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Preferential transfer of certain plasma membrane proteins onto T and B cells by trogocytosis.

Daubeuf S, Aucher A, Bordier C, Salles A, Serre L, Gaibelet G, Faye JC, Favre G, Joly E, Hudrisier D - PLoS ONE (2010)

Bottom Line: For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRgamma found among the most efficiently transferred proteins.We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells.The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.

ABSTRACT
T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRgamma found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.

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An efficiently transferred protein can be used in immunomonitoring assays to detect reactive T cells in TRAP assays.A) Splenocytes from B6 animals immunized (right panels) or not (left panels) with CyaA-OVA were co cultured with the B8 HEK-FcRγ-GFP cells (stably expressing high levels of FcRγ-GFP) transiently transfected 48 hours earlier with a vector encoding covalent H-2Kb-β2m-OVA (bottom panels) or left untransfected (top panels). At the end of the co-culture, cells were analyzed by flow cytometry and GFP fluorescence is plotted as a function of CD8 staining. Numbers represent the percentage of CD8+ cells expressing GFP (present in the gate drawn in the graph). B) As in A except that splenocytes were incubated with EL4 cells labelled with the fluorescent lipophilic probe CellVue Claret loaded (bottom panels) or not (top panels) with the OVA peptide. The Fluorescence signal from CellVue Claret was analyzed as a function of CD8 staining. Numbers represent the percentage of CD8+ cells expressing CellVue Claret fluorescence (falling within the gate drawn in the graph).
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pone-0008716-g006: An efficiently transferred protein can be used in immunomonitoring assays to detect reactive T cells in TRAP assays.A) Splenocytes from B6 animals immunized (right panels) or not (left panels) with CyaA-OVA were co cultured with the B8 HEK-FcRγ-GFP cells (stably expressing high levels of FcRγ-GFP) transiently transfected 48 hours earlier with a vector encoding covalent H-2Kb-β2m-OVA (bottom panels) or left untransfected (top panels). At the end of the co-culture, cells were analyzed by flow cytometry and GFP fluorescence is plotted as a function of CD8 staining. Numbers represent the percentage of CD8+ cells expressing GFP (present in the gate drawn in the graph). B) As in A except that splenocytes were incubated with EL4 cells labelled with the fluorescent lipophilic probe CellVue Claret loaded (bottom panels) or not (top panels) with the OVA peptide. The Fluorescence signal from CellVue Claret was analyzed as a function of CD8 staining. Numbers represent the percentage of CD8+ cells expressing CellVue Claret fluorescence (falling within the gate drawn in the graph).

Mentions: In previous studies, we found it possible to identify reactive CTL via the capture of lipophilic probes incorporated in the PM of APC expressing their cognate antigen [7], [9], [28]. In the context of the findings reported here, we determined if the capture of one of the more efficiently captured molecule could be used in TRAP assays instead of lipophilic probes. For this, we used CTL responding to an immunization with an adenylate-cyclase (CyaA) vector carrying the immunodominant H-2Kb-restricted OVA 257-264 peptide of ovalbumin [7], [9], [28]. Total splenocytes from immunized or control naïve B6 mice were incubated with HEK cells stably expressing FcRγ-GFP, which had been transiently transfected or not with a construct encoding H-2Kb-OVA-β2m construct [38]. As a control, we also used EL4 cells labelled with a fluorescent lipophilic probe and pulsed with the OVA peptide as target cells. As shown in Figure 6A, we indeed found that we could identify around 20% of CD8+ T cells displaying FcRγ-GFP fluorescence after exposure of splenocytes from immunized mice to HEK-FcRγ-GFP cells expressing the H-2Kb-OVA-β2m antigen. Interestingly, this proportion of OVA reactive CTL was quite comparable to that of CTL displaying DiO staining after exposure to DiO-labelled EL4 cells pulsed with the OVA antigen (Figure 6B) or to CTL stained with the H-2Kb-OVA tetramer (not shown) or producing IFN-γ upon antigenic stimulation (not shown). Thus our results show that TRAP assays based on the capture of preferentially transferred GFP-tagged proteins permit the identification of reactive CTL within a complex mixture of effector cells with similar efficiencies to other previously documented methods.


Preferential transfer of certain plasma membrane proteins onto T and B cells by trogocytosis.

Daubeuf S, Aucher A, Bordier C, Salles A, Serre L, Gaibelet G, Faye JC, Favre G, Joly E, Hudrisier D - PLoS ONE (2010)

An efficiently transferred protein can be used in immunomonitoring assays to detect reactive T cells in TRAP assays.A) Splenocytes from B6 animals immunized (right panels) or not (left panels) with CyaA-OVA were co cultured with the B8 HEK-FcRγ-GFP cells (stably expressing high levels of FcRγ-GFP) transiently transfected 48 hours earlier with a vector encoding covalent H-2Kb-β2m-OVA (bottom panels) or left untransfected (top panels). At the end of the co-culture, cells were analyzed by flow cytometry and GFP fluorescence is plotted as a function of CD8 staining. Numbers represent the percentage of CD8+ cells expressing GFP (present in the gate drawn in the graph). B) As in A except that splenocytes were incubated with EL4 cells labelled with the fluorescent lipophilic probe CellVue Claret loaded (bottom panels) or not (top panels) with the OVA peptide. The Fluorescence signal from CellVue Claret was analyzed as a function of CD8 staining. Numbers represent the percentage of CD8+ cells expressing CellVue Claret fluorescence (falling within the gate drawn in the graph).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2806835&req=5

pone-0008716-g006: An efficiently transferred protein can be used in immunomonitoring assays to detect reactive T cells in TRAP assays.A) Splenocytes from B6 animals immunized (right panels) or not (left panels) with CyaA-OVA were co cultured with the B8 HEK-FcRγ-GFP cells (stably expressing high levels of FcRγ-GFP) transiently transfected 48 hours earlier with a vector encoding covalent H-2Kb-β2m-OVA (bottom panels) or left untransfected (top panels). At the end of the co-culture, cells were analyzed by flow cytometry and GFP fluorescence is plotted as a function of CD8 staining. Numbers represent the percentage of CD8+ cells expressing GFP (present in the gate drawn in the graph). B) As in A except that splenocytes were incubated with EL4 cells labelled with the fluorescent lipophilic probe CellVue Claret loaded (bottom panels) or not (top panels) with the OVA peptide. The Fluorescence signal from CellVue Claret was analyzed as a function of CD8 staining. Numbers represent the percentage of CD8+ cells expressing CellVue Claret fluorescence (falling within the gate drawn in the graph).
Mentions: In previous studies, we found it possible to identify reactive CTL via the capture of lipophilic probes incorporated in the PM of APC expressing their cognate antigen [7], [9], [28]. In the context of the findings reported here, we determined if the capture of one of the more efficiently captured molecule could be used in TRAP assays instead of lipophilic probes. For this, we used CTL responding to an immunization with an adenylate-cyclase (CyaA) vector carrying the immunodominant H-2Kb-restricted OVA 257-264 peptide of ovalbumin [7], [9], [28]. Total splenocytes from immunized or control naïve B6 mice were incubated with HEK cells stably expressing FcRγ-GFP, which had been transiently transfected or not with a construct encoding H-2Kb-OVA-β2m construct [38]. As a control, we also used EL4 cells labelled with a fluorescent lipophilic probe and pulsed with the OVA peptide as target cells. As shown in Figure 6A, we indeed found that we could identify around 20% of CD8+ T cells displaying FcRγ-GFP fluorescence after exposure of splenocytes from immunized mice to HEK-FcRγ-GFP cells expressing the H-2Kb-OVA-β2m antigen. Interestingly, this proportion of OVA reactive CTL was quite comparable to that of CTL displaying DiO staining after exposure to DiO-labelled EL4 cells pulsed with the OVA antigen (Figure 6B) or to CTL stained with the H-2Kb-OVA tetramer (not shown) or producing IFN-γ upon antigenic stimulation (not shown). Thus our results show that TRAP assays based on the capture of preferentially transferred GFP-tagged proteins permit the identification of reactive CTL within a complex mixture of effector cells with similar efficiencies to other previously documented methods.

Bottom Line: For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRgamma found among the most efficiently transferred proteins.We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells.The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.

View Article: PubMed Central - PubMed

Affiliation: CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse, France.

ABSTRACT
T and B cells capture antigens via membrane fragments of antigen presenting cells (APC) in a process termed trogocytosis. Whether (and how) a preferential transfer of some APC components occurs during trogocytosis is still largely unknown. We analyzed the transfer onto murine T and B cells of a large panel of fluorescent proteins with different intra-cellular localizations in the APC or various types of anchors in the plasma membrane (PM). Only the latter were transferred by trogocytosis, albeit with different efficiencies. Unexpectedly, proteins anchored to the PM's cytoplasmic face, or recruited to it via interaction with phosphinositides, were more efficiently transferred than those facing the outside of the cell. For proteins spanning the PM's whole width, transfer efficiency was found to vary quite substantially, with tetraspanins, CD4 and FcRgamma found among the most efficiently transferred proteins. We exploited our findings to set immunodiagnostic assays based on the capture of preferentially transferred components onto T or B cells. The preferential transfer documented here should prove useful in deciphering the cellular structures involved in trogocytosis.

Show MeSH
Related in: MedlinePlus