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INHIBITORS OF Src-FAMILY TYROSINE KINASES FAVOUR Th2 DIFFERENTIATION
Article No. cyto.1998.0416, available online at http://www.idealibrary.com on
INHIBITORS OF Src-FAMILY TYROSINE KINASES FAVOUR Th2 DIFFERENTIATION Ulrike Gimsa, Avrion Mitchison, Rodger Allen* Two potent pyrazolo-pyrimidine inhibitors of Src-family kinases were found to enhance interleukin 4 (IL-4) and reduce interferon ã (IFN-ã) production in cultures of splenocytes from ovalbumin-specific TCR-transgenic BALB/c mice. The effect was increased by addition of a monoclonal antibody binding to domains 3/4 of CD4, while other antibodies binding to domain 1 had the opposite effect. The inhibitors suppressed CD40 ligand (CD40L) expression on activated CD4 T cells, which may explain this Th2 differentiating effect. More generally, the effect fits within the overall framework of signal attenuation in T cells driving this form of differentiation. The inhibitors did not revert previously induced Th1 differentiation in serial cultures of the TCR-transgenic cells. Drugs with this activity are of obvious interest as probes and potential therapeutic agents in autoimmune disease. 1999 Academic Press
The purpose of this study was to identify novel agents able to influence T-cell subset differentiation. CD4 + T cells are divided into Th1 and Th2 types according to their cytokine production and function; although this division may be over-simplified, the concept of balance between Th1 and Th2 cells in the immune system is now well established.1 To that end we have used cells transgenic for an áâ T-cell receptor (TCR) able to recognize the ovalbumin (OVA) peptide 323–339, on a BALB/c background.2 Upon stimulation in vitro by OVA peptide these cells produce interferon ã (IFN-ã) and interleukin 4 (IL-4), the cytokines characteristic of Th1 and Th2 cells. This system has been widely used to investigate Th1/Th2 differentiation as influenced by different APC, antigen-dose and cytokines.3 Bacteria and parasites are known to drive differentiation selectively. Listeria monocytogenes induces differentiation of Th1 cells4 while helminths induce that of Th2 cells.1 Both major histocompatibility complex (MHC) and genetic background influence differentiation.5–7 Imbalance in favour of Th1 cells occurs in the major human autoimmune diseases, such as rheumatoid arthritis8 and multiple sclerosis.9 Agents From the Deutsches RheumaForschungsZentrum Berlin, Germany; *Celltech Therapeutics Ltd, Slough, UK Correspondence to: Ulrike Gimsa, Institute of Anatomy, HumboldtUniversity Clinic Charite´, Dept. of Cell and Neurobiology, D-10098 Berlin, Germany Received 18 February 1998; accepted for publication 21 June 1998 1999 Academic Press 1043–4666/99/030208+08 $30.00/0 KEY WORDS: anti-CD4/CD40L/Lck/Th2/ tyrosine kinase inhibitor 208
able to restore balance are needed, as probes to test the possibility that the imbalance itself contributes to pathogenesis, and as potential therapeutic agents. A growing list of agents has been found to drive differentiation of Th2 cells preferentially: salbutamol,10,11 monomethylfumarate,12 thalidomide13 (but see also Ref. 14) and prostaglandin-E2.15 Additional agents have been found able to prolong allograft survival in association with Th2 differentiation.16 Some of these agents have been identified as acting on antigen-presenting cells (APC), others appear to act within the responding T cells themselves, while for the majority the target cell is uncertain. For those agents which do act within T cells the underlying question remains open of whether they modify signal strength from the TCR complex, or whether they act on the process of differentiation independently of this pathway. Agents likely to interfere with signalling from CD4 molecules offer a promising means of modulating signal strength, as anti-CD4 antibody can drive Th2 differentiation in vitro17 and in vivo. Treatment with this type of antibody facilitates the induction of CD4 T cell populations able to protect against autoimmunity18 through a mechanism suspected of involving cytokine imbalance. This form of treatment has a long history of promising results in clinical organ transplantation16 and more recently in rheumatoid arthritis,19 although it has not found general use. Surprisingly, CD4 / mice show a bias away from Th2 differentiation,20,21 presumably reflecting selective events in the immune system as it develops in these mice. CYTOKINE, Vol. 11, No. 3 (March), 1999: pp 208–215
Src family inhibitors favour Th2 differentiation / 209
% maximum IL-4 production
pg/ml
4000 2000
0
50 25 0
125 % maximum IFN-γ production
pg/ml
15 000 10 000
Figure 1.
75
100 200 300 400 500 600 700 800 900 1000 PP2 (nM)
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100 200 300 400 500 600 700 800 900 1000 PP2 (nM)
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125
A
6000
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100 75 50 25 0
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100 200 300 400 500 600 700 800 900 1000 PP2 (nM)
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PP2 promotes Th2 differentiation in a dose-dependent manner.
Two experiments representative of four. (A) Dose-dependent IL-4 production of TCR transgenic T cells in the presence of PP2. (B) Half-maximum IL-4 production (EC50) at 36858 nM (meansSEM in four experiments). Data were normalized as described in the Materials and Methods section. (C) Dose-dependent IFN-ã production of TCR transgenic T cells in the presence of PP2. (D) Half-maximum IFN-ã production (EC50) of TCR transgenic T cells at 26366 nM (meansSEM in four experiments). Data were normalized as described in the Materials and Methods section.
The Src-family tyrosine kinase p56lck (Lck) is physically associated with the cytoplasmic tail of CD4 and is the only molecule known to transmit signals from CD4.22 Lck also regulates tyrosine phosphorylation of the T cell receptor (TCR)-æ chain, CD3-å, and ZAP-70. Lck / mice have greatly reduced numbers of thymocytes and peripheral áâ + T cells.23 p59fyn (Fyn) is another member of the same family that also phosphorylates proteins of the T cell receptor complex, although it does not bind to the CD4 molecule. The immune system of Fyn / mice is impaired, but to a much lesser extent than that of Lck / mice.24 The Src family specific tyrosine kinase inhibitors tested here were developed as agents for blocking the kinase activity of Lck and Fyn.25 A possible mechanism through which an agent that interferes with T cell signalling could influence Th1/Th2 differentiation is via an effect on CD40L expression. TCR ligation leads to upregulation of CD40L which then signals back into APC via ligation of CD40.26–29
RESULTS Increased IL-4 production and reduced IFN-ã production in the presence of PP1 and PP2 As used here, PP2 significantly enhanced IL-4 production (Fig. 1A,B) and reduced IFN-ã production
(Fig. 1C,D); see also Figure 2. Above 1000 nM, proliferation was significantly reduced and cells changed their appearance (data not shown). The method used compensated for any reduced proliferation over the range shown, as described above. Cells from individual mice varied markedly in the maximum level of IL-4 production that could be elicited with the inhibitor (Fig. 1A), but consistent EC50 values were obtained, for PP2 as shown in Figure 1B and for PP1 at 260 nM (not shown). The same applied to the reduction of IFN-ã production (Fig. 1C,D). Intracellular FACS staining indicated that this Th2 shift was brought about by an increase in number of IL-4 producing cells rather than in production per cell (Table 1).
Th2 directing effect of PP2 is dependent on endogenous IL-4 and is overridden by IL-12 To test whether the Th2 shift depends on endogenously produced IL-4, and whether IL-12 is involved, anti-cytokine antibodies were applied as shown in Figure 2. As expected, anti-IL-4 enhanced Th1 differentiation. PP2 lost its Th2 driving effect when combined with this antibody, showing that its effect depends on secreted IL-4. Anti-IL-12 antibody diminished production of both IFN-ã and IL-4 but in combination with PP2 reduced the production of IFN-ã further while slightly increasing IL-4 production. IL-4 induced almost exclusive Th2 differentiation, and its
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CYTOKINE, Vol 11, No. 3 (March, 1999: 208–215)
10 000
pg/ml 5000 0
IFN-γ
Control
Fig. 2) that these inhibitors are not as effective as either IL-4 or IL-12 themselves in driving Th1/Th2 shifts.
5000 IL-4
PP1 and PP2 synergize with an anti-CD4 antibody which binds to the membrane-proximal domain of the CD4 molecule
PP2
Since PP1 and PP2 inhibit Lck, they might be expected to act in synergy with monoclonal anti-CD4 antibodies. To analyse synergy, six successive experiments were performed, and the IL4/IFN-ã ratio analysed. Because the controls (cells treated with antigen only) varied, the data were normalized with respect to them according to the following formula:
anti-IL-4 PP2 + anti-IL-4 anti-IL-12 PP2 + anti-IL-12
IL-4
Th2 differentiation index=(IL-4 in the treated group/ IL4 in antigen only controls)/(IFN-ã in the treated group/IFN-ã in antigen only controls).
IL-4 + PP2 IL-4 + anti-IL-12 IL-4 + anti-IL-12 + PP2
IL-12 IL-12 + PP2 IL-12 + anti-IL-4 IL-12 + anti-IL-4 + PP2 Figure 2. Regulation of T helper cell differentiation in the presence and absence of PP2. Added to the cultures at day 0:1 ìM PP2, 200 U/ml IL-4, 1 ng/ml IL-12, 10 ìg/ml anti-IL-4 or 10 ìg/ml anti-IL-12. Representative data from one of two such experiments.
effect was enhanced by PP2. IL-12 induced Th1 differentiation accompanied by greatly reduced IL-4 production; in its presence IL-4 production could be partially rescued by addition of PP2. This effect, although small, is also obtained in the presence of both IL-12 and anti-IL-4 antibody at the time of priming. Thus PP2 does not inhibit the effect of IL-12, and must therefore act prior to the secretion of this cytokine, perhaps affecting its level of expression. It is also clear (see TABLE 1.
PP2 at 800 nM yielded an index of 1.70.03 (P<10 3 in comparison with the controls which were stimulated with antigen alone). The anti CD4 antibody RmCD4 at 2 ìg/ml yielded an index of 0.890.07 (not significantly different from controls). The two combined yielded an index of 5.30.55 (P<10 3 in comparison with the controls). Controls treated with IL-4 and with IL-12 yielded indices of 22.87.4 and 0.260.08, respectively. The anti-CD4 antibodies RmCD4.4, GK1.5, H129.19, YTS177.9 and YTS 191.1 had an opposite effect, which was not shown by YTA3.12 (Table 2). PP1 behaved similarly (data not shown). Binding experiments revealed that RmCD4.2 and YTA 3.12 bind to overlapping epitopes not recognized by the other anti-CD4 antibodies (not shown). The overlap is incomplete, since YTA3.12 completely blocks staining with RmCD4.2 while RmCD4.2 allows weak staining with YTA3.12. YTA 3.12 is known to bind to the membrane-proximal domains 3/4 of CD4, while GK1.5, YTS191.1 and H129.19 bind to the outermost domain 1.30 These data suggest that RmCD4.2 also binds to domains 3/4, while YTS177.9 and RmCD4.4 bind to domain 1. Thus binding to the
Cytokine production by single cells analysed by intracellular FACS staining
Cells were stimulated with OVA-peptide OVA-peptide+1 ìM PP2 OVA-peptide+1 ìM PP2+2 ìg/ml RmCD4.2
Percentage of cells producing IL-4
P
IFN-ã
P
Th2 differentiation index
P
3.31.0 4.50.9 5.01.2
0.13 0.04
11.52.9 7.41.3 7.51.0
0.10 0.14
1 2.30.5 2.50.7
0.03 0.05
Splenocytes of TCR transgenic mice were cultivated for 7 days, washed and restimulated with PMA and ionomycin. Means and SEM from five independent experiments. Th2 differentiation indices were calculated from ratios of IL-4/IFN-ã producing cells which were normalized with respect to the controls (stimulated with antigen only) according to the following formula: Th2 differentiation index=(%IL-4 producing cells in the treated group/%IL-4 producing cells in antigen-only controls)/(%IFN-ã producing cells in the treated group/%IFN-ã producing cells in antigen-only controls). The P value (Student’s t-test) compares the means of treated cells with the means of antigen-only controls.
Src family inhibitors favour Th2 differentiation / 211
TABLE 2. Monoclonal antibody RmCD4.2 YTA3.12 YTS191.1 YTS177.9 H129.19 GK1.5 RmCD4.4
Th2 differentiation indices Th2 differentiation index (meansSEM)
P
Isotype
Epitope (CD4 domain)
5.300.55 1.550.38 0.460.16 0.330.05 0.290.05 0.250.09 0.160.03
510 4 0.38 0.04 10 3 <10 3 10 3 <10 3
IgG2b IgG2b IgG2b IgG2a IgG2a IgG2b IgG2a
3/4 3/4 1 1 1 1 1
Calculated from IL-4/IFN-ã ratios produced by different monoclonal anti-CD4 antibodies in combination with 800 nM PP2 which were normalized with respect to the controls (stimulated with antigen only) according to the following formula: Th2 differentiation index=(IL-4 in the treated group/IL-4 in antigen-only controls)/(IFN-ã in the treated group/IFN-ã in antigen-only controls). The P value (Student’s t-test) compares the means of treated cells with the means of antigen-only controls.
membrane-proximal domains of CD4 appears to be mandatory for obtaining a Th2 shift in the system used here.
CD40L expression is suppressed in the presence of PP1 and PP2 To test for a CD40L-mediated mechanism, expression of this molecule was examined in anti-CD3stimulated CD4 T cells in the presence and absence of PP1 or PP2. CD40L staining was quantified as percentage of anti-CD3 stimulated, treated or untreated cells expressing CD40L above the level of unstimulated cells. Cells which were stimulated in the absence of PP1 and PP2 were taken to represent 100% staining (Fig. 3, data of PP2 not shown). IC50 values were calculated as inhibitor concentrations yielding 50% of control cell staining. PP1 and PP2 yielded IC50 values of 38.12.0 nM (n=3) and 36.74.2 nM (n=3), respectively.
PP2 cannot revert Th1 differentiation Once cells differentiated into Th1 cells their phenotype could not be reverted by PP2 treatment to a detectable extent, as shown by cells which were primed for 1 week with antigen and IL-12 and then washed and restimulated with antigen and APC. The presence of PP2 in the second culture, with or without addition of RmCD4.2, did not detectably affect the cytokine secretion profile (Fig. 4).
DISCUSSION The in vitro differentiation system used here has once again proved its value in detecting factors that influence CD4 T cell subset differentiation. The pyrazolo-pyrimidine inhibitors of Src-family kinases that are shown here to drive Th2 differentiation are unlikely to be either the best drugs of this type, or to
represent the only type able to drive Th2 differentiation, so the assay can be recommended for further screening purposes; it has proved robust, fast and inexpensive. Variation between individual cell donors, although marked (Fig. 1A and C), had little effect on the final estimate of EC50 in the 200–400 nM range (Fig. 1B and D). The cellular assay used here is less sensitive than the enzyme-inhibition assay used previously (see Materials and Methods), as expected since in live cells the inhibitors have to compete with intracellular ATP at mM concentrations. The effect of concentrations of inhibitor which by themselves had little effect could be enhanced by adding the anti-CD4 antibody RmCD4.2, although this antibody had little effect on its own. The fact that the two agents acted synergistically indicates that this antibody had an inhibitory rather than an agonistic effect. Both of the antibodies that bind to the membrane-proximal domains had a similar effect, contrasting with that shown by the five other antibodies tested. Evidently, ligation of CD4 can transmit into the cell different Th2-influencing signals, a negative one from domain 1 and a positive one from domains 3/4. Treatment of cells with GK1.5 has indeed previously been found to increase Lck activity.31 Crystals of human CD4 molecule have a dimeric structure with interchain binding at domain 4 thought to mediate signal transduction.32 An antibody to domains 3/4 would therefore be likely to inhibit signal transduction. In addition, it has been speculated, that domains 3/4 may interact with the TCR complex.33 Synergy between Lck inhibitor and antibody opens the possibility of using the antibody to sharpen the specificity of the inhibitor for in vivo use. An attractively simple possibility is that the reduced CD40L expression observed here (Fig. 3) lowers the back-signal from T cells into APC and thereby suppressed IL-12 induction.26,28,29 IL-12 has been identified as a key agent in directing CD4 + T cells
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CYTOKINE, Vol 11, No. 3 (March, 1999: 208–215)
2000
A
IL-4
0 pg/ml
Relative no. of cells
1000
30
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101
2
10
3
10
4
10
Fluorescence intensity
Control cell staining (%)
100
B
+ PP2 + RmCD4.2
100
+ PP2
0
Control
IFN-γ 4000
Figure 4. No detectable reversal of committed Th1 cells by PP2 and anti-CD4 antibody.
75
Cells were primed with antigen and IL-12 (1 ng/ml) and restimulated at day 7 with antigen or antigen and PP2 (800 nM) alone or in combination with RmCD4.2 (4 ìg/ml). Culture supernatants were taken at day 16 after restimulation with antigen at day 14. Representative data from one of two experiments. ( ), IL-4; ( ), IFN-ã.
50
25
0 0
50
100 PP1 (nM)
150
200
Figure 3. CD40L expression of CD4 T cells following overnight stimulation with plate-bound anti-CD3 (10 ìg/ml) is suppressed by PP1. (A) FACS analysis of CD40L expression of cells which were stimulated in presence (thin solid line) or absence of PP1 (bold solid line). The dashed line shows cells which were not stimulated with antiCD3. (B) Dose–response for inhibition of CD40L expression. The IC50 in three experiments was 38.12.0 nM.
to differentiate into Th1 cells.34–36 It is true that blocking with anti-IL12 antibodies or with IL-12p40 homodimer in these reports inhibits development of IFN-ã producing BALB/c T cells most effectively only in serial cultures, whereas in primary response inhibition is in the range of 0–50%, as was observed here (Fig. 2). This discrepancy is unexplained: possibly the blocking proteins penetrate the contact region between T cell and APC poorly. Over a wide range of antigen concentrations and costimulatory activity, lowering signal-strength has been found to favour Th2 differentiation.3 This may indeed be how the inhibitors used here work. This does
not reduce their potential value; on the contrary, a drug which lowers signal strength might be more useful in practice than strategies aimed at lowering antigen concentration or reducing costimulation. In addition to any clinical significance, drugs of this type may be useful for experimental purposes. For instance, it has been proposed that Lck has a special role in the differentiation of CD4 and CD8 cells in the thymus.37,38 This raises the possibility that thymic cultures might provide the ultimate scaled-down bioassay. In the light of current concepts of immunoregulation in autoimmunity39,40 a major therapeutic strategy is to develop novel methods of modulating the T cell cytokine balance in favour of protective T cells. The major finding here of Th2-differentiation induced by an inhibitory drug in concert with certain monoclonal antibodies has implications for therapeutic strategies and represents a step forward towards the ultimate goal of finding a well-tolerated drug which has this form of activity in vivo. The particular kinase inhibitors tested here provide a lead, but obvious problems remain concerning the need for higher affinity so as to compete more effectively with intracellular ATP, and sharper specificity for the target enzyme. It is likely that anti-CD4 antibodies will be relatively potent in vivo, where much is already known about their use in organ transplantation,16,41 and where an encouraging
Src family inhibitors favour Th2 differentiation / 213
trial has just been reported in rheumatoid arthritis.42 We foresee a fruitful interplay between in vivo and in vitro screening. It will be important to bear in mind that Th2 cells are neither the only nor perhaps the optimum protective T cell subpopulations,43,44 although it is likely that the TCR-transgenic model will prove equally useful for characterizing these other subpopulations. As regards the mode of action of these and other future inhibitors, it is unlikely that the inhibition of CD40L expression described here will constitute either the only or the optimum mechanism. What is described here is no more than a first step. Doubtless the ultimate goal is to find a well-tolerated drug which has this form of activity.
MATERIALS AND METHODS Src family specific tyrosine kinase inhibitors PP1 (4-amino-5-[4-methylphenyl]-7-[t-butyl]pyrazolo [3,4-d]pyrimidine) and PP2 (4-amino-5-[4-chlorophenyl]-7[t-butyl]pyrazolo[3,4-d]pyrimidine) were shown to be selective for Lck (PP1: IC50 =0.005 ìM; PP2: IC50 =0.004 ìM) and Fyn (PP1: IC50 =0.006 ìM; PP2: IC50 =0.005 ìM) in in vitro kinase assays. The IC50 of PP1 for ZAP-70, JAK2 and EGF-R is >100 ìM, >50 ìM and 0.25 ìM, respectively, the IC50 of PP2 for these kinases is >100 ìM, >50 ìM and 0.48 ìM, respectively.25 PP1 and PP2 used here were synthesized by Celltech Therapeutics Ltd. (Slough, UK). PP1 is now available from Calbiochem (San Diego, CA).
Transgenic mouse cell assay Splenocytes of DO 10 mice (<12 weeks of age) were isolated by Ficoll (Histopaque-1083, Sigma, Deisenhofen, Germany) gradient centrifugation and cultured at 2106 cells/ml in complete medium (RPMI 1640-Medium, Gibco, BRL, Life Technologies, Eggenstein, Germany; supplemented with 10% FCS, Sigma; 2 mM -glutamine, Gibco; 50 ìM mercaptoethanol and 100 U/ml antibioticantimycotic, Gibco) together with 0.3 ìM OVA peptide 323-339 (TIB Molbiol, Berlin, Germany). As a control for Th2 differentiation, recombinant mouse IL-4 (BioSource International, Camarillo, CA) was added at a concentration of 200 U/ml; and as a control for Th1 differentiation recombinant mouse IL-12 (Hoffmann-La Roche, Nutley, NJ) was added at a concentration of 1 ng/ml. Cells plus OVA peptide alone were a further control. The inhibitors (dissolved in DMSO) were added to further wells, with or without monoclonal anti-CD4 antibodies, to a final DMSO concentration of 0.1%. At day 4 cells were split and 100 U/ml recombinant IL-2 (Chiron, Ratingen, Germany) was added. At day 7 cells were washed thoroughly and restimulated with 0.3 ìM OVA peptide at 0.5106 cells/ml together with 1106 APC/ml. Cell numbers were adjusted by counting viable cells so as to ensure that the secondary cultures used to monitor cytokine production contained equal numbers of T cells, thus compensating for any inhibition of proliferation in the primary culture. APC were splenocytes from BALB/c mice: erythrocytes were lysed with 0.83% NH4Cl for 5 min at room
temperature, and the remaining cells washed and irradiated with 26 Gy. After 48 h the culture supernatants were assayed by ELISA (DuoSeT mouse IFN-ã and DuoSeT mouse IL-4, Genzyme, Cambridge, MA).
Data analysis The IL-4 detected in the supernatant of cells treated only with OVA peptide was taken as the minimum level. The maximum was the mean level from cells treated with the IL-4-inducing drug at concentrations of 60–1000 nM, i.e. at the plateau of the dose–response curve shown in Figure 1A. Data were then calculated as % maximum IL-4 production [(observed level–minimum level)/(maximum level–minimum level)100)]. A polynomial curve was fitted to data obtained in this way and the intercept with half-maximum IL-4 production (effective concentration, EC50) found as shown in Figure 1B. IFN-ã data were analysed in the same way (Fig. 1D).
Antibodies The following gifts of rat-anti-mouse anti-CD4 antibodies were tested: RmCD4.2 (IgG2b) and RmCD4.4 (IgG2a)45 from S. Thierfelder (GSF-Forschungszentrum fu¨r Umwelt und Gesundheit GmbH, Munich, Germany); YTA3.12 (IgG2b), YTS177.9 (IgG2a) and YTS191.1 (IgG2b) from H. Waldmann (Oxford University, UK); H129.19 (IgG2a) from M. Pierres (CNRS/INSERM, Marseille, France) and GK1.5 (IgG2b) from F. Fitch (Chicago, IL). The anti-IL-4 monoclonal antibody (11B11) was from M. Lo¨hning (Deutsches Rheumaforschungszentrum Berlin, Germany) and the goat anti-IL-12 antibody from M. Gately (Hoffmann-La Roche).
CD4 binding assays and FACS analysis Lymph node cells (0.5105) of BALB/c mice were incubated with blocking anti-CD4 antibodies (see above) at a concentration of 10 ìg/ml in PBS/0.5% bovine serum albumin (BSA)/0.01% NaN3 on ice for 30 min. Biotinylated (labelling kit from Boehringer Mannheim, Germany) antiCD4 antibodies YTS191.1, YTA3.12 or RmCD4.2 were then added as staining antibodies, and incubation continued for a further 30 min on ice. As controls, cells were incubated with blocking or staining antibodies alone. Cells were washed and then incubated with streptavidin–FITC (Dako Diagnostika, Hamburg, Germany) for 30 min on ice. After washing cells, CD4 staining was analysed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Blocking of staining indicated that both the blocking antibody and the biotinylated staining antibody bind to overlapping epitopes.
Intracellular cytokine staining DO11.10 splenocytes after 7 days of culture as above were stimulated for 5 h with PMA (5 ng/ml; Sigma) plus ionomycin (1 ìg/ml; Sigma). Two hours before harvesting, brefeldin A (10 ìg/ml; Sigma) was added. After harvesting, cells were washed and fixed in 2% formaldehyde for 20 min at room temperature, and then stained with anti-IL-4–PE (11B11; 5 ìg/ml; Pharmingen, Hamburg, Germany) and antiIFN-ã-FITC (XMG1.2; 2 ìg/ml; Pharmingen) for 10 min at
214 / Gimsa et al.
room temperature. Washing and staining procedures were carried out in PBS/1% BSA/0.5% saponin (Sigma). Finally, cells were resuspended in PBS/0.5% BSA/0.01% NaN3 for FACS analysis. Samples were gated on live lymphocytes based on forward and side scatter parameters (10,000 events/ sample) and analysed using Cellquest 3.0 software (Becton Dickinson).
CD40L expression BALB/c spleen and lymph node cells were depleted of B cells and macrophages by incubation on a prewarmed nylon wool column filled with complete medium for 1.5 h. CD4 + and CD4 cells were then separated by high gradient magnetic cell sorting. Briefly, cells were labeled with superparamagnetic microparticles conjugated to monoclonal antibodies (anti-CD8, anti-B220 and anti-CD11b; Miltenyi Biotec GmbH; Bergisch-Gladbach, Germany), and then separated on a MidiMACS column allowing nonmagnetic cells to pass. FACS analysis showed about 86% CD4 + cells contaminated with about 2% B cells and 6% macrophages. These cells were then incubated overnight with plate-bound anti-CD3 (10 ìg/ml; Pharmingen) with or without PP1, and then harvested and stained with anti-CD40L-PE (MR1; 10 ìg/ml; Pharmingen) for 30 min on ice. CD40L expression was then analysed by FACS.
Acknowledgements Founder DO10 mice were a gift from D. Loh. This work was supported by the Deutsche Forschungsgemeinschaft and the Senate Administration for Research and Education of the City of Berlin.
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CYTOKINE, Vol 11, No. 3 (March, 1999: 208–215) 10. Coqueret O, Petit FC, Lagente V, Moumen M, Mencia HJ, Braquet P (1994) Modulation of IgE production in the mouse by beta 2-adrenoceptor agonist. Int Arch Allergy Immunol 105:171– 176. 11. Panina-Bordignon P, Mazzeo D, Di Lucia P, D’Ambrosio D, Lang R, Fabbri L, Self C, Sinigaglia F (1998) â2-agonists prevent Th1 development by selective inhibition of IL-12. J Clin Invest 100:1513–1519. 12. de Jong P, Bezemer AC, Zomerdijk T, Vandepouwkraan T, Ottenhoff T, Nibbering PH (1996) Selective stimulation of T helper 2 cytokine responses by the anti psoriasis agent monomethylfumarate. Eur J Immunol 26:2067–2074. 13. McHugh SM, Rifkin IR, Deighton J, Wilson AB, Lachmann PJ, Lockwood CM, Ewan PW (1995) The immunosuppressive drug thalidomide induces T helper cell type 2 (Th2) and concomitantly inhibits Th1 cytokine production in mitogen- and antigen-stimulated human peripheral blood mononuclear cell cultures. Clin Exp Immunol 99:160–167. 14. Hauschild A, Kro¨ger H, Mitchison NA, Ugrinovic S, Zwingenberger K (1997) Thalidomide therapy of established collagen-induced arthritis not accompanied by an evident Th2 shift. Clin Exp Immunol 108:428–431. 15. van der Pouw-Kraan TC, Boeije LC, Smeenk RJ, Wijdenes J, Aarden LA (1995) Prostaglandin-E2 is a potent inhibitor of human interleukin 12 production. J Exp Med 181:775–779. 16. Gimsa U, Mitchison NA (1997) New perspectives on the Th1/Th2 paradigm. Curr Opin Organ Transpl 2:18–22. 17. Stumbles P, Mason D (1995) Activation of CD4+ T cells in the presence of a nondepleting monoclonal antibody to CD4 induces a Th2-type response in vitro. J Exp Med 182:5–13. 18. Hutchings P, Parish N, O’Reilly L, Dawe K, Roitt IM, Cooke A (1993) The regulation of autoimmunity through CD4+ T cells. Autoimmunity 15:21–23. 19. van der Lubbe P, Reiter C, Breedveld FC, Kru¨ger K, Schattenkirchner M, Sanders ME, Riethmu¨ller G (1993) Chimeric CD4 monoclonal antibody cM-T412 as a therapeutic approach to rheumatoid arthritis. Arthritis Rheum 36:1375–1379. 20. Brown DR, Moskowitz NH, Killeen N, Reiner SL (1997) A role for CD4 in peripheral T cell differentiation. J Exp Med 186:101–107. 21. Fowell DJ, Magram J, Turck CW, Killeen N, Locksley RM (1997) Impaired Th2 subset development in the absence of CD4. Immunity 6:559–569. 22. Perlmutter RM, Levin SD, Appleby MW, Anderson SJ, Alberola IJ (1993) Regulation of lymphocyte function by protein phosphorylation. Annu Rev Immunol 11:451–499. 23. Molina TJ, Kishihara K, Siderovski DP, van Ewijk W, Narendran A, Timms E, Wakeham A, Paige CJ, Hartmann KU, Veillette A, Davidson D, Mak TW (1992) Profound block in thymocyte development in mice lacking p56lck. Nature 357:161– 164. 24. Appleby MW, Kerner JD, Chien S, Maliszewski CR, Bondadaa S, Perlmutter RM (1995) Involvement of p59fynT in interleukin-5 receptor signaling. J Exp Med 182:811–820. 25. Hanke JH, Gardner JP, Dow RL, Changelian PS, Brissette WH, Weringer EJ, Pollok BA, Connelly PA (1996) Discovery of a novel, potent, and src family-selective tyrosine kinase inhibitor. J Biol Chem 271:695–701. 26. Kato T, Hakamada R, Yamane H, Nariuchi H (1996) Induction of IL-12 p40 messenger RNA expression and IL-12 production of macrophages via CD40-CD40 ligand interaction. J Immunol 156:3932–3938. 27. Shu U, Kiniwa M, Wu CY, Maliszewski C, Vezzio N, Hakimi J, Gately M, Delespesse G (1995) Activated T cells induce interleukin-12 production by monocytes via CD40-CD40 ligand interaction. Eur J Immunol 25:1125–1128. 28. Koch F, Stanzl U, Jennewein P, Janke K, Heufler C, Kampgen E, Romani N, Schuler G (1996) High level IL-12 production by murine dendritic cells:upregulation via MHC class II and CD40 molecules and downregulation by IL-4 and IL-10. J Exp Med 184:741–746. 29. Cella M, Scheidegger D, Palmer-Lehmann K, Lane P, Lanzavecchia A, Alber G (1996) Ligation of CD40 on dendritic cells
Src family inhibitors favour Th2 differentiation / 215 triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity:T-T help via APC activation. J Exp Med 184:747–752. 30. Dianzani U, Shaw A, al-Ramadi BK, Kubo RT, Janeway CAJ (1992) Physical association of CD4 with the T cell receptor. J Immunol 148:678–688. 31. Tamura T, Nakano H, Nagase H, Morokata T, Igarashi O, Oshimi Y, Miyazaki S, Nariuchi H (1995) Early activation signal transduction pathways of Th1 and Th2 cell clones stimulated with anti-CD3. J Immunol 155:4692–4701. 32. Wu H, Kwong PD, Hendrickson WA (1997) Dimeric association and segmental variability in the structure of human CD4. Nature 387:527–530. 33. Lange G, Lewis SJ, Murshudov GN, Dodson GG, Moody PC, Turkenburg JP, Barclay AN, Brady RL (1994) Crystal structure of an extracellular fragment of the rat CD4 receptor containing domains 3 and 4. Structure 2:469–481. 34. Seder RA, Gazzinelli R, Sher A, Paul WE (1993) Interleukin 12 acts directly on CD4+ T cells to enhance priming for interferon gamma production and diminishes interleukin 4 inhibition of such priming. Proc Natl Acad Sci USA 90:10188–10192. 35. Heufler C, Koch F, Stanzl U, Topar G, Wysocka M, Trinchierit G, Enk A, Steinman RM, Romani N, Schuler G (1996) Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-gamma production by T helper 1 cells. Eur J Immunol 26:659–668. 36. Piccotti JR, Chan SY, Li K, Eichwald EJ, Bishop DK (1997) Differential effects of IL-12 receptor blockade with IL-12 p40 homodimer on the induction of CD4+ and CD8+ IFN-gammaproducing cells. J Immunol 158:643–648.
37. Eichmann K (1995) A signal strength hypothesis of thymic selection:preliminary considerations. Immunol Lett 44:87–90. 38. Matechak EO, Killeen N, Hedrick SM, Fowlkes BJ (1996) MHC class II-specific T cells can develop in the CD8 lineage when CD4 is absent. Immunity 4:337–347. 39. Segal BM, Dwyer BK, Shevach EM (1998) An interleukin (IL) 10/12 immunoregulatory circuit controls susceptibility to autoimmune disease. J Exp Med 187:in press. 40. Mu¨ller B, Gimsa U, Mitchison NA, Radbruch A, Sieper J, Yin Z (1998) Modulating the Th1/Th2 balance in inflammatory arthritis. Springer Seminars in Immunopathology 20:181–196. 41. Hutchings PR, Cooke A, Dawe K, Waldmann H, Roitt IM (1993) Active suppression induced by anti-CD4. Eur J Immunol 23:965–968. 42. Schulze-Koop SH, Davis LS, Haverty TP, Wacholtz MC, Lipsky PE (1997) Reduction of Th1 cell activity in patients with rheumatoid arthritis after treatment with a non-depleting monoclonal antibody to CD4. Arthritis Rheum (Suppl). 40:S191. 43. Powrie F, Carlino J, Leach MW, Mauze S, Coffman RL (1996) A critical role for transforming growth factor-beta but not interleukin 4 in the suppression of T helper type 1-mediatied colitis by CD45RB(low) CD4+ T cells. J Exp Med 183:2669–2674. 44. Groux H, O’Garra A, Bigler M, Rouleau M, Antonenko S, de Vries JE, Roncarlo MG (1997) A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature 389:737–741. 45. Reinecke K, Mysliwietz J, Thierfelder S (1994) Single as well as pairs of synergistic anti-CD4+CD8 antibodies prevent graftversus-host disease in fully mismatched mice. Transplantation 57:458–461.
INHIBITORS OF Src-FAMILY TYROSINE KINASES FAVOUR Th2 DIFFERENTIATION Ulrike Gimsa, Avrion Mitchison, Rodger Allen* Two potent pyrazolo-pyrimidine inhibitors of Src-family kinases were found to enhance interleukin 4 (IL-4) and reduce interferon ã (IFN-ã) production in cultures of splenocytes from ovalbumin-specific TCR-transgenic BALB/c mice. The effect was increased by addition of a monoclonal antibody binding to domains 3/4 of CD4, while other antibodies binding to domain 1 had the opposite effect. The inhibitors suppressed CD40 ligand (CD40L) expression on activated CD4 T cells, which may explain this Th2 differentiating effect. More generally, the effect fits within the overall framework of signal attenuation in T cells driving this form of differentiation. The inhibitors did not revert previously induced Th1 differentiation in serial cultures of the TCR-transgenic cells. Drugs with this activity are of obvious interest as probes and potential therapeutic agents in autoimmune disease. 1999 Academic Press
The purpose of this study was to identify novel agents able to influence T-cell subset differentiation. CD4 + T cells are divided into Th1 and Th2 types according to their cytokine production and function; although this division may be over-simplified, the concept of balance between Th1 and Th2 cells in the immune system is now well established.1 To that end we have used cells transgenic for an áâ T-cell receptor (TCR) able to recognize the ovalbumin (OVA) peptide 323–339, on a BALB/c background.2 Upon stimulation in vitro by OVA peptide these cells produce interferon ã (IFN-ã) and interleukin 4 (IL-4), the cytokines characteristic of Th1 and Th2 cells. This system has been widely used to investigate Th1/Th2 differentiation as influenced by different APC, antigen-dose and cytokines.3 Bacteria and parasites are known to drive differentiation selectively. Listeria monocytogenes induces differentiation of Th1 cells4 while helminths induce that of Th2 cells.1 Both major histocompatibility complex (MHC) and genetic background influence differentiation.5–7 Imbalance in favour of Th1 cells occurs in the major human autoimmune diseases, such as rheumatoid arthritis8 and multiple sclerosis.9 Agents From the Deutsches RheumaForschungsZentrum Berlin, Germany; *Celltech Therapeutics Ltd, Slough, UK Correspondence to: Ulrike Gimsa, Institute of Anatomy, HumboldtUniversity Clinic Charite´, Dept. of Cell and Neurobiology, D-10098 Berlin, Germany Received 18 February 1998; accepted for publication 21 June 1998 1999 Academic Press 1043–4666/99/030208+08 $30.00/0 KEY WORDS: anti-CD4/CD40L/Lck/Th2/ tyrosine kinase inhibitor 208
able to restore balance are needed, as probes to test the possibility that the imbalance itself contributes to pathogenesis, and as potential therapeutic agents. A growing list of agents has been found to drive differentiation of Th2 cells preferentially: salbutamol,10,11 monomethylfumarate,12 thalidomide13 (but see also Ref. 14) and prostaglandin-E2.15 Additional agents have been found able to prolong allograft survival in association with Th2 differentiation.16 Some of these agents have been identified as acting on antigen-presenting cells (APC), others appear to act within the responding T cells themselves, while for the majority the target cell is uncertain. For those agents which do act within T cells the underlying question remains open of whether they modify signal strength from the TCR complex, or whether they act on the process of differentiation independently of this pathway. Agents likely to interfere with signalling from CD4 molecules offer a promising means of modulating signal strength, as anti-CD4 antibody can drive Th2 differentiation in vitro17 and in vivo. Treatment with this type of antibody facilitates the induction of CD4 T cell populations able to protect against autoimmunity18 through a mechanism suspected of involving cytokine imbalance. This form of treatment has a long history of promising results in clinical organ transplantation16 and more recently in rheumatoid arthritis,19 although it has not found general use. Surprisingly, CD4 / mice show a bias away from Th2 differentiation,20,21 presumably reflecting selective events in the immune system as it develops in these mice. CYTOKINE, Vol. 11, No. 3 (March), 1999: pp 208–215
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% maximum IL-4 production
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4000 2000
0
50 25 0
125 % maximum IFN-γ production
pg/ml
15 000 10 000
Figure 1.
75
100 200 300 400 500 600 700 800 900 1000 PP2 (nM)
C
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PP2 promotes Th2 differentiation in a dose-dependent manner.
Two experiments representative of four. (A) Dose-dependent IL-4 production of TCR transgenic T cells in the presence of PP2. (B) Half-maximum IL-4 production (EC50) at 36858 nM (meansSEM in four experiments). Data were normalized as described in the Materials and Methods section. (C) Dose-dependent IFN-ã production of TCR transgenic T cells in the presence of PP2. (D) Half-maximum IFN-ã production (EC50) of TCR transgenic T cells at 26366 nM (meansSEM in four experiments). Data were normalized as described in the Materials and Methods section.
The Src-family tyrosine kinase p56lck (Lck) is physically associated with the cytoplasmic tail of CD4 and is the only molecule known to transmit signals from CD4.22 Lck also regulates tyrosine phosphorylation of the T cell receptor (TCR)-æ chain, CD3-å, and ZAP-70. Lck / mice have greatly reduced numbers of thymocytes and peripheral áâ + T cells.23 p59fyn (Fyn) is another member of the same family that also phosphorylates proteins of the T cell receptor complex, although it does not bind to the CD4 molecule. The immune system of Fyn / mice is impaired, but to a much lesser extent than that of Lck / mice.24 The Src family specific tyrosine kinase inhibitors tested here were developed as agents for blocking the kinase activity of Lck and Fyn.25 A possible mechanism through which an agent that interferes with T cell signalling could influence Th1/Th2 differentiation is via an effect on CD40L expression. TCR ligation leads to upregulation of CD40L which then signals back into APC via ligation of CD40.26–29
RESULTS Increased IL-4 production and reduced IFN-ã production in the presence of PP1 and PP2 As used here, PP2 significantly enhanced IL-4 production (Fig. 1A,B) and reduced IFN-ã production
(Fig. 1C,D); see also Figure 2. Above 1000 nM, proliferation was significantly reduced and cells changed their appearance (data not shown). The method used compensated for any reduced proliferation over the range shown, as described above. Cells from individual mice varied markedly in the maximum level of IL-4 production that could be elicited with the inhibitor (Fig. 1A), but consistent EC50 values were obtained, for PP2 as shown in Figure 1B and for PP1 at 260 nM (not shown). The same applied to the reduction of IFN-ã production (Fig. 1C,D). Intracellular FACS staining indicated that this Th2 shift was brought about by an increase in number of IL-4 producing cells rather than in production per cell (Table 1).
Th2 directing effect of PP2 is dependent on endogenous IL-4 and is overridden by IL-12 To test whether the Th2 shift depends on endogenously produced IL-4, and whether IL-12 is involved, anti-cytokine antibodies were applied as shown in Figure 2. As expected, anti-IL-4 enhanced Th1 differentiation. PP2 lost its Th2 driving effect when combined with this antibody, showing that its effect depends on secreted IL-4. Anti-IL-12 antibody diminished production of both IFN-ã and IL-4 but in combination with PP2 reduced the production of IFN-ã further while slightly increasing IL-4 production. IL-4 induced almost exclusive Th2 differentiation, and its
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10 000
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Fig. 2) that these inhibitors are not as effective as either IL-4 or IL-12 themselves in driving Th1/Th2 shifts.
5000 IL-4
PP1 and PP2 synergize with an anti-CD4 antibody which binds to the membrane-proximal domain of the CD4 molecule
PP2
Since PP1 and PP2 inhibit Lck, they might be expected to act in synergy with monoclonal anti-CD4 antibodies. To analyse synergy, six successive experiments were performed, and the IL4/IFN-ã ratio analysed. Because the controls (cells treated with antigen only) varied, the data were normalized with respect to them according to the following formula:
anti-IL-4 PP2 + anti-IL-4 anti-IL-12 PP2 + anti-IL-12
IL-4
Th2 differentiation index=(IL-4 in the treated group/ IL4 in antigen only controls)/(IFN-ã in the treated group/IFN-ã in antigen only controls).
IL-4 + PP2 IL-4 + anti-IL-12 IL-4 + anti-IL-12 + PP2
IL-12 IL-12 + PP2 IL-12 + anti-IL-4 IL-12 + anti-IL-4 + PP2 Figure 2. Regulation of T helper cell differentiation in the presence and absence of PP2. Added to the cultures at day 0:1 ìM PP2, 200 U/ml IL-4, 1 ng/ml IL-12, 10 ìg/ml anti-IL-4 or 10 ìg/ml anti-IL-12. Representative data from one of two such experiments.
effect was enhanced by PP2. IL-12 induced Th1 differentiation accompanied by greatly reduced IL-4 production; in its presence IL-4 production could be partially rescued by addition of PP2. This effect, although small, is also obtained in the presence of both IL-12 and anti-IL-4 antibody at the time of priming. Thus PP2 does not inhibit the effect of IL-12, and must therefore act prior to the secretion of this cytokine, perhaps affecting its level of expression. It is also clear (see TABLE 1.
PP2 at 800 nM yielded an index of 1.70.03 (P<10 3 in comparison with the controls which were stimulated with antigen alone). The anti CD4 antibody RmCD4 at 2 ìg/ml yielded an index of 0.890.07 (not significantly different from controls). The two combined yielded an index of 5.30.55 (P<10 3 in comparison with the controls). Controls treated with IL-4 and with IL-12 yielded indices of 22.87.4 and 0.260.08, respectively. The anti-CD4 antibodies RmCD4.4, GK1.5, H129.19, YTS177.9 and YTS 191.1 had an opposite effect, which was not shown by YTA3.12 (Table 2). PP1 behaved similarly (data not shown). Binding experiments revealed that RmCD4.2 and YTA 3.12 bind to overlapping epitopes not recognized by the other anti-CD4 antibodies (not shown). The overlap is incomplete, since YTA3.12 completely blocks staining with RmCD4.2 while RmCD4.2 allows weak staining with YTA3.12. YTA 3.12 is known to bind to the membrane-proximal domains 3/4 of CD4, while GK1.5, YTS191.1 and H129.19 bind to the outermost domain 1.30 These data suggest that RmCD4.2 also binds to domains 3/4, while YTS177.9 and RmCD4.4 bind to domain 1. Thus binding to the
Cytokine production by single cells analysed by intracellular FACS staining
Cells were stimulated with OVA-peptide OVA-peptide+1 ìM PP2 OVA-peptide+1 ìM PP2+2 ìg/ml RmCD4.2
Percentage of cells producing IL-4
P
IFN-ã
P
Th2 differentiation index
P
3.31.0 4.50.9 5.01.2
0.13 0.04
11.52.9 7.41.3 7.51.0
0.10 0.14
1 2.30.5 2.50.7
0.03 0.05
Splenocytes of TCR transgenic mice were cultivated for 7 days, washed and restimulated with PMA and ionomycin. Means and SEM from five independent experiments. Th2 differentiation indices were calculated from ratios of IL-4/IFN-ã producing cells which were normalized with respect to the controls (stimulated with antigen only) according to the following formula: Th2 differentiation index=(%IL-4 producing cells in the treated group/%IL-4 producing cells in antigen-only controls)/(%IFN-ã producing cells in the treated group/%IFN-ã producing cells in antigen-only controls). The P value (Student’s t-test) compares the means of treated cells with the means of antigen-only controls.
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TABLE 2. Monoclonal antibody RmCD4.2 YTA3.12 YTS191.1 YTS177.9 H129.19 GK1.5 RmCD4.4
Th2 differentiation indices Th2 differentiation index (meansSEM)
P
Isotype
Epitope (CD4 domain)
5.300.55 1.550.38 0.460.16 0.330.05 0.290.05 0.250.09 0.160.03
510 4 0.38 0.04 10 3 <10 3 10 3 <10 3
IgG2b IgG2b IgG2b IgG2a IgG2a IgG2b IgG2a
3/4 3/4 1 1 1 1 1
Calculated from IL-4/IFN-ã ratios produced by different monoclonal anti-CD4 antibodies in combination with 800 nM PP2 which were normalized with respect to the controls (stimulated with antigen only) according to the following formula: Th2 differentiation index=(IL-4 in the treated group/IL-4 in antigen-only controls)/(IFN-ã in the treated group/IFN-ã in antigen-only controls). The P value (Student’s t-test) compares the means of treated cells with the means of antigen-only controls.
membrane-proximal domains of CD4 appears to be mandatory for obtaining a Th2 shift in the system used here.
CD40L expression is suppressed in the presence of PP1 and PP2 To test for a CD40L-mediated mechanism, expression of this molecule was examined in anti-CD3stimulated CD4 T cells in the presence and absence of PP1 or PP2. CD40L staining was quantified as percentage of anti-CD3 stimulated, treated or untreated cells expressing CD40L above the level of unstimulated cells. Cells which were stimulated in the absence of PP1 and PP2 were taken to represent 100% staining (Fig. 3, data of PP2 not shown). IC50 values were calculated as inhibitor concentrations yielding 50% of control cell staining. PP1 and PP2 yielded IC50 values of 38.12.0 nM (n=3) and 36.74.2 nM (n=3), respectively.
PP2 cannot revert Th1 differentiation Once cells differentiated into Th1 cells their phenotype could not be reverted by PP2 treatment to a detectable extent, as shown by cells which were primed for 1 week with antigen and IL-12 and then washed and restimulated with antigen and APC. The presence of PP2 in the second culture, with or without addition of RmCD4.2, did not detectably affect the cytokine secretion profile (Fig. 4).
DISCUSSION The in vitro differentiation system used here has once again proved its value in detecting factors that influence CD4 T cell subset differentiation. The pyrazolo-pyrimidine inhibitors of Src-family kinases that are shown here to drive Th2 differentiation are unlikely to be either the best drugs of this type, or to
represent the only type able to drive Th2 differentiation, so the assay can be recommended for further screening purposes; it has proved robust, fast and inexpensive. Variation between individual cell donors, although marked (Fig. 1A and C), had little effect on the final estimate of EC50 in the 200–400 nM range (Fig. 1B and D). The cellular assay used here is less sensitive than the enzyme-inhibition assay used previously (see Materials and Methods), as expected since in live cells the inhibitors have to compete with intracellular ATP at mM concentrations. The effect of concentrations of inhibitor which by themselves had little effect could be enhanced by adding the anti-CD4 antibody RmCD4.2, although this antibody had little effect on its own. The fact that the two agents acted synergistically indicates that this antibody had an inhibitory rather than an agonistic effect. Both of the antibodies that bind to the membrane-proximal domains had a similar effect, contrasting with that shown by the five other antibodies tested. Evidently, ligation of CD4 can transmit into the cell different Th2-influencing signals, a negative one from domain 1 and a positive one from domains 3/4. Treatment of cells with GK1.5 has indeed previously been found to increase Lck activity.31 Crystals of human CD4 molecule have a dimeric structure with interchain binding at domain 4 thought to mediate signal transduction.32 An antibody to domains 3/4 would therefore be likely to inhibit signal transduction. In addition, it has been speculated, that domains 3/4 may interact with the TCR complex.33 Synergy between Lck inhibitor and antibody opens the possibility of using the antibody to sharpen the specificity of the inhibitor for in vivo use. An attractively simple possibility is that the reduced CD40L expression observed here (Fig. 3) lowers the back-signal from T cells into APC and thereby suppressed IL-12 induction.26,28,29 IL-12 has been identified as a key agent in directing CD4 + T cells
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+ PP2
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IFN-γ 4000
Figure 4. No detectable reversal of committed Th1 cells by PP2 and anti-CD4 antibody.
75
Cells were primed with antigen and IL-12 (1 ng/ml) and restimulated at day 7 with antigen or antigen and PP2 (800 nM) alone or in combination with RmCD4.2 (4 ìg/ml). Culture supernatants were taken at day 16 after restimulation with antigen at day 14. Representative data from one of two experiments. ( ), IL-4; ( ), IFN-ã.
50
25
0 0
50
100 PP1 (nM)
150
200
Figure 3. CD40L expression of CD4 T cells following overnight stimulation with plate-bound anti-CD3 (10 ìg/ml) is suppressed by PP1. (A) FACS analysis of CD40L expression of cells which were stimulated in presence (thin solid line) or absence of PP1 (bold solid line). The dashed line shows cells which were not stimulated with antiCD3. (B) Dose–response for inhibition of CD40L expression. The IC50 in three experiments was 38.12.0 nM.
to differentiate into Th1 cells.34–36 It is true that blocking with anti-IL12 antibodies or with IL-12p40 homodimer in these reports inhibits development of IFN-ã producing BALB/c T cells most effectively only in serial cultures, whereas in primary response inhibition is in the range of 0–50%, as was observed here (Fig. 2). This discrepancy is unexplained: possibly the blocking proteins penetrate the contact region between T cell and APC poorly. Over a wide range of antigen concentrations and costimulatory activity, lowering signal-strength has been found to favour Th2 differentiation.3 This may indeed be how the inhibitors used here work. This does
not reduce their potential value; on the contrary, a drug which lowers signal strength might be more useful in practice than strategies aimed at lowering antigen concentration or reducing costimulation. In addition to any clinical significance, drugs of this type may be useful for experimental purposes. For instance, it has been proposed that Lck has a special role in the differentiation of CD4 and CD8 cells in the thymus.37,38 This raises the possibility that thymic cultures might provide the ultimate scaled-down bioassay. In the light of current concepts of immunoregulation in autoimmunity39,40 a major therapeutic strategy is to develop novel methods of modulating the T cell cytokine balance in favour of protective T cells. The major finding here of Th2-differentiation induced by an inhibitory drug in concert with certain monoclonal antibodies has implications for therapeutic strategies and represents a step forward towards the ultimate goal of finding a well-tolerated drug which has this form of activity in vivo. The particular kinase inhibitors tested here provide a lead, but obvious problems remain concerning the need for higher affinity so as to compete more effectively with intracellular ATP, and sharper specificity for the target enzyme. It is likely that anti-CD4 antibodies will be relatively potent in vivo, where much is already known about their use in organ transplantation,16,41 and where an encouraging
Src family inhibitors favour Th2 differentiation / 213
trial has just been reported in rheumatoid arthritis.42 We foresee a fruitful interplay between in vivo and in vitro screening. It will be important to bear in mind that Th2 cells are neither the only nor perhaps the optimum protective T cell subpopulations,43,44 although it is likely that the TCR-transgenic model will prove equally useful for characterizing these other subpopulations. As regards the mode of action of these and other future inhibitors, it is unlikely that the inhibition of CD40L expression described here will constitute either the only or the optimum mechanism. What is described here is no more than a first step. Doubtless the ultimate goal is to find a well-tolerated drug which has this form of activity.
MATERIALS AND METHODS Src family specific tyrosine kinase inhibitors PP1 (4-amino-5-[4-methylphenyl]-7-[t-butyl]pyrazolo [3,4-d]pyrimidine) and PP2 (4-amino-5-[4-chlorophenyl]-7[t-butyl]pyrazolo[3,4-d]pyrimidine) were shown to be selective for Lck (PP1: IC50 =0.005 ìM; PP2: IC50 =0.004 ìM) and Fyn (PP1: IC50 =0.006 ìM; PP2: IC50 =0.005 ìM) in in vitro kinase assays. The IC50 of PP1 for ZAP-70, JAK2 and EGF-R is >100 ìM, >50 ìM and 0.25 ìM, respectively, the IC50 of PP2 for these kinases is >100 ìM, >50 ìM and 0.48 ìM, respectively.25 PP1 and PP2 used here were synthesized by Celltech Therapeutics Ltd. (Slough, UK). PP1 is now available from Calbiochem (San Diego, CA).
Transgenic mouse cell assay Splenocytes of DO 10 mice (<12 weeks of age) were isolated by Ficoll (Histopaque-1083, Sigma, Deisenhofen, Germany) gradient centrifugation and cultured at 2106 cells/ml in complete medium (RPMI 1640-Medium, Gibco, BRL, Life Technologies, Eggenstein, Germany; supplemented with 10% FCS, Sigma; 2 mM -glutamine, Gibco; 50 ìM mercaptoethanol and 100 U/ml antibioticantimycotic, Gibco) together with 0.3 ìM OVA peptide 323-339 (TIB Molbiol, Berlin, Germany). As a control for Th2 differentiation, recombinant mouse IL-4 (BioSource International, Camarillo, CA) was added at a concentration of 200 U/ml; and as a control for Th1 differentiation recombinant mouse IL-12 (Hoffmann-La Roche, Nutley, NJ) was added at a concentration of 1 ng/ml. Cells plus OVA peptide alone were a further control. The inhibitors (dissolved in DMSO) were added to further wells, with or without monoclonal anti-CD4 antibodies, to a final DMSO concentration of 0.1%. At day 4 cells were split and 100 U/ml recombinant IL-2 (Chiron, Ratingen, Germany) was added. At day 7 cells were washed thoroughly and restimulated with 0.3 ìM OVA peptide at 0.5106 cells/ml together with 1106 APC/ml. Cell numbers were adjusted by counting viable cells so as to ensure that the secondary cultures used to monitor cytokine production contained equal numbers of T cells, thus compensating for any inhibition of proliferation in the primary culture. APC were splenocytes from BALB/c mice: erythrocytes were lysed with 0.83% NH4Cl for 5 min at room
temperature, and the remaining cells washed and irradiated with 26 Gy. After 48 h the culture supernatants were assayed by ELISA (DuoSeT mouse IFN-ã and DuoSeT mouse IL-4, Genzyme, Cambridge, MA).
Data analysis The IL-4 detected in the supernatant of cells treated only with OVA peptide was taken as the minimum level. The maximum was the mean level from cells treated with the IL-4-inducing drug at concentrations of 60–1000 nM, i.e. at the plateau of the dose–response curve shown in Figure 1A. Data were then calculated as % maximum IL-4 production [(observed level–minimum level)/(maximum level–minimum level)100)]. A polynomial curve was fitted to data obtained in this way and the intercept with half-maximum IL-4 production (effective concentration, EC50) found as shown in Figure 1B. IFN-ã data were analysed in the same way (Fig. 1D).
Antibodies The following gifts of rat-anti-mouse anti-CD4 antibodies were tested: RmCD4.2 (IgG2b) and RmCD4.4 (IgG2a)45 from S. Thierfelder (GSF-Forschungszentrum fu¨r Umwelt und Gesundheit GmbH, Munich, Germany); YTA3.12 (IgG2b), YTS177.9 (IgG2a) and YTS191.1 (IgG2b) from H. Waldmann (Oxford University, UK); H129.19 (IgG2a) from M. Pierres (CNRS/INSERM, Marseille, France) and GK1.5 (IgG2b) from F. Fitch (Chicago, IL). The anti-IL-4 monoclonal antibody (11B11) was from M. Lo¨hning (Deutsches Rheumaforschungszentrum Berlin, Germany) and the goat anti-IL-12 antibody from M. Gately (Hoffmann-La Roche).
CD4 binding assays and FACS analysis Lymph node cells (0.5105) of BALB/c mice were incubated with blocking anti-CD4 antibodies (see above) at a concentration of 10 ìg/ml in PBS/0.5% bovine serum albumin (BSA)/0.01% NaN3 on ice for 30 min. Biotinylated (labelling kit from Boehringer Mannheim, Germany) antiCD4 antibodies YTS191.1, YTA3.12 or RmCD4.2 were then added as staining antibodies, and incubation continued for a further 30 min on ice. As controls, cells were incubated with blocking or staining antibodies alone. Cells were washed and then incubated with streptavidin–FITC (Dako Diagnostika, Hamburg, Germany) for 30 min on ice. After washing cells, CD4 staining was analysed on a FACScan flow cytometer (Becton Dickinson, San Jose, CA). Blocking of staining indicated that both the blocking antibody and the biotinylated staining antibody bind to overlapping epitopes.
Intracellular cytokine staining DO11.10 splenocytes after 7 days of culture as above were stimulated for 5 h with PMA (5 ng/ml; Sigma) plus ionomycin (1 ìg/ml; Sigma). Two hours before harvesting, brefeldin A (10 ìg/ml; Sigma) was added. After harvesting, cells were washed and fixed in 2% formaldehyde for 20 min at room temperature, and then stained with anti-IL-4–PE (11B11; 5 ìg/ml; Pharmingen, Hamburg, Germany) and antiIFN-ã-FITC (XMG1.2; 2 ìg/ml; Pharmingen) for 10 min at
214 / Gimsa et al.
room temperature. Washing and staining procedures were carried out in PBS/1% BSA/0.5% saponin (Sigma). Finally, cells were resuspended in PBS/0.5% BSA/0.01% NaN3 for FACS analysis. Samples were gated on live lymphocytes based on forward and side scatter parameters (10,000 events/ sample) and analysed using Cellquest 3.0 software (Becton Dickinson).
CD40L expression BALB/c spleen and lymph node cells were depleted of B cells and macrophages by incubation on a prewarmed nylon wool column filled with complete medium for 1.5 h. CD4 + and CD4 cells were then separated by high gradient magnetic cell sorting. Briefly, cells were labeled with superparamagnetic microparticles conjugated to monoclonal antibodies (anti-CD8, anti-B220 and anti-CD11b; Miltenyi Biotec GmbH; Bergisch-Gladbach, Germany), and then separated on a MidiMACS column allowing nonmagnetic cells to pass. FACS analysis showed about 86% CD4 + cells contaminated with about 2% B cells and 6% macrophages. These cells were then incubated overnight with plate-bound anti-CD3 (10 ìg/ml; Pharmingen) with or without PP1, and then harvested and stained with anti-CD40L-PE (MR1; 10 ìg/ml; Pharmingen) for 30 min on ice. CD40L expression was then analysed by FACS.
Acknowledgements Founder DO10 mice were a gift from D. Loh. This work was supported by the Deutsche Forschungsgemeinschaft and the Senate Administration for Research and Education of the City of Berlin.
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