The functional outcome of a T cell response to Ag is the result of a balance between coactivation and inhibitory signals. In this study we have investigated the effects of the CD85/leukocyte Ig-like receptor (LIR)-1/Ig-like transcript (ILT) 2 and of CD152 (CTLA-4) inhibitory receptors on the modulation of cell-mediated immune responses to specific Ags, both at the effector and at the resting/memory cell level. Proliferation and cytokine production of CD4+ T lymphocytes stimulated by recall Ags have been evaluated. Cross-linking of CD85/LIR-1/ILT2 or CD152 molecules on cultured T cells using specific mAb and goat anti-mouse antiserum inhibits Ag-specific T cell proliferation. This inhibition is always paralleled by increased production of cytokines that down-regulate immune responses, e.g., IL-10 and TGF-β. In contrast, the production of cytokines that support T cell expansion and function (e.g., IL-2, IFN-γ, and IL-13) is significantly decreased. A long-term effect of CD85/LIR-1/ILT2 and of CD152 occurs during Ag-specific T cell activation and expansion. T cells, primed in the presence of anti-CD85/LIR-1/ILT2 and anti-CD152 blocking mAb (but in the absence of cross-linking), proliferate at higher rates and produce higher amounts of IL-2, IFN-γ, and IL-13, in comparison with T cells stimulated with the Ag alone. We also show that the inhibitory receptors exert a similar effect during Ag activation of specific CD4+ effector T cells. Ag-specific polyclonal CD4+ T cell lines exhibit increased proliferation and IL-2, IFN-γ, and IL-13 production when the CD85/LIR-1/ILT2 receptor is blocked by specific mAb. In contrast, cross-linking of this receptor down-regulates Ag-specific CD4+ T cell proliferation and increases IL-10 and TGF-β production.

The cell-mediated arm of the immune system is finely tuned in its ability to maintain a balance between reactivity and quiescence, thus controlling potentially harmful responses (1, 2, 3, 4). An example is provided by the specificity of the immune responses against non-self molecules, while sparing the self. In contrast, the ability to limit or quench an immune response by inactivation or deletion of the effector cells is essential when these cells are no longer required (3). Thus, the outcome of Ag-specific T cell responses results from a balance between coactivation and inhibitory signals. A variety of inhibitory molecules has been described that down-regulate T cell proliferation and effector functions (5, 6, 7). Some of these receptors are expressed on small subsets of T cells, whereas others are shared by a large variety of hemopoietic cell types (8, 9, 10, 11, 12).

The CD85/leukocyte Ig-like receptor (LIR)3-1/Ig-like transcript (ILT) 2 molecule binds to the nonclassical class I HLA-G protein, to some alleles of HLA-A, -B, and -C loci, and to the human CMV UL18 gene product, a viral homolog of HLA-class I (8, 11, 13). It is a transmembrane molecule that bears four immunoreceptor tyrosine-based inhibition motifs in its cytoplasmic domain (14, 15). Tyrosine phosphorylation of immunoreceptor tyrosine-based inhibition motifs establishes docking sites for the Src homology domain 2-containing phosphatase SHP-1 that subsequently transduces inhibitory signals by dephosphorylating and inactivating downstream tyrosine kinases (16). We have shown that CD85/LIR-1/ILT2 is expressed on the surface of the majority of T cells and in cytoplasmic transport vesicles of all T lymphocytes (6). Although membrane expression may vary at times, there is no apparent relationship between cell activation and CD85/LIR-1/ILT2 expression. CD85/LIR-1/ILT2 down-regulates the Ag-specific cytolytic activity of CD8+ T cells, and its cross-linking results in inhibition of proliferative responses to recall Ags by CD4+ T cells (6).

CD152 (CTLA-4) is an inhibitory molecule homologous to CD28 that delivers a costimulatory signal and is an inducible receptor. Its mRNA is detectable within 1 h after T cell activation, and cell surface expression of the protein, although weak, reaches a peak 1–3 days later (17, 18, 19). Moreover, its engagement triggers phosphatases that dephosphorylate molecules of the CD3/TCR activation cascade (1, 3, 20). In addition, it has been shown that engagement of CD152 inhibits specific target cell lysis mediated by CD8+ T lymphocytes (5), down-regulates proliferation of T cells as well as IL-2, IFN-γ, IL-4 (21, 22, 23, 24), and IL-13 production (7), and up-regulates the production of IL-10 (25, 26, 27) and TGF-β (28, 29). Conversely, blockade of CD152 by specific mAb increases proliferation of CD4+ T cell blasts and clones (5, 21, 22).

In this study we have analyzed the function of resting/memoryand effector CD4+ T lymphocytes with the aim of understanding the effects of CD85/LIR-1/ILT2 and of CD152 (CTLA-4) on T cell proliferation and cytokine production in response to Ags. We show that these inhibitory receptors exert a dual function on T cell proliferation and that they modulate cytokine production when blocked or cross-linked.

Abs for immunophenotypic analyses were anti-CD4, anti-CD8, anti-CD20 mAb (BD PharMingen, Hamburg, Germany), anti-CD3 (clone OKT3; American Type Culture Collection, Manassas, VA), anti-MHC class II mAb (clone D1.12; kindly provided by R. S. Accolla, Unit of Cellular and Molecular Genetics, Advanced Biotechnology Center, Genoa, Italy), anti-CD152 mAb (kindly provided by A. Lanzavecchia, Institute for Research in Biomedicine, Bellinzona, Switzerland), HP-F1 (kindly provided by M. Lopez-Botet, Servicio de Immunologia, Hospital Universitario de la Princesa, Madrid, Spain), and anti-p58.2 (A3 mAb; produced in our laboratory). Ags used as stimulators were tetanus toxoid (TT), Candida albicans bodies (Ca), and Cryptococcus heat-inactivated yeasts (Cr).

Freshly isolated PBMCs were isolated from heparinized venous blood of healthy donors. CD4+ T lymphocytes were negatively selected from PBMCs by MACS (Miltenyi Biotec, Milan, Italy) and incubated with irradiated autologous feeder cells pulsed with Ags (TT, Ca, and Cr), in the presence or absence of mAb to inhibitory receptors (anti-CD152 or HP-F1) either soluble or cross-linked by goat anti-mouse (GAM) antiserum. An isotype-matched mAb (A3, IgG1) was used as a control. Supernatants were collected on day 2 (Fig. 1,A) and cytokine production was measured by an ELISA. After 10 days of culture, cells were harvested, washed three times with PBS, and restimulated with irradiated autologous PBMCs pulsed with the Ags used previously. No inhibitory mAb were used in this latter phase of the experiments. Supernatants were collected on day 12 to measure cytokine production. Proliferation indexes were evaluated as [3H]thymidine uptake on days 2 and 12 (as shown in Fig. 1 A).

FIGURE 1.

Plan of the experiments. A, Short-term (T2) and long-term (T12) effects of CD152 and CD85/LIR-1/ILT2 blockade or cross-linking performed at the time of priming (T0) of resting/memory T lymphocytes by recall Ags. B, Generation of Ag-specific CD4+ T cell lines and evaluation of the short-term (T2) effects of CD152 and CD85/LIR-1/ILT2 blockade or cross-linking of these effector/memory T cells.

FIGURE 1.

Plan of the experiments. A, Short-term (T2) and long-term (T12) effects of CD152 and CD85/LIR-1/ILT2 blockade or cross-linking performed at the time of priming (T0) of resting/memory T lymphocytes by recall Ags. B, Generation of Ag-specific CD4+ T cell lines and evaluation of the short-term (T2) effects of CD152 and CD85/LIR-1/ILT2 blockade or cross-linking of these effector/memory T cells.

Close modal

Ag-specific CD4+ T cell lines were generated from total PBMCs by repeated stimulation with autologous irradiated PBMCs pulsed with the appropriate Ag (Fig. 1 B). Human rIL-2 (final concentration, 20 U/ml) was added to the cell cultures every 2–3 days. Specificity of the response indicated by cell proliferation at each stimulatory cycle point was evaluated by [3H]thymidine uptake. Cultures were analyzed for cytokine production when the cell phenotype was >97% CD4+, MHC class II+, CD8, CD20, and CD16 (data not shown). Clones were produced by plating T cells at a limiting dilution of 10 and 1 cells/well, in the presence of 3 × 104 autologous irradiated Ag-pulsed PBMCs.

Ag-specific CD4+ T cell lines were diluted at 2 × 106/ml and dispensed into 96-well plates in a volume of 200 μl/well. Each well was precoated with anti-CD3 mAb (1 μg/ml) in 1× Dulbecco’s PBS (pH 7) at 4°C overnight. Ag-specific CD4+ T cell lines were subsequently incubated in the presence or absence of one of the inhibitory mAb (anti-CD152 or HP-F1 mAb) either soluble (receptor blockade) or cross-linked by GAM antiserum. An isotype-matched mAb (A3, IgG1) was used as a control. T cell proliferation was evaluated on day 2 by [3H]thymidine uptake and by flow cytometric DNA content measurements. Supernatants were collected on day 2 and cytokine production was measured by an ELISA.

Cells were activated by recall Ags as described above. Forty-eight hours after activation, cells were harvested, fixed with 70% ethanol, and stained with 30 μg/ml propidium iodide (Sigma-Aldrich, St. Louis, MO) in the presence of 0.5 mg/ml RNase for 30 min at room temperature. Flow cytometric measurements were performed using a FACSCalibur (BD Biosciences, Mountain View, CA). Frequency distributions of DNA content were analyzed for the evaluation of apoptosis and percentage of cells in the various phases of the cell cycle.

IL-2, IFN-γ, IL-10, IL-13, and TGF-β ELISAs were performed as indicated by the manufacturer (Diaclone Research, Besançon, France). Supernatants were tested in triplicate adding a volume of 100 μl to each well. Deviation between triplicates was <10% for any reported value. The lower sensitivity threshold of the ELISA is 10 pg/ml for IL-2, 5 pg/ml for IFN-γ and IL-10, 1.5 pg/ml for IL-13, and 1.9 pg/ml for TGF-β.

Differences in cell proliferation and cytokine production between control and mAb-treated cells were observed. To assess their statistical significance, the Student’s t test was used with a level of p < 0.05.

The role of CD85/LIR-1/ILT2 and CD152 receptors on Ag-specific T cell activation was assessed by measuring proliferative responses to recall Ags and the pattern of cytokines produced 48 h after Ag stimulation.

PBMCs from healthy donors proliferated in the presence of Ags (TT, Ca, and Cr).

Addition of soluble mAb to the inhibitory receptors enhances several T cell functions (Fig. 2). This cannot be due to activation via FcR, as control mAb of the same isotype as that of HP-F1 and anti-CD152 mAb (i.e., A3) have no effect. Furthermore, when isotype-matched mAb against other surface receptors (i.e., anti-CD4, anti-CD8, anti-class I, and anti-HLA-DR) are included in the assay, no proliferative or functional enhancement is observed (Refs. 5, 6 , and 24 , and data not shown). Hence, this augmented T cell function is due to the interaction between mAb and inhibitory molecules. Therefore, it is conceivable that engagement of the inhibitory receptors by the specific mAb leads to a blockade of receptor-ligand interaction due to steric hindrance. Thus, although direct experimental evidence is not available, in Results we refer to this effect as a receptor blockade.

FIGURE 2.

CD85/LIR-1/ILT2 and CD152 modulate recall Ag-induced proliferation and cytokine production during priming of CD4+ T lymphocytes. CD4+ T lymphocytes (T) isolated from PBMCs were stimulated using autologous feeder cells (APCs) pulsed with recall Ags TT, Ca, and Cr, in the absence or in the presence of the indicated mAb (HP-F1, CD152, and A3 mAb, respectively, specific for CD85/LIR-1/ILT2, CD152, and p58.2 receptors). Proliferation and cytokine production were measured 48 h after stimulation (T2 in Fig. 1 A). Data are from one representative donor. A, Mean values of [3H]thymidine uptake from two independent experiments are reported. Bars represent the range/2. B, IL-2 and IFN-γ mean values. C, IL-10 and TGF-β mean values. Supernatants were tested in triplicate and deviation between triplicates was <10% for any reported value. Results are representative of four experiments with similar results. ∗, Samples that are significantly different (p < 0.05) from the control culture (T + APC + Ag); ▨, samples where receptor blockade is exerted by soluble mAb; ▪, samples with receptors cross-linked by mAb plus GAM antiserum.

FIGURE 2.

CD85/LIR-1/ILT2 and CD152 modulate recall Ag-induced proliferation and cytokine production during priming of CD4+ T lymphocytes. CD4+ T lymphocytes (T) isolated from PBMCs were stimulated using autologous feeder cells (APCs) pulsed with recall Ags TT, Ca, and Cr, in the absence or in the presence of the indicated mAb (HP-F1, CD152, and A3 mAb, respectively, specific for CD85/LIR-1/ILT2, CD152, and p58.2 receptors). Proliferation and cytokine production were measured 48 h after stimulation (T2 in Fig. 1 A). Data are from one representative donor. A, Mean values of [3H]thymidine uptake from two independent experiments are reported. Bars represent the range/2. B, IL-2 and IFN-γ mean values. C, IL-10 and TGF-β mean values. Supernatants were tested in triplicate and deviation between triplicates was <10% for any reported value. Results are representative of four experiments with similar results. ∗, Samples that are significantly different (p < 0.05) from the control culture (T + APC + Ag); ▨, samples where receptor blockade is exerted by soluble mAb; ▪, samples with receptors cross-linked by mAb plus GAM antiserum.

Close modal

When in the assay HP-F1 or anti-CD152 soluble mAb were added, an increased Ag-induced T cell proliferation, as measured by [3H]thymidine uptake, was observed (Fig. 2,A shows results from one representative experiment, as planned in Fig. 1,A). Increased proliferation varied according to the Ags used (from 20 to 126% increase), and it was particularly relevant in low responders (Fig. 2,A; the donor is a low responder to TT). The positive effect of the inhibitory receptor blockade was even sharper when the production of cytokines that sustain cell-mediated immune responses was analyzed. As shown in Fig. 2,B, IL-2 and IFN-γ were detectable in Ag-stimulated cultures after 48 h. IL-2 production ranged from 25 to 45 pg/ml and IFN-γ production ranged from 5 to 95 pg/ml, according to the Ag used. Addition of either HP-F1 or anti-CD152 mAb yielded an increase of cytokine production varying from 50 to 270% for IL-2, and from 20 to 1400% for IFN-γ after 48 h (Fig. 2 B). In these experimental conditions, IL-13 was undetectable in the culture medium.

Cytokines that down-regulate proliferation of Ag-specific T cells were also evaluated. Blocking of CD85/LIR-1/ILT2 and CD152 inhibitory receptors by specific mAb resulted in a reduction of IL-10 production, whereas TGF-β was undetectable after 48 h of culture (Fig. 2 C). The effect on IL-10 production was specific for CD85/LIR-1/ILT2 and CD152, as an irrelevant isotype-matched mAb (i.e., A3) did not influence cell proliferation and cytokine production.

We also investigated the outcome of a physiological engagement of CD85/LIR-1/ILT2 and CD152 inhibitory receptors, mimicking such ligand/receptor interaction by cross-linking the two specific mAb (HP-F1 and anti-CD152) using a GAM antiserum. CD85/LIR-1/ILT2 or CD152 cross-linking resulted in decreased [3H]thymidine uptake by anti-CD3-stimulated T cells (data not shown). Ag-mediated proliferation of specific T cells is shown in Fig. 2,A. Inhibition of proliferation ranged between 40 and 75%, according to the Ag used. Moreover, cross-linking of CD85/LIR-1/ILT2 or CD152 reduced IL-2 and IFN-γ production (Fig. 2 B). Altogether, these data suggest that an inhibition of the events associated with the progression of resting T cell activation is related to the engagement of the two inhibitory receptors.

As determined by flow cytometric analyses of the cell cycle distribution, inhibition of CD4+ T cell activation by CD85/LIR-1/ILT2 or CD152 is due to a restricted transit from the G0/G1 to the S/G2M phase of the cell cycle, rather than to an enhancement of cell death (Table I). Accordingly, no significant increase of Ag-specific CD4+ T cell apoptosis by cross-linking of HP-F1 or anti-CD152 mAb by GAM antiserum was detected 48 h after Ag-specific restimulation, but an accumulation of cells in G1 occurred (Table I).

Table I.

CD85/LIR-1/ILT2 and CD152 affect transit from the G0/G1 to the S/G2M phase of the cell cycle in Ag-specific CD4+ T lymphocytes stimulated by recall Agsa

ControlAgAg + HP-F1Ag + HP-F1 + GAMAg + Anti-CD152Ag + Anti-CD152 + GAMAg + GAM
ApobG0/G1cS/G2McApoG0/G1S/G2MApoG0/G1S/G2MApoG0/G1S/G2MApoG0/G1S/G2MApoG0/G1S/G2MApoG0/G1S/G2M
TT 14.7 95.8 4.2 30.2 82.0 18.0 33 83.9 16.1 20.1 96.5d 3.5 30.1 80.1 19.9 20.6 93.2 6.8 31 83.9 16.1 
Ca 10.8 98.7 1.3 20.1 75.1 24.9 23.2 75.9 24.1 9.9 92.2 7.8 23.1 74.6 25.4 10.2 99.8 0.2 19.5 77.1 22.9 
Cr 14.7 95.7 4.3 30.8 82.2 17.8 28.8 78.9 21.1 25 98.9 1.1 30.6 80.6 19.4 22.9 96.7 3.3 32.2 83.2 16.8 
ControlAgAg + HP-F1Ag + HP-F1 + GAMAg + Anti-CD152Ag + Anti-CD152 + GAMAg + GAM
ApobG0/G1cS/G2McApoG0/G1S/G2MApoG0/G1S/G2MApoG0/G1S/G2MApoG0/G1S/G2MApoG0/G1S/G2MApoG0/G1S/G2M
TT 14.7 95.8 4.2 30.2 82.0 18.0 33 83.9 16.1 20.1 96.5d 3.5 30.1 80.1 19.9 20.6 93.2 6.8 31 83.9 16.1 
Ca 10.8 98.7 1.3 20.1 75.1 24.9 23.2 75.9 24.1 9.9 92.2 7.8 23.1 74.6 25.4 10.2 99.8 0.2 19.5 77.1 22.9 
Cr 14.7 95.7 4.3 30.8 82.2 17.8 28.8 78.9 21.1 25 98.9 1.1 30.6 80.6 19.4 22.9 96.7 3.3 32.2 83.2 16.8 
a

Data are from flow cytometric DNA histograms.

b

Percentage of T lymphocytes in the sub-G1 peak. Apo, Apoptosis.

c

Percentage of T cells in the various phases of cell cycle, normalized to the viable cell population.

d

Numbers in bold indicate samples that have a cell cycle distribution significantly different from that of control samples (i.e., Ag).

A possible role of IL-10 and TGF-β that down-regulate T cell responses was taken into account. IL-10 has been detected in all of the experimental conditions, and its production increased when the two inhibitory receptors were cross-linked by GAM antiserum. Increase of IL-10 production ranged from 31 to 108% when CD85/LIR-1/ILT2 was cross-linked, and from 23 to 308% following CD152 cross-linking (Fig. 2,C), depending on the Ag tested. At variance, although recall Ags did not increase detectable TGF-β in the supernatants in comparison with unstimulated cell cultures, a striking increase of this cytokine above controls was induced by HP-F1 or anti-CD152 cross-linked with GAM antiserum (Fig. 2 C).

Addition in the assays of irrelevant mAb (i.e., A3), with or without cross-linking by GAM antiserum, had no effect on proliferation or cytokine production, thus providing a specificity control for the above experiments.

The long-term effect of blocked or cross-linked inhibitory receptors was also investigated. To this end, resting Ag-specific T cells were activated by their respective recall Ags, in the presence of soluble (“blocking”) or cross-linked HP-F1 or anti-CD152 mAb. Subsequently, as shown in Fig. 1,A, secondary restimulations were performed by Ags only, without further mAb addition. Forty-eight hours later, proliferation and cytokine production were measured and compared with those of T lymphocytes previously primed by recall Ags in the absence of HP-F1 or anti-CD152 mAb (Fig. 1,A). Proliferation of T cells primed in a condition of inhibitory receptor blockade was higher than that of T cells previously mAb untreated or treated with an irrelevant mAb (Fig. 3,A). Accordingly, the antecedent blocking of inhibitory receptors increased the production of IL-2, IFN-γ, and IL-13, as compared with control cultures primed in the absence of the mAb (Fig. 3,B). It is of note that a secondary stimulation induced higher production of these cytokines, particularly IL-13, than the first priming (compare mAb untreated controls in Fig. 2,B with the same controls in Fig. 3 B).

FIGURE 3.

Proliferation and cytokine production during secondary Ag stimulation are modulated by previous blockade or cross-linking of CD85/LIR-1/ILT2 and CD152 during the priming phase of CD4+ T lymphocytes. CD4+ T lymphocytes (T) isolated from PBMCs were stimulated using autologous feeder cells (APCs) pulsed with recall Ags, in the absence or in the presence of the indicated mAb (HP-F1, CD152, and A3 mAb, respectively, specific for CD85/LIR-1/ILT2, CD152, and p58.2 receptors). Cells were then stimulated again 10 days later without further mAb addition. Proliferation and cytokine production were measured 48 h later (T12 in Fig. 1 A). Data from one representative donor are shown. A, Mean values of [3H]thymidine uptake from two independent experiments. Bars represent the range/2. B, IL-2, IFN-γ, and IL-13 mean values. C, IL-10 and TGF-β mean values. Supernatants were tested in triplicate and deviation between triplicates was <10% for any reported value. Results are representative of four experiments with similar results. ∗, Samples that are significantly different (p < 0.05) from the control culture (T + APC + Ag); ▨, samples where receptor blockade is exerted by soluble mAb; ▪, samples with receptors cross-linked by mAb plus GAM antiserum.

FIGURE 3.

Proliferation and cytokine production during secondary Ag stimulation are modulated by previous blockade or cross-linking of CD85/LIR-1/ILT2 and CD152 during the priming phase of CD4+ T lymphocytes. CD4+ T lymphocytes (T) isolated from PBMCs were stimulated using autologous feeder cells (APCs) pulsed with recall Ags, in the absence or in the presence of the indicated mAb (HP-F1, CD152, and A3 mAb, respectively, specific for CD85/LIR-1/ILT2, CD152, and p58.2 receptors). Cells were then stimulated again 10 days later without further mAb addition. Proliferation and cytokine production were measured 48 h later (T12 in Fig. 1 A). Data from one representative donor are shown. A, Mean values of [3H]thymidine uptake from two independent experiments. Bars represent the range/2. B, IL-2, IFN-γ, and IL-13 mean values. C, IL-10 and TGF-β mean values. Supernatants were tested in triplicate and deviation between triplicates was <10% for any reported value. Results are representative of four experiments with similar results. ∗, Samples that are significantly different (p < 0.05) from the control culture (T + APC + Ag); ▨, samples where receptor blockade is exerted by soluble mAb; ▪, samples with receptors cross-linked by mAb plus GAM antiserum.

Close modal

TGF-β was not detectable during the 48 h that followed the secondary Ag stimulation (Fig. 3,C), as it occurred after the first priming (Fig. 2,C). In contrast, IL-10 was detected at significant amounts, but its level was lower when T cells were primed previously in the presence of soluble HP-F1 or anti-CD152 mAb, which blocked the ligand/inhibitory receptor interaction (Fig. 3 C).

Cross-linking of CD85/LIR-1/ILT2 or CD152 by GAM antiserum during T cell priming inhibited Ag-specific T cell proliferation after secondary restimulation (Fig. 3,A). Likewise, IL-2, IFN-γ and IL-13 production was reduced (Fig. 3 B).

Conversely, IL-10 production was higher than that detected when T cells were primed in the absence of mAb, and the amount of TGF-β, otherwise undetectable, was sharply increased (Fig. 3 C).

As blockade of CD85/LIR-1/ILT2 triggered cytokine polarization in Ag-stimulated resting/memory T cells, we next evaluated whether the HP-F1 mAb, soluble or cross-linked by GAM antiserum, could modulate IL-2, IFN-γ, IL-13, IL-10, and TGF-β production by effector/memory human CD4+ polyclonal T cell lines generated by repeated Ag stimulation (Fig. 1 B).

To this end, Ag-specific CD4+ T cell lines were restimulated by autologous irradiated PBMCs pulsed with the respective Ags. Simultaneous addition in the culture of HP-F1 mAb, cross-linked by GAM antiserum, inhibited proliferation as shown by a reduced [3H]thymidine uptake (Fig. 4,A). In contrast, addition of soluble HP-F1, which blocks the receptors, increased specific Ag-induced proliferation by 60–120% (Fig. 4,A). Controls with GAM antiserum alone, or with an isotype-matched irrelevant Ab (A3), had no effect on Ag-induced proliferation (Fig. 4 A), both when added as soluble mAb and when cross-linked by GAM antiserum.

FIGURE 4.

Blockade or cross-linking of CD85/LIR-1/ILT2 modulates proliferation and cytokine production of Ag-specific CD4+ T cell lines. CD4+ T cell lines (T) were generated as shown in Fig. 1,B. The fourth stimulation with autologous irradiated feeder cells (APCs) pulsed with recall Ags was performed in the absence or in the presence of the indicated mAb (HP-F1, CD152, and A3 mAb, respectively, specific for CD85/LIR-1/ILT2, and p58.2 receptors). Proliferation and cytokine production were measured 48 h later (T2 of Fig. 1 B). Data are from one representative donor. A, Mean values of [3H]thymidine uptake from two independent experiments. Bars represent the range/2. B, IL-2, IFN-γ, and IL-13 mean values. C, IL-10 and TGF-β mean values. Supernatants were tested in triplicate and deviation between triplicates was <10% for any reported value. Results are representative of five experiments with similar results. ∗, Samples that are significantly different (p < 0.05) from the control culture (T + APC + Ag); ▨, samples where receptor blockade is exerted by soluble mAb; ▪, samples with receptors cross-linked by mAb plus GAM antiserum.

FIGURE 4.

Blockade or cross-linking of CD85/LIR-1/ILT2 modulates proliferation and cytokine production of Ag-specific CD4+ T cell lines. CD4+ T cell lines (T) were generated as shown in Fig. 1,B. The fourth stimulation with autologous irradiated feeder cells (APCs) pulsed with recall Ags was performed in the absence or in the presence of the indicated mAb (HP-F1, CD152, and A3 mAb, respectively, specific for CD85/LIR-1/ILT2, and p58.2 receptors). Proliferation and cytokine production were measured 48 h later (T2 of Fig. 1 B). Data are from one representative donor. A, Mean values of [3H]thymidine uptake from two independent experiments. Bars represent the range/2. B, IL-2, IFN-γ, and IL-13 mean values. C, IL-10 and TGF-β mean values. Supernatants were tested in triplicate and deviation between triplicates was <10% for any reported value. Results are representative of five experiments with similar results. ∗, Samples that are significantly different (p < 0.05) from the control culture (T + APC + Ag); ▨, samples where receptor blockade is exerted by soluble mAb; ▪, samples with receptors cross-linked by mAb plus GAM antiserum.

Close modal

A possible regulatory role of CD85/LIR-1/ILT2 on cytokine production by Ag-specific CD4+ T cell lines was also investigated. Whereas feeder cells alone did not stimulate T cells, the presence of Ags (TT, Ca, or Cr) increased production of IL-2, IFN-γ, and IL-13. Blockade of the inhibitory receptors by the HP-F1 mAb resulted in a further increased production of cytokines. The range of increase was 25–200% for IL-2 and 28–45% for IFN-γ (Fig. 4,B). The increase in IL-13 production was not significant (Fig. 4 B).

In all T cell lines tested, cross-linking of HP-F1 mAb by GAM antiserum led to an almost complete inhibition of IL-2 production (Fig. 4,B). In addition, we observed an inhibition of IFN-γ production ranging from 50 to 80% and of IL-13 production ranging from 25 to 46.5% (Fig. 4,B). Addition of an irrelevant mAb used as control (i.e., A3, both soluble or cross-linked) had no effect on cytokine production (Fig. 4 B).

The presence of soluble HP-F1 mAb during Ag-mediated restimulation of the T cell lines decreased the amount of IL-10 production (Fig. 4,C). TGF-β was not detectable during the first 48 h of Ag restimulation, when cells were challenged both with the Ag alone and in the presence of mAb blocking ligand/inhibitory receptor interactions (Fig. 4,C). In contrast, when HP-F1 mAb was cross-linked by GAM antiserum, the amount of IL-10 was significantly increased, by 120–300%, depending on the Ag (Fig. 4,C), and TGF-β, otherwise undetectable, was produced at high amounts (Fig. 4 C).

Similar data were obtained when the CD152 inhibitory receptor was blocked or cross-linked (data not shown), suggesting a similar role for this molecule in the regulation of the immune response at the effector cell level.

The CD85/LIR-1/ILT2 inhibitory receptor is expressed by both T cells and APCs. Therefore, we asked whether the effects observed for Ag-specific T cells, which require APCs for appropriate Ag stimulation, are due to mechanisms directly related to T cells or to a CD85/LIR-1/ILT2-mediated down-regulation of Ag processing and presentation by APCs, which would indirectly down-regulate T cell function.

To this end, we performed experiments in a T cell activation model that does not require APCs. Proliferation and cytokine production after stimulation via CD3 were evaluated, in the absence or in the presence of soluble or cross-linked HP-F1 mAb.

As shown in Fig. 5, an Ag-specific CD4+ T cell clone (RP.TT) increased its anti-CD3-induced proliferation in response to soluble HP-F1 mAb and decreased it when the mAb was cross-linked. Moreover, addition of soluble mAb increased IL-2 and IFN-γ production, while its cross-linking decreased it (Fig. 5). Conversely, TGF-β production was significantly increased when anti-CD3 stimulation was performed in the presence of cross-linked CD85/LIR-1/ILT2.

FIGURE 5.

CD85/LIR-1/ILT2 and CD152 modulate CD3-induced proliferation and cytokine production of Ag-specific CD4+ T lymphocytes. The Ag-specific CD4+ T cell clone RP.TT was stimulated via CD3 (OKT3 mAb) in the absence or in the presence of the indicated mAb (HP-F1, CD152, and A3 mAb, respectively, specific for CD85/LIR-1/ILT2, CD152, and p58.2 receptors). Proliferation ([3H]thymidine uptake) and cytokine production (IL-2, IFN-γ, and TGF-β) were measured 48 h later. Supernatants were tested in triplicate and deviation between triplicates was <10% for any reported value. Results are representative of three experiments with similar results. ▨, Samples where receptor blockade is exerted by soluble mAb; ▪, samples with receptors cross-linked by mAb plus GAM antiserum.

FIGURE 5.

CD85/LIR-1/ILT2 and CD152 modulate CD3-induced proliferation and cytokine production of Ag-specific CD4+ T lymphocytes. The Ag-specific CD4+ T cell clone RP.TT was stimulated via CD3 (OKT3 mAb) in the absence or in the presence of the indicated mAb (HP-F1, CD152, and A3 mAb, respectively, specific for CD85/LIR-1/ILT2, CD152, and p58.2 receptors). Proliferation ([3H]thymidine uptake) and cytokine production (IL-2, IFN-γ, and TGF-β) were measured 48 h later. Supernatants were tested in triplicate and deviation between triplicates was <10% for any reported value. Results are representative of three experiments with similar results. ▨, Samples where receptor blockade is exerted by soluble mAb; ▪, samples with receptors cross-linked by mAb plus GAM antiserum.

Close modal

These data parallel those obtained when recall Ag stimulation was used, thus indicating that the effects of HP-F1 mAb on Ag-specific T cells is mainly due to interaction of such Ab with CD85/LIR-1/ILT2 expressed by T cells.

We have shown that cross-linking of inhibitory receptors during T cell activation leads to decreased IL-2, IFN-γ, and IL-13 production, paralleled by decreased proliferation, and to an increase of IL-10 and TGF-β production. To determine whether such effects were caused by decreased IL-2 production, exogenous IL-2 (50 UI/ml) was added to cultures of CD4+ T cell lines during the experiments. Cell cultures were restimulated with anti-CD3 in the absence or presence of HP-F1 mAb, soluble or cross-linked by GAM antiserum. An isotype-matched mAb (A3) was used as a control.

Addition of IL-2 prevented the decrease of IFN-γ and IL-13 production and the increase of IL-10 and TGF-β production, which occurred when CD85/LIR-1/ILT2 was cross-linked (Fig. 6).

FIGURE 6.

Exogenous IL-2 prevents modulation of cytokine production mediated by cross-linked CD85/LIR-1/ILT2. The CD4+ T cell line RP10.57 was stimulated via CD3 (OKT3 mAb) in the absence or in the presence of the indicated mAb (HP-F1 and A3 mAb, respectively, specific for CD85/LIR-1/ILT2, and p58.2 receptors). Exogenous IL-2 (50 UI/ml) was added where indicated. Supernatants were harvested 48 h after stimulation and tested for IFN-γ, IL-13, IL-10, and TGF-β production. Supernatants were analyzed in triplicate and deviation among samples was <10% for any reported value. ∗, Samples that are significantly different (p < 0.05) from the control culture (T + OKT3); ▨, samples where receptor blockade is exerted by soluble mAb; ▪, samples with receptors cross-linked by mAb plus GAM antiserum.

FIGURE 6.

Exogenous IL-2 prevents modulation of cytokine production mediated by cross-linked CD85/LIR-1/ILT2. The CD4+ T cell line RP10.57 was stimulated via CD3 (OKT3 mAb) in the absence or in the presence of the indicated mAb (HP-F1 and A3 mAb, respectively, specific for CD85/LIR-1/ILT2, and p58.2 receptors). Exogenous IL-2 (50 UI/ml) was added where indicated. Supernatants were harvested 48 h after stimulation and tested for IFN-γ, IL-13, IL-10, and TGF-β production. Supernatants were analyzed in triplicate and deviation among samples was <10% for any reported value. ∗, Samples that are significantly different (p < 0.05) from the control culture (T + OKT3); ▨, samples where receptor blockade is exerted by soluble mAb; ▪, samples with receptors cross-linked by mAb plus GAM antiserum.

Close modal

Accordingly, addition of IL-2 in the assays was able to restore proliferative responses of the CD4+ T cell clone (RP10.57) down-regulated via inhibitory receptor cross-linking (Table II). Exogenous IL-2 removed the inhibitory effect of CD85/LIR-1/ILT2 cross-linking and allowed transition to the S/G2M phases of the cell cycle (Table II).

Table II.

CD85/LIR-1/ILT2-induced cell accumulation in the G0/G1 phase of the cell cycle is prevented by addition of exogenous IL-2a

ControlOKT3OKT3 + HP-F1OKT3 + HP-F1 + GAM
G0/G1bS/G2MbG0/G1S/G2MG0/G1S/G2MG0/G1S/G2M
RP10.57 96.3 3.7 57.3 42.7 60.1 39.9 88.6d 11.4 
RP10.57+ IL-2c 90.9 9.1 62.0 38.0 62.7 37.3 57.2 42.8 
ControlOKT3OKT3 + HP-F1OKT3 + HP-F1 + GAM
G0/G1bS/G2MbG0/G1S/G2MG0/G1S/G2MG0/G1S/G2M
RP10.57 96.3 3.7 57.3 42.7 60.1 39.9 88.6d 11.4 
RP10.57+ IL-2c 90.9 9.1 62.0 38.0 62.7 37.3 57.2 42.8 
a

Data are from flow cytometric DNA histograms.

b

Percentage of T cells in the various phases of cell cycle, normalized to the viable cell population.

c

50 IU/ml.

d

Numbers in bold indicate samples that have a cell cycle distribution significantly different from that of control samples (i.e., OKT3).

Similar results were obtained using specific anti-CD152 mAb (data not shown).

In conclusion, it appears that the low response to Ag of T cells due to CD85/LIR-1/ILT2 and/or CD152 can be reverted by addition of IL-2.

Several experimental models have been used to assess the inhibitory role of CD152 on T cell responses to Ags both in vitro (5, 21, 22) and in vivo (30, 31, 32), whereas the CD85/LIR-1/ILT2 inhibitory receptor has not been analyzed to the same extent. Moreover, the majority of studies on CD152 have been conducted in an in vitro system using polyclonal stimulators, such as anti-CD3 mAb. In this study, the effects of blockade of CD85/LIR-1/ILT2 and CD152 using mAb specific for the two inhibitory receptors and their cross-linking by GAM antiserum has been evaluated in the course of Ag-specific T cell responses.

In all experiments, the inhibitory role of these molecules has been demonstrated. It is of note that the production of IL-2, IFN-γ, and IL-13 was sharply reduced following cross-linking of the receptors; in contrast, the production of IL-10 and TGF-β was significantly increased. The biological roles played by these cytokines are known (25, 28, 29, 33). IL-2 is a non-Ag-specific proliferation factor for T cells that prompts cell cycle progression of resting cells, thus allowing clonal expansion of activated T lymphocytes. IFN-γ sustains the response of T cells to mitogens and Ags; in addition, it acts in synergy with IL-2 and promotes the expression of IL-2R on the membrane of T lymphocytes. IL-13 acts in concert with IL-2 for the regulation of IFN-γ synthesis. IL-10 inhibits Ag- or anti-CD3-induced proliferation of T cells and down-regulates the production of IL-2 and IFN-γ. Finally, TGF-β inhibits the proliferation of T lymphocytes mainly by reducing IL-2-mediated signals (34). It follows that the outcome of CD85/LIR-1/ILT2 and CD152 engagement results in a control of cell-mediated immunity.

Our observations suggest that the inhibitory receptors play a dual role and, while the biochemical pathways of inhibition are well understood (2, 3), the mechanisms that enhance the production of IL-10 and TGF-β remain to be elucidated (25, 28, 35, 36). In our experimental model, the induction of IL-10 and TGF-β production could suggest an additional role of the inhibitory receptors in the regulation of the intensity of immune responses. It is of note that resting/memory T cells primed with Ag in a condition of inhibitory receptor cross-linking were down-regulated also during the secondary antigenic stimulation in the absence of CD85/LIR-1/ILT2 engagement. These cells were still able to proliferate, although at a lower rate than mAb untreated ones, and to produce cytokines such as IL-10 and TGF-β that may control other T cell clones specific for the same Ag. Such a mechanism could revert effector T cells to a resting/memory condition when the immune response is no longer needed.

The inhibitory effect of CD85/LIR-1/ILT2 and CD152 cross-linking on T lymphocyte proliferation could be due to an enhancement of apoptotic cell death or to an arrest in the G1 phase of cell cycle. Interestingly, during early stages of T cell activation induced by recall Ags or anti-CD3 mAb in the absence of exogenous IL-2, analyses of DNA histograms revealed no increased induction of apoptosis by CD85/LIR-1/ILT2 or CD152 engagement. Rather, the reduction of [3H]thymidine uptake is related to an accumulation of T cells in the G0/G1 phase of the cell cycle. It is of note that CD85/LIR-1/ILT2 or CD152 engagement even reduced the percentage of T cells undergoing apoptosis, possibly because of the lower proliferation as an effect of CD85/LIR-1/ILT2 and CD152 cross-linking.

Because expression of CD85/LIR-1/ILT2 is not restricted to T lymphocytes but is also found in APCs, it was important to rule out the possibility that anti-CD85/LIR-1/ILT2 mAb had a direct effect on T lymphocytes or an indirect effect on Ag presentation by APCs. With regard to this, it is important to assess that CD85/LIR-1/ILT2 is certainly inhibitory on T cell functions as shown in non-Ag-specific assays (6). In these models, such as that of the redirected killing assays, inhibition of OKT3-induced proliferation and of cytokine production does not require the presence of APCs. Nevertheless, the function of CD85/LIR-1/ILT2 on APCs is not completely understood, and there is no evidence as to the ability of CD85/LIR-1/ILT2 to down-regulate endogenous pathways of Ag presentation by APCs.

In this work we show that blockade of the inhibitory receptors during the initial Ag priming of T lymphocytes up-regulates their response. CD85/LIR-1/ILT2 and CD152 blockade during Ag restimulation resulted in higher proliferative responses and increased IL-2, IFN-γ, and IL-13 production, as well as decreased IL-10 and TGF-β release. These results are in support of a critical involvement of inhibitory receptors during Ag-specific activation of resting/memory T lymphocytes. Although the molecular bases of such phenomena are not defined yet, it seems that the possibility to use mAb blocking the inhibitory receptors as an adjuvant in the planning of vaccines, especially for individuals that are low responders to exogenous Ags, bears some relevance.

Finally, several signal pathways have been shown to induce secretion of various cytokines. Some of them, i.e., IL-4 and IFN-γ, have been shown to be IL-2 dependent (25). In addition, inhibition of priming mediated by CD152 cross-linking has been reversed by addition of IL-2 (25). In our experimental models, a prevention of the inhibitory effect obtained by cross-linking of HP-F1 or anti-CD152 mAb occurs after addition of exogenous IL-2 to activated T cells. It is of note that IL-2 blocks the production of IL-10 and TGF-β, thus preventing their negative effects on T lymphocyte functions. We confirm that IL-2 plays a critical role in the activation pathway of memory/effector CD4+ T cells, and we show that it contrasts with CD85/LIR-1/ILT2- and CD152-mediated inhibition.

1

This study was supported by grants from Ministero della Università e Ricerca Scientifica e Tecnologica and from Associazione Italiana Ricerca sul Cancro (to C.E.G. and E.C.). A.M. is supported by a fellowship from the Italian Foundation for Cancer Research.

3

Abbreviations used in this paper: LIR, leukocyte Ig-like receptor; ILT, Ig-like transcript; GAM, goat anti-mouse; TT, tetanus toxoid; Ca, Candida albicans bodies; Cr, Cryptococcus heat-inactivated yeasts.

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