TAP-independent Pathway Research Articles

In Vivo Cross-Priming of MHC Class I–Restricted Antigens Requires the TAP Transporter

Recent in vitro evidence suggests two alternative mechanisms by which bone marrow–derived APCs may process exogenous antigens for presentation to CTL in vivo, a phenomenon termed cross-priming. Although in vitro studies have suggested that both TAP-dependent and TAP-independent pathways exist, we have now demonstrated an absolute requirement for a functional TAP for cross-priming to occur in vivo. Bone marrow chimeras reconstituted with marrow from TAP-defective donors develop functional CD8 + CTL, but have APCs with disrupted TAP function. In such chimeras, in vivo priming of naive CTL was observed when antigen was targeted to the ER in a TAP-independent fashion, but cross-priming could not be demonstrated. These results support the TAP-dependent mechanism of cross-priming.

Characterization of TAP‐Independent and Brefeldin A‐Resistant Presentation of Sendai Virus Antigen to CD8+ Cytotoxic T Lymphocytes

H-2Kb-transfected T2 cells, which lack both TAP1/2 and LMP2/7 genes, are able to efficiently process and present Sendai virus Antigen to Kb-restricted Sendai virus-specific CTL. This presentation is not inhibited by Brefeldin A (BFA). Here we extend our analysis of this novel antigen presentation pathway. We show that presentation of Sendai virus antigen was not due to sensitization of T2Kb cells by peptides in the virus preparation or peptides released from virus infected cells. Also, the ability to present Sendai virus in a BFA resistant fashion was specific for cells of the T2 lineage. Re-expression of TAP1/2 genes in T2Kb cells did not alter the capability to present antigen in a BFA resistant fashion, i.e. the presence of a functional TAP transporter complex did not relocate (all) peptides to the classical pathway for antigen processing and presentation. We found that co-infection of T2Kb cells with either Sendai virus plus influenza virus or Sendai virus plus VSV did not relocate presentation of influenza or VSV antigen to the TAP independent BFA resistant antigen presentation pathway. Peptide elution experiments and studies with peptide-specific CTL firmly demonstrated that the antigen presented by T2Kb cells after infection with Sendai virus was the natural Sendai virus epitope NP324-332. The same epitope, when expressed as a minigene in vaccinia virus, could be presented also by T2Kb cells but this presentation could be blocked by BFA. Thus, the TAP independent BFA resistant presentation of antigen seem cell (T2 lineage) and virus (Sendai virus) specific, but not epitope specific. The ability of T2Kb cells to present Sendai virus antigen in a TAP independent BFA resistant fashion was only partially blocked by lysosomal inhibitors such as methylamine, ammonium chloride and chloroquine. These findings demonstrate that TAP1/2-independent and BFA-resistant class I processing is only expressed in certain cell types, in parallel with classical MHC class I processing, and that Sendai virus selectively can enter this pathway. Hypothetical models for the TAP-independent class I processing are discussed.

TAP1-independent loading of class I molecules by exogenous viral proteins.

Presentation of peptides derived from endogenous proteins on class I molecules needs functional TAP peptide transporters. To reveal whether class I-associated presentation of exogenous proteins also required the presence of TAP transporters, we assessed in vitro the ability of spleen cells and macrophages from TAP1-deficient mice (TAP1-/-) to present peptides derived from exogenous recombinant viral proteins on their class I molecules. We found that recombinant glyco- and nucleoprotein from lymphocytic choriomeningitis virus and nucleoprotein of vesicular stomatitis virus were presented as efficiently by TAP1-/- cells as by control cells. Peptide regurgitation was not involved. Since particulate, non-replicating antigens can efficiently prime anti-viral cytotoxic T cells in vivo, this new, TAP-independent pathway of class I-associated antigen presentation may be applicable for vaccine strategies.

Nonclassical behavior of the thymus leukemia antigen: peptide transporter-independent expression of a nonclassical class I molecule.

The thymus leukemia (TL) antigen is a major histocompatibility complex-encoded nonclassical class I molecule. Here we present data demonstrating that expression of the TL antigen, unlike other class I molecules, is completely independent of the function of the transporter associated with antigen processing (TAP). The TL antigen is expressed by transfected TAP-2-deficient RMA-S cells when these cells are grown at 37 degrees C. In transfected RMA cells, the kinetics of arrival of TL antigen on the cell surface are similar to those of a classical class I molecule. The kinetics are not altered in TAP-deficient RMA-S cells, demonstrating that surface TL expression in TAP-deficient cells is not due to the stable expression of a few molecules that leak out by a TAP-independent pathway. Soluble TL molecules produced by Drosophila melanogaster cells are highly resistant to thermal denaturation, unlike peptide-free classical class I molecules synthesized by these insect cells. In addition, these soluble TL molecules are devoid of detectable bound peptides. The results demonstrate that the TL antigen is capable of reaching the surface without bound peptide, although acquisition of peptide or some other ligand through a TAP-independent pathway cannot be formally excluded. We speculate that the ability of the TL antigen to reach the cell surface, under conditions in which other class I molecules do not, may be related to a specialized function of the TL molecule in the mucosal immune system, and possibly in the stimulation of intestinal gamma delta T cells.

Heat-inactivated Sendai virus can enter multiple MHC class I processing pathways and generate cytotoxic T lymphocyte responses in vivo.

We have earlier described an alternative MHC class I processing pathway for Sendai virus (SV) in H-2Kb-transfected T2 cells (T2Kb). These cells have deleted genes for transporters associated with Ag processing (TAP1/2) and proteasome subunits LMP2/7 but can still process SV for the presentation of an immunodominant nucleoprotein CTL epitope (nucleoprotein peptide 324-332, FAPGNYPAL, SV9), even in the presence of the fungal metabolite brefeldin A (BFA). Presently we have compared live and heat-inactivated SV to investigate whether infectious virus, including early events such as binding and fusion at the host cell membrane, is important for nonclassical MHC class I processing and immunogenicity. We have found that heated virus (56 degrees C, boiled or autoclaved) with no fusion and hemagglutinin-neuraminidase activities, behaves similar to live SV in T2kb cells by entering a TAP-independent and BFA-resistant pathway. In EL-4 cells, which do not express this nonclassical TAP-independent and BFA-resistant pathway, heat-treated SV is processed in a BFA-sensitive way. In T1Kb- and TAP1/2-transfected T2Kb cells, as in T2Kb cells, processing of heat-inactivated SV was completely BFA resistant. Heat-inactivated SV was also found to prime CTLs in vivo. We conclude that heat-inactivated SV can enter both BFA-sensitive and -resistant MHC class I processing pathways and that SV in this respect may be particularly efficient. What property in the SV that is important for this characteristic is presently not clear but might be useful for the deliberate generation of CTL responses in vivo.