Radiation modulates the peptide repertoire, enhances MHC class I expression, and induces successful antitumor immunotherapy

Radiotherapy is one of the most successful cancer therapies. Here the effect of irradiation on antigen presentation by MHC class I molecules was studied. Cell surface expression of MHC class I molecules was increased for many days in a radiation dose-dependent manner as a consequence of three responses. Initially, enhanced degradation of existing proteins occurred which resulted in an increased intracellular peptide pool. Subsequently, enhanced translation due to activation of the mammalian target of rapamycin pathway resulted in increased peptide production, antigen presentation, as well as cytotoxic T lymphocyte recognition of irradiated cells. In addition, novel proteins were made in response to γ-irradiation, resulting in new peptides presented by MHC class I molecules, which were recognized by cytotoxic T cells. We show that immunotherapy is successful in eradicating a murine colon adenocarcinoma only when preceded by radiotherapy of the tumor tissue. Our findings indicate that directed radiotherapy can improve the efficacy of tumor immunotherapy

Cross-presentation by intercellular peptide transfer through gap junctions

Major histocompatibility complex (MHC) class I molecules present peptides that are derived from endogenous proteins. These antigens can also be transferred to professional antigen-presenting cells in a process called cross-presentation, which precedes initiation of a proper T-cell response; but exactly how they do this is unclear. We tested whether peptides can be transferred directly from the cytoplasm of one cell into the cytoplasm of its neighbour through gap junctions. Here we show that peptides with a relative molecular mass of up to approximately 1,800 diffuse intercellularly through gap junctions unless a three-dimensional structure is imposed. This intercellular peptide transfer causes cytotoxic T-cell recognition of adjacent, innocent bystander cells as well as activated monocytes. Gap-junction-mediated peptide transfer is restricted to a few coupling cells owing to the high cytosolic peptidase activity. We present a mechanism of antigen acquisition for cross-presentation that couples the antigen presentation system of two adjacent cells and is lost in most tumours: gap-junction-mediated intercellular peptide coupling for presentation by bystander MHC class I molecules and transfer to professional antigen presenting cells for cross-primin

Blockade of ErbB2 and PD-L1 using a bispecific antibody to improve targeted anti-ErbB2 therapy

A significant proportion of human epidermal growth factor receptor 2 (Her2/ErbB2)-positive metastatic breast cancer patients are refractory to Her2-targeted trastuzumab-like therapy. Some of this resistance has been attributed to the upregulation of immune checkpoints such as programmed cell death-1 (PD-1) and its ligand, PD-L1 in Her2-positive breast cancer patients. Therefore, therapies targeting both the PD-1/PD-L1 interaction and oncogenic Her2 signaling are of significant clinical interest. Here, we constructed a mouse bispecific antibody targeting PD-L1 and rat Her2 (referred to as BsPD-L1xrErbB2) aiming to redirect the anti-PD-L1 response toward Her2-expressing tumor cells. BsPD-L1xrErbB2 demonstrated additive binding to interferon (IFN)-gamma treated Her2(+) TUBO tumor cells, but it did not affect the proliferation of tumor cells in-vitro. BsPD-L1xrErbB2 also blocked the PD-1/PD-L1 interaction. This bispecific antibody was constructed with a mouse IgG2a Fc backbone and interacted with Fc gamma receptors and resulted in complement deposition (C3). ADCC and complement action could be potential mechanisms of action of this molecule. BsPD-L1xrErbB2 successfully reduced TUBO tumor growth and increased tumor rejection rate compared to the monovalent anti-PD-L1, monovalent anti-ErbB2 or the combination of anti-PD-L1 and anti-ErbB2 monotherapies. The enhanced anti-tumor effect of BsPD-L1xrErbB2 was dependent on CD8(+) T lymphocytes and IFN-gamma, as depletion of CD8(+) T lymphocytes and neutralization of IFN-gamma completely abolished the antitumor activity of the bispecific antibody. Consistently, BsPD-L1xrErbB2 treatment also increased the frequency of intratumor CD8(+) T lymphocytes. Taken together, our data support a bispecific antibody approach to enhance the anti-tumor efficacy of PD-1/PD-L1 checkpoint blockade in Her2-positive metastatic breast cancers

Cutting edge: HLA-B27 acquires many N-terminal dibasic peptides: coupling cytosolic peptide stability to antigen presentation

Ag presentation by MHC class I is a highly inefficient process because cytosolic peptidases destroy most peptides after proteasomal generation. Various mechanisms shape the MHC class I peptidome. We define a new one: intracellular peptide stability. Peptides with two N-terminal basic amino acids are more stable than other peptides. Such peptides should be overrepresented in the peptidome of MHC class I-associated peptides. HLA-B27 binding peptides use anchor residue R at P2 and, although most amino acids are allowed, particular amino acids are overrepresented at P1, including R and K. We show that such N-terminal dibasic peptides are indeed more efficiently presented by HLA-B27. This suggests that HLA-B27 can present peptides from Ags present in fewer copies than required for successful peptide generation for other MHC class I molecule

Puromycin-sensitive aminopeptidase limits MHC class I presentation in dendritic cells but does not affect CD8 T cell responses during viral infections

Previous experiments using enzyme inhibitors, cell lysates, and purified enzyme have suggested that puromycin-sensitive aminopeptidase (PSA) plays a role in creating and destroying MHC class I-presented peptides although its precise contribution to these processes is unknown. To examine the importance of this enzyme in MHC class I Ag presentation, we have generated PSA-deficient mice and cell lines from these animals. PSA-deficient mice are smaller and do not reproduce as well as wild type mice. In addition, dendritic cells from PSA-deficient mice display more MHC class I molecules on the cell surface, suggesting that PSA normally limits Ag presentation by destroying certain peptides in these key APCs. Surprisingly, MHC class I levels are not altered on other PSA-deficient cells and the processing and presentation of peptide precursors in PSA-deficient fibroblasts is normal. Moreover, PSA-deficient mice have normal numbers of T cells in the periphery, and respond as well as wild type mice to eight epitopes from three viruses. These data indicate that PSA may play a role in limiting MHC class I Ag presentation in dendritic cells in vivo but that it is not essential for generating most MHC class I-presented peptides or for stimulating CTL responses to several Ags

Leucine aminopeptidase is not essential for trimming peptides in the cytosol or generating epitopes for MHC class I antigen presentation

To detect viral infections and tumors, CD8+ T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation. Leucine aminopeptidase (LAP) is an IFN-inducible cytosolic aminopeptidase that can trim precursor peptides to mature epitopes and has been thought to play an important role in Ag presentation. To examine the role of LAP in generating MHC class I peptides in vivo, we generated LAP-deficient mice and LAP-deficient cell lines. These mutant mice and cells are viable and grow normally. The trimming of peptides in LAP-deficient cells is not reduced under basal conditions or after stimulation with IFN. Similarly, there is no reduction in presentation of peptides from precursor or full-length Ag constructs or in the overall supply of peptides from cellular proteins to MHC class I molecules even after stimulation with IFN. After viral infection, LAP-deficient mice generate normal CTL responses to seven epitopes from three different viruses. These data demonstrate that LAP is not an essential enzyme for generating most MHC class I-presented peptides and reveal redundancy in the function of cellular aminopeptidases