The negative regulators Foxj1 and Foxo3a are up-regulated by a peptide that inhibits systemic lupus erythematosus-associated T cell responses
Version of Record online: 19 OCT 2006
Copyright © 2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
European Journal of Immunology
Volume 36, Issue 11, pages 2971–2980, November 2006
How to Cite
Sela, U., Dayan, M., Hershkoviz, R., Cahalon, L., Lider, O. and Mozes, E. (2006), The negative regulators Foxj1 and Foxo3a are up-regulated by a peptide that inhibits systemic lupus erythematosus-associated T cell responses. Eur. J. Immunol., 36: 2971–2980. doi: 10.1002/eji.200636137
- Issue online: 30 OCT 2006
- Version of Record online: 19 OCT 2006
- Manuscript Accepted: 13 SEP 2006
- Manuscript Revised: 14 AUG 2006
- Manuscript Received: 6 APR 2006
- TEVA Pharmaceutical Industries, Israel (E.M.)
- Immunomodulatory peptide;
- T cells;
- Transcription factors
A peptide (hCDR1) based on the complementarity determining region-1 of an anti-DNA antibody ameliorates systemic lupus erythematosus (SLE) in induced and spontaneous lupus models. Our objectives were to determine the effects of hCDR1 on TCR signaling and on its negative regulators, Foxj1 and Foxo3a. BALB/c mice were immunized with the SLE-inducing anti-DNA antibody, designated 16/6Id, and treated with hCDR1. hCDR1 treatment specifically inhibited IFN-γ secretion by T cells in association with down-regulated T-bet expression and NF-κB activation; however, GATA-3 expression was not affected. Furthermore, TCR signaling (ZAP-70 phosphorylation) was inhibited, and the mRNA expression of the two modulators of Th1 activation, Foxj1 and Foxo3a, was significantly up-regulated. The latter were also elevated in SLE-afflicted (NZB×NZW)F1 mice that were treated with hCDR1. Addition of TGF-β, which was elevated following treatment with hCDR1, to T cells from 16/6Id immunized mice, up-regulated Foxj1 and Foxo3a mRNA expression, similarly to hCDR1. In contrast, anti-TGF-β antibodies added to hCDR1-treated T cells abrogated its effect. Thus, hCDR1 elevates TGF-β, which contributes to the up-regulation of T cell Foxj1 and Foxo3a expression, leading to inhibition of NF-κB activation and IFN-γ secretion, which is required for the maintenance of SLE.
systemic lupus erythematosus
Experimental systemic lupus erythematosus (SLE) can be induced in naïve, non-SLE prone mice, by their immunization with anti-DNA mAb that express the major idiotype (Id), designated 16/6Id 1, 2. The 16/6Id-induced disease in mice is manifested by high levels of autoantibodies (anti-dsDNA and anti-nuclear protein Ab), and by SLE-associated clinical symptoms 1, 2. Furthermore, a peptide based on the sequences of the complementarity determining region (CDR)1 of the human 16/6Id was shown to down-regulate autoreactive T cell responses in vitro and in vivo, and to ameliorate the clinical (e.g., proteinuria, leukopenia) and serological (anti-dsDNA Ab) manifestations, histopathological findings and kidney IgG deposits of spontaneous (NZB/NZW)F1 and induced models of SLE in mice 3–7. The latter was associated with down-regulation of the cytokines that play a key role in the pathogenesis of lupus (e.g., IFN-γ, IL-10, IL-1) and with an up-regulation of the immunosuppressive cytokine TGF-β 4, 5, 7. Moreover, treatment with hCDR1 inhibited T cell interactions with the ECM including adhesion and chemotaxis by down-regulating ERK phosphorylation 8. The inhibition of phosphorylation of this kinase was also found to be involved in 16/6Id-stimulated proliferation via down-regulating the expression and function of a pair of adhesion and costimulatory molecules, leukocytes function antigen-1 (LFA-1) and CD44. IFN-γ was also found to play an important role in 16/6Id-stimulated proliferation 9.
TCR ligation by an Ag induces phosphorylation of immunoreceptor tyrosine-based activation motif (ITAM) that serves to recruit ZAP-70. The latter activates down-stream signaling components, leading to dissociation of IκB from NF-κB and degradation of IκB. This results in activation of NF-κB (p65) and its subsequent translocation to the nucleus, where it affects many genes including Th1 type cytokines 10. Another Th1-associated transcription factor is T-bet, which serves as a master gene for IFN-γ secretion and is also activated following TCR ligation by an Ag 11, 12.
To prevent destructive effects of the active immune system, T cell stimulation by an Ag is accompanied by attenuation of its activation signal 13. Foxj1 and Foxo3a are members of the forkhead (Fox) transcription factors that were recently found to be important negative regulators of T cell activation through inhibition of NF-κB activity, and as a result, Th1 cytokine secretion is down-regulated 14. Both factors were shown to be diminished in MRL or BXSB lupus prone mice 15, 16
The objectives of the present study were to determine the effect of the hCDR1 on TCR signaling and regulation. We show here that treatment with hCDR1 down-regulated the 16/6Id-stimulated IFN-γ secretion by T cells already 10 days after immunization concomitant with hCDR1 treatment. The latter was associated with inhibition of T-bet expression and NF-κB activity. Furthermore, up-stream TCR signaling (ZAP-70 phosphorylation) was inhibited and the mRNA expression of the two modulators of Th1 activation, Foxj1 and Foxo3a, was significantly up-regulated.
hCDR1 immunomodulates mRNA expression and 16/6Id-stimulated secretion of IFN-γ by T cells
We have recently shown 9 that treatment with hCDR1 specifically down-regulated the 16/6Id-stimulated proliferation in association with up-regulation of TGF-β secretion. To find out whether the hCDR1-induced inhibition of proliferation is associated with down-regulation of IFN-γ, we determined the secretion of IFN-γ in mice that were immunized with 16/6Id and treated with hCDR1. Three groups of mice were used: a control group injected with PBS in CFA, a group immunized with 16/6Id in CFA, and a group that was treated with hCDR1 concomitant with 16/6Id immunization. At 10 days after immunization, LN cells from the three groups were incubated (48 h) in vitro in the presence or absence of 16/6Id (25 μg/mL). As shown in Fig. 1A, the levels of IFN-γ secretion were low in the control group (108 pg/mL and 151 pg/mL in the absence or presence of in vitro stimulation with 16/6Id, respectively) compared to the 16/6Id-immunized mice in which the constitutive secretion of IFN-γ was 628 pg/mL, and the in vitro 16/6Id-stimulated secretion was 1047 pg/mL. In vivo treatment with hCDR1 down-regulated significantly both the constitutive (384 pg/mL, p<0.05) and the 16/6Id-stimulated secreted IFN-γ (242 pg/mL = 4.3-fold decrease, p<0.001). Table 1 demonstrates that the inhibitory effect of hCDR1 is specific since in vivo treatment with a control peptide (scrambled) did not inhibit IFN-γ secretion from 16/6Id-immunized mice. Furthermore, when the hCDR1 was injected as treatment to hIgG immunized mice, it did not down-regulate the IFN-γ secretion.
|Immunization||Treatment||% IFN-γ secretion|
To determine whether the change in cytokine secretion by LN cells is due to the effect of hCDR1 on T cells, purified T cells from all groups were incubated with irradiated splenocytes from normal mice (as a source for APC), in the presence or absence of 16/6Id stimulation. Supernatants were collected and levels of IFN-γ were measured by ELISA. As shown in Fig. 1B, in vivo treatment with hCDR1 significantly down-regulated the in vitro 16/6Id-stimulated IFN-γ secretion by T cells (1907 pg/mL versus 165 pg/mL in the absence or presence of in vivo treatment with hCDR1, respectively). As shown in Fig. 1C, in vivo treatment with hCDR1 also significantly decreased the mRNA expression of IFN-γ. Thus, treatment with hCDR1 down-regulated IFN-γ secretion from T cells in association with inhibition of its mRNA expression.
hCDR1 down-regulates T-bet expression and NF-κB activity
T-bet is a master gene that plays an important role in coordinating Th1 differentiation and induction of IFN-γ expression 11. Therefore, we studied the effect of in vivo treatment with hCDR1 on the 16/6Id-stimulated expression of T-bet in T cells. To this end, T cells derived from the various groups of mice were incubated with irradiated splenocytes derived from normal mice, and in the presence or absence of 16/6Id stimulation (25 μg/mL). The extent of T-bet expression was examined in the T cell lysates and total ERK was used as a constitutively expressed control protein for quantification of the amount of the protein. As shown in Fig. 2A, in vivo treatment with hCDR1 markedly inhibited both the constitutive and the in vitro 16/6Id-stimulated expression of T-bet in T cells, as compared to its expression in T cells derived from 16/6Id-immunized mice in the absence of treatment.
To determine whether the down-regulation in T-bet could reflect enhanced Th2 stimulation, we studied the effect of treatment with hCDR1 on GATA-3 expression and IL-10 secretion. As shown in Fig. 2B, treatment with hCDR1 did not up-regulate the expression of this transcription factor. In addition, levels of 16/6Id stimulated IL-10 secretion were not affected following hCDR1 treatment (206 pg/mL and 196 pg/mL by T cells from 16/6Id-immunized mice with or without concomitant treatment with hCDR1, respectively).
NF-κB is another Th1-associated factor that has a pivotal role in the specific regulation of Th1 development 10. Therefore, we studied the effect of in vivo hCDR1 treatment on NF-κB translocation to the nucleus by probing nuclear and cytoplasmic T cell extracts using an mAb specific for the p65 subunit of NF-κB. The cytoplasmic ERK and nuclear protein lamin B were used as constitutively expressed controls for the quantification of protein amounts. Fig. 2C and D demonstrates results of one experiment out of three performed, all yielding similar data. It is shown that in vitro stimulation with 16/6Id of T cells derived from 16/6Id-immunized mice, down-regulated NF-κB in the cytoplasmic compartment (Fig. 2C) due to NF-κB activation and translocation to the nucleus (Fig. 2D). On the other hand, in vivo treatment with hCDR1 inhibited the NF-κB activation as it down-regulated its translocation to the nucleus (Fig. 2D), while the p65 subunit of this nuclear factor remained in the cytoplasmic compartment (Fig. 2C). The down-regulation of NF-κB activation was associated with up-regulation of IκB expression (Fig. 2E). Thus, both the constitutive and the in vitro 16/6Id-induced IκB expression of T cells derived from hCDR1-treated mice were up-regulated as compared to IκB expression by T cells from 16/6Id-immunized mice that were not concomitantly treated with hCDR1.
hCDR1 down-regulates ZAP-70 phosphorylation by T cells
To determine whether the inhibition of NF-κB activation and T-bet expression by hCDR1 is associated with its effect on the up-stream TCR signaling, we studied the phosphorylation of ZAP-70. To this end, T cells derived from 16/6Id-immunized mice that were either treated or not treated with hCDR1, were incubated with irradiated splenocytes derived from normal mice in the presence or absence of 16/6Id stimulation (25 μg/mL). The extent of ZAP-70 phosphorylation was examined in the T cell lysates. Fig. 3 demonstrates that in vivo treatment with hCDR1 markedly down-regulated ZAP-70 phosphorylation (from 43% to 19.8% in the absence or presence of in vivo treatment with hCDR1, respectively, based on desitometric measurement). Thus, the down-regulation of NF-κB activity and T-bet expression following hCDR1 treatment is associated with inhibition of ZAP-70 phosphorylation.
hCDR1 up-regulates Foxj1 and Foxo3a mRNA expression by T cells
Foxj1 and Foxo3a are transcription factors that participate in negative regulation of Th1 activation, including IFN-γ secretion 14; both can down-regulate Th1 activity through up-regulation of IκB expression, which results in inhibition of activation and nuclear translocation of Rel-A (p65) NF-κB subunit 15, 16. To find out whether the down-regulation in NF-κB activity following in vivo treatment with hCDR1 is also due to its effect on these transcription factors, we measured the mRNA levels of the latter in T cells derived from 16/6Id-immunized mice that were either concomitantly treated or not treated with hCDR1, 10 days after immunization, using real-time RT-PCR. As shown in Fig. 4A, treatment with hCDR1 induced a significant up-regulation of about 6-fold in Foxj1 expression compared with T cells derived from mice immunized with 16/6Id without concomitant treatment with hCDR1 (considered as 100%). Likewise, treatment with hCDR1 significantly up-regulated (7.7-fold, Fig. 4B) Foxo3a expression compared with T cells derived from 16/6Id-immunized mice that were not treated with hCDR1. To verify the observed up-regulation of Foxj1 and Foxo3a following in vivo treatment with hCDR1, we studied the expression levels of these transcription factors in lupus prone (NZB×NZW)F1 mice in which treatment with hCDR1 was previously shown 3–7 to ameliorate SLE manifestations in association with down-regulation of IFN-γ secretion. To this end, T cells were purified from splenocytes of (NZB×NZW)F1 mice that were harvested after 10 weeks of treatment (once a week) with either hCDR1 or the vehicle (PBS). Fig. 4C and D demonstrates significant up-regulation in the mRNA expression of these factors also in (NZB×NZW)F1 mice following treatment with hCDR1 (4.3- and 9.8-fold for Foxj1 and Foxo3a mRNA, respectively, as compared with non-treated mice, considered 100%).
hCDR1 immunomodulates Foxj1 and Foxo3a mRNA expression by up-regulating TGF-β
We have previously shown that hCDR1 up-regulated the TGF-β secretion by LN cells 8. Fig. 5A shows that in vivo treatment with hCDR1 up-regulated TGF-β secretion by T cells ten days after immunization. The up-regulated secretion was associated with an increase (Fig. 5B) in mRNA expression of TGF-β (171% as compared with T cells of mice immunized with 16/6Id without a concomitant treatment with hCDR1, considered as 100%).
To determine whether the up-regulated TGF-β may contribute to the hCDR1 induced increase in Foxj1 and Foxo3a expression, T cells purified from 16/6Id-immunized mice were stimulated in vitro with 16/6Id in the presence or absence of TGF-β. Fig. 5C and D demonstrates the significant up-regulation in the expression of these two transcription factors following their incubation with TGF-β. To confirm the role of TGF-β in up-regulation of these two transcription factors, T cells purified from mice that were treated with hCDR1 concomitant with 16/6Id immunization were incubated with anti-TGF-β mAb or its isotype control. Fig. 6 shows that an in vitro addition of anti- TGF-β mAb, but not its isotype control, abrogated the in vivo up-regulatory effect of hCDR1 on the expression of Foxj1 and Foxo3a. Thus, the hCDR1 induced up-regulation in TGF-β contributed to the up-regulation of these negative regulators of TCR activation.
The main findings of this study are that hCDR1 (Edratide) inhibited the secretion of IFN-γ by T cells in association with down-regulated T-bet expression and NF-κB activation. The latter was accompanied by inhibition of TCR signaling (ZAP-70 phosphorylation) and up-regulation of the mRNA expression of two negative regulators of TCR activation, Foxj1 and Foxo3a. This effect is mediated by hCDR1-induced up-regulation of TGF-β by T cells. To the best of our knowledge, this is the first demonstration that the negative regulators Foxj1 and Foxo3a can be immunomodulated.
The beneficial effects of hCDR1 on the clinical manifestations of SLE were shown to be associated with down-regulation of pathogenic cytokines including IFN-γ, and with an increase in the immunosuppressive cytokine TGF-β 7. We have recently shown 8 that already 10 days following immunization with 16/6Id, much before the appearance of serological and clinical manifestations of experimental lupus, treatment with hCDR1 specifically diminished T cell responses, including chemotaxis, adhesion, and proliferation in association with inhibition of ERK phosphorylation. IFN-γ was found to play an important role in 16/6Id-stimulated proliferation, because its addition to cell cultures abrogated the hCDR1-induced inhibition of proliferation 9. Based on these observations, we carried out the present study, in an attempt to further elucidate the mode of action of hCDR1, especially in relation to TCR signaling and its negative regulators, Foxj1 and Foxo3a.
IFN-γ plays a key role in the pathogenesis of lupus 17, and its levels are elevated in serum of lupus patients 18. Its early secretion by infiltrating T cells was essential for a later tissue destruction 19, whereas treatment with anti-IFN-γ mAb 20 was beneficial in (NZB×NZW)F1 mice. We have previously demonstrated that treatment with hCDR1 down-regulated IFN-γ secretion by LN cells or splenocytes of mice with a full-blown disease. Here we showed that hCDR1 inhibited mainly the 16/6Id-stimulated IFN-γ secretion and mRNA expression by T cells. This effect was observed already 10 days after immunization with 16/6Id, and was shown to be specific for hCDR1 (Table 1).
T-bet is a major transcription factor that directs Th1 lineage commitment and transactivates the IFN-γ gene. The expression of T-bet is correlated with IFN-γ expression in Th1 cells 11. Peng et al. 12 reported that T-bet regulates IgG class switching and pathogenic autoantibody production by B cells in murine lupus, but little is known about the role of T-bet in SLE-associated T cells. Here we demonstrated that treatment with hCDR1 concomitant with 16/6Id immunization down-regulated T-bet expression in T cells (Fig. 2A) in association with the significant decrease in IFN-γ secretion. GATA-3, which induces Th2 lineage 21, may be involved in silencing the opposing Th1 regulator. Here we demonstrated that hCDR1 did not up-regulate either the expression of GATA-3 or IL-10 secretion. Therefore, the inhibition of IFN-γ secretion probably does not reflect enhanced Th2 stimulation. Further, the inhibitory effect of hCDR1 on IFN-γ did not result in up-regulated IL-17 mRNA expression, but rather the latter was down-regulated by about threefold (unpublished results)
NF-κB is a transcription factor that has been shown to control the amount of IFN-γ produced by a differentiated Th1 population 22. Its main activated form is a heterodimer of p65 subunit that translocates to the nucleus 23, where it activates the expression of many genes including IFN-γ. Indeed, the increased IFN-γ secretion by T cells derived from 16/6Id-immunized mice was associated with a marked up-regulation of the p65 subunit activation and translocation to the nucleus (Fig. 2D). However, following treatment with hCDR1, the p65 subunit of this NF mainly remained in the cytoplasmic compartment (Fig. 2C) apparently due to up-regulation of IkB expression (Fig. 2E).
The classical signaling pathway leads to activation of the p65 subunit of the NF-κB pathway, and triggers of this pathway include TCR stimulation 24, 25, which is also involved in inducing the expression of T-bet 26, 27. We show here that treatment with hCDR1 markedly inhibited TCR activation as manifested by down-regulation of ZAP-70 phosphorylation (Fig. 3), compared to T cells derived from non-treated 16/6Id-immunized mice. This result supports the involvement of TCR signaling in the above observed inhibitory effects of the hCDR1.
Foxj1 and Foxo3a are two members of the forkhead family that have been shown to be actively involved in the negative regulation of T cells by maintaining NF-κB in an inactive state, and both negative regulators are down-regulated in the lymphoid cells derived from lupus prone mice 15, 16. In this study we demonstrated that in vivo treatment with hCDR1 significantly up-regulated these two transcription factors, both 10 days after 16/6Id immunization in BALB/c mice and in lupus prone (NZB/NZW)F1 mice with a full-blown disease (Fig. 4). After TCR signaling Foxj1 and Foxo3a are inactivated and their expression is dramatically down-regulated 13. Therefore, the down-regulated ZAP-70 phosphorylation following in vivo treatment with hCDR1 may also account for the increased expression of these two transcription factors.
TGF-β is an immunosuppressive cytokine that is associated with amelioration of inflammatory reactions and autoimmune diseases 28, 29. Both constitutive and stimulated levels of TGF-β were shown to be low in SLE patients, especially when the disease was active 30. Treatment of SLE prone mice, which led to up-regulation of LN cell-derived TGF-β, ameliorated the serological and clinical manifestations 4, 7, 31. Furthermore, the inhibition of 16/6Id-stimulated proliferation by hCDR1 10 days after immunization with 16/6Id was also associated with specific up-regulation of LN cell-derived TGF-β secretion, and in vitro addition of TGF-β inhibited the 16/6Id-stimulated cell proliferation to an extent similar to that of in vivo treatment with hCDR1 9. Here we showed that hCDR1 up-regulated TGF-β secretion and mRNA expression by T cells. These results might suggest that hCDR1, which inhibited T cell proliferation 9 and TCR signaling along with modulation of the cytokine profile, is probably acting as a partial agonist 32, 33.
TGF-β has been found to down-regulate ZAP-70 phosphorylation by activation of the protein tyrosine phosphatase SHP-1 34, 35, and therefore it is possible that hCDR1-induced up-regulation in TGF-β reinforced the action of hCDR1 and contributed to the observed down-regulation of TCR signaling. Moreover, similarly to the in vivo treatment with hCDR1, incubation of 16/6Id-immunized murine T cells with TGF-β significantly up-regulated Foxj1 and Foxo3a expression (Fig. 5). The role of TGF-β was further confirmed when addition of anti-TGF-β mAb abrogated the hCDR1-induced up-regulation of these transcription factors. The TGF-β induced up-regulation in Foxj1 and Foxo3a expression suggests a novel mechanism whereby this cytokine exerts its immunosuppressive effect. However, it is not clear whether TGF-β exerts its effect directly on these transcription factors or indirectly by down-regulating TCR signaling and subsequently the expression of these factors.
Taken together, our results suggest the existence of a mechanism whereby hCDR1 exerts its specific inhibitory capacities in vivo. The apparent down-regulation of murine T cell IFN-γ secretion is probably a consequence of the peptide's ability to down-regulate TCR activation (ZAP-70 phosphorylation). This leads to inhibition of T-bet expression and NF-κB activation. The inhibition of the latter may be mediated by down-regulation of TCR signaling directly and/or indirectly by up-regulation of the negative regulators of TCR activation Foxj1 and Foxo3a. The up-regulation in TGF-β secretion following treatment with hCDR1 reinforces the direct effect of the peptide by up-regulating Foxj1 and Foxo3a expression in T cells. Our findings of the hCDR1-induced down-regulation of IFN-γ secretion and the up-regulation of Foxj1 and Foxo3a resulted from experiments performed both early in disease induction (10 days after immunization with 16/6Id), and in (NZB×NZW)F1 mice with full-blown disease. Therefore, these mechanisms probably play a role in the amelioration of SLE manifestations following treatment with hCDR1. Moreover, the ability to modulate the expression of the negative regulators of T cell activation, Foxj1 and Foxo3a, may lead to future studies in which immunomodulation of these transcription factors will be used for the treatment of other autoimmune diseases.
Materials and methods
Mice of the BALB/c inbred strain were obtained from Harlan, and female mice were used at the age of 8–10 weeks. (NZB×NZW)F1 female mice were purchased from Jackson Laboratory (Bar Habor, ME). This study has been approved by the Animal Care and Use Committee of the Weizmann Institute of Science.
A peptide based on the sequence of the CDR1 36 of the human mAb anti-DNA that bears the major 16/6Id idiotype (GYYWSWIRQPPGKGEEWIG) was synthesized by Polypeptide laboratories (Los Angeles, CA) using solid-phase synthesis by Fmoc chemistry. The peptide was designed based on the information obtained from previous studies using peptides with sequences of a murine anti-DNA mAb that bears the 16/6Id 37, 38. hCDR1 (Edratide) is under clinical development for the treatment of SLE by TEVA Pharmaceutical Industries. As a control, we used a peptide containing the amino acids of hCDR1 (SKGIPQYGGWPWEGWRYEI) in a scrambled order (designated scrambled peptide). This peptide binds MHC class II with a similar avidity to that of hCDR1 (unpublished data).
Antibodies and reagents
The human anti-DNA mAb that bears the 16/6Id (IgG1/κ) has been described previously 36. The Ab is secreted by hybridoma cells that are grown in culture and purified using a protein G-Sepharose column (Pharmacia). Polyclonal Ab anti-total (t)ERK1/2 was obtained from Sigma-Aldrich (Rehovot, Israel); anti-NF-κB p65, anti-T-bet (39D), anti-GATA-3, anti-lamin B1 (C-20), anti-IκBβ (C-20) and anti-phospho (p)ZAP-70 (Tyr 319) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Recombinant human Transforming Growth Factor-β1 (TGFβ1 was obtained from Pepro Tech (Rocky Hill, NJ).
Immunization and treatment of mice
Mice were immunized (intradermally, hind footpad) with 1 µg of the human mAb 16/6Id, hIgG or PBS (as a control) in CFA. Additional groups of mice were injected subcutaneously (back of the neck) with hCDR1 (50 µg/mouse) in PBS concomitant with 16/6Id or hIgG immunization or with the scrambled peptide (control) concomitant with 16/6Id immunization. Mice of all groups were killed at day 10, and their LN cells or LN-derived T cells were studied. (NZB×NZW)F1 mice with a full-blown disease (7 months old) were treated for 10 weeks, once a week, with either PBS (vehicle) or hCDR1 (50 μg/mouse).
T cell purification
Preparation of an enriched population of LN-derived T cells was performed as follows: petri dishes were pre-coated (overnight, 4ºC) with goat anti-mouse immunoglobulin (15 µg/mL in 5 mL PBS), and then washed three times. Inguinal murine LN cells were incubated (70 min, 4ºC, in RPMI containing 10% FCS and antibiotics) on the coated plates. The purified cells, which were mainly T cells (>92% as assessed by FACS analysis), were then collected and washed in RPMI.
In vitro cell stimulation with 16/6Id
LN-derived cells were removed from mice 10 days after immunization. Cells (5×106) were incubated in the presence of 16/6Id or hIgG (25 μg/mL). Purified T cells were stimulated by incubating them with irradiated (3000 rad) syngeneic splenocytes from normal mice (as a source APC), in the presence of 16/6Id or hIgG (25 μg/mL).
Detection of IFN-γ and TGF-β secretion
LN-derived cells or purified T cells were stimulated with 16/6Id as described above. Their supernatants were collected after 48 and 72 h. The levels of IFN-γ in the supernatants were determined by ELISA according to the manufacturer's instructions (PharMingen). The levels of TGF-β1 were measured by ELISA using recombinant human TGF-β1 (sRII/Fc chimera; R&D systems; Minneapolis, MN). Supernatants were added after activation of latent TGF-β1 to immunoreactive TGF-β1 by adding HCl and then neutralizing the acidified sample with NaOH (manufacturer's instructions).
The levels of mRNA of IFN-γ, TGF-β, Foxj1, and Foxo3a were determined by quantitative real-time RT-PCR using LightCycler (Roche, Mannheim, Germany). Reverse transcription into complementary DNA was performed using Molony murine leukemia virus reverse transcriptase (Promega). Real-time RT-PCR was performed according to the manufacturer's instructions. Briefly, 20 µL of reaction volume contained 3 mM MgCl2, LightCycler HotStart DNA SYBR Green I mix (Roche), specific primer pairs, and 5 µL cDNA. PCR cycling conditions were 10 min at 95°C followed by 45 cycles of 95°C for 15 s, 60°C for 15 s, and 72°C for 15 s. The following primer sequences were used (forward and reverse respectively): IFN-γ (5′-gaacgctacacactgc-3′ and 5′-ctggacctgtgggttg-3′), TGF-β (5′-gaacccccattgctgt-3′ and 5′-gccctgtattccgtct-3′), Foxj1 (5′-ccatacacacagtgtttca-3′ 5′-gagcgtttgttgtacct-3′) Foxo3a (5′-atgggccacgataagt-3′ 5′-cagtaacagtccgcct-3′) and β-actin (5′-gtgacgttgacatccg-3′ 5′-gagcgtttgttgtacct-3′). The levels of β-actin were used for normalization while calculating the expression levels of other genes. The results are expressed as the relative expression levels for each gene.
Western blot analysis of T cell whole cell cytoplasmic and nuclear extracts
For preparation of whole cell lysate, and separation of cytoplasmic and nuclear extracts, T cells that were either unstimulated or in vitro stimulated with16/6Id, were lysed as previously described 8, 39. Samples containing equal amounts of proteins were separated on 10% SDS-PAGE gel and transferred to nitrocellulose membranes. The membranes were blocked and probed with the following mAb: anti-pZap-70, anti-T-bet, anti-GATA-3, anti-IκB, anti-tERK, and anti-tZAP-70 for membranes prepared from whole cell lysate. Anti-NF-κB and anti-tERK mAb, were used for membranes prepared from cytoplasmic extract and anti-NF-κB and anti-lamin B for membranes prepared from nuclear extract. Immunoreactive protein bands were visualized using labeled secondary Ab and the enhanced ECL system. The results were also demonstrated as densitometric histograms that were calculated as percent of total protein using the NIH image program.
The nonparametric Mann–Whitney test was used for statistical analyses of the data. A value of p⩽0.05 was considered statistically significant.
This study was supported by TEVA Pharmaceutical Industries, Israel (E.M.).
- 13The yins of T cell activation. SciSTKE 2005. 265.