of autologous as well as allogeneic PBM. The pattern of inhibition by anti-DR moAbs was not altered whether the responding cells were autologous or allogeneic with the stimulatory clones. The same pattern was also observed when irradiated PBM were used instead of TLC 28. This observation suggests that the responder cells (either autologous or allogeneic) recognize the same class II determinants present on the stimulatory clone or irradiated PBM. Moreover, blocking experiments by incubating either the responder cells or the irradiated TLC 1 hr with D1-12 or VI-15 C (Table 3) indicate that the stimulatory molecules were predominantly D R antigens on the irradiated TLC. In addition, these antigens on TLC were also capable of stimulating a secondary proliferative response in PLT. In our laboratory, work is in progress to address the question of the structural analysis o f D R antigens expressed on activated TLC that are capable o f inducing autologous lymphoproliferative responses. Antigen presentation by cloned T cells represent an ideal model to study the association of class II molecules and the antigen, the role of ILl and the processing of the antigen in the events leading to activation of the responding TLC. In our hands, few TLC were able to present antigen efficiently and in an antigen-specific and MHC-restricted fashion. Antigen presentation by a particular TLC may be linked to a certain expression (either qualitatively or quantitatively) of class II molecules or to certain intrinsic properties such as secretion of ILl or the possibility to process antigens to some extent. Initially ascribed to macrophages, the function of APC has been extended recently to other cell types including dendritic [21], B cells [22], a number of Ia-positive tumor cell lines [23,24], and mouse L cells transfected with A~ k and A3k cloned genes [25]. These findings suggest that any cell that can express an appropriate amount o f class II antigens and process antigen, represents an APC for a TLC. Moreover, experiments with murine B cell hybridomas seem to indicate that the efficiency of antigen presentation correlates quantitatively with cell surface Ia antigen expression [26]. In this context, the present study on the expression o f class II antigens on TLC provides additive information to the
314
F. Triebel et al.
concept that the qualificative and quantitative aspects of class II antigens expression on TLC may be pivotal in the control o f T cell responsiveness during a proliferative response against a soluble or allogeneic antigen.
ACKNOWLEDGMENTS
The authors wish to thank Marie-Claude Couty for performing the PLT assay and Val~rie Perseil for typing the manuscript.
REFERENCES 1. Ko HS, Fu SM, Winchester RJ, Yu DTY, Kunkel HG: Ia determinants on stimulated human T lymphocytes. Occurence on mitogen and antigen-activated T cells. J Exp Med 150:246, 1979. 2. Uchiyama T, Broder S, Waldmann TA: A monoclonal antibody (anti-Tac) reactive with activated and functionnaly mature human T cells. I. Production of anti-Tac monoclonal antibody and distribution of Tac + cells. J Immunol 126:1393, 1981. 3. Shaw S, Johnson AH, Shearer GM: Evidence for a new segregant series of B cell antigens that are encoded in the HLA-D region and that stimulate secondary allogeneic proliferative cytotoxic responses. J Exp Med 152:565, 1980. 4. Goyert S, ShivelyJ, Silver J: Biochemical characterization of a second family of human Ia molecules, HLA-DS, equivalent to murine I-A subregion molecules. J Med 156:550, 1982. 5. Nunez G, Giles RC, Ball EJ, Hurley CK, Capra JD, Stastny P: Expression of HLA-DR, MT and SB antigens on human mononuclear cells: identification of two phenotypically distinct monocyte populations. J Immunol 133:1300, 1984. 6. Engleman EG, Benike CJ, Charron DJ: Ia antigen on peripheral blood mononuclear leukocytes in man. II Functional studies of HLA-DR positive cells activated in mixed lymphocyte reactions. J Exp Med 152:114, 1980. 7. Triebel F, Missenard-Leblond V, Couty MC, Charron DJ, Debr~ P: Differential inhibition of human antigen-specific T cell clone proliferative responses by distinct monoclonal anti-HLA-DR antibodies. J Immunol 132:1773, 1984. 8. Triebel F, Missenard-Leblond V, Autran B, Couty MC, Charron DJ, Debr~ P: Antigen-specific proliferative human T cell clones with specificity for diphtheria toxo~d: genetic and molecular restriction by class II antigens. EurJ Immunol 14:697, 1984. 9. Watson AJ, DeMars R, Trowbridge IS, Bach FH: Detection of a novel human class II HLA antigen. Nature 304:358, 1983. 10. Cantrell DA, Smith KA: Transient expression of interleukin 2 receptors. Consequences for T cell growth. J Exp Med 158:1895, 1983. 11. Reske-Kunz AB, Steldern DV, Rude E, Osawa H, Diamantstein T: Interleukin 2 receptors on an insulin-specific T cell line: dynamics of receptors expression. J Immunol 133;1356, 1984. 12. Hemler ME, Brenner MB, McLean JM, Strominger JL: Antigenic stimulation regulates the level of expression of interleukin 2 receptor on human T cells. Proc Natl Acad Sci USA 81:2172, 1984. 13. Welte K, Andreeff M, Platzer E, Holloway K, Rubin BV, Moore MAS, and Mertelsman R: interleukin 2 regulates the expression of Tac antigen on peripheral blood T lymphocytes. J Exp Med 160:1390, 1984.
Activated TLC Can Stimulate in MLR and Present Antigen
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14. Robb RJ, Greene WC, Rusk CM: Low and high affinity cellular receptors for interleukin 2. J Exp Med 160:1126, 1984. 15. Gonwa TL, Picker L, Raft H, Goyert S, Silver J, Stobo J: Antigen presenting capabilities of human monocytes correlated with their expression of HLA-DS, an la determinant distinct from HLA-DR. J Immunol 130:706, 1983. 16. Collins T, Korman AJ, Wake CT, BossJM, Kappes DJ, Fiers W, Ault KA, Gimbrone MA, Strominger JL, Pober JS: Immune interferon activates multiple class II major histocompatibility complex genes and the associated invariant chain gene in human endothelial cells and dermal fibroblasts. Proc Natl Acad Sci USA 84:4917, 1984. 17. Faille A, Turmel P, Charron DJ: Differential expression of HLA-DR and HLA-DC/DS molecules in a patient with Hairy cell leukemia: restoration of HLA-DC/DS expression by (12-0-tetradecanoyl phorbol-13-acetate), 5 azacytidine, and sodium butyrate. Blood 64:37, 1984. 18. Wollman E, Cohen D, Fradelizi D, Sasportes M, Dausset J: Different stimulating capacity of B and T lymphocytes in primary and secondary allogenic reactions: cellular detection of HLA-D products on T lymphocytes. J Immunol 125:2039, 1980. 19. Indiveri F, Wilson BS, Russo C, Quaranta V, Pellegrino MA, Ferrone S: Ia-like antigens on human T lymphocytes: relationship to other surface markers, role in mixed lymphocyte reactions and structural profile. J Immunol 125:2673, 1980. 20. Pawelec G, Schneider EM, Wernet P: Cloned human T lymphocytes with lymphostimulatory capacity preferentially activate suppressor cells. Eur. J Immunol 14:335, 1984. 21. Steinman RN, Witmer MG: Lymphoid dendritic cells are potent stimulators of the primary mixed leukocyte reaction in mice. Proc Natl Acad Sci USA 75:5132, 1978. 22. Chestnut RW, Grey HM: Studies on the capacity of B cells to serve as antigenpresenting cells. J Immunol 126:1075, 1981. 23. McKean DJ, Infante AJ, Nilson A, Kimoto M, Fathman CG, Walker E, Warner N: Major histocompatibility complex-restricted antigen presentation to antigen-reactive T cells by lymphocyte tumor cells. J Exp Med 154:1419, 1981. 24. Glimcher LH, Kim KJ, Green I, Paul WE: Ia antigen-bearing B cell tumor lines can present protein antigen and alloantigen in a major histocompatibility complex-restricted fashion to antigen-reactive T cells. J Exp Med 155:445, 1982. 25. Malissen B, Peele Price M, GovermanJM, McMillan M, White J, Kappler J, Marrack P, Pierres A, Pierres M, Hood L: Gene transfer of H-2 class II genes: antigen presentation by mouse fibroblast and hamster B cell lines. Cell 36:319, 1984. 26. Bekkhoucha F, Naquet P, Pierres A, Marchetto S, Pierres M: Efficiency of antigen presentation of T cell clones by (B cell × B cell lymphoma) hybridomas correlates quantitatively with cell surface Ia antigen expression. Eur J Immunol 14:807, 1984.
ABSTRACT: The expression of interleukin 2 (IL2) receptor and HLA-DR, DQ, and DP antigens on the surface of four diphtheria toxoid (DT)-specific T lymphocyte clones (TLC) and two TLC specific for an allogeneic EBV-transformed cell line was investigated with the use of monoclonal antibodies (MoAbs) that recognize defined molecules or epitopes. Incubation of a resting TLC with IL2 resulted in a 1O- to 30-fold increase in the level of DR, DQ, and Tac antigen expression. On the other hand, incubation of an activated TLC with IL2 decreasedfor i to 6 hr the level of expression of these three antigens. Anti-FA MoAbs did not react with any of the TLC tested suggesting that the expression of DR, DQ, and DP antigens is dissociated on activated TLC. Surface-marker analysis with anti-DR MoAbs indicated that DR epitopes were differently expressed at some activation stages of the TLC. Functional studies showed that activated TLC can stimulate in MLR, AMLR, and PLT. These proliferative responses were inhibited by preincubating the TLC with anti-DR MoAbs suggesting that the stimulatory determinants were predominantly DR molecules. In addition, some TLC can act as antigen presenting cells in DTspecific proliferative responses. These results indicate that MHC class II molecules on activated T lymphocytes may be relevant for the control of specific immunologic responses in vivo.
ABBREVIATIONS DT diphtheria toxoid TLC T lymphocyte clone IL2 interleukin 2 MoAb monoclonal antibody MLR mixed lymphocyte reaction
PLT PBM Var
primed lymphocyte testing peripheral blood mononuclear cells varidase
INTRODUCTION The surface membrane of activated lymphocytes expresses a number of gene products that differ from those found on resting T cells. Among them, Tac and Ia antigens represent common markers of T cell activation. These surface molecules have been detected on T cells activated in vitro by antigen or lectin stimulation [1,2]. From the Laboratoire d'Immunog~n~tique, D~partement d'H~matologie, C.H.U. Pitie-Salpetriere, 91, Boulevard de l'H$pital, 75013 Paris, France. Address reprint requests to Docteur Frederic Triebel, Laboratoire D'Immogenetique, Dept. d'Hematologie, CHU PITIE SALPETRIERE 91, Blvd. de l'H~pital, 75013 Paris, France. Received April 18, 1985; acceptedJuly 13, 1985.
3O2 0198-8859/86/$3.50~
H u m a n Immunology 15, 3 0 2 - 3 1 5 (1986) © Elsevier Science Publishing Co., Inc., 1986 52 Vanderbilt Ave., N e w York, N Y 10017
Activated TLC Can Stimulate in MLR and Present Antigen
303
Class II MHC antigens appear to regulate cell-cell interactions that are necessary for the initiation of immune responses including interactions between T cells and antigen presenting cells (APC). Recent progress in the understanding of class II antigens has provided evidence for the existence of at least three types of Ia-like molecules, namely DR, DQ, and DP antigens [3,4]. Class II antigens are expressed primarily on B lymphocytes, monocytes, macrophages, and dendritic cells. None of these antigens were expressed on resting T cells, whereas T cells activated by soluble antigens or alloantigens were found to express DR, DQ, and DP determinants [5]. In addition, the biosynthesis of DR antigens by T cells during the course of their activation has been demonstrated [6]. However, many questions concerning the physiological role of class II molecules on activated T cells have not been resolved. We established different T lymphocyte clones (TLC) that proliferate either after stimulation with diphtheria toxoid (DT) or with an allogeneic EBV-transformed cell line. These TLC required repetitive antigenic stimulation to resume growth. Studying the regulation of cell growth of these activated T cells, we observed that the cell surface density of Tac and class II antigens was affected by the addition every 3 to 4 days of an interleukin 2 (IL2) supplemented medium used for their growth. In the present report we describe experiments designed to monitor the kinetics of DR, DP, DQ antigens appearance and disappearance after stimulation of different TLC by IL2 supplemented medium. Furthermore, in order to relate the expression of class II molecules to their functional properties, we studied their capacity to stimulate in mixed lymphocyte reaction (MLR) autologous MLR (AMLR) primed lymphocyte testing (PLT) and for their capacity to present a soluble antigen. MATERIALS AND METHODS
Cell culture. TLC 24, 28, 29, and 30 are specific for DT and are restricted by the DR7 haplotype in their proliferative response to DT in the presence of APC [7]. They are derived from a DR 6/7 individual and were tested 2 to 3 weeks after the last stimulation with antigen and autologous filler cells. Allogeneic TLC P10 (T4 ÷, T 8 - ) and P16 (T4-, T8 ÷) were derived from a MLR between PBL of a DR2/5 individual and a DR 6/6 EBV-transformed cell line. The cloning procedure, the long-term culture, and the preparation of the growth factor, were described previously [7,8].
Monoclonal antibodies (moAbs). The first group of anti-DR moAbs, CA 141, D112, and VI-15C immunoprecipitates only DR molecules on DR 1-8 and detects very similar epitopic specificities [7]. The second group, CA 206 and CA 135, reacts with DR molecules on DR 1-6, whereas in DR7 individuals DR but also DQ molecules are recognized [7]. CA 206 and CA 135 detect very similar epitopic specificities using reciprocal binding inhibition to target cells in radioimmunoassay. SG 465 (a gift of Dr. Silver) detects DQ molecules on DR 1-7 and DR molecules on DR2 haplotype. Anti-FA MoAb (a gift of DR F.H. Bach) recognizes a determinant present only on DP molecules [9]. Anti-Tac MoAbs was kindly provided by Dr. Waldmann (NIH, Bethesda, MA).
Immunofluorescence analysis. All antibodies were used in antibody excess. D1-12, 141, 206, 135, and SG 465 were used as ascites fluid at a 1:100 dilution and anti-Tac at a 1 : 1000 dilution. The TLC were suspended for 1 hr at room temperature with pure AB serum to reduce back ground fluorescence levels and 3
304
F. Triebel et al. to 5 x 104 cells were then incubated in 50 /zl of MoAb or control ascite of irrelevant specificity. After incubation for 15 min at room temperature, the cells were washed twice in PBS-BSA containing 0.02% sodium azide and fluorescein labeled goat anti-mouse IgG (Cappel laboratories, Cochranville, PA) was added for 15 min at room temperature. The cells were then washed twice and analyzed on a Epics C cytofluorograph (Coulter Electronics, Hialeah, FL). All samples from a single kinetic were analyzed on the same day and back-ground fluorescence in the cytofluorograph was determined with a control antibody by using the same settings as those used for the relevant antibodies. Emission of fluorescence was logarithmically amplified and stored into histograms with a resolution of 256 channels. A minimum of 5000 cells were analyzed.
Proliferative assays. Autologous and allogeneic peripheral blood mononuclear cells (PBM) were tested in MLR (1 × 105 cells) with decreasing numbers of 2500 rad irradiated TLC. Cultures were incubated for 6 days in RPMI 1640 supplemented with 10% pooled human serum and glutamine. During the last 18 hr of culture, cells were pulsed with 3H thymidine (3HTdR) (1 /~Ci -- 37 kBq/well). The cells were harvested onto glass fiber filter paper by using an automatic cell harvester and the incorporated radioactivity was determined by liquid scintillation counting. The results are expressed as the mean counts/min of triplicate determinations. The standard error of the mean was less than 10%. The effect of anti-DR moAbs on these proliferative responses was tested by adding the respective antibody at tenfold dilution steps to appropriate wells at the beginning of cell culture. Only the data obtained with the first dilution (1 : 150) are expressed in the result section. Secondary MLR was performed by testing 10-day cultured lymphocytes (5 x 104 cells) primed against the autologous PBM with different irradiated TLC (1 × 104 cells) from the same individual. The secondary MLR were cultured for 48 hr and 3HTdR was added during the last 8 hr of the culture. TLC were also tested for their capacity to present a soluble antigen. 2500 rad irradiated TLC cells (1 × 104) were used as APC in the presence of the relevant antigen and these cells were incubated with DT and TLC responder cells (2 x 104) in a 3-day culture. Varidase (Streptokinase-Streptodornase) was used as a control antigen [8].
RESULTS Fluorescence Analysis of the Expression of DR, DP, DQ, and Tac Antigens on the ~urface of IL2-Stimulated TLC These studies utilized 4 DT-specific TLC (clones 24, 28, 29, and 30) and two allogeneic TLC (clones P10 and P16) which were maintained by serial stimulation with the antigen every 3 weeks alternating every 3 to 4 days with subculture in IL2-supplemented medium. The kinetics were started at least 15 days after the last antigenic stimulation and 5 days after the addition of IL2. The growth factor preparation was added 1, 6, 24, 48, 72, or 96 hr before the fluorescence analysis as indicated in Figures 1, 2, and 3. All the clones expressed variable amounts of DR and DQ antigens depending upon their activation stage whereas no DP antigen can be detected by the anti-FA MoAbs (see below). Without addition of IL2, these activation antigens were poorly expressed and the TLC cells finally died 3-5 days after the beginning of the kinetic. After addition of IL2, the same kinetic of activation was reproducibly obtained for a given TLC when the kinetics were started at least 15 days after the antigenic stimulation. Each clone exhibited
Activated TLC Can Stimulate in MLR and Present Antigen
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a different time course o f acquisition or loss of these activation antigens. The peak o f activation was observed either at 1, 2, or 3 days and even at day 4 for P10. Clones 24 and P16 were poorly activated. Clone 29 expressed a high density o f activation antigens before the start of the kinetic and the addition of IL2 diminished the density of its activation antigens by at least a factor of 10 at time points 1 or 6 hr. On the whole, the kinetics of expression o f DR, DQ, and Tac antigens were similar except for TLC, P10, and P16 for which a dissociation between the level o f Tac and D R and D Q antigens was observed. The different mean fluorescence intensities observed on some time points may be accounted for by: (1) the fact that 206 and 135 recognize D R but also D Q antigens on DR7 cells (TLC 24, 28, 29, and 30) and that 206, 135, 141, and D1-12 belong to two different groups o f epitopic specificities on Ia molecules; (2) or alternatively, for the differential expression o f epitopic determinants on Ia molecules which are gradually expressed or lossed at the surface o f the TLC. D i f f e r e n t i a l D e t e c t i o n o f D R Epitopes R e c o g n i z e d by 141 and D1-12 m o A b s The differential expression of two closely related epitopes detected by 141 and D1-12 MoAbs was suggested by the mean fluorescence values obtained at 72 hr with TLC 28 and 30 and also at 24, 48, 72, and 96 hr with TLC 24. It was important to verify that these two MoAbs reacted in the same conditions with a EBV-transformed cell line and a PHA-stimulated T cell line maintained 1 month in culture, both cell lines from the same individual P H (DR 6/7) from which the clones were derived. The same mean fluorescence intensities were obtained with these two cell lines (Table 1). Kinetic studies on the time course of the appearance
306
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FIGURE 3 Analysis of the time-dependent expression of IL2 receptor, DR, and DQ antigens after stimulation of TLC P10 and P16 by IL2. For each time point the cells were labeled with anti-DR MoAb (D1-12,141,206, 135), anti-DQ MoAb (465), anti-DP MoAb (anti-FA), and anti-IL2 receptor (anti-Tac). ,,,
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TABLE
1
M e a n f l u o r e s c e n c e i n t e n s i t y o f d i f f e r e n t cell l i n e s d e r i v e d f r o m i n d i v i d u a l P H w i t h D 1 - 1 2 , 141, a n d a n t i - F A M o A b s Mean fluorescence intensity ascites
Cell linesa
Control
141
D 1-12
anti-FA
PHA-PH EBV-PH TLC 24 (96 hr) TLC 28 (72 hr) TLC 30 (96 hr)
0.78 1.12 0.54 0.97 0.87
7.74 12.42 2.29 1.34 4.56
7.96 12.36 4.16 6.25 7.56
4.77 11.04 0.61 1.11 0.92
~A EBV-transformed cell line and a PHA-stimulated T cell line cultured with IL2 for 1 month were tested with an optimal dilution of MoAb (1:100) 141, D1-12, and FA. These two lines were used as controls to demonstrate the differential expression of some DR epitopes and the absence of FA-related epitope on TLC 24, 28 and 30 (derived from the same individual PH).
F I G U R E 4 Differential d e t e c t i o n o f D R e p i t o p e s recognized by 141 and D 1-12 M o A b on TLC 28. S h o w n are the fluorescence-activated cell sorter histograms o f reactivity o f 141 and D 1 - 1 2 anti-Dr M o A b with TLC 28 cells 0, 24, 48, and 72 hr after stimulation by IL2.
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308
F. Triebel et al. and loss o f the epitopes recognized by 141 and D1-12 are illustrated in Figure 4 where cytofluorograph histograms o f TLC cells incubated from 0 to 72 hr in IL2-supplemented medium are shown. A significant increase of D R molecules was detected after 24 hr o f incubation. The level of D R antigen continued to increase until 72 hr as detected by moAb D1-12, whereas at this time point 141 did not stain TLC 28.
A n t i - F A m o A b s D o N o t R e a c t w i t h DT-Specific T L C or A l l o g e n e i c T L C This moAb did not recognize any surface antigen during the course o f activation o f the four DT-specific TLC or of P10 (Figures 1, 2, and 3). It is important to note that this moAb reacted with either a EBV-transformed cell line or a P H A stimulated T cell line derived from the same individual D R 6/7. The mean fluorescence intensity was much lower with the PHA-stimulated T cell line than with the EBV-transformed B cell line (Table 1). T L C S t i m u l a t e P r i m a r y L y m p h o p r o l i f e r a t i v e Response o f A u t o l o g o u s and A l l o g e n e i c P B M Forty-eight-hour IL2-activated TLC possessing high number of D R and D Q molecules at their surface (Figures 1 and 2) were irradiated (2500 rad) and titrated against a fixed number of normal PBM to test their stimulating ability in primary MLR. Figure 5 shows results obtained when using TLC 24, 28, and 29 to stimulate autologous PBM (individual A) or allogeneic PBM (individuals B, C, D). These results were compared to a classical MLR directed against irradiated PBM from individual A. Responses caused by irradiated TLC were equally high either with
FIGURE 5 TLC stimulate primary lymphoproliferative response of autologous and allogeneic PBM. 105-103 2500 tad irradiated PBM (MLR) or 104-102 irradiated TLC (clone 24, 28, and 30) were titrated against a fixed number of autologous PBM (A) or three allogeneic PBM (B, C, D) in a 5-day assay. o
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37.8 ( - 2 2 % ) 26.4 ( - 10%) 12.6 (8%)
141 29.5 (5%) 26.4 ( - 10%) 11.6 (15%)
135 9.2 (70%) 2.7 (89%) 3.1 (77%)
D1-12
3HTdR incorporation in A cpm × 10 _3 (% inhibition) Ascites b
M o A b s on the MLR a
7.5 (75%) 6.3 (74%) 3.7 (73%)
VI-15 C
35.6 ( - 15%) 17.0 (29%) 12.0 (12%)
BM 50
bCells were cultured with a 1:150 dilution of each of the indicated anti-DR ascites or with a ascite of irrelevant specificity (control).
al05 PBM of individual D (DR 4/6) and A (DR 6/7) were cultured for 5 days with 105 irradiated (2500 rads) PBM from individual A or with 104 irradiated cells from TLC 28 which is derived from individual A.
Control
Effect o f A N T I - D R
MLR
TABLE 2
27.0 (13%) 17.0 (29%) 17.2 ( - 2 5 % )
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F. Triebel et al. autologous or allogeneic responder cells. Strong proliferative responses were observed even at a ratio of 1 : 100. Kinetic study indicates a peak proliferative response at 120 hr, consistent with the proliferative time course in classical MLR (data not shown). Very similar results were obtained using eight other TLC either allogeneic or specific for DT and derived from three other individuals (data not shown), indicating that this stimulatory ability was not restricted to the DR 6/7 haplotypes of A (individual PH).
Blocking with MoAb Indicates T h a t DR Antigens Are Involved in Primary Lymphoproliferative Stimulation by TLC The ability of TLC 28 to stimulate autologous or allogeneic PBM in the presence of different class II specific MoAbs was compared to the ability of PBM from the clone donor to stimulate allogeneic PBM. D 1-12 and VI- 15 C that recognized only DR molecules, strongly inhibited the proliferative responses of individual D against A, of D against TLC 28, and of A against TLC 28, at a dilution of 1:150 (Table 2), and 1:1500 (data not shown). 206 and 135, which recognize DR and DQ antigens on TLC 28, did not block the response. Kinetic experiments indicated that 1-hr incubation of the cells with either D1-12 or VI-15 C, followed by three washings with medium, was sufficient to inhibit the response (data not shown). These experiments suggest that early activation events are linked to the stimulatory signals in the MLR. In order to discriminate the functional epitopes of the DR molecule, we incubated either the stimulatory cells (TLC) or the responder cells with one of these moAb. Table 3 shows that maximum inhibition effect was obtained by the incubation of TLC 28 with D1-12 or VI-15 C for 1 hr at 37°C. On this basis, the stimulatory antigens present on activated TLC would appear to be DR determinants. The mechanisms of the stimulation in the MLR and AMLR is under study in our laboratory. TABLE 3
Stimulatory determinants in the MLR are primarily DR molecules on the target cells a 3HTdR incorporation in A cpm (% inhibition) Ascites
MLR D vs. TLC 28 b D vs. TLC 28 (5 days)
D1-12 6258 (0%) 564 (91%)
VI-15C 6258 (0%) 1840 (70%)
D vs. TLC 28 (1 hr)
922 (86%)
D vs. TLC 28 (1 hr)
2561 (60%)
D vs. TLC 28 (1 hr)
518 (92%)
1127 (82%)
6850 (0%) 802 (89%)
6850 (0%) 423 (94%)
A vs. TLC 28 A vs. TLC 28 (5 days)
1183 (81%) 7472 ( - 19%)
A vs. TLC 28 (1 hr)
196 (97%)
655 (90%)
A vs. TLC 28 (1 hr)
5314 (22%)
7048 ( - 2 % )
A vs. TLC 28 (1 hr)
1030 (85%)
999 (96%)
a105 PBM of individual D (Dr 4/6) and A (autologous) were cultured for 5 days with 104 irradiated cells from TLC 28. t~Fhe underlined responder or stimulatory cells were incubated for the indicated time with either Dl-12 or VI-15C.
Activated TLC Can Stimulate in MLR and Present Antigen
311
A c t i v ated TLC Stimulate Secondary Responses Directed Against H L A - D Antigens All TLC were found to stimulate primary lymphoproliferative responses of autologous as well as allogeneic PBM (see above). Moreover, blocking experiments indicated that the stimulatory molecules were predominantly DR antigens on the irradiated TLC. Our results (Table 4) clearly show that different TLC activated for 48 hr with IL2 can induce secondary proliferative responses. TLC 24, 28, and 29 stimulated cells primed against DR determinants present on the autologous PBM (as shown by blocking experiments in Table 3). An I r r a d i a t e d TLC Can Present D T A n t i g e n to A n o t h e r Autologous DT-Specific TLC To test the capacity of activated TLC to present antigen, we incubated the presenting irradiated TLC with antigen and the responder TLC in a 3-day culture. These studies were performed after 3 weeks of clonal expansion with the addition of 5000 rad irradiated feeder cells and antigen at the beginning of the culture. Therefore we tested T-T interactions in the absence of contaminating macrophages or dendritic cells. Table 5 shows an example of stimulation of DT-specific TLC 29 by 2500 rad irradiated TLC 28, acting as APC. TLC 28 was unable to present antigen to resting TLC including itself (TLC 28) or TLC 24, even when the stimulating clone was activated for 1 or 2 days with IL2. Among a series of 20 DT-specific TLC derived from three different individuals, we found two other examples of antigen presentation by irradiated TLC. Surface expression of class II antigens on these TLC was not carefully studied and these results are not presented here. TLC 28 when used as on APC could reproducibly present antigen to TLC 29 and not vice versa. Because TLC 28 reacts with the B fragment of the toxin and TLC 29 reacts with the A fragment [8], it is possible that the epitope recognized by TLC 28 on the B chain is not accessible to TLC 28 when the intact DT molecule is presented by TLC 29.
DISCUSSION In this report, we examined the expression and some of the functional properties of MHC class II molecules on the surface of DT-specific TLC and of TLC derived from a MLR against an EBV-transformed cell line. Incubation of a resting TLC that expressed a low level of activation antigens with a IL2-supplemented medium resulted in a 10-30-fold increase in the level of DR, DQ, and IL2 receptor expression, 24-48 hr after stimulation. The kinetics
TABLE 4
Activated TLC can induce secondary proliferative responses a Irradiated stimulatory cells 3HTdR incorporation (mean ÷ SD)
PLT
A
D
TLC 24
TLC 28
TLC 29
TLC N 4
Dvs. A
55679 ± 3248
1034 ± 201
5913 -+ 6038
20158 ± 4192
32388 ± 6623
892 ± 524
~5 × l04 PI3M of individual D (DR 4/6), primed during 10 days with cells of individual A were cultured for 48 hr with 105 irradiated (2500 rad) PBM or with 104 irradiated TLC cells from A (TLC 24, 28, and 29) or from a different donor (TLC N4) as a control of specificity.
312
F. Triebel et al. TABLE 5
DT-specific proliferative responses of TLC 24, 28, and 29 in response to antigen and 2500 rad irradiated TLC acting as APC a Mean 3HTdR incorporation (± SD) APC
TLC
PBM
TLC 24
TLC 28
TLC 29
TLC 24 + DT TLC 24 + Var TLC 24
28382 (2962) 1002 (76) 348 (136)
188 (33) 190 (90) 146 (45)
322 (65) 264 (57) 185 (35)
141 (27) 191 (94) 123 (23)
TLC 28 + DT TLC 28 + Var TLC 28
47943 (5149) 887 (154) 377 (227)
324 (164) 301 (78) 256 (78)
413 (197) 278 (73) 509 (226)
208 (84) 260 (126) 276 (66)
TLC 29 + DT TLC 29 + Var TLC 29
49264 (4210) 1620 (334) 350 (167)
1901 (139) 475 (378) 582 (412)
17847 (2635) 347 (66) 374 (69)
757 (289) 134 (15) 202 (15)
~2 × 104 TLC cells were cultured for 3 days with 105 irradiated PBM or 104 irradiated TLC to study the antigen presentation capacity of irradiated TLC.
of expression of these antigens on the surface of a single TLC were generally similar except in two occasions where high numbers of DR and DQ molecules were found on cells expressing very low levels of IL2 receptors. The coordinate expression of these three antigens on activated TLC suggests that the regulation of IL2-receptor expression is not readily distinguishable from other antigens that appear on activated T cells. In addition, our results support the concept that IL2 receptor expression is regulatable in antigen-specific TLC and depend not only on the periodic stimulation by antigen [10-12], but also on the addition of exogeneous IL2. This observation with TLC is consistent with previous results, which showed that the expression of Tac antigen on peripheral blood T lymphocytes activated by low, nonmitogenic concentrations of OKT 3 moAb, is dependent upon exogenous IL2, suggesting that IL2 might induce its own receptor [13]. Another example of the modulation of these surface antigens is the decrease of Tac, DR, and DQ antigens 1-6 hr after stimulation by IL2 (Figure 2). The rapid modulation of Tac antigens by their ligand was surprising considering that a large portion of the Tac proteins on activated T cells possess a low affinity for IL2 that would exclude any interactions between these proteins and their ligand [14]. Our results would be explanable if human T-cell clones express a relative prevalence of high-affinity IL2 binding sites. Indeed, the proportion of high and low-affinity binding sites for the different cell types was found to vary [14]. The transient decrease of DR and DQ antigens at the surface of activated TLC could not be related to any changes in the expression of T3, T4, or T l l molecules as determined by immunofluorescence analysis (data not shown). Of interest is that FA moAb do not detect any DP determinant on the surface of the activated TLC. Dissociation in the expression of the three class II antigens has been reported for normal monocytes [5,15], endothelial cells [16], and hairy cell leukemia [ 17]. The question of the absence of DP molecules or more simply of its FA associated epitope on the surface of activated TLC has not been resolved in the absence of other known monospecific DP reagents. Many questions concerning the transport across the cell membrane and the maturation of class II molecules on the surface of the cell have not been resolved.
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In order to follow the expression of two closely related epitopes detected on a single molecule, we chose two anti-DR moAbs 141 and D1-12, which recognized very similar epitopes on D R molecules as determined by reciprocal binding inhibition to target cells in radioimmunoassay [7]. Our results suggest a differential expression o f the epitopic determinants recognized by these two moAbs on the surface o f some TLC at the end of the kinetic of activation. This study underlines that differential cell surface detection in selected cell populations is a tool more sensitive than competitive radioimmunoassay to dissect the fine topology of Ia determinants. Relatively little is known about the function of class II antigens on T cells. In particular, functional studies are lacking in mice, simply because activated murine T cells do not express Ia antigens. It is probable that this study with TLC may be relevant to the dissection of the functional properties of class II molecules on normal activated T cells. Several laboratories have reported that activated human T cells can stimulate in MLR and even present antigen [6,18,19]. However, in these experiments the T cell populations were arguably contaminated with monocytic or dendritic cells which could have accounted for the observed stimulation. Experiments with long-term cultured T cells were reported by Pawelec et al. [20] who observed that some but not all allogeneic TLC could stimulate rapid primary but not secondary lymphoproliferative responses. These TLC were found to be strongly suppressive when titrated directly into MLR and this suppression could account for the diverse effects reported in this study. All TLC tested were found to stimulate primary lymphoproliferative responses of autologous as well as allogeneic PBM. The pattern of inhibition by anti-DR moAbs was not altered whether the responding cells were autologous or allogeneic with the stimulatory clones. The same pattern was also observed when irradiated PBM were used instead of TLC 28. This observation suggests that the responder cells (either autologous or allogeneic) recognize the same class II determinants present on the stimulatory clone or irradiated PBM. Moreover, blocking experiments by incubating either the responder cells or the irradiated TLC 1 hr with D1-12 or VI-15 C (Table 3) indicate that the stimulatory molecules were predominantly D R antigens on the irradiated TLC. In addition, these antigens on TLC were also capable of stimulating a secondary proliferative response in PLT. In our laboratory, work is in progress to address the question of the structural analysis o f D R antigens expressed on activated TLC that are capable o f inducing autologous lymphoproliferative responses. Antigen presentation by cloned T cells represent an ideal model to study the association of class II molecules and the antigen, the role of ILl and the processing of the antigen in the events leading to activation of the responding TLC. In our hands, few TLC were able to present antigen efficiently and in an antigen-specific and MHC-restricted fashion. Antigen presentation by a particular TLC may be linked to a certain expression (either qualitatively or quantitatively) of class II molecules or to certain intrinsic properties such as secretion of ILl or the possibility to process antigens to some extent. Initially ascribed to macrophages, the function of APC has been extended recently to other cell types including dendritic [21], B cells [22], a number of Ia-positive tumor cell lines [23,24], and mouse L cells transfected with A~ k and A3k cloned genes [25]. These findings suggest that any cell that can express an appropriate amount o f class II antigens and process antigen, represents an APC for a TLC. Moreover, experiments with murine B cell hybridomas seem to indicate that the efficiency of antigen presentation correlates quantitatively with cell surface Ia antigen expression [26]. In this context, the present study on the expression o f class II antigens on TLC provides additive information to the
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concept that the qualificative and quantitative aspects of class II antigens expression on TLC may be pivotal in the control o f T cell responsiveness during a proliferative response against a soluble or allogeneic antigen.
ACKNOWLEDGMENTS
The authors wish to thank Marie-Claude Couty for performing the PLT assay and Val~rie Perseil for typing the manuscript.
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