Calcium responses elicited in human T cells and dendritic cells by cell–cell interaction and soluble ligands

Mônica Montes, Dorian McIlroy1, Anne Hosmalin2 and Alain Trautmann

Laboratoire d'Immunologie Cellulaire, UMR CNRS 7627, CERVI, 83 Boulevard de l'Hôpital, 75013 Paris France

Correspondence to: A. Trautmann


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The interactions between a human CD4+ T cell clone and monocyte-derived human dendritic cells (DC) were analyzed with an imaging system. The first question addressed was the relationship between the formation of a contact zone and the triggering of a Ca2+ response in the T cells, in the presence or absence of antigen. Interaction of T cells with DC pulsed with the antigen led to the formation of a stable contact zone, followed by the appearance in the T cells of large and sustained Ca2+ oscillations. In the absence of antigen, contact zones formed normally and, surprisingly, Ca2+ responses were also observed, characterized by rare and small transients. Antigen-independent Ca2+ responses were not MHC restricted. The possible influence of Ca2+ responses in the DC on the efficiency of antigen presentation was then investigated. In DC, Ca2+ responses can be elicited by a variety of stimuli: cell adhesion, platelet-activating factor, UTP and chemotactic molecules (formyl-Met–Leu–Pro, RANTES, MIP-1ß and SDF-1{alpha}). Importantly, Ca2+ responses were also induced in ~30% of DC as a result of their interaction with T cells. However, the efficiency of antigen presentation (as judged by the percentage of T cells presenting a Ca2+ response) was independent of the Ca2+ level in DC. Thus, imaging the interactions between human T cells and DC led us to observe two novel phenomena: DC-induced but antigen-independent Ca2+ responses in T cells and T cell-induced Ca2+ responses in DC.

Keywords: calcium imaging, dendritic cells, T cells


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In vivo, the place where a naive T cell has the best chance of meeting its antigen (e.g. following an infection) is at the surface of mature dendritic cells (DC) fully loaded with many antigens, which have migrated into the T zone of secondary lymphoid organs. The particularly high efficiency of DC as antigen-presenting cells (APC) in vivo results from a combination of factors. One is the large number of peptide–MHC complexes at the DC surface, due (i) to the intense activity of DC in antigen capture and processing before their maturation, and (ii) to the expression of high levels of MHC at the surface of mature DC. In addition, the high expression of adhesion and co-stimulation molecules and their ability to release T cell-attracting chemokines also contribute to the overall efficiency of antigen presentation by DC to T cells, including naive T cells (reviewed in 1–3). The exceptional performance of DC as APC has been exploited by a number of groups using adoptive transfer of DC to stimulate immune responses, e.g. in strategies of cancer immunotherapy (46).

The existence of these clinical trials points to the importance of fully understanding how human DC work as APC. Our knowledge of DC biology is increasing rapidly, but one missing piece of information is the analysis at the single cell level of the interaction between normal human DC and T cells. The imaging system used in this study allowed us to analyze a number of parameters during T–DC interactions: conjugate formation, morphological changes, percentage of responding cells and Ca2+ responses of both cell types. The Ca2+ response is of particular interest since, being a very early response, even abortive signals (not followed by later responses) can be detected. An important finding is the existence of Ca2+ responses that are observed in the absence of antigen and even when MHC haplotypes are mismatched. In addition, we have analyzed Ca2+ responses that can be observed in DC either as a result of the interaction with T cells or in response to various soluble ligands. Finally, we have shown that the efficiency of antigen presentation by DC is independent of their Ca2+ level during the T–DC interaction.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Reagents
Antibodies anti-CD1a–phycoerythrin (T6) and anti-CD14–FITC (My4) were from Immunotech (Marseille, France). Diphtheria toxoid (Chiron Technologies, Clayton, Victoria, Australia). IL-2 (Boehringer Mannheim, Meylan, France), IL-4 (PeproTech, London, UK), granulocyte macrophage colony stimulating factor (GM-CSF; Schering Plough, Dardilly, France). Formyl-Met–Leu–Pro (fMLP), platelet-activating factor (PAF) and UTP (Sigma, St Quentin Fallavier, France). Solutions containing 1 mM UTP also included 2 mM MgCl2, since UTP is a powerful chelator of Mg2+. MIP-1ß, SDF-1{alpha} and RANTES were kindly provided by Dr F. Arenzana (Institut Pasteur, Paris, France).

Cells
The human CD4+ clone P28D, specific for diphtheria toxoid, was derived from a healthy HLA-DR6/7 individual as previously described (7). The clone was propagated in complete culture medium (RPMI 1640 supplemented with 10% pooled human AB serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin) by periodic re-stimulation, with the antigen presented by an autologous Epstein–Barr virustransformed B cell line. In addition, 10 U/ml IL-2 containing medium was added every 3 days to maintain cell growth and viability. Cells were used 12–18 days after antigenic re-stimulation.

Monocytes from human peripheral blood lymphocytes HLA-DR7 and non-DR7 were obtained by a 2 h adhesion on six-well plates (10x106 cells/well) and differentiated in vitro in complete medium (RPMI 1640 with 10% FCS, 2 mM L-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin) supplemented with 200 U/ml (18 ng/ml) of GM-CSF and 100 U/ml (20 ng/ml) of IL-4 for 5–8 days (810). As shown by flow cytometry (FACScan; Becton Dickinson), after 1 week of culture, two-thirds of the cells (67 ± 14%) were CD1a+, CD14. However, we expect that practically all the cells used in the present study were CD1a+. Indeed, the morphology of the purified cells was heterogeneous, with some cells presenting a typical, rounded shape, whereas others had an elongated shape. The elongated cells were strongly adherent to the glass, whereas the rounded ones did not adhere, unless the dish was coated with poly-lysine. By immunofluorescence, we observed non-adherent, rounded cells were CD1a+, whereas elongated cells were systematically CD1a. These cells were never taken into account in the rest of the study.

Single-cell calcium measurements and video imaging
For Fura-2 loading, T cells were incubated for 20 min at 37°C with 1 µM Fura-2/AM (Molecular Probes, Eugene, OR), whereas DC were incubated with 0.5 µM Fura-2/AM at room temperature for 20 min. Measurements of intracellular calcium concentration ([Ca2+]i) were performed at 37°C in MS buffer (140 mM NaCl, 5 mM KCl, 10 mM HEPES, pH 7.2, 1 mM CaCl2, 1 mM MgCl2) with a Nikon Diaphot 300 microscope and an IMSTAR imaging system as previously described (11). Briefly, each [Ca2+]i image (every 12 s) was calculated from the average of four fluorescence images after 340 nm excitation and four fluorescence images after 380 nm excitation. Transmitted light images (Nomarski optics) were taken every 12 s between Ca2+ images. Calibrations for DC and T cells were done as previously reported (12).

To monitor T–DC interactions, Fura-2-loaded T cells were added to DC previously immobilized on the glass dish coated with poly-D-lysine (2 µg/ml). For antigen processing, DC were cultured overnight with 50 µg/ml diphtheria toxoid. DC were plated at a density low enough to follow single-cell interactions. Since a fraction of T cells fell too far from DC to interact with any of them, to evaluate the percent of responding T cells (conjugate formation and Ca2+ response), only the T cells located in the immediate vicinity of a DC were taken into account. The immediate vicinity was defined as the surface extending to two T cell diameters beyond the DC.

Proliferation assays
T cell proliferation assays were performed in 96-well U-bottomed microtiter plates in a final volume of 200 µl in complete medium, 1.5x105 cells/well were cultivated in the presence of irradiated DC (3:1 ratio) and variable concentrations of diphtheria toxoid. Proliferation was measured on a Microbeta scintillation counter (Wallac, Evry, France) on day 3 of culture after a 16 h pulse with 1 µCi/well [3H]thymidine (Amersham, Les Ulis, France).

Statistics
Data are expressed as mean ± SD and the significance of difference between two series of results was assessed using two-tailed Student's t-test.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Early events triggered in human T cells by their interaction with DC
When diphtheria toxoid-specific CD4+ T cells were added to DC pulsed overnight with the antigen, 68 ± 14% (in five independent experiments) of the T cells falling in the immediate vicinity of DC (see Methods) formed a contact zone with at least one DC. After a delay of a few seconds following the initial T–DC contact, a Ca2+ response was observed in 100% of the T cells having formed a contact zone (Fig. 1AGo). This response was characterized by a fast [Ca2+]i increase followed in many cases by sustained oscillations (Fig. 1BGo). Although not all T cell types show oscillating Ca2+ responses, this particular human T cell clone has been shown to frequently present an oscillating Ca2+ response, following anti-CD3 stimulation (12) as well as after antigen-specific stimulation by fibroblasts transfected with MHC class II (13). Once the Ca2+ response had started, the T cell never detached from the DC (during a 20 min recording), i.e. the contact zone behaved as a stable structure. However, the T cell retained its capability of deformation around the contact zone, where an intense membrane ruffling was always observed. This persistent mobility allowed the T cell, in some cases, to form additional contact zones with several DC (Fig. 1CGo). Thus, an initial T–DC contact did not inhibit the formation of additional contact zones.



View larger version (72K):
[in this window]
[in a new window]
 
Fig. 1. Interactions between T cells and DC. (A and B) Response of a T cell interacting with an antigen-pulsed DC. (A) Sequence of events observed when a Fura-2 loaded T cell reaches a DC. Image interval: 48 s. (B) Two examples of single T cell Ca2+ responses. At time zero, the T cells appeared in the focal plane of the DC. The dotted line corresponds to the cell shown in (A). (C) A T cell can interact with more than one DC. Top: time zero of the interaction. Bottom: 9 min later. Left: fluorescence of the Fura-2-loaded cell used to visualize its shape. Right: superimposition of Nomarski and Ca2+ images (same colour coding as in A). (D and E) Response of a T cell interacting with a DC in the absence of antigen. (D) Sequence of events observed with an image interval of 48 s. (E) Three examples of single T cell antigen-independent Ca2+ responses.

 
Pretreating DC with tumor necrosis factor (TNF)-{alpha} does not markedly alter the efficacy with which they present the antigen to a T cell clone
It has been reported that addition of TNF-{alpha} to DC differentiated in GM-CSF and IL-4 results in a more mature DC phenotype, closer to the phenotype found in secondary lymphoid organs, where antigen presentation takes place (14,15). We examined whether TNF-{alpha}-treated cells behaved as better APC for T cells under our experimental conditions. First, we confirmed that DC treated with TNF-{alpha} for 24 h had a phenotype typical of mature DC, as judged from surface marker expression (DR and B7-2). In addition, TNF-{alpha}-treated DC exhibited more dendrite-like structures which are often mobile, including during conjugate formation with T cells. We then compared the T cell Ca2+ responses triggered by TNF-{alpha}-treated or untreated DC. Neither the percentage of responding T cells nor the amplitude of the mean initial Ca2+ transient was altered when T cells interacted with antigen-pulsed, TNF-{alpha}-treated DC. It is known that immature DC such as Langerhans cells are poor APC for naive T cells, but that they can present antigen to activated (memory/effector) T cells (16). Our data show that immature DC behave as quite efficient APC for a T cell clone.

T cell responses elicited by DC in the absence of antigen
Next, we examined the antigen specificity of the events described above. When T cells were left to adhere onto DC in the absence of antigen, formation of contact zones was still observed. The fraction of T cells forming conjugates with DC was 60 ± 3.4% (n = 4 experiments), i.e. not significantly different from the percentage observed in the presence of antigen. This observation is in line with previous reports concerning the formation of T–DC conjugates in the absence of antigen (1720). More surprising was the fact that in T cells which had formed a conjugate with a DC in the absence of antigen, Ca2+ responses were observed in 100% of the cases. Like most antigen-dependent Ca2+ responses, antigen-independent Ca2+ responses were oscillating (Fig. 1D and EGo). However, both the amplitude and the frequency of these oscillations were much lower than those observed in the presence of antigen.

Figure 1(D)Go also illustrates the fact that formation of a contact zone in the absence of antigen did not prevent the crawling of the T cell on the DC. It was a typical observation that during antigen independent Ca2+ responses, T cells retained a mobility even higher than that observed when antigen recognition triggered a full blown Ca2+ response.

Next, we asked whether the antigen-independent, DC-induced Ca2+ responses could be followed by more distal responses like T cell proliferation. Indeed, in the absence of antigen, thymidine incorporation was significantly (6 times) higher in T cells cultured with DC than in control T cells cultured without APC (data not shown). Not surprisingly, this response was much lower than that obtained in the presence of antigen, where thymidine incorporation was 60 times higher than in control T cells, with a half maximal stimulation at ~0.5 µg/ml of diphtheria toxoid (data not shown).

We then examined if antigen-independent Ca2+ responses depended upon the MHC haplotype (DR7 in our case). When the same T cells were made to interact with DR7 DR4 DC (since there is some cross-reactivity between DR7 and DR4), formation of contact zones and small oscillating Ca2+ responses were observed (data not shown), just like with DR7+ DC in the antigen-independent experiments. The only difference was that in 4% of the cases (n = 54), formation of a contact zone with DR7 DC was not followed by a Ca2+ response, a situation which was never observed when T cells and DC were MHC-matched. As expected, T cell Ca2+ responses were similar with antigen-pulsed or unpulsed DR7 DC. These data show that neither contact zone formation nor the initiation of antigen-independent Ca2+ responses depend upon the MHC class II haplotype of the DC; however, this does not rule out an haplotype-independent role of MHC class II molecules (see Discussion).

Different stimuli can trigger Ca2+ responses in DC
Having characterized DC-induced T cell Ca2+ responses, we examined a phenomenon which has been much less explored, i.e. Ca2+ responses in DC. Of particular interest would be the existence of Ca2+ responses that would be observed in DC during T–DC interactions. However, before getting to this point, it seemed useful to test first the effects of cell adhesion and of a series of soluble ligands on the DC [Ca2+]i level.

A light adhesion of DC to glass was sufficient to trigger [Ca2+]i transients in 10% of the DC. A stronger adhesion to poly-lysine (see Methods) triggered a sustained Ca2+ increase, superimposed with Ca2+ transients, in 65% of the DC (Fig. 2Go). Both adhesion and Ca2+ transients were reversible, Ca2+ transients stopping well before cell detachment. Indeed, the intervals between the Ca2+ transients progressively increased with time and, generally, after 3 h they were scanty, whereas most DC were still adherent. DC detachment was observed after an overnight incubation.



View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2. Ca2+ responses elicited in DC by adhesion, UTP, PAF or IL-4 (A), or by chemotactic molecules such as fMLP, RANTES, MIP-1ß and SDF-1{alpha} (B). The trace marked adhesion corresponds to a single cell; its time calibration bar is marked 10 min. All the other traces are the averages of six to 15 individual curves; the corresponding calibration is marked in the left corner of each panel. The addition of the ligands is marked by arrows.

 
The presence on human DC of different G-protein coupled, serpentine receptors, has been examined. We first looked if purinergic receptors, already described in monocytes and macrophages (21), were also functional in DC. UTP (1 mM) triggered a strong Ca2+ response in DC, revealing for the first time the presence of P2U receptors in DC (22). We checked that this receptor was not functionally expressed in our T cell clone, a feature that was subsequently exploited to trigger Ca2+ responses specifically in DC (see below). The inflammatory molecule PAF is another molecule able to evoke a Ca2+ response in DC (Fig. 2Go). The cytokine IL-4 used to differentiate peripheral monocytes into DC also triggered Ca2+ responses in DC (Fig. 2Go). Additionally, a number of chemotactic agents were able to trigger Ca2+ responses in DC and to affect their migration (23,24). As shown in Fig 2Go, four of these molecules, fMLP, RANTES, MIP-1ß and SDF-1{alpha}, were indeed able to elicit an increase in [Ca2+]i in human DC.

In other cell types, Ca2+ signals have been shown to be associated with MHC class II signaling, on one hand, and with endocytosis, on the other (15,25). We thus tested if Ca2+ responses could be evoked in DC following MHC class II cross-linking or following stimulation with BSA-mannose, which is endocytosed via the DC mannose receptor (10,15). None of these stimuli triggered Ca2+ responses in DC.

Antigen recognition by T cell is not altered by the DC Ca2+ level
Having shown that the Ca2+ level in DC could be modulated by many physiological ligands, we wondered whether a Ca2+ rise in the DC could modify its efficiency in triggering a Ca2+ response in T cells. As mentioned above, the question could be properly addressed by using UTP. To our disappointment, the percentage of T cells responding to antigen presentation was not significantly altered when UTP was added together with T cells (86% responding T cells versus 80% in this series of experiments).

It could be argued that treating DC with UTP, besides causing a Ca2+ rise, might have negative effects on the efficiency of antigen presentation. Thus, instead of triggering DC Ca2+ responses with UTP, we made a statistical comparison of antigen recognition by T cells interacting with DC having a low Ca2+ level and with DC having a high Ca2+ level (e.g. corresponding to adhesion-induced Ca2+ responses). Again, the efficiency with which antigen is recognized on DC appeared independent of their [Ca2+]i level.

Can T cells also trigger Ca2+ responses in DC?
Having observed that DC could present Ca2+ increases in response to cell adhesion and to a variety of soluble ligands, we next examined if the interaction with T cells could also trigger a Ca2+ response in DC. As shown in Fig. 3(A and B)Go, in some instances, shortly after the initial contact between the T cell and an antigen-pulsed DC, a Ca2+ response was triggered almost simultaneously in the two cells, the T cell response always preceding the response in the DC. In other cases, the Ca2+ response was only observed in the T cell (Fig. 3C and DGo). Note that non-oscillating T cell Ca2+ responses could be observed when the DC presented a Ca2+ response (Fig. 3AGo) as well as when the DC failed to give a Ca2+ response (not shown). In 58 T–DC pairs where the Ca2+ level of both cells was monitored simultaneously and where the DC did not show adhesion-induced Ca2+ transients, DC-induced Ca2+ responses were observed in 100% of the T cells, whereas T cell-induced Ca2+ responses were observed in 31% of the DC (Fig. 3Go).



View larger version (81K):
[in this window]
[in a new window]
 
Fig. 3. Ca2+ responses can also be triggered in DC by T cell contact. (A) Example of Ca2+ responses observed both in the T cell (continuous line) and in the antigen-pulsed DC (dotted line). (B) Sequence of images (2 min. interval) showing a Ca2+ response in the smaller T cell and then in the DC, following cell–cell interaction. (C) Example where a Ca2+ response was observed only in the T cell (same symbols as in A). (D) Sequence of images (2 min. interval) showing a Ca2+ response only in the T cell following T–DC interaction. Note the marked flattening of the T cell in the last image. The fluorescence of the flattened area is too dim to be detected.

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
This report constitutes the first analysis, at the single-cell level, of the initial events occurring during the interaction of a human CD4+ T cell with human DC. The first detectable event is the appearance of a contact zone, a structure which is both stable (it does not break) and dynamic (membrane ruffling is sustained). In 100% of the cases, with or without antigen, formation of the contact zone is followed by a Ca2+ response. However, the presence of the antigen strongly influenced the amplitude of the Ca2+ response and of subsequent T cell proliferation.

An important finding of this paper is thus the existence of several responses triggered by DC in an antigen-independent fashion: full adhesion, small Ca2+ responses and weak proliferation. The last response, presumably controlled by the first two, has not been studied in detail. The absence of MHC haplotype dependence of the early responses might suggest that they do not require the interaction of MHC with the TCR. Previous studies have shown that the interaction between LFA-1 and ICAM-1 was of major importance in the formation of conjugates between human T cells and DC (19,26). However, we observed that the mean antigen-independent Ca2+ response of responding T cells interacting with DC was not altered in the presence of 2 µg/ml anti-ICAM-1 (data not shown). In addition, we have observed similar antigen-independent Ca2+ responses induced by DC in a transgenic murine model. In this system, we have confirmed that, although antigen-independent Ca2+ responses do not depend upon the MHC haplotype, they are abolished when DC are purified from mice deficient in MHC class II molecules (20).

In the absence of antigen, the Ca2+ response in the T cell is composed of largely spaced oscillations of small amplitude. Following antigen recognition, both the amplitude and the frequency of these oscillations is increased. The functional relevance of these oscillations is unclear. In this T cell clone, the Ca2+ response is frequently oscillating, whereas in other T cells, the initial Ca2+ peak is followed by a plateau of lower amplitude (e.g., see 27). It is likely that in both situations, the T cells are endowed with two feed-back mechanisms (12,28). A positive feed-back is responsible for the large Ca2+ increases (initial peak plus oscillations when they exist), and, when [Ca2+]i reaches levels which might become cytotoxic, a negative feed-back pulls [Ca2+]i towards its resting level. It may well be that different ways of synchronizing these two mechanisms lead either to an oscillating response or to a smoothly inactivating one.

We have shown that the Ca2+ response in the T cell reduced its mobility but did not abolish it and that a high [Ca2+]i level in a T cell interacting with a DC is not accompanied by a rounding of the T cell similar to that observed when the same T cell interacts with an antigen-pulsed L cell (11). This suggests that a DC delivers a signal promoting T cell membrane ruffling, in addition to triggering a Ca2+ signal which tends to round up and immobilize the T cell. In addition, we have observed that formation of one T–DC contact zone does not prevent the T cell from forming additional contact zones with other DC and that T cells interacting with unpulsed DC are highly mobile. These facts may be important when considering interactions between T cells and interdigitating cells in the T zone of secondary lymphoid organs. Functionally, when T cells are interacting with DC, searching their cognate antigen, it is not desirable that the interaction between a DC and a T cell lasts for too long if the DC does not present the `correct' antigen. If our in vitro observations are valid for the in vivo situation, an intimate but mobile and probably transient interaction between the T cell and the DC (although we never observed detachment of T cells over our usual 20 min period of observation) is ideally suited for a thorough and fast scanning of successive DC by T cells (29).

Looking now at DC, we have shown that Ca2+ responses can be evoked in DC by a number of stimuli. First, Ca2+ responses are elicited by cell adhesion. This feature is shared with a number of other cells, and in particular endothelial cells and neutrophils (30,31). In neutrophils, this response may result from the cross-linking of CD11b/CD18 integrin. We have not examined the surface molecule involved in eliciting this response in DC. The presence of P2U receptors in DC was revealed by UTP-induced Ca2+ responses in vitro. In vivo, these receptors can be stimulated by ATP. The level of ATP in interstitial fluids is usually very low. However, ATP levels high enough to stimulate these receptors and thus to cause a Ca2+ increase in DC can be found in the vicinity of purinergic nerve terminals; interestingly, Langerhans cells are intimately associated with nerve endings (2). Probably more importantly, P2U receptors in DC could be activated in the vicinity of dying cells when they lose their cellular ATP (for a review, see 32).

Thus, it is very likely that in vivo the Ca2+ level in a DC is not always at its resting level, given the number of physiological ligands able to trigger Ca2+ responses in these cells. Such Ca2+ changes are expected to trigger Ca2+-dependent phenomena such as the exocytotic release of cytokines or chemokines. This prompted us to examine the possible role of such variations in [Ca2+]i on the efficiency of antigen presentation. However, in our experimental conditions, an instantaneous modulation could not be evidenced. Finally, we have observed than in one-third of the T–DC conjugates, a Ca2+ response could be observed not only in the T cell but also in the DC. As mentioned, this T cell-induced Ca2+ response in the DC is unlikely to affect the efficiency of antigen recognition by the T cell interacting with the DC. However, one cannot exclude that in vivo it could lead to chemokine secretion by the DC, helping the recruitment of additional T cells. In addition, repeated stimulations of the DC by interacting T cells may contribute to the maturation process of the DC.

In conclusion, the interaction between a human CD4+ T cell clone and monocyte-derived human DC have the following characteristics: high probability of conjugate formation, even in the absence of antigen; existence of Ca2+ responses, even in the absence of antigen, but with a much larger amplitude when the antigen is recognized; persistence of a T cell mobility after the formation of stable T–DC conjugates, allowing a scanning of the DC surface by the T cell. We have recently observed similar features with murine ex vivo splenic DC interacting with ex vivo naive T cells (20), which underlines their general relevance for T–DC interactions.


    Acknowledgments
 
This study was supported by CNRS, INSERM, Association pour la Recherche contre le Cancer and Ministère de la Recherche. M. M. was supported by a fellowship from Colciencias (Colombia). The authors thank Hélène Gary-Gouy and Cecile Arrieumerlou for their help with P28 cells, and Georges Bismuth, Jérôme Delon and Roland Liblau for critical reading of the manuscript.


    Abbreviations
 
APCantigen-presenting cell
[Ca2+]iintracellular free calcium level
DCdendritic cell
fMLPformyl-Met–Leu–Pro
GM-CSFgranulocyte macrophage colony stimulating factor
PAFplatelet-activating factor
TNFtumor necrosis factor

    Notes
 
1 Present address: Department of Genetics, University of Osaka Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan Back

2 Present address: INSERM U445, Institut Cochin de Génétique Moléculaire, 27 rue du Faubourg Saint Jacques, 75014 Paris, France Back

Transmitting editor: J. Blanchereau

Received 9 July 1998, accepted 14 December 1998.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Steinman, R. M. 1991. The dendritic cell system and its role in immunogenicity. Annu. Rev. Immunol. 9:271.[ISI][Medline]
  2. Hart, D. N. 1997. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 90:3245.[Free Full Text]
  3. Banchereau, J. and Steinman, R. M. 1998. Dendritic cells and the control of immunity. Nature 392:245.[ISI][Medline]
  4. Young, J. W. and Inaba, K. 1996. Dendritic cells as adjuvants for Class I Major Histocompatibility complex-restricted antitumor immunity. J. Exp. Med. 183:7.[ISI][Medline]
  5. Girolomoni, G. and Ricciardi-Castagnoli, P. 1997. Dendritic cells hold promise for immunotherapy. Immunol. Today 18:102.[ISI][Medline]
  6. Hsu, F. J., Benike, C., Fagnoni, F., Liles, T. M., Czerwinski, D., Taidi, B., Engleman, E. G. and Levy, R. 1996. Vaccination of patients with B-cell lymphoma using autologous antigen-pulsed dendritic cells. Nat. Med. 2:52.[ISI][Medline]
  7. Triebel, F., Missenard-Leblond, V., Autran, B., Couty, M. C., Charron, D. J. and Debré, P. 1984. Specific proliferative human T cell clones with specificity for diphtheria toxoid: genetic and molecular restriction by class II antigens. Eur. J. Immunol. 14:697.[ISI][Medline]
  8. Sallusto, F. and Lanzavecchia, A. 1994. Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony stimulating factor plus interleukin-4 and down regulated by tumor necrosis factor. J. Exp. Med. 179:1109.[Abstract]
  9. Romani, S., Gruners, S., Brang, D., Kampgen, E., Lenz, A., Trockenbacher, B., Konwalinka, G., Fritsch, P. O., Steinman, R. M. and Schuler, G. 1994. Proliferating dendritic cell progenitors in human blood. J. Exp. Med. 180:83.[Abstract]
  10. Avrameas, A., McIlroy, D., Hosmalin, A., Autran, B., Debré, P., Monsigny, M., Roche, A. C. and Midoux, P. 1996. Expression of a mannose/fucose membrane lectin on human dendritic cells. Eur. J. Immunol. 26:394.[ISI][Medline]
  11. Donnadieu, E., Bismuth, G. and Trautmann, A. 1994. Antigen recognition by helper T cells elicits a sequence of distinct changes of their shape and intracellular calcium. Curr. Biol. 4:584.[ISI][Medline]
  12. Donnadieu, E., Bismuth, G. and Trautmann, A. 1992. Calcium fluxes in T lymphocytes. J. Biol. Chem. 267:25864.[Abstract/Free Full Text]
  13. Donnadieu, E., Cefai, D., Tan, Y. P., Paresys, G., Bismuth, G. and Trautmann, A. 1992. Imaging early steps of human T cell activation by antigen-presenting cells. J. Immunol. 148:2643.[Abstract/Free Full Text]
  14. Winzler, C., Rovere, P., Rescigno, M., Granucci, F., Penna, G., Adorini, L., Zimmermann, V. S., Davoust, J. and Riccardi-Castagnoli, P. 1997. Maturation stages of mouse dendritic cells in growth factor-dependent long term cultures. J. Exp. Med. 185:317.[Abstract/Free Full Text]
  15. Sallusto, F., Cella, M., Danieli, C. and Lanzavecchia, A. 1995. Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. J. Exp. Med. 182:389.[Abstract]
  16. Inaba, K., Schuler, G., Witmer, M. D., Valinsky, J., Atassi, B. and Steinman, R. M. 1986. Immunologic properties of purified epidermal Langerhans cells. Distinct requirements for stimulation of unprimed and sensitized T lymphocytes. J. Exp. Med. 164:605.[Abstract]
  17. Inaba, K., Inaba, M., Romani, N., Aya, H., Deguchi, M., Ikehara, S., Muramatsu, S. and Steinman, R. M. 1992. Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony stimulating factor. J. Exp. Med. 176:1693.[Abstract]
  18. Inaba, K. and Steinman, R. M. 1986. Accessory cell-T lymphocyte interactions. Antigen-dependent and -independent clustering. J. Exp. Med. 163:247.[Abstract]
  19. Hauss, P., Selz, F., Cavazzana-Calvo, M. and Fischer, A. 1995. Characteristic of antigen-independent and antigen-dependent interaction of dendritic cells with CD4+ T cells. Eur. J. Immunol. 25:2285.[ISI][Medline]
  20. Delon, J., Bercovici, N., Raposo, G., Liblau, R. and Trautmann, A. 1998. Antigen-dependent and antigen-independent Ca2+ responses triggered in T cells by dendritic cells compared to B cells. J. Exp. Med., 188:1473.[Abstract/Free Full Text]
  21. Greenberg, S., Di Virgilio, F., Steinberg, T. H. and Silverstein, S. C. 1988. Extracellular nucleotide mediate Ca2+ fluxes in J774 macrophages by two distinct mechanisms. J. Biol. Chem. 263:10337.[Abstract/Free Full Text]
  22. Alonso-Torre, S. R. and Trautmann, A. 1993. Calcium responses elicited by nucleotides in macrophages. Interaction between two receptor subtypes. J. Biol. Chem. 268:18640.[Abstract/Free Full Text]
  23. Sozzani, S., Sallusto, F., Luini, W., Zhou, D., Piemonti, L., Allavena, P., Van Damme, J., Valitutti, S., Lanzavecchia, A. and Mantovani, A. 1995. Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokines. J. Immunol. 155:3292.[Abstract]
  24. Sozzani, S., Luini, W., Borsatti, A., Polentarutti, N., Zhou, D., Piemonti, L., D'Amico, G., Power, C., Wells, T., Gobbi, M., Allavena, P. and Mantovani, A. 1997. Receptor expression and responsiveness of human dendritic cells to a defined set of CC and CXC chemokines. J. Immunol. 159:1993.[Abstract]
  25. Lew, D. P., Anderson, T., Hed, J., Di Virgilio, F., Pozzan, T. and Stendhal, O. 1985. Ca2+-dependent and Ca2+-independent phagocytosis in human neutrophils. Nature 315:509.[ISI][Medline]
  26. Scheeren, R. A., Koopman, G., Van der Baan, S., Meijer, C. J. and Pals, S. T. 1991. Adhesion receptors involved in clustering of blood dendritic cells and T lymphocytes. Eur. J. Immunol. 21:1101.[ISI][Medline]
  27. Delon, J., Bercovici, N., Liblau, R. and Trautmann, A. 1998. Imaging antigen recognition by naive CD4+ T cells: compulsory cytoskeletal alterations for the triggering of a Ca2+ response. Eur. J. Immunol. 28:716.[ISI][Medline]
  28. Dolmetsch, R. E. and Lewis, R. S. 1994. Signaling between intracellular Ca2+ stores and depletion-activated Ca2+ channels generates [Ca2+]i oscillations in T lymphocytes. J. Gen. Physiol. 103:365.[Abstract]
  29. Ingulli, E., Mondino, A., Khoruts, A. and Jenkins, M. K. 1997. In vivo detection of dendritic cell antigen presentation to CD4+ T cells. J. Exp. Med. 185:2133.[Abstract/Free Full Text]
  30. Schwartz, M. A. 1993. Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium. J. Cell. Biol. 120:1003.[Abstract]
  31. Richter, J., Ng-Sikorski, J., Olsson, I. and Andersson, T. 1990. Tumor necrosis factor-induced degranulation in adherent human neutrophils is dependent on CD11b/CD18 integrin-triggered oscillations of cytosolic free Ca2+. Proc. Natl Acad. Sci. USA 87:9472.[Abstract]
  32. Gordon, J. L. 1986. Extracellular ATP: effects, source and fate. Biochem. J. 233:309.[ISI][Medline]