Ferry Ossendorp

CTLs are targeted to kill β cells in patients with type 1 diabetes through recognition of a glucose-regulated preproinsulin epitope

The final pathway of beta cell destruction leading to insulin deficiency, hyperglycemia, and clinical type 1 diabetes is unknown. Here we show that circulating CTLs can kill beta cells via recognition of a glucose-regulated epitope. First, we identified 2 naturally processed epitopes from the human preproinsulin signal peptide by elution from HLA-A2 (specifically, the protein encoded by the A*0201 allele) molecules. Processing of these was unconventional, requiring neither the proteasome nor transporter associated with processing (TAP). However, both epitopes were major targets for circulating effector CD8+ T cells from HLA-A2+ patients with type 1 diabetes. Moreover, cloned preproinsulin signal peptide-specific CD8+ T cells killed human beta cells in vitro. Critically, at high glucose concentration, beta cell presentation of preproinsulin signal epitope increased, as did CTL killing. This study provides direct evidence that autoreactive CTLs are present in the circulation of patients with type 1 diabetes and that they can kill human beta cells. These results also identify a mechanism of self-antigen presentation that is under pathophysiological regulation and could expose insulin-producing beta cells to increasing cytotoxicity at the later stages of the development of clinical diabetes. Our findings suggest that autoreactive CTLs are important targets for immune-based interventions in type 1 diabetes and argue for early, aggressive insulin therapy to preserve remaining beta cells.

Reorganization of multivesicular bodies regulates MHC class II antigen presentation by dendritic cells

Immature dendritic cells (DCs) sample their environment for antigens and after stimulation present peptide associated with major histocompatibility complex class II (MHC II) to naive T cells. We have studied the intracellular trafficking of MHC II in cultured DCs. In immature cells, the majority of MHC II was stored intracellularly at the internal vesicles of multivesicular bodies (MVBs). In contrast, DM, an accessory molecule required for peptide loading, was located predominantly at the limiting membrane of MVBs. After stimulation, the internal vesicles carrying MHC II were transferred to the limiting membrane of the MVB, bringing MHC II and DM to the same membrane domain. Concomitantly, the MVBs transformed into long tubular organelles that extended into the periphery of the cells. Vesicles that were formed at the tips of these tubules nonselectively incorporated MHC II and DM and presumably mediated transport to the plasma membrane. We propose that in maturing DCs, the reorganization of MVBs is fundamental for the timing of MHC II antigen loading and transport to the plasma membrane.

Antigen-Antibody Immune Complexes Empower Dendritic Cells to Efficiently Prime Specific CD8+ CTL Responses In Vivo

Dendritic cells (DCs) require a maturation signal to acquire efficient CTL-priming capacity. In vitro FcγR-mediated internalization of Ag-Ab immune complexes (ICs) can induce maturation of DCs. In this study, we show that IC-induced DC maturation in vitro enables DCs to prime peptide-specific CD8+ CTLs in vivo, independently of CD4+ Th cells. Importantly, OVA/anti-OVA IC-treated DCs not only primed CD8+ CTLs to an exogenously loaded peptide nonrelated to OVA, but also efficiently primed CTLs against the dominant CTL epitope derived from the OVA Ag present in the ICs. Our studies show that ICs fulfill a dual role in priming of CD8+ CTL responses to exogenous Ags: enhancement of Ag uptake by DCs and activation of DCs, resulting in “license to kill.” These findings indicate that the presence of specific Abs can crucially affect the induction of cytotoxic cellular responses.

A Single Residue Exchange Within a Viral CTL Epitope Alters Proteasome-Mediated Degradation Resulting in Lack of Antigen Presentation

CTL epitope (KSPWFTTL) encoded by AKV/MCF type of murine leukemia virus (MuLV) differs from the sequence in Friend/Moloney/Rauscher (FMR) type in one residue (RSPWFTTL). CTL experiments indicated defective processing of the FMR peptide in tumor cells. Proteasome-mediated digestion of AKV/MCF-type 26-mer peptides resulted in the early generation and higher levels of epitope-containing fragments than digestion of FMR-type peptides, explained by prominent cleavage next to R in the FMR sequence. The fragments were identified as 10- and 11-mer peptides and were efficiently translocated by TAP. The naturally presented AKV/MCF peptide is the 8-mer, indicating ER peptide trimming. In conclusion, a single residue exchange can cause CTL epitope destruction by specific proteasomal cleavage.

Vaccines for established cancer: overcoming the challenges posed by immune evasion

Therapeutic vaccines preferentially stimulate T cells against tumour-specific epitopes that are created by DNA mutations or oncogenic viruses. In the setting of premalignant disease, carcinoma in situ or minimal residual disease, therapeutic vaccination can be clinically successful as monotherapy; however, in established cancers, therapeutic vaccines will require co-treatments to overcome immune evasion and to become fully effective. In this Review, we discuss the progress that has been made in overcoming immune evasion controlled by tumour cell-intrinsic factors and the tumour microenvironment. We summarize how therapeutic benefit can be maximized in patients with established cancers by improving vaccine design and by using vaccines to increase the effects of standard chemotherapies, to establish and/or maintain tumour-specific T cells that are re-energized by checkpoint blockade and other therapies, and to sustain the antitumour response of adoptively transferred T cells.

Therapeutic cancer vaccines

The clinical benefit of therapeutic cancer vaccines has been established. Whereas regression of lesions was shown for premalignant lesions caused by HPV, clinical benefit in cancer patients was mostly noted as prolonged survival. Suboptimal vaccine design and an immunosuppressive cancer microenvironment are the root causes of the lack of cancer eradication. Effective cancer vaccines deliver concentrated antigen to both HLA class I and II molecules of DCs, promoting both CD4 and CD8 T cell responses. Optimal vaccine platforms include DNA and RNA vaccines and synthetic long peptides. Antigens of choice include mutant sequences, selected cancer testis antigens, and viral antigens. Drugs or physical treatments can mitigate the immunosuppressive cancer microenvironment and include chemotherapeutics, radiation, indoleamine 2,3-dioxygenase (IDO) inhibitors, inhibitors of T cell checkpoints, agonists of selected TNF receptor family members, and inhibitors of undesirable cytokines. The specificity of therapeutic vaccination combined with such immunomodulation offers an attractive avenue for the development of future cancer therapies.

MHC II in dendritic cells is targeted to lysosomes or T cell-induced exosomes via distinct multivesicular body pathways.

Dendritic cells (DCs) express major histocompatibility complex class II (MHC II) to present peptide antigens to T cells. In immature DCs, which bear low cell surface levels of MHC II, peptide‐loaded MHC II is ubiquitinated. Ubiquitination drives the endocytosis and sorting of MHC II to the luminal vesicles of multivesicular bodies (MVBs) for lysosomal degradation. Ubiquitination of MHC II is abrogated in activated DCs, resulting in an increased cell surface expression. We here provide evidence for an alternative MVB sorting mechanism for MHC II in antigen‐loaded DCs, which is triggered by cognately interacting antigen‐specific CD4+ T cells. At these conditions, DCs generate MVBs with MHC II and CD9 carrying luminal vesicles that are secreted as exosomes and transferred to the interacting T cells. Sorting of MHC II into exosomes was, in contrast to lysosomal targeting, independent of MHC II ubiquitination but rather correlated with its incorporation into CD9 containing detergent‐resistant membranes. Together, these data indicate two distinct MVB pathways: one for lysosomal targeting and the other for exosome secretion.

Distinct Uptake Mechanisms but Similar Intracellular Processing of Two Different Toll-like Receptor Ligand-Peptide Conjugates in Dendritic Cells *

Covalent conjugation of Toll-like receptor ligands (TLR-L) to synthetic antigenic peptides strongly improves antigen presentation in vitro and T lymphocyte priming in vivo. These molecularly well defined TLR-L-peptide conjugates, constitute an attractive vaccination modality, sharing the peptide antigen and a defined adjuvant in one single molecule. We have analyzed the intracellular trafficking and processing of two TLR-L conjugates in dendritic cells (DCs). Long synthetic peptides containing an ovalbumin cytotoxic T-cell epitope were chemically conjugated to two different TLR-Ls the TLR2 ligand, Pam3CysSK4 (Pam) or the TLR9 ligand CpG. Rapid and enhanced uptake of both types of TLR-L-conjugated peptide occurred in DCs. Moreover, TLR-L conjugation greatly enhanced antigen presentation, a process that was dependent on endosomal acidification, proteasomal cleavage, and TAP translocation. The uptake of the CpG∼conjugate was independent of endosomally-expressed TLR9 as reported previously. Unexpectedly, we found that Pam∼conjugated peptides were likewise internalized independently of the expression of cell surface-expressed TLR2. Further characterization of the uptake mechanisms revealed that TLR2-L employed a different uptake route than TLR9-L. Inhibition of clathrin- or caveolin-dependent endocytosis greatly reduced uptake and antigen presentation of the Pam-conjugate. In contrast, internalization and antigen presentation of CpG∼conjugates was independent of clathrin-coated pits but partly dependent on caveolae formation. Importantly, in contrast to the TLR-independent uptake of the conjugates, TLR expression and downstream TLR signaling was required for dendritic cell maturation and for priming of naïve CD8+ T-cells. Together, our data show that targeting to two distinct TLRs requires distinct uptake mechanism but follows similar trafficking and intracellular processing pathways leading to optimal antigen presentation and T-cell priming.

Selective cytotoxic T-lymphocyte targeting of tumor immune escape variants

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.

Design and evaluation of antigen-specific vaccination strategies against cancer

After studies in preclinical mouse models, the efficacy and safety of tumor-specific vaccination strategies is currently being evaluated in cancer patients. The first wave of clinical trials has shown that in general such vaccination strategies are safe. However examples of clinical responses, especially in conjunction with vaccine-induced immune responses, are still scarce. The fact that most trials have so far been performed with end-stage cancer patients can largely account for this deficit. Greater efficacy of anticancer vaccines is expected in patients with less-progressed disease. In addition, the detection of both natural and vaccine-induced T cell immunity needs further improvement.