Cyclic Adenosine 5′-Monophosphate and Calcium Induce CD152 (CTLA-4) Up-Regulation in Resting CD4⁺ T Lymphocytes

Media and reagents

RPMI 1640 supplemented with 2 mM l-glutamine, 1% nonessential amino acids, 1% pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin (Life Technologies, Grand Island, NY), and 10% FCS (HyClone Laboratories, Logan, UT) was used as complete medium in all cultures. Anti-CD3 (clone UCHT1) and anti-CD28 (clone CD28.2) mAbs were purchased from Immunotech (Westbrook, ME) and BD PharMingen (San Diego, CA), respectively. Cholera toxin (CT) and CT B subunit (CT-B) were purchased from Calbiochem-Novabiochem (San Diego, CA); forskolin (FSK), dibutyryl cAMP (dbcAMP), Ion, and cyclosporin A (CsA) were purchased from Sigma-Aldrich (St. Louis, MO).

Lymphocyte purification and cell cultures

PBMC were isolated from healthy donors by Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) density centrifugation. CD4⁺ T lymphocytes were purified by negative selection using an immunomagnetic cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany). Briefly, PBMC were labeled using a cocktail of hapten-conjugated mAbs anti-CD8, CD11b, CD16, CD19, CD36, and CD56 molecules and MACS MicroBeads coupled to an anti-hapten mAb. The magnetically labeled cells were depleted by retaining them on a column using MidiMACS cell separator. PBMC or CD4⁺ purified T cells (2 × 10⁶/ml) were treated either with CT (3 μg/ml), CT-B (3 μg/ml), dbcAMP (from 0.01 to 0.5 mM), FSK (50 μM), or Ion (200 ng/ml), and analyzed at different time points. PBMC were also stimulated with anti-CD3 mAb (1 μg/ml). In some experiments, CsA (1 μg/ml) was used as inhibitor of Ca²⁺/calcineurin signal pathway.

Flow cytometry analysis

FITC- or PE-conjugated anti-CD4, CD152, CD25, and CD69 mAbs and their isotype-matched controls were purchased from BD PharMingen (San Diego, CA) and used for direct immunofluorescence staining. CD152 detection was performed by intracellular staining. Briefly, cells were washed twice in PBS, 1% BSA, and 0.1% sodium azide and stained with anti-CD4 mAb on the membrane for 15 min at 4°C. Samples were then fixed in 4% paraformaldehyde for 5 min at 4°C; incubated with anti-CD152 mAb; and diluted in PBS, 1% BSA, and 0.5% saponin. The cells were finally washed twice in PBS, 1% BSA, and 0.1% saponin, and acquired on a FACSCalibur instrument running CellQuest software.

CD152 mRNA analysis by RT-PCR

Total RNA was extracted using RNAFast reagent (Life Technologies), according to the manufacturer’s recommendations. The single-stranded cDNA was synthesized using 1 μg of RNA by reverse transcription using random examers (Invitrogen, San Diego, CA). PCR were performed with cDNA corresponding to 10 ng of RNA, Taq polymerase (Invitrogen), and primers designed to amplify the entire coding sequence of CD152: 5′-ATGGCTTGCCTTGGATTTCAGCGGCACAAGG-3′ and 5′-TCAATTGATGGGAATAAAATAAGGCTGAAATTGC-3′. PCR was as follows: 94°C for 5 min, 30 cycles 94°C for 30 s, 58°C for 30 s, and 72°C for 30 s, followed by a final extension at 72°C for 7 min. The amplified fragments were separated on 1% agarose gel and visualized by ethidium bromide. RNA integrity and cDNA synthesis were verified by amplifying β₂-microglobulin cDNA. The intensity of the revealed bands was directly quantified by Image QuaNT software (Amersham Pharmacia Biotech, Piscataway, NJ), which gives rise to a volume report (expressed as arbitrary units, a.u.) by integrating the area of the bands and their OD. CD152 mRNA values were normalized to those of β₂-microglobulin and expressed as a.u.

Electrophoretic mobility shift assay

Binding reactions containing 12 μg cell extracts, 10 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, 0.5 mM EDTA, 10% glycerol, 0.5 mg poly(dI-dC), and 1 μg BSA were incubated for 30 min with 5000 cpm of ³²P-end-labeled double-stranded oligonucleotide in a total volume of 10 ml. The probe was 5′-AGTTGAGGGGACTTTCCCAGGC-3′. Labeling of the probe was obtained by incubating 10 pmol of oligonucleotide with 10 pmol [γ-³²P]ATP and 10 U T4 polynucleotide kinase for 30 min at 37°C. The probe was then purified with Micro BIO-Spin P-30 columns. Complexes were separated on 6% polyacrylamide gel. After drying, gels were exposed on phosphor screen and were then analyzed by phospho/fluorescence imager STORM 840 (Amersham Pharmacia Biotech). The intensity of the revealed bands was directly quantified by Image QuaNT software (Amersham Pharmacia Biotech).

cAMP up-regulates CD152 in resting T cells

To investigate whether intracellular increase of cAMP had any effects on the expression of CD152, we treated human PBMC with an analog of cAMP, dbcAMP, or with agents that increase intracellular cAMP and analyzed CD152 expression in CD4⁺ T lymphocytes by intracellular staining (Fig. 1⇓). An increase in the percentage of CD4⁺CD152⁺ cells was evident already after 3 h of incubation with dbcAMP and, after 24 h, 45% of the CD4⁺ T cells were CD152⁺ (Fig. 1⇓A). The up-regulation of CD152 by dbcAMP was dose dependent, as shown in Fig. 1⇓B. The increase of CD152 expression was also observed with FSK, which directly activates adenylyl cyclase, and with CT, a bacterial toxin that causes intracellular cAMP increase by ADP ribosylation of Gs proteins (Fig. 1⇓A). In contrast, treatment with CT-B, the B subunit of CT, which lacks the ADP-ribosyl transferase activity, did not affect the up-regulation of CD152. Finally, anti-CD3 mAb-activated cells, which were included in the experiments as a positive control, showed a high CD152 up-regulation, as expected (Fig. 1⇓, A and B).

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FIGURE 1.

Up-regulation of CD152 induced by cAMP in the absence of T cell activation. A, CD152 expression at different time points after incubation of PBMC in the presence of medium alone (ctr), dbcAMP, FSK, CT, CT-B, or anti-CD3 mAb. The cells were stained with anti-CD4 mAb on the membrane, fixed, permeabilized, and stained with anti-CD152 at time zero and after 3, 6, and 24 h of culture. B, Dose-response curve of CD152 up-regulation with dbcAMP after 24 h of culture. The control of CD152 up-regulation by anti-CD3 mAb is also shown. C–F, PBMC incubated with dbcAMP (C, D) and with anti-CD3 mAb (E, F) were double stained with anti-CD4 and anti-CD69 mAbs after 24 h of culture or with anti-CD4 and anti-CD25 mAbs after 48 h. Cytofluorometric analysis was performed on CD4⁺-gated population. The data shown are from one representative experiment of three performed.

To rule out the possibility that dbcAMP, FSK, and CT up-regulate CD152 molecule by inducing activation of T cells, we analyzed the expression of the activation markers CD69 and CD25, after 24 and 48 h, respectively, of culture. Fig. 1⇑ (C–F) shows that the treatment with dbcAMP did not alter the expression of the early activation marker CD69, nor that of CD25, whereas activation of the cells with anti-CD3 mAb induced up-regulation of CD69 and CD25 molecules, as expected. Furthermore, while anti-CD3-activated cells proliferated, those treated with dbcAMP remained in a resting state (data not shown). Similar results were obtained with the cAMP inducers FSK and CT (data not shown).

To evaluate whether cAMP-dependent up-regulation of CD152 correlated with an increase of mRNA, we analyzed the CD152 mRNA in PBMC cultured in the presence of dbcAMP (Fig. 2⇓). We observed an increase of two mRNA transcripts that correspond to the membrane (672-bp) and the soluble (550-bp) molecule of CD152 (14). Altogether, these results show that the increase of intracellular cAMP leads to increased CD152 mRNA and protein levels in T lymphocytes, and that this occurs in the absence of full T cell activation.

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FIGURE 2.

CD152 mRNA expression in T lymphocytes. CD152 mRNA was evaluated in human PBMC either unstimulated (ctr) or stimulated with dbcAMP, Ion, or both. RNA integrity and cDNA synthesis were verified by amplifying β₂-microglobulin cDNA. The graph represents CD152 mRNA quantification of the two mRNA transcripts, both normalized to those of β₂-microglobulin.

Ca²⁺ influx enhances cAMP-induced CD152 expression

To investigate whether the effect of cAMP on CD152 expression was affected by increased Ca²⁺ levels in T cells, purified CD4⁺ T lymphocytes were treated with Ion in the presence or absence of dbcAMP or cAMP inducers. We found that Ion alone up-regulated CD152 to a similar degree as dbcAMP, FSK, or CT (Fig. 3⇓A). However, a stronger induction of CD152 was observed by concomitant treatment of the cells with Ion and either dbcAMP, FSK, or CT, with >70% of the CD4⁺ T cells expressing CD152 after 24 h of culture (Fig. 3⇓A). The expression level of CD152 relative to the single cell, expressed as mean fluorescence intensity (MFI), was 14 in the absence of any stimuli and increased to 30–38 with the different stimuli (Fig. 3⇓B). However, an increase of the MFI value to 175–261 was observed after concomitant treatment with Ion and dbcAMP, FSK, or CT (Fig. 3⇓B). These data show that the effects of cAMP and Ca²⁺ on purified CD4⁺ T cells are more than additive, suggesting an active integration of the two signaling pathways. At the mRNA level, a stronger up-regulation of the transcripts corresponding to the membrane and the soluble CD152 molecule was observed with the combination of dbcAMP and Ion as compared with the single treatments (Fig. 2⇑). Finally, the up-regulation of CD152 mediated by Ion alone or by the combination of Ion and the cAMP inducers occurred in the absence of proliferation and expression of the activation markers CD25 and CD69 (data not shown).

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FIGURE 3.

Ion and cAMP synergize for the induction of CD152 expression. Purified CD4⁺ T lymphocytes were cultured with Ion, dbcAMP, FSK, or CT, or with Ion in combination with each of the others. Twenty-four hours later, the cells were stained with anti-CD4 mAb on the membrane, and with anti-CD152 mAb after fixation and permeabilization. A, The results are expressed as percentage of CD4⁺CD152⁺ T cells. B, The cytofluorometric profiles of CD152 expression are shown for untreated CD4⁺ T cells (thin line), cells cultured either with Ion, dbcAMP, FSK, or CT alone (bold line), or with the combination of Ion and dbcAMP, FSK, or CT (dotted line). Numbers in parentheses indicate MFI values. The data shown are from one representative experiment of four performed.

CsA prevents Ca²⁺- but not cAMP-induced CD152 up-regulation

It has been described that cAMP can influence Ca²⁺ levels in different cell types, and that many adenylyl cyclase isoforms are Ca²⁺ dependent. In the attempt to understand the relationship between Ca²⁺ and cAMP signaling pathways in the up-regulation of CD152, purified CD4⁺ T cells were treated with CsA, which is known to interfere with the Ca²⁺/calcineurin signaling pathway. CsA fully prevented the Ion-induced up-regulation of CD152 (Fig. 4⇓A), confirming that Ca²⁺-mediated signals are involved in CD152 up-regulation. However, CsA did not affect the induction of CD152 mediated by dbcAMP or FSK (Fig. 4⇓B), and only partially prevented the up-regulation of CD152 in the presence of both Ion and dbcAMP or Ion and FSK (Fig. 4⇓C). These data suggest that the up-regulation of CD152 mediated by cAMP is independent from Ca²⁺ signals, and that at least two signaling pathways may regulate CD152 expression in T lymphocytes.

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FIGURE 4.

CsA prevents the up-regulation of CD152 mediated by Ion, but not that mediated by cAMP. Purified CD4⁺ T lymphocytes were cultured with Ion (A), dbcAMP or FSK (B), or with the combination of Ion and dbcAMP or FSK (C), in the presence or absence of CsA. After 24 h, the cells were stained with anti-CD4 and anti-CD152 mAbs, as indicated in the legend of Fig. 3⇑, and the percentage of CD4⁺CD152⁺ T cells was analyzed. The data shown are from one representative experiment of three performed.

CD152 molecules up-regulated by cAMP and Ion are functional

To verify whether CD152 up-regulation induced by cAMP and Ion results in inhibitory effects on T cells, purified CD4⁺ T cells were cultured with anti-CD3, anti-CD28, and anti-CD152 mAbs in the presence or absence of CT, Ion, or both. Cell extracts were probed with a radiolabeled NF-κB consensus sequence and resolved by EMSA. The data in Fig. 5⇓ show that CT and Ion alone induced NF-κB activation and that their combination resulted in a slightly higher stimulation. Furthermore, the engagement of membrane CD152 by mAbs in CT- or Ion-treated cells inhibited NF-κB activation by 48 and 33%, respectively, and in cells treated with both CT and Ion inhibition of NF-κB activation was 54%. Altogether, these results show that CD152 molecules that have been up-regulated by cAMP and Ion either alone or in combination are expressed on the cell membrane, and that their engagement by anti-CD152 mAb results in inhibitory effects in T lymphocytes.

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FIGURE 5.

Engagement of membrane CD152 by specific mAb leads to NF-κB inhibition. Purified CD4⁺ T cells were cultured with anti-CD3, anti-CD28, and anti-CD152 mAbs, or with the isotype-matched control anti-trinitrophenol mAb, in the presence or absence of CT, Ion, or both. Cell extracts were probed with radiolabeled NF-κB consensus sequences and resolved by EMSA. The phosphor screen was analyzed by Image QuaNT software, which gives rise to a volume report (expressed as a.u.) by integrating the area of the bands and their OD.