CELLULAR
IMMUNOLOGY
141, 189-199 (1992)
CD7 Augments T Cell Proliferation via the Interleukin-2 Autocrine Pathway LAWRENCE K.
L. JUNG, AMIT K. ROY, AND HRISHEKPSH R. CHAKKALATH
Division of Pediatric Immunology, Department of Pediatrics, University of MassachusettsMedical Center, 55 Lake Avenue North, Worcester.Massachusetts 01655 Received October 7, 1991; acceptedDecember 30, 1991 The role of CD7, a T cell differentiation antigen, in T cell function is not known at present; this study evaluates the effect of anti-CD7 mAb in PBMC cultures activated with suboptimal concentrations of lectins, antigens, and anti-CD3 mAb. We found that the inclusion of anti-CD7 resulted in increased IL-2 production and IL-2R-(U expression in these cultures. H-7, a protein kinase C (PKC) inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, significantly suppressedthe proliferation of T cells in comitogenic assays.This suggestedthat the comitogenic effectmediated by CD7 molecule involved both the PKC and the PTK pathways of T cell activation. Thesedrugs appeared to affect the CD7-mediated effectsby inhibiting the IL-2 autocrine pathway, especially the up-regulation of IL-ZR-cusince inhibition was not relieved with exogenous rIL-2. Taken together, our results suggestthat CD7 augments T cell function by up-regulating IL-2R01expression and IL-2 production via multiple pathways of protein phosphorylation. o 1992 Academic
Press, Inc.
INTRODUCTION The function of CD7,’ a 40-kDa human T cell specific glycoprotein ( l-4) is of great interest in studies of T cell ontogeny becauseit is the earliest T cell associatedantigen to appear and persists in T cell differentiation (5). Although the precise role that CD7 plays in T cell activation is not known, earlier studies have shown that it is a useful marker of T cell activation, since CD7 expression is up-regulated on PHA- or Con Astimulated T cells (6). On the other hand, CD7 is down-regulated on T cells after treatment with phorbol esters(7). Some studies have reported that soluble anti-CD7 mAb may inhibit in vitro PHA and tetanus toxoid response of PBMC (8) as well as the allogeneic mixed lymphocyte reaction (9). On the other hand, a recent study has suggestedthat immobilized anti-CD7 synergizeswith CD3 mAb to induce T cell activation (IO). One mechanism by which this occurs is that crosslinked anti-CD7 could induce transmembrane calcium flux in T cells (11, 12). CD7 molecule has also been implicated to be the Fc receptor for IgM (13). However, transfection experiments with CD7 cDNA did not confirm this observation (14). Defective expression of CD7 has been noted in a number of diseasessuggestingthat decreasedexpressionin vivo of this glycoprotein may have important pathophysiological consequences( 15, 16). For example, we have found that the T cells of a child with ’ Abbreviations used: CD, cluster designation; kDa, kilodaltons; PKC, protein kinase C; PTK, protein tyrosine kinase; PPD, purified protein derivative. 189 0008-8749192$3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
190
JUNG,
ROY,
AND
CHAKKALATH
severecombined immunodeficiency were selectively deficient in CD7; their proliferative responsesto T cell mitogens were also defective ( 15). In addition, the patient’s T cells were deficient in IL-2R expression. Although a direct role for CD7 in the immunopathogenesis of this case remains to be proven, this observation suggeststhat CD7 may play an important role in T cell ontogeny. Our study was undertaken to examine if CD7 is involved in peripheral blood T cell functions. In the course of these experiments we observed that immobilized mAb to CD7 induced a signal(s) that in conjunction with submitogenic doses of lectins or anti-CD3 mAb triggered T cell proliferation. The antigenic response in PBMC was also augmented at higher dose of the antigen. In the present study, we also showed that the comitogenic activity of anti-CD7 involved an increase in IL-2 production and IL-2R-a expression that depended on multiple pathways of protein phosphorylation. MATERIALS AND METHODS CeZlseparation. Peripheral blood used in this study was from healthy volunteers of both sexes,ages25-50. PBMC were isolated on Ficoll-Hypaque (density 1.077 g/ml; Pharmacia, Uppsala, Sweden) gradients. After three washings in PBS (pH 7.2), the cells were resuspended in culture medium: RPM1 1640 (GIBCO, Grand Island, NY) supplemented with 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 pg/ ml) (GIBCO), and 10%FCS (Hyclone Laboratories, Logan, UT). Viability of the cells was checked by trypan blue dye exclusion technique and was found to be greater than 96% viable. Stimulation of mononuclear cells. PBMC at a density of 1 X lo5 cells/well in a total volume of 200 ~1culture medium were seededin antibody pretreated microtiter tissue culture plates. The pretreatment of the plates was done as follows: 50 ~1 of the mAbs anti-CD3, 235 (17), and anti-CD7, 69 (18), diluted in PBS (pH 8.0) was added to 96well round-bottom microtiter plates. After an overnight incubation, the plates were washed three times with PBS (pH 6.5). Mitogens including phytohemagglutinin-P (Difco Laboratories, Detroit, MI), Concanavalin A, protein A (Sigma Chemicals, St. Louis, MO), and purified protein derivative (Connaught Laboratories Ltd., Willowdale, Canada) were added at the indicated concentrations. In some experiments, either the protein kinase inhibitor drug H-7 (Seikagaku America, Inc., St. Petersburg, FL) or 4’,5,7-trihydroxyisoflavone (genistein; Biomol Research Laboratories, Plymouth Meeting, PA) dissolved in DMSO (Sigma) was added at the appropriate dose to the plates at the same time as the cells. Finally for certain experiments, cultures were supplemented with 50 U/ml of rIL-2 (Genzyme, Boston, MA). The cultures were maintained in a humidified atmosphere containing 5% COZ for 72 hr. Cell proliferation was estimated by [3H]TdR (0.5 &i = 24 kBq, New England Nuclear, Boston, MA, sp act 6.7 Ci/mmol) incorporation during the last 16 hr of culture. The percentage inhibition was calculated as follows: o/ inhibition = 1 - cpm in cultures treated with drugs 0 x 100. cpm in control cultures without dtugs Immunofluorescence studies. Cells were stimulated with the appropriate mAb in presenceor absenceof the drugs. At the end of 72 hr in culture, the cells were harvested using a rubber policeman and washed twice in culture medium. Cells were stained with either anti-IL-2R-a! mAb conjugated to FITC (Becton Dickinson, Mountain View, CA) or a classspecific control mAb reagent at 4°C for 30 min. After extensive washing,
CD7 AND T CELL ACTIVATION
191
the cells were analyzed on a FACScan flow cytometer, (Becton Dickinson). Log fluorescenceof the gated population was measured and 5000 cells were collected and analyzed using FACScan Research software. IL-2 assay. IL-2 production by PBMC cultures stimulated with mAb was detected by monitoring the ability of the culture supernatants to support the growth of the murine IL-2 dependent cell line CTLL-2 (American Type Tissue Collection). Briefly, culture supernatants were subjected to one freeze-thaw cycle before analysis and 100 ~1of the supernatant was used per well. CTLL-2 cells were extensively washed (four times) with medium devoid of IL-2 and added at a concentration of 1 X lo4 cells in 100 ,ul per well. As reference, rIL-2 (Genzyme) serially diluted with RPM1 was also included. Cultures were incubated for 24 hr with [3H]thymidine being added 4 hr before harvest. Thymidine incorporation was determined at 24 hr by liquid scintillation counting and the IL-2 present in each sample was determined in terms of units per milliliter as compared to the reference rIL-2. Agents used to stimulate PBMC such as mAb separately or in combination did not support the growth of CTLL-2. Statistical analysis. Statistical analysis was done by Student’s t test. RESULTS Monoclonal Antibody to CD7 Enhances PBMC Proliferation To understand the functional role CD7 plays in T cell activation, we examined the effectsof immobilized mAb to CD7 (mAb 69) on the proliferative capacity of PBMC. The cells were incubated with varying dosesof PHA-P, Con A, protein A, and PPD in a 96-well round-bottom culture plate precoated with mAb 69 (1 pg/well). Uptake of [3H]thymidine was measured after 3 (mitogen cultures) and 6 (antigen cultures) days in culture. Four experiments were done with PBMC from different donors and the result of one representative experiment is shown in Fig. 1. The presence of immobilized anti-CD7 in the incubation mixture resulted in an enhancement of the proliferation of PBMC in response to mitogens. This effect is best appreciated with submitogenic dosesof the lectins in culture. The comitogenic effect of anti-CD7 was not detectable at high dosesof the lectins. However, with most donors similar comitogenic effect was observed even with a low dose of anti-CD7 (0.1 pg/well; data not shown). Anti-CD7 mAb was also found to enhance the proliferation to PPD at higher antigenic dose (50 pg/ml). We also observed that the comitogenic effectswere lessevident when the anti-CD7 mAb was in soluble form instead of bound to the plastic surface. However, soluble anti-CD7 mediated a similar enhancing effect when added to wells precoated with a crosslinking antibody (anti-mouse Ig). Isotype mouse Ig controls and anti-T1 1, antibodies, immobilized in culture plates, were without effecton mitogen aswell asantigenstimulated PBMC (data not shown). To study the effect of anti-CD7 on the CD3-Ti complex, we investigated the comitogenie effect of anti-CD7 and anti-CD3 mAb. To this end, PBMC or purified T cells were added to culture plates precoated with varying doses of anti-CD3 mAb and a dose of 1 pg/well of anti-CD7. Two representative experiments are shown in Fig. 2. The anti-CD3 mAb at higher doseswas capable by itself to induce significant cellular proliferation, whereasat the lower dose minimal proliferation was observed. However, the crosslinking of CD3 with anti-CD7 caused a strong increase in proliferation. The effect was more evident with the lower dose of anti-CD3 mAb. Similar effectsof antiCD3/CD7 were observed in purified T cell cultures (data not shown). Taken together,
192
JUNG, ROY, AND CHAKKALATH
lOO--
0
1
5
CONC
10
20
15
25
(w/ml)
OF PHA-P
CONC
OF CON-A
&g/ml)
50-
150~ ,25-.
0-o
ProtIm
o-,.,.0
Prctaln
C
A alma A + antl-CD7
40--
0-o
PPD dona
~...‘a
PPD + cdl-CD7
II
lOO--
30 -75 -,;* .,... p
09//, 0
t
p. . ..p
,
,
,
,
5
10
15
20
20 --
T
, 25
0
10
CONC. OF PROTEIN A (pg/ml)
20
30
40
50
60
0
CONC OF PPD &g/ml)
FIG. 1. Enhancement of the proliferation of PBMC by the mAb to CD7. Cells (1 X IO’) in triplicates were stimulated in a total volume of 200 ~1,with varying dosesof PHA-P (A), Con A (B), protein A (C), and PPD (D) in the presenceand absenceof anti-CD7 (69; 1 &well) precoated on a 96-well round-bottom tissue culture plate. Of four separate experiments with four donors, the results from one experiment are represented as mean counts per minute (cpm). The mean cpm in cultures stimulated with 69 alone (278) was comparable to the values obtained in control cultures with media alone (266). The mitogen cultures (PHA-P, Con A, and protein A) were terminated on Day 3 and the antigen culture (PPD) was terminated on Day 6.
the above results suggestedthat CD7 molecule may have a regulatory role in T cell activation. Role of IL-2 Pathway in Anti-CD7-Induced Comitogenesis We tested whether the comitogenic effect by anti-CD7 mAb involved the IL-2 pathway. Therefore, we studied the expression of IL-2R-(r and the production of IL-2 in PBMC stimulated by solid-phaseimmobilized anti-CD7 either alone or in combination with anti-CD3 mAb. As shown in Fig. 3 neither the mAb to CD7 nor submitogenic dose of anti-CD3 mAb by themselves induced the expression of IL-2R-a. However, the combination of both the mAbs induced very significant levels of IL-2R-a expression. It was com-
193
CD7 AND T CELL ACTIVATION O-O
anti-CD3
O..... 0 anti-CD3
A---A
500
T
anti-CD3 alone
A.. .. A antl-CD3
400
alone + anti-CD7
+ anti-CD7
EXPT 1 EXPT 2
300
200
100
0
CONC OF IMMOBILIZED anti-CD3
&g/well)
FIG. 2. Comitogenic effect of anti-CD7 on T cell activation via CD3. Cells (1 X 105)in triplicates were stimulated in a total volume of 200 ~1,with varying dosesof solid-phase immobilized anti-CD3 in presence and absenceof immobilized mAb 69 (I pg/well) also immobilized on a 96-well round-bottom tissue culture microtiter plate. Incubation with solid-phaseimmobilized 69 mAb alone resulted in no proliferative activity over background (lessthan 300 cpm). Data are from two representative experiments obtained with PBMC of four separatedonors.
parable to those observedin positive control of PBMC cultures stimulated with optimal dose of PHA-P (data not shown). To determine the effect of anti-CD7 on IL-2 production, supernatants from PBMC stimulated with anti-CD7 or submitogenic dose of anti-CD3 were examined in the CTLL-2 bioassay. Anti-CD7 and anti-CD3 by themselves induced little or no IL-2 production (Table 1). However, the supernatants from the comitogenic assay had significant amounts of IL-2 produced by PBMC in responseto stimulation with antiCD7 and anti-CD3. PKC and PTK Inhibitors Block the Comitogenic Efect of Anti-CD7 Activation of protein kinase C (PKC) and protein tyrosine kinase (PTK) has been shown to be an important signal-transduction mechanism for T cells in response to mitogens or anti-CD3 mAb stimulation. Therefore, to determine whether anti-CD7 induced comitogenesisinvolves these signal-transduction pathways, we tested the effect of PKC inhibitor H-7 and PTK inhibitor genistein in PBMC cultures. Figure 4 shows the results of three such experiments on [3H]thymidine incorporation. Proliferation of PBMC stimulated with anti-CD3 or in a combination of anti-CD3 and anti-CD7 was inhibited in a dose-dependent manner by both kinase inhibitors. The level of suppression in cultures stimulated with anti-CD3 and anti-CD7 using H-7 at 10 pA4 was 63 & 8%, at 20 PALM was 84 f 2%, and at 40 PM was 97 * 1% (Fig. 4A). Data in Fig. 4B show that percentage of suppression was 47 t lo%, 8 1 f 0. l%, and 99 + 1%
194
KING, ROY, AND CHAKKALATH Medium
anti-CD7
anti-CD3
C
A
D
am-(CDS+CDI)
II L L m’2 IAL.L IL Exp UX3
IMMUNOFLUORESCENCE
FIG. 3. Anti-CD7 costimulated with CD3 to upregulate IL-2R-a/IL-2 pathway. IL-2 receptor expression was determined by direct immunofluorescence assay as described under Materials and Methods. Data are from three separate experiments. A, B, and C indicate absenceof IL-2R-a expression in PBMC stimulated with media, anti-CD3 (0.05 rig/well), and anti-CD7 69 (1 pg/well), respectively. D shows an increase in IL2R-a expression in PBMC stimulated with a combination of submitogenic anti-CD3 (0.05 &well) and 69 ( 1 &well).
with genistein at dosesof 1, 10, and 50 pM, respectively. A similar effect of H-7 and genistein was also observed in PBMC cultures stimulated with submitogenic dosesof anti-CD3 (Figs. 4A and B). Altogether, these results suggestthat the biochemical pathways of T cell activation, namely the PKC and PTK pathways, are also involved in T cells stimulated via CD7 molecule, directly or indirectly with anti-CD3.
TABLE 1 Effect of Anti-CD7 on IL-2 Secretion in Peripheral Blood T Cells Stimulated with Submitogenic Doses of Anti-CD3 IL-2 secretion (U/ml) as measured by IL-2-dependent murine CTLL-2 Stimulus
Expt. 1
Expt. 2
Expt. 3
Unstimulated Anti-CD3 mAb Anti-CD7 mAb Anti-(CD3 + CD7) mAb PHA-P
IMMUNOLOGY
141, 189-199 (1992)
CD7 Augments T Cell Proliferation via the Interleukin-2 Autocrine Pathway LAWRENCE K.
L. JUNG, AMIT K. ROY, AND HRISHEKPSH R. CHAKKALATH
Division of Pediatric Immunology, Department of Pediatrics, University of MassachusettsMedical Center, 55 Lake Avenue North, Worcester.Massachusetts 01655 Received October 7, 1991; acceptedDecember 30, 1991 The role of CD7, a T cell differentiation antigen, in T cell function is not known at present; this study evaluates the effect of anti-CD7 mAb in PBMC cultures activated with suboptimal concentrations of lectins, antigens, and anti-CD3 mAb. We found that the inclusion of anti-CD7 resulted in increased IL-2 production and IL-2R-(U expression in these cultures. H-7, a protein kinase C (PKC) inhibitor, and genistein, a protein tyrosine kinase (PTK) inhibitor, significantly suppressedthe proliferation of T cells in comitogenic assays.This suggestedthat the comitogenic effectmediated by CD7 molecule involved both the PKC and the PTK pathways of T cell activation. Thesedrugs appeared to affect the CD7-mediated effectsby inhibiting the IL-2 autocrine pathway, especially the up-regulation of IL-ZR-cusince inhibition was not relieved with exogenous rIL-2. Taken together, our results suggestthat CD7 augments T cell function by up-regulating IL-2R01expression and IL-2 production via multiple pathways of protein phosphorylation. o 1992 Academic
Press, Inc.
INTRODUCTION The function of CD7,’ a 40-kDa human T cell specific glycoprotein ( l-4) is of great interest in studies of T cell ontogeny becauseit is the earliest T cell associatedantigen to appear and persists in T cell differentiation (5). Although the precise role that CD7 plays in T cell activation is not known, earlier studies have shown that it is a useful marker of T cell activation, since CD7 expression is up-regulated on PHA- or Con Astimulated T cells (6). On the other hand, CD7 is down-regulated on T cells after treatment with phorbol esters(7). Some studies have reported that soluble anti-CD7 mAb may inhibit in vitro PHA and tetanus toxoid response of PBMC (8) as well as the allogeneic mixed lymphocyte reaction (9). On the other hand, a recent study has suggestedthat immobilized anti-CD7 synergizeswith CD3 mAb to induce T cell activation (IO). One mechanism by which this occurs is that crosslinked anti-CD7 could induce transmembrane calcium flux in T cells (11, 12). CD7 molecule has also been implicated to be the Fc receptor for IgM (13). However, transfection experiments with CD7 cDNA did not confirm this observation (14). Defective expression of CD7 has been noted in a number of diseasessuggestingthat decreasedexpressionin vivo of this glycoprotein may have important pathophysiological consequences( 15, 16). For example, we have found that the T cells of a child with ’ Abbreviations used: CD, cluster designation; kDa, kilodaltons; PKC, protein kinase C; PTK, protein tyrosine kinase; PPD, purified protein derivative. 189 0008-8749192$3.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
190
JUNG,
ROY,
AND
CHAKKALATH
severecombined immunodeficiency were selectively deficient in CD7; their proliferative responsesto T cell mitogens were also defective ( 15). In addition, the patient’s T cells were deficient in IL-2R expression. Although a direct role for CD7 in the immunopathogenesis of this case remains to be proven, this observation suggeststhat CD7 may play an important role in T cell ontogeny. Our study was undertaken to examine if CD7 is involved in peripheral blood T cell functions. In the course of these experiments we observed that immobilized mAb to CD7 induced a signal(s) that in conjunction with submitogenic doses of lectins or anti-CD3 mAb triggered T cell proliferation. The antigenic response in PBMC was also augmented at higher dose of the antigen. In the present study, we also showed that the comitogenic activity of anti-CD7 involved an increase in IL-2 production and IL-2R-a expression that depended on multiple pathways of protein phosphorylation. MATERIALS AND METHODS CeZlseparation. Peripheral blood used in this study was from healthy volunteers of both sexes,ages25-50. PBMC were isolated on Ficoll-Hypaque (density 1.077 g/ml; Pharmacia, Uppsala, Sweden) gradients. After three washings in PBS (pH 7.2), the cells were resuspended in culture medium: RPM1 1640 (GIBCO, Grand Island, NY) supplemented with 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 pg/ ml) (GIBCO), and 10%FCS (Hyclone Laboratories, Logan, UT). Viability of the cells was checked by trypan blue dye exclusion technique and was found to be greater than 96% viable. Stimulation of mononuclear cells. PBMC at a density of 1 X lo5 cells/well in a total volume of 200 ~1culture medium were seededin antibody pretreated microtiter tissue culture plates. The pretreatment of the plates was done as follows: 50 ~1 of the mAbs anti-CD3, 235 (17), and anti-CD7, 69 (18), diluted in PBS (pH 8.0) was added to 96well round-bottom microtiter plates. After an overnight incubation, the plates were washed three times with PBS (pH 6.5). Mitogens including phytohemagglutinin-P (Difco Laboratories, Detroit, MI), Concanavalin A, protein A (Sigma Chemicals, St. Louis, MO), and purified protein derivative (Connaught Laboratories Ltd., Willowdale, Canada) were added at the indicated concentrations. In some experiments, either the protein kinase inhibitor drug H-7 (Seikagaku America, Inc., St. Petersburg, FL) or 4’,5,7-trihydroxyisoflavone (genistein; Biomol Research Laboratories, Plymouth Meeting, PA) dissolved in DMSO (Sigma) was added at the appropriate dose to the plates at the same time as the cells. Finally for certain experiments, cultures were supplemented with 50 U/ml of rIL-2 (Genzyme, Boston, MA). The cultures were maintained in a humidified atmosphere containing 5% COZ for 72 hr. Cell proliferation was estimated by [3H]TdR (0.5 &i = 24 kBq, New England Nuclear, Boston, MA, sp act 6.7 Ci/mmol) incorporation during the last 16 hr of culture. The percentage inhibition was calculated as follows: o/ inhibition = 1 - cpm in cultures treated with drugs 0 x 100. cpm in control cultures without dtugs Immunofluorescence studies. Cells were stimulated with the appropriate mAb in presenceor absenceof the drugs. At the end of 72 hr in culture, the cells were harvested using a rubber policeman and washed twice in culture medium. Cells were stained with either anti-IL-2R-a! mAb conjugated to FITC (Becton Dickinson, Mountain View, CA) or a classspecific control mAb reagent at 4°C for 30 min. After extensive washing,
CD7 AND T CELL ACTIVATION
191
the cells were analyzed on a FACScan flow cytometer, (Becton Dickinson). Log fluorescenceof the gated population was measured and 5000 cells were collected and analyzed using FACScan Research software. IL-2 assay. IL-2 production by PBMC cultures stimulated with mAb was detected by monitoring the ability of the culture supernatants to support the growth of the murine IL-2 dependent cell line CTLL-2 (American Type Tissue Collection). Briefly, culture supernatants were subjected to one freeze-thaw cycle before analysis and 100 ~1of the supernatant was used per well. CTLL-2 cells were extensively washed (four times) with medium devoid of IL-2 and added at a concentration of 1 X lo4 cells in 100 ,ul per well. As reference, rIL-2 (Genzyme) serially diluted with RPM1 was also included. Cultures were incubated for 24 hr with [3H]thymidine being added 4 hr before harvest. Thymidine incorporation was determined at 24 hr by liquid scintillation counting and the IL-2 present in each sample was determined in terms of units per milliliter as compared to the reference rIL-2. Agents used to stimulate PBMC such as mAb separately or in combination did not support the growth of CTLL-2. Statistical analysis. Statistical analysis was done by Student’s t test. RESULTS Monoclonal Antibody to CD7 Enhances PBMC Proliferation To understand the functional role CD7 plays in T cell activation, we examined the effectsof immobilized mAb to CD7 (mAb 69) on the proliferative capacity of PBMC. The cells were incubated with varying dosesof PHA-P, Con A, protein A, and PPD in a 96-well round-bottom culture plate precoated with mAb 69 (1 pg/well). Uptake of [3H]thymidine was measured after 3 (mitogen cultures) and 6 (antigen cultures) days in culture. Four experiments were done with PBMC from different donors and the result of one representative experiment is shown in Fig. 1. The presence of immobilized anti-CD7 in the incubation mixture resulted in an enhancement of the proliferation of PBMC in response to mitogens. This effect is best appreciated with submitogenic dosesof the lectins in culture. The comitogenic effect of anti-CD7 was not detectable at high dosesof the lectins. However, with most donors similar comitogenic effect was observed even with a low dose of anti-CD7 (0.1 pg/well; data not shown). Anti-CD7 mAb was also found to enhance the proliferation to PPD at higher antigenic dose (50 pg/ml). We also observed that the comitogenic effectswere lessevident when the anti-CD7 mAb was in soluble form instead of bound to the plastic surface. However, soluble anti-CD7 mediated a similar enhancing effect when added to wells precoated with a crosslinking antibody (anti-mouse Ig). Isotype mouse Ig controls and anti-T1 1, antibodies, immobilized in culture plates, were without effecton mitogen aswell asantigenstimulated PBMC (data not shown). To study the effect of anti-CD7 on the CD3-Ti complex, we investigated the comitogenie effect of anti-CD7 and anti-CD3 mAb. To this end, PBMC or purified T cells were added to culture plates precoated with varying doses of anti-CD3 mAb and a dose of 1 pg/well of anti-CD7. Two representative experiments are shown in Fig. 2. The anti-CD3 mAb at higher doseswas capable by itself to induce significant cellular proliferation, whereasat the lower dose minimal proliferation was observed. However, the crosslinking of CD3 with anti-CD7 caused a strong increase in proliferation. The effect was more evident with the lower dose of anti-CD3 mAb. Similar effectsof antiCD3/CD7 were observed in purified T cell cultures (data not shown). Taken together,
192
JUNG, ROY, AND CHAKKALATH
lOO--
0
1
5
CONC
10
20
15
25
(w/ml)
OF PHA-P
CONC
OF CON-A
&g/ml)
50-
150~ ,25-.
0-o
ProtIm
o-,.,.0
Prctaln
C
A alma A + antl-CD7
40--
0-o
PPD dona
~...‘a
PPD + cdl-CD7
II
lOO--
30 -75 -,;* .,... p
09//, 0
t
p. . ..p
,
,
,
,
5
10
15
20
20 --
T
, 25
0
10
CONC. OF PROTEIN A (pg/ml)
20
30
40
50
60
0
CONC OF PPD &g/ml)
FIG. 1. Enhancement of the proliferation of PBMC by the mAb to CD7. Cells (1 X IO’) in triplicates were stimulated in a total volume of 200 ~1,with varying dosesof PHA-P (A), Con A (B), protein A (C), and PPD (D) in the presenceand absenceof anti-CD7 (69; 1 &well) precoated on a 96-well round-bottom tissue culture plate. Of four separate experiments with four donors, the results from one experiment are represented as mean counts per minute (cpm). The mean cpm in cultures stimulated with 69 alone (278) was comparable to the values obtained in control cultures with media alone (266). The mitogen cultures (PHA-P, Con A, and protein A) were terminated on Day 3 and the antigen culture (PPD) was terminated on Day 6.
the above results suggestedthat CD7 molecule may have a regulatory role in T cell activation. Role of IL-2 Pathway in Anti-CD7-Induced Comitogenesis We tested whether the comitogenic effect by anti-CD7 mAb involved the IL-2 pathway. Therefore, we studied the expression of IL-2R-(r and the production of IL-2 in PBMC stimulated by solid-phaseimmobilized anti-CD7 either alone or in combination with anti-CD3 mAb. As shown in Fig. 3 neither the mAb to CD7 nor submitogenic dose of anti-CD3 mAb by themselves induced the expression of IL-2R-a. However, the combination of both the mAbs induced very significant levels of IL-2R-a expression. It was com-
193
CD7 AND T CELL ACTIVATION O-O
anti-CD3
O..... 0 anti-CD3
A---A
500
T
anti-CD3 alone
A.. .. A antl-CD3
400
alone + anti-CD7
+ anti-CD7
EXPT 1 EXPT 2
300
200
100
0
CONC OF IMMOBILIZED anti-CD3
&g/well)
FIG. 2. Comitogenic effect of anti-CD7 on T cell activation via CD3. Cells (1 X 105)in triplicates were stimulated in a total volume of 200 ~1,with varying dosesof solid-phase immobilized anti-CD3 in presence and absenceof immobilized mAb 69 (I pg/well) also immobilized on a 96-well round-bottom tissue culture microtiter plate. Incubation with solid-phaseimmobilized 69 mAb alone resulted in no proliferative activity over background (lessthan 300 cpm). Data are from two representative experiments obtained with PBMC of four separatedonors.
parable to those observedin positive control of PBMC cultures stimulated with optimal dose of PHA-P (data not shown). To determine the effect of anti-CD7 on IL-2 production, supernatants from PBMC stimulated with anti-CD7 or submitogenic dose of anti-CD3 were examined in the CTLL-2 bioassay. Anti-CD7 and anti-CD3 by themselves induced little or no IL-2 production (Table 1). However, the supernatants from the comitogenic assay had significant amounts of IL-2 produced by PBMC in responseto stimulation with antiCD7 and anti-CD3. PKC and PTK Inhibitors Block the Comitogenic Efect of Anti-CD7 Activation of protein kinase C (PKC) and protein tyrosine kinase (PTK) has been shown to be an important signal-transduction mechanism for T cells in response to mitogens or anti-CD3 mAb stimulation. Therefore, to determine whether anti-CD7 induced comitogenesisinvolves these signal-transduction pathways, we tested the effect of PKC inhibitor H-7 and PTK inhibitor genistein in PBMC cultures. Figure 4 shows the results of three such experiments on [3H]thymidine incorporation. Proliferation of PBMC stimulated with anti-CD3 or in a combination of anti-CD3 and anti-CD7 was inhibited in a dose-dependent manner by both kinase inhibitors. The level of suppression in cultures stimulated with anti-CD3 and anti-CD7 using H-7 at 10 pA4 was 63 & 8%, at 20 PALM was 84 f 2%, and at 40 PM was 97 * 1% (Fig. 4A). Data in Fig. 4B show that percentage of suppression was 47 t lo%, 8 1 f 0. l%, and 99 + 1%
194
KING, ROY, AND CHAKKALATH Medium
anti-CD7
anti-CD3
C
A
D
am-(CDS+CDI)
II L L m’2 IAL.L IL Exp UX3
IMMUNOFLUORESCENCE
FIG. 3. Anti-CD7 costimulated with CD3 to upregulate IL-2R-a/IL-2 pathway. IL-2 receptor expression was determined by direct immunofluorescence assay as described under Materials and Methods. Data are from three separate experiments. A, B, and C indicate absenceof IL-2R-a expression in PBMC stimulated with media, anti-CD3 (0.05 rig/well), and anti-CD7 69 (1 pg/well), respectively. D shows an increase in IL2R-a expression in PBMC stimulated with a combination of submitogenic anti-CD3 (0.05 &well) and 69 ( 1 &well).
with genistein at dosesof 1, 10, and 50 pM, respectively. A similar effect of H-7 and genistein was also observed in PBMC cultures stimulated with submitogenic dosesof anti-CD3 (Figs. 4A and B). Altogether, these results suggestthat the biochemical pathways of T cell activation, namely the PKC and PTK pathways, are also involved in T cells stimulated via CD7 molecule, directly or indirectly with anti-CD3.
TABLE 1 Effect of Anti-CD7 on IL-2 Secretion in Peripheral Blood T Cells Stimulated with Submitogenic Doses of Anti-CD3 IL-2 secretion (U/ml) as measured by IL-2-dependent murine CTLL-2 Stimulus
Expt. 1
Expt. 2
Expt. 3
Unstimulated Anti-CD3 mAb Anti-CD7 mAb Anti-(CD3 + CD7) mAb PHA-P
