This study shows that induction of tumor-specific CD4+ T cells by vaccination with a specific viral T helper epitope, contained within a synthetic peptide, results in protective immunity against major histocompatibility complex (MHC) class II negative, virus-induced tumor cells. Protection was also induced against sarcoma induction by acutely transforming retrovirus. In contrast, no protective immunity was induced by vaccination with an unrelated T helper epitope. By cytokine pattern analysis, the induced CD4+ T cells were of the T helper cell 1 type. The peptide-specific CD4+ T cells did not directly recognize the tumor cells, indicating involvement of cross-priming by tumor-associated antigen-presenting cells. The main effector cells responsible for tumor eradication were identified as CD8+ cytotoxic T cells that were found to recognize a recently described immunodominant viral gag-encoded cytotoxic T lymphocyte (CTL) epitope, which is unrelated to the viral env-encoded T helper peptide sequence. Simultaneous vaccination with the tumor-specific T helper and CTL epitopes resulted in strong synergistic protection. These results indicate the crucial role of T helper cells for optimal induction of protective immunity against MHC class II negative tumor cells. Protection is dependent on tumor-specific CTLs in this model system and requires cross-priming of tumor antigens by specialized antigen-presenting cells. Thus, tumor-specific T helper epitopes have to be included in the design of epitope-based vaccines.

Defects in major histocompatibility complex (MHC) class I-restricted antigen presentation are frequently observed in human cancers and result in escape of tumors from cytotoxic T lymphocyte (CTL) immune surveillance in mice. Here, we show the existence of a unique category of CTLs that can prevent this escape. The CTLs target an alternative repertoire of peptide epitopes that emerge in MHC class I at the surface of cells with impaired function of transporter associated with antigen processing (TAP), tapasin or the proteasome. These peptides, although derived from self antigens such as the commonly expressed Lass5 protein (also known as Trh4), are not presented by normal cells. This explains why they act as immunogenic neoantigens. The newly discovered epitopes can be exploited for immune intervention against processing-deficient tumors through adoptive T-cell transfer or peptide vaccination.

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Dendritic cells (DCs) are crucial for priming of naive CD8 ؉ T lymphocytes to exogenous antigens, so-called ''cross-priming.'' We report that exogenous protein antigen can be conserved for several days in mature DCs, coinciding with strong cytotoxic T lymphocyte crosspriming potency in vivo. After MHC class I peptide elution, protein antigen-derived peptide presentation is efficiently restored, indicating the presence of an intracellular antigen depot. We characterized this depot as a lysosome-like organelle, distinct from MHC class II compartments and recently described early endosomal compartments that allow acute antigen presentation in MHC class I. The storage compartments we report here facilitate continuous supply of MHC class I ligands. This mechanism ensures sustained crosspresentation by DCs, despite the short-lived expression of MHC class I-peptide complexes at the cell surface.antigen processing ͉ cross-presentation ͉ endocytosis ͉ Fc receptor ͉ MHC class I D endritic cells (DCs) are crucial in the initiation and orchestration of the T cell immune response (1-3). DCs operate as sentinels of an infection in the periphery and subsequently as conductors of the T cell response in the lymph nodes. To exert these functions, DCs are specialized in the ingestion, processing, and presentation of antigens acquired by receptor-independent pinocytosis or by receptor-mediated endocytosis (4-6). To this end, they are equipped with a diverse set of receptors for uptake of antigen, such as scavenger receptors, lectin receptors, or IgG (Fc␥) receptors (7).Immature DCs have the capacity to efficiently acquire antigen, but a poor capacity to migrate and to stimulate T cells. Proper T cell response initiation requires maturation of the DCs, a process that is triggered by contact with infectious or inflammatory signals. Mature DCs characteristically show enhanced migratory capacity, up-regulation of the MHC class I processing machinery, and enhanced expression of MHC I and II and costimulatory molecules.DCs present antigenic peptides in either MHC class I to induce CD8 T cell responses and in MHC class II to induce CD4 T cell responses. Whereas MHC class I ligands are commonly derived from breakdown products of endogenous proteins that are degraded by the proteasome (8), DCs also have the unique capacity to present peptides derived from exogenous antigens in MHC class I to CD8 ϩ T cells, a process called ''crosspresentation.'' This process is crucial for induction of effective cytotoxic T lymphocyte (CTL) immunity against tumors, which lack direct priming capacity themselves, but also against microorganisms including viruses (1).DCs have dedicated organelles to facilitate efficient loading of antigenic peptides in MHC class II molecules. These MHC class II compartments (MIIC) are multivesicular endosomes that express high levels of MHC class II and invariant chain and are abundantly present in immature DCs (9). On maturation of the DC, rapid reorganization of the MIIC takes place that facilitates transport of MHC class II...

The efficiency of antigen (Ag) processing by dendritic cells (DCs) is vital for the strength of the ensuing T-cell responses. Previously, we and others have shown that in comparison to protein vaccines, vaccination with synthetic long peptides (SLPs) has shown more promising (pre-)clinical results. Here, we studied the unknown mechanisms underlying the observed vaccine efficacy of SLPs. We report an in vitro processing analysis of SLPs for MHC class I and class II presentation by murine DCs and human monocyte-derived DCs. Compared to protein, SLPs were rapidly and much more efficiently processed by DCs, resulting in an increased presentation to CD4 + and CD8 + T cells. The mechanism of access to MHC class I loading appeared to differ between the two forms of Ag. Whereas whole soluble protein Ag ended up largely in endolysosomes, SLPs were detected very rapidly outside the endolysosomes after internalization by DCs, followed by proteasomeand transporter associated with Ag processing-dependent MHC class I presentation. Compared to the slower processing route taken by whole protein Ags, our results indicate that the efficient internalization of SLPs, accomplished by DCs but not by B or T cells and characterized by a different and faster intracellular routing, leads to enhanced CD8 + T-cell activation.Keywords: Antigen presentation/processing r Cellular immunology r CD8 + T cells r Dendritic cells Additional supporting information may be found in the online version of this article at the publisher's web-site lower efficiency compared to SLP-loaded DCs (Fig. 1B). Prestimulation of DCs with the TLR4 ligand LPS had no effect on the MHC class I presentation of OVA-protein but improved Ag presentation of SSP-OVA 8aa (data not shown) and long peptide Ag (Fig. 1C). HLA-B7-restricted presentation by human monocyte-derived DCs (MoDCs) of HIV-derived protein and SLPs was also studied. We were unable to detect cytokine production by CD8 + T cells cocultured with GAG-protein-loaded DCs. In contrast, SLP-GAG 22aa induced significant CD8 + T-cell activation (see Fig. 2 and below).Together, these data show that cross-presentation of SLPs is superior to that of proteins as examined with both mouse and human DCs. Rapid Ag presentation of SLPs by murine and human DCsThe efficiency of SLP-processing was assessed by studying the time required for DCs to present Ag on MHC class I (H2-K b )molecules. Murine DCs were incubated with a single concentration of SLPs, synthetic short peptides (SSPs) or protein for the indicated time periods. The minimal peptide, SSPs, was rapidly presented to CD8 + T cells resulting in strong activation already after 1 h. DCs loaded with SLP also activated CD8 + T cells 1 h after Ag loading but with lower potency. We excluded that SLPs were cleaved extracellularly, processed, and loaded on MHC class I and II molecules by incubating paraformaldehyde (PFA) fixed cells with the peptide Ag and observed no cross-presentation (data not shown). DCs loaded with 10 μM OVA-protein failed to induce significant CD8 + T-ce...