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Monoclonal antibody OKT3-induced T cell proliferation: Differential role of HLA class II determinants expressed by T cells and monocytes
CELLULARIMMUNOLOGY
125,79-91 (1990)
Monoclonal Antibody OKT34nduced T Cell Proliferation: Differential Role of HLA Class II Determinants Expressed by T Cells and Monocytes’ CIRO MANZO,* GIUSEPPINA RuCcmo,t~* LUIGI DEL VECCHIO,$ LUIGI RAcroppI,t22 GIUSEPPE PIROZZI,* MASSIMO TEMPONI,~ SOLDANO FERRONE,~ SILVIA FONTANA,” AND SERAFINO ZAPPACosTAt *Divisione di Immunologia. Istituto Tumori Fondazione Pascale, 80131 Napoli. Italy; TCattedra di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare. Universitci di Napoli Federico II, 80131 Napoli. Italy; SDivisione di Immunoematologia. Ospedale A. Cardarelli, 80131 Napoli, Italy; SDepartment ofMicrobiology & Immunology, New York Medical College, Valhalla, New York 10595; and I’Centro di Endocrinologia ed Oncologia Sperimentale de1 CNR, 80131 Napoli, Italy Receiwd April 25, 1989; accepted August 28, 1989 Monoclonal antibodies (MAb) to monomorphic determinants of HLA Class II antigens inhibit monocyte-dependent T cell proliferation induced by MAb OKT3 to a different extent, suggesting a differential regulatory role ofthe corresponding determinants in T cell proliferation. To elucidate the mechanism(s) underlying this pattern, the MAb CRlO-343 and Q5/6 with high inhibitory effect and MAb CR1 l-462 and CR12-356 with low inhibitory effect were characterized. Cross-inhibition studies showed that the four MAb recognize distinct determinants. The determinants recognized by MAb CR 1O-343 and CR 12-462 are spatially close. The determinants recognized by the four MAb appear to be functionally independent in MAb OKT3-induced T cell proliferation, since the inhibitory effect ofthe combination of MAb CR IO-343 and Q5/6 and of the MAb CR1 I-462 and CR 12-356 was additional but not synergistic. To compare the functional activity of HLA Class II determinants expressed by monocytes and by activated T cells in MAb OKT3-induced T cell proliferation, the effect of the four MAb on MAb OKT3induced T cell proliferation in a monocyte-dependent and in a monocyte-free system was studied. Dose-response and proliferation kinetics studies showed that the four MAb display a similar inhibitory effect on MAb OKT3-induced T cell proliferation in a monocyte-free system. These results suggest fine differences in the role played by monocyte- and T cell-bound HLA Class II determinants in the regulation of MAb OKT3-induced T cell proliferation. This functional heterogeneity may enhance the flexibility of HLA Class II antigens to mediate cell-cell interactions involved in the proliferative response to a variety of mitogenic stimuli. 0 1990 Academic Press, Inc.
’ Supported in part by grants from the Cons&ho Nazionale delle Ricerche (CNR), Roma (Progetti Finalizzati “Oncologia” (87.0 1490.44 and 88.00927.44) and “Ingegneria Genetica e Basi Molecolari delle Malattie Ereditarie” (86.00109.5 1 and 87.00876.5 I)), by grants from the Minister0 della Pubblica Istruzione, Roma, by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milano, and by NIH Grants AI2 I384 and CA39559. 2 Recipient of a research fellowship from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milano. 79 0008-8749/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.
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INTRODUCTION T cell proliferation induced by anti-CD3 monoclonal antibodies (MAb) represents a useful model for investigating the mechanisms underlying T cell proliferation triggered by antigen stimulation. In this system, T cell proliferation is induced by the binding of anti-CD3 MAb to a subunit of the antigen recognition structure complex (CD3-Ti). This effect is accessory-cell-dependent ( 1,2) and involves interleukin 1 (IL 1) (3,4) and interleukin 2 (IL 2) production as well as IL 2 receptors’ (IL 2R) induction on activated T cells ($6). HLA Class II antigens are involved in this phenomenon, since anti-HLA Class II MAb inhibit MAb OKT3-induced T cell proliferation (7,8). The effect is mediated by the interaction of MAb with the corresponding determinants expressed by monocytes and is not Fc-mediated, as the same effect is displayed by pepsin fragments and by the whole Ig of the MAb. MAb to distinct determinants of HLA Class II antigens display differential effects on IL 2 production and inhibit T cell proliferation with different kinetics and to a different extent, suggesting that the corresponding determinants expressed on monocytes play a differential regulatory role in the sequence of events leading to T cell proliferation. The aim of this study was threefold: first, we analyzed the spatial relationship of determinants defined by anti-HLA Class II MAb with high and low inhibitory effects on MAb OKT3induced T cell proliferation, to determine whether regions with distinct functional properties could be identified on HLA Class II molecules. Second, we tested the effect on MAb OKT3-induced T cell proliferation of combinations of MAb recognizing distinct determinants of HLA Class II antigens, to evaluate if the effect is additive or synergistic. Last, we compared the effect of anti-HLA Class II MAb with high and low inhibitory effects on the proliferation of MAb OKT3-activated T cells in a monocyte-free system, to determine if the differential regulatory role in T cell proliferation played by monocyte-bound determinants is also played by T cell-bound determinants. MATERIALS
AND METHODS
Cells Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood of healthy donors by Ficoll-Paque (Pharmacia Fine Chemicals AB, Uppsala, Sweden) density gradient centrifugation (400g for 30 min). Following three washings, PBMC were resuspended in medium RPM1 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), glutamine (2 mM), penicillin (100 U/ml), and streptomycin ( 100 pg/ml). MAb OKT3-preactivated T cells were obtained by sequential incubation of PBMC with MAb OKT3 (2 rig/ml) for 4 hr at 37°C with carbonyl iron for 30 min at 37°C removal of phagocytizing cells by a magnet, and washing and incubation for 30 min at 4°C with MAb OKB7 and Leu M3 and for 1 additional hr at 37°C with rabbit complement, diluted 1:2. Under these experimental conditions, T cells did not undergo lysis when incubated with rabbit complement. The purity of the resulting cell population, as evaluated in indirect immunofluorescence (IIF) with the MAb OK3, OKM5, anti-Ia, OKB7, Q5/13, and CRlO-343 was at least 95%. Cultured B lymphoid cells WALK were grown in medium RPM1 1640, supplemented with 10% FCS and glutamine (2 mM).
DIFFERENTIAL
Interleukin
ROLE
OF
HLA
CLASS
II EPITOPES
81
2
Human recombinant IL 2 was a gift of Dr. F. Sinigaglia (Hoffmann-La Co., Basel, Switzerland).
Roche &
MA6 and ConventionaE Antisera The anti-CD3 MAb OKT3 (IgG2a), OKB7 (IgGl), and OKM5 (IgGl) were purchased from Ortho Diagnostic Systems (Milan, Italy). The MAb Leu M3 (IgG2b), fluorescein isothiocyanate (FITC)-conjugated MAb anti-Tat (IgG2a), and phycoerythrin (PE)-conjugated MAb anti-HLA-DR (IgG2a) were obtained from BectonDickinson Italia SpA (Milan, Italy). The MAb anti-Ia (IgG2a) was obtained from Technogenetics SpA (S. Mauro Torinese, Italy). The anti HLA-DQw 1 MAb KS 11 (IgM); the anti HLA-DQw3 MAb KS 13 (IgG2b); the MAb Q5/6 (IgGZa) to a determinant expressed on all HLA-DR allospecificities but HLA-DR7; the anti-HLA-DR MAb CL4 13 (IgG2a), the anti-HLA-DP MAb B7/ 21 (IgG2a); the anti-HLA-DR, DP MAb CRlO-343 (IgG2a), CR1 l-462 (IgGl), and CR12-356 (IgGl); the anti HLA-DR, DQ, DP MAb Q5/13 (IgG2a), the anti-arsonate MAb R16.7 (IgGl); and the MAb 345.134 (IgG2a) to a 115-kDa glycoprotein (115K GP) expressed by a variety of cells were developed and characterized as described (9- 13). FITC-conjugated goat anti-mouse IgG antibody was purchased from Ortho Diagnostic Systems. MAb were purified from ascites fluid either by sequential precipitation with caprylic acid and ammonium sulfate (14) or by affinity chromatography on a protein A-Sepharose column ( 15). The purity of antibody preparations was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ( 16). Protein concentration was determined by the Lowry method (17). MAb were labeled with “‘1 utilizing the iodogen method (18). Serological Assays The competition assay to map the determinants recognized by MAb was performed in 96-well microtiter plates, as described (19). Direct immunofluorescence (IF) and IIF were performed as described (8), by using a cytofluorograph Spectrum III (Ortho Diagnostic Systems, Raritan, NJ). Proliferation
Assays
They were performed in 96-microwell plates. Cells (2 X 105) in 200 ~1 of medium RPM1 1640, supplemented with 10% FCS, anti-HLA Class II MAb, and MAb OKT3 (2 rig/ml), PHA-P (Sigma Chemical Co., St. Louis, MO) (4 pg/ml), or IL 2 (25 U/ ml) were incubated for 3 days in a 5% CO2 atmosphere. Then, 1 FCi = 37 kBq of [3H]thymidine ([3H]dThd, sp act, 47 Ci/mmol, Amersham International plc, Amersham, England) was added to each well. At the end of an additional 3-hr incubation at 37°C cultures were harvested and [3H]dThd incorporation was measured. Mixed Lymphocyte
Cultures
PBMC ( 1 X 1OS)were mixed with irradiated (3000 rad) allogeneic PBMC ( I X 10’) in a total volume of 200 11 of medium RPM1 1640 supplemented with 10% FCS.
82
MANZO
ET AL.
I*
IB
5c
a 4
8 cold
12 MAb
0 (j~glmll
4
a
12
FIG. 1. Mapping of the determinants recognized by anti-HLA Class II MAb CRIO-343, CR1 l-462, CR12-356, and Q5/6. Cultured B lymphoid cells WALK (1 X 105) were incubated with increasing concentrations of cold anti-HLA-DR MAb Q5/6 (0), anti-HLA-DR, DP MAb CRlO-343 (0), CR1 l-462 (0) and CR12-356 (A) and with “‘1-MAb Q5/6 (A), CRlO-343 (B), CR1 l-462 (C), and CR12-356 (D) (2 X lo5 cpm) for 3 hr at 4°C. Then cells were spun down and washed three times with cold medium, and pellets were counted in a gamma counter.
Cultures were incubated in the presence of anti-HLA MAb at 37°C in a 5% CO;! atmosphere for 7 days. Sixteen hours before harvesting the cultures, 1 PCi of [3H]dThd was added to each well and radioactivity incorporation was then measured. RESULTS Mapping OfDeterminants on MAb OKT3-Induced
Defined by Anti-HLA T Cell Proliferation
Class II MAb with D@erential
Efect
Screening of nine anti-HLA Class II MAb for their effect on MAb OKT3-induced T cell proliferation showed that the MAb CRlO-343, Q5/6, and Q5/13 displayed strong inhibitory effects on T cell proliferation, while the MAb CR1 l-462, CR1 2356, KS13, CL413, B7/21, and KS1 1 displayed weak inhibitory effects. The MAb CRlO-343 and Q5/6 on one hand and the MAb CR1 l-462 and CR12-356 on the other hand were selected for additional investigations. To determine the spatial relationship among determinants defined by the above MAb, competition experiments were performed utilizing cultured B lymphoid cells WALK as targets. As shown in Fig. 1, the MAb CRlO-343 and CR1 l-462 crossinhibited each other in their binding to targets, but were not affected in their binding
DIFFERENTIAL
ROLE
OF
HLA
TABLE Additive
Inhibitory
MAb 345.134
R16.7
48
-
-
48 4 8 -
-
8 16 16
16 32 -
83
II EPITOPES
1
Effect of the Combination of MAb Q5/6 OKT3-Induced T Cell Proliferation”
and CR lo-343
on MAb
(rig/ml) Q5/6
CRIO-343
8 8 16 16 32
4 4 8 -
32 32 -
CLASS
32
16 16 16
[‘H]dThd incorporation 88.7 86.6 91.3 71.6? 76.1 60.4 54.3 26.2 42.6 42.2 37.9 40.2 6.1
f 10.1” k 5.6 k 9.6 6.6 f 7.9 f 4.5 f 6.3 f 2.9 f 4.5 f 3.5 f 3.2 f 3.6 f 0.4
Inhibition (S)
19.2’ 14.2 31.9 38.8 70.5 52.0 52.5 57.3 54.7 93.2
” PBMC (2 X IO’) were incubated with MAb OKT3 (2 ng/mI) and with anti-HLA Class II MAb total volume of 200 ~1 for 72 hr at 37°C and then [3H]dThd incorporation was measured. ’ cpm X 10 ‘; mean f SD. ‘ As compared to [ 3H]dThd incorporation by PBMC incubated without MAb.
in a
by MAb Q5/6. While the binding of MAb CR 1 l-462 was not affected by MAb CR 12356, that of MAb CRlO-343 was partially inhibited by high concentrations of MAb CR 12-356. The binding of the latter was not affected by any of the other three MAb. The binding of MAb Q5/6 was partially inhibited by high concentrations of MAb CR 1 l-462 and CR 12-356. These results suggest that the four MAb recognize distinct determinants. The one recognized by MAb CR 1 l-462 is spatially close to that defined by MAb CRlO-343, but distant from that defined by MAb CR12-356. The determinants defined by MAb CRlO-343 and Q5/6 are spatially distant. Provided that the partial inhibition induced by high concentrations of MAb CR 1 l-462 and CR 12-356 does not reflect changes in conformation induced by the binding of the MAb, the determinant defined by MAb CRlO-343 is in spatial proximity to that defined by MAb CR12-356 and that defined by MAb Q5/6 with those recognized by MAb CR1 l-462 and CR12-356. Additive Inhibitory Proliferation
Eflect ofAnti-HLA
Class II MA6 on MA6 OKT3-Induced
T Cell
To evaluate the possible cooperative effect of inhibitory anti-HLA Class II MAb, PBMC were cultured with MAb OKT3 in the presence of the combination of the two MAb with a low inhibitory activity and of the two MAb with a high inhibitory activity. In either case, the effect of the combination was compared to the inhibition exerted by each MAb of the combination. At each of the concentrations tested, the inhibitory effect of the combination of MAb Q5/6 and CRlO-343 corresponded to the sum of the effect exerted by the same concentration of each MAb tested individu-
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TABLE 2 Additive Inhibitory Effect ofthe Combination of MAb CR1 l-462 and CRl2-356 on MAb OKT3-Induced T Cell Proliferation” MAb (r&ml) 345.134 -
1600 800 800
R16.7 -
1600 200 200 400 400 -
CR 1l-462
CRl2-356
-
-
200 200 400 400 800 800
200 200 400 400 800 800
[3H]dThd incorporation 86.1 f 5.3h 88.3 f 4.1 90.4 f 6.2 61.1 k 3.0 64.3 i 2.2 4 1.7 * 2.2 46.1 k2.1 60.7 f 4.3 34.7 f 2.6 38.4 f 2.1 49.6 + 2.8 20.6 f 1.6
Inhibition @)
30.9‘ 27.2 52.9 47.6 31.3 61.7 56.5 43.8 76.7
0 PBMC (2 X 105) were incubated with MAb OKT3 (2 rig/ml) and with anti-HLA Class II MAb in a total volume of 200 ~1 for 72 hr at 37°C and then [3H]dThd incorporation was measured. ’ cpm X 10-j; mean + SD. ‘ As compared to [3H]dThd incorporation by PBMC incubated without MAb.
ally. The same conclusion was reached for the combination of MAb CR1 l-462 and CR12-356, except for the highest concentration tested (800 fig/ml). At the latter concentration, the inhibition of T cell proliferation was less than the sum of the values obtained with each MAb. Representative results of at least three experiments are shown in Tables 1 and 2. The inhibition induced by the combination of the two MAb does not reflect a nonspecific effect due to high protein concentration, since no inhibition of proliferation was detected when cultures were added with high concentrations of anti- 115K GP MAb 345.134 and anti-arsonate MAb R 16.7. Furthermore, addition to cultures of either control MAb with one of the anti-Class II MAb did not affect the inhibitory activity of the latter. Efect of Monocytes on the Inhibition Induced T Cell Proliferation
by Anti-HLA
Class II MAb of MA6 OKT3-
To determine whether the differential inhibitory effect of anti-HLA Class II MAb on MAb OKT3-induced T cell proliferation reflected their interaction with different target cells, both sets of MAb were tested with purified monocytes and T cells. When monocytes pretreated with MAb CRlO-343 or CR12-356 were added to purified T cells stimulated with MAb OKT3, proliferation was inhibited only by MAb CR 1O-343 (Table 3). The effect was dose-dependent. To determine the direct effect of MAb on T cells, they were purified from PBMC incubated for 4 hr at 37°C with MAb OKT3 (2 rig/ml) and were cultured with IL 2 (25 U/ml) for up to 72 hr. Anti-HLA Class II MAb were added to the cultures at concentrations ranging from 1.2 to 40 pg/ml. The proliferation obtained under these
DIFFERENTIAL
85
ROLE OF HLA CLASS 11 EPITOPES TABLE 3
Effect of Monocytes Preincubated with Anti-HLA Class II MAb on MAb OKT3 Induced T Cell Proliferation” Preincubation with MAbh
T cells
Monocytes
+ +
+
R16.7
+
+
CR lo-343
+
+
CRl2-356
MAb (rg/W 40
108.8 f 9.3‘ (0)” 2.5 * 0.3 (97.8) 80.5 f 8.2 (27.0)
10
115.6 f 8.8 (0) 30.7 -t 3.8 (72.2) 95.8 * 10.2 (13.1)
2.5
0
113.4 f 12.6 (0) 83.3 k 7.2 (24.4) 111.zi 9.4 (0)
2.1 + 0.2 1.3 * 0.2 110.2 5 12. I (0)
il T cells (2 X IO’/200 ~1) and monocytes (2 X 104/200 ~1) were cultured with MAb OKT3 (2 rig/ml) for 3 days at 37°C. Then F3H]dThd incorporation was measured. ” Monocytes were incubated with MAb for 90 min at 37°C and washed three times with medium warmed at 37’C. ‘ cpm x 10 -j; mean f SD. “ Percentage inhibition as compared to [ ‘H]dThd uptake by cultures of T cells and untreated monocytes stimulated with MAb OKT3 (2 rig/ml).
experimental conditions was compared to that of unfractionated PBMC stimulated with MAb OKT3. The inhibitory effect of the four MAb on T cell proliferation was influenced by the presence of monocytes. While the MAb CR 1O-343 and Q5/6 inhibited the proliferation of unfractionated PBMC more than the MAb CR1 l-462 and CR 12-356, the four MAb did not differ in the extent of inhibition of the proliferation of purified T cells. In both culture systems, the inhibitory effect was dose-dependent, higher concentrations of MAb being required to inhibit purified T cell proliferation than to inhibit PBMC proliferation. In addition, while the proliferation of PBMC was completely inhibited by the four MAb, complete inhibition of purified T cell proliferation was never reached, even at high MAb concentrations (Fig. 2). To analyze the mechanism(s) underlying the latter observations, the kinetics of appearance of HLA Class II antigens (Table 4) as well as the distribution of IL 2 receptors expression in relation to these antigens on purified T cells (Table 5) was studied. Double IF staining with anti-Ia MAb and anti-CD25 MAb showed that about 10% of activated T cells express the CD25 antigen, but lack Class II antigens; the large majority lack both types of antigens; about 20% express both types of antigens; and about 10% express only HLA Class II antigens. Efect of Monocytes on the Kinetics of the Inhibition MAb OKT3-Induced T Cell Proliferation
by Anti-HLA
Class II A4Ab of
The kinetics of the inhibitory activity of the four MAb was influenced by the presence of monocytes in the culture system (Fig. 3). When the MAb were tested with unfractionated PBMC, the kinetics of the inhibitory activity of MAb CR 1 l-462 and CR 12-356 was different from that of MAb CR 1O-343 and Q5/6. The inhibitory activity of the latter two was reduced by about 50% when they were added to the culture
86
MANZO
ET
AL.
loor-
Of 0.1
0.5
1.0
2.5
MAb
5.0
10.0
25.0
1 50.0
@g/ml)
FIG. 2. Dose-dependent inhibition by anti-HLA Class II MAb of MAb OKT3-induced T cell proliferation in the presence of monocytes and in a monocyte-free system. Increasing amounts of anti-HLA-DR MAb Q5/6 (circle), anti-HLA-DR, DP MAb CRlO-343 (rhomboid), CR1 l-462 (square), CRl2-356 (triangle) were added with MAb OKT3 (2 rig/ml) to PBMC (2 X 10’) (closed symbols) and to IL 2 (25 U/ml)stimulated T cells (2 X 105) (open symbols) purified from PBMC, which had been pulsed for 4 hr at 37°C with MAb OKT3 (2 rig/ml). The anti- 115K GP MAb 345.134 and the anti-arsonate MAb R 16.7 were used as specificity controls (data not shown). At the end of a 3-day incubation at 37°C [3H]dThd incorporation was measured.
after a 6- to 8-hr incubation, while that of the former two was reduced by only 15%. The degree of inhibition caused by the four MAb did not markedly change when they were added up to 24 hr following the beginning of the incubation; the inhibition TABLE Kinetics
4
of Appearance of HLA Class II Antigens and of IL 2 Receptors on Activated T Cells Incubated with IL 2” Incubation
MAb
specificity
W Leu M3 CD14 Leu 12CDl9 OKTl 1 CD2 anti-Ia DR anti-Tat CD25
24
0 0.2h(23)C 0.5 (29) 2 (25) 93 (117) 6 (97) 4 (31)
(hr)
0.4 0 1 92 7 22
(29) (40) (111) (104) (89)
0 PBMC (2 X 106/ml) were incubated with MAb OKT3 isolated and cultured at the concentration of 2 X 10’/200 indicated times, cells were harvested, sequentially incubated bodies, and analyzed with a cytofluorograph. h Percentage stained cells. ’ Mean fluorescence intensity.
72
48 0.3 0 2 94 15 26
(21) (65) (108) (110) (120)
0.1 0 2 93 23 30
(20) (50) (111) (132) (138)
(2 &ml) for 4 hr at 37°C. Then T cells were ~1 in the presence of IL 2 (25 U/ml). At the with MAb and FITC-anti mouse Ig xenoanti-
DIFFERENTIAL
ROLE
OF TABLE
Expression
CD25
ofCD25
HLA-DR
-
+
+ +
+ -
HLA
CLASS
87
II EPITOPES
5
and HLA-DR Antigens on Activated for 72 hr in the Presence of IL 2“ First experiment 8.5” 63.3 17.5 10.7
T Cells Cultured
Second experiment 9.5 68.0 14.0 8.5
” PBMC (2 X IOh/ml) were incubated with MAb OKT3 (2 rig/ml) for 4 hr at 37°C and then T cells were isolated and cultured at a concentration of 2 X 106/ml in the presence of IL 2 (25 U/ml). After a 72-hr incubation, cells were incubated with FITC-MAb anti-Tat and with PE-MAb anti-HLA-DR and analyzed with a cytofluorograph. ” Percentage stained cells.
displayed a gradual additional reduction as the time of addition of the MAb to the cultures was progressively delayed. On the other hand, no difference was found in the kinetics of inhibition of the four MAb, when they were tested in a monocyte-free system. In addition, at variance with the results obtained with PBMC, the degree of inhibition gradually decreased as the time of addition of MAb to the culture was delayed. Efect ofAnti-HLA Class II MAb on the Proliferation and Allogeneic Lymphocytes
of T Cells Induced by PHA-P
To determine whether the differential inhibitory effect of each anti-HLA Class II MAb is a general phenomenon, the four MAb were tested for their effect on the proliferation of T cells in two other systems, i.e., stimulation with PHA-P and with allogeneic lymphocytes, in which the inhibitory effect of anti-HLA Class II MAb had been already reported (7,20). The four MAb inhibited PHA-P-induced T cell proliferation to a similar extent, no appreciable difference being detected between MAb with high and low inhibitory activity on MAb OKT3-induced T cell proliferation. The four MAb inhibited proliferation to the same extent; in allogeneic MLR, the effect was dose-dependent (Table 6). DISCUSSION The present study analyzes several characteristics of four monomorphic determinants of HLA Class II antigens to elucidate the mechanism(s) underlying the marked difference in the extent of the inhibitory effect of the corresponding MAb on MAb OKT3-induced T cell proliferation. Results of cross-blocking experiments suggest that determinants recognized by MAb with marked difference in their inhibitory effect on MAb OKT3-induced T cell proliferation are spatially close to each other. This finding argues in favor of the presence of hot “points” with regulatory function rather than of hot “regions” on HLA Class II molecules. This possibility is also consistent with the observation that the combination of two MAb recognizing distinct determinants of HLA Class II antigens has an additive and not a synergistic inhibitory effect on MAb OKT3-induced
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B
lo<
t i me
(hr)
FIG. 3. Kinetics ofthe inhibition by anti-HLA Class II MAb of MAb OKT3-induced T cell proliferation in the presence of monocytes and in a monocyte-free system. (A) Anti-HLA-DR MAb Q5/6 (0; 200 ng/ ml), anti-HLA-DR, DP MAb CRIO-343 (+; 200 @ml), CR1 l-462 (m; I &ml), and CR12-356 (A; 1 rg/ ml) were added at the indicated times to PBMC (2 X 105) incubated with MAb OKT3 (2 rig/ml). (B) MAb Q5/6 (0; 20 pg/ml), CRlO-343 (+; 20 pg/ml), CR1 I-462 (m; 20 &/ml), and CR12-356 (A; 20 pg/ml) were added at the indicated times to T cells (2 X 1OS)incubated with IL 2 (25 U/ml), following purification from PBMC pulsed for 4 hr at 37°C with MAb OKT3 (2 &ml). Cell cultures were incubated for a total of 72 hr at 37°C. Then [3H]dThd incorporation was measured.
T cell proliferation. Whether the latter phenomenon reflects the regulation by the two MAb of different steps in the sequence of events leading to T cell proliferation or the partial effect of each MAb on a given step of the proliferative machinery remains to be determined. This differential effect of the four anti-HLA Class II MAb on MAb OKT3-induced T cell proliferation is not a general phenomenon, since they inhibit T cell proliferation induced by PHA-P and by alloantigens to a similar extent. These results suggest that monomorphic determinants of HLA Class II antigens display a different regulatory role in T cell proliferation triggered by different stimuli. Furthermore, the differential inhibitory effect of the four anti-HLA Class II MAb is not likely to be an isotype-mediated phenomenon, since in our system the F(ab’), of the anti-HLA Class II MAb display an inhibitory effect similar to that of the whole Ig (8). This conclusion parallels that reached by others who have studied the effect of anti-HLA Class I MAb (2 1) and of anti-CD4 MAb (22) on MAb OKT3-induced T cell proliferation. Whether the affinity of the four anti-HLA Class II MAb tested plays a role in the phenomenon we have described could not be assessed, since the labeling of the MAb with rz51 utilizing various methods resulted in some loss of their immunoreactive fraction, therefore, rendering the results of the assay to measure the affinity constants of the four anti-HLA Class II MAb questionable. However, we believe, also in consid-
DIFFERENTIAL
ROLE
OF
HLA
TABLE Effect of Anti-HLA
CLASS
6
Class II MAb on PHA-P-Induced and Allogeneic MLR Responseh
T Cell Proliferation”
PHA-P MAb
(rg/ml) -
R16.7 345.134
(20.0) (20.0)
Q5/6
(20.0) (10.0) (5.0) (2.5)
cpm X 10-j f SD
MLR % Inhibition’
85.6 t 9.6 86.4 +- 1.9 84.3 f 7.8 43.3
(1.2)
CRlO-343 CRIO-343 CRlO-343 CR lo-343 CRIO-343
(20.0) (10.0) (5.0) (2.5) (1.2)
44.0 f 5.0 n.d. 76.1 f 6.9 nd. n.d.
42.7
CR 1 l-462 CR1 1-462 CR1 l-462 CR I l-462 CR 1 l-462
(20.0) (10.0) (5.0) (2.5) ( 1.2)
50.5 f 5.6 n.d. 72.6 f 6.5 n.d. nd.
41.1
CR 12-356 CR12-356 CR12-356 CR 12-356 CR12-356
(20.0) (10.0) (5.0) (2.5) (1.2)
53.3 +- 4.6 n.d. 73.8 f 1.4 n.d. n.d.
37.1
Q5/6 Q5/6 Q5/6
cpm X 10-j k SD
% Inhibition”
23.9 f 3.1 25.8 f 2.7 22.0 + 2.6
48.6 f 4.2 n.d.” 74.2 I!Z 6.9 n.d. nd.
Q5/6
89
II EPITOPES
13.3
Il.1
11.7
13.8
6.9 7.3 10.6 13.2 17.0
f f f f +
1.1 1.2 1.5 1.7 1.8
7 1.o 69.1 55.7 45.0 29.3
5.6 7.0* 8.3 12.2 17.3
+ 1.0 1.3 f 1.9 f 1.5 f 2.6
76.7 71.9 66.2 49.2 28.0
2.3 3.1 4.9 8.6 17.3
f + f -+ k
1.2 1.0 1.6 1.4 1.6
90.5 87.0 79.5 63.9 28.0
1.6kO.9 2.5 + 0.9 4.5 f 1.1 5.8 -t 1.2 10.1 + 1.8
93.3 89.6 81.2 75.5 57.8
” PBMC (2 X 105/200 ~1) were cultured with PHA-P (4 pg/ml) for 72 hr at 37°C. [jH]dThd was added 4 hr before harvesting the cultures. ’ PBMC (1 X 105/100 ~1) were cultured with 1 X 10’ irradiated (3000 rad) allogeneic PBMC (1 X 105/ 100 ~1) for 7 days at 37°C in the presence of anti-HLA Class II MAb. [‘H]dThd was added 16 hr before harvesting the cultures. ’ Measured by comparison with [‘H]dThd mean incorporation by PBMC incubated with control MAb 345.134 or R16.7. I’ Measured by comparison with [‘H]dThd mean incorporation by MLR incubated with control MAb 345.134 or R16.7. ” Not done.
eration of binding curves (data not shown), that the four anti-HLA Class II MAb do not differ markedly in their affinity constants, since all of them immunoprecipitate Class II antigens from B lymphoid cells (9- 12) and display a similar inhibitory effect on T cell proliferation induced by mitogenic stimuli other than MAb OKT3. Furthermore, even if the anti-HLA Class II MAb differ in their affinity, a crucial role of this variable in their differential effect on MAb OKT3-induced T cell proliferation is not supported by the results obtained in mice, where differences in the affinity of antiMHC Class II antibodies have been reported not to be sufficient to account for different patterns of inhibition of T cell proliferation (23).
90
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ET AL.
As T cells express HLA Class II antigens upon activation (24), we studied the effect of our MAb on MAb OKT3-induced T proliferation in a monocyte-free system. In this regard, the information in the literature is conflicting (25, 26). This may reflect the differences in the characteristics of the anti-HLA Class II MAb used and/or in the proliferative systems. Under our experimental conditions, the four anti-HLA Class II MAb tested inhibited T cell proliferation to a similar extent, suggesting a different functional activity of HLA Class II determinants expressed by activated T cells and monocytes. Thus, when T cell proliferation is induced by MAb OKT3 in the presence of monocytes, HLA Class II determinants can be divided into two functional groups, whereas such a difference is not detected when T cell proliferation is induced by other types of monocyte-dependent mitogenic stimuli and when MAb OKT3-induced T cell proliferation occurs in a monocyte-free system. Furthermore, two lines of evidence indicate that the regulatory activity of HLA Class II determinants expressed by activated T cells is markedly less susceptible to modulation by MAb than that of those expressed by monocytes. First, the amount of MAb required to induce a certain degree of inhibition of T cell proliferation is higher in a monocyte-free system than in a monocyte-dependent one, likely reflecting differences in sensitivity of the proliferative steps controlled by monocytes and by activated T cells. Second, HLA Class II determinants expressed by activated T cells do not mediate complete inhibition of MAb OKT3-induced T cell proliferation, while those expressed by monocytes do. Since some activated T cells may not express HLA Class II antigens, they may not be susceptible to the inhibitory effect of MAb. This possibility is consistent with our finding that some T cells express the activation marker CD25 but lack HLA Class II antigens, in agreement with other data of the literature (27). From our results it can be concluded that the fine regulatory role of HLA Class II determinants in T cell proliferation can be modulated by a number of variables, enhancing the functional heterogeneity of such antigens to mediate cell-cell interactions. ACKNOWLEDGMENTS The authors thank Drs. Elena Cosentini and Giorgio Fratellanza of the Blood Bank of the 2nd Medical School of the Naples University for providing the blood specimens.
REFERENCES I. Van Wauwe, J. P., De Mey, J. R., and Goossens, J. G., .I Immunol. 124,2708, 1980. 2. Landegren, U., Anderson, J., and Wigzell, H., Eur. J. Immunol. 14,325, 1984. 3. Smith, K. A., Lachman, L. B., Oppenheim, J. J., and Favata, M. F., J. Exp. Med. 151, 155 1, 1984. 4. Mizel, S. B., Immunol. Rev. 63,51, 1982. 5. Robb, R. J., Munk, A., and Smith, K. A., J. Exp.Med. 154, 1455, 1981. 6. Depper, J. M., Leonard, W. J., Robb, R. J., Waldmann, W. A., and Greene, W. A., J. Immunol. 131, 690,1983.
7. Akiyama, Y., Zicht, R., Ferrone, S., Bonnard, G. D., and Herbennan, R. B., Cell. Immunol.
91,477,
1985. 8.
Ruggiero, G., Manzo, C., Fontana, S., Scala, G., Pirozzi, G., Fen-one, S., and Zappacosta, S., Eur. J. Immunol.
17,1585,
1987.
Quaranta, V., Pellegrino, M. A., and Ferrone, S., J. Immunol. 126, 548, 198 1. 10. Watson, A. J., DeMars, R., Trowbridge, I. S., and Bach, F. H., Nature (London) 304,358, 1983. I I. Russo, C., Nepo, A., Lanza, E., Igarashi, M., and Ferrone, S., J. Immunol. 138,3152, 1987. 9.
DIFFERENTIAL
ROLE
OF
HLA
CLASS
II EPITOPES
91
12. Imai, K., Natali, P. G., Kay, N. E., Wilson, B. S., and Ferrone, S., Cancer Irnmunol. Immunother. 12, 159, 1982. 13. Hirai, Y., Lamoy, E., Dohi, Y., and Nisonoff, A., J. Immunoi. 126,71, 198 1. 14. Temponi, M., Kageshita, T., Perosa, F., Ono, R., Okada, H., and Ferrone, S., Hybridoma 8,85, 1989. 15. Ey, P. L., Prowse, S. J., and Jenkin, C. R., Immunochemistry 15,429, 1978. 16. Laemmli, U. K., Nature (London) 227,265, 1970. 17. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J., J. Biol. Chem. 193,265, 1951. 18. Fraker, P. J., and Speck, J. C., Jr., Biochem. Biophys. Res. Commun. 80,849, 1987. 19. Perosa, F., and Ferrone, S., Hum. Immunol. 23,255, 1988. 20. Kalil, J., and Wollman, E. E., Cell. Immunol. 79,367, 1983. 21. Turco, M. C., De Felice, M., Corbo, L., Morrone, G., Mertelsmann, R., Ferrone, S., and Venuta, S.,
J. Immunol. 135,2268, 1985. 22. Geppert, 23. Lemonnier, 1982. 24. Ko, H.-S., 25. Moretta, 26. Tsoukas,
T. D., and Lipsky, P. E., J. Immunol. 137,3065, 1986. F. A., Birnbaum, D. Z., Dubreuil, P. C., and Caillol,
D. H.,
Stand. J. Immunol. 16,233,
Fu, S. M., Winchester, R. J., Yu, D. T. Y., and Kunkel, H. G., J. Exp. Med. 150,246, 1979. A., Accolla, R. S., and Cerottini, J. C., J. Exp. Med. 155,599, 1982. C. D., Landgraf, B., Bentin, J., Valentine, M., Lotz, M., Vaughan, J. H., and Carson, D. A., J. Immunol. 135, 1719, 1985. 27. Yachie, A., Miyawaki, T.. Uwadana, N., Ohzeki, S., and Taniguchi, N., J. Immunol. 131,73 1, 1983.
125,79-91 (1990)
Monoclonal Antibody OKT34nduced T Cell Proliferation: Differential Role of HLA Class II Determinants Expressed by T Cells and Monocytes’ CIRO MANZO,* GIUSEPPINA RuCcmo,t~* LUIGI DEL VECCHIO,$ LUIGI RAcroppI,t22 GIUSEPPE PIROZZI,* MASSIMO TEMPONI,~ SOLDANO FERRONE,~ SILVIA FONTANA,” AND SERAFINO ZAPPACosTAt *Divisione di Immunologia. Istituto Tumori Fondazione Pascale, 80131 Napoli. Italy; TCattedra di Immunologia, Dipartimento di Biologia e Patologia Cellulare e Molecolare. Universitci di Napoli Federico II, 80131 Napoli. Italy; SDivisione di Immunoematologia. Ospedale A. Cardarelli, 80131 Napoli, Italy; SDepartment ofMicrobiology & Immunology, New York Medical College, Valhalla, New York 10595; and I’Centro di Endocrinologia ed Oncologia Sperimentale de1 CNR, 80131 Napoli, Italy Receiwd April 25, 1989; accepted August 28, 1989 Monoclonal antibodies (MAb) to monomorphic determinants of HLA Class II antigens inhibit monocyte-dependent T cell proliferation induced by MAb OKT3 to a different extent, suggesting a differential regulatory role ofthe corresponding determinants in T cell proliferation. To elucidate the mechanism(s) underlying this pattern, the MAb CRlO-343 and Q5/6 with high inhibitory effect and MAb CR1 l-462 and CR12-356 with low inhibitory effect were characterized. Cross-inhibition studies showed that the four MAb recognize distinct determinants. The determinants recognized by MAb CR 1O-343 and CR 12-462 are spatially close. The determinants recognized by the four MAb appear to be functionally independent in MAb OKT3-induced T cell proliferation, since the inhibitory effect ofthe combination of MAb CR IO-343 and Q5/6 and of the MAb CR1 I-462 and CR 12-356 was additional but not synergistic. To compare the functional activity of HLA Class II determinants expressed by monocytes and by activated T cells in MAb OKT3-induced T cell proliferation, the effect of the four MAb on MAb OKT3induced T cell proliferation in a monocyte-dependent and in a monocyte-free system was studied. Dose-response and proliferation kinetics studies showed that the four MAb display a similar inhibitory effect on MAb OKT3-induced T cell proliferation in a monocyte-free system. These results suggest fine differences in the role played by monocyte- and T cell-bound HLA Class II determinants in the regulation of MAb OKT3-induced T cell proliferation. This functional heterogeneity may enhance the flexibility of HLA Class II antigens to mediate cell-cell interactions involved in the proliferative response to a variety of mitogenic stimuli. 0 1990 Academic Press, Inc.
’ Supported in part by grants from the Cons&ho Nazionale delle Ricerche (CNR), Roma (Progetti Finalizzati “Oncologia” (87.0 1490.44 and 88.00927.44) and “Ingegneria Genetica e Basi Molecolari delle Malattie Ereditarie” (86.00109.5 1 and 87.00876.5 I)), by grants from the Minister0 della Pubblica Istruzione, Roma, by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milano, and by NIH Grants AI2 I384 and CA39559. 2 Recipient of a research fellowship from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Milano. 79 0008-8749/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form resewed.
80
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INTRODUCTION T cell proliferation induced by anti-CD3 monoclonal antibodies (MAb) represents a useful model for investigating the mechanisms underlying T cell proliferation triggered by antigen stimulation. In this system, T cell proliferation is induced by the binding of anti-CD3 MAb to a subunit of the antigen recognition structure complex (CD3-Ti). This effect is accessory-cell-dependent ( 1,2) and involves interleukin 1 (IL 1) (3,4) and interleukin 2 (IL 2) production as well as IL 2 receptors’ (IL 2R) induction on activated T cells ($6). HLA Class II antigens are involved in this phenomenon, since anti-HLA Class II MAb inhibit MAb OKT3-induced T cell proliferation (7,8). The effect is mediated by the interaction of MAb with the corresponding determinants expressed by monocytes and is not Fc-mediated, as the same effect is displayed by pepsin fragments and by the whole Ig of the MAb. MAb to distinct determinants of HLA Class II antigens display differential effects on IL 2 production and inhibit T cell proliferation with different kinetics and to a different extent, suggesting that the corresponding determinants expressed on monocytes play a differential regulatory role in the sequence of events leading to T cell proliferation. The aim of this study was threefold: first, we analyzed the spatial relationship of determinants defined by anti-HLA Class II MAb with high and low inhibitory effects on MAb OKT3induced T cell proliferation, to determine whether regions with distinct functional properties could be identified on HLA Class II molecules. Second, we tested the effect on MAb OKT3-induced T cell proliferation of combinations of MAb recognizing distinct determinants of HLA Class II antigens, to evaluate if the effect is additive or synergistic. Last, we compared the effect of anti-HLA Class II MAb with high and low inhibitory effects on the proliferation of MAb OKT3-activated T cells in a monocyte-free system, to determine if the differential regulatory role in T cell proliferation played by monocyte-bound determinants is also played by T cell-bound determinants. MATERIALS
AND METHODS
Cells Peripheral blood mononuclear cells (PBMC) were separated from heparinized blood of healthy donors by Ficoll-Paque (Pharmacia Fine Chemicals AB, Uppsala, Sweden) density gradient centrifugation (400g for 30 min). Following three washings, PBMC were resuspended in medium RPM1 1640 supplemented with 10% heat-inactivated fetal calf serum (FCS), glutamine (2 mM), penicillin (100 U/ml), and streptomycin ( 100 pg/ml). MAb OKT3-preactivated T cells were obtained by sequential incubation of PBMC with MAb OKT3 (2 rig/ml) for 4 hr at 37°C with carbonyl iron for 30 min at 37°C removal of phagocytizing cells by a magnet, and washing and incubation for 30 min at 4°C with MAb OKB7 and Leu M3 and for 1 additional hr at 37°C with rabbit complement, diluted 1:2. Under these experimental conditions, T cells did not undergo lysis when incubated with rabbit complement. The purity of the resulting cell population, as evaluated in indirect immunofluorescence (IIF) with the MAb OK3, OKM5, anti-Ia, OKB7, Q5/13, and CRlO-343 was at least 95%. Cultured B lymphoid cells WALK were grown in medium RPM1 1640, supplemented with 10% FCS and glutamine (2 mM).
DIFFERENTIAL
Interleukin
ROLE
OF
HLA
CLASS
II EPITOPES
81
2
Human recombinant IL 2 was a gift of Dr. F. Sinigaglia (Hoffmann-La Co., Basel, Switzerland).
Roche &
MA6 and ConventionaE Antisera The anti-CD3 MAb OKT3 (IgG2a), OKB7 (IgGl), and OKM5 (IgGl) were purchased from Ortho Diagnostic Systems (Milan, Italy). The MAb Leu M3 (IgG2b), fluorescein isothiocyanate (FITC)-conjugated MAb anti-Tat (IgG2a), and phycoerythrin (PE)-conjugated MAb anti-HLA-DR (IgG2a) were obtained from BectonDickinson Italia SpA (Milan, Italy). The MAb anti-Ia (IgG2a) was obtained from Technogenetics SpA (S. Mauro Torinese, Italy). The anti HLA-DQw 1 MAb KS 11 (IgM); the anti HLA-DQw3 MAb KS 13 (IgG2b); the MAb Q5/6 (IgGZa) to a determinant expressed on all HLA-DR allospecificities but HLA-DR7; the anti-HLA-DR MAb CL4 13 (IgG2a), the anti-HLA-DP MAb B7/ 21 (IgG2a); the anti-HLA-DR, DP MAb CRlO-343 (IgG2a), CR1 l-462 (IgGl), and CR12-356 (IgGl); the anti HLA-DR, DQ, DP MAb Q5/13 (IgG2a), the anti-arsonate MAb R16.7 (IgGl); and the MAb 345.134 (IgG2a) to a 115-kDa glycoprotein (115K GP) expressed by a variety of cells were developed and characterized as described (9- 13). FITC-conjugated goat anti-mouse IgG antibody was purchased from Ortho Diagnostic Systems. MAb were purified from ascites fluid either by sequential precipitation with caprylic acid and ammonium sulfate (14) or by affinity chromatography on a protein A-Sepharose column ( 15). The purity of antibody preparations was assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ( 16). Protein concentration was determined by the Lowry method (17). MAb were labeled with “‘1 utilizing the iodogen method (18). Serological Assays The competition assay to map the determinants recognized by MAb was performed in 96-well microtiter plates, as described (19). Direct immunofluorescence (IF) and IIF were performed as described (8), by using a cytofluorograph Spectrum III (Ortho Diagnostic Systems, Raritan, NJ). Proliferation
Assays
They were performed in 96-microwell plates. Cells (2 X 105) in 200 ~1 of medium RPM1 1640, supplemented with 10% FCS, anti-HLA Class II MAb, and MAb OKT3 (2 rig/ml), PHA-P (Sigma Chemical Co., St. Louis, MO) (4 pg/ml), or IL 2 (25 U/ ml) were incubated for 3 days in a 5% CO2 atmosphere. Then, 1 FCi = 37 kBq of [3H]thymidine ([3H]dThd, sp act, 47 Ci/mmol, Amersham International plc, Amersham, England) was added to each well. At the end of an additional 3-hr incubation at 37°C cultures were harvested and [3H]dThd incorporation was measured. Mixed Lymphocyte
Cultures
PBMC ( 1 X 1OS)were mixed with irradiated (3000 rad) allogeneic PBMC ( I X 10’) in a total volume of 200 11 of medium RPM1 1640 supplemented with 10% FCS.
82
MANZO
ET AL.
I*
IB
5c
a 4
8 cold
12 MAb
0 (j~glmll
4
a
12
FIG. 1. Mapping of the determinants recognized by anti-HLA Class II MAb CRIO-343, CR1 l-462, CR12-356, and Q5/6. Cultured B lymphoid cells WALK (1 X 105) were incubated with increasing concentrations of cold anti-HLA-DR MAb Q5/6 (0), anti-HLA-DR, DP MAb CRlO-343 (0), CR1 l-462 (0) and CR12-356 (A) and with “‘1-MAb Q5/6 (A), CRlO-343 (B), CR1 l-462 (C), and CR12-356 (D) (2 X lo5 cpm) for 3 hr at 4°C. Then cells were spun down and washed three times with cold medium, and pellets were counted in a gamma counter.
Cultures were incubated in the presence of anti-HLA MAb at 37°C in a 5% CO;! atmosphere for 7 days. Sixteen hours before harvesting the cultures, 1 PCi of [3H]dThd was added to each well and radioactivity incorporation was then measured. RESULTS Mapping OfDeterminants on MAb OKT3-Induced
Defined by Anti-HLA T Cell Proliferation
Class II MAb with D@erential
Efect
Screening of nine anti-HLA Class II MAb for their effect on MAb OKT3-induced T cell proliferation showed that the MAb CRlO-343, Q5/6, and Q5/13 displayed strong inhibitory effects on T cell proliferation, while the MAb CR1 l-462, CR1 2356, KS13, CL413, B7/21, and KS1 1 displayed weak inhibitory effects. The MAb CRlO-343 and Q5/6 on one hand and the MAb CR1 l-462 and CR12-356 on the other hand were selected for additional investigations. To determine the spatial relationship among determinants defined by the above MAb, competition experiments were performed utilizing cultured B lymphoid cells WALK as targets. As shown in Fig. 1, the MAb CRlO-343 and CR1 l-462 crossinhibited each other in their binding to targets, but were not affected in their binding
DIFFERENTIAL
ROLE
OF
HLA
TABLE Additive
Inhibitory
MAb 345.134
R16.7
48
-
-
48 4 8 -
-
8 16 16
16 32 -
83
II EPITOPES
1
Effect of the Combination of MAb Q5/6 OKT3-Induced T Cell Proliferation”
and CR lo-343
on MAb
(rig/ml) Q5/6
CRIO-343
8 8 16 16 32
4 4 8 -
32 32 -
CLASS
32
16 16 16
[‘H]dThd incorporation 88.7 86.6 91.3 71.6? 76.1 60.4 54.3 26.2 42.6 42.2 37.9 40.2 6.1
f 10.1” k 5.6 k 9.6 6.6 f 7.9 f 4.5 f 6.3 f 2.9 f 4.5 f 3.5 f 3.2 f 3.6 f 0.4
Inhibition (S)
19.2’ 14.2 31.9 38.8 70.5 52.0 52.5 57.3 54.7 93.2
” PBMC (2 X IO’) were incubated with MAb OKT3 (2 ng/mI) and with anti-HLA Class II MAb total volume of 200 ~1 for 72 hr at 37°C and then [3H]dThd incorporation was measured. ’ cpm X 10 ‘; mean f SD. ‘ As compared to [ 3H]dThd incorporation by PBMC incubated without MAb.
in a
by MAb Q5/6. While the binding of MAb CR 1 l-462 was not affected by MAb CR 12356, that of MAb CRlO-343 was partially inhibited by high concentrations of MAb CR 12-356. The binding of the latter was not affected by any of the other three MAb. The binding of MAb Q5/6 was partially inhibited by high concentrations of MAb CR 1 l-462 and CR 12-356. These results suggest that the four MAb recognize distinct determinants. The one recognized by MAb CR 1 l-462 is spatially close to that defined by MAb CRlO-343, but distant from that defined by MAb CR12-356. The determinants defined by MAb CRlO-343 and Q5/6 are spatially distant. Provided that the partial inhibition induced by high concentrations of MAb CR 1 l-462 and CR 12-356 does not reflect changes in conformation induced by the binding of the MAb, the determinant defined by MAb CRlO-343 is in spatial proximity to that defined by MAb CR12-356 and that defined by MAb Q5/6 with those recognized by MAb CR1 l-462 and CR12-356. Additive Inhibitory Proliferation
Eflect ofAnti-HLA
Class II MA6 on MA6 OKT3-Induced
T Cell
To evaluate the possible cooperative effect of inhibitory anti-HLA Class II MAb, PBMC were cultured with MAb OKT3 in the presence of the combination of the two MAb with a low inhibitory activity and of the two MAb with a high inhibitory activity. In either case, the effect of the combination was compared to the inhibition exerted by each MAb of the combination. At each of the concentrations tested, the inhibitory effect of the combination of MAb Q5/6 and CRlO-343 corresponded to the sum of the effect exerted by the same concentration of each MAb tested individu-
84
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ET AL.
TABLE 2 Additive Inhibitory Effect ofthe Combination of MAb CR1 l-462 and CRl2-356 on MAb OKT3-Induced T Cell Proliferation” MAb (r&ml) 345.134 -
1600 800 800
R16.7 -
1600 200 200 400 400 -
CR 1l-462
CRl2-356
-
-
200 200 400 400 800 800
200 200 400 400 800 800
[3H]dThd incorporation 86.1 f 5.3h 88.3 f 4.1 90.4 f 6.2 61.1 k 3.0 64.3 i 2.2 4 1.7 * 2.2 46.1 k2.1 60.7 f 4.3 34.7 f 2.6 38.4 f 2.1 49.6 + 2.8 20.6 f 1.6
Inhibition @)
30.9‘ 27.2 52.9 47.6 31.3 61.7 56.5 43.8 76.7
0 PBMC (2 X 105) were incubated with MAb OKT3 (2 rig/ml) and with anti-HLA Class II MAb in a total volume of 200 ~1 for 72 hr at 37°C and then [3H]dThd incorporation was measured. ’ cpm X 10-j; mean + SD. ‘ As compared to [3H]dThd incorporation by PBMC incubated without MAb.
ally. The same conclusion was reached for the combination of MAb CR1 l-462 and CR12-356, except for the highest concentration tested (800 fig/ml). At the latter concentration, the inhibition of T cell proliferation was less than the sum of the values obtained with each MAb. Representative results of at least three experiments are shown in Tables 1 and 2. The inhibition induced by the combination of the two MAb does not reflect a nonspecific effect due to high protein concentration, since no inhibition of proliferation was detected when cultures were added with high concentrations of anti- 115K GP MAb 345.134 and anti-arsonate MAb R 16.7. Furthermore, addition to cultures of either control MAb with one of the anti-Class II MAb did not affect the inhibitory activity of the latter. Efect of Monocytes on the Inhibition Induced T Cell Proliferation
by Anti-HLA
Class II MAb of MA6 OKT3-
To determine whether the differential inhibitory effect of anti-HLA Class II MAb on MAb OKT3-induced T cell proliferation reflected their interaction with different target cells, both sets of MAb were tested with purified monocytes and T cells. When monocytes pretreated with MAb CRlO-343 or CR12-356 were added to purified T cells stimulated with MAb OKT3, proliferation was inhibited only by MAb CR 1O-343 (Table 3). The effect was dose-dependent. To determine the direct effect of MAb on T cells, they were purified from PBMC incubated for 4 hr at 37°C with MAb OKT3 (2 rig/ml) and were cultured with IL 2 (25 U/ml) for up to 72 hr. Anti-HLA Class II MAb were added to the cultures at concentrations ranging from 1.2 to 40 pg/ml. The proliferation obtained under these
DIFFERENTIAL
85
ROLE OF HLA CLASS 11 EPITOPES TABLE 3
Effect of Monocytes Preincubated with Anti-HLA Class II MAb on MAb OKT3 Induced T Cell Proliferation” Preincubation with MAbh
T cells
Monocytes
+ +
+
R16.7
+
+
CR lo-343
+
+
CRl2-356
MAb (rg/W 40
108.8 f 9.3‘ (0)” 2.5 * 0.3 (97.8) 80.5 f 8.2 (27.0)
10
115.6 f 8.8 (0) 30.7 -t 3.8 (72.2) 95.8 * 10.2 (13.1)
2.5
0
113.4 f 12.6 (0) 83.3 k 7.2 (24.4) 111.zi 9.4 (0)
2.1 + 0.2 1.3 * 0.2 110.2 5 12. I (0)
il T cells (2 X IO’/200 ~1) and monocytes (2 X 104/200 ~1) were cultured with MAb OKT3 (2 rig/ml) for 3 days at 37°C. Then F3H]dThd incorporation was measured. ” Monocytes were incubated with MAb for 90 min at 37°C and washed three times with medium warmed at 37’C. ‘ cpm x 10 -j; mean f SD. “ Percentage inhibition as compared to [ ‘H]dThd uptake by cultures of T cells and untreated monocytes stimulated with MAb OKT3 (2 rig/ml).
experimental conditions was compared to that of unfractionated PBMC stimulated with MAb OKT3. The inhibitory effect of the four MAb on T cell proliferation was influenced by the presence of monocytes. While the MAb CR 1O-343 and Q5/6 inhibited the proliferation of unfractionated PBMC more than the MAb CR1 l-462 and CR 12-356, the four MAb did not differ in the extent of inhibition of the proliferation of purified T cells. In both culture systems, the inhibitory effect was dose-dependent, higher concentrations of MAb being required to inhibit purified T cell proliferation than to inhibit PBMC proliferation. In addition, while the proliferation of PBMC was completely inhibited by the four MAb, complete inhibition of purified T cell proliferation was never reached, even at high MAb concentrations (Fig. 2). To analyze the mechanism(s) underlying the latter observations, the kinetics of appearance of HLA Class II antigens (Table 4) as well as the distribution of IL 2 receptors expression in relation to these antigens on purified T cells (Table 5) was studied. Double IF staining with anti-Ia MAb and anti-CD25 MAb showed that about 10% of activated T cells express the CD25 antigen, but lack Class II antigens; the large majority lack both types of antigens; about 20% express both types of antigens; and about 10% express only HLA Class II antigens. Efect of Monocytes on the Kinetics of the Inhibition MAb OKT3-Induced T Cell Proliferation
by Anti-HLA
Class II A4Ab of
The kinetics of the inhibitory activity of the four MAb was influenced by the presence of monocytes in the culture system (Fig. 3). When the MAb were tested with unfractionated PBMC, the kinetics of the inhibitory activity of MAb CR 1 l-462 and CR 12-356 was different from that of MAb CR 1O-343 and Q5/6. The inhibitory activity of the latter two was reduced by about 50% when they were added to the culture
86
MANZO
ET
AL.
loor-
Of 0.1
0.5
1.0
2.5
MAb
5.0
10.0
25.0
1 50.0
@g/ml)
FIG. 2. Dose-dependent inhibition by anti-HLA Class II MAb of MAb OKT3-induced T cell proliferation in the presence of monocytes and in a monocyte-free system. Increasing amounts of anti-HLA-DR MAb Q5/6 (circle), anti-HLA-DR, DP MAb CRlO-343 (rhomboid), CR1 l-462 (square), CRl2-356 (triangle) were added with MAb OKT3 (2 rig/ml) to PBMC (2 X 10’) (closed symbols) and to IL 2 (25 U/ml)stimulated T cells (2 X 105) (open symbols) purified from PBMC, which had been pulsed for 4 hr at 37°C with MAb OKT3 (2 rig/ml). The anti- 115K GP MAb 345.134 and the anti-arsonate MAb R 16.7 were used as specificity controls (data not shown). At the end of a 3-day incubation at 37°C [3H]dThd incorporation was measured.
after a 6- to 8-hr incubation, while that of the former two was reduced by only 15%. The degree of inhibition caused by the four MAb did not markedly change when they were added up to 24 hr following the beginning of the incubation; the inhibition TABLE Kinetics
4
of Appearance of HLA Class II Antigens and of IL 2 Receptors on Activated T Cells Incubated with IL 2” Incubation
MAb
specificity
W Leu M3 CD14 Leu 12CDl9 OKTl 1 CD2 anti-Ia DR anti-Tat CD25
24
0 0.2h(23)C 0.5 (29) 2 (25) 93 (117) 6 (97) 4 (31)
(hr)
0.4 0 1 92 7 22
(29) (40) (111) (104) (89)
0 PBMC (2 X 106/ml) were incubated with MAb OKT3 isolated and cultured at the concentration of 2 X 10’/200 indicated times, cells were harvested, sequentially incubated bodies, and analyzed with a cytofluorograph. h Percentage stained cells. ’ Mean fluorescence intensity.
72
48 0.3 0 2 94 15 26
(21) (65) (108) (110) (120)
0.1 0 2 93 23 30
(20) (50) (111) (132) (138)
(2 &ml) for 4 hr at 37°C. Then T cells were ~1 in the presence of IL 2 (25 U/ml). At the with MAb and FITC-anti mouse Ig xenoanti-
DIFFERENTIAL
ROLE
OF TABLE
Expression
CD25
ofCD25
HLA-DR
-
+
+ +
+ -
HLA
CLASS
87
II EPITOPES
5
and HLA-DR Antigens on Activated for 72 hr in the Presence of IL 2“ First experiment 8.5” 63.3 17.5 10.7
T Cells Cultured
Second experiment 9.5 68.0 14.0 8.5
” PBMC (2 X IOh/ml) were incubated with MAb OKT3 (2 rig/ml) for 4 hr at 37°C and then T cells were isolated and cultured at a concentration of 2 X 106/ml in the presence of IL 2 (25 U/ml). After a 72-hr incubation, cells were incubated with FITC-MAb anti-Tat and with PE-MAb anti-HLA-DR and analyzed with a cytofluorograph. ” Percentage stained cells.
displayed a gradual additional reduction as the time of addition of the MAb to the cultures was progressively delayed. On the other hand, no difference was found in the kinetics of inhibition of the four MAb, when they were tested in a monocyte-free system. In addition, at variance with the results obtained with PBMC, the degree of inhibition gradually decreased as the time of addition of MAb to the culture was delayed. Efect ofAnti-HLA Class II MAb on the Proliferation and Allogeneic Lymphocytes
of T Cells Induced by PHA-P
To determine whether the differential inhibitory effect of each anti-HLA Class II MAb is a general phenomenon, the four MAb were tested for their effect on the proliferation of T cells in two other systems, i.e., stimulation with PHA-P and with allogeneic lymphocytes, in which the inhibitory effect of anti-HLA Class II MAb had been already reported (7,20). The four MAb inhibited PHA-P-induced T cell proliferation to a similar extent, no appreciable difference being detected between MAb with high and low inhibitory activity on MAb OKT3-induced T cell proliferation. The four MAb inhibited proliferation to the same extent; in allogeneic MLR, the effect was dose-dependent (Table 6). DISCUSSION The present study analyzes several characteristics of four monomorphic determinants of HLA Class II antigens to elucidate the mechanism(s) underlying the marked difference in the extent of the inhibitory effect of the corresponding MAb on MAb OKT3-induced T cell proliferation. Results of cross-blocking experiments suggest that determinants recognized by MAb with marked difference in their inhibitory effect on MAb OKT3-induced T cell proliferation are spatially close to each other. This finding argues in favor of the presence of hot “points” with regulatory function rather than of hot “regions” on HLA Class II molecules. This possibility is also consistent with the observation that the combination of two MAb recognizing distinct determinants of HLA Class II antigens has an additive and not a synergistic inhibitory effect on MAb OKT3-induced
88
MANZO
ET AL.
B
lo<
t i me
(hr)
FIG. 3. Kinetics ofthe inhibition by anti-HLA Class II MAb of MAb OKT3-induced T cell proliferation in the presence of monocytes and in a monocyte-free system. (A) Anti-HLA-DR MAb Q5/6 (0; 200 ng/ ml), anti-HLA-DR, DP MAb CRIO-343 (+; 200 @ml), CR1 l-462 (m; I &ml), and CR12-356 (A; 1 rg/ ml) were added at the indicated times to PBMC (2 X 105) incubated with MAb OKT3 (2 rig/ml). (B) MAb Q5/6 (0; 20 pg/ml), CRlO-343 (+; 20 pg/ml), CR1 I-462 (m; 20 &/ml), and CR12-356 (A; 20 pg/ml) were added at the indicated times to T cells (2 X 1OS)incubated with IL 2 (25 U/ml), following purification from PBMC pulsed for 4 hr at 37°C with MAb OKT3 (2 &ml). Cell cultures were incubated for a total of 72 hr at 37°C. Then [3H]dThd incorporation was measured.
T cell proliferation. Whether the latter phenomenon reflects the regulation by the two MAb of different steps in the sequence of events leading to T cell proliferation or the partial effect of each MAb on a given step of the proliferative machinery remains to be determined. This differential effect of the four anti-HLA Class II MAb on MAb OKT3-induced T cell proliferation is not a general phenomenon, since they inhibit T cell proliferation induced by PHA-P and by alloantigens to a similar extent. These results suggest that monomorphic determinants of HLA Class II antigens display a different regulatory role in T cell proliferation triggered by different stimuli. Furthermore, the differential inhibitory effect of the four anti-HLA Class II MAb is not likely to be an isotype-mediated phenomenon, since in our system the F(ab’), of the anti-HLA Class II MAb display an inhibitory effect similar to that of the whole Ig (8). This conclusion parallels that reached by others who have studied the effect of anti-HLA Class I MAb (2 1) and of anti-CD4 MAb (22) on MAb OKT3-induced T cell proliferation. Whether the affinity of the four anti-HLA Class II MAb tested plays a role in the phenomenon we have described could not be assessed, since the labeling of the MAb with rz51 utilizing various methods resulted in some loss of their immunoreactive fraction, therefore, rendering the results of the assay to measure the affinity constants of the four anti-HLA Class II MAb questionable. However, we believe, also in consid-
DIFFERENTIAL
ROLE
OF
HLA
TABLE Effect of Anti-HLA
CLASS
6
Class II MAb on PHA-P-Induced and Allogeneic MLR Responseh
T Cell Proliferation”
PHA-P MAb
(rg/ml) -
R16.7 345.134
(20.0) (20.0)
Q5/6
(20.0) (10.0) (5.0) (2.5)
cpm X 10-j f SD
MLR % Inhibition’
85.6 t 9.6 86.4 +- 1.9 84.3 f 7.8 43.3
(1.2)
CRlO-343 CRIO-343 CRlO-343 CR lo-343 CRIO-343
(20.0) (10.0) (5.0) (2.5) (1.2)
44.0 f 5.0 n.d. 76.1 f 6.9 nd. n.d.
42.7
CR 1 l-462 CR1 1-462 CR1 l-462 CR I l-462 CR 1 l-462
(20.0) (10.0) (5.0) (2.5) ( 1.2)
50.5 f 5.6 n.d. 72.6 f 6.5 n.d. nd.
41.1
CR 12-356 CR12-356 CR12-356 CR 12-356 CR12-356
(20.0) (10.0) (5.0) (2.5) (1.2)
53.3 +- 4.6 n.d. 73.8 f 1.4 n.d. n.d.
37.1
Q5/6 Q5/6 Q5/6
cpm X 10-j k SD
% Inhibition”
23.9 f 3.1 25.8 f 2.7 22.0 + 2.6
48.6 f 4.2 n.d.” 74.2 I!Z 6.9 n.d. nd.
Q5/6
89
II EPITOPES
13.3
Il.1
11.7
13.8
6.9 7.3 10.6 13.2 17.0
f f f f +
1.1 1.2 1.5 1.7 1.8
7 1.o 69.1 55.7 45.0 29.3
5.6 7.0* 8.3 12.2 17.3
+ 1.0 1.3 f 1.9 f 1.5 f 2.6
76.7 71.9 66.2 49.2 28.0
2.3 3.1 4.9 8.6 17.3
f + f -+ k
1.2 1.0 1.6 1.4 1.6
90.5 87.0 79.5 63.9 28.0
1.6kO.9 2.5 + 0.9 4.5 f 1.1 5.8 -t 1.2 10.1 + 1.8
93.3 89.6 81.2 75.5 57.8
” PBMC (2 X 105/200 ~1) were cultured with PHA-P (4 pg/ml) for 72 hr at 37°C. [jH]dThd was added 4 hr before harvesting the cultures. ’ PBMC (1 X 105/100 ~1) were cultured with 1 X 10’ irradiated (3000 rad) allogeneic PBMC (1 X 105/ 100 ~1) for 7 days at 37°C in the presence of anti-HLA Class II MAb. [‘H]dThd was added 16 hr before harvesting the cultures. ’ Measured by comparison with [‘H]dThd mean incorporation by PBMC incubated with control MAb 345.134 or R16.7. I’ Measured by comparison with [‘H]dThd mean incorporation by MLR incubated with control MAb 345.134 or R16.7. ” Not done.
eration of binding curves (data not shown), that the four anti-HLA Class II MAb do not differ markedly in their affinity constants, since all of them immunoprecipitate Class II antigens from B lymphoid cells (9- 12) and display a similar inhibitory effect on T cell proliferation induced by mitogenic stimuli other than MAb OKT3. Furthermore, even if the anti-HLA Class II MAb differ in their affinity, a crucial role of this variable in their differential effect on MAb OKT3-induced T cell proliferation is not supported by the results obtained in mice, where differences in the affinity of antiMHC Class II antibodies have been reported not to be sufficient to account for different patterns of inhibition of T cell proliferation (23).
90
MANZO
ET AL.
As T cells express HLA Class II antigens upon activation (24), we studied the effect of our MAb on MAb OKT3-induced T proliferation in a monocyte-free system. In this regard, the information in the literature is conflicting (25, 26). This may reflect the differences in the characteristics of the anti-HLA Class II MAb used and/or in the proliferative systems. Under our experimental conditions, the four anti-HLA Class II MAb tested inhibited T cell proliferation to a similar extent, suggesting a different functional activity of HLA Class II determinants expressed by activated T cells and monocytes. Thus, when T cell proliferation is induced by MAb OKT3 in the presence of monocytes, HLA Class II determinants can be divided into two functional groups, whereas such a difference is not detected when T cell proliferation is induced by other types of monocyte-dependent mitogenic stimuli and when MAb OKT3-induced T cell proliferation occurs in a monocyte-free system. Furthermore, two lines of evidence indicate that the regulatory activity of HLA Class II determinants expressed by activated T cells is markedly less susceptible to modulation by MAb than that of those expressed by monocytes. First, the amount of MAb required to induce a certain degree of inhibition of T cell proliferation is higher in a monocyte-free system than in a monocyte-dependent one, likely reflecting differences in sensitivity of the proliferative steps controlled by monocytes and by activated T cells. Second, HLA Class II determinants expressed by activated T cells do not mediate complete inhibition of MAb OKT3-induced T cell proliferation, while those expressed by monocytes do. Since some activated T cells may not express HLA Class II antigens, they may not be susceptible to the inhibitory effect of MAb. This possibility is consistent with our finding that some T cells express the activation marker CD25 but lack HLA Class II antigens, in agreement with other data of the literature (27). From our results it can be concluded that the fine regulatory role of HLA Class II determinants in T cell proliferation can be modulated by a number of variables, enhancing the functional heterogeneity of such antigens to mediate cell-cell interactions. ACKNOWLEDGMENTS The authors thank Drs. Elena Cosentini and Giorgio Fratellanza of the Blood Bank of the 2nd Medical School of the Naples University for providing the blood specimens.
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