Robert Tampé

Early phagosomes in dendritic cells form a cellular compartment sufficient for cross presentation of exogenous antigens

Conventionally, MHC class I-restricted antigen (Ag) processing requires the action of the multimolecular peptide-loading complex within the endoplasmic reticulum (ER). Here we show that early phagosomes from human dendritic cells (DCs) contain the peptide-loading complex, incorporating MHC class I, β2 microglobulin, transporter associated with Ag processing (TAP), calreticulin, tapasin, and ERp57. Antigenic peptides could be translocated into purified phagosomes by TAP and loaded onto cognate class I molecules, inducing their specific dissociation from the loading complex. Endoglycosidase H-sensitive class I molecules were detected at the DC cell surface, suggesting that these molecules traffic there directly from phagosomes. Macropinocytosis also allowed internalized soluble Ags access to an ER-like compartment containing the class I loading complex. Blockade of TAP by endocytosis of a soluble derivative of human cytomegalovirus protein US6 confirmed that, although retrotranslocation into the cytosol is critical for processing, efficient association of class I molecules with peptides derived from exogenous Ags occurs within a compartment directly accessible to internalized proteins. Together, this evidence suggests that early phagosomes and pinosomes facilitate cross presentation of exogenous Ags by DCs.

Molecular mechanism and species specificity of tap inhibition by herpes simplex virus protein icp47

The immediate early protein ICP47 of herpes simplex virus (HSV) inhibits the transporter for antigen processing (TAP)‐mediated translocation of antigen‐derived peptides across the endoplasmic reticulum (ER) membrane. This interference prevents assembly of peptides with class I MHC molecules in the ER and ultimately recognition of HSV‐infected cells by cytotoxic T‐lymphocytes, potentially leading to immune evasion of the virus. Here, we demonstrate that recombinant, purified ICP47 containing a hexahistidine tag inhibits peptide import into microsomes of insect cells expressing human TAP, whereas inhibition of peptide transport by murine TAP was much less effective. This finding indicates an intrinsic species‐specificity of ICP47 and suggests that no additional proteins interacting specifically with either ICP47 or TAP are required for inhibition of peptide transport. Since neither purified nor induced ICP47 inhibited photocrosslinking of 8‐azido‐ATP to TAP1 and TAP2 it seems that ICP47 does not prevent ATP from binding to TAP. By contrast, peptide binding was completely blocked by ICP47 as shown both by photoaffinity crosslinking of peptides to TAP and peptide binding to microsomes from TAP‐transfected insect cells. Competition experiments indicated that ICP47 binds to human TAP with a higher affinity (50 nM) than peptides whereas the affinity to murine TAP was 100‐fold lower. Our data suggest that ICP47 prevents peptides from being translocated by blocking their binding to the substrate‐binding site of TAP.

A sequential model for peptide binding and transport by the transporters associated with antigen processing.

The TAP proteins translocate antigenic peptides into the endoplasmic reticulum. Investigation of the specificity of this process has been complicated by TAP-independent factors that influence the amount of peptide that accumulates in the ER in transport assays. We have developed an overexpression system in which binding of peptides to the TAP substrate-binding site and peptide transport by TAP can be quantified separately. Efficiency of peptide accumulation in the ER parallels affinity for the TAP substrate-binding site, but can be modified by interaction with the glycosylation system within the ER and, probably, peptide efflux. Random peptide mixtures of 9-16 aa display significantly higher affinity for the binding site than mixtures of shorter or longer peptides. Peptide binds to TAP heteromers in the absence of ATP and is released by the binding of ATP, suggesting a model for TAP function.

Structure and mechanism of ABC transporters

ATP-binding cassette (ABC) transporters are central to many physiological processes, including the uptake of nutrients, the non-classical secretion of signaling molecules and toxins, multidrug resistance and the development of human disease. As one might expect from this spectrum of translocation events, these ubiquitous, ATP-dependent pumps or channels are capable of transporting an enormous variety of substrates, ranging from small ions to large proteins. Recently determined structures of full-length ABC transporters and isolated ABC domains have increased our understanding of the functional mechanism of these proteins.

Stochastic sensing of proteins with receptor-modified solid-state nanopores

Solid-state nanopores are capable of the label-free analysis of single molecules. It is possible to add biochemical selectivity by anchoring a molecular receptor inside the nanopore, but it is difficult to maintain single-molecule sensitivity in these modified nanopores. Here, we show that metallized silicon nitride nanopores chemically modified with nitrilotriacetic acid receptors can be used for the stochastic sensing of proteins. The reversible binding and unbinding of the proteins to the receptors is observed in real time, and the interaction parameters are statistically analysed from single-molecule binding events. To demonstrate the versatile nature of this approach, we detect His-tagged proteins and discriminate between the subclasses of rodent IgG antibodies.

Dynamic Superresolution Imaging of Endogenous Proteins on Living Cells at Ultra-High Density

Versatile superresolution imaging methods, able to give dynamic information of endogenous molecules at high density, are still lacking in biological science. Here, superresolved images and diffusion maps of membrane proteins are obtained on living cells. The method consists of recording thousands of single-molecule trajectories that appear sequentially on a cell surface upon continuously labeling molecules of interest. It allows studying any molecules that can be labeled with fluorescent ligands including endogenous membrane proteins on living cells. This approach, named universal PAINT (uPAINT), generalizes the previously developed point-accumulation-for-imaging-in-nanoscale-topography (PAINT) method for dynamic imaging of arbitrary membrane biomolecules. We show here that the unprecedented large statistics obtained by uPAINT on single cells reveal local diffusion properties of specific proteins, either in distinct membrane compartments of adherent cells or in neuronal synapses.

Identifying MHC Class I Epitopes by Predicting the TAP Transport Efficiency of Epitope Precursors

We are able to make reliable predictions of the efficiency with which peptides of arbitrary lengths will be transported by TAP. The pressure exerted by TAP on Ag presentation thus can be assessed by checking to what extent MHC class I (MHC-I)-presented epitopes can be discriminated from random peptides on the basis of predicted TAP transport efficiencies alone. Best discriminations were obtained when N-terminally prolonged epitope precursor peptides were included and the contribution of the N-terminal residues to the score were down-weighted in comparison with the contribution of the C terminus. We provide evidence that two factors may account for this N-terminal down-weighting: 1) the uncertainty as to which precursors are used in vivo and 2) the coevolution in the C-terminal sequence specificities of TAP and other agents in the pathway, which may vary among the various MHC-I alleles. Combining predictions of MHC-I binding affinities with predictions of TAP transport efficiency led to an improved identification of epitopes, which was not the case when predictions of MHC-I binding affinities were combined with predictions of C-terminal cleavages made by the proteasome.

Functional expression and purification of the ABC transporter complex associated with antigen processing (TAP) in insect cells

Using the baculovirus expression system the gene products of human tap1 and tap2 were over‐expressed as wild‐type as well as oligohistidine fusion proteins in Spodoptera frugiperda (Sf9) insect cells. Both gene products were co‐expressed within the same cells and were found enriched in microsomal membranes. Immunoprecipitation and immobilized metal affinity chromatography revealed complex formation between TAP1 and TAP2. The expressed TAP complex was shown to be functional by peptide translocation into microsomes of Sf9 cells. Peptide transport strictly requires TAP1 and TAP2 as well as ATP. For the first time the functional expression of the human TAP complex in insect cells has been demonstrated, indicating that additional cofactors of a highly developed immune system are not essential for peptide transport across microsomal membranes.

Access of soluble antigens to the endoplasmic reticulum can explain cross-presentation by dendritic cells

In dendritic cells (DCs), peptides derived from internalized particulate substrates are efficiently cross-presented by major histocompatibility complex (MHC) class I molecules. Exogenous soluble antigens are also presented by DCs but with substantially lower efficiency. Here we show that particulate and soluble antigens use different transport pathways. Particulate antigens have been shown to access peripheral endoplasmic reticulum (ER)–like phagosomes that are competent for cross-presentation, whereas we show here that soluble proteins that escape proteolysis enter the lumen of the ER. From there, they may be translocated into the cytosol by the pathway established for ER-associated degradation and their derived peptides may be transported back into the ER for binding by MHC class I molecules. MHC class I presentation involving the constitutive retrograde transport of soluble proteins to the ER by DCs may facilitate DC tolerance to components of their extracellular environment.

Cross-presenting human γδ T cells induce robust CD8+ αβ T cell responses

γδ T cells are implicated in host defense against microbes and tumors but their mode of function remains largely unresolved. Here, we have investigated the ability of activated human Vγ9Vδ2+ T cells (termed γδ T-APCs) to cross-present microbial and tumor antigens to CD8+ αβ T cells. Although this process is thought to be mediated best by DCs, adoptive transfer of ex vivo antigen-loaded, human DCs during immunotherapy of cancer patients has shown limited success. We report that γδ T-APCs take up and process soluble proteins and induce proliferation, target cell killing and cytokine production responses in antigen-experienced and naïve CD8+ αβ T cells. Induction of APC functions in Vγ9Vδ2+ T cells was accompanied by the up-regulation of costimulatory and MHC class I molecules. In contrast, the functional predominance of the immunoproteasome was a characteristic of γδ T cells irrespective of their state of activation. γδ T-APCs were more efficient in antigen cross-presentation than monocyte-derived DCs, which is in contrast to the strong induction of CD4+ αβ T cell responses by both types of APCs. Our study reveals unexpected properties of human γδ T-APCs in the induction of CD8+ αβ T effector cells, and justifies their further exploration in immunotherapy research.