Pancreatic cancer-associated fibroblasts modulate macrophage differentiation via sialic acid-Siglec interactions.

Despite recent advances in cancer immunotherapy, pancreatic ductal adenocarcinoma (PDAC) remains unresponsive due to an immunosuppressive tumor microenvironment, which is characterized by the abundance of cancer-associated fibroblasts (CAFs). Once identified, CAF-mediated immune inhibitory mechanisms could be exploited for cancer immunotherapy. Siglec receptors are increasingly recognized as immune checkpoints, and their ligands, sialic acids, are known to be overexpressed by cancer cells. Here, we unveil a previously unrecognized role of sialic acid-containing glycans on PDAC CAFs as crucial modulators of myeloid cells. Using multiplex immunohistochemistry and transcriptomics, we show that PDAC stroma is enriched in sialic acid-containing glycans compared to tumor cells and normal fibroblasts, and characterized by ST3GAL4 expression. We demonstrate that sialic acids on CAF cell lines serve as ligands for Siglec-7, -9, -10 and -15, distinct from the ligands on tumor cells, and that these receptors are found on myeloid cells in the stroma of PDAC biopsies. Furthermore, we show that CAFs drive the differentiation of monocytes to immunosuppressive tumor-associated macrophages in vitro, and that CAF sialylation plays a dominant role in this process compared to tumor cell sialylation. Collectively, our findings unravel sialic acids as a mechanism of CAF-mediated immunomodulation, which may provide targets for immunotherapy in PDAC.

Deciphering the Chemical Language of the Immunomodulatory Properties of Veillonella parvula Lipopolysaccharide.

Veillonella parvula, prototypical member of the oral and gut microbiota, is at times commensal yet also potentially pathogenic. The definition of the molecular basis tailoring this contrasting behavior is key for broadening our understanding of the microbiota-driven pathogenic and/or tolerogenic mechanisms that take place within our body. In this study, we focused on the chemistry of the main constituent of the outer membrane of V. parvula, the lipopolysaccharide (LPS). LPS molecules indeed elicit pro-inflammatory and immunomodulatory responses depending on their chemical structures. Herein we report the structural elucidation of the LPS from two strains of V. parvula and show important and unprecedented differences in both the lipid and carbohydrate moieties, including the identification of a novel galactofuranose and mannitol-containing O-antigen repeating unit for one of the two strains. Furthermore, by harnessing computational studies, in vitro human cell models, as well as lectin binding solid-phase assays, we discovered that the two chemically diverse LPS immunologically behave differently and have attempted to identify the molecular determinant(s) governing this phenomenon. Whereas pro-inflammatory potential has been evidenced for the lipid A moiety, by contrast a plausible "immune modulating" action has been proposed for the peculiar O-antigen portion.

The immunological landscape of peripheral blood in glioblastoma patients and immunological consequences of age and dexamethasone treatment.

Background: Glioblastomas manipulate the immune system both locally and systemically, yet, glioblastoma-associated changes in peripheral blood immune composition are poorly studied. Age and dexamethasone administration in glioblastoma patients have been hypothesized to limit the effectiveness of immunotherapy, but their effects remain unclear. We compared peripheral blood immune composition in patients with different types of brain tumor to determine the influence of age, dexamethasone treatment, and tumor volume. Methods: High-dimensional mass cytometry was used to characterise peripheral blood mononuclear cells of 169 patients with glioblastoma, lower grade astrocytoma, metastases and meningioma. We used blood from medically-refractory epilepsy patients and healthy controls as control groups. Immune phenotyping was performed using FlowSOM and t-SNE analysis in R followed by supervised annotation of the resulting clusters. We conducted multiple linear regression analysis between intracranial pathology and cell type abundance, corrected for clinical variables. We tested correlations between cell type abundance and survival with Cox-regression analyses. Results: Glioblastoma patients had significantly fewer naive CD4+ T cells, but higher percentages of mature NK cells than controls. Decreases of naive CD8+ T cells and alternative monocytes and an increase of memory B cells in glioblastoma patients were influenced by age and dexamethasone treatment, and only memory B cells by tumor volume. Progression free survival was associated with percentages of CD4+ regulatory T cells and double negative T cells. Conclusion: High-dimensional mass cytometry of peripheral blood in patients with different types of intracranial tumor provides insight into the relation between intracranial pathology and peripheral immune status. Wide immunosuppression associated with age and pre-operative dexamethasone treatment provide further evidence for their deleterious effects on treatment with immunotherapy.

The transcriptional landscape of glycosylation-related genes in cancer.

Changes in glycosylation patterns have been associated with malignant transformation and clinical outcomes in several cancer types, prompting ongoing research into the mechanisms involved and potential clinical applications. In this study, we performed an extensive transcriptomic analysis of glycosylation-related genes and pathways, using publicly available bulk and single cell transcriptomic datasets from tumor samples and cancer cell lines. We identified genes and pathways strongly associated with different tumor types, which may represent novel diagnostic biomarkers. By using single cell RNA-seq data, we characterized the contribution of different cell types to the overall tumor glycosylation. Transcriptomic analysis of cancer cell lines revealed that they present a simplified landscape of genes compared to tissue. Lastly, we describe the association of different genes and pathways with the clinical outcome of patients. These results can serve as a resource for future research aimed to unravel the role of the glyco-code in cancer.

The sialic acid-Siglec immune checkpoint: an opportunity to enhance immune responses and therapy effectiveness in melanoma.

Modulation of immune responses through immune checkpoint blockade has revolutionized cutaneous melanoma treatment. However, it is still the case that not all patients respond successfully to these therapies, indicating the presence of as yet unknown resistance mechanisms. Hence, it is crucial to find novel targets to improve therapy efficacy. One of the described resistance mechanisms is regulated by immune inhibitory Siglec receptors, which are engaged by the carbohydrates sialic acids expressed on tumour cells, contributing to programmed cell death protein-1 (PD1)-like immune suppression mechanisms. In this review, we provide an overview on the regulation of sialic acid synthesis, its expression in melanoma, and the contribution of the sialic acid-Siglec axis to tumour development and immune suppressive mechanisms in the tumour microenvironment. Finally, we highlight potential sialic acid-Siglec axis-related therapeutics to improve the treatment of melanoma.

Sialic acid-modified der p 2 allergen exerts immunomodulatory effects on human PBMCs.

Background: House dust mite extract-based allergen immunotherapy (AIT) to treat house dust mite allergy is substantially effective but still presents some safety and efficacy concerns that warrant improvement. Several major allergen-based approaches to increase safety and efficacy of AIT have been proposed. One of them is the use of the group 2 allergen, Der p 2. Objective: We sought to investigate the immunomodulatory effects of sialic acid-modified major allergen recombinant Der p 2 (sia-rDer p 2) on PBMCs from healthy volunteers. Methods: We activated PBMCs with anti-CD3/CD28 antibodies and incubated them at 37°C for 6 days in the presence or absence of either native rDer p 2 or α2-3 sialic acid-modified rDer p 2 (sia-rDer p 2). We assessed the changes in CD4+ T-cell activation and proliferation by flow cytometry and changes in T-lymphocyte cytokine production in cell culture supernatant by ELISA. Results: We observed that PBMCs treated with sia-rDer p 2 presented with a markedly decreased expression of CD69 and an increased abundance of LAG-3+ lymphocytes compared with cells treated with rDer p 2. Moreover, PBMCs treated with sia-rDer p 2 showed a reduced production of IL-4, IL-13, and IL-5 and displayed a higher IL-10/IL-5 ratio compared with rDer p 2-treated PBMCs. Conclusions: We demonstrate that sia-rDer p 2 might be a safer option than native rDer p 2 for Der p 2-specific AIT. This is most relevant in the early phase of AIT that is often characterized by heightened TH2 responses, because sia-rDer p 2 does not enhance the production of TH2 cytokines.

Targeting myeloid cells for cancer immunotherapy: Siglec-7/9/10/15 and their ligands.

Advances in immunotherapy have revolutionized cancer treatment, yet many patients do not show clinical responses. While most immunotherapies target T cells, myeloid cells are the most abundant cell type in solid tumors and are key orchestrators of the immunosuppressive tumor microenvironment (TME), hampering effective T cell responses. Therefore, unraveling the immune suppressive pathways within myeloid cells could unveil new avenues for cancer immunotherapy. Over the past decade, Siglec receptors and their ligand, sialic acids, have emerged as a novel immune checkpoint on myeloid cells. In this review, we highlight key findings on how sialic acids modify immunity in the TME through engagement of Siglec-7/9/10/15 expressed on myeloid cells, and how the sialic acid-Siglec axis can be targeted for future cancer immunotherapies.

Platelets interact with CD169+ macrophages and cDC1 and enhance liposome-induced CD8+ T cell responses.

Historically platelets are mostly known for their crucial contribution to hemostasis, but there is growing understanding of their role in inflammation and immunity. The immunomodulatory role of platelets entails interaction with pathogens, but also with immune cells including macrophages and dendritic cells (DCs), to activate adaptive immune responses. In our previous work, we have demonstrated that splenic CD169+ macrophages scavenge liposomes and collaborate with conventional type 1 DCs (cDC1) to induce expansion of CD8+ T cells. Here, we show that platelets associate with liposomes and bind to DNGR-1/Clec9a and CD169/Siglec-1 receptors in vitro. In addition, platelets interacted with splenic CD169+ macrophages and cDC1 and further increased liposome internalization by cDC1. Most importantly, platelet depletion prior to liposomal immunization resulted in significantly diminished antigen-specific CD8+ T cell responses, but not germinal center B cell responses. Previously, complement C3 was shown to be essential for platelet-mediated CD8+ T cell activation during bacterial infection. However, after liposomal vaccination CD8+ T cell priming was not dependent on complement C3. While DCs from platelet-deficient mice exhibited unaltered maturation status, they did express lower levels of CCR7. In addition, in the absence of platelets, CCL5 plasma levels were significantly reduced. Overall, our findings demonstrate that platelets engage in a cross-talk with CD169+ macrophages and cDC1 and emphasize the importance of platelets in induction of CD8+ T cell responses in the context of liposomal vaccination.

Unraveling the impact of sialic acids on the immune landscape and immunotherapy efficacy in pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers. Despite the successful application of immune checkpoint blockade in a range of human cancers, immunotherapy in PDAC remains unsuccessful. PDAC is characterized by a desmoplastic, hypoxic and highly immunosuppressive tumor microenvironment (TME), where T-cell infiltration is often lacking (immune desert), or where T cells are located distant from the tumor islands (immune excluded). Converting the TME to an immune-inflamed state, allowing T-cell infiltration, could increase the success of immunotherapy in PDAC. METHOD: In this study, we use the KPC3 subcutaneous PDAC mouse model to investigate the role of tumor-derived sialic acids in shaping the tumor immune landscape. A sialic acid deficient KPC3 line was generated by genetic knock-out of the CMAS (cytidine monophosphate N-acetylneuraminic acid synthetase) enzyme, a critical enzyme in the synthesis of sialic acid-containing glycans. The effect of sialic acid-deficiency on immunotherapy efficacy was assessed by treatment with anti-programmed cell death protein 1 (PD-1) and agonistic CD40. RESULT: The absence of sialic acids in KPC3 tumors resulted in increased numbers of CD4+ and CD8+ T cells in the TME, and reduced frequencies of CD4+ regulatory T cells (Tregs) within the T-cell population. Importantly, CD8+ T cells were able to infiltrate the tumor islands in sialic acid-deficient tumors. These favorable alterations in the immune landscape sensitized sialic acid-deficient tumors to immunotherapy, which was ineffective in sialic acid-expressing KPC3 tumors. In addition, high expression of sialylation-related genes in human pancreatic cancer correlated with decreased CD8+ T-cell infiltration, increased presence of Tregs, and poorer survival probability. CONCLUSION: Our results demonstrate that tumor-derived sialic acids mediate T-cell exclusion within the PDAC TME, thereby impairing immunotherapy efficacy. Targeting sialic acids represents a potential strategy to enhance T-cell infiltration and improve immunotherapy outcomes in PDAC.

Spatial immune composition of tumor microenvironment in patients with pancreatic cancer.

This study examined the composition of the immune microenvironment at different sites within resected pancreas specimens from patients with pancreatic ductal adenocarcinoma (PDAC). Therefore, single-cell suspensions were made from fresh tumor and non-tumorous tissue. Fourteen patients were included from whom twelve PDAC and five non-tumorous samples were obtained. These samples were analyzed with a nineteen marker panel on the Aurora spectral flow cytometer. Furthermore, slides from formalin-fixed paraffine PDACs of eight additional patients were stained with eight markers and analyzed by multispectral imaging. These corresponded to central tumor, periphery of the tumor, i.e., invasive front and resected lymph node and were divided into tumor and adjacent tissue. In the single-cell suspension, a decreased ratio between lymphoid and myeloid cells and between M1 and M2 macrophages was observed in the tumor tissue compared to non-tumorous tissue. Furthermore, an increase in CD169 + macrophages in patients undergoing neoadjuvant therapy was found. Using immunofluorescence, more macrophages compared to T cells were observed, as well as a lower ratio of CD8 to M2 macrophage, a higher ratio of CD4-CD8 T cells and a higher ratio of immune-suppressive cells to pro-inflammatory cells in the PDAC area compared to the adjacent non-tumorous tissue. Finally, there were more immune-suppressive cells in the central tumor area compared to the invasive front. In conclusion, we show a gradient in the immune-suppressive environment in PDAC from most suppressive in the central tumor to least suppressive in distant non-tumorous tissue.

The immune microenvironment after neoadjuvant therapy compared to upfront surgery in patients with pancreatic cancer.

BACKGROUND: Patients with resectable and borderline resectable pancreatic ductal adenocarcinoma increasingly receive neoadjuvant therapy prior to surgery. However, the effect of neoadjuvant therapy on the immune microenvironment remains largely unknown. We analyzed the immune microenvironment in pancreatic cancer tumor tissue samples from patients treated with neoadjuvant therapy compared to patients after upfront surgery to gain knowledge about the immunological environment after therapy. METHODS: Multispectral imaging was performed on tissue from resected specimens from patients with PDAC who underwent upfront surgery (n = 10), neoadjuvant FOLFIRINOX (n = 10) or gemcitabine + radiotherapy (gem-RT) (n = 9) followed by surgery. The samples were selected by a dedicated pancreas pathologist from both the central part and the invasive front of the tumor (by the resected vein or venous surface) and subsequently analyzed using the Vectra Polaris. RESULTS: Patients receiving neoadjuvant FOLFIRINOX display a more pro-inflammatory immune profile, with less regulatory T cells and more CD8 T cells in the tumor tissue compared to patients receiving neoadjuvant gem-RTgem-RT or undergoing upfront surgery. Furthermore, CD163+ macrophages were decreased, and a higher CD163- macrophages versus CD163+ macrophages ratio was found in patients with neoadjuvant FOLFIRINOX. In all treatment groups, percentage of FoxP3+ B cells was significantly higher in tumor tissue compared to adjacent tissue. Furthermore, an increase in regulatory T cells in the tumor tissue was found in patients undergoing upfront surgery or receiving neoadjuvant gem-RT. In the gem-RT group, less CD8 T cells and a higher CD163+ macrophages to CD8 ratio were noted in the tumor tissue, suggesting a more immune suppressive profile in the tumor tissue. CONCLUSION: Patients receiving neoadjuvant FOLFIRINOX display a more pro-inflammatory immune profile compared to patients receiving neoadjuvant gem-RT or undergoing upfront surgery. Furthermore, in all treatment groups, a more immune suppressive microenvironment was found in the tumor tissue compared to the adjacent non-tumorous tissue.

Simple engineering of hybrid cellulose nanocrystal-gold nanoparticles results in a functional glyconanomaterial with biomolecular recognition properties.

Cellulose nanocrystal and gold nanoparticles are assembled, in a unique way, to yield a novel modular glyconanomaterial whose surface is then easily engineered with one or two different headgroups, by exploiting a robust click chemistry route. We demonstrate the potential of this approach by conjugating monosaccharide headgroups to the glyconanomaterial and show that the sugars retain their binding capability to C-type lectin receptors, as also directly visualized by cryo-TEM.

Human Milk Oligosaccharide 2'-Fucosyllactose Inhibits Ligand Binding to C-Type Lectin DC-SIGN but Not to Langerin.

Human milk oligosaccharides (HMOs) and their most abundant component, 2'-Fucosyllactose (2'-FL), are known to be immunomodulatory. Previously, it was shown that HMOs and 2'-FL bind to the C-type lectin receptor DC-SIGN. Here we show, using a ligand-receptor competition assay, that a whole mixture of HMOs from pooled human milk (HMOS) and 2'-FL inhibit the binding of the carbohydrate-binding receptor DC-SIGN to its prototypical ligands, fucose and the oligosaccharide Lewis-B, (Leb) in a dose-dependent way. Interestingly, such inhibition by HMOS and 2'-FL was not detected for another C-type lectin, langerin, which is evolutionarily similar to DC-SIGN. The cell-ligand competition assay using DC-SIGN expressing cells confirmed that 2'-FL inhibits the binding of DC-SIGN to Leb. Molecular dynamic (MD) simulations show that 2'-FL exists in a preorganized bioactive conformation before binding to DC-SIGN and this conformation is retained after binding to DC-SIGN. Leb has more flexible conformations and utilizes two binding modes, which operate one at a time via its two fucoses to bind to DC-SIGN. Our hypothesis is that 2'-FL may have a reduced entropic penalty due to its preorganized state, compared to Leb, and it has a lower binding enthalpy, suggesting a better binding to DC-SIGN. Thus, due to the better binding to DC-SIGN, 2'-FL may replace Leb from its binding pocket in DC-SIGN. The MD simulations also showed that 2'-FL does not bind to langerin. Our studies confirm 2'-FL as a specific ligand for DC-SIGN and suggest that 2'-FL can replace other DC-SIGN ligands from its binding pocket during the ligand-receptor interactions in possible immunomodulatory processes.

Incorporation of Toll-Like Receptor Ligands and Inflammasome Stimuli in GM3 Liposomes to Induce Dendritic Cell Maturation and T Cell Responses.

Cancer vaccination aims to activate immunity towards cancer cells and can be achieved by delivery of cancer antigens together with immune stimulatory adjuvants to antigen presenting cells (APC). APC maturation and antigen processing is a subsequent prerequisite for T cell priming and anti-tumor immunity. In order to specifically target APC, nanoparticles, such as liposomes, can be used for the delivery of antigen and adjuvant. We have previously shown that liposomal inclusion of the ganglioside GM3, an endogenous ligand for CD169, led to robust uptake by CD169-expressing APC and resulted in strong immune responses when supplemented with a soluble adjuvant. To minimize the adverse effects related to a soluble adjuvant, immune stimulatory molecules can be incorporated in liposomes to achieve targeted delivery of both antigen and adjuvant. In this study, we incorporated TLR4 (MPLA) or TLR7/8 (3M-052) ligands in combination with inflammasome stimuli, 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (PGPC) or muramyl dipeptide (MDP), into GM3 liposomes. Incorporation of TLR and inflammasome ligands did not interfere with the uptake of GM3 liposomes by CD169-expressing cells. GM3 liposomes containing a TLR ligand efficiently matured human and mouse dendritic cells in vitro and in vivo, while inclusion of PGPC or MDP had minor effects on maturation. Immunization with MPLA-containing GM3 liposomes containing an immunogenic synthetic long peptide stimulated CD4+ and CD8+ T cell responses, but additional incorporation of either PGPC or MDP did not translate into stronger immune responses. In conclusion, our study indicates that TLRL-containing GM3 liposomes are effective vectors to induce DC maturation and T cell priming and thus provide guidance for further selection of liposomal components to optimally stimulate anti-cancer immune responses.

Uptake Kinetics Of Liposomal Formulations of Differing Charge Influences Development of in Vivo Dendritic Cell Immunotherapy.

Dendritic cells (DCs) control adaptive immunity and are therefore attractive for in vivo targeting to either induce immune activation or tolerance, depending on disease. Liposomes, nanoparticles comprised of a lipid bi-layer, provide a nanoplatform for loading disease-relevant antigen, adjuvant and DC-targeting molecules simultaneously. However, it is yet not fully understood how liposomal formulations affect uptake by DCs and DC function. Here, we examined monocyte-derived DC (moDC) and skin DC uptake of six different liposomal formulations, together with their DC-modulating effect. Contrary to literature, we show using imaging flow cytometry that anionic or neutral liposomes are taken up more efficiently than cationic liposomes by moDCs, or by skin DCs after intradermal injection. None of the formulations yielded significant modulation of DC function as determined by the upregulation of maturation markers and cytokine production. These results suggest that anionic liposomes would be more suitable as vaccine carriers for a dermal application.

Analysis of the glyco-code in pancreatic ductal adenocarcinoma identifies glycan-mediated immune regulatory circuits.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a 5-year survival rate of only 9%. Despite the fact that changes in glycosylation patterns during tumour progression have been reported, no systematic approach has been conducted to evaluate its potential for patient stratification. By analysing publicly available transcriptomic data of patient samples and cell lines, we identified here two specific glycan profiles in PDAC that correlated with progression, clinical outcome and epithelial to mesenchymal transition (EMT) status. These different glycan profiles, confirmed by glycomics, can be distinguished by the expression of O-glycan fucosylated structures, present only in epithelial cells and regulated by the expression of GALNT3. Moreover, these fucosylated glycans can serve as ligands for DC-SIGN positive tumour-associated macrophages, modulating their activation and inducing the production of IL-10. Our results show mechanisms by which the glyco-code contributes to the tolerogenic microenvironment in PDAC.

CD169 Defines Activated CD14+ Monocytes With Enhanced CD8+ T Cell Activation Capacity.

Monocytes are antigen-presenting cells (APCs) that play diverse roles in promoting or regulating inflammatory responses, but their role in T cell stimulation is not well defined. In inflammatory conditions, monocytes frequently show increased expression of CD169/Siglec-1, a type-I interferon (IFN-I)-regulated protein. However, little is known about the phenotype and function of these CD169+ monocytes. Here, we have investigated the phenotype of human CD169+ monocytes in different diseases, their capacity to activate CD8+ T cells, and the potential for a targeted-vaccination approach. Using spectral flow cytometry, we detected CD169 expression by CD14+ CD16- classical and CD14+ CD16+ intermediate monocytes and unbiased analysis showed that they were distinct from dendritic cells, including the recently described CD14-expressing DC3. CD169+ monocytes expressed higher levels of co-stimulatory and HLA molecules, suggesting an increased activation state. IFNα treatment highly upregulated CD169 expression on CD14+ monocytes and boosted their capacity to cross-present antigen to CD8+ T cells. Furthermore, we observed CD169+ monocytes in virally-infected patients, including in the blood and bronchoalveolar lavage fluid of COVID-19 patients, as well as in the blood of patients with different types of cancers. Finally, we evaluated two CD169-targeting nanovaccine platforms, antibody-based and liposome-based, and we showed that CD169+ monocytes efficiently presented tumor-associated peptides gp100 and WT1 to antigen-specific CD8+ T cells. In conclusion, our data indicate that CD169+ monocytes are activated monocytes with enhanced CD8+ T cell stimulatory capacity and that they emerge as an interesting target in nanovaccine strategies, because of their presence in health and different diseases.

Myeloid-Specific Acly Deletion Alters Macrophage Phenotype In Vitro and In Vivo without Affecting Tumor Growth.

Cancer cells rely on ATP-citrate lyase (Acly)-derived acetyl-CoA for lipid biogenesis and proliferation, marking Acly as a promising therapeutic target. However, inhibitors may have side effects on tumor-associated macrophages (TAMs). TAMs are innate immune cells abundant in the tumor microenvironment (TME) and play central roles in tumorigenesis, progression and therapy response. Since macrophage Acly deletion was previously shown to elicit macrophages with increased pro- and decreased anti-inflammatory responses in vitro, we hypothesized that Acly targeting may elicit anti-tumor responses in macrophages, whilst inhibiting cancer cell proliferation. Here, we used a myeloid-specific knockout model to validate that absence of Acly decreases IL-4-induced macrophage activation. Using two distinct tumor models, we demonstrate that Acly deletion slightly alters tumor immune composition and TAM phenotype in a tumor type-dependent manner without affecting tumor growth. Together, our results indicate that targeting Acly in macrophages does not have detrimental effects on myeloid cells.

Palmitoylated antigens for the induction of anti-tumor CD8+ T cells and enhanced tumor recognition.

Induction of tumor-specific cytotoxic CD8+ T cells (CTLs) via immunization relies on the presentation of tumor-associated peptides in major histocompatibility complex (MHC) class I molecules by dendritic cells (DCs). To achieve presentation of exogenous peptides into MHC class I, cytosolic processing and cross-presentation are required. Vaccination strategies aiming to induce tumor-specific CD8+ T cells via this exogenous route therefore pose a challenge. In this study, we describe improved CD8+ T cell induction and in vivo tumor suppression of mono-palmitic acid-modified (C16:0) antigenic peptides, which can be attributed to their unique processing route, efficient receptor-independent integration within lipid bilayers, and continuous intracellular accumulation and presentation through MHC class I. We propose that this membrane-integrating feature of palmitoylated peptides can be exploited as a tool for quick and efficient antigen enrichment and MHC class I loading. Importantly, both DCs and non-professional antigen-presenting cells (APCs), similar to tumor cells, facilitate anti-tumor immunity by efficient CTL priming via DCs and effective recognition of tumors through enhanced presentation of antigens.