Metabolic stress in cancer cells induces immune escape through a PI3K-dependent blockade of IFN? receptor signaling.
ABSTRACT: BACKGROUND:T-cell mediated immunotherapy brought clinical success for many cancer patients. Nonetheless, downregulation of MHC class I antigen presentation, frequently occurring in solid cancers, limits the efficacy of these therapies. Unraveling the mechanisms underlying this type of immune escape is therefore of great importance. We here investigated the immunological effects of metabolic stress in cancer cells as a result of nutrient deprivation. METHODS:TC1 and B16F10 tumor cell lines were cultured under oxygen- and glucose-deprivation conditions that mimicked the tumor microenvironment of solid tumors. Presentation of peptide antigens by MHC class I molecules was measured by flow cytometry and via activation of tumor-specific CD8 T cell clones. The proficiency of the IFNy-STAT1 pathway was investigated by Western blots on phosphorylated proteins, transfection of constitutive active STAT1 constructs and qPCR of downstream targets. Kinase inhibitors for PI3K were used to examine its role in IFNy receptor signal transduction. RESULTS:Combination of oxygen- and glucose-deprivation resulted in decreased presentation of MHC class I antigens on cancer cells, even in the presence of the stimulatory cytokine IFNy. This unresponsiveness to IFNy was the result of failure to phosphorylate the signal transducer STAT1. Forced expression of constitutive active STAT1 fully rescued the MHC class I presentation. Furthermore, oxygen- and glucose-deprivation increased PI3K activity in tumor cells. Pharmacological inhibition of this pathway not only restored signal transduction through IFNy-STAT1 but also improved MHC class I presentation. Importantly, PI3K inhibitors also rendered tumor cells sensitive for recognition by CD8 T cells in culture conditions of metabolic stress. CONCLUSIONS:These data revealed a strong impact of metabolic stress on the presentation of tumor antigens by MHC class I and suggest that this type of tumor escape takes place at hypoxic areas even during times of active T cell immunity and IFNy release.
Project description:Antigen presentation to T cells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. Maturation is driven by the phosphoinositide kinase PIKfyve. Blocking PIKfyve activity by small molecule inhibitors caused a delay in the conversion of phagosomes into lysosomes and in phagosomal acidification, whereas production of reactive oxygen species (ROS) increased. Elevated ROS resulted in reduced activity of cathepsin S and B, but not X, causing a proteolytic defect of MHC class II chaperone invariant chain Ii processing. We developed a novel universal MHC class II presentation assay based on a bio-orthogonal "clickable" antigen and showed that MHC class II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4+ T cells. Our results demonstrate a key role of PIKfyve in the processing and presentation of antigens, which should be taken into consideration when targeting PIKfyve in autoimmune disease and cancer.
Project description:Autophagy is known to be important in presentation of cytosolic antigens on MHC class II (MHC II). However, the role of autophagic process in antigen presentation in vivo is unclear. Mice with dendritic cell (DC)-conditional deletion in Atg5, a key autophagy gene, showed impaired CD4(+) T cell priming after herpes simplex virus infection and succumbed to rapid disease. The most pronounced defect of Atg5(-/-) DCs was the processing and presentation of phagocytosed antigens containing Toll-like receptor stimuli for MHC class II. In contrast, cross-presentation of peptides on MHC I was intact in the absence of Atg5. Although induction of metabolic autophagy did not enhance MHC II presentation, autophagic machinery was required for optimal phagosome-to-lysosome fusion and subsequent processing of antigen for MHC II loading. Thus, our study revealed that DCs utilize autophagic machinery to optimally process and present extracellular microbial antigens for MHC II presentation.
Project description:Tumor antigen-specific CD4(+) T cells that directly recognize cancer cells are important for orchestrating antitumor immune responses at the local tumor sites. However, the mechanisms of direct MHC class II (MHC-II) presentation of intracellular tumor antigen by cancer cells are poorly understood. We found that two functionally distinct subsets of CD4(+) T cells were expanded after HLA-DPB1*04 (DP04)-binding NY-ESO-1157-170 peptide vaccination in patients with ovarian cancer. Although both subsets recognized exogenous NY-ESO-1 protein pulsed on DP04(+) target cells, only one type recognized target cells with intracellular expression of NY-ESO-1. The tumor-recognizing CD4(+) T cells more efficiently recognized the short 8-9-mer peptides than the non-tumor-recognizing CD4(+) T cells. In addition to endosomal/lysosomal proteases that are typically involved in MHC-II antigen presentation, several pathways in the MHC class I presentation pathways, such as the proteasomal degradation and transporter-associated with antigen-processing-mediated peptide transport, were also involved in the presentation of intracellular NY-ESO-1 on MHC-II. The presentation was inhibited significantly by primaquine, a small molecule that inhibits endosomal recycling, consistent with findings that pharmacologic inhibition of new protein synthesis enhances antigen presentation. Together, our data demonstrate that cancer cells selectively present peptides from intracellular tumor antigens on MHC-II by multiple nonclassical antigen-processing pathways. Harnessing the direct tumor-recognizing ability of CD4(+) T cells could be a promising strategy to enhance antitumor immune responses in the immunosuppressive tumor microenvironment.
Project description:Tumor cells frequently escape from CD8+ T cell recognition by abrogating MHC-I antigen presentation. Deficiency in processing components, like the transporter associated with antigen processing (TAP), results in strongly decreased surface display of peptide/MHC-I complexes. We previously identified a class of hidden self-antigens known as T cell epitopes associated with impaired peptide processing (TEIPP), which emerge on tumor cells with such processing defects. In the present study, we analyzed thymus selection and peripheral behavior of T cells with specificity for the prototypic TEIPP antigen, the "self" TRH4 peptide/Db complex. TEIPP T cells were efficiently selected in the thymus, egressed with a naive phenotype, and could be exploited for immunotherapy against immune-escaped, TAP-deficient tumor cells expressing low levels of MHC-I (MHC-Ilo). In contrast, overt thymus deletion and functionally impaired TEIPP T cells were observed in mice deficient for TAP1 due to TEIPP antigen presentation on all body cells in these mice. Our results strongly support the concept that TEIPPs derive from ubiquitous, nonmutated self-antigens and constitute a class of immunogenic neoantigens that are unmasked during tumor immune evasion. These data suggest that TEIPP-specific CD8+ T cells are promising candidates in the treatment of tumors that have escaped from conventional immunotherapies.
Project description:CD4+ T cells have been shown to reject tumor cells with no detectable expression of major histocompatibility complex class II (MHC II). However, under certain circumstances, induction of ectopic MHC II expression on tumor cells has been reported.To confirm that CD4+ T cell-mediated anti-tumor immunity can be successful in the complete absence of antigen display on the tumor cells themselves, we eliminated MHC II on tumor cells using CRISPR/Cas9. Our results demonstrate that ablation of the relevant MHC II (I-Ed) in multiple myeloma cells (MOPC315) does not hinder rejection by tumor-specific CD4+ T cells. These findings provide conclusive evidence that CD4+ T cells specific for tumor antigens can eliminate malignant cells in the absence of endogenous MHC class II expression on the tumor cells. This occurs through antigen uptake and indirect presentation on tumor-infiltrating macrophages.
Project description:Atmospheric oxygen (?20% O(2)) has been the universal condition employed to culture tumor cells used as vaccine antigen. We tested the hypothesis that reducing oxygen tension would increase the efficacy of tumor cell lysate vaccines.GL261 glioma cells and EMT6 breast carcinoma cells were grown in 5% or 20% O(2). Syngeneic tumor-bearing mice were vaccinated with these tumor cell lysates mixed with CpG oligodeoxynucleotides as an adjuvant. Tumor infiltrating T cells and apoptotic GL261 cells were quantified by immunohistochemistry. Tumor-reactive immunoglobulin was detected by Western blot. Ovalbumin and gp100-derived peptides were mixed with GL261 lysates as marker antigens to detect changes in presentation of exogenous antigen on MHC class I in vitro, and in vivo following adoptive transfer of gp100-specific CD8(+) T cells.Mice bearing orthotopic glioma and breast carcinoma survived significantly longer when vaccinated with 5% O(2) lysates. Antigen-specific CTL activation was significantly enhanced following stimulation with lysates derived from GL261 cells grown in 5% O(2) versus 20% O(2) through a mechanism that involved enhanced cross-presentation of exogenous antigen on MHC I. Vaccination with 5% O(2) GL261 cell lysates caused a significant increase in CTL proliferation, tumoricidal function, and trafficking into brain tumor sites, whereas 20% O(2) lysate vaccines predominantly evoked an antibody response.Tissue culture oxygen functions as an "immunologic switch" by dictating the cellular and humoral immune responses elicited by tumor cell lysates. These results have profound implications for cancer vaccines that utilize tumor cells as the source of antigen.
Project description:Dendritic cells (DCs) can initiate immune responses by presenting exogenous antigens to T cells via both major histocompatibility complex (MHC) class I pathways and MHC class II pathways. Lysosomal activity has an important role in modulating the balance between these two pathways. The transcription factor TFEB regulates lysosomal function by inducing lysosomal activation. Here we report that TFEB expression inhibited the presentation of exogenous antigen by MHC class I while enhancing presentation via MHC class II. TFEB promoted phagosomal acidification and protein degradation. Furthermore, we found that the activation of TFEB was regulated during DC maturation and that phagosomal acidification was impaired in DCs in which the gene encoding TFEB was silenced. Our data indicate that TFEB is a key participant in the differential regulation of the presentation of exogenous antigens by DCs.
Project description:Influenza A virus (IAV) infection is normally controlled by adaptive immune responses initiated by dendritic cells (DCs). We investigated the consequences of IAV infection of human primary DCs on their ability to function as antigen-presenting cells. IAV was internalized by both myeloid DCs (mDCs) and plasmacytoid DCs but only mDCs supported viral replication. Although infected mDCs efficiently presented endogenous IAV antigens on MHC class II, this was not the case for presentation on MHC class I. Indeed, cross-presentation by uninfected cells of minute amounts of endocytosed, exogenous IAV was -300-fold more efficient than presentation of IAV antigens synthesized by infected cells and resulted in a statistically significant increase in expansion of IAV-specific CD8 T cells. Furthermore, IAV infection also impaired cross-presentation of other exogenous antigens, indicating that IAV infection broadly attenuates presentation on MHC class I molecules. Our results suggest that cross-presentation by uninfected mDCs is a preferred mechanism of antigen-presentation for the activation and expansion of CD8 T cells during IAV infection.
Project description:Peptides presented by MHC class I molecules are mostly derived from proteins synthesized by the antigen-presenting cell itself, while peptides presented by MHC class II molecules are predominantly from materials acquired by endocytosis. External antigens can also be presented by MHC class I molecules in a process referred to as cross-presentation. Here, we report that mouse dendritic cell (DC) engagement to a phagocytic target alters endocytic processing and inhibits the proteolytic activities. During phagocytosis, endosome maturation is delayed, shows less progression toward the lysosome, and the endocytosed soluble antigen is targeted for MHC class I cross-presentation. The antigen processing in these arrested endosomes is under the control of NAPDH oxidase associated ROS. We also show that cathepsin S is responsible for the generation of the MHC class I epitope. Taken together, our results suggest that in addition to solid structure uptake, DC phagocytosis simultaneously modifies the kinetics of endosomal trafficking and maturation. As a consequence, external soluble antigens are targeted into the MHC class I cross-presentation pathway.
Project description:Human cancers are known to downregulate Major Histocompatibility Complex (MHC) class I expression thereby escaping recognition and rejection by anti-tumor T cells. Here we report that oxygen tension in the tumor microenvironment (TME) serves as an extrinsic cue that regulates antigen presentation by MHC class I molecules. In support of this view, hypoxia is shown to negatively regulate MHC expression in a HIF-dependent manner as evidenced by (i) lower MHC expression in the hypoxic TME in vivo and in hypoxic 3-dimensional (3D) but not 2-dimensional (2D) tumor cell cultures in vitro; (ii) decreased MHC in human renal cell carcinomas with constitutive expression of HIF due to genetic loss of von Hippel-Lindau (VHL) function as compared with isogenically paired cells with restored VHL function, and iii) increased MHC in tumor cells with siRNA-mediated knockdown of HIF. In addition, hypoxia downregulated antigen presenting proteins like TAP 1/2 and LMP7 that are known to have a dominant role in surface display of peptide-MHC complexes. Corroborating oxygen-dependent regulation of MHC antigen presentation, hyperoxia (60% oxygen) transcriptionally upregulated MHC expression and increased levels of TAP2, LMP2 and 7. In conclusion, this study reveals a novel mechanism by which intra-tumoral hypoxia and HIF can potentiate immune escape. It also suggests the use of hyperoxia to improve tumor cell-based cancer vaccines and for mining novel immune epitopes. Furthermore, this study highlights the advantage of 3D cell cultures in reproducing hypoxia-dependent changes observed in the TME.