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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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Related in: MedlinePlus

HEK-FcγRII cells and their transient transfectants are targets of murine T and B cells in redirected trogocytosis experiments.A) HEK cells (grey histogram) or HEK-FcγRII (white histogram) were stained with the anti-murine FcγRII/RIII mAb 2.4G2 and analyzed by flow cytometry. B) HEK-FcγRII cells were incubated 1 hour at 37°C with activated OT-I T cells in the presence (white histogram) or absence (grey histogram) of the Y3 mAb triggering trogocytosis or in the presence of its isotype control (dashed line) before analysis by flow cytometry using anti-CD8 and anti-FcγRII/RIII mAb. Shown are histograms of FcγRII/RII staining on gated CD8+ T cells. C) as in B) except that OT-II T cells were used instead of OT-I cells and were detected using anti-CD4 mAb. D) As in B) except that MD4 B cells coated or not with anti-κ chain mAb or its isotype control were used instead of OT-I T cells and that FcγRII/RIII capture was detected with the anti-Flag Ab on gated B220+ B cells. E) As in B) except that capture of FcγRII was analyzed after co-culture of OT-I T cells with HEK-FcγRII that were transiently transfected 48 hours earlier with vectors encoding the indicated proteins fused to GFP. Capture of FcγRII from HEK-FcγRII transfected with the indicated constructs is shown as fold induction, which was calculated as indicated in the Materials and Methods section and normalized with fold induction obtained in cocultures with untransfected HEK-FcγRII. Represented are means plus standard deviation from 3 independent experiments (no statistically significant differences emerged from student t-test analysis).
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pone-0008716-g001: HEK-FcγRII cells and their transient transfectants are targets of murine T and B cells in redirected trogocytosis experiments.A) HEK cells (grey histogram) or HEK-FcγRII (white histogram) were stained with the anti-murine FcγRII/RIII mAb 2.4G2 and analyzed by flow cytometry. B) HEK-FcγRII cells were incubated 1 hour at 37°C with activated OT-I T cells in the presence (white histogram) or absence (grey histogram) of the Y3 mAb triggering trogocytosis or in the presence of its isotype control (dashed line) before analysis by flow cytometry using anti-CD8 and anti-FcγRII/RIII mAb. Shown are histograms of FcγRII/RII staining on gated CD8+ T cells. C) as in B) except that OT-II T cells were used instead of OT-I cells and were detected using anti-CD4 mAb. D) As in B) except that MD4 B cells coated or not with anti-κ chain mAb or its isotype control were used instead of OT-I T cells and that FcγRII/RIII capture was detected with the anti-Flag Ab on gated B220+ B cells. E) As in B) except that capture of FcγRII was analyzed after co-culture of OT-I T cells with HEK-FcγRII that were transiently transfected 48 hours earlier with vectors encoding the indicated proteins fused to GFP. Capture of FcγRII from HEK-FcγRII transfected with the indicated constructs is shown as fold induction, which was calculated as indicated in the Materials and Methods section and normalized with fold induction obtained in cocultures with untransfected HEK-FcγRII. Represented are means plus standard deviation from 3 independent experiments (no statistically significant differences emerged from student t-test analysis).

Mentions: With the aim of understanding if some proteins were more efficiently transferred than others during trogocytosis, we chose an approach whereby fluorescent forms of proteins representative of various subcellular localizations and various anchoring to the plasma membrane were expressed in transiently transfected cells. We would then co-culture these transiently transfected cells with either T or B cells, and analyse the efficiency of transfer of the GFP-tagged proteins by trogocytosis. Because only a small percentage of the components present on the target cells are captured by effector cells, we needed a system where the fluorescent proteins could be expressed at high levels and in a large proportion of target cells. Transient transfection of HEK cells is one of the more efficient and versatile systems for the expression of plasmid-encoded recombinant proteins in a mammalian cell. Therefore, we used HEK cells engineered to express a flagged form of FcγRII [31] (Figure 1A), which allows them to be targeted by T or B cells coated with appropriate antibodies (an approach termed «redirected trogocytosis”) [32], [33]. For OT-I CD8+ or OT-II CD4+ T, trogocytosis on the HEK-FcγRII cells was triggered very efficiently by the Y3 anti-H-2Kb mAb, as shown by the capture of the FcγRII receptor but not in the absence of mAb nor in the presence of a mouse IgG2a isotype control (Figure 1B and C). The capture of FcγRII receptor by T cells was simply detected with the 2.4.G2 anti-FcγRII/III mAb since T cells do not express these receptors endogenously. For MD4 B cells, we found that HEK-FcγRII cells could also be used as targets for redirected trogocytosis when the co-culture was performed in the presence of the anti-BCR κ chain mAb but not in the absence of mAb nor in the presence of the rat IgG1 isotype control (Figure 1D). In B cells, which constitutively express FcγRII, we used the Flag epitope carried by the recombinant FcγRII protein expressed by HEK cells to monitor trogocytosis with anti-Flag antibodies. Since, as in previous studies [32], we could not detect any differences (i.e. no trogocytosis was triggered) when isotypic controls were used as compared with no mAb added (Figure 1B–D) we chose to use controls simply performed in the absence of mAb for the bulk of our experiments. We obtained or generated a series of 32 different constructs encoding proteins fused to GFP or chromatic variants. The identity, source and expected subcellular location are summarized in Tables 1 and 2. When each one of these constructs was transiently transfected in HEK-FcγRII, a high proportion (>40%) of the cells consistently over-expressed the protein (see Figure S1). The vast majority of these constructs which encode proteins fused to AFP have been described before and extensively used by various laboratories worldwide (see reference for each of them in Table 1 and 2). For each of these constructs, we thus simply confirmed the expected localization of each fluorescent protein by fluorescence microscopy (Figure 2 and data not shown). For the few constructs generated in our laboratory, a more detailed analysis of their localization was performed using co-localization experiments with plasma membrane or nucleus markers (not shown). Furthermore, we confirmed the correct topology of the various fusion proteins using a polyclonal anti-GFP antibody in flow cytometry experiments, by checking that permeabilization was required in order to detect AFP moieties located intracellularly whereas this step was not necessary when the AFP moiety was accessible extracellularly (not shown). Importantly, we found no marked alteration in the overall efficiency of trogocytosis by OT-I CTL for any of the plasmids used, as detected by the capture of FcγRII (Figure 1E), indicating that the overexpression of these proteins does not detectably alter the interaction between target cells and effector cells. Similar results were obtained with OT-II cells or MD4 B cells (not shown). Thus, using redirected trogocytosis, HEK-FcγRII cells provide a versatile cellular system to study the capture of overexpressed, fluorescent proteins by T or B cells and the capture of FcγRII can be used to control for trogocytosis efficiency.


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)

HEK-FcγRII cells and their transient transfectants are targets of murine T and B cells in redirected trogocytosis experiments.A) HEK cells (grey histogram) or HEK-FcγRII (white histogram) were stained with the anti-murine FcγRII/RIII mAb 2.4G2 and analyzed by flow cytometry. B) HEK-FcγRII cells were incubated 1 hour at 37°C with activated OT-I T cells in the presence (white histogram) or absence (grey histogram) of the Y3 mAb triggering trogocytosis or in the presence of its isotype control (dashed line) before analysis by flow cytometry using anti-CD8 and anti-FcγRII/RIII mAb. Shown are histograms of FcγRII/RII staining on gated CD8+ T cells. C) as in B) except that OT-II T cells were used instead of OT-I cells and were detected using anti-CD4 mAb. D) As in B) except that MD4 B cells coated or not with anti-κ chain mAb or its isotype control were used instead of OT-I T cells and that FcγRII/RIII capture was detected with the anti-Flag Ab on gated B220+ B cells. E) As in B) except that capture of FcγRII was analyzed after co-culture of OT-I T cells with HEK-FcγRII that were transiently transfected 48 hours earlier with vectors encoding the indicated proteins fused to GFP. Capture of FcγRII from HEK-FcγRII transfected with the indicated constructs is shown as fold induction, which was calculated as indicated in the Materials and Methods section and normalized with fold induction obtained in cocultures with untransfected HEK-FcγRII. Represented are means plus standard deviation from 3 independent experiments (no statistically significant differences emerged from student t-test analysis).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2806835&req=5

pone-0008716-g001: HEK-FcγRII cells and their transient transfectants are targets of murine T and B cells in redirected trogocytosis experiments.A) HEK cells (grey histogram) or HEK-FcγRII (white histogram) were stained with the anti-murine FcγRII/RIII mAb 2.4G2 and analyzed by flow cytometry. B) HEK-FcγRII cells were incubated 1 hour at 37°C with activated OT-I T cells in the presence (white histogram) or absence (grey histogram) of the Y3 mAb triggering trogocytosis or in the presence of its isotype control (dashed line) before analysis by flow cytometry using anti-CD8 and anti-FcγRII/RIII mAb. Shown are histograms of FcγRII/RII staining on gated CD8+ T cells. C) as in B) except that OT-II T cells were used instead of OT-I cells and were detected using anti-CD4 mAb. D) As in B) except that MD4 B cells coated or not with anti-κ chain mAb or its isotype control were used instead of OT-I T cells and that FcγRII/RIII capture was detected with the anti-Flag Ab on gated B220+ B cells. E) As in B) except that capture of FcγRII was analyzed after co-culture of OT-I T cells with HEK-FcγRII that were transiently transfected 48 hours earlier with vectors encoding the indicated proteins fused to GFP. Capture of FcγRII from HEK-FcγRII transfected with the indicated constructs is shown as fold induction, which was calculated as indicated in the Materials and Methods section and normalized with fold induction obtained in cocultures with untransfected HEK-FcγRII. Represented are means plus standard deviation from 3 independent experiments (no statistically significant differences emerged from student t-test analysis).
Mentions: With the aim of understanding if some proteins were more efficiently transferred than others during trogocytosis, we chose an approach whereby fluorescent forms of proteins representative of various subcellular localizations and various anchoring to the plasma membrane were expressed in transiently transfected cells. We would then co-culture these transiently transfected cells with either T or B cells, and analyse the efficiency of transfer of the GFP-tagged proteins by trogocytosis. Because only a small percentage of the components present on the target cells are captured by effector cells, we needed a system where the fluorescent proteins could be expressed at high levels and in a large proportion of target cells. Transient transfection of HEK cells is one of the more efficient and versatile systems for the expression of plasmid-encoded recombinant proteins in a mammalian cell. Therefore, we used HEK cells engineered to express a flagged form of FcγRII [31] (Figure 1A), which allows them to be targeted by T or B cells coated with appropriate antibodies (an approach termed «redirected trogocytosis”) [32], [33]. For OT-I CD8+ or OT-II CD4+ T, trogocytosis on the HEK-FcγRII cells was triggered very efficiently by the Y3 anti-H-2Kb mAb, as shown by the capture of the FcγRII receptor but not in the absence of mAb nor in the presence of a mouse IgG2a isotype control (Figure 1B and C). The capture of FcγRII receptor by T cells was simply detected with the 2.4.G2 anti-FcγRII/III mAb since T cells do not express these receptors endogenously. For MD4 B cells, we found that HEK-FcγRII cells could also be used as targets for redirected trogocytosis when the co-culture was performed in the presence of the anti-BCR κ chain mAb but not in the absence of mAb nor in the presence of the rat IgG1 isotype control (Figure 1D). In B cells, which constitutively express FcγRII, we used the Flag epitope carried by the recombinant FcγRII protein expressed by HEK cells to monitor trogocytosis with anti-Flag antibodies. Since, as in previous studies [32], we could not detect any differences (i.e. no trogocytosis was triggered) when isotypic controls were used as compared with no mAb added (Figure 1B–D) we chose to use controls simply performed in the absence of mAb for the bulk of our experiments. We obtained or generated a series of 32 different constructs encoding proteins fused to GFP or chromatic variants. The identity, source and expected subcellular location are summarized in Tables 1 and 2. When each one of these constructs was transiently transfected in HEK-FcγRII, a high proportion (>40%) of the cells consistently over-expressed the protein (see Figure S1). The vast majority of these constructs which encode proteins fused to AFP have been described before and extensively used by various laboratories worldwide (see reference for each of them in Table 1 and 2). For each of these constructs, we thus simply confirmed the expected localization of each fluorescent protein by fluorescence microscopy (Figure 2 and data not shown). For the few constructs generated in our laboratory, a more detailed analysis of their localization was performed using co-localization experiments with plasma membrane or nucleus markers (not shown). Furthermore, we confirmed the correct topology of the various fusion proteins using a polyclonal anti-GFP antibody in flow cytometry experiments, by checking that permeabilization was required in order to detect AFP moieties located intracellularly whereas this step was not necessary when the AFP moiety was accessible extracellularly (not shown). Importantly, we found no marked alteration in the overall efficiency of trogocytosis by OT-I CTL for any of the plasmids used, as detected by the capture of FcγRII (Figure 1E), indicating that the overexpression of these proteins does not detectably alter the interaction between target cells and effector cells. Similar results were obtained with OT-II cells or MD4 B cells (not shown). Thus, using redirected trogocytosis, HEK-FcγRII cells provide a versatile cellular system to study the capture of overexpressed, fluorescent proteins by T or B cells and the capture of FcγRII can be used to control for trogocytosis efficiency.

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