Kinetics and extent of protein tyrosine kinase activation in individual T cells upon antigenic stimulation

Abstract

Using human CD4⁺ T-cell clones and peptide-pulsed antigen-presenting cells (APC) we measured, at the single cell level, different steps in the T-cell activation cascade. Simultaneous analysis of T-cell antigen receptor (TCR) down-regulation and interferon-γ (IFN-γ) production shows that both the level of TCR occupancy and the amount of IFN-γ produced by single T cells increase in an antigen dose-dependent fashion. Conversely, commitment of T cells to IFN-γ production does not occur as soon as a defined number of TCR have been engaged, but requires the same duration of sustained signalling at low as well as at high antigen concentrations. Measurement of phosphotyrosine levels by flow cytometry reveals that, upon conjugation with APC, individual T cells undergo an antigen dose-dependent activation of protein tyrosine kinases (PTK), which parallels the level of TCR occupancy. In antigen-stimulated T cells the increased phosphotyrosine staining is localized in the area of contact with APC, as shown by confocal microscopy. PTK activation is sustained for at least 2 hr after conjugation, and is required to maintain a sustained increase in intracellular Ca²⁺ concentration. Our results show, for the first time, a direct correlation between the level of TCR occupancy and the activation of PTK in individual T cells and offer an explanation for how the number of triggered TCR can be ‘counted’ and integrated in a corresponding biological response.

INTRODUCTION

The interaction of the T-cell receptor (TCR) with specific peptide–major histocompatibility complex (MHC) complexes, displayed on the surface of antigen-presenting cells (APC), triggers a complex cascade of signalling events that ultimately leads to cellular responses, such as interleukin production and cell-cycle progression.¹ The earliest detectable event is the recruitment and activation of two families of protein tyrosine kinases (PTK) followed by the activation of downstream signalling effectors, including calcineurin and Ras pathways.²

Upon conjugation with APC, T cells undergo a sustained intracellular Ca²⁺ concentration ([Ca²⁺]_i) increase which is required for activation of interleukin gene transcription³ and results from serial engagement of TCR with a limited number of specific peptide–MHC complexes.^4–6 We have previously proposed that, in antigen-stimulated T cells, the level of TCR occupancy could control the type and magnitude of T-cell biological response.⁵ More recently it has been shown, by using single cell multiparameter approaches, that the number of engaged TCR regulates the frequency of interferon-γ-(IFN-γ) and interleukin-2- (IL-2) producing cells,⁷ as well as the frequency of T cells that enter the proliferative pool.⁸

The observation that the level of TCR occupancy correlates with the magnitude of T-cell biological responses is difficult to reconcile with the notion that productive TCR engagement results in swift TCR internalization and targeting to the lysosomes.⁹ It is, indeed, not clear how internalized and promptly degraded receptors can be ‘counted’.

In this work we investigated the mechanisms by which signals generated in the T-cell–APC area of contact are transmitted to the nucleus of the T cell leading to a biological response which reflects the level of antigenic stimulation. We report that, even though sustained signalling is essential for T-cell commitment to interleukin production, the amplitude of the T-cell biological response is already determined in single T cells at the level of PTK activation.

MATERIALS AND METHODS

T-cell clones and APC

A DRBI*1104-restricted T-cell clone (KS140) specific for the tetanus toxin peptide (TT830–843; QYIKANSKFIGITE) and DRBI*0101-restricted T-cell clones (6396p5.1.2 and SDM3.5) specific for the measles virus fusion protein peptide (F254–268; GDLLGILESRGIKAR) were used. Autologous Epstein–Barr virus (EBV)-transformed B cells (EBV-B; KS-EBV, DRBI* 1104 and LG-2, DRBI*0101) were used as APC. T-cell clones and EBV-B-cell lines were generated and maintained as described.⁴

Single cell analysis for IFN-γ production and TCR surface expression

EBV-B cells were pulsed with specific peptides and conjugated with T cells as described.⁵ Immediately after conjugate formation 10 μg/ml brefeldin A (kindly provided by Dr G. Engel, Sandoz Pharma, Basel, Switzerland) was added. After 4 hr culture, cells were washed with ice-cold phosphate-buffered saline (PBS) containing 0·5 mm ethylenediamine tetraacetic acid (EDTA), to brake T-cell–APC conjugates. Surface TCR/CD3 complexes were stained with anti-CD3 (OKT3, American Type Culture Collection, Rockville, MD) followed by a goat anti-mouse IgG2a phycoerythrin (PE)-labelled antibody [Southern Biotechnology Associates Inc. (SBA), Birmingham, AL]. After washing, cells were fixed with 3% paraformaldehyde for 10 min at room temperature, washed twice with PBS containing 3% bovine serum albumin (BSA), permeabilized for 10 min with washing buffer (HEPES-buffered PBS containing 0·1% saponin, 3% BSA) and stained with anti-IFN-γ antibodies kindly provided by Dr G. Garotta (γ73A, γ69B, γ123A 5 μg/ml each)¹⁰ and a goat anti-human immunoglobulin biotin-labelled antibody (SBA) in HEPES-buffered PBS containing 0·1% saponin and 5% BSA, followed by a goat anti-mouse IgG1 fluorescein isothiocyanate (FITC)-labelled antibody (SBA) and Tri-color-labelled (TRIC-labelled) streptavidin (Caltag Laboratories, San Francisco, CA). The CD3 and the IFN-γ fluorescences were analysed on a fluorescence-activated cell sorter (FACScan; Becton Dickinson, Mountain View, CA). EBV-B cells were gated out using both forward side scatter/side scatter (FSC/SSC) parameters and TRIC fluorescence.

TCR down-regulation and IFN-γ release

TCR down-regulation and IFN-γ release in the culture medium were measured at different time-points after conjugate formation as previously described.⁵

Intracellular staining for phosphotyrosine

Peptide-pulsed or unpulsed EBV-B cells were loaded with 1 μm 2′,7-bis-(carboxyethyl)-5(6′)-carboxyfluorescin (BCECF-AM; Calbiochem, San Diego, CA) and conjugated with T cells as previously described.⁵ In some experiments T cells were pretreated for 10 min with 10 μm 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP1; Calbiochem),¹¹ the drug was present throughout the assay. At different times of culture, the cells were washed in ice cold PBS 0·5 mm EDTA containing 2 mm Na₃VO₄ (Sigma, St Louis, MO) and fixed in 3% paraformaldehyde, 2 mm Na₃VO₄. After two washes in PBS, 3% BSA, 2 mm Na₃VO₄, the cells were permeabilized with HEPES-buffered PBS containing 0·1% saponin, 2 mm Na₃VO₄ and stained with anti-phosphotyrosine (PTyr) (either 4G10, Upstate Biotechnology, Lake Placid, NY, or PY99, Santa Cruz Biotechnology, Santa Cruz, CA), in HEPES-buffered PBS containing 0·1% saponin, 5% BSA, 2 mm Na₃VO₄, followed by a goat anti-mouse PE-labelled antibody (SBA). The PTyr fluorescence was analysed on a FACScan. EBV-B cells were gated out using both FSC/SSC parameters and green BCECF fluorescence. Comparable results were obtained when EBV-B cells were loaded with the red dye PKH26 (2 μm) (Sigma) as described¹⁰ and the anti-PTyr antibodies were followed by a goat anti-mouse FITC-labelled antibody (SBA). In some experiments T cells were conjugated with unstained peptide-pulsed EBV-B cells. After 30 min of incubation at 37° the cells where fixed, permeabilized and stained with anti-PTyr, anti-ζchain (Santa Cruz) and goat anti-human immunoglobulin biotin-labelled antibody (SBA) followed by a goat anti-mouse immunoglobulin G2b (IgG2b) PE-labelled antibody, a goat anti-mouse IgG1 FITC-labelled antibody (SBA) and TRIC-labelled streptavidin (Caltag Laboratories). The PTyr and the ζ-chain fluorescences were analysed on a FACScan. EBV-B cells were gated out using both FSC/SSC parameters and TRIC fluorescence.

In some experiments 50 mm phenylphosphate was added during the first step of staining.

For confocal microscopy, T cells were conjugated with unstained peptide-pulsed EBV-B cells. At different times of incubation at 37°, the cells were gently resuspended and laid on poly l-lysine-coated slides for 10 min at 37°. The cells were fixed with PBS containing 3% paraformaldehyde, 2 mm Na₃VO_4, permeabilized with washing buffer and stained with an anti-PTyr antibody (Santa Cruz) and an anti-human immunoglobulin biotin-labelled antibody (SBA) in HEPES-buffered PBS containing 0·1% saponin, 5% BSA, 2 mm Na₃VO_4, followed by a FITC-labelled goat anti-mouse antibody (SBA) and Texas Red-labelled streptavidin (SBA). The samples were mounted in 90% glycerol–PBS containing 2·5% 1-4-diazabicyclo (2.2.2) octane (DABCO, Fluka AG, Buchs, Switzerland) and were examined using a Carl Zeiss, LSM 410, confocal microscope (Carl Zeiss, Jena, Germany).

[Ca²⁺]_i measurement

[Ca²⁺]_i was measured as previously described.⁴ Only live, Indo-1-loaded and conjugated T cells were included in the analysis.

RESULTS

In individual T cells TCR occupancy and IFN-γ production increase in an antigen dose-dependent fashion

We have previously shown that measurement of TCR down-regulation is a convenient method to estimate the level of TCR occupancy in T-cell–APC conjugates.⁵ We have also shown that the amount of IFN-γ released in the cell culture medium correlates with the level of TCR down-regulation.⁵ To investigate whether the extent of T-cell activation could reflect the level of TCR occupancy in single cells, we measured TCR down-regulation in parallel with IFN-γ production, by FACS analysis, in antigen-stimulated human T-cell clones.

In agreement with a recent report,⁷ we found that, even at high levels of TCR occupancy, a heterogeneity exists among individual cells, regarding the amount of IFN-γ produced (Fig. 1a–d). However, as shown in Fig. 1(e),(f,) IFN-γ production increased in single cells in an antigen dose-dependent fashion, with increasing TCR occupancy. These results indicate that not only the release of IFN-γ in the culture medium,⁵ but also the amount of IFN-γ produced by individual T cells of a clonal population, increases with the strength of antigenic stimulation.

Figure 1.

Parallel increase of IFN-γ production and TCR down-regulation in single T cells as a function of antigenic stimulation. T cells were conjugated with autologous APC. After 4 hr of culture, cells were surface stained with anti-CD3, then fixed, permeabilized and stained with anti-IFN-γ. (a, b) Clone 6396p5.1.2 conjugated with APC either unpulsed (a) or pulsed with 1 μm peptide (b). (c,d) Clone KS140 conjugated with APC either unpulsed (c) or pulsed with 10 μm peptide (d). The mean FL1 and FL2 intensity of the dot plots are indicated. (e,f) CD3 and IFN-γ staining as a function of antigenic stimulation in clone 6396p5.1.2 (e) and KS140 (f). A representative experiment out of four is shown. Similar results were obtained when T cells were conjugated with APC for longer time periods (5–16 hr, data not shown).

The time of signalling required for induction of IFN-γ production is not dependent on the strength of antigenic stimulation

We next investigated the mechanisms by which the strength of antigenic stimulation could be transduced in a corresponding T-cell biological response.

We first investigated whether T cells could simply ‘count’ the number of TCR engaged at the T-cell–APC contact area and be committed to produce IFN-γ once a critical number of TCR have been triggered. T cells were conjugated with autologous EBV-B cells pulsed with different concentrations of antigenic peptide and IFN-γ production was measured by FACS analysis. At different time-points after conjugation 1 mm EGTA was added to interrupt TCR-mediated signalling at the level of the Ca²⁺ pathway. This treatment resulted in a time-dependent inhibition of IFN-γ production by individual T cells at all the antigen concentrations used for pulsing. Indeed at high as well as at low antigen concentrations EGTA addition, as late as 30–60 min after conjugate formation, resulted in a profound inhibition of IFN-γ production (Fig. 2a). Similar results were observed when IFN-γ release was measured in supernatants of T-cell–APC cocultures treated at different time-points after conjugation with either 1 mm EGTA or 1 μm cyclosporin A (data not shown). Interestingly, at time-points where commitment to IFN-γ production is not yet achieved (30–60 min), T cells stimulated with high concentrations of the specific peptides have already engaged and down-regulated a large number of receptors (refs 5 and 10 and Fig. 2b). Taken together these results indicate that T cells are not committed to IFN-γ production as soon as a given number of TCR have been engaged; yet a minimal time of signalling is required to trigger interleukin production, regardless of the strength of antigenic stimulation.

Figure 2.

The time of signalling required for commitment to IFN-γ production is not dependent on the strength of antigenic stimulation. (a) T cells (clone KS140) were conjugated with autologous APC either unpulsed or pulsed with different peptide concentrations. After 5 hr culture cells were fixed, permeabilized and stained with anti-IFN-γ; 1 mm EGTA was added to the cultures at indicated time-points. The median fluorescence intensity (MFI) is shown. (b) Time kinetics of TCR–CD3 complex down-regulation in T cells (KS140) conjugated with APC pulsed with the indicated concentrations of the specific peptide. The median fluorescence intensity (MFI) is shown as percentage of unstimulated. Data from one representative experiment out of three.

The extent of PTK activation correlates, at the single cell level, with the level of TCR occupancy

Since most of TCR down-regulation occurs earlier than commitment to interleukin production, it is not clear how internalized and promptly degraded receptors could be ‘counted’ and integrated in a corresponding biological response.

To address this question, we investigated the possibility that, in single T cells, the number of engaged TCR could be promptly transduced in a proportional activation of the signalling cascade.

To test this hypothesis we set up an assay to measure, in single T-cell–APC conjugates, the level of PTK activation.

As shown in Fig. 3(a–c) in T cells conjugated for 5 min with peptide-pulsed APC the level of PTyr staining, measured by FACS analysis, increased in an antigen dose-dependent fashion. Treatment of T cells with the src-PTK inhibitor PP1 (10 μm),¹¹ resulted in a profound inhibition of the antigen dose-dependent increase of PTyr staining as well as in a reduction of the basal level of tyrosine phosphorylation in T cells (Fig. 3b). Interestingly, PTK activation in single T cells correlates with the antigen dose-dependent TCR down-regulation measured a short time after conjugate formation (ref. 10 and Fig. 3b). One should note that ligand-induced TCR down-regulation is itself dependent on protein tyrosine phosphorylation mediated by p56lck and p59fyn.¹² Accordingly we found that TCR down-regulation is profoundly inhibited by PP1 (data not shown).

Figure 3.

Antigen dose-dependent increase of phosphotyrosine staining in single T cells. (a–c) T cells were conjugated for 5 min with APC at 37°. Cells were fixed, permeabilized and stained with anti-PTyr. In parallel experiments (b) CD3 down-regulation was measured in fresh T-cell–APC conjugates 5 min after conjugation. (a) Staining for PTyr in clone SDM3·5 (left) and clone 6396p.5.1.2 (right) conjugated with unpulsed or peptide-pulsed APC. (b) PTyr staining as a function of antigenic stimulation in clone SDM3·5 either untreated or treated with 10 μm PP1 and CD3 down-regulation in clone SDM3·5 as a function of antigenic stimulation. (c) PTyr staining in antigen stimulated clone 6396p.5.1.2 and KS140. The median fluorescence intensity (MFI) is shown as percentage of unstimulated. (d) T cells were conjugated with APC either unpulsed or pulsed with 1 μm or 100 μm peptide. Cells were fixed, permeabilized and stained with anti-PTyr either in the absence (left) or in the presence of 50 mm phenylphosphate (right). Data from one representative experiment out of at least three for each T-cell clone. (e) T cells were conjugated for 30 min with APC at 37°. Cells were fixed, permeabilized and stained with anti-PTyr and anti-ζ chain. T cells were conjugated with unpulsed APC (i, iii), or with APC pulsed with 100 μm peptide (ii, iv). In (iii) and (iv) 50 mm phenylphosphate was added during the first step of staining. The mean FL1 and FL2 intensity of the dot plots is indicated. Data from one representative experiment out of three.

In some experiments we added in the staining procedure phenylphosphate, an analogue of phosphotyrosines. This treatment completely abolished PTyr staining in resting, as well as in activated cells (Fig. 3d), indicating that the increased staining for phosphotyrosine measured in activated T cells does not result from unspecific binding of anti-PTyr antibodies.

To better correlate the level of TCR engagement with the activation of PTK in single T cells we measured by FACS analysis the level of PTyr staining and of CD3-ζ expression in T cells conjugated for 30 min with APC. T cells conjugated with peptide-pulsed APC undergo degradation of ζ-chain which is paralleled by an increase in PTyr staining (Fig. 3e, i and ii.)Addition of phenylphosphate did not affect the staining for ζ-chain, but completely abolished PTyr staining (Fig. 3e, iii and iv).

Taken together the above results demonstrate that in individual T cells, the strength of antigenic stimulation and therefore the level of TCR occupancy, are promptly integrated in a corresponding activation of PTK.

Morphological evidence of PTK activation in T-cell–APC conjugates

T cells were conjugated with peptide-pulsed or unpulsed APC, the conjugates were fixed, permeabilized and stained with anti-phosphotyrosine antibodies. In unstimulated T cells most phosphotyrosines were detected in association with the plasma membrane (Fig. 4a), indicating that a basal level of membrane-associated PTK activation is maintained in T-cell clones, as reported for Jurkat cells.^13,14 Upon conjugation with peptide-pulsed APC a remarkable increase of PTyr staining was observed in T cells, which was localized in the T-cell–APC area of contact (Fig. 4b,c). This increase of staining was blocked by treatment with 10 μm PP1 (Fig. 4d). These results support those obtained by FACS analysis (Fig. 3) and indicate that localized activation of PTK in the area of cell contact is the first event of T-cell polarization towards APC.¹⁵ Additional studies are required for an exhaustive identification of the signalling components that could be enriched in the area of cell contact where the pronounced staining for PTyr is detected. Initial observations made on this line indicate that p56lck and ZAP-70 are not accumulated at the cell–cell contact area (data not shown), suggesting that the strong PTyr signal observed, results from an activation of PTK enzymatic activity rather than from their enrichment at the site of cell contact.

Figure 4.

Activation of PTK occurs at the T-cell–APC contact area. T cells were conjugated with either unpulsed APC (a) or APC pulsed with 10 μm peptide (b) or 100 μm peptide (c,d). Cells were either untreated (a–c) or pretreated 10 min with 10 μm PP1 (d), the drug was left in culture throughout the assay. After 5 min at 37°, the conjugates were laid on polylysine-coated slides, fixed, permeabilized and stained with anti-PTyr. T cells were identified by being negative for anti-human immunoglobulin staining (not shown) and are pointed by arrows. Similar results were obtained using three different T-cell clones.

Sustained activation of PTK is required for sustained [Ca²⁺]_i increase

Based on snapshot measurements of the level of tyrosine phosphorylation in single T cells, the above reported data indicate that the level of TCR engagement in the T cell–APC area of contact elicits a proportional activation of PTK. Since a sustained [Ca²⁺]_i increase is required for T-cell activation,^3,4 we tested whether it results from a sustained activation of PTK.

T cells were conjugated with peptide-pulsed APC and the level of PTyr was measured by FACS analysis in single T-cell–APC conjugates at different time-points. As shown in Fig. 5(a), T cells conjugated with peptide-pulsed APC undergo a sustained PTK activation which parallels the time kinetics of TCR down-regulation and [Ca²⁺]_i increase, in antigen stimulated T cells.^5,10 Addition of 10 μm PP1 to T-cell–APC conjugates 10 min before PTyr staining completely abolished the antigen-induced increase of tyrosine phosphorylation at all time-points of the kinetics (Fig. 5a).

Figure 5.

Sustained activation of PTKs is required for sustained [Ca²⁺]_i increase and IFN-γ production. (a) T cells were conjugated with APC pulsed either with 10 μm or 100 μm peptide. At different time-points after conjugation cells were fixed, permeabilized and stained with anti-PTyr. In some samples 10 μm PP1 was added 10 min before Ptyr staining. The median fluorescence intensity (MFI) is shown as percentage of unstimulated. (b) [Ca²⁺]_i increase in T cells conjugated with APC pulsed with 10 μm peptide, in the absence (i) or in the presence of 10 μm PP1 added 2 min (ii) or 7 min (iii) after beginning of sample recording. (c) T cells were conjugated with autologous APC either unpulsed or pulsed with 100 nm and 10 μm peptide. After 5 hr cells were fixed, permeabilized and stained with anti-IFN-γ; 10 μm PP1 was added to the cultures at the time-points indicated. The median fluorescence intensity (MFI) is shown. Data from one representative experiment out of three.

To verify whether a sustained activation of PTK is required for sustained [Ca²⁺]_i increase, we measured intracellular calcium levels in T cells conjugated with peptide-pulsed APC in absence or in presence of PP1. As shown in Fig. 5(b), [Ca²⁺]_i increase in T cells was completely blocked by the addition of 10 μm PP1 at different times after conjugate formation (Fig. 5b, i–iii). In parallel experiments we measured IFN-γ production by antigen-stimulated T cells treated at different times after conjugate formation with 10 μm PP1. This treatment resulted, at low as well as at high antigen concentrations, in a time-dependent inhibition of IFN-γ production by individual T cells (Fig. 5c).

Taken together these results indicate that the number of TCR engaged, at any given time-point, in the T-cell–APC contact area, is integrated in a corresponding level of PTK activation, which in turn results in a sustained [Ca²⁺]_i, that is required for interleukin production.

DISCUSSION

The recruitment and activation of PTK is the earliest known event of the TCR-associated signal transduction cascade, since it is measurable within seconds after TCR triggering and is mandatory for the activation of downstream signalling pathways.²

The measurement of phosphotyrosines by FACS analysis in T-cell–APC conjugates has an important advantage over the detection of tyrosine-phosphorylated substrates by Western blot analysis on total cell lysates, since it allows a quantitative measurement of PTK activation in individual T cells.

In the present work we employed this method to investigate, in antigen-stimulated T cells, the level of PTK activation in parallel with TCR engagement, calcium mobilization and IFN-γ production. We report that, at any given time of sustained T-cell–APC interaction, the strength of antigenic stimulation is transduced, in single T cells, in a corresponding activation of PTK, which in turn maintains sustained [Ca²⁺]_i increase required for interleukin production.

We also show that antigen-induced phosphotyrosine staining is localized in the T-cell–APC area of contact (Fig. 4), indicating that activation of PTK by engaged TCR drives polarization of T-cell cytoskeleton and secretory machinery towards the APC.¹⁵ This is in agreement with recently reported data showing that the induction of cytoskeletal rearrangement requires the presence of at least one intact ITAM motif in the TCR–CD3-ζ complex.¹⁶

Previous studies have shown that, in single T cells, the frequency and amplitude of calcium oscillations correlate with the strength of antigenic stimulation.^17,18 However, intracellular calcium mobilization is a relatively late event with regard to TCR engagement, since it results from PLC-γ phosphorylation, followed by phosphoinositide breakdown, calcium release from the intracellular stores and, finally, opening of calcium channels in the plasma membrane.² Our work extends these observations, indicating that the number of TCR immediately at work in the T-cell–APC contact area is integrated in a corresponding activation of PTK. In addition we demonstrate that activation of src-PTK is required not only to elicit the early spike of Ca²⁺ mobilization but also for the sustained phase of TCR-mediated [Ca²⁺]_i increase.

We also show that the level of phosphotyrosine staining in single T cells tends to increase during the initial time of T-cell–APC interaction, peaking in about 30 min (Fig. 5a). This is probably due to the time required to recruit TCR and signalling components to the area of cell contact and is consistent with a model of activation of downstream signalling pathways based on accumulation of signalling intermediates.¹⁸ At later time points (2–3 hr), PTyr staining decreases to levels even lower than those of unstimulated cells (Fig. 5a). This result may be explained by the observation that following antigenic stimulation, p56lck is inactivated and ZAP-70 is degraded (ref. 19 and D. Penna, S. Müller and S. Valitutti, in preparation) within a few hours.

We also find that the heterogeneity observed among individual T cells at the level of IFN-γ production (ref. 7 and Fig. 1) is not detectable at the early steps of TCR-mediated signal transduction, such as PTK activation and [Ca²⁺]_i increase (Fig 3 and Fig 5). These observations indicate that, in individual T cells, thresholds for interleukin production are determined downstream of TCR engagement and signalling.

Our results offer an explanation for how, in individual T cells, the number of engaged TCR can be ‘counted’ and integrated in a corresponding T-cell biological response.²⁰ We find that commitment of T cells to IFN-γ production does not occur as soon as a defined number of TCR have been engaged. Indeed we show that in conditions where a large number of TCR has been already productively engaged (i.e. 60 min stimulation with high antigen concentrations, Fig. 2b), the block of sustained [Ca²⁺]_i increase with three different treatments (PP1, EGTA and cyclosporin A) acting at distinct levels of Ca²⁺/calcineurin pathway, results in a profound inhibition of IFN-γ production.

Conversely, we show that in individual T cells the level of PTK activation immediately reflects the level of TCR occupancy. This indicates that even though triggered TCR are promptly internalized and degraded, the strength of antigenic stimulation is already integrated in a corresponding activation of the signalling cascade.

We propose that our results are compatible with the following model of signal transmission from the T-cell–APC contact area to the T-cell nucleus. T cells form random conjugates with APC which are mediated by adhesion molecules.²¹ Upon recognition of specific peptide–MHC complexes, cell–cell adhesion increases²¹ and T cells polarize toward APC.^15,16 TCR are serially engaged by peptide–MHC complexes and the number of triggered TCR is integrated, at any given time, in a corresponding activation of the signalling cascade. Productively engaged TCR are promptly removed by internalization and degradation whereas new ones take their place at the T-cell–APC contact area.^5,9 As a consequence, the magnitude of the signal transduction cascade reflects, for a sustained time, the rate of TCR engagement. This allows maintenance of correspondingly elevated concentrations of regulatory factors in the nucleus of T cells for the time required for gene transcription.²²

Abbreviations

[Ca²⁺]_i

intracellular Ca²⁺ concentration

ITAM

immunoreceptor tyrosine-based activation motif

MFI

median fluorescence intensity

PTK

protein tyrosine kinase

PTyr

phosphotyrosine