Control of CD4 T cell fate by antigen re-stimulation with or without CTLA-4 engagement 24 h after priming

Yukiko Nakata, Akiko Uzawa and Gen Suzuki

Division of Radiation Health, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba-city, Chiba 263-8555, Japan

Correspondence to: G. Suzuki


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
After two consecutive inoculations with Staphylococcus enterotoxin B (SEB) at 24 h intervals in vivo, CD4 T cells became anergic to the antigen challenge in vitro. Administration of anti-CTLA-4 mAb in conjunction with the second SEB inoculation 24 h after antigen priming interfered with anergy and CD4 T cells became Th2 cells. However, the anergy induction was not ablated when SEB and anti-CTLA-4 mAb were administered 48 or 72 h after antigen priming. Moreover, anti-CTLA-4 mAb without SEB did not interfere with anergy nor promoted the Th2 differentiation. T–antigen-presenting cell (APC) interaction in vitro in the presence of high doses of antigen and anti-CTLA-4 mAb induced a Th2-polarizing cytokine IL-6 and IL-10. IL-10 then down-modulated a Th1-polarizing cytokine IL-12. The results demonstrate that 24 h after the initial antigen stimulation, CD4 T cells enter the critical activation phase where antigen re-stimulation with or without CTLA-4 engagement alters the fate of the cell, anergy or differentiation respectively. Once anergy is interfered with, Th2-polarizing cytokines produced upon prolonged T–APC interaction favor the Th2 differentiation.

Keywords: anergy, IL-6, IL-10, Staphylococcus enterotoxin B, Th2


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The Th1/Th2 balance has been known to determine the nature of immune responses to infection by various microorganisms (13) and to autoantigens (4,5). Development of an inappropriate immune response is unprotective against infections and could be causative of autoimmune diseases.

It is well documented that cytokines in the microenvironment skew the differentiation of naive CD4 T cells (6,7). IL-4 and IL-12/IFN-|{gamma} polarize the T cell differentiation to Th2 and Th1 respectively. IL-6 is now listed as an inducing factor for Th2 (8). Other factors potentially skewing Th differentiation are the dose of antigen, the affinity of antigen, the type of antigen-presenting cell (APC), the genetic background, and/or the co-stimulatory pathways (912). The relationship between polarizing cytokines and the other factors is still unknown. Low and moderate doses of protein antigen induce Th2 and Th1 respectively (9,10). High doses of antigen, however, induce Th2 again (9). The reason why antigen alters the Th1/Th2 balance in a dose-dependent manner has not been fully elucidated.

The interaction between CD28 and CD80/CD86 activates signal cascades important for T cell proliferation, differentiation and survival (1315). After antigen stimulation, T cells express CTLA-4 that interacts with CD80 with higher affinity than does CD28 (16). In contrast to CD28, CTLA-4 is thought to play an inhibitory role in the immune response, especially in the induction of tolerance (1723). Since high doses of antigen induce T cell tolerance, which may upset the Th1/Th2 balance, we have attempted to interfere with tolerance by administering anti-CTLA-4 mAb and to evaluate the Th1/Th2 balance.

High doses of antigen may work on T cells by two ways: antigen occupies more TCR at the same time and it repeatedly stimulates cell for longer period. In order to dissect the late T–APC interaction after the inoculation of high-dose antigen, we consecutively inoculated mice with two moderate doses of Staphylococcus enterotoxin B (SEB) instead of one high dose of SEB. It was demonstrated that two consecutive inoculations of SEB at 24 h intervals induced Th2 in vivo if tolerance was interfered with by anti-CTLA-4 mAb. The second inoculation of SEB was essential for CD4 T cells to escape from tolerance and to differentiate into Th2 as well. Our data are the first to show that antigen re-stimulation with or without CTLA-4 engagement 24 h after antigen priming alters the fate of T cells.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents and antibodies
SEB was obtained from Sigma Israel Chemicals (Jerusalem, Israel). Low-Tox-M rabbit complement was purchased from Cedarlane (Hornby, Ontario, Canada). PharM Lyse, hamster IgG (A19-4), anti-mouse CD28 (37.51), anti-mouse CD152 (CTLA-4) (UC10-4F10-11), biotin–anti-mouse CD3 (145-2C11), phycoerythrin (PE)–anti-mouse CD4 (L3T4), biotin–anti-mouse CD11c (HL3), biotin–anti-mouse CD45R/B220 (RA3-6B2) and PE–anti-mouse CD45R/B220 mAb were purchased from PharMingen (San Diego, CA). Anti-mouse TCR Vß8 (F23.1) mAb was purified from ascites by affinity chromatography using Protein A—Sepharose 4FF (Pharmacia Biotech, Uppsala, Sweden). Biotinylation of anti-mouse TCR Vß8 mAb was performed in our laboratory. Fluorescein avidin DCS was purchased from Vector (Burlingame, CA). SPHERO streptavidin magnetic particles and Dynabeads-pan T were from Spherotech (Libervilli, IL) and Dynal (Oslo, Norway) respectively. Anti-mouse IL-10 mAb were purified from SXC-1 and -2 supernatants by affinity chromatography using CM-Affigel-Blue (BioRad, Richmond, CA).

Mice and T cell preparations
C57BL/6 (B6) male mice were purchased from SLC (Shizuoka, Japan) and used at 8–14 weeks of age. Mice were primed i.p. with 10 µg of SEB. After 24, 48 or 72 h, they were injected with 1 µg of SEB and 75 µg of anti-CTLA-4 mAb or control IgG i.p. Six days after the second SEB inoculation, a single-cell suspension of spleen cells was prepared and loaded on a nylon wool column to enrich the T cell fraction. CD4 T cells were prepared by sequential treatment with a cocktail of anti-mouse CD8{alpha} (83-12-5), anti-mouse heat stable antigen (J11d.2) and anti-mouse I-Ab (M5/114) mAb, followed by Low-Tox-M rabbit complement.

Short-term T cell lines for the measurement of T cell cytokines
To investigate cytokine production, we created a three-step culture system. In the first culture, CD4 T cells (2x106/well) were cultured for 3 days with 1 µg/ml of SEB in the presence of 30 Gy irradiated syngeneic spleen cells (2x106/well) as a source of APC in 24-well plates. Then, cells were harvested, washed and re-cultured in fresh medium for 1 day as the second culture. Finally, SEB-reactive Vß8+CD4+ T cells were re-stimulated with plate-coated mAb in the third culture to investigate the cytokine production. The cell culture medium was {alpha}-MEM supplemented with 10% FCS, 2 mM L-glutamine, 5x10—5 M 2-mercaptoethanol, 15 mM HEPES/NaOH (pH 7.2) and antibiotics (complete medium).

Culture plates were pre-coated with anti-TCR Vß8 mAb (1 µg/ml final) in 50 mM Tris-HCl (pH 8.0) for several hours at room temperature. Anti-CD28 mAb (5 µg/ml final) was added and the plate incubated overnight at 4°C. Plates were washed 6 times with PBS before use. To assay the production of cytokines, Vß8+CD4+ T cells (0.5—2.0x106/well/0.5—2.0 ml) were cultured in the mAb-coated 48- or 24-well plates for 1 day at 37°C.

Co-cultivation of T cells and APC for the measurement of cytokines
T- and B-depleted APC were enriched by the negative immunomagnetic sorting of unirradiated spleen cells. Red blood cells were lysed by PharM Lyse. B and T cells were depleted using biotin–anti-CD45R/B220 mAb in combination with SPHERO streptavidin magnetic particles and Dynabeads-pan T by MACS (Militenyi Biotec, Bergisch Gladbach, Germany). The purity of APC was checked by FACStar (Becton Dickinson, Mountain View, CA).

Splenic T cells (2.5x105/well) from mice immunized with SEB (10 µg) 24 h before were co-cultured with APC (2.2x105/well) and SEB in 96-well flat-bottomed plates. Anti-CTLA-4 mAb or control IgG (1 µg/ml each) or anti-IL-10 mAb (10 µg/well each) were added to the culture and cytokine in the supernatants was analyzed by ELISA.

ELISA
Cytokine levels in the supernatants were determined by a sandwich ELISA technique according to the manufacturer's instructions. Specific mAb pairs for each cytokine were obtained from PharMingen: IL-2 (JES6-1A12 and JES6-5H4), IL-4 (BVD4-1D11 and BVD4-24G2), IL-6 (MP5-20F3 and MP5-32C11), IL-10 (JES5-2A5 and SXC-1), IL-12 (C15.6 and C17.8) and IFN-{gamma} (R4-6A2 and XMG1.2). Recombinant cytokines were purchased from the following sources and used as a standard; mIL-2, mIL-12 and mIFN-{gamma} from Genzyme (Cambridge, MA); mIL-4 from Becton Dickinson; mIL-10 from PeproTech (Rocky Hill, NJ); mIL-6 from PharMingen.

Proliferation assay
CD4 T cells (2x105/well) were cultured with SEB in the presence of 30 Gy-irradiated syngeneic APC (2x105/well) for 3 days in 96-well flat-bottomed plates. At the end of culture, T cells were pulse-labeled for 16 h with 18.5 kBq of [3H]methyl-thymidine (New England Nuclear, Boston, MA). [3H]methyl-thymidine incorporation into DNA was measured by a scintillation counter.

Flow cytometry
CD4 T cells were dually stained with biotin–anti-TCR Vß8 and PE–anti-CD4 mAb at 4°C for 20 min. They were then washed and reacted with fluorescein avidin DCS. To ascertain the purity of APC, cells were stained with biotin–anti-CD11c mAb or with the combination of PE–anti-CD45R/B220 and biotin–anti-CD3{varepsilon} mAb at 4°C for 20 min. The cells were again washed and reacted with fluorescein avidin DCS. The washing solution was PBS containing 1% FCS and 0.1% sodium azide. Cells were analyzed on a FACStar.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Administration of anti-CTLA-4 mAb 24 h after antigen priming interferes with peripheral tolerance
After the injection of a high dose of SEB, Vß8+CD4+ T cells proliferate in response to antigen and then die by apoptosis, a phenomenon known as activation-induced cell death (AICD) (24). The Vß8+CD4+ T cells that survive AICD are anergic to subsequent antigen challenge (2427). In the present study, we have shown that two consecutive injections of moderate doses of SEB at 24 h intervals also induced anergy. B6 mice were inoculated with 10 µg of SEB on the first day and 1 µg of SEB 24 h later (2xSEB mice) (Fig. 1AGo). As seen in Fig. 1Go(B), the proliferation of CD4 T cells in response to SEB was significantly suppressed in 2xSEB mice. Since a negative regulatory molecule, CTLA-4, is expressed on the cell surface after antigen stimulation, we injected anti-CTLA-4 mAb into mice concurrently with the second SEB inoculation. The induction of anergy was interfered with by anti-CTLA-4 mAb but not by control IgG.



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Fig. 1. Anti-CTLA-4 mAb treatment in vivo prevents SEB-induced T cell tolerance. (A) Mice were treated as indicated. C57BL/6 mice were primed with 10 µg of SEB i.p. and on the next day injected with 1 µg of SEB together with either 75 µg of anti-CTLA-4 mAb or control IgG i.p. Six days after the second inoculation, CD4 T cells were prepared. (B) CD4 T cells from anti-CTLA-4 mAb-treated mice (n = 7), control IgG-treated mice (n = 6), SEB-treated mice (n = 2) and untreated mice (n = 4) were cultured with 30 Gy-irradiated APC in the presence of various concentrations of SEB.

 
The proportion of Vß8+CD4+ T cells in splenic T cells was 19.3 and 18.5% in CTLA-4- and control IgG-treated 2xSEB mice respectively (Table 1Go). Although repeated administration of anti-CTLA-4 mAb for 7 days prevented AICD (21), a single injection of the mAb could interfere with anergy but not with AICD.


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Table 1. Anti-CTLA-4 mAb treatment in vivo fails to prevent AICD after SEB inoculation
 
In order to determine critical hours after the first SEB inoculation, we varied the injection time of the second SEB and anti-CTLA-4 mAb from 24 to 48 or 72 h (Fig. 2A–CGo). As shown in Fig. 2Go(D), the administration of the second SEB and anti-CTLA-4 mAb 48 or 72 h after the first SEB inoculation failed to interfere with tolerance.



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Fig. 2. Twenty-four hours after the first SEB inoculation in vivo is a critical time to prevent SEB-induced T cell tolerance. C57BL/6 mice were primed with 10 µg of SEB i.p. and injected with 1 µg of SEB 24 (A), 48 (B) or 72 (C) h later together with either 75 µg of anti-CTLA-4 mAb or control IgG i.p. Six days after the second inoculation, CD4 T cells were prepared. (D) CD4 T cells from 24 (n = 7), 48 (n = 3) and 72 (n = 2) h interval mice were cultured with 30 Gy-irradiated APC in the presence of various concentrations of SEB.

 
Next, we investigated the importance of the second SEB inoculation. B6 mice were inoculated with 10 µg of SEB (1xSEB mice) and 24 h later mice received anti-CTLA-4 mAb alone (Fig. 3AGo). In contrast to 2xSEB mice in Fig. 1Go, the proliferative responses of CD4 T cells in anti-CTLA-4 mAb-injected 1xSEB mice were not significantly greater than those in control IgG-injected 1xSEB mice (Fig. 3CGo). While, CD4 T cells from mice receiving anti-CTLA-4 mAb alone proliferate significantly in response to syngeneic APC. Their responses to SEB after subtracting with those to syngeneic APC, however, were similar to those of CD4 T cells from mice receiving control IgG (Fig. 3B and CGo). Moreover, the magnitude of T cell proliferation in response to APC alone is significantly elevated in Fig. 1Go. These results indicated that the anti-CTLA-4 mAb treatment augmented the response to syngeneic APC as well.



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Fig. 3. The second SEB inoculation is essential to ablate the tolerance induction by anti-CTLA-4 mAb. C57BL/6 mice were primed with 10 µg of SEB i.p. (A) or unprimed (B) and the next day injected with either 75 µg of anti-CTLA-4 mAb or control IgG i.p. Six days after the last inoculation, CD4 T cells were prepared. (C) CD4 T cells from SEB-treated mice (n = 6) and SEB-non-treated mice (n = 6) were cultured with 30 Gy-irradiated APC in the presence of various concentrations of SEB.

 
Collectively, it is demonstrated that CD4 T cells enter a critical phase 24 h after the first inoculation; the second antigen re-stimulation without CTLA-4 engagement causes Th differentiation while that with CTLA-4 engagement induces tolerance.

Second antigen stimulation without CTLA-4 engagement promotes Th differentiation to Th2
To investigate the Th1/Th2 balance, short-term T cell lines were established from 1xSEB and 2xSEB mice. As shown in Fig. 4, GoVß8+CD4+ T cells from 2xSEB- and 2xSEB with control IgG-treated mice produced IL-2 and IFN-|{gamma} but little IL-4, suggesting that SEB-reactive T cells, although most of the cells were anergized, differentiated into Th1. In contrast, Vß8+CD4+ T cells from 2xSEB with anti-CTLA-4 mAb- treated mice differentiated into IL-4- and IL-10-producing Th2. Simultaneous injection of the second SEB and anti-CTLA-4 mAb was essential for the induction of Th2, since neither treatment alone promoted Th2 differentiation (Fig. 5Go). Thus, in order to differentiate into Th2, CD4 T cells must be re-stimulated with antigen in the absence of CTLA-4 engagement 24 h after the initial antigen priming.



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Fig. 4. Anti-CTLA-4 mAb treatment in vivo skews the immune response to Th2. Short-term T cell lines were established from mice used in the experiments in Fig. 1Go. Cell lines were stimulated with plate-coated anti-TCR Vß8 (1 µg/ml) and anti-CD28 (5 µg/ml) mAb. Cytokine productions were measured by ELISA. Data represent the mean ± SE. Statistical analysis was performed by Student's t-test (**P < 0.001, *P < 0.05).

 


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Fig. 5. Both the second SEB stimulation and anti-CTLA-4 mAb are needed to promote Th2 differentiation. Short-term T cell lines were established from mice used in experiments in Fig. 3Go. Cell lines were stimulated with plate-coated anti-TCR Vß8 (1 µg/ml) and anti-CD28 (5 µg/ml) mAb. Cytokine productions were measured by ELISA. Data represent the mean ± SE. Note that anti-CTLA-4 mAb without concurrent SEB stimulation failed to promote Th2 differentiation.

 
Antigen dose-dependent alteration of polarizing cytokine in culture
For mimicking in vivo situation after the second antigen inoculation, dendritic cells were enriched from normal spleen and co-cultured with splenic T cells from mice immunized with SEB 24 h before. SEB was titered in the culture and the kinetics of cytokine production was investigated. Along with the SEB concentration, the levels of IL-6 and IL-10 in the culture supernatant increased (Fig. 6AGo). Moreover, the addition of anti-CTLA-4 mAb augmented the levels of these cytokines ~2-fold. On the contrary, IL-12 production was suppressed along with the SEB concentration. Anti-CTLA-4 mAb further suppressed the IL-12 production. These data suggested that high doses of antigen altered the balance of Th1- and Th2-polarizing cytokines. As shown in Fig. 6Go(B), the kinetics of cytokine production is unique to each cytokine. Levels of IL-6 gradually increased during culture, while those of IL-10 steeply increased at a later time. Levels of IL-12, however, decreased with time. Addition of anti-CTLA-4 mAb exaggerated their kinetics.




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Fig. 6. Differential regulation of polarizing cytokines by antigen. B- and T-depleted APC (2.2x105/well) and splenic T cells (2.5x105/well) from mice inoculated with 10 µg of SEB 24 h before were co-cultured with the indicated concentration (A) or 3 µg/ml (B) of SEB in the presence of 1 µg/ml anti-CTLA-4 mAb or control IgG. Supernatants were harvested after 84 h (A, n = 4) or the indicated time (B, 48 and 72 h; n = 6, 84 h; n = 4). The proportion of CD11c+ cells in B- and T-depleted APC was 57.9 ± 3.3%. Cytokine production was measured by ELISA. Data are representative of four independent experiments. Values are the mean ± SE.

 
Since IL-10 is a regulatory cytokine, it might be responsible for the suppression of IL-12 at high concentrations of antigen. To verify this possibility, we added anti-IL-10 mAb at 24 h of culture. As shown in Fig. 7Go, the neutralization of IL-10 significantly enhanced the IL-12 production, suggesting that IL-10 was a negative regulator of these cytokines. It was noted that the levels of IL-12 decreased along with the SEB concentration even in the presence of anti-IL-10 mAb. Therefore, IL-12 production was regulated by an IL-10- independent mechanism(s) as well.



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Fig. 7. IL-10 down-modulates IL-12 production. B- and T-depleted APC (2.2x105/well) and splenic T cells (2.5x105/well) were co-cultured with 3 µg/ml of SEB for 72 h. Anti-IL-10 mAb were added (10 µg/well each) at 24 h of culture. Supernatants were harvested and analyzed for IL-12 production by ELISA. Data are representative of two independent experiments. Values are the mean ± SE of five or six samples.

 
These results demonstrated that the prolonged T–APC interaction in the presence of anti-CTLA-4 mAb increased a Th2-polarizing cytokine IL-6 (8) but decreased a Th1-polarizing cytokine IL-12 through IL-10-dependent and -independent mechanisms.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In this communication, we have described that CD4 T cells enter a critical activation phase 24 h after the initial antigen priming. In the phase, antigen re-stimulation without CTLA-4 engagement ablates tolerance and promotes T cell differentiation into Th2 cell. In contrast, antigen re-stimulation with CTLA-4 engagement anergizes most T cells. The reason for the Th2 differentiation is obscure, but both a Th2-polarizing cytokine IL-6 and a regulatory cytokine IL-10 are produced by APC after prolonged T–APC interaction. Moreover, IL-10 down-modulates a Th1-polarizing cytokine IL-12. Thus, once the tolerance induction is interfered with by anti-CTLA-4 mAb, Th2-polarizing cytokines may promote Th2 differentiation.

Antigen controls the Th1/Th2 balance in a dose-dependent manner. Low and moderate doses of protein antigen induce Th2 and Th1 respectively (9,10). However, high doses of antigen induce Th2 (9). In a previous study, we demonstrated that a concentration of antigen, which induced Th1 in wild-type mice, was able to induce Th2 if APC was from IRF-1-defective mice (28). Therefore, the moderate degree of TCR occupancy with antigen per se did not inhibit the differentiation of Th2. Rather, moderate dose of antigen induced the production of a Th1-polarizing cytokine IL-12 by APC in an IRF-1-dependent manner. It remained to be elucidated why high doses of antigen favored the differentiation of Th2. In the present study, we demonstrated that high doses of antigen in vitro increased IL-6 and IL-10 but decreased IL-12. Neutralization of IL-10 significantly but not completely restored the levels of IL-12 at high antigen concentrations, indicating IL-12 was regulated by both IL-10-dependent and -independent mechanisms. A negative regulatory role for IL-10 in IL-12 production was also observed in other experimental models (2931).

There is evidence that CTLA-4 plays an important role in peripheral tolerance. Disruption of the CTLA-4 gene resulted in aggressive and fatal autoimmune diseases in mice (17,18). Administration of anti-CTLA-4 mAb aggravated autoimmune disorders (32), facilitated the rejection of tumor (33) and cardiac allograft (22), prevented oral tolerance (20), and interfered with ovalbumin-induced peripheral tolerance (19). However, it is controversial at which phase of T cell activation CTLA-4 suppresses the immune response. Moreover, it is unknown why CTLA-4 skews the Th1/Th2 balance; Walunas and Bluestone reported that the continuous administration of anti-CTLA-4 mAb skewed the immune response to Th2 (21). In this study, we demonstrate that CTLA-4 engagement is critical at the time of the antigen re-stimulation 24 h after the initial antigen priming. If re-stimulation occurs without CTLA-4 engagement, CD4 T cells differentiate into Th cells. However, if re-stimulation occurs with CTLA-4 engagement, T cells are anergized. APC are heterogeneous in terms of CD80 and CD86 expression. Since CTLA-4 binds more strongly to CD80 (16), it is anticipated that CTLA-4+ pre-activated CD4 T cells will be anergized upon re-stimulation with CD80+ APC, while they will differentiate into Th2 upon re-stimulation with CD80 APC. We demonstrated in the previous study that the administration of IL-1 24 h but not 48 h after high-dose SEB inoculation interfered with anergy induction (34). Vella et al. demonstrated that the administration of lipopolysaccharide 24 but not 48 h after SEA inoculation interfered with AICD (35). These results suggest the existence of critical activation phase at 24 h after antigen priming. It remains to be elucidated in future works whether CD28 is engaged in this re-stimulation, since T cell activation in CTLA-4-deficient mice depends on B7–CD28 interaction (36,37).


    Acknowledgments
 
This work was partially supported by a Grant-in-Aid from the Ministry of Education, Science, and Culture in Japan. The authors thank Ms E. Hazawa and Ms M. Nakamura for technical assistance with ELISA, and Ms M. Nakamura for care of the animals.


    Abbreviations
 
APC antigen-presenting cell
AICD activation-induced cell death.
PE phycoerythrin
SEB Staphylococcus enterotoxin B

    Notes
 
Transmitting editor: M. Taniguchi

Received 21 October 1999, accepted 15 December 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
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