Laboratoire d'Immunologie Cellulaire, UMR CNRS 7627, CERVI, 83 Boulevard de l'Hôpital, 75013 Paris France
Correspondence to: A. Trautmann
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Abstract |
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Keywords: calcium imaging, dendritic cells, T cells
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Introduction |
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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 TDC 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 TDC interaction.
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Methods |
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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 EpsteinBarr 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 1218 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 58 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 TDC 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.
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Results |
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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 TDC 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 E). However, both the amplitude and the frequency of these oscillations were much lower than those observed in the presence of antigen.
Figure 1(D) 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 TDC 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. 2). 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.
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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), 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 D
). Note that non-oscillating T cell Ca2+ responses could be observed when the DC presented a Ca2+ response (Fig. 3A
) as well as when the DC failed to give a Ca2+ response (not shown). In 58 TDC 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. 3
).
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Discussion |
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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 TDC 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 TDC 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 TDC 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 TDC interactions.
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Acknowledgments |
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Abbreviations |
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APC | antigen-presenting cell |
[Ca2+]i | intracellular free calcium level |
DC | dendritic cell |
fMLP | formyl-MetLeuPro |
GM-CSF | granulocyte macrophage colony stimulating factor |
PAF | platelet-activating factor |
TNF | tumor necrosis factor |
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Notes |
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2 Present address: INSERM U445, Institut Cochin de Génétique Moléculaire, 27 rue du Faubourg Saint Jacques, 75014 Paris, France
Transmitting editor: J. Blanchereau
Received 9 July 1998, accepted 14 December 1998.
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References |
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