Microparticle-mediated transfer of the viral receptors CAR and CD46, and the CFTR channel in a CHO cell model confers new functions to target cells

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins

Dynamics of Macrophage Trogocytosis of Rituximab-Coated B Cells

Macrophages can remove antigen from the surface of antibody-coated cells by a process termed trogocytosis. Using live cell microscopy and flow cytometry, we investigated the dynamics of trogocytosis by RAW264.7 macrophages of Ramos B cells opsonized with the anti-CD20 monoclonal antibody rituximab. Spontaneous and reversible formation of uropods was observed on Ramos cells, and these showed a strong enrichment in rituximab binding. RAW-Ramos conjugate interfaces were highly enriched in rituximab, and transfer of rituximab to the RAW cells in submicron-sized puncta occurred shortly after cell contact. Membrane from the target cells was concomitantly transferred along with rituximab to a variable extent. We established a flow cytometry-based approach to follow the kinetics of transfer and internalization of rituximab. Disruption of actin polymerization nearly eliminated transfer, while blocking phosphatidylinositol 3-kinase activity only resulted in a delay in its acquisition. Inhibition of Src family kinase activity both slowed acquisition and reduced the extent of trogocytosis. The effects of inhibiting these kinases are likely due to their role in efficient formation of cell-cell conjugates. Selective pre-treatment of Ramos cells with phenylarsine oxide blocked uropod formation, reduced enrichment of rituximab at cell-cell interfaces, and reduced the efficiency of trogocytic transfer of rituximab. Our findings highlight that dynamic changes in target cell shape and surface distribution of antigen may significantly influence the progression and extent of trogocytosis. Understanding the mechanistic determinants of macrophage trogocytosis will be important for optimal design of antibody therapies

Trans-endocytosis of CD80 and CD86:a molecular basis for the cell-extrinsic function of CTLA-4

Cytotoxic T lymphocyte antigen 4 (CTLA-4) is an essential negative regulator of T cell immune responses whose mechanism of action is the subject of debate. CTLA-4 shares two ligands (CD80 and CD86) with a stimulatory receptor, CD28. Here, we show that CTLA-4 can capture its ligands from opposing cells by a process of trans-endocytosis. After removal, these costimulatory ligands are degraded inside CTLA-4-expressing cells, resulting in impaired costimulation via CD28. Acquisition of CD86 from antigen-presenting cells is stimulated by T cell receptor engagement and observed in vitro and in vivo. These data reveal a mechanism of immune regulation in which CTLA-4 acts as an effector molecule to inhibit CD28 costimulation by the cell-extrinsic depletion of ligands, accounting for many of the known features of the CD28-CTLA-4 system

Intercellular Transfer of Oncogenic H-Ras at the Immunological Synapse

Immune cells establish dynamic adhesive cell–cell interactions at a specific contact region, termed the immunological synapse (IS). Intriguing features of the IS are the formation of regions of plasma membrane fusion and the intercellular exchange of membrane fragments between the conjugated cells. It is not known whether upon IS formation, intact intracellular proteins can transfer from target cells to lymphocytes to allow the transmission of signals across cell boundaries. Here we show by both FACS and confocal microscopy that human lymphocytes acquire from the cells they scan the inner-membrane protein H-Ras, a G-protein vital for common lymphocyte functions and a prominent participant in human cancer. The transfer was cell contact-dependent and occurred in the context of cell-conjugate formation. Moreover, the acquisition of oncogenic H-RasG12V by natural killer (NK) and T lymphocytes had important biological functions in the adopting lymphocytes: the transferred H-RasG12V induced ERK phosphorylation, increased interferon-γ and tumor necrosis factor-α secretion, enhanced lymphocyte proliferation, and augmented NK-mediated target cell killing. Our findings reveal a novel mode of cell-to-cell communication—allowing lymphocytes to extend the confines of their own proteome—which may moreover play an important role in natural tumor immunity

Fas Signalling Promotes Intercellular Communication in T Cells

Cell-to-cell communication is a fundamental process for development and maintenance of multicellular organisms. Diverse mechanisms for the exchange of molecular information between cells have been documented, such as the exchange of membrane fragments (trogocytosis), formation of tunneling nanotubes (TNTs) and release of microvesicles (MVs). In this study we assign to Fas signalling a pivotal role for intercellular communication in CD4+ T cells. Binding of membrane-bound FasL to Fas expressing target cells triggers a well-characterized pro-apoptotic signalling cascade. However, our results, pairing up flow cytometric studies with confocal microscopy data, highlight a new social dimension for Fas/FasL interactions between CD4+ T cells. Indeed, FasL enhances the formation of cell conjugates (8 fold of increase) in an early time-frame of stimulation (30 min), and this phenomenon appears to be a crucial step to prime intercellular communication. Our findings show that this communication mainly proceeds along a cytosolic material exchange (ratio of exchange >10, calculated as ratio of stimulated cells signal divided by that recorded in control cells) via TNTs and MVs release. In particular, inhibition of TNTs genesis by pharmacological agents (Latruculin A and Nocodazole) markedly reduced this exchange (inhibition percentage: >40% and >50% respectively), suggesting a key role for TNTs in CD4+ T cells communication. Although MVs are present in supernatants from PHA-activated T cells, Fas treatment also leads to a significant increase in the amount of released MVs. In fact, the co-culture performed between MVs and untreated cells highlights a higher presence of MVs in the medium (1.4 fold of increase) and a significant MVs uptake (6 fold of increase) by untreated T lymphocytes. We conclude that Fas signalling induces intercellular communication in CD4+ T cells by different mechanisms that seem to start concomitantly with the main pathway (programmed cell death) promoted by FasL

Plasmodium falciparum Adhesion on Human Brain Microvascular Endothelial Cells Involves Transmigration-Like Cup Formation and Induces Opening of Intercellular Junctions

Cerebral malaria, a major cause of death during malaria infection, is characterised by the sequestration of infected red blood cells (IRBC) in brain microvessels. Most of the molecules implicated in the adhesion of IRBC on endothelial cells (EC) are already described; however, the structure of the IRBC/EC junction and the impact of this adhesion on the EC are poorly understood. We analysed this interaction using human brain microvascular EC monolayers co-cultured with IRBC. Our study demonstrates the transfer of material from the IRBC to the brain EC plasma membrane in a trogocytosis-like process, followed by a TNF-enhanced IRBC engulfing process. Upon IRBC/EC binding, parasite antigens are transferred to early endosomes in the EC, in a cytoskeleton-dependent process. This is associated with the opening of the intercellular junctions. The transfer of IRBC antigens can thus transform EC into a target for the immune response and contribute to the profound EC alterations, including peri-vascular oedema, associated with cerebral malaria

Bone Marrow Transplantation Results in Human Donor Blood Cells Acquiring and Displaying Mouse Recipient Class I MHC and CD45 Antigens on Their Surface

Background: Mouse models of human disease are invaluable for determining the differentiation ability and functional capacity of stem cells. The best example is bone marrow transplants for studies of hematopoietic stem cells. For organ studies, the interpretation of the data can be difficult as transdifferentiation, cell fusion or surface antigen transfer (trogocytosis) can be misinterpreted as differentiation. These events have not been investigated in hematopoietic stem cell transplant models. Methodology/Principal Findings: In this study we investigated fusion and trogocytosis involving blood cells during bone marrow transplantation using a xenograft model. We report that using a standard SCID repopulating assay almost 100 % of the human donor cells appear as hybrid blood cells containing both mouse and human surface antigens. Conclusion/Significance: Hybrid cells are not the result of cell-cell fusion events but appear to be due to efficient surface antigen transfer, a process referred to as trogocytosis. Antigen transfer appears to be non-random and includes all donor cells regardless of sub-type. We also demonstrate that irradiation preconditioning enhances the frequency of hybrid cell

Dendritic cell-specific delivery of Flt3L by coronavirus vectors secures induction of therapeutic antitumor immunity

Efficacy of antitumor vaccination depends to a large extent on antigen targeting to dendritic cells (DCs). Here, we assessed antitumor immunity induced by attenuated coronavirus vectors which exclusively target DCs in vivo and express either lymphocyte- or DC-activating cytokines in combination with a GFP-tagged model antigen. Tracking of in vivo transduced DCs revealed that vectors encoding for Fms-like tyrosine kinase 3 ligand (Flt3L) exhibited a higher capacity to induce DC maturation compared to vectors delivering IL-2 or IL-15. Moreover, Flt3L vectors more efficiently induced tumor-specific CD8(+) T cells, expanded the epitope repertoire, and provided both prophylactic and therapeutic tumor immunity. In contrast, IL-2- or IL-15-encoding vectors showed a substantially lower efficacy in CD8(+) T cell priming and failed to protect the host once tumors had been established. Thus, specific in vivo targeting of DCs with coronavirus vectors in conjunction with appropriate conditioning of the microenvironment through Flt3L represents an efficient strategy for the generation of therapeutic antitumor immunity

T cells at the site of autoimmune inflammation show increased potential for trogocytosis

CD4+ T cells acquire membrane fragments from antigen-presenting-cells via a process termed trogocytosis. Identifying which CD4+ T cells undergo trogocytosis in co-culture with Ag-loaded APC can enrich for antigen-reactive T cells without knowledge of their fine specificity or cytokine-production profiles. We sought to assess the suitability of this method to identify disease relevant effector and regulatory T cells during autoimmune inflammation. Trogocytosis efficiently identified MBP-reactive T cells in vitro and ex-vivo following immunization. However, Foxp3+ regulatory T cells constitutively displayed a higher rate of trogocytosis than their Foxp3- counterparts which limits the potential of trogocytosis to identify antigen-reactive Treg cells. During inflammation a locally elevated rate of trogocytosis (seen in both effector and regulatory T cells isolated from the inflamed CNS) precludes the use of trogocytosis as a measure of antigenic reactivity among cells taken from inflammatory sites. Our results indicate trogocytosis detection can enrich for Ag-reactive conventional T cells in the periphery but is limited in its ability to identify Ag-reactive Treg or T effector cells at sites of inflammation. Increased trogocytosis potential at inflammatory sites also draws into the question the biological significance of this phenomenon during inflammation, in Treg mediated suppression and for the maintenance of tolerance in health and disease