Tumor-associated dendritic cells (DCs) lose the ability to cross-present tumor antigens, and Veglia et al. (Nature Communications, 2017) traced this defect to lipid bodies (LB) enriched in electrophilic oxidatively truncated (ox-tr) neutral lipids that covalently sequester the chaperone HSP70, trapping peptide-MHC class I complexes inside late endosomes/lysosomes. The problem that remains open is to complete the causal chain by (i) establishing the origin and biogenesis of the ox-tr lipids in DC lipid bodies (extracellular uptake, including transfer from polymorphonuclear myeloid-derived suppressor cells, versus in situ oxidation or peroxidation of locally synthesized lipids, and their relative contributions); (ii) establishing the mechanistic role of the integrin CD103 in forming the large HSP70-binding LB of CD103+ DCs; (iii) chemically mapping HSP70 lipidation in DCs in vivo, including the identity of modified residues; and (iv) testing whether specifically preventing ox-tr-lipid formation or blocking the HSP70-LB interaction restores cross-presentation and antitumor immunity in intact tumor-bearing hosts.

Background

Dendritic cells (DCs) are antigen-presenting cells that can cross-present exogenous tumor antigens on major histocompatibility complex (MHC) class I molecules to activate CD8+ cytotoxic T lymphocytes; this function is required for antitumor immunity and for the efficacy of cancer immunotherapy, including checkpoint blockade (antibody therapies that release inhibitory signals on T cells). In mice, cross-presentation in tumors and draining lymph nodes is carried out mainly by the CD103+ subset of conventional type 1 DCs, the dominant cross-presenting lineage (CD103, also called integrin alpha E, is a cell-adhesion receptor protein). Cross-presentation proceeds by two routes: a cytosolic route, in which internalized antigen is exported to the cytosol for proteasomal degradation, and a vacuolar route, in which peptides are loaded onto MHC class I molecules within endosomes and lysosomes and the resulting peptide-MHC class I (pMHC) complexes are trafficked to the cell surface. Lipid bodies (LB, also called lipid droplets) are neutral-lipid storage organelles composed mainly of triacylglycerols (TAG) and cholesterol esters (CE); in most healthy cells they are small (~0.1-0.2 micrometers), but DCs in tumor-bearing hosts accumulate abnormally large LB, and this accumulation correlates with defective cross-presentation (Herber et al., 2010; Cao et al., 2014). Herber et al. (2010) implicated the scavenger receptor Msr1/CD204 (a receptor that internalizes modified, including oxidized, lipoproteins and lipids) in lipid uptake by tumor-associated DCs, and Cubillos-Ruiz et al. (2015) showed that the endoplasmic-reticulum-stress transcription factor XBP1 drives triglyceride biosynthesis and lipid peroxidation in DCs inside tumors. Veglia et al. (2017) identified the molecular mechanism: the LB of tumor-associated DCs are enriched in electrophilic oxidatively truncated (ox-tr) lipids — neutral lipids whose polyunsaturated fatty acyl chains have undergone oxidative truncation to reactive aldehydes such as 4-hydroxy-2-nonenal (4-HNE) — and these electrophilic lipids form covalent adducts (Michael-type and Schiff-base) with nucleophilic amino-acid side chains, principally lysine, of the stress-inducible chaperone heat shock protein 70 (HSP70; a chaperone is a protein that assists the folding and transport of other proteins, and HSP70 carries antigens and peptides within the cross-presentation pathway). Sequestration of HSP70 on ox-tr-LB prevents translocation of pMHC from late endosomes/lysosomes to the cell surface, disabling the vacuolar pathway of cross-presentation. Coarse-grained molecular dynamics and docking (computational simulation and binding-prediction methods) in that study were performed with the Escherichia coli HSP70 orthologue DnaK, because the full-length structure of human HSP70 had not been published, and predicted that the ox-tr-TAG anchor the N-terminal ATPase domain of HSP70 through conserved lysine residues (lys55 and lys245, reported as conserved lysines after aligning the E. coli and human HSP70 sequences; Lys294, named in the docking figure legend; all three positions are numbered in the E. coli DnaK sequence used for the modeling). No residue-level mapping of HSP70 lipidation was performed: covalent adducts were detected only in vitro, using adduct-specific antibodies (4-HNE, MDA, hexanoyl-lysine) on recombinant HSP70 incubated with 4-HNE or with MPO/H2O2/NaCl-oxidized TAG, and not in DCs in vivo. The paper left explicit gaps that define the present problem: the origin of the ox-tr lipids (uptake of pre-formed oxidized lipids from the tumor microenvironment versus in situ oxidation within DCs) was hypothesized but not determined; the contribution of CD103 to large-LB formation remained the paper's own speculation, resting on its observation that CD103-deficient DCs generated in vitro from CD103-knockout mice accumulated lipids equally well but showed a significantly lower proportion of cells with large LB; the residue-level chemistry of HSP70 lipidation was not established in DCs in vivo; and the reversibility of the block by interventions acting specifically on ox-tr-lipid formation or the HSP70-LB interaction was not tested in intact tumor-bearing animals (complicated by the finding that HSP70 inhibition itself blocks cross-presentation). Its human evidence was limited to monocyte-derived DCs from healthy donors exposed to tumor-conditioned medium ex vivo.

Problem Statement

How do tumors drive the accumulation of electrophilic oxidatively truncated lipids in dendritic-cell lipid bodies, how does this disable cross-presentation at the molecular level, and can it be reversed? Specifically: (i) determine the origin and biogenesis of ox-tr lipids in DC lipid bodies — the relative contributions of extracellular uptake of pre-formed oxidized neutral lipids from the tumor microenvironment (including scavenger-receptor-dependent uptake and transfer of myeloperoxidase (MPO)-generated ox-tr lipids from polymorphonuclear myeloid-derived suppressor cells (PMN-MDSC)) versus in situ enzymatic or non-enzymatic oxidation and peroxidation of lipids synthesized or internalized by the DCs themselves; (ii) establish the mechanism by which CD103 (integrin alpha E), alone or among other integrins, promotes formation of the large lipid bodies that bind heat shock protein 70 (HSP70) in CD103+ DCs, and its relevance in vivo; (iii) map the chemical lipidation of HSP70 in DCs under physiological tumor conditions in vivo — the identity of the modified residues (including whether the conserved lysine positions predicted by the 2017 DnaK-based modeling — lys55/lys245 and the Lys294 of the docking figure, in E. coli DnaK numbering — are among them), and the structures of the lipid adducts; and (iv) test whether specifically preventing ox-tr-lipid formation or blocking the HSP70-lipid-body interaction restores trafficking of peptide-MHC class I (pMHC) complexes to the cell surface, cross-presentation, and CD8+ T-cell-mediated antitumor immunity in intact tumor-bearing hosts, given that global HSP70 inhibition itself impairs cross-presentation. Each of these four components is an independently assessable sub-question that stands on its own; jointly they complete one causal account — from tumor-derived lipid to molecular target to immune failure to therapeutic reversal — of the mechanism discovered by Veglia et al. (2017).

References

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