Immunotherapy offers a promising novel approach for the treatment of cancer and both adoptive T-cell transfer and immune modulation lead to regression of advanced melanoma. However, the potential synergy between these two strategies remains unclear.

We describe a new mechanism regulating the tumor endothelial barrier and T cell infiltration into tumors. We detected selective expression of the death mediator Fas ligand (FasL, also called CD95L) in the vasculature of human and mouse solid tumors but not in normal vasculature. In these tumors, FasL expression was associated with scarce CD8(+) infiltration and a predominance of FoxP3(+) T regulatory (Treg) cells. Tumor-derived vascular endothelial growth factor A (VEGF-A), interleukin 10 (IL-10) and prostaglandin E2 (PGE2) cooperatively induced FasL expression in endothelial cells, which acquired the ability to kill effector CD8(+) T cells but not Treg cells because of higher levels of c-FLIP expression in Treg cells. In mice, genetic or pharmacologic suppression of FasL produced a substantial increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells. Pharmacologic inhibition of VEGF and PGE2 produced a marked increase in the influx of tumor-rejecting CD8(+) over FoxP3(+) T cells that was dependent on attenuation of FasL expression and led to CD8-dependent tumor growth suppression. Thus, tumor paracrine mechanisms establish a tumor endothelial death barrier, which has a critical role in establishing immune tolerance and determining the fate of tumors.

Bone marrow-derived endothelial progenitor cells (EPCs) infiltrate into sites of neovascularization in adult tissues and mature into functional blood endothelial cells (BECs) during a process called vasculogenesis. Human marrow-derived EPCs have recently been reported to display a mixed myeloid and lymphatic endothelial cell (LEC) phenotype during inflammation-induced angiogenesis; however, their role in cancer remains poorly understood. We report the in vitro differentiation of human cord blood CD133+CD34+ progenitors into podoplanin+ cells expressing both myeloid markers (CD11b, CD14) and the canonical LEC markers vascular endothelium growth factor receptor 3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and prospero homeobox 1 (PROX-1). These podoplanin+ cells displayed sprouting behavior comparable to that of LECs in vitro and a dual hemangiogenic and lymphangiogenic activity in vivo in an endothelial cell sprouting assay and corneal vascularization assay, respectively. Furthermore, these cells expressed vascular endothelium growth factor (VEGF) family members A, -C, and -D. Thus, bone-marrow derived EPCs stimulate hemangiogenesis and lymphangiogenesis through their ability to differentiate into LECs and to produce angiogenic factors. Importantly, plasma from patients with breast cancer induced differentiation of CD34+ cord blood progenitors into hemangiogenic and lymphangiogenic CD11b+ myeloid cells, whereas plasma from healthy women did not have this effect. Consistent with these findings, circulating CD11b+ cells from breast cancer patients, but not from healthy women, displayed a similar dual angiogenic activity. Taken together, our results show that marrow-derived EPCs become hemangiogenic and lymphangiogenic upon exposure to cancer plasma. These newly identified functions of bone-marrow derived EPCs are expected to influence the diagnosis and treatment of breast cancer.

While T cell-based immunotherapies are steadily improving, there are still many patients who progress, despite T cell-infiltrated tumors. Emerging evidence suggests that T cells themselves may provoke immune escape of cancer cells. Here, we describe a well-controlled co-culture system for studying the dynamic T cell - cancer cell interplay, using human melanoma as a model. We explain starting material, controls, and culture parameters to establish reproducible and comparable cultures with highly heterogeneous tumor cells. Low passage melanoma cell lines and melanoma-specific CD8+ T cell clones generated from patient blood were cultured together for up to 3 days. Living melanoma cells were isolated from the co-culture system by fluorescence-activated cell sorting. We demonstrate that the characterization of isolated melanoma cells is feasible using flow cytometry for protein expression analysis as well as an Agilent whole human genome microarray and the NanoString technology for differential gene expression analysis. In addition, we identify five genes (ALG12, GUSB, RPLP0, KRBA2, and ADAT2) that are stably expressed in melanoma cells independent of the presence of T cells or the T cell-derived cytokines IFNγ and TNFα. These genes are essential for correct normalization of gene expression data by NanoString. Further to the characterization of melanoma cells after exposure to CTLs, this experimental system might be suitable to answer a series of questions, including how the affinity of CTLs for their target antigen influences the melanoma cell response and whether CTL-induced gene expression changes in melanoma cells are reversible. Taken together, our human T cell - melanoma cell culture system is well suited to characterize immune-related mechanisms in cancer cells.

CD160 is a T cell coinhibitory molecule that interacts with the herpes virus entry mediator (HVEM) on antigen-presenting cells to provide an inhibitory signal to T cells. To date, the structure of CD160 and its complex with HVEM are unknown. Here, we have identified the fragments of CD160 interacting with HVEM using ELISA tests, hydrogen/deuterium studies, affinity chromatography and mass spectrometry (MS). By combining hydrogen/deuterium exchange and mass spectrometry (HDX-MS) we obtained key information about the tertiary structure of CD160, predicting the 3D structure of the CD160-HVEM complex. Our results provide insights into the molecular architecture of this complex, serving as a useful basis for designing inhibitors for future immunotherapies.

Despite the promising therapeutic effects of immune checkpoint blockade (ICB), most patients with solid tumors treated with anti-PD-1/PD-L1 monotherapy do not achieve objective responses, with most tumor regressions being partial rather than complete. It is hypothesized that the absence of pre-existing antitumor immunity and/or the presence of additional tumor immune suppressive factors at the tumor microenvironment are responsible for such therapeutic failures. It is therefore clear that in order to fully exploit the potential of PD-1 blockade therapy, antitumor immune response should be amplified, while tumor immune suppression should be further attenuated. Cancer vaccines may prime patients for treatments with ICB by inducing effective anti-tumor immunity, especially in patients lacking tumor-infiltrating T-cells. These "non-inflamed" non-permissive tumors that are resistant to ICB could be rendered sensitive and transformed into "inflamed" tumor by vaccination. In this article we describe a clinical study where we use pancreatic cancer as a model, and we hypothesize that effective vaccination in pancreatic cancer patients, along with interventions that can reprogram important immunosuppressive factors in the tumor microenvironment, can enhance tumor immune recognition, thus enhancing response to PD-1/PD-L1 blockade. We incorporate into the schedule of standard of care (SOC) chemotherapy adjuvant setting a vaccine platform comprised of autologous dendritic cells loaded with personalized neoantigen peptides (PEP-DC) identified through our own proteo-genomics antigen discovery pipeline. Furthermore, we add nivolumab, an antibody against PD-1, to boost and maintain the vaccine's effect. We also demonstrate the feasibility of identifying personalized neoantigens in three pancreatic ductal adenocarcinoma (PDAC) patients, and we describe their optimal incorporation into long peptides for manufacturing into vaccine products. We finally discuss the advantages as well as the scientific and logistic challenges of such an exploratory vaccine clinical trial, and we highlight its novelty.

The efficacy of T cells depends on their functional avidity, i. e., the strength of T cell interaction with cells presenting cognate antigen. The overall T cell response is composed of multiple T cell clonotypes, involving different T cell receptors and variable levels of functional avidity. Recently, it has been proposed that the presence of low avidity tumor antigen-specific CD8 T cells hinder their high avidity counterparts to protect from tumor growth. Here we analyzed human cytotoxic CD8 T cells specific for the melanoma antigen Melan-A/MART-1. We found that the presence of low avidity T cells did not result in reduced cytotoxicity of tumor cells, nor reduced cytokine production, by high avidity T cells. In vivo in NSG-HLA-A2 mice, the anti-tumor effect of high avidity T cells was similar in presence or absence of low avidity T cells. These data indicate that low avidity T cells are not hindering anti-tumor T cell responses, a finding that is reassuring because low avidity T cells are an integrated part of natural T cell responses.

Humans and other mammals are colonized by microbial agents across the kingdom which can represent a unique microbiome pattern. Dysbiosis of the microbiome has been associated with pathology including cancer. We have identified a microbiome signature unique to ovarian cancers, one of the most lethal malignancies of the female reproductive system, primarily because of its asymptomatic nature during the early stages in development. We screened ovarian cancer samples along with matched, and non-matched control samples using our pan-pathogen array (PathoChip), combined with capture-next generation sequencing. The results show a distinct group of viral, bacterial, fungal and parasitic signatures of high significance in ovarian cases. Further analysis shows specific viral integration sites within the host genome of tumor samples, which may contribute to the carcinogenic process. The ovarian cancer microbiome signature provides insights for the development of targeted therapeutics against ovarian cancers.

Immune cells infiltrating tumors can have important impact on tumor progression and response to therapy. We present an efficient algorithm to simultaneously estimate the fraction of cancer and immune cell types from bulk tumor gene expression data. Our method integrates novel gene expression profiles from each major non-malignant cell type found in tumors, renormalization based on cell-type-specific mRNA content, and the ability to consider uncharacterized and possibly highly variable cell types. Feasibility is demonstrated by validation with flow cytometry, immunohistochemistry and single-cell RNA-Seq analyses of human melanoma and colorectal tumor specimens. Altogether, our work not only improves accuracy but also broadens the scope of absolute cell fraction predictions from tumor gene expression data, and provides a unique novel experimental benchmark for immunogenomics analyses in cancer research (http://epic.gfellerlab.org).

The remarkable clinical efficacy of the immune checkpoint blockade therapies has motivated researchers to discover immunogenic epitopes and exploit them for personalized vaccines. Human leukocyte antigen (HLA)-binding peptides derived from processing and presentation of mutated proteins are one of the leading targets for T-cell recognition of cancer cells. Currently, most studies attempt to identify neoantigens based on predicted affinity to HLA molecules, but the performance of such prediction algorithms is rather poor for rare HLA class I alleles and for HLA class II. Direct identification of neoantigens by mass spectrometry (MS) is becoming feasible; however, it is not yet applicable to most patients and lacks sensitivity. In an attempt to capitalize on existing immunopeptidomics data and extract information that could complement HLA-binding prediction, we first compiled a large HLA class I and class II immunopeptidomics database across dozens of cell types and HLA allotypes and detected hotspots that are subsequences of proteins frequently presented. About 3% of the peptidome was detected in both class I and class II. Based on the gene ontology of their source proteins and the peptide's length, we propose that their processing may partake by the cellular class II presentation machinery. Our database captures the global nature of the in vivo peptidome averaged over many HLA alleles, and therefore, reflects the propensity of peptides to be presented on HLA complexes, which is complementary to the existing neoantigen prediction features such as binding affinity and stability or RNA abundance. We further introduce two immunopeptidomics MS-based features to guide prioritization of neoantigens: the number of peptides matching a protein in our database and the overlap of the predicted wild-type peptide with other peptides in our database. We show as a proof of concept that our immunopeptidomics MS-based features improved neoantigen prioritization by up to 50%. Overall, our work shows that, in addition to providing huge training data to improve the HLA binding prediction, immunopeptidomics also captures other aspects of the natural in vivo presentation that significantly improve prediction of clinically relevant neoantigens.

Induction of an effective tumor immunity is a complex process that includes the appropriate presentation of the tumor antigens, activation of specific T cells, and the elimination of malignant cells. Potent and efficient T cell activation is dependent on multiple factors, such as timely expression of co-stimulatory molecules, the differentiation state of professional antigen presenting cells (e.g., dendritic cells; DCs), the functionality of the antigen processing and presentation machinery (APPM), and the repertoire of HLA class I and II-bound peptides (termed immunopeptidome) presented to T cells. So far, how molecular perturbations underlying DCs maturation and differentiation affect the in vivo cross-presented HLA class I and II immunopeptidomes is largely unknown. Yet, this knowledge is crucial for further development of DC-based immunotherapy approaches. We applied a state-of-the-art sensitive MS-based immunopeptidomics approach to characterize the naturally presented HLA-I and -II immunopeptidomes eluted from autologous immune cells having distinct functional and biological states including CD14+ monocytes, immature DC (ImmDC) and mature DC (MaDC) monocyte-derived DCs and naive or activated T and B cells. We revealed a presentation of significantly longer HLA peptides upon activation that is HLA allotype specific. This was apparent in the self-peptidome upon cell activation and in the context of presentation of exogenously loaded antigens, suggesting that peptide length is an important feature with potential implications on the rational design of anti-cancer vaccines.

Pembrolizumab is an immune checkpoint inhibitor against programmed cell death protein-1 (PD-1) approved for therapy in metastatic melanoma. PD-1 expression is associated with a diminished functionality in HIV-1 specific-CD8+ T cells. It is thought that PD-1 blockade could contribute to reinvigorate antiviral immunity and reduce the HIV-1 reservoir.

High T-cell infiltration in colorectal cancer (CRC) correlates with a favorable disease outcome and immunotherapy response. This, however, is only observed in a small subset of CRC patients. A better understanding of the factors influencing tumor T-cell responses in CRC could inspire novel therapeutic approaches to achieve broader immunotherapy responsiveness. Here, we investigated T cell-suppressive properties of different myeloid cell types in an inducible colon tumor mouse model. The most potent inhibitors of T-cell activity were tumor-infiltrating neutrophils. Gene expression analysis and combined in vitro and in vivo tests indicated that T-cell suppression is mediated by neutrophil-secreted metalloproteinase activation of latent TGFβ. CRC patient neutrophils similarly suppressed T cells via TGFβ in vitro, and public gene expression datasets suggested that T-cell activity is lowest in CRCs with combined neutrophil infiltration and TGFβ activation. Thus, the interaction of neutrophils with a TGFβ-rich tumor microenvironment may represent a conserved immunosuppressive mechanism in CRC.

Recent clinical developments in antitumor immunotherapy involving T-cell related therapeutics have led to a renewed interest for human leukocyte antigen class I (HLA-I) binding peptides, given their potential use as peptide vaccines. Databases of HLA-I binding peptides hold therefore information on therapeutic targets essential for understanding immunity. In this work, we use in depth and accurate HLA-I peptidomics datasets determined by mass-spectrometry (MS) and analyze properties of the HLA-I binding peptides with structure-based computational approaches. HLA-I binding peptides are studied grouping all alleles together or in allotype-specific contexts. We capitalize on the increasing number of structurally determined proteins to (1) map the 3D structure of HLA-I binding peptides into the source proteins for analyzing their secondary structure and solvent accessibility in the protein context, and (2) search for potential differences between these properties in HLA-I binding peptides and in a reference dataset of HLA-I motif-like peptides. This is performed by an in-house developed heuristic search that considers peptides across all the human proteome and converges to a collection of peptides that exhibit exactly the same motif as the HLA-I peptides. Our results, based on 9-mers matched to protein 3D structures, clearly show enriched sampling for HLA-I presentation of helical fragments in the source proteins. This enrichment is significant, as compared to 9-mer HLA-I motif-like peptides, and is not entirely explained by the helical propensity of the preferred residues in the HLA-I motifs. We give possible hypothesis for the secondary structure biases observed in HLA-I peptides. This contribution is of potential interest for researchers working in the field of antigen presentation and proteolysis. This knowledge refines the understanding of the rules governing antigen presentation and could be added to the parameters of the current peptide-MHC class I binding predictors to increase their antigen predictive ability.

Patient derived organoids (PDOs) can be established from colorectal cancers (CRCs) as in vitro models to interrogate cancer biology and its clinical relevance. We applied mass spectrometry (MS) immunopeptidomics to investigate neoantigen presentation and whether this can be augmented through interferon gamma (IFNγ) or MEK-inhibitor treatment.

Efforts to precisely identify tumor human leukocyte antigen (HLA) bound peptides capable of mediating T cell-based tumor rejection still face important challenges. Recent studies suggest that non-canonical tumor-specific HLA peptides derived from annotated non-coding regions could elicit anti-tumor immune responses. However, sensitive and accurate mass spectrometry (MS)-based proteogenomics approaches are required to robustly identify these non-canonical peptides. We present an MS-based analytical approach that characterizes the non-canonical tumor HLA peptide repertoire, by incorporating whole exome sequencing, bulk and single-cell transcriptomics, ribosome profiling, and two MS/MS search tools in combination. This approach results in the accurate identification of hundreds of shared and tumor-specific non-canonical HLA peptides, including an immunogenic peptide derived from an open reading frame downstream of the melanoma stem cell marker gene ABCB5. These findings hold great promise for the discovery of previously unknown tumor antigens for cancer immunotherapy.

The maintenance of homeostasis in the gut is a major challenge for the immune system. Here we demonstrate that the transcription factor MAF plays a central role in T cells for the prevention of gastro-intestinal inflammation. Conditional knock out mice lacking Maf in all T cells developed spontaneous late-onset colitis, correlating with a decrease of FOXP3+RORγt+ T cells proportion, dampened IL-10 production in the colon and an increase of inflammatory TH17 cells. Strikingly, FOXP3+ specific conditional knock out mice for MAF did not develop colitis and demonstrated normal levels of IL-10 in their colon, despite the incapacity of regulatory T cells lacking MAF to suppress colon inflammation in Rag1-/- mice transferred with naïve CD4+ T cells. We showed that one of the cellular sources of IL-10 in the colon of these mice are TH17 cells. Thus, MAF is critically involved in the maintenance of the gut homeostasis by regulating the balance between Treg and TH17 cells either at the level of their differentiation or through the modulation of their functions.

Liquid biopsies allow monitoring of cancer progression and detection of relapse, but reliable biomarkers in melanoma are lacking. Because secreted factors preferentially drain to lymphatic vessels before dilution in the blood, we hypothesized that lymph should be vastly enriched in cancer biomarkers. We characterized postoperative lymphatic exudate and plasma of metastatic melanoma patients after lymphadenectomy and found a dramatic enrichment in lymphatic exudate of tumor-derived factors and especially extracellular vesicles containing melanoma-associated proteins and miRNAs, with unique protein signatures reflecting early versus advanced metastatic spread. Furthermore, lymphatic exudate was enriched in memory T cells, including tumor-reactive CD137+ and stem cell-like types. In mice, lymph vessels were the major route of extracellular vesicle transport from tumors to the systemic circulation. We suggest that lymphatic exudate provides a rich source of tumor-derived factors for enabling the discovery of novel biomarkers that may reflect disease stage and therapeutic response.

The CD8 T cell response to the HLA-A2-restricted epitope LLWNGPMAV (LLW) of the non-structural protein 4b of Yellow Fever Virus (YFV) is remarkably immunodominant, highly prevalent and powerful in YFV-vaccinated humans. Here we used a combinatorial peptide library screening in the context of an A2/LLW-specific CD8 T cell clone to identify a superagonist that features a methionine to isoleucine substitution at position 7. Based on in silico modeling, the functional enhancement of this LLW-7I mutation was associated with alterations in the structural dynamics of the peptide in the major histocompatibility complex (pMHC) binding with the T cell receptor (TCR). While the TCR off-rate of LLW-7I pMHC is comparable to the wild type peptide, the rigidity of the 7I peptide seems to confer less entropy loss upon TCR binding. This LLW-7I superagonist is an example of improved functionality in human CD8 T cells associated with optimized ligand rigidity for TCR binding and not with changes in TCR:pMHC off-rate kinetics.

The understanding of the role of B cells in patients with solid tumors remains insufficient. We found that circulating B cells produced TNFα and/or IL-6, associated with unresponsiveness and poor overall survival of melanoma patients treated with anti-CTLA4 antibody. Transcriptome analysis of B cells from melanoma metastases showed enriched expression of inflammatory response genes. Publicly available single B cell data from the tumor microenvironment revealed a negative correlation between TNFα expression and response to immune checkpoint blockade. These findings suggest that B cells contribute to tumor growth via the production of inflammatory cytokines. Possibly, these B cells are different from tertiary lymphoid structure-associated B cells, which have been described to correlate with favorable clinical outcome of cancer patients. Further studies are required to identify and characterize B cell subsets and their functions promoting or counteracting tumor growth, with the aim to identify biomarkers and novel treatment targets.