EP1704413A1

EP1704413A1 - Examination method for phagocytosis

Info

Publication number: EP1704413A1
Application number: EP04804202A
Authority: EP
Inventors: Marion Schneider; Jörg WIEDENMANN
Original assignee: Universitaet Ulm
Current assignee: Universitaet Ulm
Priority date: 2003-12-22
Filing date: 2004-12-22
Publication date: 2006-09-27
Also published as: DE10361243B4; DE10361243A1; WO2005062043A1

Abstract

The invention relates to a method for the quantification and qualification of cell activities, phagocytized particles to be processed and/or fragments of phagocytized particles to be presented, wherein particles marked with fluorescence are initially provided, said particles comprising at least one pH-dependent fluorescent dye. The marked particles are then incubated with cells which are to be examined and finally analysed in a fluorescence microscopic, fluorimetric and/or fluorescence cytometric manner. Particles which are marked with fluorescence can be detected at least partially over at least 12 hours.

Description

description

Examination procedure for phagocytosis

The invention relates to a method for examining the activity of cells to process phagocytized particles and / or to present fragments of phagocytated particles.

An important part of the immune response of mammals is the uptake, breakdown and processing of pathogens, dead or aberrant body cells and other foreign substances. Here, the particles recognized as foreign or undesirable are absorbed by certain cells in the blood and broken down intracellularly. The cells capable of this so-called phagocytosis essentially comprise myeloid cells, macrophages, granulocytes, monocytes, endothelial cells and dendritic cells as well as precursors and differentiation products derived from these cell types.

A prerequisite for the phagocytosis of the undesired particles is that the cells capable of phagocytosis bind the particle and as

Detect "undesired". Only then is the particle enclosed and absorbed by the cell. An important factor for the detection of the particles is the so-called opsonization, in which the

Surface of the particles Opsόnine, d. H. z. B. antibodies or complements. The absorbed particles are broken down into fragments by the corresponding cells and in the

Context with molecules of class MHC class II and in individual cases also of MHC class 1 as antigens presented again on the surface. This degradation process, called processing, takes place primarily by means of modification or degradation by pH-dependent denaturation, by reactive oxygen species and / or proteolysis. These processing reactions are carried by enzymes from the corresponding immune cells.

After recognition, phagocytosis and processing, the modified and fragmented foreign particles are made accessible by the antigen-presenting cells on the cell surface for subsequent processes of the immune reaction.

A disturbance in the immune response can therefore lie in opsonization, phagocytosis, processing and / or in the antigen presentation. Corresponding diseases that are based on such defects in the immune system can manifest themselves, for example, through a lack of immune response to pathogens or else through an immunological overreaction. These defects can therefore z. B. manifest as immune insufficiency, tumor formation, allergy or autoimmune disease. These diseases can be caused, for example, by genetic defects, which can impair the functionality of the enzymes involved, or by a disturbed regulation of the enzymes involved, e.g. B. as a result of an infection. Possible therapy for this is either stimulation of the immune system or suppression of the immune response. However, these therapies require a precise diagnosis of the underlying anomaly (s), since every intervention in the immune system has very far-reaching consequences and a corresponding therapy should therefore intervene as specifically as possible. Therefore, for the diagnosis and research of such diseases as well as for the search for therapeutically usable substances that act, for example, as immunostimulants, a precise examination of the processes taking place in this area of the immune response is necessary. Furthermore, an investigation of these processes also plays a role in the analysis of potentially pathogenic agents, such as. B. allergens, as well as for one Monitoring and reviewing possible effects in therapeutic treatment play an important role.

Various methods for examining the phagocytosis performance of immune cells have already been described. For example, whole blood or isolated granulocytes were incubated with microorganisms or particles that were themselves labeled with fluorescent substances. Subsequent measurement of the recorded fluorescence allowed conclusions to be drawn about the phagocytic activity. For example, EP 0 435 226 B1 describes a method in which mammalian leukocytes are incubated in whole blood with fluorescence-labeled Gram-negative bacteria. FITC (fluorescein isothiocyanate) is used here as the fluorescent dye. Phagocytic activity could be demonstrated here by an increase in fluorescence in the phagocytic granulocytes and monocytes.

Such methods can be used to diagnose disorders which relate to the uptake of particles recognized as foreign to the body or as undesirable, that is to say phagocytosis in the true sense. The diagnosable disorders are primarily in the area of non-specific immunity, in which monocytes and granulocytes absorb and break down undesirable particles in order to eliminate them. Faults in the subsequent processes, in particular the processing of the ingested particles and the subsequent presentation on the surface of the phagocytic cells, are not recognized using conventional methods. In particular, disorders of the dendritic cells, which are primarily responsible for the antigen presentation as a prerequisite for induction of the primary cytotoxic T cell response, are not covered by this. The object of the invention is therefore to provide a method with which the processes downstream of phagocytosis, that is to say in particular the processing and the antigen presentation, can be examined. Above all, this method is intended to make it possible to diagnose corresponding diseases, in order then to treat the defect in a targeted manner if necessary. In particular, the new method is intended to detect disorders of the dendritic cell functions which are crucial for the generation of a primary cytotoxic T cell response. The aim of the new method is to be able to detect the cellular initiators of a specific immune response. These are, in particular, macrophages and the various representatives of the dendritic cell series, such as lymphoid and myeloid dendritic cells, and their phenotypically definable developmental stages circulating in the blood. These different cells are responsible for the sensitization and recruitment of memory cells of the specific immunity and are therefore important target points for diagnosing and treating possible disorders.

Furthermore, the new method should above all be able to investigate the ability to process these various phagocytic cells and their ability to present antigens, or should it be possible to detect corresponding disorders. With the new method, the diagnosis of clinical clinical pictures, in particular of severe clinical pictures, is to be made possible, which are based on defects in molecular "trafficking", whereby the phagocytosis of foreign particles, infectious agents or agents, dead or aberrant endogenous cells, such as e.g. B. tumor cells, and the subsequent processing and presentation of these particles is disturbed or impaired. - α -

Furthermore, the new method is intended to enable screening and / or analysis of potential therapeutically active substances which change the immune response in the desired manner. The aim of the new procedure is also to be able to investigate and analyze the effects of therapeutic interventions in the event of immune response disorders. In addition, the new method should make it possible to screen and / or analyze potential pathogenic or allergenic substances which cause disorders in the above-mentioned processes of the immune response.

This object is achieved by a method as described in claim 1. Preferred embodiments are set out in dependent claims 2 to 26. Claims 27 to 29 relate to various uses of this method. Claims 30 and 31 claim a reagent kit which is suitable for carrying out the method according to the invention. The wording of all claims is hereby incorporated by reference into the content of the description.

The method according to the invention is used to investigate the activity of cells to process phagocytized particles as a result of the fusion of the endosome and lyosome and / or to present fragments of phagocytosed particles. For this purpose, particles are first provided which are marked by fluorescence. These particles contain at least one pH-dependent or pH-sensitive fluorescent dye. The labeled particles are incubated with the cells whose activity is to be examined. The cells are analyzed by fluorescence microscopy, fluorimetric and / or fluorescence cytometric methods. It is crucial for this method that the fluorescence is at least partially detectable over at least 12 hours. The temporal persistence of fluorescence depends on the experimental conditions chosen. For example, the activity of the cells has an influence on the detectability of the fluorescence. Therefore, the time of 12 hours is an indication, which can be subject to certain fluctuations.

The method according to the invention is therefore based on the absorption of phagocytable particles which represent, for example, a model exciter. These particles are marked by pH-dependent fluorescent dyes and in particular by pH-dependent fluorescent proteins, so that the phagocytosis of these pH-dependent fluorescent particles makes an accumulation of the fluorescence in the cell interior visible and quantifiable. In the course of processing the particles or the model exciter, the fluorescence inside the cell decreases due to the cell-specific degradation of the pH-dependent fluorescent dyes or their inactivation. If, for example, there is a disturbance in the processing capacity after phagocytosis in the cells to be examined, there is no decrease in the fluorescence inside the cell compared to suitable control cells, so that conclusions can be drawn about the processing activity or the processing capacity. In addition, a disruption of the phagocytosis activity can of course also be ascertained, in which reduced or missing fluorescence is observed in comparison with controls inside the cell. In addition, the ability of the cells to present the phagocytized particles after fragmentation on the surface can be examined, which is the prerequisite for the development of specific immunity. The pH-dependent fluorescence used for this method can be at least partially detected over at least 12 hours. This means that the fluorescence used can still be detected in the cells after 12 hours, provided that cellular influences do not reduce the fluorescence. The cells to be examined are advantageously cells which are capable of phagocytosis. Mammalian cells, in particular cells of the immune system, are particularly suitable. These are advantageously myeloid cells, that is to say cells whose precursors are generated in the bone marrow, as well as monocytes, macrophages, granulocytes, endothelial cells and dendritic cells. In particular, the dendritic cells are of particular interest for the method according to the invention, since these are extremely potent antigen-presenting cells which essentially induce the primary cytotoxic T cell response and are responsible for the activation of regulatory T cells (T _reg ). Furthermore, differentiation products and / or precursors or precursors of these cells derived from these cells can be investigated with the method according to the invention. On the other hand, the method according to the invention can also be used to examine other cells insofar as they are phagocytotically active, for example slime molds, unicellular lower organisms or the like.

The detection of the pH-dependent fluorescence or of the pH-dependent fluorescent dyes in the course of the analysis of the cells can be carried out both by light microscopy and fluorescence microscopy and in particular by automated methods. Flow cytometry or fluorimetry is particularly preferred, in particular in plate measuring devices.

To carry out the method according to the invention, the labeled particles or the model pathogens are advantageously incubated with cells in whole blood or in isolated form in a manner which enables normal physiological processes to take place. For this purpose, suitable buffer solutions are used, for example, which provide a physiological milieu. During this incubation, the labeled particles are removed by the cells according to their - o -

Phagocytosis activity recorded, so that after phagocytosis, the cells are marked by the pH-dependent fluorescence of the recorded particles. Already this phagocytosis activity of the cells to be examined can be evaluated by appropriate analysis of the fluorescence of the cells, for example by using a fluorescence cytometer or by fluorimetric plate measuring devices. In the further course of the method or the incubation, the phagocytosed particles are located within the cells to be examined and come into contact with the degradation apparatus of the cells. The degradation or degradation or fragmentation of the ingested particles depends on the fusion of the primary endosomes, within which the phagocytized particles are initially enclosed, with lysosomes in the phagocytic cells. These lysosomes are equipped with different enzymes, which among other things cause the destruction or fragmentation of the phagocytosed particles and the components of the particles. In a typical sequence of the method according to the invention, the particles or the model exciters are still recognizable as granular structures under the fluorescence microscope after phagocytosis. If the particle is broken down further in the course of processing, the fluorescence signal appears to be distributed uniformly in the cytoplasm of the phagocytic line. This ability to analyze the further fate of the phagocytosed particles represents a particular advantage of the method according to the invention, since this is not possible with conventional methods.

The fate of a particle after phagocytosis in the cell depends in particular on the stability of the components to be processed or the proteins of the particle. First of all, the biochemical nature of the protein and the enzyme activation in the cell to be examined play a role. The kinetics of protein degradation is denatured in Influenced by the acidic pH conditions in the lysosomes and by degradation or degradation as a result of proteolysis and / or oxidation. Although the pH-dependent fluorescence or the corresponding pH-dependent fluorescent dyes used in the process according to the invention, which are essentially pH-dependent fluorescent proteins, are essentially stable due to their tertiary structure, functional phagocytic cells occur over time degradation of the pH-dependent fluorescent proteins, in particular through the action of acidic pH, reactive oxygen species and / or proteases. The associated destruction of the tertiary structure of the pH-dependent fluorescent proteins or the pH-dependent fluorescent dyes leads to a loss of fluorescence. According to the invention, the capacity of the cells capable of phagocytosis to break down proteins can thus be shown and quantified. By analyzing the cells in the flow cytometer or in other suitable measuring devices, this capacity can be quantified on the basis of the loss of fluorescence of the cells to be examined. This is preferably done after a sufficient incubation period within which the corresponding phagocytosis and processing processes in the cell have taken place. These incubation times are preferably standardized. Furthermore, different incubation times can advantageously be used in order to be able to draw conclusions about the temporal courses of the corresponding processes.

Particular advantages of the method according to the invention lie in the fact that the method can be automated and in the inexpensive and simple provision options for the required detection substances. The particles which are to be phagocytosed by the cells to be examined are advantageously membrane-coated structures. The particles can have, for example, a double membrane, for example as in eukaryotes, certain viruses, artificial vesicles or exosomes, or a single membrane, for example in prokaryotes. It is particularly preferred if the particles have receptors which facilitate phagocytosis by so-called opsonization. In preferred embodiments of the method according to the invention, the particles are bacteria, cyanobacteria, fungi and / or eukaryotic cells such as, for example, tumor cells, and particles derived from tumor cells, such as, for example, exosomes or organelles, which are marked by pH-dependent fluorescence. Furthermore, the phagocytosis, processing and / or presentation of viruses can also be advantageously examined with the method according to the invention.

In a preferred embodiment of the method according to the invention, synthetic particles such as lipid vesicles or latex beads are used. These particles primarily serve as carrier substances for the pH-dependent fluorescent dyes or antigens, the further processing of which is followed in the course of the method according to the invention.

The particles are advantageously infectious particles which play a role in immune reactions. In order to investigate corresponding immune reactions, such infectious particles can be used in the method according to the invention. On the other hand, it can also be preferred if particles derived from such particles are used which are not infectious, in particular for humans. This has the advantage that the method according to the invention can be carried out without security risks. The most important infectious particles or pathogens To cover, the following model pathogens are preferably used as particles in the process according to the invention: Escherichia coli as a gram-negative pathogen, which plays a role in abdominal surgery, for example, and / or staphylococci as representatives of gram-positive pathogens, which are particularly involved in pneumonia. On the other hand, viruses can also be used which can trigger acute, chronic and / or latent infections. In particular for the investigation of infectious viruses, recombinant, non-infectious and / or transformation-defective organisms are advantageously used which, for example, express properties of common viruses such as CMV or EBV or of tumor antigens on the surface. When using eukaryotic cells as particles, in particular those cells come into question which can form tumors in mammalian organisms. These can also be autologous tumor cells, for example, in order to use the method according to the invention to check the immunity situation against a tumor. In addition to the particles mentioned by way of example, fragments of these particles are also suitable for the process according to the invention.

The particles or the model exciter can be used both in the living and / or functional state and in the inactivated state. Inactivation can e.g. B. by fixing the microorganisms used and has the advantage that safety risks are largely avoided, for example, in the case of infectious particles.

Different pH-dependent fluorescent dyes, in particular different pH-dependent fluorescent proteins and / or peptides, can be used to provide particles labeled with pH-dependent fluorescence. In principle, all pH-dependent fluorescent proteins or peptides are suitable for use in the method according to the invention have sufficient long-term detectability or a corresponding stability. In addition to naturally occurring pH-dependent fluorescent proteins and peptides, it is also possible to use proteins and peptides that have been modified by chemical and / or molecular biological methods. Both naturally occurring and artificially modified pH-dependent fluorescent proteins or peptides can be produced by recombinant expression and linked to the particles to be phagocytosed, or can even be expressed within the particles by recombinant expression. In the following, the term “protein” should also be understood to mean a peptide, even if this is not expressly mentioned.

In a particularly preferred embodiment, the fluorescent dye used is the green fluorescent protein from Aequorea victo a or a protein or peptide which can be derived therefrom and which has a pH-dependent fluorescence emission spectrum. In this context, reference is made, for example, to German published patent application DE 197 18 640, which describes suitable fluorescent proteins. So far, these fluorescent proteins have usually been used to detect the location of fusion proteins or to measure gene activity. According to the present invention, these fluorescent dyes can now be used to label phagocytable particles, for example bacteria or other microorganisms. Furthermore, these fluorescent dyes can also be used to label eukaryotic cells, in particular tumor cells, and to use them for the method according to the invention. The particular advantage of using this green fluorescent protein or proteins or peptides derived therefrom that have a pH-dependent fluorescence emission spectrum is the extraordinary stability of these fluorescent dyes (green fluorescent protein - _ | ^ 5 -

Properties, Applications and Protocols. Martin Chalfie and Steven Kain (eds.). Wiley-Liss, New York, 1998). The green fluorescent protein from Aequorea victoria and proteins and peptides that can be derived therefrom and have a pH-dependent fluorescence emission spectrum are therefore particularly suitable for the process according to the invention owing to their extraordinary stability.

In a particularly preferred embodiment of the method according to the invention, the pH-dependent fluorescent dye is a protein or peptide with a fluorophore, which consists of at least three amino acids. The second amino acid of these at least three amino acids of the fluorophore is preferably tyrosine and / or the third amino acid is glycine. The first position is variable.

In a further preferred embodiment of the method according to the invention, the pH-dependent fluorescent dye is a photosynthetic pigment, e.g. B. chlorophyll, or accessory photosynthetic pigments, such as phycobiliproteins. The use of phycoerythrin as a pH-dependent fluorescent dye is very particularly preferred.

In a particularly preferred embodiment of the method according to the invention, the fluorescent dye used is a pH-dependent fluorescent protein or peptide. Certain immune defects are associated with an abnormal pH environment in the lysosomes of the phagocytic cells. In order to be able to detect such defects, it is particularly advantageous to use particles or model exciters in the method according to the invention which are labeled with pH-dependent fluorescent proteins or peptides. Such fluorescent proteins or peptides preferably show one at an acidic pH Decrease in fluorescence. The content of lysosomes normally shows an acidic pH of around pH 4-5. When the method according to the invention is carried out using pH-dependent fluorescent proteins or peptides, there is therefore a direct decrease in fluorescence in the normal, acidic pH environment in the lysosomes after the primary endosome has fused with the lysosomes. If this immediate decrease in fluorescence is absent, an abnormal pH environment in the lysosomes and a corresponding immune deficiency can be concluded. The fluorescent protein lhFP516 / 581 (EosFP) can be used as a particularly suitable pH-dependent fluorescent protein (Schmitt, F. (2003). A novel, green to red photoconverting fluorescent protein from the indopacific stone coral Lobophyllia hemprichii, Ehrenberg, 1834 (Cnidaria, Anthozoa ) Diploma thesis, Ulm University Library; Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Röcker, C, Salih, A., Spindler, KD, and Nienhaus, GU (2004). EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion. Proc Natl Acad Sei USA, 101 (45), 15905-15910.).

In a particularly preferred embodiment of the method according to the invention, the particles labeled with pH-dependent fluorescence have at least one naturally expressed pH-dependent fluorescent dye. Particularly preferred particles are autofluorescent microorganisms, in particular bacteria, cyanobacteria (blue-green algae) and / or algae. The dinoflagellates, in particular endosymbiotic dinoflagellates, are particularly advantageous here. Fragments of these particles can also be used. This auto- or self-fluorescence of the microorganisms has the advantage that these particles are used without further labeling reactions , _. - 15 -

can be because they inherently have the fluorescence required for the inventive method.

It is further preferred if the particles labeled with pH-dependent fluorescence have at least one recombinantly expressed pH-dependent fluorescent dye. This is made possible in particular by an autocatalyzed formation of the fluorophore in pH-dependent fluorescent proteins or peptides. In a particularly preferred embodiment of the method according to the invention, at least one pH-dependent fluorescent protein or peptide is expressed within the particles. They can be particles which naturally express one or more pH-dependent fluorescent proteins or peptides, or else particles in which a corresponding expression takes place due to genetic engineering manipulation. For a suitable recombinant expression of the pH-dependent fluorescent proteins or peptides within the particles, in particular within the model pathogen, the corresponding coding DNA is introduced into the model pathogen in such a way that expression of the protein or peptide in the organism itself is possible. For this purpose, the corresponding coding DNA is placed under the control of a suitable promoter, for example, and is introduced into the organism, which is used as a particle, in particular in the form of a suitable vector. Corresponding proteins or peptides are preferably expressed stably and over a longer period of time, so that the particles can be used for the method according to the invention without further preparation. On the other hand, it can also be advantageous if the particles express the corresponding protein or peptides transiently, ie temporarily. In this case, the corresponding particles or model exciters usually have to be carried out before the method according to the invention is carried out molecular biological methods are treated. Appropriate procedures open up to the expert.

In another preferred embodiment, one or more pH-dependent fluorescent dyes, in particular pH-dependent fluorescent proteins or peptides, are bound to the particles, for example by being coupled to the surface of the particle. For this, e.g. B. naturally occurring pH-dependent fluorescent proteins and / or peptides can be cleaned or enriched in order to then bind them to the particles to be phagocytized using conventional methods. On the other hand, recombinantly produced pH-dependent fluorescent proteins and / or peptides can also be used for this purpose. By binding to comparatively large particles, which have a diameter of 1-2 μm, for example, the purification of the pH-dependent fluorescent proteins and / or peptides and the labeling of the particles can, for. B. advantageously by filtration and / or centrifugation techniques in one step. A preferred example of a coupling of the pH-dependent fluorescent proteins or peptides is a coupling via biotin-streptavidin bonds.

It is particularly preferred if pH-dependent fluorescent proteins and / or peptides, in particular recombinant proteins and / or peptides, are specifically bound to the particles. For example, the pH-dependent fluorescent proteins are bound or immobilized via metal-affine peptide tags, which are attached to the fluorescent protein in particular by molecular biological methods. Such peptide tags are commonly used in affinity chromatography. An example of this is the so-called 6x histidine tag. In addition to recombinantly produced pH dependent fluorescent proteins and / or peptides, which in particular can carry further functionalities such as the peptide tags mentioned, which are advantageous for binding to the particles or for immobilization, can also bind naturally occurring pH-dependent fluorescent proteins or peptides to corresponding particles become. An example of this is the phycobiliprotein phycoerythrin.

In a further preferred embodiment, the pH-dependent fluorescent dyes, that is to say in particular pH-dependent fluorescent proteins or peptides, are coupled with further peptides and / or proteins. This coupling is preferably carried out by a fusion of the peptides or proteins, so that the pH-dependent fluorescent dye is expressed as a fusion protein. If, for example, viruses are to be used as particles or model pathogens, their fluorescent labeling is preferably carried out by a suitable host organism. For this purpose, the genetic information of the virus is changed to the extent that one or more viral proteins are expressed in fusion with pH-dependent fluorescent proteins or peptides. The already mentioned combination of the pH-dependent fluorescent proteins or peptides with peptide tags or the like, which enables or facilitates binding of the pH-dependent fluorescent proteins or peptides to the particles, can also be achieved by fusing the pH-dependent fluorescent proteins or peptides with corresponding ones Peptides or proteins can be achieved which already carry the corresponding functionalities.

In a particularly preferred embodiment of the method according to the invention, the particles labeled with pH-dependent fluorescence have at least two different pH-dependent fluorescent dyes. You can either different types of particles each carry different pH-dependent fluorescent dyes, or one type of particle has different pH-dependent fluorescent dyes. It is particularly preferred if one type of particle, for example bacteria, cyanobacteria and / or algae, has a naturally expressed pH-dependent fluorescent dye and at least one recombinantly expressed pH-dependent fluorescent dye. Of course, the invention also encompasses particles which have two or more recombinantly expressed different pH-dependent fluorescent dyes.

It is particularly advantageous if the different pH-dependent fluorescent dyes, in particular as a result of different pH-dependent emission spectra, produce distinguishable fluorescences. This can advantageously be used for various aspects of the method according to the invention. In a preferred embodiment of this aspect according to the invention, the various pH-dependent fluorescent dyes which bring about the distinguishable fluorescence react differently to the intracellular activities of the cells to be examined. For example, one of these pH-dependent fluorescent dyes, in particular one of the pH-dependent fluorescent proteins, is broken down more quickly by the intracellular enzymes than the other pH-dependent fluorescent protein. This can be caused by the fact that, for example, one of the pH-dependent fluorescent proteins has specific interfaces for intracellular proteases, whereas the other protein does not contain these interfaces. Furthermore, for example, one of the fluorescent proteins can be a pH-dependent fluorescent protein and the other one can be a protein which is essentially unaffected by the pH within the cell. In addition, one of the pH-dependent fluorescent proteins can be a protein act, the fluorescence of which decreases in contact with reactive oxygen species faster than with the other protein, the fluorescence of which remains largely stable under these conditions.

It is particularly preferred that the pH-dependent fluorescent dyes or, in particular, the pH-dependent fluorescent proteins, which bring about the distinguishable fluorescence, have different stabilities. This can be, for example, compartment-specific stabilities. The conditions (e.g. pH value, protease equipment) in the different compliments (e.g. exosomes, lysosomes) can be very different. By using pH-dependent fluorescent dyes, which each react to certain of these conditions, statements can be made about compartment-specific processes.

A particular advantage of using different and distinguishable pH-dependent fluorescent dyes is that, for example, the use of pH-dependent fluorescent proteins with different colors and stability can improve the quantification of the processing capacity of the cells to be examined. The use of two different pH-dependent fluorescent proteins, which differ in their sensitivity to cellular degradation systems, makes it possible to measure the processing capacity of phagocytic cells, such as is caused by the protease activity of the enzymes involved. The more stable protein serves as an internal standard for quantifying the amount of protein incorporated, whereas the more unstable protein serves as a pointer for, for example, protease-related degradation. This form of test enables, for example, the diagnosis of immunodeficiencies that are triggered by an abnormal or missing protease activity. For this you can for example, differences between naturally occurring pH-dependent fluorescent proteins or peptides can be exploited. In addition, pH-dependent fluorescent proteins can be used, in which such differences were generated artificially, in particular by molecular biological methods. This is done, for example, by targeted stabilization by removing suitable protease interfaces and / or by targeted destabilization by adding suitable protease interfaces.

A similar parallel approach can be carried out with a pH-dependent fluorescent protein and a differently colored, more stable fluorescent protein, which serves as an internal standard. In this way, statements about disturbances in the pH environment of the lysosomes can be made in a particularly advantageous manner, as has already been described above. In this case, the protein or peptide, which serves as an internal standard, must have little or no pH dependence of the fluorescence. An example of a stable protein which is essentially pH-independent is the red fluorescent protein eqFP61 1 (Wiedenmann et al. (2002) Proc Natl Acad Sei 99: 1 1646-1 1651). The protein lhFP516 / 581 can be used as the pH-dependent fluorescent protein.

In a comparable parallel approach, pH-dependent fluorescent proteins or peptides are used, which differ both in terms of the color of the fluorescence and in terms of the stability towards reactive oxygen species. In this way, statements about the involvement of reactive oxygen species in degradation processes can be made in a particularly advantageous manner. In a preferred embodiment of the method according to the invention, in which the particles have at least two distinguishable pH-dependent fluorescences, these fluorescences are brought about by at least two different pH-dependent fluorescent dyes, in particular by at least two pH-dependent fluorescent proteins or peptides. On the other hand, it can also be very advantageous if the distinguishable fluorescences are brought about by at least two distinguishable pH-dependent fluorescent states of a protein. These different fluorescent states of a protein preferably differ in terms of their stability. An example of this is the pH-dependent fluorescent protein lhFP516 / 581 from Lobophyllia hemprichii. This protein can be specifically and irreversibly converted from a green fluorescent to a red fluorescent form by irradiation with short-wave light. The degree of photoconversion and thus the quantitative ratio between the green and red fluorescent form can be controlled, for example, by the intensity and duration of the irradiation and can also be carried out within a particle, in particular within a model exciter. Both conditions are pH dependent. The red fluorescent form shows a lower resistance to reactive oxygen species. If both the green and the red fluorescence are quantified, for example in a fluorescence cytometer, the degradation capacity and thus the processing activity of the cells to be examined can be calculated from their ratio.

In a preferred embodiment of the method according to the invention, the presentation of fragments of the phagocytosed particles is examined by the cells to be examined. This examination of the so-called antigen presentation either takes place in addition to the analysis of the processing capacity or the processing activity of the cells or becomes independent of this carried out. To examine the antigen presentation, fragments of the pH-dependent fluorescent proteins or peptides and / or fragments of the peptides and / or proteins fused with these proteins or peptides are analyzed, which appear on the surface of the cells to be examined during the course of the antigen presentation. The processed components or fragments are presented as peptides of a relatively constant length by certain immune cells, in particular dendritic cells, on their surface. The understanding of these relationships has been discussed above all in connection with considerations regarding vaccination strategies (Hung and Wu (2003) Gurr Opin Mol Ther 5 (1): 20-4). The antigen presentation is the prerequisite for the progress of the further immune reactions of a mammalian organism. The method according to the invention is particularly advantageously suitable for examining these processes and in particular the capacity of the phagocytic cells to present antigens. Defined antigens are introduced into the phagocytic cells by the pH-dependent fluorescent proteins or peptides used in the method according to the invention. Since these proteins or peptides do not normally occur naturally in the mammalian organism, antigens derived from them can be detected with great certainty.

For this purpose, the fragments presented are preferably detected using antibodies which recognize fragments of the pH-dependent fluorescent proteins or peptides. In principle, polyclonal and monoclonal antibodies can be used. Monoclonal antibodies are particularly preferred because they show a particularly high specificity for the antigens to be recognized. In a particularly advantageous embodiment, these antibodies are labeled, which makes it easier to detect the binding of the antibodies to the respective antigens. For example, you can for this, the antibodies must be labeled with distinguishable pH-dependent fluorescent dyes. Other methods of antibody detection can also be used. Advantageously, the antibodies by suitable second antibodies z. B. carry a detectable enzymatic function can be detected. Details of this are available to the person skilled in the art.

Furthermore, the fragments presented can also be detected using cellular receptors. Preferred cellular receptors are, in particular, those receptors which recognize and bind the respective fragment presented as physiological structures. These receptors can be used as very specific and effective tools for the detection and detection of antigen presentation.

In the event that the pH-dependent fluorescent proteins or pH-dependent fluorescent peptides are fused with other peptides or proteins, fragments of these fusion partners can be detected with suitable antibodies. Furthermore, other specific antigens which are derived from constituents of the particles, in particular the model pathogen, can also be detected using suitable antibodies. These antigens, e.g. B. tumor antigens, can occur naturally or by artificial change in the particles or model pathogens.

In a particularly preferred embodiment of the method according to the invention, in which the

If the antigen presentation capacity of the cells is investigated, the pH-dependent fluorescent proteins or peptides or the peptides or proteins fused with them are labeled with pH-dependent fluorescent dyes in such a way that they retain their fluorescence even after processing. This causes the processed fragment, which is presented on the surface of the cells, itself continues to fluoresce and can thus be detected directly on the cell surface. Fluorescent dye is to be understood here as the fluorescent component of a protein or peptide which gives the respective protein or peptide the fluorescent properties. The detection of these fluorescences on the surface can be carried out using customary methods, in particular using fluorescence microscopy and / or flow cytometry. In addition, the specific localization on the cell surface can be checked, for example, after adding suitable cell-impermeable quenching substances.

A particular advantage of the method according to the invention is that this method can be carried out with isolated phagocytic cells as well as with whole blood or in the complete bone marrow. When using whole blood, it is generally advantageous to remove the non-phagocytic cells, in particular erythrocytes, before the fluorescence measurement. This can be done by physical methods and / or for example by lysis. The lysis can be carried out with the aid of a non-isotonic solution, that is to say a hypotonic solution, the conditions advantageously being chosen such that above all the erythrocytes and not the phagocytic cells are destroyed. This can be achieved, for example, by means of suitable buffer and / or temperature conditions.

Furthermore, in isolated cells as well as in whole blood or bone marrow, it is advantageous that the phagocytic cells are fixed before the fluorescence measurement. The fixation takes place in the solution and ensures that the phagocytotic and subsequent processes in the cells are stopped or at least impaired or slowed down. Such fixation or locking can be achieved in particular by adding formaldehyde, for example about 1% Paraformaldehyde. By locking the cells with subsequent fluorescence analysis, precise information can be made about the processes in the cell that are related to phagocytosis.

The method according to the invention can be used in a particularly advantageous manner to clarify which cells of whole blood (e.g. macrophages, dendritic cells, other myeloid cells, endothelial cells) or of the bone marrow are active in relation to phagocytosis and the downstream processes. For this, the whole blood, the bone marrow or certain isolated cells are incubated with the fluorescence-labeled particles in the presence of line-specific antibodies that recognize the respective cell types. Monoclonal antibodies are preferably used for this because of their special specificity. These antibodies are also fluorescence-labeled, the fluorescence advantageously differing from the fluorescence with which the particles are labeled. Subsequent localization of both fluorescences allows conclusions to be drawn about the phagocytotic, processing and / or antigen presentation function of certain cell types.

Particular advantages of the method according to the invention are the great sensitivity of the detection of the fluorescent marking, the cost-effective production, storage and shipping of the particles to be used or the model exciter and the automatable quantification, which can be carried out, for example, with the aid of flow cytometry and / or with suitable plate measuring devices. Above all, this enables the evaluation of large sample quantities. In addition to the use in diagnostics, the method according to the invention can therefore also be used with great advantage in the search and evaluation of active substances. The invention therefore also includes the use of a method as described above for the screening and / or for the characterization of immunostimulating or immunosuppressive substances. Here, for example, the efficiency and / or the mechanism of action of immunostimulants and immunosuppressants can be examined in vivo and in vitro. For this purpose, the phagocytic cells can be mixed with potential immunostimulating or immunosuppressive substances. The effect on the phagocytosis activity and the processing activity and / or the antigen presentation capacity is advantageously analyzed in each case in comparison with control cells according to the method according to the invention. In the case of an immunostimulating substance, for example, an increase in phagocytic activity, an increase in processing activity and / or an increase in antigen presentation can be demonstrated. The method according to the invention is therefore particularly suitable for finding and evaluating therapeutically active substances which, for. B. stimulate the phagocytosis and processing of bacteria, viruses and / or tumor cells or their fragments. On the other hand, for example with regard to autoimmune diseases, for example from artherosclerosis, or to allergies, the method according to the invention can be used to find and evaluate therapeutically active substances which dampen or prevent overreactions of the immune response. An example of an overreaction is an increased antigen presentation. The ability to automate the method according to the invention is particularly advantageous since this enables use in the high-throughput method. Since the immune system of humans and other mammals is comparable in many respects, the method according to the invention can be used both with cells of human origin and with cells from other animals, especially from other mammals. This is particularly advantageous for the screening and / or the characterization of immunostimulating or immunosuppressive substances, since corresponding substances can be examined in an animal model by using the method according to the invention. The method according to the invention can also be used in non-mammalian organisms or their cells, provided that these cells are able to carry out phagocytosis, processing and / or presentation of fragments or antigens.

In a particularly advantageous manner, the method according to the invention can be used to diagnose immunodeficiencies that can be congenital or acquired. As mentioned at the beginning, the defects detectable with the method according to the invention can manifest themselves, for example, as immune deficiencies, tumor formation, allergies or autoimmune diseases, for example from atherosclerosis. These immune defects can be caused, for example, by functional defects in definable antigen-presenting cells. The method according to the invention is particularly advantageous for the detection of genetic defects in intracellular trafficking. Congenital immunodeficiencies can e.g. B. as Griscelli syndrome (mutated Rab27a, Bahadoran et al. (2003) J Bio! Chem 278 (13): 11386-92), as Herrmann Pudlack syndrome (mutated vsp33, Chiang et al. (2003) J Biol Chem 278 (22): 20332-7) or as Chediak Higashi syndrome (mutated Lyst, Huizing et al. (2001) Thromb Haemost 86 (1): 233-45). In the case of these syndromes, for example, the method according to the invention can be used to make an immediate diagnosis for the existence of such a defect. Standardization by examining healthy controls and / or testing healthy parents or siblings is advantageous for evaluation. Furthermore, the diagnosis of chronic viral infections is particularly possible, for example. chronic African virus infections also appear to be defective

Phagocytic performance and defects in the subsequent processes, especially in macrophages and dendritic cells. Genetic HIV receptor mutations can also lead to changes in the immune response. Patients with chronic virus infections and resulting immune defects or autoimmune phenomena can therefore be examined with the aid of the method according to the invention and the defects can be diagnosed quickly.

Furthermore, the method according to the invention is suitable with great advantage for monitoring a therapy of immune defects. In particular, the immune defects that have already been mentioned above come into question for this. In general, both congenital and acquired immune defects can be both diagnosed and monitored during therapy with the aid of the method according to the invention. Therapy that is intended to stimulate a reduced immune response, for example, or to dampen an overreacting immune response, can be controlled with the aid of the method. For this purpose, blood cells from patients and / or test subjects who have undergone immunomodulatory therapy are advantageously used in an in vitro test method. The therapeutic effect is checked on the basis of the phagocytosis activity and the processing activity and / or the presentation activity using the method according to the invention in the manner described above. Of particular interest are phagocytic dendritic cells and macrophages.

Finally, the invention comprises a reagent kit for examining the activity of cells to process phagocytized particles and / or to present fragments of phagocytosed particles. This kit includes at least pH-dependent Fluorescence-labeled particles, which are in particular appropriately labeled bacteria, cyanobacteria, fungi, viruses and / or eukaryotic cells or fragments of these different particles. These particles are characterized in that they have at least one pH-dependent fluorescent dye. This fluorescent dye produces a cytoplasmic fluorescence, in particular, which can be detected for at least 12 hours. It is particularly preferred if the particles have or show at least two distinguishable pH-dependent fluorescences. Furthermore, the reagent kit advantageously contains suitable customary buffers. With regard to further features of these particles marked with fluorescence or of the reagent kit, reference is made to the above description.

Further features of the invention result from the subclaims and the examples in connection with the figures. The various features can be implemented individually or in combination with one another.

The figures show:

Fig. 1 fluorescence activity after phagocytosis by intact, enriched and isolated dendritic cells from whole blood.

Fig. 2 fluorescence activity after phagocytosis by leukocytes in whole blood.

Fig. 3 microscopic images of an E. coli bacteria phagocytizing single cell.

Fig. 4 emission spectrum of photoconverted pH-dependent lhFP516 / 581. 5 reaction of the green and red fluorescent forms of pH-dependent lhFP516 / 581 to an incubation at different pH values over a period of 12 h.

Examples

1. Investigation of phagocytosis and processing on isolated dendritic cells

Phagocytic cells from the blood of a healthy donor are enriched and isolated and incubated with recombinant, fluorescent bacteria (ash chia coli). Fluorescent labels which are homologous to the green fluorescent protein (GFP) from Aequorea victo a and have a pH-dependent fluorescence emission spectrum are used as the fluorescent label.

To isolate the phagocytic cells, 10 ml of whole blood is mixed with 50 IU Na-heparin / ml. The blood is diluted with 10 ml PBS and overlaid on Ficoll (density 1.077 g / l). The mononuclear cells are centrifuged at 840 xg for 20 min and washed twice in Hank's BSS. The mononuclear cells are brought into cell culture at a cell concentration of 2-4 × 10 ⁵ / ml (RPMI 1640, 25 mM Hepes, antibiotics, L-glutamine, 10% FCS, endotoxin-free). After 10 days, the floating cells are roughly removed. The remaining weakly adherent cells are cultivated in fresh medium for a further 14 days. After a further 14 to 28 days, well-enriched phagocytic cultures can be obtained Remove dendritic cells that are used for the method according to the invention.

Bacteria (E. coli) which express fluorescent proteins (GFP) are used as particles to be phagocytosed. For this purpose, the bacteria were transformed with plasmids (pQE32, Qiagen), which carry the coding sequences for the pH-dependent fluorescent GFP-like proteins lhFP516 / 581 (Schmitt, 2003) and eqFP61 1 (Wiedenmann et al., 2002). After expression, the pH-dependent fluorescent protein lhFP516 / 581 shows green fluorescence with an emission maximum at 516 nm. The maximum excitability is 506 nm. After irradiation with UV light (366 nm), the emission maximum is shifted to 581 nm. At the same time, the excitation maximum shifts to 571 nm. In order to test the applicability of this red fluorescent form of the protein, the bacterial solution was irradiated with UV light (366 nm) for 30 min. These now red fluorescent bacteria were used in parallel to bacteria which contain the protein in the green fluorescent starting form. The red fluorescent protein eqFP611 has an emission maximum at 611 nm and is most strongly excited at 559 nm. The green or red fluorescence is detected using suitable excitation and emission filters. 1 x 10 ⁵ dendritic cells / ml are incubated with 1 x 10 ⁷ fluorescent bacteria / ml, which express the pH-dependent fluorescent protein lhFP516 / 581. The stages of phagocytosis, in particular from the uptake into primary endosomes to the release of the pH-dependent fluorescent dye into the cytoplasm of the dendritic cells, which differentiate with the phagocytic act into antigen-presenting dendritic cells, can subsequently be microscopically and / or follow flow cytometry. The phagocytic activity of individual dendritic cells can be between 6 and 12 hours. It can take 12 to 48 hours for the pH-dependent fluorescent dye to be released into the cytoplasm. The processes taking place can be analyzed by measuring and localizing the fluorescence.

2. Examination of phagocytosis and processing in whole blood or bone marrow

In a further experiment, whole blood is incubated with recombinant E. coli, which express the pH-dependent fluorescent protein IhFP 516/581, and the fluorescence of the nuclear cells is examined.

0.1 ml of heparinized whole blood (50 IU Na-heparin / ml) are incubated with 1 -5 μl fluorescent bacteria (1 x 10 ⁹ / ml) (see above). The incubation period depends on the question of the test protocol. To investigate the processing activity, the incubation can be terminated, for example, if the release of the pH-dependent fluorescent dye into the cytoplasm is expected, for example after 12 to 48 hours. The incubation can also be ended earlier, especially if the pH-dependent fluorescent dyes of the phagocytized particles in certain endosomes lose their fluorescence, for example due to organelle-specific conditions and enzymes.

To end the incubation and before measuring the pH-dependent fluorescence, the erythrocytes are replaced by physical

Methods or removed by lysis. For the lysis of the erythrocytes, 2 ml of hypotonic ammonium chloride solution in sodium phosphate buffer (0.05 M, pH 7.4) with 1% paraformaldehyde and 0.1% Tween 20 added to 0.1 ml of blood. The incubation is carried out on ice until the erythrocytes are lysed. The cell suspension, consisting of intact, fixed white blood cells and membrane residues of the erythrocytes, is washed twice with PBS and then measured in a flow cytometer. A similar approach can be done with bone marrow. The advantage of using whole blood or bone marrow (Vboll) is, in particular, that the phagocytic activity in all available white cells can be examined simultaneously and evaluated differentially.

These two experimental approaches show that the method according to the invention can be carried out both with isolated cells and with whole blood. The pH-dependent fluorescence is not lost if the non-phagocytic erythrocytes are lysed and the phagocytic cells are fixed at the same time. The system enables the experimenter to stop the process at any time and to investigate whether the pH-dependent fluorescence is present in the primary or secondary endosomes, the lysosomes or in the cytoplasm. In this way, informative statements can be made about the processes after phagocytosis.

Fig. 1 shows the phagocytosis performance, which is measured based on the fluorescence activity after 24 hours (gray histogram b). As a control, phagocytic cells without bacteria are incubated and measured accordingly (black histogram a). The x-axis of the flow cytometric measurement (FACScalibur, BD-Europe, Heidelberg) records the green fluorescence on a logarithmic scale. The y-axis shows the number of cells measured (counts). 2 shows that cells which are incubated without E. coli show a weak intrinsic fluorescence (black histogram a). The blood cells incubated with E. coli show three populations (gray histogram b): i) non-fluorescent cells that have not phagocytized and are below the black negative curve, ii) weakly fluorescent cells that have little E. coli phagocytosed (fluorescence between channels 80 and about 700), and iii) a highly fluorescent population that has more phagocytosed and peaks between the fluorescence intensity channels 1,000 and 10,000.

FIG. 3 shows microscopic images of a single cell which E. coli bacteria have taken up by phagocytosis (A). The bacteria express the pH-dependent protein lhFP516 / 581. The bacterial cells show both green (B) and red fluorescence (C) due to the partial photoconversion of the protein. The rod shape of the bacteria is clearly visible.

4 shows the emission spectrum of photoconverted pH-dependent lhFP516 / 581. In addition to the pronounced red fluorescence (581 nm, dotted line), 516 nm green fluorescence from unconverted protein can be seen. When treated with singlet oxygen ( ¹ 0 ₂ ), the red fluorescence at 581 nm disappears (solid line). The maximum at 516 nm remains unchanged. This indicates a higher stability of the non-converting, green fluorescent form towards reactive oxygen species. Singlet oxygen was generated by the thermal decay of 3,3 '- (1,4-naphthylidenes) dipropionate endoperoxide (NDP02).

5 shows the response of the green and red fluorescent forms of pH-dependent lhFP516 / 581 to an incubation at different pH Values over a period of 12 hours. The fluorescence of the green form remains stable, while the fluorescence of the red form decreases sharply at a pH <6.5. The selective degradation of the red dye compared to the fluorescence intensity of the green form can be used to draw conclusions about the pH, preferably within cells. The investigation of the pH value and its change is possible both in cells which produce the protein and in cells into which the dyes are introduced from the outside, for example by expressing bacteria.

Claims

claims

1. A method for examining the activity of cells to process phagocytized particles as a result of the fusion of the endosome and lysosome and / or to present fragments of phagocytosed particles, comprising the following method steps: providing particles marked with fluorescence which are at least one pH-dependent Have fluorescent dye, incubate the labeled particles with the cells, fluorescence microscopic, fluorimetric and / or fluorescence cytometric analysis of the cells, the fluorescence being at least partially detectable over at least 12 hours.

2. The method according to claim 1, characterized in that the cells are mammalian cells.

3. The method according to claim 1 or claim 2, characterized in that the cells are myeloid cells, monocytes, macrophages, granulocytes, endothelial cells, dendritic cells and / or precursors of these cells.

4. The method according to any one of the preceding claims, characterized in that the particles are bacteria, cyanobacteria, fungi, viruses and / or eukaryotic cells, in particular tumor cells, and / or fragments thereof.

5. The method according to any one of the preceding claims, characterized in that the particles are infectious particles.

6. The method according to any one of the preceding claims, characterized in that the particles are bacteria, in particular Escherichia coli and / or Staphylococcus spec.

7. The method according to any one of the preceding claims, characterized in that the fluorescent dye is the green fluorescent protein from Aequorea victo a or a protein or peptide derived therefrom, which has a pH-dependent fluorescence emission spectrum.

8. The method according to any one of the preceding claims, characterized in that the fluorescent dye is a protein or peptide with a fluorophore of at least three amino acids, preferably the second amino acid is tyrosine and / or the third amino acid is glycine.

9. The method according to any one of the preceding claims, characterized in that the fluorescent dye is a fluorescent phycobiliprotein, in particular phycoerythrin.

10. The method according to any one of the preceding claims, characterized in that the fluorescent dye is a pH-dependent fluorescent protein or peptide, wherein preferably the fluorescent protein or peptide shows a decrease in fluorescence at an acidic pH.

1 1. The method according to any one of the preceding claims, characterized in that the fluorescent dye is a pH-dependent fluorescent protein or peptide, which shows an increased sensitivity to proteases and / or reactive oxygen species, preferably the fluorescent protein or peptide in the presence of proteases and / or at Exposure to reactive oxygen species shows a decrease in fluorescence.

12. The method according to any one of the preceding claims, characterized in that the particles marked with fluorescence have at least one naturally expressed pH-dependent fluorescent dye.

13. The method according to any one of the preceding claims, characterized in that the particles are autofluorescent microorganisms, in particular bacteria, cyanobacteria and / or algae, in particular dinoflagellates, and / or fragments thereof.

14. The method according to any one of the preceding claims, characterized in that the particles marked with fluorescence have at least one recombinantly expressed pH-dependent fluorescent dye.

15. The method according to any one of the preceding claims, characterized in that at least one pH-dependent fluorescent protein or peptide is expressed within the particles to provide particles marked with fluorescence.

16. The method according to any one of the preceding claims, characterized in that at least one pH-dependent fluorescent protein or peptide, preferably phycoerythrin, is bound to the particles in order to provide particles marked with fluorescence.

17. The method according to any one of the preceding claims, characterized in that at least one pH-dependent fluorescent protein or peptide is coupled, in particular fused, with peptides and / or proteins of the particles in order to provide particles marked with fluorescence.

18. The method according to any one of the preceding claims, characterized in that the particles marked with fluorescence have at least two pH-dependent fluorescent dyes, in particular a naturally expressed and at least one recombinantly expressed fluorescent dye.

19. The method according to any one of the preceding claims, characterized in that the particles show at least two distinguishable pH-dependent fluorescence.

20. The method according to claim 19, characterized in that the distinguishable pH-dependent fluorescence is influenced differently by the intracellular activity of the cells to be examined.

21. The method according to claim 19 or claim 20, characterized in that the distinguishable pH-dependent fluorescences have different stabilities.

22. The method according to any one of claims 19 to 21, characterized in that the distinguishable pH-dependent fluorescence is caused by at least two proteins and / or peptides.

23. The method according to any one of claims 19 to 22, characterized in that the distinguishable pH-dependent Fluorescence can be caused by distinguishable pH-dependent fluorescent states of a protein or peptide.

24. The method according to any one of the preceding claims, characterized in that for examining the presentation of fragments of phagocytosed particles fragments of at least one pH-dependent fluorescent protein or peptide and / or fragments of at least one peptide fused with a pH-dependent fluorescent protein or peptide and / or protein are analyzed.

25. The method according to claim 24, characterized in that the fragments presented are detected with antibodies and / or with cellular receptors.

26. The method according to claim 24 or claim 25, characterized in that the fragments presented are labeled with pH-dependent fluorescence.

27. Use of a method according to any one of claims 1 to 26 for screening and / or for the characterization of immunostimulating or immunosuppressive substances.

28. Use of a method according to one of claims 1 to 26 for screening and / or characterizing substances which influence, in particular increase or inhibit, the phagocytosis, processing and / or presentation of bacteria, viruses and / or tumor cells and / or their fragments ,

29. Use of a method according to one of claims 1 to 26 for the diagnosis and / or monitoring of a therapy of immune defects.

30. Reagent kit for examining the activity of cells to process phagocytized particles and / or to present fragments of phagocytized particles, at least comprising particles marked with fluorescence, in particular bacteria, cyanobacteria, fungi, viruses and / or eukaryotic cells, and / or Fragments thereof which have at least one pH-dependent fluorescent dye, the fluorescence being at least partially detectable over at least 12 hours.

31. Reagent kit according to claim 30, characterized in that the particles show at least two distinguishable fluorescences.