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(Journal of Leukocyte Biology. 2002;72:921-931.)
© 2002 by Society for Leukocyte Biology

Role of CD80, CD86, and CTLA4 on mouse CD4+ T lymphocytes in enhancing cell-cycle progression and survival after activation with PMA and ionomycin

Sambuddho Mukherjee, Prasanta K. Maiti and Dipankar Nandi

Department of Biochemistry, Indian Institute of Science, Bangalore, India

Correspondence: Dipankar Nandi, Ph.D., Assistant Professor, Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India. E-mail: nandi{at}biochem.iisc.ernet.in


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell surface interactions between the T cell costimulatory receptors, CD28 and cytotoxic T-lymphocyte antigen-4 (CTLA4), with their cognate ligands, CD80 and CD86, on antigen-presenting cells play an important role in T cell activation. Although CD80 and CD86 are induced on T cells after activation, not much is known about their role in modulating T cell function. We show that CD80, CD86, and CTLA4 are induced on purified CD4+ T cells after in vitro activation with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and they play an essential role for proliferation and survival. Blockade of CTLA4-CD80/CD86 interactions greatly reduces PMA and ionomycin-mediated mouse CD4+ T cell activation. The three key features of this inhibition of activation are: First, late events in T cell activation (after 18 h) are affected; second, these cells do not undergo anergy; and third, CD4+CD25+ regulatory T cells are not responsible. Activation of T cells with PMA and ionomycin together with CTLA4-CD80/CD86 blockade results in decreased induction of CD25 and Bcl-XL, reduced interleukin (IL)-2, and enhanced transforming growth factor-ß (TGF-ß) production. Furthermore, extended CTLA4-CD80/CD86 blockade results in decreased cell-cycle progression and enhanced apoptosis in a large proportion of cells. This inhibition of T cell proliferation can be rescued completely with anti-CD28 or IL-2 and partially with TGF-ß antagonists. This study reveals a functional role for CD80, CD86, and CTLA4 on CD4+ T lymphocytes and sheds light on the mechanisms by which these molecules enhance activation and survival with PMA and ionomycin.

Key Words: costimulation • apoptosis • transforming growth factor-ß • antigen-presenting cells


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
T cell activation is a complex process requiring two signals for optimal proliferation. The first signal via the T cell receptor (TCR) is an antigen-specific signal, whereas the second involves interactions between costimulatory receptors and their ligands. CD28, cytotoxic T-lymphocyte antigen 4 (CTLA4), CD80, and CD86 constitute the best-known family of costimulatory molecules. T cell costimulatory receptors, CD28 and CTLA4, are homologous to each other and bind their ligands, CD80 and CD86, with different affinities and kinetics (reviewed in refs. [1 2 3 ]). The interaction of CD28 with CD80 and CD86, together with TCR signaling, results in interleukin (IL)-2 production, T cell proliferation, and rescue from anergy [1 2 3 4 5 6 ]. Conversely, CTLA4 triggering inhibits TCR-mediated IL-2 production and proliferation [5 6 7 8 ]. Blockade of CTLA4 interaction with CD80 and CD86 enhances T cell proliferative response and differentiation of T helper cell type 2 (Th2) effectors [9 10 11 ]. Moreover, ctla4-/- mice die within 3–4 weeks [12 13 14 ], and hyperproliferation of CD4+ T cells in these mice is dependent on TCR [15 , 16 ] and CD28-CD80/CD86 interaction [17 , 18 ]. These evidences clearly demonstrate that interaction between costimulatory receptors (CD28 and CTLA4) with their ligands (CD80 and CD86) plays an important role in regulating mouse T cell homeostasis and activation.

The mechanisms by which CTLA4 inhibits T cell activation are controversial [1 2 3 , 19 ]. CTLA4 is expressed on the cell surface after T cell activation and binds CD80/CD86 with higher affinity, which results in sequestration of B7 from binding to CD28. In addition, CTLA4-triggering reduces "raft" formation and inhibits T cell activation and cell-cycle progression [20 21 22 23 24 25 ]. However, it is unclear whether CTLA4 mediates its actions directly or indirectly via transforming growth factor-ß (TGF-ß) [7 , 26 , 27 ]. TGF-ß is known to inhibit T (Treg) cell activation by decreasing IL-2 receptor expression and IL-2 production and by inducing apoptosis [28 , 29 ]. In fact, a subset of CD4+ T cells known as regulatory CD25+CD4+ T cells produces TGF-ß in a CTLA4-dependent manner and suppresses autoreactive T cells [30 , 31 ]. CTLA4 interaction with CD80 and CD86 may be important in maintenance of peripheral tolerance by induction of anergy [32 33 34 ]; however, other reports have demonstrated the induction of anergy in the absence of CTLA4-CD80/CD86 interaction [35 , 36 ]. CTLA4 triggering of naïve T cells results in inhibition of T cell proliferation without the induction of apoptosis [6 , 37 , 38 ], whereas activated T cells undergo Fas-independent apoptosis on CTLA4 cross-linking [38 , 39 ]. It is interesting that T cells from ctla4-/- mice are resistant to {gamma}-irradiation-induced apoptosis [40 ], suggesting a role for CTLA4 in enhancing apoptosis under some conditions.

The combination of phorbol 12-myristate 13-acetate (PMA; P) + ionomycin (I) is routinely used as a TCR-independent model to study T cell activation and proliferation [41 ]. In addition, the combination of P + I has been shown to positively select CD4+ T cells in thymocyte cultures [42 ], modulate differentiation of Th and T cytotoxic (Tc) subsets [43 ], and study molecules involved in T cell death [44 ]. However, the role of cell surface interactions in enhancing proliferation of T cells with P + I is not known. We purified mouse CD4+ T cells and studied the effect of soluble antibodies (Ab) to CD80, CD86, CD28, CTLA4 [5 ], and the fusion protein mCTLA4h immunoglobulin G (IgG)1 [45 ] on T cell proliferation after activation with P + I in vitro. We demonstrate that CTLA4 interaction with CD80 and CD86 on freshly isolated mouse CD4+ T cells is required for optimal proliferation of T cell activated with P + I. In the absence of this interaction, T cell activation is inhibited, followed by decreased cell-cycle progression and enhanced apoptosis in a large percentage of cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
C57BL/6 mice, aged 6–10 weeks, were obtained from the Central Animal Facility, Indian Institute of Science (IISc; Bangalore), and were housed in our departmental facility, according to institutional guidelines.

Media, Ab, 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 (Sigma Chemical Co.), 100 µg/ml penicillin, 250 µg/ml streptomycin, 50 µg/ml gentamycin (HiMedia Labs, Mumbai, India), and 5% heat-inactivated fetal bovine serum (FBS; Sigma Chemical Co.). Anti-CD3 (145-2C11), anti-CD28 (37.51), anti-CTLA4 (9H10), anti-CD80 (16-10A1), anti-CD86 (GL1), and anti-trinitrophenol (TNP; control Ab) were from Pharmingen (San Diego, CA). Ascites containing anti-CD28, anti-CTLA4, and mCTLA4hIgG1 were generated by priming 6- to 8-week-old BALB/c mice with pristane (Sigma Chemical Co.), followed by immunosuppression with hydrocortisone (3 mg/mouse; HiMedia Labs) and low-dose (500 rad) irradiation. After this treatment, ~2 x 106 hybridomas/mouse were injected intraperitoneally every 2–3 days. For flow cytometry, fluorescein isothiocyanate (FITC) conjugates of anti-CD3, anti-CD80, anti-CD86, and anti-CD69 and phycoerythrin (PE) conjugates of anti-CD28 and anti-CTLA4 were obtained from eBioscience (San Diego, CA). Anti-CD4-FITC and control-FITC/PE Ab were obtained from Pharmingen. Anti-CD25 (PC61-5.3) and the irrelevant rat (11B11) isotype controls were used as culture supernatant for flow cytometry experiments. Secondary Ab were from Jackson ImmunoResearch Laboratories (West Grove, PA). Antibodies to major histocompatibility complex (MHC) class II (BP107), CD8 (3.155), and heat-stable antigen (J11D) culture supernatants were used to purify lymph node CD4+ T cells. Antibodies to TGF-ß1, -2, and -3 [46 ] and CCL-64, a TGF-ß sensitive cell line [47 ], were kindly provided by P. Kondaiah, IISC, Bangalore, and CTEV-2 cells, a derivative of the IL-2 responsive cell line CTLL-2, were provided by A. Sarin, NCBS, Bangalore.

Preparation of lymph node T cells
CD4+ T cells were purified by complement-mediated lysis of MHC class II+, heat stable antigen, and CD8+ cells. Live cells were collected by Histopaque 1083 (Sigma Chemical Co.) gradient centrifugation and were subjected to panning over a T25 flask coated with 100 µg/ml anti-mouse Ig. CD4+ T cell preparations were typically ~98% pure, as measured by flow cytometry. Purification of CD4+CD25- T cell populations was carried out by initially purifying CD4+ T cells, followed by complement-mediated depletion of CD25+ cells using 7D4 culture supernatant. Flow cytometric analysis using anti-CD25 (PC61) failed to detect CD25 on the cell surface after this treatment.

T cell activation assays
Purified T cells were plated at ~6 x 104 cells/well in 96-well U-bottom plates (Costar, Corning Inc., Corning, NY) in a final volume of 100 µl/well. For most experiments, all wells were precoated with RPMI 1640 containing 5% FBS to minimize nonspecific adhesion of monoclonal Ab (mAb) to the plate. Cells were activated with 10 ng/ml PMA (Sigma Chemical Co.) and 0.1 µM I (Sigma Chemical Co.). Cross-linking was performed using purified anti-CD3 (50 µl 0.1 µg/ml), anti-TNP (50 µl 1 µg/ml), or anti-CTLA4 (50 µl 1 µg/ml) in phosphate-buffered saline (PBS) for ~8 h at 6–8°C followed by extensive washes and blocking of wells with RPMI + 5% FBS to prevent nonspecific adhesion of mAb added subsequently [5 ]. Anti-CD28, anti-CTLA4, and mCTLA4hIgG ascites were used at 1:100 throughout, whereas the control Ab (anti-TNP) was used at 20 µg/ml. Other purified Ab were used at the indicated concentration. Fetuin (Sigma Chemical Co.) or the control protein, ovalbumin (ova; Sigma Chemical Co.), was titered and used at 0.5 mg/ml, and recombinant (r)IL-2 [National Institute for Biological Standards and Control (NIBSC), UK] was used at 50 U/ml. Unless otherwise mentioned, T cell cultures were pulsed 36 h after activation with 0.25 or 0.5 µCi/well [3H]-thymidine (TdT; NEN, Perkin-Elmer, Boston, MA, or BRIT, Mumbai, India) and were harvested 12 h later. Incorporated radioactivity was measured using a liquid scintillation counter (Wallac, Perkin-Elmer, Foster City, CA) to assess levels of proliferation. The data are presented as mean ± SD of triplicates in one representative of at least three individual experiments.

Cytokine assays
Supernatants from T cell assays were collected 36 h after activation, and cytokine-specific bioassays were performed for IL-2 and TGF-ß using the cell lines CTEV-2 and CCL-64, respectively. The amount of cytokine in the supernatants was determined using an equation derived from values obtained from known amounts of standard cytokines. Specific T cell secretion of cytokines was determined by deducting appropriate controls, e.g., supernatants containing Ab, P + I, etc., only. CTEV-2 were grown in 25 U/ml IL-2, starved of IL-2 for 10 h, and cultured at 3000 cells/well with standard amounts of rIL-2 (NIBSC) or various dilutions of culture supernatants. Typically, the linear detection range of the IL-2 bioassay was 3.1–50 U/ml. TGF-ß was measured as an index of growth inhibition of CCL-64 cells [47 ], which were cultured (~3000 cells/well) with supernatants or with known amounts of rTGF-ß1 (R&D Systems, Mineapolis, MN). The linear detection range of the TGF-ß bioassay was 7.5–500 pg/ml. Active TGF-ß present in the supernatants was directly measured by this assay. To measure total levels of TGF-ß, heat activation of supernatants at 80°C for 15 min was performed to convert all latent TGF-ß into its active form.

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 pretitred amounts of culture supernatants or direct conjugates, washed, and incubated with the appropriate FITC-conjugated, preadsorbed secondary Ab. Flow cytometry was performed on FACScan or FACSVantage (Becton Dickinson, San Jose, CA) using CellQuest (Becton Dickinson) software for acquisition and WinList (Verity, Topsham, ME) software for analysis. To detect changes in mitochondrial potential, cells were stained with 40 nM DiOC6 (Molecular Probes, Eugene, OR) in PBS for 20 min, washed in excess PBS, and acquired immediately [48 ]. Cell-cycle analysis was performed as previously reported [49 ]. Briefly, ~2 x 105 cells were centrifuged, and the pellet was resuspended in ice-cold permeabilization buffer consisting of 0.1% sodium citrate and 0.1% Triton X-100 followed by the addition of propidium iodide (PI; 1 mg/ml; Sigma Chemical Co.). The tubes were incubated for 18–24 h at 6–8°C before performing flow cytometry.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Expression of cell surface molecules on activation of CD4+ T cells with P + I
Pure CD4+ T cells were isolated from lymph nodes of C57BL/6 mice, activated with P + I, and cell surface expression of different molecules was studied by flow cytometry. As observed in Figure 1 , the levels of CD3 or CD4 were high on freshly isolated cells, and these levels were not modulated after 12 or 42 h of activation with P + I. Conversely, the levels of the activation markers CD25 and CD69 were induced. The surface levels of CD28, CD80, and CD86 were also increased with activation with no significant difference in their expression profile after 12 or 42 h of activation. However, cell surface expression of CTLA4 was detected at 12 h and increased after 42 h of activation with P + I. Thus, the induction of cell surface CTLA4 was slower than CD28, CD80, and CD86. Previous reports have demonstrated increased expression of cell surface CD28, CTLA4, CD80, and CD86 after activation with anti-CD3 [5 , 50 ]. We have shown that CD4+ mouse T cells expressed CD28 and CTLA4, as well as their ligands, CD80 and CD86, on activation with P + I.



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  		Figure 1. Flow cytometric analysis of cell surface markers on unactivated and activated CD4+ mouse T cells. Freshly isolated, unactivated (U) mouse lymph node CD4+ T cells or P + I-activated for 12 h (A-12h) or 42 h (A-42h) were stained with specific Ab and acquired on a FACSVantage cytometer. The light gray dotted lines indicate control Ab; solid light gray lines indicate unactivated cells; black stippled lines indicate 12-h activated cells; and solid black lines indicate cells activated for 42 h.

 
Anti-CTLA4 or mCTLA4hIgG1 enhances or inhibits CD4+ T cell activation depending on activation conditions
The effect of soluble Ab to costimulatory molecules on T cell proliferation under different conditions of activation was studied (Fig. 2A ). We used anti-CD28, which activates T cells in the presence of a first signal [5 ], and the fusion protein mCTLA4hIgG1, which binds CD80 and CD86 and blocks their interactions with CD28 and CTLA4 [45 ]. Anti-CTLA4 can inhibit or enhance T cell proliferation depending on the experimental conditions [5 , 51 ]. We used soluble anti-CTLA4, which enhances T cell proliferation by blocking the negative signals as a result of CTLA4-CD80/CD86 interaction [5 , 9 10 11 , 51 ]; however, anti-CTLA4 on cross-linking and in combination with the first signal triggers a negative signal that inhibits T cell proliferation [5 6 7 8 ]. Activation of CD4+ T cells with plate-bound anti-CD3 resulted in increased proliferation, which was further enhanced by ~1.7-fold with anti-CTLA4 and mCTLA4hIgG1 and ~sixfold with anti-CD28. Similarly, anti-CD28 + P greatly enhanced proliferation of CD4+ T cells. Anti-CTLA4 or mCTLA4hIgG1 in combination with P did not significantly enhance T cell proliferation but synergized with P + anti-CD28. The combination of P + I resulted in proliferation, which was enhanced by the addition of anti-CD28 (Fig. 2A) . Surprisingly, addition of anti-CTLA4 or mCTLA4hIgG1 in these cultures greatly reduced proliferation by ~sevenfold. The effects of anti-CTLA4 and mCTLA4hIgG1 with plate-bound anti-CD3 and P + anti-CD28 were consistent with the modest increase in proliferation above the first signal alone, as reported earlier [5 ]. However, the inhibition of P + I-mediated T cell proliferation by anti-CTLA4 or mCTLA4hIgG1 was intriguing, and we decided to study this observation in depth. Kinetic studies (Fig. 2B) revealed that there was no great difference between cultures treated with P + I alone or P + I along with anti-CD28, anti-CTLA4, or mCTLA4hIgG1 after 24 h activation with P + I. Proliferation of T cells with P + I and control Ab increased up to 36 h after activation. Increased proliferation was observed in cultures treated with anti-CD28 until 72 h, in keeping with the known role of anti-CD28 to increase and sustain proliferation [4 5 6 ]. However, there was no increase in T cell proliferation after 24 h of activation in cultures treated with anti-CTLA4 or mCTLA4hIgG1 compared with cultures treated with control Ab. These results suggest that initially, T cells were activated with P + I and anti-CTLA4 or mCTLA4hIgG1 to similar extents compared with control, but the subsequent increase in proliferation was not observed. As CTLA4, CD80, and CD86 are induced on T cell activation with P + I (Fig. 1) , we wished to determine the effect of adding Ab at different time points after activation. Addition of control Ab or anti-CD28 at 0 h or 36 h after activation did not affect overall proliferation (Fig. 2C) . However, maximal inhibition of proliferation with anti-CTLA4 or mCTLA4hIgG1 was observed between 0 and 12 h after activation with P + I, although cell surface CTLA4 levels were much higher at 42 h than 12 h. These results demonstrate that CTLA4 interaction with CD80 and CD86 was required during the initial 12-h period for maximal activation with P + I.



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  		Figure 2. Anti-CTLA4 and mCTLA4hIgG1 inhibits P + I-mediated T cell proliferation. (A) Freshly isolated CD4+ T cells were cultured with mCTLA4hIgG1 and soluble Ab to CD28 and CTLA4 (ascites 1:100) in wells coated with plate-bound ({alpha}CD3pc) anti-CD3 (0.1 µg/ml) or in the presence of P (PMA; 10 ng/ml) or P (10 ng/ml) + I (0.1 µM). Cells were pulsed with TdT at 36 h and harvested after 12 h. The all-minus group is cells alone, whereas the control group was treated with anti-TNP (20 µg/ml). (B) CD4+ T cells were cultured under different conditions and pulsed with TdT during the last 12 h of the indicated time points and harvested. (C) CD4+ T cells were cultured in the presence of P + I, and the indicated Ab were added at different time points. Cells were pulsed with TdT at 36 and harvested after 12 h.

 
Blockade of CTLA4 interaction with CD80 and CD86 greatly reduces T cell activation with P + I
Next, we wished to understand the mechanism by which anti-CTLA4 was inhibiting the proliferation of T cells activated with P + I, i.e., triggering a negative signal or blocking CTLA4-CD80/CD86 interaction. Cross-linking purified anti-CTLA4 inhibited the proliferation of CD4+ T cells activated with soluble anti-CD3 and anti-CD28 (Fig. 3A ) as previously reported [5 ]. However, cross-linked anti-CTLA4 did not inhibit the proliferation of T cells activated with P + I, suggesting that the observed inhibition of proliferation with P + I and soluble anti-CTLA4 was unlikely to be a result of the generation of a negative signal by anti-CTLA4 cross-linking. To assess whether the inhibition of proliferation was a result of blockade of CD80 and CD86 interaction with CTLA4, soluble Ab to CD80 and CD86 were used, singly or in combination. Dose-dependent studies (Fig. 3B) indicated that anti-CD80 or anti-CD86 inhibited T cell proliferation with P + I after 48 h of activation, and maximal inhibition of T cell responses was observed with the use of both Ab. These experiments clearly demonstrate that blocking CTLA4 interaction with CD80 and CD86 inhibited P + I-mediated T cell proliferation. CD25 is a well-known marker for T cell activation (Fig. 1) . Activation of CD4+ T cells with P + I (Fig. 3C) for 18 h resulted in expression of CD25 on ~50% of cells [mean fluorescence intensity (MFI), ~40], and by 42 h of activation, ~70% of cells expressed high levels of CD25 (MFI, ~40). Anti-CD28 enhanced CD25 expression on ~90% of CD4+ T cells activated with P + I for 42 h (MFI, ~150), as previously reported [5 ]. It is interesting that activation of CD4+ T cells with P + I in the presence of anti-CTLA4 (~44% positive; MFI, ~25) or mCTLA4hIgG1 (~47% positive; MFI, ~20) resulted in transient induction (18 h) of CD25. However, the number of cells expressing CD25 decreased after 42 h of activation with anti-CTLA4 (~20% positive; MFI, 25) and mCTLA4hIgG1 (~28% positive; MFI, ~25). These data together with Figure 2B demonstrate that blockade of CTLA4-CD80/CD86 interactions after activation of CD4+ T cells with P + I results in inhibition of T cell activation and proliferation at a late stage.



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  		Figure 3. Blockade of CTLA4-CD80/CD86 interaction inhibits P + I activation of CD4+ mouse T cells. (A) Freshly isolated CD4+ T cells were activated in wells coated (pc) with purified anti-TNP (1 µg/ml) or anti-CTLA4 (1 µg/ml) together with soluble anti-CD3 (5 µg/ml) + anti-CD28 ascites (1:1000) or P + I. Cells were pulsed with TdT at 36 h and harvested after 12 h. (B) Freshly isolated CD4+ T cells were cultured with P + I and soluble Ab [pure (pur) or ascites (asc)], as indicated. Purified Ab to TNP and CTLA4 were used at 20 µg/ml, and anti-CD28 was used at 5 µg/ml. All ascites were used at a 1:100 dilution. Cells were pulsed with TdT at 36 h and harvested after 12 h. (C) CD4+ T cells were cultured with P + I and the indicated Ab for 18 h and 42 h and were stained for CD25.

 
Treg cells are not responsible for the inhibition of proliferation of CD4+ T cells activated with P + I together with CTLA4-CD80/CD86 blockade
We wished to study whether CD4+CD25+ regulatory T cells [30 , 31 ] were responsible for the above effect. Undepleted or CD25+-depleted CD4+ T cells were activated with P + I together with soluble Ab to costimulatory molecules (Fig. 4 ). Anti-CTLA4 and mCTLA4hIgG1 inhibited T cell proliferation with P + I to a similar extent in both cell populations. CD4+CD25+ T cells did not play a major role, as the results observed with undepleted or CD25-depleted CD4+ T cell populations were identical.



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  		Figure 4. CD4+CD25+ T cells are not responsible for the inhibition of proliferation of mouse T cells activated with P + I and anti-CTLA4 or mCTLA4hIgG1. Freshly isolated CD4+ T cells, with or without depletion of CD25+ cells, were cultured with P + I in the presence of the indicated Ab (1:100 ascites). Cells were pulsed with TdT at 36 h and harvested after 12 h.

 
CD4+ T cells activated with P + I together with CTLA4-CD80/CD86 blockade can be restimulated
We investigated whether CD4+ T cells activated with P + I together with CTLA4-CD80/CD86 blockade could be restimulated. Briefly, freshly isolated CD4+ T cells were activated with P + I in the presence of different Ab for 48 h, washed, and rested for ~42 h. The percentage of viable cells recovered (measured by trypan blue exclusion) after the rest period under different conditions as compared with the initial number of cells added was P + I 75 ± 14%, P + I + anti-CD28 182 ± 16%, P + I + anti-CTLA4 34 ± 8%, and P + I + mCTLA4hIgG1 33 ± 9%. Equal numbers of viable cells recovered under different conditions were restimulated with P + I or plate-bound anti-CD3 in the absence or presence of anti-CD28. Cells initially stimulated with P + I + anti-CD28 responded well to restimulation with anti-CD3 or P + I (Fig. 5 ). There was no major difference in restimulation with P + I between cells initially stimulated with P + I in the presence or absence of CTLA4-CD80/CD86 blockade. However, cells initially stimulated with P + I along with CTLA4-CD80/CD86 blockade restimulated less efficiently with anti-CD3 compared with P + I-stimulated cells. Further studies are required to assess whether these differences are a result of lowered ability of these T cells (initially activated with P + I and subjected to CTLA4-CD80/CD86 blockade) to respond to restimulation via the TCR or as a result of differences in sensitivity to the degree of TCR cross-linking. However, the fact that T cells responded to restimulation under all conditions demonstrated that the inhibition of proliferation observed in Figures 2 3 4 was possibly a result of the secretion of inhibitory factors, e.g., TGF-ß, and not the induction of anergy.



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  		Figure 5. T cells initially activated with P + I and anti-CTLA4 or mCTLA4hIgG1 can be reactivated. Cells were activated under the enlisted primary stimulation conditions for 48 h, washed, and rested for 42 h. Live cells/well (~5x104) were restimulated under different conditions: A, plate-bound {alpha}CD3; B, plate-bound {alpha}CD3 + {alpha}CD28; C, P + I; D, P + I + {alpha}CD28. Cells were pulsed with TdT at 36 h and harvested after 12 h. Cells alone, without any restimulation, gave 280, 338, 210, and 212 cpm for the four primary stimulation conditions (left to right), respectively.

 
CTLA4-CD80/CD86 blockade in CD4+ T cells activated with P + I results in production of low levels of IL-2 but high levels of TGF-ß
We quantified the amounts of IL-2 and TGF-ß produced under different conditions of activation (Table 1 ). P + I activation of CD4+ T cells resulted in production of high amounts of IL-2, which were further enhanced by anti-CD28. However, CTLA4-CD80/CD86 blockade resulted in seven- to tenfold lower amounts of IL-2 produced, as compared with P + I-activated cells. Concurrently, the levels of active or total TGF-ß secreted under CTLA4-CD80/CD86 blockade were significantly higher than the levels produced by P + I-activated T cells. At the same time, treatment with anti-CD28 resulted in decreased amounts of TGF-ß produced. Thus, there appears to be differential induction of IL-2 and TGF-ß depending on the activation conditions.


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  		Table 1. Anti-CTLA4 or mCTLA4hIgG1 Treatment of CD4+ T Cells Activated with P + I Resulted in Decreased IL-2 Production and Enhanced TGF-ß Production

 
Extended CTLA4-CD80/CD86 blockade results in decreased cell-cycle progression and enhanced apoptosis of CD4+ T cells activated with P + I
Cell-cycle analysis was performed after activating CD4+ T cells under different conditions for various time points. As observed in Figure 6A , CTLA4-CD80/CD86 blockade by anti-CTLA4 or mCTLA4hIgG1 resulted in a time-dependent increase in the hypodiploid population together with decreased cell-cycle progression, and maximal effects were observed after 72 h of activation with P + I. Anti-CD28-treated cell populations displayed maximum numbers of cycling cells and the least numbers of hypodiploid cells, which correlates with the known antiapoptotic function of CD28 [52 , 53 ]. In addition, CTLA4-CD80/CD86 blockade in CD4+ T cells activated with P + I resulted in lower mitochondrial potential [48 ], another marker for apoptosis (Fig. 6B) . The percentage of DiOC6low cells in P + I, P + I + anti-CD28, P + I + anti-CTLA4, and P + I + mCTLA4hIgG1 after 60 h of activation was 6.0 ± 1.9, 6.6 ± 2.3, 33.9 ± 3.5, and 38.5 ± 0.7, respectively (n=3). Thus, the induction of apoptosis as a result of CTLA4-CD80/CD86 blockade was confirmed by two independent methods. Next, we studied the expression of Bcl-XL, an antiapoptotic protein [37 , 52 , 53 ] in our culture system (Fig. 6C) . The expression of intracellular Bcl-XL increased with time in P + I-stimulated cells (three- to fourfold at 18 h or 42 h over the MFI obtained with control Ab). Treatment with P + I + anti-CD28 enhanced Bcl-XL expression (threefold at 18 h and 7.5-fold at 42 h). It is interesting that intracellular Bcl-XL levels increased by ~2.5-fold over control values in anti-CTLA4 and mCTLA4hIgG1-treated cells after 18 h of activation followed by decreased levels (~1.5-fold over control values) by 42 h of activation. The decreased level of Bcl-XL is probably one of the mechanisms contributing to increased apoptosis in this system. The ability of anti-CD28, IL-2, or fetuin, a protein that blocks TGF-ß activity [54 ], to rescue cell-cycle progression was studied. Briefly, CD4+ T cells were activated under different conditions in the absence or presence of the three molecules for 72 h followed by cell-cycle analysis. As observed in Table 2 , all three lowered the amount of apoptotic cells compared with controls, although fetuin was comparatively less effective than IL-2 or anti-CD28. As compared with cultures treated with the control protein ova, no significant increase was observed in the numbers of cycling cells with fetuin. Strikingly, anti-CD28 or IL-2 treatment increased the proportion of actively cycling S/G2M phase cells.



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  		Figure 6. Prolonged CTLA4-B7 blockade together with P + I activation leads to lower levels of Bcl-XL followed by lower cell-cycle progression and enhanced apoptosis. (A) CD4+ T cells were incubated under the conditions described above for the indicated time points, after which they were stained with PI and analyzed by flow cytometry. The numbers indicate the percentage of cells in G0/G1 (top) phase, G2/M (middle) phase, and the hypodiploid (bottom) population, respectively. (B) Treatment of CD4+ T cells with P + I and anti-CTLA4 or mCTLA4hIgG1 (1:100 ascites) for 60 h resulted in loss of mitochondrial potential. The numbers indicate the percentage of normal DiOC6high (above) and DiOC6low cells (below). (C) CD4+ T cells were activated with P + I and Ab to different costimulatory molecules for 18 and 42 h. After activation, the cells were washed and stained for intracellular Bcl-XL. The dotted lines are controls, and the gray and black lines indicate levels of Bcl-XL at 18 and 42 h, respectively.

 

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  		Table 2. Rescue by anti-CD28 or IL-2 Results in Increased Cell-Cycle Progression in CD4+ Mouse T Cells Activated with P + I and Anti-CTLA4 or mCTLA4hIgG1

 
Differential effects of anti-CD28, IL-2, and fetuin in rescuing proliferation of CD4+ T cells activated with P + I together with CTLA4-CD80/CD86 blockade
The above results led us to investigate whether the inhibition of proliferation of T cells could be rescued with IL-2, anti-CD28, fetuin, or neutralizing Ab against TGF-ß. The addition of ova, fetuin, or anti-TGF-ß Ab did not affect the extent of proliferation on activation with P + I, although some increase was observed with IL-2 and anti-CD28 (Fig. 7A ). The inhibition of proliferation with anti-CTLA4 or mCTLA4hIgG1 was rescued with the addition of anti-CD28 or IL-2 to levels observed with P + I. Conversely, dose-dependent studies revealed that fetuin increased the level of proliferation to about 40% of control values. Similarly, anti-TGF-ß1 alone resulted in partial rescue (~40%), and the addition of neutralizing Ab to all three isoforms did not increase proliferation over that with anti-TGF-ß1 alone. Thus, the inhibition of proliferation observed in this system (Figs. 2 3 4) is partially a result of the production of TGF-ß1. There is a possibility that CD80/CD86 binding to CTLA-4 provides positive signals to the T cells that are not mimicked by the blocking reagents used, which may explain the partial rescue by anti-TGF-ß. Next, we determined whether the addition of anti-CD28, IL-2, or fetuin at different time points after P + I activation and CTLA4-B7 blockade modulated the degree of rescue (Fig. 7B) . Maximum rescue with anti-CD28 and IL-2 is observed at 0 h addition, and the degree of rescue decreased at later time points of addition. However, the addition of fetuin at 0 or 24 h did not affect the extent of rescue, which suggested that the TGF-ß-mediated effects were late-acting.



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  		Figure 7. Differences in the levels of rescue and kinetics between IL-2/anti-CD28 are observed as compared with TGF-ß antagonists. (A) CD4+ T cells were activated with P + I and anti-CTLA4, mCTLA4hIgG1, or control Ab, together with anti-CD28 (1:100 ascites), IL-2 (50 U/ml), ova (1.0 mg/ml), fetuin (0.1, 0.5, 1.0 mg/ml), or anti-TGF-ß (1:500). (B) CD4+ T cells were activated with P + I + anti-CTLA4 or mCTLA4hIgG1. Ova, fetuin, IL-2, or anti-CD28 was added at different time points, as indicated. Cells were pulsed with TdT at 36 h and harvested 12 h later. The mean cpm of P + I-activated cells was 14,372.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We studied the effect of soluble Ab to CD28, CTLA4, and mCTLA4hIgG1 on proliferation of freshly isolated, purified CD4+ T cells under different activation conditions. Anti-CD28 enhanced the proliferation of T cells in combination with a first signal under all conditions (Fig. 2A) . However, anti-CTLA4 and mCTLA4hIgG1 enhanced proliferation of T cells in response to plate-bound anti-CD3 and P + anti-CD28 but inhibited proliferation of T cells activated with P + I (Fig. 2A) . This latter observation has been the focus of this study. The inhibition of proliferation observed with anti-CTLA4 and P + I is a result of blockade of CTLA4-CD80/CD86 interaction: Blocking Ab to receptor (CTLA4) and ligands (CD80 and CD86) inhibited proliferation (Fig. 3B) . The inhibition is unlikely to be a result of negative signal generation via cross-linking of anti-CTLA4, as this does not decrease the level of proliferation in this system (Fig. 3A) . There are several reports on differential effects of CD80 and CD86 in modulating the immune response [1 ]. However, in keeping with some reports [17 , 55 , 56 ] in this system, there was no major difference between CD80 and CD86. It is unlikely that signaling via CD80 or CD86 is playing a role, as Ab to these molecules were used under soluble conditions, whereas studies on signaling via CD80 and CD86 have used cross-linked Ab [57 , 58 ]. Finally, most studies on the role of CD80/CD86 in this system were performed using mCTLA4hIgG1 [45 ], which cannot be cross-linked, as it is a single polypeptide chain. Together, we conclude that CTLA4-CD80/CD86 interactions enhance P + I-mediated T cell activation, whereas these interactions inhibit anti-CD3 and P + anti-CD28-mediated T cell activation. CTLA4-CD80/CD86 interaction, under some conditions, has been reported to enhance T cell activation [59 60 61 62 ]. Our results suggest that CTLA4-CD80/CD86 interactions can enhance or inhibit T cell proliferation depending on the activation conditions.

All receptor-ligand interactions in this system involved binding CD4+ T cells to each other. CTLA4-CD80/CD86 interactions are specifically required to enhance P + I-mediated T cell activation, as blockade of lymphocyte function-associated antigen-1-intercellular adhesion molecule-1/2 interaction did not have any effect on T cell proliferation (data not shown). The initial effects of activation with P + I in the presence and absence of CTLA4-CD80/CD86 blockade are similar with respect to three different readouts: TdT incorporation (Fig. 2B) , CD25 (Fig. 3C) , and Bcl-XL (Fig. 6C) . Thus, CTLA4, CD80, and CD86 are induced by P + I, and their interaction in the first 12 h results in production of IL-2 and decrease in the amounts of TGF-ß and other inhibitory factors, which results in increased cell-cycle progression and survival (Fig. 8 ). However, the effects of blockade of CTLA4-CD80/CD86 interaction were manifested later (42–48 h) during T cell activation. Our results are different from the reported inhibition of proliferation of human T cells with P + I and Chinese hamster ovary cells expressing CD80 in which CD28-CD80 and CTLA4-CD80 interactions play a negative role [63 ]. In our system, CD28-CD80/CD86 interaction was unable to rescue cells from the dominant inhibitory effects of in vitro CTLA4-CD80/CD86 blockade, although triggering CD28 using an Ab rescued T cell proliferation. This difference between the effects of anti-CD28 and CD28-CD80/CD86 binding may be because CD80 and CD86 molecules expressed by mouse T cells are unable to bind CD28 but are capable of functionally binding CTLA4 [64 ]. In addition, there are other functional differences between CD80 and CD86 on APC and T cells [65 , 66 ]. Also, there is evidence to suggest that the effects of CD28-CD80/CD86 binding may be different from those observed using anti-CD28 [67 , 68 ].



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  		Figure 8. A model depicting the essential role of CTLA4-CD80/CD86 interaction in activation and survival of CD4+ T cells with P + I. CTLA4-CD80/CD86 interaction during the first 12 h of activation with P + I lowers TGF-ß production and induction of apoptosis but enhances IL-2 production and Bcl-XL levels, leading to increased cell-cycle progression and survival.

 
The inhibition of T cell proliferation by CTLA4-CD80/CD86 blockade was possibly a result of production of inhibitory factors by CD4+ T cells and not the induction of anergy. In addition, this inhibition of proliferation was mediated by bulk CD4+ T cells and not regulatory CD4+CD25+ T cells. Several reports have demonstrated that CTLA4-CD80/CD86 interaction results in TGF-ß production [26 , 27 , 30 ]. In contrast, CTLA4-CD80/CD86 interaction prevented TGF-ß production by CD4+ T cells activated with P + I. Indeed, blocking CD80 or CD86 alone (Fig. 3B) resulted in significant inhibition, possibly as a result of the production of factors that inhibit proliferation of other T cells in a paracrine manner (Fig. 3B) . In this system, TGF-ß was partly responsible for the inhibition of T cell activation: First, TGF-ß was produced in greater amounts under conditions in which T cell proliferation was inhibited (Table 1) . Secondly, TGF-ß antagonists rescued proliferation of inhibited cells by ~40% (Fig. 7A) . Finally, TGF-ß-mediated actions were late-acting (Fig. 7B) and partially contributed to increased apoptosis (Table 2) . These results also suggest the involvement of yet-to-be-identified, non-TGF-ß-dependent mediator/s or mechanisms. Anti-IL-10 did not rescue proliferation of inhibited cells, thereby ruling out IL-10 as a likely candidate (data not shown). It is interesting that TGF-ß and unidentified mediators are involved in an in vivo cytokine-mediated model of suppression [69 ], and future studies will attempt to identify the mediators of the non-TGF-ß pathway. The inverse correlation between production of IL-2 and TGF-ß in our system was striking. Decreased IL-2 production or a delay in adding exogenous IL-2 resulted in the inability to rescue cells completely from the inhibitory effects of CTLA4-CD80/CD86 blockade. It is possible that high levels of IL-2 were required early to counteract the action of the TGF-ß and the non-TGF-ß-dependent pathway. Although fetuin partially rescued proliferation in inhibited cultures, its action was insufficient to increase the numbers of cycling cells 72 h after activation. Conversely, anti-CD28 or IL-2 completely rescued the inhibition of proliferation on CTLA4 blockade by reducing the hypodiploid population and increasing numbers of cycling cells, which were detected 72 h after activation. This suggests that an additional mitogenic stimulus (e.g., IL-2), in addition to P + I-mediated activation, was required to fully counteract the effects of CTLA4-CD80/CD86 blockade. In our system, the induction of apoptosis as a result of CTLA4-CD80/CD86 blockade may be because of decreased Bcl-XL levels after 42 h of activation and low levels of IL-2 produced, which could contribute to lowered mitochondrial potential [52 , 53 ]. Also, TGF-ß is known to be produced by apoptotic cells [70 ], but it is not clear whether the increased TGF-ß levels observed in this system are a result of CTLA4-CD80/CD86 blockade or release by cells undergoing apoptosis. It is known that the combination of P + I induces Fas ligand on the surface of T cells [44 ]; however, the role of Fas and additional mediators of apoptosis in this system needs to be investigated further.

The roles of CD80 and CD86 in mouse T cells are not well studied. High expression of CD80 or CD86 on naïve CD4+ T cells results in proliferation in response to low concentrations of anti-CD3 alone without exogenous costimulation [71 ]. TCR signal strength appears to modulate CTLA4 expression [72 ] and response [61 , 62 ]. The present work demonstrates a role for CD80, CD86, and CTLA4 on CD4+ T cells in enhancing or inhibiting proliferation, depending on activation conditions. Our findings demonstrate that after activation with P + I, CD80 and CD86 on mouse CD4+ T lymphocytes bind to CTLA4 in a T:T cell interaction manner to enhance activation and survival. These results shed new light on the role of CD80, CD86, and CTLA4 on CD4+ T cells in modulating the induction of Bcl-XL, IL-2, and TGF-ß. Further studies are underway to understand the mechanisms involved in enhancement or inhibition of T cell responses by CD80, CD86, and CTLA4 under different activation conditions. These studies will enhance our knowledge of the role of costimulatory interactions and modulation of the T cell-immune response.


   ACKNOWLEDGEMENTS
 
This work was supported by funds from the Director’s Start-up grant, IISc, and the Department of Biotechnology (DBT), Government of India. P. K. M. was supported with postdoctoral fellowships from DBT and the Council for Scientific and Industrial Research, India. We thank Drs. J. Allison, J. Monaco, R. Manjunath, A. Karande, P. Kondaiah, D. Danielpour, A. Sarin, K. B. Sainis, S. Malu, S. Rath, and V. Bal for encouragement and gift of cell lines/reagents. Dr. O. Joy and the IISc FACS facility deserve a special mention.

Received April 9, 2002; accepted July 23, 2002.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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