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Originally published online as doi:10.1189/jlb.0505260 on April 19, 2006

Published online before print April 19, 2006
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(Journal of Leukocyte Biology. 2006;80:66-74.)
© 2006 by Society for Leukocyte Biology

Modulation of cell cycle progression by CTLA4-CD80/CD86 interactions on CD4+ T cells depends on strength of the CD3 signal: critical role for IL-2

Sambuddho Mukherjee, Asma Ahmed, Shruti Malu and Dipankar Nandi1

Department of Biochemistry, Indian Institute of Science, Bangalore

1 Correspondence: #126, Department of Biochemistry, Indian Institute of Science, Bangalore, India 560012. E-mail: nandi{at}biochem.iisc.ernet.in


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cytotoxic T-lymphocyte antigen 4 (CTLA4) is a well-studied T cell costimulatory receptor that is known to inhibit T cell activation. In this study, the relationship between strength of the first signal and costimulatory interactions on primary mouse CD4+ T cells was investigated. CTLA4-CD80/CD86 interactions differentially modulate T cell cycling based on the mode of CD3 signal: Activation with plate-bound (pb) anti-CD3 generates a strong signal compared with a weak signal with soluble (sol) anti-CD3, resulting in approximately sevenfold higher amounts of interleukin (IL)-2 and an increase in cell cycling. Activation of T cells with sol anti-CD3 (weak signal) together with CTLA4-CD80/CD86 blockade lowers IL-2 production and cell cycling, demonstrating an enhancing role for these interactions. Conversely, blockade of CTLA4-CD80/CD86 interactions on T cells activated with pb anti-CD3 (strong signal) increases proliferation, which is consistent with CTLA4 as a negative regulator. Also, coculture of T cells with Chinese hamster ovary cells expressing CD80 or CD86 demonstrates that the strength of the primary signal plays an important role. It is important that modulation of IL-2 amounts leads to distinct alterations in the functional effects of CTLA4-CD80/CD86 interactions. On increasing IL-2 amounts, activation of T cells stimulated with sol anti-CD3 (weak signal) and CTLA4-CD80/CD86 blockade is greater compared with control. Concurrently, neutralization of IL-2 greatly reduces activation of T cells stimulated with pb anti-CD3 (strong signal) and CTLA4-CD80/CD86 blockade compared with control. These results underscore the importance of strength of first signal, CTLA4-CD80/CD86 interactions, and IL-2 amounts in modulating primary CD4+ T cell responses.

Key Words: T cell activation • costimulation • cell cycle progression


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T cell activation is a complex process that requires two distinct sets of signals: the primary signal through the T cell receptor (TCR)-CD3 complex and a costimulatory signal [1 2 3 ]. The strength of the primary TCR signal regulates several cellular responses and is modulated by multiple factors, e.g., the affinity and avidity of the major histocompatibility complex (MHC)-peptide complexes, duration of interaction, and others [4 5 6 7 ]. The CD28/cytotoxic T-lymphocyte antigen 4 (CTLA4) and CD80/CD86 family of receptors and ligands constitute the best-known family of costimulatory molecules. CD28 and CTLA4 (CD152) bind to the same ligands, CD80 and CD86; however, their functional properties are often strikingly different [8 , 9 ]. CD28-CD80/86 interactions synergize with TCR signaling to enhance T cell responses, resulting in higher amounts of interleukin (IL)-2 and survival factors. Consequently, cd28–/– T cells can initiate but cannot sustain T cell proliferation. Conversely, CTLA4 is thought to attenuate T cell responses, as blockade of CTLA4-CD80/CD86 interactions potentiates T cell activation. In addition, cross-linking CTLA4 reduces mitogen-activated protein kinase and nuclear factor of activated T cell activation, leading to low IL-2 amounts and a decrease in cell cycling. It is notable that ctla4–/– mice die within 4 weeks as a result of uncontrolled CD4+ T cell reactivity to environmental antigens (reviewed in refs. [1 2 3 ]).

Although CTLA4 plays key roles in T cell biology, the mechanisms by which CTLA4 functions are controversial, CTLA4 ligation increases transforming growth factor-ß (TGF-ß), although the functional role of this TGF-ß is not clear [10 , 11 ]. CTLA4 may enhance [12 13 14 ] or inhibit [15 , 16 ] T cell survival. To complicate matters, there are recent studies that demonstrate a role for CTLA4 in enhancing T cell responses [13 , 17 18 19 20 21 ]. A bispecific single-chain Fv reagent to CTLA4 alone increases association between CTLA4 and protein phosphatase 2A, leading to IL-2 production and T cell proliferation [19 ]. The central role of the CTLA4-CD80/CD86 pathway in costimulation has made it a favorite target for immune intervention [2 ]. However, CTLA4 blockade during autoimmune disease may result in differential outcomes, ameliorating or increasing T cell responses [17 , 18 ]. It is, therefore, important to gain insights into the mechanisms responsible for these differences and evolve a predictive model to determine when CTLA4 would act as an enhancer or an inhibitor of T cell responses.

To study the functional roles of CTLA4-CD80/CD86 interactions, we use an in vitro system consisting of highly purified mouse CD4+ T cells. In this T:T cell interaction system, CTLA4-CD80/CD86 interactions play a dominant role, as CD28 is unable to bind and signal effectively upon interacting with hypoglycosylated B7 molecules expressed on mouse T cells [22 , 23 ]. CTLA4-CD80/CD86 interactions enhance CD4+ T cell activation with the combination of phorbol 12-myristate 13-acetate (PMA), a phorbol ester, and ionomycin (I), a Ca2+ ionophore [13 ], or mitogenic doses of concanavalin A (Con A) [21 ]; however, these interactions inhibit T cell activation with suppressive doses of Con A [21 ]. In our previous studies, T cells were activated using pharmacological agents that bypass TCR triggering [13 ] or the lectin Con A, which binds to several cell surface molecules, including the TCR [21 ]. It was, therefore, important to address whether these interactions modulate T cell activation via surface TCR-CD3 signaling. Here, we demonstrate that the functional effects of CTLA4-CD80/CD86 interactions depend on the strength of the primary CD3 signal. Also, IL-2 plays a key role in mediating the strength of the primary CD3 signal, thereby setting a basis for a predictive model to determine the functional outcomes of CTLA4-CD80/CD86 interactions.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Media, antibodies, and cell lines
Cells were cultured in RPMI 1640 supplemented with 25 mM HEPES (Sigma Chemical Co., St. Louis, MO), 2 mM L-glutamine (Life Technologies, Gaithersburg, MD), 5 µM ß-mercaptoethanol (E Merck, San Diego, CA), 5% heat-inactivated fetal bovine serum (FBS; Sigma Chemical Co.), and antibiotics [13 , 21 ]. Anti-CD3 (145-2C11), anti-CD28 (37.51), anti-CTLA4 (9H10), anti-CD80 (16-10A1), anti-CD86 (GL1), and anti- trinitrophenol (TNP) hamster control antibody were from eBioScience (San Diego, CA). The generation and use of ascites containing anti-CTLA4 and murine CTLA4-human immunoglobulin G1 (mCTLA4hIgG1) were as described previously [13 ].

Preparation of mouse lymph node CD4+ T cells and activation assays
Lymph nodes from C57BL/6 mice (6–8 weeks) were dissected, and CD4+ T cells were purified using complement-mediated lysis of CD24+ and CD8+ cells followed by panning over a T25 flask coated with anti-mouse Ig [13 , 21 ]. CD4+ T cells (>95% pure) were plated at 6–7 x 104 cells/well in 96-well U-bottom plates (Costar, Corning Inc., NY) in a final volume of 100 µl/well. For most experiments, wells were precoated with RPMI 1640 containing 5% FBS to minimize nonspecific adhesion of monoclonal antibodies (mAb). Cells were activated with indicated amounts of plate-bound (pb) or soluble (sol) anti-CD3 or the combination of PMA (10 ng/ml) and I (0.1 µM; Sigma Chemical Co.). Purified anti-CD3 (50 µl 0.1–0.5 µg/ml) in phosphate-buffered saline (PBS) was bound to wells for ~6 h at 6–8°C, followed by extensive washes and blocking of wells with RPMI 5% FBS to prevent nonspecific adhesion of mAb. Together with the primary activation signal via pb or sol anti-CD3, T cells were activated with purified 5–20 µg/ml anti-TNP hamster IgG (referred to as control in all figures), anti-CD28 was used at a suboptimal concentration of 0.2 µg/ml, and anti-CTLA4 and mCTLA4hIgG ascites were used at 1:100. Unless otherwise mentioned, T cell cultures were pulsed 36 h after activation with 0.4 µCi/well [3H]Thymidine (BRIT, Mumbai, India) and harvested 12 h later. The data are presented as mean ± SD of replicates within one experiment and/or SE across multiple experiments. The Student’s t-test was performed, and the statistical significance obtained after analysis of variation is displayed in appropriate figure legends.

Cytokine assays
Supernatants from T cell assays were collected 36 h after activation, and cytokine-specific enzyme-linked immunosorbent assay (ELISA) or bioassays were performed [13 , 21 ]. ELISA was performed with standard amounts of recombinant IL-2 and various dilutions of culture supernatants. Active TGF-ß was measured as an index of growth inhibition of CCL64, a mink lung epithelial cell line (~3000 cells/well). Typically, the linear detection range of IL-2 was 30–900 pg/ml, and TGF-ß was 80–1250 pg/ml.

Flow cytometric analysis
For surface staining, ~2 x 105 cells were washed in cold Hanks’ balanced saline solution (Sigma Chemical Co.), containing 0.5% FBS, stained with pretiterd amounts of culture supernatants or direct conjugates, washed, and incubated with the appropriate fluorescein isothiocyanate-conjugated, preadsorbed secondary antibodies. Flow cytometry was performed on FACScan (Becton Dickinson, San Jose, CA) using CellQuest (Becton Dickinson) software for acquisition and WinList (Verity, Topsham, ME) software for analysis. Cell cycle analysis was performed as reported previously [13 ]. Flow cytometry experiments were depicted as a single representative of three or more independent experiments with similar fold differences in mean fluorescence intensity (MFI) values.

Chinese hamster ovary (CHO) cell transfectants and use in T cell proliferation assays
Mouse cDNAs encoding CD80 [24 ] and CD86 [25 ] were released using XbaI from their parent clones in pCDNA1 and pcDM8 and ligated to XbaI-linearized pCDNA3. Plasmid DNA from appropriate clones were purified using QIAprep (Qiagen, Valencia, CA), and lipofectamine (Life Technologies)-mediated transfections were performed. After 48 h, G418 (750 ug/ml) was added, and resistant colonies were allowed to expand for 8 days. Cells were cloned three times by the limiting dilution method and selected for stable expression of CD80 and CD86. Approximately 106 transfectants were resuspended in 1 ml sterile PBS and fixed with 0.005% glutaraldehyde for 2 min. They were then washed twice with complete RPMI medium containing 5% fetal calf serum, and ~104 fixed CHO cells were added to ~6 x 104 CD4+ T cells/well.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CTLA4-CD80/CD86 interactions on CD4+ T cells modulate T cell activation depending on the strength of the primary signal mediated by anti-CD3
CD4+ T cells were activated in vitro with pb or sol anti-CD3 in the absence or presence of antibodies to different costimulatory molecules. Anti-CD3 in the pb mode is immobilized to a solid matrix and generates a strong signal by cross-linking CD3 (Fig. 1A ). The degree of cross-linking is much lower and flexible with sol anti-CD3, which generates a weaker signal [26 , 27 ]. These differences were manifested in lower proliferation of T cells activated with sol versus pb anti-CD3 (Fig. 1B) . Suboptimal amounts of anti-CD28 increased proliferation of T cells activated with low concentrations of pb anti-CD3 and sol anti-CD3 [1 , 3 ]. The roles of CTLA4-CD80/CD86 interactions in this system were studied, using sol anti-CTLA4, which blocks the interaction between CTLA4 and CD80/CD86 [9 ], and the monovalent reagent, mCTLA4hIgG1, which binds to CD80 and CD86 and blocks their interactions with CD28 and CTLA4 [28 ]. Increased proliferation was observed in T cellsactivated with pb anti-CD3 and anti-CTLA4 or mCTLA4 compared with the control, which demonstrated that CTLA4-CD80/CD86 interactions inhibited T cell activation [9 , 13 ]. However, with low concentrations of sol anti-CD3, a small but consistent decrease in proliferation was observed on CTLA4-CD80/CD86 blockade. To widen the operational window, T cells were activated with sol anti-CD3 and suboptimal amounts of anti-CD28 together with CTLA4-CD80/CD86 blockade, which decreased T cell proliferation (Fig. 1B) . Similar results were obtained with 40 µg/ml purified anti-CTLA4 or a combination of blocking antibodies to CD80 and CD86 (data not shown). Also, complement-mediated depletion of CD25+ T cells gave identical results with both modes of signaling via anti-CD3, thereby ruling out the involvement of T regulatory cells (data not shown). The proportion of actively cycling and hypodiploid cells was determined in T cells activated under different conditions (Fig. 1C) . Activation with anti-CD3 and anti-CD28 increased the proportion of cycling cells but reduced the proportion of hypodiploid cells [1 , 3 ]. However, reduced proportion of actively dividing cells was observed in T cells activated with sol anti-CD3 + anti-CD28 and CTLA4-CD80/CD86 blockade.


Figure 1
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  		Figure 1. Modulation of CD4+ T cell activation by CTLA4-CD80/CD86 interactions depends on the mode of stimulation by anti-CD3. (A) Diagrammatic representation of pb (left) or sol (right) anti-CD3-mediated activation of T cells. (B) Primary lymph node CD4+ T cells were activated with different concentrations of pb or sol anti-CD3, together with control antibodies (5 µg/ml), anti-CD28 (aCD28; 0.2 µg/ml), anti-CTLA4 (1:100), or mCTLA4hIgG1 (mCTLA4; 1:100) for 36 h and pulsed for 12 h with [3H]Thymidine. Analysis of the fold difference in [3H]Thymidine incorporation in CD4+ T cells activated with 0.1 µg/ml pb anti-CD3 + control antibody (normalized to 1) compared with pb anti-CD3 activation with anti-CTLA4, mCTLA4, and anti-CD28 averages 2.8, 2.8, and 2.3, respectively, and P < 0.005. Similarly, the fold difference in [3H]Thymidine incorporation in CD4+ T cells activated with 0.1 µg/ml sol anti-CD3 + control antibody (normalized to 1) compared with sol anti-CD3 activated with anti-CTLA4, mCTLA4, anti-CD28, anti-CD28 + anti-CTLA4, and anti-CD28 + mCTLA4 averages 0.55, 0.58, 22, 14, and 14, respectively, and P < 0.001. These data are representative of more than three independent experiments. cpm, Counts per minute. (C) The mean fold difference ± SE of the S/G2M and hypodiploid populations of CD4+ T cells activated with pb or sol anti-CD3 (0.1 µg/ml) for 48 h together with the indicated antibodies from three independent experiments, normalized to control antibody-treated cells, is also depicted. *, P < 0.05; **, P < 0.01.

 
Next, the amounts of IL-2 and active TGF-ß secreted by T cells under different activation conditions were determined (Table 1 ). Active TGF-ß produced by T cells activated with anti-CD3 was much lower compared with PMA + I [13 ]. T cells activated with pb anti-CD3 and control antibody, consistent with its greater signal strength, produced over sevenfold more IL-2 compared with cells activated with sol anti-CD3. The amount of IL-2 produced by T cells activated with pb anti-CD3 and CTLA4-CD80/CD86 blockade was comparable with control. However, in keeping with the proliferation data (Fig. 1B) , ~55% decreased IL-2 production was observed in cells activated with sol anti-CD3 + anti-CD28 and CTLA4-CD80/CD86 blockade. Together, CTLA4-CD80/CD86 interactions on primary CD4+ T cells activated with pb or sol anti-CD3 modulate IL-2 production and proliferation.


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  		Table 1. Differences in Production of IL-2 and TGF-ß on Blockade of CTLA4-CD80/CD86 Interactions by CD4+ T Cells Stimulated with pb or sol Anti-CD3 with PMA + I for Comparison

 
Expression and functional roles of costimulatory molecules on CD4+ T cells activated with anti-CD3
The expression of cell surface molecules on CD4+ T cells before and after activation with pb or sol anti-CD3 was studied (Fig. 2 ). CD28 was constitutively expressed and increased upon T cell activation. CTLA4 was not detected on unstimulated cells but was greatly induced after 42 h of activation. CD80, which was present at low levels on unactivated T cells, and CD86 were enhanced upon activation. Thus, costimulatory receptors and ligands were present on CD4+ T cells after activation with anti-CD3. Next, T cells were activated with pb or sol anti-CD3, and reagents that block CTLA4-CD80/CD86 interactions were added at different time-points. Blockade of CTLA4-CD80/CD86 interactions increased proliferation of T cells activated with pb anti-CD3 at 0 h (Fig. 2B) ; however, delay in addition of these reagents reduced the fold increase. Similarly, anti-CTLA4 and mCTLA4 reduced proliferation of T cells activated with sol anti-CD3 + anti-CD28. However, addition of these blocking reagents 12 h after activation with sol anti-CD3 greatly reduced this effect. Although maximal CTLA4 cell surface expression was detected 42 h after activation with pb and sol anti-CD3 (Fig. 2A) , CTLA4-CD80/CD86 interactions were functionally required early (before 12 h) during T cell activation by anti-CD3.


Figure 2
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  		Figure 2. Expression and functional roles of CTLA4, CD80, and CD86 on primary CD4+ T cells (A), which were activated with pb or sol aCD3 (0.1 µg/ml) for 12 or 42 h and stained with specific mAb to different cell surface markers. The innermost gray lines indicate control antibodies; solid gray lines indicate unactivated cells (0 h); thin black lines indicate 12 h-activated cells; and solid black lines indicate cells activated for 42 h. The numbers (top to bottom) indicate fluorescence intensities at 0, 12, and 42 h, respectively. This figure is representative of four independent experiments, and similar fold differences in MFI values were observed. (B) T cells were activated with pb or sol anti-CD3 (0.1 µg/ml), and control antibodies (Ab; 5 µg/ml), anti-CD28 (0.2 µg/ml), anti-CTLA4 (1:100), or mCTLA4hIgG1 (1:100) were added at different time-points. After 36 h of culture, [3H]Thymidine was added, and cells were harvested 12 h later.

 
IL-2 amounts modulate T cell proliferation in CD4+ T cells activated with anti-CD3 and blockade of CTLA4-CD80/CD86 interactions
The direct role of IL-2 was studied as major differences were observed in IL-2 amounts secreted by T cells activated with sol versus pb anti-CD3 (Table 1) . T cells were activated under indicated conditions with sol anti-CD3 together with different cytokines (Fig. 3A ). All the conditions were compared with the index population activated with sol anti-CD3 and control antibody. There was no difference in the activation profile with no exogenous cytokine or IFN-{gamma}. However, the inhibition of proliferation (~45%) observed with CTLA4-CD80/CD86 blockade with sol anti-CD3 or sol anti-CD3 + anti-CD28 was abrogated completely by 10 U/ml IL-2. Indeed, at higher concentrations of IL-2 (100 U/ml), a consistent increase in proliferation (~25%) was observed over sol anti-CD3 + control antibody. Also, an increased proportion of cycling cells was observed in T cells activated with sol anti-CD3 + anti-CD28 and CTLA4-CD80/CD86 blockade compared with sol anti-CD3 + anti-CD28 in the presence of 50 U/ml IL-2 (data not shown). IL-4 addition also ameliorated the inhibition of proliferation; however, the switch in activation profile was not observed even at 100 U/ml IL-4, suggesting that IL-2 was the major player in this system.


Figure 3
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  		Figure 3. IL-2 amounts modulate the effects of CTLA4-CD80/CD86 interactions in CD4+ T cells activated with pb or sol anti-CD3. (A) Fold differences in [3H]Thymidine count after activation of T cells with sol anti-CD3, and indicated conditions are shown. The values from control antibody-treated cells, without exogenous IL-2, were normalized to unity, and fold differences with ± SE from four independent experiments are depicted. The inset magnifies the first three conditions in cells with no exogenous cytokines. These data are representative of three independent experiments. *, P < 0.05; IFNg, interferon-{gamma} (IFN-{gamma}). (B) T cells were activated with pb anti-CD3 under the indicated conditions. [3H]Thymidine counts from control antibody-treated cells were normalized to unity, and fold differences with ± SE from three independent experiments are depicted. *, P < 0.05.

 
Next, CD4+ T cells were activated with pb anti-CD3 under various conditions in the absence or presence of different amounts of neutralizing antibodies to IL-2, and all counts were normalized to cells treated with control antibody alone (Fig. 3B) . The addition of anti-IL-2 to the control antibody-treated cells resulted in a dose-dependent decrease in proliferation. In the presence of anti-CD28, the observed decline was slight and observed only at the highest concentration of anti-IL-2. However, on CTLA4-CD80/CD86 blockade, a steep, dose-dependent decline in proliferation was observed, ~3.5-fold lower (based on slope values) compared with control antibodies. Neutralization of IL-2 reduced cell cycling in T cells activated with pb anti-CD3 and CTLA4-CD80/CD86 blockade (data not shown). Thus, the mode of anti-CD3 stimulation, CTLA4-CD80/CD86 interactions, and IL-2 amounts modulates T cell activation.

Coculture studies with CHO cells expressing CD80 or CD86 and CD4+ T cells activated with anti-CD3
To address the role of increased levels of costimulatory ligands in this system, CHO cells expressing high levels of CD80 and CD86 were obtained (Fig. 4A ). In the presence of pb anti-CD3 activation, together with control antibodies or anti-CD28, T cell proliferation was not modulated with coculture with different CHO transfectants (Fig. 4B) . The inclusion of anti-CTLA4 or mCTLA4 in this condition, together with the CHO vector, enhanced T cell proliferation, as observed previously (Figs. 1 2 and 4) . No significant effect of CHO-CD80 or CHO-CD86 was observed in T cells activated with pb anti-CD3 and anti-CTLA4. However, inclusion of mCTLA4 reduced proliferation of T cells activated with pb anti-CD3 and CHO-CD80 or CHO-CD86 cells, compared with CHO vector. As a result of the presence of excess CD80/CD86, mCTLA4 was probably limiting, and inhibition of T cell proliferation was observed as a result of binding of excess CD80/CD86 to CTLA4. A marginal increase in proliferation was observed upon coculture of T cells activated with sol anti-CD3 and CHO-CD80 or CHO-CD86 compared with CHO vector; this difference was manifested greater in T cells activated with sol anti-CD3 + anti-CD28. The inclusion of anti-CTLA4 or mCTLA4 reduced proliferation of T cell activated with sol anti-CD3 in the absence or presence of anti-CD28, demonstrating the enhancing nature of CD80/CD86-CTLA4 interactions in cells activated with sol anti-CD3.


Figure 4
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  		Figure 4. Effect of CHO cells expressing CD80 or CD86 on CD4+ T cell proliferation after activation with pb or sol anti-CD3. (A) Stable clones of CHO cells transfected with pcDNA3, pcDNA3-CD80, or pcDNA3-CD86 were stained with specific antibodies to CD80 and CD86. (B) CD4+ T cells were activated with pb (0.5 µg/ml) or sol (0.1 µg/ml) anti-CD3 in the presence or absence of anti-CD28 (0.2 µg/ml), anti-CTLA4 (1:100), or mCTLA4 (1:100). Control (vector), CD80, or CD86 expressing CHO cells were fixed with 0.005% glutaraldehyde and added at 0 h. After 36 h of activation, [3H]Thymidine was added, and cells were harvested after 12 h. No significant [3H]Thymidine incorporation was observed by unactivated CD4+ T cells or fixed CHO transfectants. Significant differences with *, P < 0.05, are highlighted.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Initially, different modes of anti-CD3 stimulation were used to deliver a weak or strong first signal to activate primary mouse lymph node CD4+ T cells. Activation with sol anti-CD3, compared with pb anti-CD3, generated a weaker signal using different criteria: slower reduction in cell surface CD62L expression (data not shown), reduced IL-2 production (Table 1) , [3H]Thymidine incorporation, and proportion of cycling cells (Fig. 1) . Subsequently, the relationship between the strength of the primary signal via TCR-CD3 and CTLA4-CD80/CD86 interactions on primary CD4+ T cells was investigated. Cell surface expression of CTLA4, CD80, and CD86 on T cells was low and induced after activation with anti-CD3 (Fig. 2A) . It was, therefore, important to determine whether higher levels of costimulatory ligands, together with different modes of the primary anti-CD3 signal, could modulate T cell function in this system. Also, weak agonists or low amounts of agonist may generate a strong T cell response in the presence of high amounts of B7 ligands as a result of B7-CD28 interactions [29 ]. Alternately, high amounts of CD80/CD86 in the presence of a strong, primary signal may enhance CTLA4 expression, leading to down-regulation of T cell function [8 , 21 ]. This study demonstrates that the strength of the primary signal and CTLA4-CD80/CD86 interactions modulates T cell activation.

Consistent with previous reports [8 , 9 , 13 ], our data demonstrate that CD80/CD86-CTLA4 interactions are inhibitory for T cell activation with pb anti-CD3 (strong signal). However, we demonstrate that upon activation with sol anti-CD3, CTLA4-CD80/CD86 interactions enhance T cell activation (Figs. 1 2 3) . IL-2 is critical, as increasing IL-2 levels during activation of T cells with sol anti-CD3 and CTLA4-CD80/CD86 blockade enhanced proliferation (Fig. 3A) . Conversely, neutralization of IL-2 in T cells activated with pb anti-CD3 and CTLA4-CD80/CD86 blockade reduced proliferation (Fig. 3B) . The roles of CTLA4-CD80/CD86 interactions on activation with anti-CD3 are summarized in Figure 5 . Binding of CTLA4 to CD80/CD86 on CD4+ T cells activated with pb anti-CD3 (strong signal) inhibited T cell activation. Conversely, in conjunction with weak TCR signals, e.g., activation with sol anti-CD3, CTLA4-CD80/CD86 interactions stimulate IL-2 and T cell proliferation. These studies highlight the relationship between signal strength by anti-CD3 and CTLA4-CD80/CD86 interactions and IL-2 in modulating T cell activation.


Figure 5
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  		Figure 5. The functional effects of CTLA4-CD80/CD86 interactions in a CD4+ T cell:T cell activation model depend on the strength of primary signal by CD3. These interactions are stimulatory with a weak, primary signal but are inhibitory with a strong, primary signal. IL-2 plays a key role as a mediator of the strength of primary signal in this system.

 
What are the possible immunological consequences of this study? To explain the roles of CTLA4, two mutually nonexclusive models have been proposed: attenuator and threshold [2 ]. In the first case, CTLA4 attenuates the T cell response and prevents the generation of T cells that express high-affinity TCRs exclusively. Conversely, CTLA4 may set a threshold, and T cell activation above a threshold is required for initiating immune responses. Modifications of both models may explain the data presented in this study. Under conditions that result in suboptimal activation of T cells, e.g., during the generation of autoreactive T cells, as a result of the binding of TCRs to low-affinity/cross-reactive peptide-MHC complexes, CTLA4-CD80/CD86 interactions may enhance T cell activation. It is, therefore, not surprising that the two reports of CTLA4-enhancing in vivo T cell responses are in autoimmune disease models [17 , 18 ]. It is also possible that by enhancing the generation of low-affinity TCR clones, CTLA4 may broaden T cell responses to cross-reactive antigens, which may play important roles as pathogens mutate to overcome the host immune response [2 , 8 ]. In summary, this study demonstrates that CTLA4 integrates the primary TCR-CD3 signal and IL-2 amounts to modulate CD4+ T cell responses.


   ACKNOWLEDGEMENTS
 
This study was supported by a grant from the Department of Biotechnology, Government of India. S. M. was awarded a research fellowship from Council of Scientific and Industrial Research. We thank Dr. S. Rath for encouraging this study and Dr. P. Kondaiah for the TGF-ß bioassay. The assistance of O. Joy and H. Krishnan, DBT-FACS facility, IISc, is highly appreciated.

Received May 12, 2005; revised February 24, 2006; accepted March 10, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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