Clin. exp. Immunol. (1991) 85, 164-167
ADONIS
0009910491002 1 OB
Inhibition of lymphocyte proliferation by a monoclonal antibody (RFT2) against CD7 Y. COSTANTINIDES, G. KINGSLEY, C. PITZALIS & G. S. PANAYI Rheumatology Unit, Division United Medical and Dental Schools, Guy's Hospital, London, England
of
Medicine,
(Acceptedfor publication 14 January 1991)
SUMMARY The effect of the monoclonal antibody RFT2, directed against CD7, on T cell proliferation in unseparated peripheral blood mononuclear cell populations induced by various stimulants was investigated. The addition of RFT2 to cell cultures inhibited the T cell proliferation induced by tuberculin PPD and OKT3 but not by phytohaemagglutinin, concanavalin A or phorbol myristic acid; RFT2 had to be present during the first 24 h of culture in order to elicit inhibition; inhibition of proliferation was not due to down regulation of interleukin-2 receptor on the surface of T cells; and suppressive effects could be transferred by mononuclear cells pre-treated with RFT2. These results are of particular relevance in view of the known in vivo suppressive effect of RFT2 in humans.
Keywords T cells CD7
suppressor
cells lymphocyte proliferation IL-2 receptor
MoAbs. It has been shown that the CD7 structure may be involved in the provision of T cell help for in vitro immunoglobulin production against erythrocyte-bound determinants (Morishma et al., 1982). The dim CD7 population contains precursor T cells proliferating in the allogeneic mixed lymphocyte reaction (MLR) as well as the precursor T cells necessaty for the in vitro generation of allogeneic cytotoxic T cells (Morishma et al., 1982). Furthermore, while MoAbs against CD7 do not inhibit phytohaemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) induced T cell proliferation (Lazarovitz et al., 1987), one anti-CD7 MoAb (7G5) is particularly effective at inhibiting the autologous and allogeneic MLR (AMLR) (Lazarovits & Karsh, 1988a; Morishma et al., 1982). There is some evidence that different MoAbs have different functional effects in vitro; this may also be true in vivo (Lazarovits et al., 1987). The intracellular mechanism of CD7 signal transduction is not known. It is known that CD7 is phosphorylated in resting T cells, unlike most similar molecules such as CD2, and, following activation, becomes hyper-phosphorylated; this is linked to progression of the cell to proliferation (Chatila & Geha, 1988). Because ofthe apparent importance of CD7 in events which lead to T cell activation, it has aroused interest in the context of autoimmune diseases in which altered T cell immunity plays a central role. For example, in rheumatoid arthritis (RA), it has been shown, using the 7G5 MoAb, that peripheral blood T cells have decreased expression of CD7 on their surface (Lazarovits & Karsh, 1988a). In the rheumatoid joint there appears to be an accumulation of brightly staining CD7+ T cells, which are activated as assessed by surface HLA-DR expression (Poulter et al., 1985). In view of the successful use of anti-CD7 MoAb in
INTRODUCTION The development of monoclonal antibodies (MoAbs) to cell surface structures on T lymphocytes has increased our knowledge of the phenotypic classification of these cells as well as the role which surface molecules play in T cell function. These advances have helped in our understanding of pathogenic events involving T lymphocytes and have also been ofclinical use in the diagnosis of malignant disease, and more recently, in the treatment of allograft rejection and rheumatoid arthritis (Poulter et al., 1985; Carrera et al., 1988). One T cell surface structure is CD7 which is a 40-KD glycoprotein, a member of the immunoglobulin superfamily, expressed on the surface of thymocytes and most peripheral blood T cells (Morishma et al., 1982; Tax et al., 1984; Lazarovits & Karsh, 1988b). Its gene has been cloned and has a structural homology with genes of known adhesion molecules (Aruffo & Seed, 1987; Snadrin et al., 1987). Its ligand is unknown. On mature peripheral T lymphocytes two populations can be defined, one with high and the other with low expression of CD7 (bright and dim, respectively) (Morishma et al., 1982; Tax et al., 1984; Lazarovitz et al., 1987; Lazarovitz & Karsh, 1988a). These may represent resting (dim) and activated (bright) T cells since, following stimulation in vitro of resting T cells, the surface expression of CD7 increases; subsequently, however, the molecule modulates from the surface of the cell (Lazarovitz & Karsh, 1988a, 1988b). The function of the CD7 molecule, and of the T cells which express it, has been investigated using a variety of anti-CD7 Correspondence: Professor G. S. Panayi, Rheumatology Unit, Division of Medicine, United Medical and Dental Schools, Guy's Hospital, London SE1 9RT, UK.
164
CD7 and T cell proliferation preventing human allograft rejection, we have used one such MoAb (RFT2) for the treatment of patients with RA (Kirkham et al., 1988). We have found that the peripheral blood T cells in such patients show decreased proliferation in vitro to mitogenic stimulation after treatment (Kirkham et al., 1987, 1988). Consequently, we decided to investigate further the effects of this MoAb on lymphocyte function in vitro. We show that RFT2 has potent immunosuppressive effects in vitro and that this appears to be due to the induction of a suppressor effect in a subpopulation of T cells.
165
Table 1. The effect of RFT2 (5 ug/ml) on the T cell proliferation induced by tuberculin PPD (10 pg/ml)
Experiment no. 1 2 3 4
Stimulus (d/min)
Stimulus (d/min)
TCM 1294 1735 2444 1635
TCM + RFT2 794 1274 2269 907
Inhibition (%)*
139 27 7 45
(55 + 58)
MATERIALS AND METHODS Mononuclear cell separation Peripheral blood mononuclear cells (MNC) from healthy subjects were separated from heparinized blood by gradient centrifugation on Lymphoprep and cultured, at 106/ml, in tissue culture medium (TCM) consisting of RPMI 1640 containing 10% heat-inactivated fetal calf serum, antibiotics and bicarbonate as previously described (Kingsley et al., 1988). Mononuclear cell culture For mitogenic studies, cells were cultured for 3 days with TCM alone (control wells), phorbol-12-myristate-13-acetate (PMA; 50 g/ml, Sigma), PHA (10 g/ml, Sigma), ConA (15 g/ml, Sigma) and MoAb OKT3 hybridoma supernatant (0 1 pg/ml; derived from OKT3 hybridoma, American Type Culture Collection). For antigenic studies cells were cultured with tuberculin PPD (10 pg/ml; Central Veterinary Laboratory) or TCM alone for 6 days. Optimal doses were established in preliminary experiments. The MoAb RFT2 (anti-CD7), a kind gift of Professor G. Janossy, Royal Free Hospital, London, was added to cultures where indicated at various doses from 0-02 to 5 pg/ml. An irrelevant monoclonal antibody (anti-herpes simplex virus, American Type Culture Collection) was used as a control in parallel experiments. Pre-culture experiments In experiments involving the pre-incubation of cells with MoAbs (pre-culture experiments), a proportion of the freshly separated MNC were cultured for 24 h with RFT2 or irrelevant
MoAb (pretreated cell) while the remainder of the cells (fresh cells) were stored at 4°C for 24 h until required. Pretreated cells were then mixed with the fresh cells at several ratios and then cultured with TCM, PPD or OKT3. All cultures were performed in 96-well microtitre plates, in triplicate, in a humidified incubator in the presence of 5% CO2. Tritiated thymidine (3H-TdR) (0-2 Ci per well; Amersham) was added during the last 24 h of culture. Cultures were harvested on a semi-automatic harvester (Skatron) and incorporated 3H-TdR measured by liquid scintillation in a beta counter (LKB, Bromma, Sweden). Cytofluorographic analysis Immunofluorescence and cytofluorographic analysis was performed as previously described (Pitzalis et al., 1988). Briefly, MNC pre-incubated with 2% normal human serum, were labelled in suspension with anti-Tac (anti-IL-2 receptor (IL-2R) MoAb, Becton Dickinson). The MNC were then labelled with FITC-conjugated goat anti-mouse IgG (Becton Dickinson) and, after blocking with 2% normal mouse serum, stained with
1 2 3 4
PPD 33 180 65 100 35 581 3468
PPD + RFT2 11918 36654 7649 1571
64 44 79 55 (61 + 15)
* Mean + 1 s.d. in parentheses.
an anti-CD3 MoAb (Leu4; Becton Dickinson). Analysis was carried out on a cytofluorograph (FACScan, Becton Dickinson). Ten-thousand cells were analysed per sample; dead and non-lymphoid cells were excluded by appropriate setting of the forward and 90° light scatter gates and appropriate single-stain and negative controls were performed.
Statistical analysis Results are expressed as mean + 1 s.e.m. Statistical analysis was by the paired Student's t-test. RESULTS The effect of RFT2 on the T cell proliferation induced by tuberculin PPD RFT2 (5 pg/ml) was added at the initiation of culture to peripheral blood MNC stimulated for 6 days with tuberculin PPD and the effect on proliferation was noted. As can be seen from Table 1, there was a marked (61 %) inhibition of proliferation. The inhibition was highly significant (P < 0-02). Additional experiments showed that there was a clear-cut doseresponse effect whereby increasing amounts of the MoAb caused increased inhibition of the tuberculin PPD response
(Fig. 1). The effect of RFT2 on the Tcellproliferation induced by mitogens As shown in Fig. 2, RFT2 was able to inhibit T cell proliferation induced by OKT3 but not that due to ConA, PHA or PMA. Again, the inhibition was dose related. The time course of the inhibitory effect of RFT2 on T cell proliferation Peripheral blood MNC cultures were set up and stimulated either for 3 days with OKT3 or for 6 days with tuberculin PPD. RFT2 (2 ,g/ml) was added, at 0, 24 and 48 h after initiation of culture. It can be seen (Table 2) that both for tuberculin PPD and for OKT3 there is inhibition of the response when RFT2 is added at the initiation of culture. Delaying the addition to 24 or 48 h leads to a complete loss of the inhibitory effect.
Y. Costantinides et al.
166 I00
Table 2. The effect of RFT2 (2 pg/ml) added at 0, 24 and 48 h of culture on PPD- and OKT3-induced proliferation
r
T
80 H
t
0
0-
60 I-
Proliferation induced by
T
Time of addition of RFT2 No RFT2 Oh 24 h 48 h
-o -c .9 C
40 I- NS
OKT3
81 925 + 36 518 24268 + 11 892t 33 137+ 10393 34925 + 10 282
43 225 + 8863 12481 +4108* 73732+25313 99 540+ 24 120
Proliferation is expressed as mean d/min + s.e.m. standard error. Results of seven experiments (PPD) and three experiments (OKT3). * P=0-01 (paired Student's (-test).
20
0
PPD
t P= 0-02 (paired Student's (-test). 0.05
0-2 0-5 RFT2 added (pLg/ml)
Fig. 1. Increasing amounts (0-05-2 yg/ml) of RFT2 causes increasing inhibition of the T cell response to tuberculin PPD. The response is shown as a percentage of that in the presence of PPD alone and represents the results of four experiments. NS, not significant; * P
ADONIS
0009910491002 1 OB
Inhibition of lymphocyte proliferation by a monoclonal antibody (RFT2) against CD7 Y. COSTANTINIDES, G. KINGSLEY, C. PITZALIS & G. S. PANAYI Rheumatology Unit, Division United Medical and Dental Schools, Guy's Hospital, London, England
of
Medicine,
(Acceptedfor publication 14 January 1991)
SUMMARY The effect of the monoclonal antibody RFT2, directed against CD7, on T cell proliferation in unseparated peripheral blood mononuclear cell populations induced by various stimulants was investigated. The addition of RFT2 to cell cultures inhibited the T cell proliferation induced by tuberculin PPD and OKT3 but not by phytohaemagglutinin, concanavalin A or phorbol myristic acid; RFT2 had to be present during the first 24 h of culture in order to elicit inhibition; inhibition of proliferation was not due to down regulation of interleukin-2 receptor on the surface of T cells; and suppressive effects could be transferred by mononuclear cells pre-treated with RFT2. These results are of particular relevance in view of the known in vivo suppressive effect of RFT2 in humans.
Keywords T cells CD7
suppressor
cells lymphocyte proliferation IL-2 receptor
MoAbs. It has been shown that the CD7 structure may be involved in the provision of T cell help for in vitro immunoglobulin production against erythrocyte-bound determinants (Morishma et al., 1982). The dim CD7 population contains precursor T cells proliferating in the allogeneic mixed lymphocyte reaction (MLR) as well as the precursor T cells necessaty for the in vitro generation of allogeneic cytotoxic T cells (Morishma et al., 1982). Furthermore, while MoAbs against CD7 do not inhibit phytohaemagglutinin (PHA), concanavalin A (ConA) and pokeweed mitogen (PWM) induced T cell proliferation (Lazarovitz et al., 1987), one anti-CD7 MoAb (7G5) is particularly effective at inhibiting the autologous and allogeneic MLR (AMLR) (Lazarovits & Karsh, 1988a; Morishma et al., 1982). There is some evidence that different MoAbs have different functional effects in vitro; this may also be true in vivo (Lazarovits et al., 1987). The intracellular mechanism of CD7 signal transduction is not known. It is known that CD7 is phosphorylated in resting T cells, unlike most similar molecules such as CD2, and, following activation, becomes hyper-phosphorylated; this is linked to progression of the cell to proliferation (Chatila & Geha, 1988). Because ofthe apparent importance of CD7 in events which lead to T cell activation, it has aroused interest in the context of autoimmune diseases in which altered T cell immunity plays a central role. For example, in rheumatoid arthritis (RA), it has been shown, using the 7G5 MoAb, that peripheral blood T cells have decreased expression of CD7 on their surface (Lazarovits & Karsh, 1988a). In the rheumatoid joint there appears to be an accumulation of brightly staining CD7+ T cells, which are activated as assessed by surface HLA-DR expression (Poulter et al., 1985). In view of the successful use of anti-CD7 MoAb in
INTRODUCTION The development of monoclonal antibodies (MoAbs) to cell surface structures on T lymphocytes has increased our knowledge of the phenotypic classification of these cells as well as the role which surface molecules play in T cell function. These advances have helped in our understanding of pathogenic events involving T lymphocytes and have also been ofclinical use in the diagnosis of malignant disease, and more recently, in the treatment of allograft rejection and rheumatoid arthritis (Poulter et al., 1985; Carrera et al., 1988). One T cell surface structure is CD7 which is a 40-KD glycoprotein, a member of the immunoglobulin superfamily, expressed on the surface of thymocytes and most peripheral blood T cells (Morishma et al., 1982; Tax et al., 1984; Lazarovits & Karsh, 1988b). Its gene has been cloned and has a structural homology with genes of known adhesion molecules (Aruffo & Seed, 1987; Snadrin et al., 1987). Its ligand is unknown. On mature peripheral T lymphocytes two populations can be defined, one with high and the other with low expression of CD7 (bright and dim, respectively) (Morishma et al., 1982; Tax et al., 1984; Lazarovitz et al., 1987; Lazarovitz & Karsh, 1988a). These may represent resting (dim) and activated (bright) T cells since, following stimulation in vitro of resting T cells, the surface expression of CD7 increases; subsequently, however, the molecule modulates from the surface of the cell (Lazarovitz & Karsh, 1988a, 1988b). The function of the CD7 molecule, and of the T cells which express it, has been investigated using a variety of anti-CD7 Correspondence: Professor G. S. Panayi, Rheumatology Unit, Division of Medicine, United Medical and Dental Schools, Guy's Hospital, London SE1 9RT, UK.
164
CD7 and T cell proliferation preventing human allograft rejection, we have used one such MoAb (RFT2) for the treatment of patients with RA (Kirkham et al., 1988). We have found that the peripheral blood T cells in such patients show decreased proliferation in vitro to mitogenic stimulation after treatment (Kirkham et al., 1987, 1988). Consequently, we decided to investigate further the effects of this MoAb on lymphocyte function in vitro. We show that RFT2 has potent immunosuppressive effects in vitro and that this appears to be due to the induction of a suppressor effect in a subpopulation of T cells.
165
Table 1. The effect of RFT2 (5 ug/ml) on the T cell proliferation induced by tuberculin PPD (10 pg/ml)
Experiment no. 1 2 3 4
Stimulus (d/min)
Stimulus (d/min)
TCM 1294 1735 2444 1635
TCM + RFT2 794 1274 2269 907
Inhibition (%)*
139 27 7 45
(55 + 58)
MATERIALS AND METHODS Mononuclear cell separation Peripheral blood mononuclear cells (MNC) from healthy subjects were separated from heparinized blood by gradient centrifugation on Lymphoprep and cultured, at 106/ml, in tissue culture medium (TCM) consisting of RPMI 1640 containing 10% heat-inactivated fetal calf serum, antibiotics and bicarbonate as previously described (Kingsley et al., 1988). Mononuclear cell culture For mitogenic studies, cells were cultured for 3 days with TCM alone (control wells), phorbol-12-myristate-13-acetate (PMA; 50 g/ml, Sigma), PHA (10 g/ml, Sigma), ConA (15 g/ml, Sigma) and MoAb OKT3 hybridoma supernatant (0 1 pg/ml; derived from OKT3 hybridoma, American Type Culture Collection). For antigenic studies cells were cultured with tuberculin PPD (10 pg/ml; Central Veterinary Laboratory) or TCM alone for 6 days. Optimal doses were established in preliminary experiments. The MoAb RFT2 (anti-CD7), a kind gift of Professor G. Janossy, Royal Free Hospital, London, was added to cultures where indicated at various doses from 0-02 to 5 pg/ml. An irrelevant monoclonal antibody (anti-herpes simplex virus, American Type Culture Collection) was used as a control in parallel experiments. Pre-culture experiments In experiments involving the pre-incubation of cells with MoAbs (pre-culture experiments), a proportion of the freshly separated MNC were cultured for 24 h with RFT2 or irrelevant
MoAb (pretreated cell) while the remainder of the cells (fresh cells) were stored at 4°C for 24 h until required. Pretreated cells were then mixed with the fresh cells at several ratios and then cultured with TCM, PPD or OKT3. All cultures were performed in 96-well microtitre plates, in triplicate, in a humidified incubator in the presence of 5% CO2. Tritiated thymidine (3H-TdR) (0-2 Ci per well; Amersham) was added during the last 24 h of culture. Cultures were harvested on a semi-automatic harvester (Skatron) and incorporated 3H-TdR measured by liquid scintillation in a beta counter (LKB, Bromma, Sweden). Cytofluorographic analysis Immunofluorescence and cytofluorographic analysis was performed as previously described (Pitzalis et al., 1988). Briefly, MNC pre-incubated with 2% normal human serum, were labelled in suspension with anti-Tac (anti-IL-2 receptor (IL-2R) MoAb, Becton Dickinson). The MNC were then labelled with FITC-conjugated goat anti-mouse IgG (Becton Dickinson) and, after blocking with 2% normal mouse serum, stained with
1 2 3 4
PPD 33 180 65 100 35 581 3468
PPD + RFT2 11918 36654 7649 1571
64 44 79 55 (61 + 15)
* Mean + 1 s.d. in parentheses.
an anti-CD3 MoAb (Leu4; Becton Dickinson). Analysis was carried out on a cytofluorograph (FACScan, Becton Dickinson). Ten-thousand cells were analysed per sample; dead and non-lymphoid cells were excluded by appropriate setting of the forward and 90° light scatter gates and appropriate single-stain and negative controls were performed.
Statistical analysis Results are expressed as mean + 1 s.e.m. Statistical analysis was by the paired Student's t-test. RESULTS The effect of RFT2 on the T cell proliferation induced by tuberculin PPD RFT2 (5 pg/ml) was added at the initiation of culture to peripheral blood MNC stimulated for 6 days with tuberculin PPD and the effect on proliferation was noted. As can be seen from Table 1, there was a marked (61 %) inhibition of proliferation. The inhibition was highly significant (P < 0-02). Additional experiments showed that there was a clear-cut doseresponse effect whereby increasing amounts of the MoAb caused increased inhibition of the tuberculin PPD response
(Fig. 1). The effect of RFT2 on the Tcellproliferation induced by mitogens As shown in Fig. 2, RFT2 was able to inhibit T cell proliferation induced by OKT3 but not that due to ConA, PHA or PMA. Again, the inhibition was dose related. The time course of the inhibitory effect of RFT2 on T cell proliferation Peripheral blood MNC cultures were set up and stimulated either for 3 days with OKT3 or for 6 days with tuberculin PPD. RFT2 (2 ,g/ml) was added, at 0, 24 and 48 h after initiation of culture. It can be seen (Table 2) that both for tuberculin PPD and for OKT3 there is inhibition of the response when RFT2 is added at the initiation of culture. Delaying the addition to 24 or 48 h leads to a complete loss of the inhibitory effect.
Y. Costantinides et al.
166 I00
Table 2. The effect of RFT2 (2 pg/ml) added at 0, 24 and 48 h of culture on PPD- and OKT3-induced proliferation
r
T
80 H
t
0
0-
60 I-
Proliferation induced by
T
Time of addition of RFT2 No RFT2 Oh 24 h 48 h
-o -c .9 C
40 I- NS
OKT3
81 925 + 36 518 24268 + 11 892t 33 137+ 10393 34925 + 10 282
43 225 + 8863 12481 +4108* 73732+25313 99 540+ 24 120
Proliferation is expressed as mean d/min + s.e.m. standard error. Results of seven experiments (PPD) and three experiments (OKT3). * P=0-01 (paired Student's (-test).
20
0
PPD
t P= 0-02 (paired Student's (-test). 0.05
0-2 0-5 RFT2 added (pLg/ml)
Fig. 1. Increasing amounts (0-05-2 yg/ml) of RFT2 causes increasing inhibition of the T cell response to tuberculin PPD. The response is shown as a percentage of that in the presence of PPD alone and represents the results of four experiments. NS, not significant; * P
