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Colorectal cancer (CRC) raises considerable clinical challenges, including a high mortality rate once the tumor spreads to distant sites. At this advanced stage, more accurate prediction of prognosis and treatment outcome is urgently needed. The role of cancer immunity in metastatic CRC (mCRC) is poorly understood. Here, we explore cellular immune cell status in patients with multi-organ mCRC. We analyzed T cell infiltration in primary tumor sections, surveyed the lymphocytic landscape of liver metastases, and assessed circulating mononuclear immune cells. Besides asking whether immune cells are associated with survival at this stage of the disease, we investigated correlations between the different tissue types; as this could indicate a dominant immune phenotype. Taken together, our analyses corroborate previous observations that higher levels of CD8+ T lymphocytes link to better survival outcomes. Our findings therefore extend evidence from earlier stages of CRC to indicate an important role for cancer immunity in disease control even after metastatic spreading to multiple organs. This finding may help to improve predicting outcome of patients with mCRC and suggests a future role for immunotherapeutic strategies.

Introduction In rheumatoid arthritis (RA), immunosuppressive therapies may achieve symptomatic relief, but do not induce long-term, drug-free remission. Meanwhile, the lifelong use of immunosuppressive drugs confers increased risk for malignancy and infections. As such, there is an unmet need for novel treatments that selectively target the pathogenic immune response in RA by inducing tolerance to autoantigens. Autologous cell therapy using antigen-loaded tolerogenic dendritic cells (tolDCs) aims to reinstate autoantigen-specific immunological tolerance in RA and could potentially meet this need.Methods and analysis We report here the design of the phase I/II, investigator-initiated, open-label, dose-escalation trial TOLERANT. In this study, we will evaluate the intranodal administration of tolDCs in patients with RA that are in remission under immunosuppressive therapy. The tolDCs in this trial are loaded with the heat shock protein 70-derived peptide mB29a, which is an effective surrogate autoantigen in animal models of arthritis. Within this study, three dose-escalation cohorts (two intranodal injections of 5×106, 10×106 and 15×106 tolDCs), each consisting of three patients, are evaluated to identify the highest safe dose (recommended dose), and an extension cohort of nine patients will be treated with the recommended dose. The (co-)primary endpoints of this study are safety and feasibility, which we assess by the number of AEs and the successful production of tolDCs. The secondary endpoints include the immunological effects of the treatment, which we assess with a variety of high-dimensional and antigen-specific immunological assays. Clinical effects are exploratory outcomes.Ethics and dissemination Ethical approval for this study has been obtained from the Netherlands Central Committee on Research Involving Human Subjects. The outcomes of the trial will be disseminated through publications in open-access, peer-reviewed scientific journals, scientific conferences and to patient associations.Trial registration numbers NCT05251870; 2019-003620-20 (EudraCT); NL71296.000.20 (CCMO register).

Abstract Autologous natural dendritic cells (nDCs) treatment can induce tumor-specific immune responses and clinical responses in cancer patients. In this phase III clinical trial (NCT02993315), 148 patients with resected stage IIIB/C melanoma were randomized to adjuvant treatment with nDCs (n = 99) or placebo (n = 49). Active treatment consisted of intranodally injected autologous CD1c+ conventional and plasmacytoid DCs loaded with tumor antigens. The primary endpoint was the 2-year recurrence-free survival (RFS) rate, whereas the secondary endpoints included median RFS, 2-year and median overall survival, adverse event profile, and immunological response The 2-year RFS rate was 36.8% in the nDC treatment group and 46.9% in the control group (p = 0.31). Median RFS was 12.7 months vs 19.9 months, respectively (hazard ratio 1.25; 90% CI: 0.88−1.79; p = 0.29). Median overall survival was not reached in both treatment groups (hazard ratio 1.32; 90% CI: 0.73−2.38; p = 0.44). Grade 3−4 study-related adverse events occurred in 5% and 6% of patients. Functional antigen-specific T cell responses could be detected in 67.1% of patients tested in the nDC treatment group vs 3.8% of patients tested in the control group (p

Abstract Tumor immunosurveillance plays a major role in melanoma, prompting the development of immunotherapy strategies. The gut microbiota composition, influencing peripheral and tumoral immune tonus, earned its credentials among predictors of survival in melanoma. The MIND-DC phase III trial (NCT02993315) randomized (2:1 ratio) 148 patients with stage IIIB/C melanoma to adjuvant treatment with autologous natural dendritic cell (nDC) or placebo (PL). Overall, 144 patients collected serum and stool samples before and after 2 bimonthly injections to perform metabolomics (MB) and metagenomics (MG) as prespecified exploratory analysis. Clinical outcomes are reported separately. Here we show that different microbes were associated with prognosis, with the health-related Faecalibacterium prausnitzii standing out as the main beneficial taxon for no recurrence at 2 years (p = 0.008 at baseline, nDC arm). Therapy coincided with major MB perturbations (acylcarnitines, carboxylic and fatty acids). Despite randomization, nDC arm exhibited MG and MB bias at baseline: relative under-representation of F. prausnitzii, and perturbations of primary biliary acids (BA). F. prausnitzii anticorrelated with BA, medium- and long-chain acylcarnitines. Combined, these MG and MB biomarkers markedly determined prognosis. Altogether, the host-microbial interaction may play a role in localized melanoma. We value systematic MG and MB profiling in randomized trials to avoid baseline differences attributed to host-microbe interactions.

BackgroundMetastatic endometrial cancer (mEC) continues to have a poor prognosis despite the introduction of several novel therapies including immune checkpoints inhibitors. Dendritic cell (DC) vaccination is known to be a safe immunotherapeutic modality that can induce immunological and clinical responses in patients with solid tumors. Platinum-based chemotherapy is known to act synergistically with immunotherapy by selectively depleting suppressive immune cells. Therefore, we investigated the immunological efficacy of combined chemoimmunotherapy with an autologous DC vaccine and carboplatin/paclitaxel chemotherapy.Study designThis is a prospective, exploratory, single-arm phase I/II study (NCT04212377) in 7 patients with mEC. The DC vaccine consisted of blood-derived conventional and plasmacytoid dendritic cells, loaded with known mEC antigens Mucin-1 and Survivin. Chemotherapy consisted of carboplatin/paclitaxel, given weekly for 6 cycles and three-weekly for 3 cycles. The primary endpoint was immunological vaccine efficacy; secondary endpoints were safety and feasibility.ResultsProduction of DC vaccines was successful in five out of seven patients. These five patients started study treatment and all were able to complete the entire treatment schedule. Antigen-specific responses could be demonstrated in two of the five patients who were treated. All patients had at least one adverse event grade 3 or higher. Treatment-related adverse events grade ≥3 were related to chemotherapy rather than DC vaccination; neutropenia was most common. Suppressive myeloid cells were selectively depleted in peripheral blood after chemotherapy.ConclusionDC vaccination can be safely combined with carboplatin/paclitaxel in patients with metastatic endometrial cancer and induces antigen-specific responses in a minority of patients. Longitudinal immunological phenotyping is suggestive of a synergistic effect of the combination.

Tumors educate their environment to prime the occurrence of suppressive cell subsets, which enable tumor evasion and favors tumor progression. Among these, there are the myeloid-derived suppressor cells (MDSCs), their presence being associated with the poor clinical outcome of cancer patients. Tumor-derived prostaglandin E2 (PGE2) is known to mediate MDSC differentiation and the acquisition of pro-tumor features. In myeloid cells, PGE2 signaling is mediated via E-prostanoid receptor type 2 (EP2) and EP4. Although the suppressive role of PGE2 is well established in MDSCs, the role of EP2/4 on human MDSCs or whether EP2/4 modulation can prevent MDSCs suppressive features upon exposure to tumor-derived PGE2 is poorly defined. In this study, using an in vitro model of human monocytic-MDSCs (M-MDSCs) we demonstrate that EP2 and EP4 signaling contribute to the induction of a pro-tumor phenotype and function on M-MDSCs. PGE2 signaling via EP2 and EP4 boosted M-MDSC ability to suppress T and NK cell responses. Combined EP2/4 blockade on M-MDSCs during PGE2 exposure prevented the occurrence of these suppressive features. Additionally, EP2/4 blockade attenuated the suppressive phenotype of M-MDSCs in a 3D coculture with colorectal cancer patient-derived organoids. Together, these results identify the role of tumor-derived PGE2 signaling via EP2 and EP4 in this human M-MDSC model, supporting the therapeutic value of targeting PGE2-EP2/4 axis in M-MDSCs to alleviate immunosuppression and facilitate the development of anti-tumor immunity.

Abstract Late-stage cancer immunotherapy trials often lead to unusual survival curve shapes, like delayed curve separation or a plateauing curve in the treatment arm. It is critical for trial success to anticipate such effects in advance and adjust the design accordingly. Here, we use in silico cancer immunotherapy trials – simulated trials based on three different mathematical models – to assemble virtual patient cohorts undergoing late-stage immunotherapy, chemotherapy, or combination therapies. We find that all three simulation models predict the distinctive survival curve shapes commonly associated with immunotherapies. Considering four aspects of clinical trial design – sample size, endpoint, randomization rate, and interim analyses – we demonstrate how, by simulating various possible scenarios, the robustness of trial design choices can be scrutinized, and possible pitfalls can be identified in advance. We provide readily usable, web-based implementations of our three trial simulation models to facilitate their use by biomedical researchers, doctors, and trialists.

IntroductionNanomedicine provides a promising platform for manipulating dendritic cells (DCs) and the ensuing adaptive immune response. For the induction of regulatory responses, DCs can be targeted in vivo with nanoparticles incorporating tolerogenic adjuvants and auto-antigens or allergens.MethodsHere, we investigated the tolerogenic effect of different liposome formulations loaded with vitamin D3 (VD3). We extensively phenotyped monocyte-derived DCs (moDCs) and skin DCs and assessed DC-induced regulatory CD4+ T cells in coculture. ResultsLiposomal VD3 primed-moDCs induced the development of regulatory CD4+ T cells (Tregs) that inhibited bystander memory T cell proliferation. Induced Tregs were of the FoxP3+ CD127low phenotype, also expressing TIGIT. Additionally, liposome-VD3 primed moDCs inhibited the development of T helper 1 (Th1) and T helper 17 (Th17) cells. Skin injection of VD3 liposomes selectively stimulated the migration of CD14+ skin DCs. DiscussionThese results suggest that nanoparticulate VD3 is a tolerogenic tool for DC-mediated induction of regulatory T cell responses.

BackgroundBicuspid aortic valve (BAV) is associated with ascending aorta aneurysms and dissections. Presently, genetic factors and pathological flow patterns are considered responsible for aneurysm formation in BAV while the exact role of inflammatory processes remains unknown.MethodsIn order to objectify inflammation, we employ a highly sensitive, quantitative immunohistochemistry approach. Whole slides of dissected, dilated and non-dilated ascending aortas from BAV patients were quantitatively analyzed.ResultsDilated aortas show a 4-fold increase of lymphocytes and a 25-fold increase in B lymphocytes in the adventitia compared to non-dilated aortas. Tertiary lymphoid structures with B cell follicles and helper T cell expansion were identified in dilated and dissected aortas. Dilated aortas were associated with an increase in M1-like macrophages in the aorta media, in contrast the number of M2-like macrophages did not change significantly.ConclusionThis study finds unexpected large numbers of immune cells in dilating aortas of BAV patients. These findings raise the question whether immune cells in BAV aortopathy are innocent bystanders or contribute to the deterioration of the aortic wall.

Background: Aim of this study was to investigate immune cells and subsets in different stages of human coronary artery disease with a novel multiplex immunohistochemistry (mIHC) technique. Methods: Human left anterior descending coronary artery specimens were analyzed: eccentric intimal thickening (N = 11), pathological intimal thickening (N = 10), fibroatheroma (N = 9), and fibrous plaque (N = 9). Eccentric intimal thickening was considered normal, and pathological intimal thickening, fibroatheroma, and fibrous plaque were considered diseased coronary arteries. Two mIHC panels, consisting of six and five primary antibodies, autofluoresence, and DAPI, were used to detect adaptive and innate immune cells. Via semi-automated analysis, (sub)types of immune cells in whole plaques and specific plaque regions were quantified. Results: Increased numbers of CD3+ T cells (P

BackgroundThe use of circulating cDC1 to generate anti-cancer vaccines is among the most promising approaches to overcome the limited immunogenicity and clinical efficacy of monocyte-derived DC. However, the recurrent lymphopenia and the reduction of DC numbers and functionality in patients with cancer may represent an important limitation of such approach. In patients with ovarian cancer (OvC) that had received chemotherapy, we previously showed that cDC1 frequency and function were reduced.MethodsWe recruited healthy donors (HD, n=7) and patients with OvC at diagnosis and undergoing interval debulking surgery (IDS, n=6), primary debulking surgery (PDS, n=6) or at relapse (n=8). We characterized longitudinally phenotypic and functional properties of peripheral DC subsets by multiparametric flow cytometry.ResultsWe show that the frequency of cDC1 and the total CD141+ DC capacity to take up antigen are not reduced at the diagnosis, while their TLR3 responsiveness is partially impaired in comparison with HD. Chemotherapy causes cDC1 depletion and increase in cDC2 frequency, but mainly in patients belonging to the PDS group, while in the IDS group both total lymphocytes and cDC1 are preserved. The capacity of total CD141+ DC and cDC2 to take up antigen is not impacted by chemotherapy, while the activation capacity upon Poly(I:C) (TLR3L) stimulation is further decreased.ConclusionsOur study provides new information about the impact of chemotherapy on the immune system of patients with OvC and sheds a new light on the importance of considering timing with respect to chemotherapy when designing new vaccination strategies that aim at withdrawing or targeting specific DC subsets.

Colorectal cancer (CRC) remains one of the most aggressive and lethal cancers, with metastasis accounting for most deaths. As such, there is an unmet need for improved therapies for metastatic CRC (mCRC). Currently, the research focus is shifting towards the reciprocal interactions within the tumor microenvironment (TME), which prevent tumor clearance by the immune system. Dendritic cells (DCs) play a key role in the initiation and amplification of anti-tumor immune responses and in driving the clinical success of immunotherapies. Dissecting the interactions between DCs and CRC cells may open doors to identifying key mediators in tumor progression, and possible therapeutic targets. This requires representative, robust and versatile models and tools. Currently, there is a shortage of such in vitro systems to model the CRC TME and its tumor-immune cell interactions. Here we develop and establish a dynamic organotypic 3D co-culture system to recapitulate and untangle the interactions between DCs and patient-derived mCRC tumor organoids. To our knowledge, this is the first study investigating human DCs in co-culture with tumor organoids in a 3D, organotypic setting. This system reveals how mCRC organoids modulate and shape monocyte-derived DCs (MoDCs) behavior, phenotype, and function, within a collagen matrix, using techniques such as brightfield and fluorescence microscopy, flow cytometry, and fluorescence-activated cell sorting. Our 3D co-culture model shows high viability and extensive interaction between DCs and tumor organoids, and its structure resembles patient tissue sections. Furthermore, it is possible to retrieve DCs from the co-cultures and characterize their phenotypic and functional profile. In our study, the expression of activation markers in both mature and immature DCs and their ability to activate T cells were impacted by co-culture with tumor organoids. In the future, this direct co-culture platform can be adapted and exploited to study the CRC-DC interplay in more detail, enabling novel and broader insights into CRC-driven DC (dys)function.

Dendritic cell (DC)-based vaccination for cancer treatment has seen considerable development over recent decades. However, this field is currently in a state of flux toward niche-applications, owing to recent paradigm-shifts in immuno-oncology mobilized by T cell-targeting immunotherapies. DC vaccines are typically generated using autologous (patient-derived) DCs exposed to tumor-associated or -specific antigens (TAAs or TSAs), in the presence of immunostimulatory molecules to induce DC maturation, followed by reinfusion into patients. Accordingly, DC vaccines can induce TAA/TSA-specific CD8+/CD4+ T cell responses. Yet, DC vaccination still shows suboptimal anti-tumor efficacy in the clinic. Extensive efforts are ongoing to improve the immunogenicity and efficacy of DC vaccines, often by employing combinatorial chemo-immunotherapy regimens. In this Trial Watch, we summarize the recent preclinical and clinical developments in this field and discuss the ongoing trends and future perspectives of DC-based immunotherapy for oncological indications.

Homologous recombination repair deficiency (HRD) is observed in 10% of patients with castrate-resistant prostate cancer (PCa). Preliminary data suggest that HRD-PCa might be more responsive to immune checkpoint inhibitors (ICIs). In this study, we compare the tumor immune landscape and peripheral T cell receptor (TCR) repertoire of patients with and without HRD-PCa to gain further insight into the immunogenicity of HRD-PCa. Immunohistochemistry was performed on tumor tissue of 81 patients, including 15 patients with HRD-PCa. Peripheral TCR sequencing was performed in a partially overlapping cohort of 48 patients, including 16 patients with HRD-PCa. HRD patients more frequently had intratumoral CD3+, CD3+CD8−FoxP3− or Foxp3+ TILs above median compared to patients without DNA damage repair alterations (DDRwt; CD3+ and Foxp3+: 77% vs 35%, p = .013; CD3+CD8−FoxP3−: 80% vs 44%, p = .031). No significant difference in CD8+ TILs or PD-L1 expression was observed. In peripheral blood, HRD patients displayed a more diverse TCR repertoire compared to DDRwt patients (p = .014). Additionally, HRD patients shared TCR clusters with low generation probability, suggesting patient-overlapping T cell responses. A pooled analysis of clinical data from 227 patients with molecularly characterized PCa suggested increased efficacy of ICIs in HRD-PCa. In conclusion, patients with HRD-PCa display increased TIL density and an altered peripheral TCR repertoire. Further research into the efficacy of ICIs and the presence of shared neoantigens in HRD-PCa is warranted.

Background Immunotherapy is currently part of the standard of care for patients with advanced-stage non-small cell lung cancer (NSCLC). However, many patients do not respond to this treatment, therefore combination strategies are being explored to increase clinical benefit. The PEMBRO-RT trial combined the therapeutic programmed cell death 1 (PD-1) antibody pembrolizumab with stereotactic body radiation therapy (SBRT) to increase the overall response rate and study the effects on the tumor microenvironment (TME).Methods Here, immune infiltrates in the TME of patients included in the PEMBRO-RT trial were investigated. Tumor biopsies of patients treated with pembrolizumab alone or combined with SBRT (a biopsy of the non-irradiated site) at baseline and during treatment were stained with multiplex immunofluorescence for CD3, CD8, CD20, CD103 and FoxP3 for lymphocytes, pan-cytokeratin for tumors, and HLA-ABC expression was determined.Results The total number of lymphocytes increased significantly after 6 weeks of treatment in the anti-PD-1 group (fold change: 1.87, 95% CI: 1.06 to 3.29) and the anti-PD-1+SBRT group (fold change: 2.29, 95% CI: 1.46 to 3.60). The combination of SBRT and anti-PD-1 induced a 4.87-fold increase (95% CI: 2.45 to 9.68) in CD103+ cytotoxic T-cells 6 weeks on treatment and a 2.56-fold increase (95% CI: 1.03 to 6.36) after anti-PD-1 therapy alone. Responders had a significantly higher number of lymphocytes at baseline than non-responders (fold difference 1.85, 95% CI: 1.04 to 3.29 for anti-PD-1 and fold change 1.93, 95% CI: 1.08 to 3.44 for anti-PD-1+SBRT).Conclusion This explorative study shows that that lymphocyte infiltration in general, instead of the infiltration of a specific lymphocyte subset, is associated with response to therapy in patients with NSCLC.Furthermore, anti-PD-1+SBRT combination therapy induces an immunological abscopal effect in the TME represented by a superior infiltration of cytotoxic T cells as compared with anti-PD-1 monotherapy.

Tolerogenic dendritic cell (tolDC) therapies aim to restore self-tolerance in patients suffering from autoimmune diseases. Phase 1 clinical trials with tolDC have shown the feasibility and safety of this approach, but have also highlighted a lack of understanding of their distribution in vivo. Fluorine-19 magnetic resonance imaging (19F-MRI) promises an attractive cell tracking method because it allows for detection of 19F-labelled cells in a non-invasive and longitudinal manner. Here, we tested the suitability of nanoparticles containing 19F (19F-NP) for labelling of therapeutic human tolDC for detection by 19F-MRI. We found that tolDC readily endocytosed 19F-NP with acceptable effects on cell viability and yield. The MRI signal-to-noise ratios obtained are more than sufficient for detection of the administered tolDC dose (10 million cells) at the injection site in vivo, depending on the tissue depth and the rate of cell dispersal. Importantly, 19F-NP labelling did not revert tolDC into immunogenic DC, as confirmed by their low expression of typical mature DC surface markers (CD83, CD86), low secretion of pro-inflammatory IL-12p70, and low capacity to induce IFN-γ in allogeneic CD4+ T cells. In addition, the capacity of tolDC to secrete anti-inflammatory IL-10 was not diminished by 19F-NP labelling. We conclude that 19F-NP is a suitable imaging agent for tolDC. With currently available technologies, this imaging approach does not yet approach the sensitivity required to detect small numbers of migrating cells, but could have important utility for determining the accuracy of injecting tolDC into the desired target tissue and their efflux rate.

Serum lactate dehydrogenase (LDH) levels are inversely related with response to immune checkpoint inhibitors (ICIs) in patients with metastatic melanoma. LDH is a key regulator of glycolysis, a pathway known to be upregulated in malignant tumors and to negatively affect antitumor immunity. We hypothesized that LDH isotype distribution in peripheral blood better reflects tumor glycolytic activity than total LDH levels and might therefore contribute to immunotherapy response prediction. LDH isotyping was performed in blood of 40 patients with metastatic melanoma and elevated LDH levels, of which 22 were treated with ipilimumab plus nivolumab. LDH-1 levels were decreased in 57.5% of patients. The percentage of LDH-2, -3 and -4, on the other hand, was elevated in 35%, 67.5% and 37.5% of patients, respectively. There was no difference in LDH isotype distribution between patients with versus patients without clinical benefit of ICIs, except for a numerically lower percentage of LDH-1 in patients without clinical benefit (median 13.3% vs. 17.6%, p = 0.1295). The percentage of LDH-1 correlated with total LDH levels and tumor burden and is therefore not likely to have strong, independent predictive value for response to ICIs. In conclusion, LDH isotyping does not contribute to ICI response prediction in melanoma patients with elevated LDH levels.

Background Immune checkpoint inhibitors (ICI) can lead to long-term responses in patients with metastatic melanoma. Still many patients with melanoma are intrinsically resistant or acquire secondary resistance. Previous studies have used primary or metastatic tumor tissue for biomarker assessment. Especially in melanoma, metastatic lesions are often present at different anatomical sites such as skin, lymph nodes, and visceral organs. The anatomical site may directly affect the tumor microenvironment (TME). To evaluate the impact of tumor evolution on the TME and on ICI treatment outcome, we directly compared paired primary and metastatic melanoma lesions for tumor mutational burden (TMB), HLA-ABC status, and tumor infiltrating lymphocytes (TILs) of patients that received ipilimumab.Methods TMB was analyzed by sequencing primary and metastatic melanoma lesions using the TruSight Oncology 500 assay. Tumor tissues were subjected to multiplex immunohistochemistry to assess HLA-ABC status and for the detection of TIL subsets (B cells, cytotoxic T cells, helper T cells, and regulatory T cells), by using a machine-learning algorithm.Results While we observed a very good agreement between TMB of matched primary and metastatic melanoma lesions (intraclass coefficient=0.921), such association was absent for HLA-ABC status, TIL density, and subsets thereof. Interestingly, analyses of different metastatic melanoma lesions within a single patient revealed that TIL density and composition agreed remarkably well, rejecting the hypothesis that the TME of different anatomical sites affects TIL infiltration. Similarly, the HLA-ABC status between different metastatic lesions within patients was also comparable. Furthermore, high TMB, of either primary or metastatic melanoma tissue, directly correlated with response to ipilimumab, whereas lymphocyte density or composition did not. Loss of HLA-ABC in the metastatic lesion correlated to a shorter progression-free survival on ipilimumab.Conclusions We confirm the link between TMB and HLA-ABC status and the response to ipilimumab-based immunotherapy in melanoma, but no correlation was found for TIL density, neither in primary nor metastatic lesions. Our finding that TMB between paired primary and metastatic melanoma lesions is highly stable, demonstrates its independency of the time point and location of acquisition. TIL and HLA-ABC status in metastatic lesions of different anatomical sites are highly similar within an individual patient.

We evaluated the immunological responses of lymph-node involved (stage III) melanoma patients to adjuvant dendritic cell vaccination with subsets of naturally occurring dendritic cells (nDCs). Fifteen patients with completely resected stage III melanoma were randomized to receive adjuvant dendritic cell vaccination with CD1c+ myeloid dendritic cells (cDC2s), plasmacytoid dendritic cells (pDCs) or the combination. Immunological response was the primary endpoint and secondary endpoints included safety and survival. In 80% of the patients, antigen-specific CD8+ T cells were detected in skin test-derived T cells and in 55% of patients, antigen-specific CD8+ T cells were detectable in peripheral blood. Functional interferon-γ-producing T cells were found in the skin test of 64% of the patients. Production of nDC vaccines meeting release criteria was feasible for all patients. Vaccination only induced grade 1–2 adverse events, mainly consisting of fatigue. In conclusion, adjuvant dendritic cell vaccination with cDC2s and/or pDCs is feasible, safe and induced immunological responses in the majority of stage III melanoma patients.

Checkpoint inhibitors targeting PD-(L)1 induce objective responses in 20% of patients with metastatic urothelial cancer (UC). CD8+ T cell infiltration has been proposed as a putative biomarker for response to checkpoint inhibitors. Nevertheless, data on spatial and temporal heterogeneity of tumor-infiltrating lymphocytes in advanced UC are lacking. The major aims of this study were to explore spatial heterogeneity for lymphocyte infiltration and to investigate how the immune landscape changes during the disease course. We performed multiplex immunohistochemistry to assess the density of intratumoral and stromal CD3+, CD8+, FoxP3+ and CD20+ immune cells in longitudinally collected samples of 49 UC patients. Within these samples, spatial heterogeneity for lymphocyte infiltration was observed. Regions the size of a 0.6 tissue microarray core (0.28 mm2) provided a representative sample in 60.6 to 71.6% of cases, depending on the cell type of interest. Regions of 3.30 mm2, the median tumor surface area in our biopsies, were representative in 58.8 to 73.8% of cases. Immune cell densities did not significantly differ between untreated primary tumors and metachronous distant metastases. Interestingly, CD3+, CD8+ and FoxP3+ T cell densities decreased during chemotherapy in two small cohorts of patients treated with neoadjuvant or palliative platinum-based chemotherapy. In conclusion, spatial heterogeneity in advanced UC challenges the use of immune cell infiltration in biopsies as biomarker for response prediction. Our data also suggests a decrease in tumor-infiltrating T cells during platinum-based chemotherapy.

Introduction The undiminished need for more effective cancer treatments stimulates the development of novel cancer immunotherapy candidates. The archetypical cancer immunotherapy would induce robust, targeted and long-lasting immune responses while simultaneously circumventing immunosuppression in the tumour microenvironment. For this purpose, we developed a novel immunomodulatory nanomedicine: PRECIOUS-01. As a PLGA-based nanocarrier, PRECIOUS-01 encapsulates a tumour antigen (NY-ESO-1) and an invariant natural killer T cell activator to target and augment specific antitumour immune responses in patients with NY-ESO-1-expressing advanced cancers.Methods and analysis This open-label, first-in-human, phase I dose-escalation trial investigates the safety, tolerability and immune-modulatory activity of increasing doses of PRECIOUS-01 administered intravenously in subjects with advanced NY-ESO-1-expressing solid tumours. A total of 15 subjects will receive three intravenous infusions of PRECIOUS-01 at a 3-weekly interval in three dose-finding cohorts. The trial follows a 3+3 design for the dose-escalation steps to establish a maximum tolerated dose (MTD) and/or recommended phase II dose (RP2D). Depending on the toxicity, the two highest dosing cohorts will be extended to delineate the immune-related parameters as a readout for pharmacodynamics. Subjects will be monitored for safety and the occurrence of dose-limiting toxicities. If the MTD is not reached in the planned dose-escalation cohorts, the RP2D will be based on the observed safety and immune-modulatory activity as a pharmacodynamic parameter supporting the RP2D. The preliminary efficacy will be evaluated as an exploratory endpoint using the best overall response rate, according to Response Evaluation Criteria in Solid Tumors V.1.1.Ethics and dissemination The Dutch competent authority (CCMO) reviewed the trial application and the medical research ethics committee (CMO Arnhem-Nijmegen) approved the trial under registration number NL72876.000.20. The results will be disseminated via (inter)national conferences and submitted for publication to a peer-reviewed journal.Trial registration number NCT04751786.

Immune checkpoint inhibitors (ICIs) have provided tremendous clinical benefit in several cancer types. However, systemic activation of the immune system also leads to several immune-related adverse events. Of these, ICI-mediated colitis (IMC) occurs frequently and is the one with the highest absolute fatality. To improve current treatment strategies, it is important to understand the cellular mechanisms that induce this form of colitis. In this review, we discuss important pathways that are altered in IMC in mouse models and in human colon biopsy samples. This reveals a complex interplay between several types of immune cells and the gut microbiome. In addition to a mechanistic understanding, patients at risk should be identifiable before ICI therapy. Here we propose to focus on T-cell subsets that interact with bacteria after inducing epithelial damage. Especially, intestinal resident immune cells are of interest. This may lead to a better understanding of IMC and provides opportunities for prevention and management.

Colorectal cancer (CRC) is the third most diagnosed malignancy and the second leading cause of cancer-related deaths worldwide. Locally advanced and metastatic disease exhibit resistance to therapy and are prone to recurrence. Despite significant advances in standard of care and targeted (immuno)therapies, the treatment effects in metastatic CRC patients have been modest. Untreatable cancer metastasis accounts for poor prognosis and most CRC deaths. The generation of a strong immunosuppressive tumor microenvironment (TME) by CRC constitutes a major hurdle for tumor clearance by the immune system. Dendritic cells (DCs), often impaired in the TME, play a critical role in the initiation and amplification of anti-tumor immune responses. Evidence suggests that tumor-mediated DC dysfunction is decisive for tumor growth and metastasis initiation, as well as for the success of immunotherapies. Unravelling and understanding the complex crosstalk between CRC and DCs holds promise for identifying key mechanisms involved in tumor progression and spread that can be exploited for therapy. The main goal of this review is to provide an overview of the current knowledge on the impact of CRC-driven immunosuppression on DCs phenotype and functionality, and its significance for disease progression, patient prognosis, and treatment response. Moreover, present knowledge gaps will be highlighted as promising opportunities to further understand and therapeutically target DC dysfunction in CRC. Given the complexity and heterogeneity of CRC, future research will benefit from the use of patient-derived material and the development of in vitro organoid-based co-culture systems to model and study DCs within the CRC TME.

Abstract Background Clinical benefit of cellular immunotherapy has been shown in patients with castration-resistant prostate cancer (CRPC). We investigated the immunological response and clinical outcome of vaccination with blood-derived CD1c+ myeloid dendritic cells (mDCs; cDC2) and plasmacytoid DCs (pDCs). Methods In this randomized phase IIa trial, 21 chemo-naive CRPC patients received maximally 9 vaccinations with mature mDCs, pDCs or a combination of mDCs plus pDCs. DCs were stimulated with protamine/mRNA and loaded with tumor-associated antigens NY-ESO-1, MAGE-C2 and MUC1. Primary endpoint was the immunological response after DC vaccination, which was monitored in peripheral blood and in T cell cultures of biopsies of post-treatment delayed-type hypersensitivity-skin tests. Main secondary endpoints were safety, feasibility, radiological PFS (rPFS) and overall survival. Radiological responses were assessed by MRIs and contrast-enhanced 68Ga-prostate-specific membrane antigen PET/CT, according to RECIST 1.1, PCWG2 criteria and immune-related response criteria. Results Both tetramer/dextramer-positive (dm+) and IFN-γ-producing (IFN-γ+) antigen specific T cells were detected more frequently in skin biopsies of patients with radiological non-progressive disease (5/13 patients; 38%) compared to patients with progressive disease (0/8 patients; 0%). In these patients with vaccination enhanced dm+ and IFN-γ+ antigen-specific T cells median rPFS was 18.8 months (n = 5) vs. 5.1 months (n = 16) in patients without IFN-γ-producing antigen-specific T cells (p = 0.02). The overall median rPFS was 9.5 months. All DC vaccines were well tolerated with grade 1–2 toxicity. Conclusions Immunotherapy with blood-derived DC subsets was feasible and safe and induced functional antigen-specific T cells. The presence of functional antigen-specific T cells correlated with an improved clinical outcome. Trial registration ClinicalTrials.gov identifier NCT02692976, registered 26 February 2016, retrospectively registered.

Abstract Dendritic cells (DCs) can initiate and direct adaptive immune responses. This ability is exploitable in DC vaccination strategies, in which DCs are educated ex vivo to present tumor antigens and are administered into the patient with the aim to induce a tumor-specific immune response. DC vaccination remains a promising approach with the potential to further improve cancer immunotherapy with little or no evidence of treatment-limiting toxicity. However, evidence for objective clinical antitumor activity of DC vaccination is currently limited, hampering the clinical implementation. One possible explanation for this is that the most commonly used monocyte-derived DCs may not be the best source for DC-based immunotherapy. The novel approach to use naturally circulating DCs may be an attractive alternative. In contrast to monocyte-derived DCs, naturally circulating DCs are relatively scarce but do not require extensive culture periods. Thereby, their functional capabilities are preserved, the reproducibility of clinical applications is increased, and the cells are not dysfunctional before injection. In human blood, at least three DC subsets can be distinguished, plasmacytoid DCs, CD141+ and CD1c+ myeloid/conventional DCs, each with distinct functional characteristics. In completed clinical trials, either CD1c+ myeloid DCs or plasmacytoid DCs were administered and showed encouraging immunological and clinical outcomes. Currently, also the combination of CD1c+ myeloid and plasmacytoid DCs as well as the intratumoral use of CD1c+ myeloid DCs is under investigation in the clinic. Isolation and culture strategies for CD141+ myeloid DCs are being developed. Here, we summarize and discuss recent clinical developments and future prospects of natural DC-based immunotherapy.

Carriers of a pathogenic germline mutations in the PTEN gene, a well-known tumor suppressor gene, are at increased risk of multiple benign and malignant tumors, e.g. breast, thyroid, endometrial and colon cancer. This is called PTEN Hamartomous Tumor Syndrome (PHTS). PHTS patients may also have an increased risk of immunological dysregulation, such as autoimmunity and immune deficiencies. The effects of PTEN on the immune system have been studied in murine knockout models demonstrating that loss of PTEN function leads to dysregulation of the immune response. This results in susceptibility to autoimmunity, impaired B cell class switching with subsequent hypogammaglobulinemia. Additionally, a decreased ability of dendritic cells to prime CD8+ T cells was observed, leading to impaired tumor eradication. Immune dysfunction in PHTS patients has not yet been extensively studied but might be a manageable contributing factor to the increased cancer risk in PHTS.

Current treatment for patients with non-small-cell lung cancer (NSCLC) is suboptimal since therapy is only effective in a minority of patients and does not always induce a long-lasting response. This highlights the importance of exploring new treatment options. The clinical success of immunotherapy relies on the ability of the immune system to mount an adequate anti-tumor response. The activation of cytotoxic T cells, the effector immune cells responsible for tumor cell killing, is of paramount importance for the immunotherapy success. These cytotoxic T cells are primarily instructed by dendritic cells (DCs). DCs are the most potent antigen-presenting cells (APCs) and are capable of orchestrating a strong anti-cancer immune response. DC function is often suppressed in NSCLC. Therefore, resurrection of DC function is an interesting approach to enhance anti-cancer immune response. Recent data from DC-based treatment studies has given rise to the impression that DC-based treatment cannot induce clinical benefit in NSCLC by itself. However, these are all early-phase studies that were mainly designed to study safety and were not powered to study clinical benefit. The fact that these studies do show that DC-based therapies were well-tolerated and could induce the desired immune responses, indicates that DC-based therapy is still a promising option. Especially combination with other treatment modalities might enhance immunological response and clinical outcome. In this review, we will identify the possibilities from current DC-based treatment trials that could open up new venues to improve future treatment.

Background Predicting treatment response or survival of cancer patients remains challenging in immuno-oncology. Efforts to overcome these challenges focus, among others, on the discovery of new biomarkers. Despite advances in cellular and molecular approaches, only a limited number of candidate biomarkers eventually enter clinical practice.Methods A computational modeling approach based on ordinary differential equations was used to simulate the fundamental mechanisms that dictate tumor-immune dynamics and to investigate its implications on responses to immune checkpoint inhibition (ICI) and patient survival. Using in silico biomarker discovery trials, we revealed fundamental principles that explain the diverging success rates of biomarker discovery programs.Results Our model shows that a tipping point—a sharp state transition between immune control and immune evasion—induces a strongly non-linear relationship between patient survival and both immunological and tumor-related parameters. In patients close to the tipping point, ICI therapy may lead to long-lasting survival benefits, whereas patients far from the tipping point may fail to benefit from these potent treatments.Conclusion These findings have two important implications for clinical oncology. First, the apparent conundrum that ICI induces substantial benefits in some patients yet completely fails in others could be, to a large extent, explained by the presence of a tipping point. Second, predictive biomarkers for immunotherapy should ideally combine both immunological and tumor-related markers, as a patient’s distance from the tipping point can typically not be reliably determined from solely one of these. The notion of a tipping point in cancer-immune dynamics helps to devise more accurate strategies to select appropriate treatments for patients with cancer.

Immunotherapeutic approaches have revolutionized the treatment of several diseases such as cancer. The main goal of immunotherapy for cancer is to modulate the anti-tumor immune responses by favoring the recognition and destruction of tumor cells. Recently, a better understanding of the suppressive effect of the tumor microenvironment (TME) on immune cells, indicates that restoring the suppressive effect of the TME is crucial for an efficient immunotherapy. Natural killer (NK) cells and dendritic cells (DCs) are cell types that are currently administered to cancer patients. NK cells are used because of their ability to kill tumor cells directly via cytotoxic granzymes. DCs are employed to enhance anti-tumor T cell responses based on their ability to present antigens and induce tumor-antigen specific CD8+ T cell responses. In preclinical models, a particular DC subset, conventional type 1 DCs (cDC1s) is shown to be specialized in cross-presenting extracellular antigens to CD8+ T cells. This feature makes them a promising DC subset for cancer treatment. Within the TME, cDC1s show a bidirectional cross-talk with NK cells, resulting in a higher cDC1 recruitment, differentiation, and maturation as well as activation and stimulation of NK cells. Consequently, the presence of cDC1s and NK cells within the TME might be of utmost importance for the success of immunotherapy. In this review, we discuss the function of cDC1s and NK cells, their bidirectional cross-talk and potential strategies that could improve cancer immunotherapy.

Tumor-specific neoantigens can be highly immunogenic, but their identification for each patient and the production of personalized cancer vaccines can be time-consuming and prohibitively expensive. In contrast, tumor-associated antigens are widely expressed and suitable as an off the shelf immunotherapy. Here, we developed a PLGA-based nanoparticle vaccine that contains both the immunogenic cancer germline antigen NY-ESO-1 and an α-GalCer analog IMM60, as a novel iNKT cell agonist and dendritic cell transactivator. Three peptide sequences (85–111, 117–143, and 157–165) derived from immunodominant regions of NY-ESO-1 were selected. These peptides have a wide HLA coverage and were efficiently processed and presented by dendritic cells via various HLA subtypes. Co-delivery of IMM60 enhanced CD4 and CD8 T cell responses and antibody levels against NY-ESO-1 in vivo. Moreover, the nanoparticles have negligible systemic toxicity in high doses, and they could be produced according to GMP guidelines. Together, we demonstrated the feasibility of producing a PLGA-based nanovaccine containing immunogenic peptides and an iNKT cell agonist, that is activating DCs to induce antigen-specific T cell responses.

Cancer immunotherapies have induced long-lasting responses in cancer patients including those with melanoma and head and neck squamous cell carcinoma (HNSCC). However, the majority of treated patients does not achieve clinical benefit from immunotherapy because of systemic tumor-induced immunosuppression. Monocytic myeloid-derived suppressor cells (M-MDSCs) are implicated as key players in inhibiting anti-tumor immune responses and their frequencies are closely associated with tumor progression. Tumor-derived signals, including signaling via STAT3-COX-2, induce the transformation of monocytic precursors into suppressive M-MDSCs. In a retrospective assessment, we observed that survival of melanoma patients undergoing dendritic cell vaccination was negatively associated with blood M-MDSC levels. Previously, it was shown that platinum-based chemotherapeutics inhibit STAT signaling. Here, we show that cisplatin and oxaliplatin treatment interfere with the development of M-MDSCs, potentially synergizing with cancer immunotherapy. In vitro, subclinical doses of platinum-based drugs prevented the generation of COX-2+ M-MDSCs induced by tumor cells from melanoma patients. This was confirmed in HNSCC patients where intravenous cisplatin treatment drastically lowered M-MDSC frequency while monocyte levels remained stable. In treated patients, expression of COX-2 and arginase-1 in M-MDSCs was significantly decreased after two rounds of cisplatin, indicating inhibition of STAT3 signaling. In line, the capacity of M-MDSCs to inhibit activated T cell responses ex vivo was significantly decreased after patients received cisplatin. These results show that platinum-based chemotherapeutics inhibit the expansion and suppressive activity of M-MDSCs in vitro and in cancer patients. Therefore, platinum-based drugs have the potential to enhance response rates of immunotherapy by overcoming M-MDSC-mediated immunosuppression.

BackgroundMaintaining health-related quality of life (HRQoL) is highly desirable during systemic therapies for patients with castration-resistant prostate cancer (CRPC). Patient-reported outcome measures (PROs) were studied in our phase IIa trial on cellular-based immunotherapy with dendritic cells (DC).MethodsWe treated 21 chemo-naive asymptomatic or minimally symptomatic patients with CRPC with maximally three cycles of DC vaccinations (ClinicalTrials.gov, NCT02692976). Here, we report the impact of DC vaccination on HRQoL. PROs were assessed using the EORTC-QLQ-C30, the EORTC-QLQ-PR25, Checklist Individual Strength (CIS20-R), and Beck Depression Inventory Primary Care questionnaires. Short-term and long-term vaccine-related effects on HRQoL were studied.ResultsQuestionnaires were collected at baseline (n=20), week 6 (n=19), week 12 (n=18), week 24 (n=13), week 50 (n=8) and week 100 (n=2). No clinically relevant differences in symptom-related outcome, functioning-related outcome, and Global Health Status were observed directly after the first cycle of DC vaccinations (week 6) and at follow-up (week 12) compared to baseline. HRQoL remained high throughout the vaccination cycle and six weeks afterward. In radiographic non-progressive patients, who continued DC vaccination, high HRQoL scores were observed up to one and two years after study enrolment.ConclusionsPatients with asymptomatic or minimally symptomatic CRPC show high HRQoL throughout DC-based immunotherapy. This is a clinically relevant finding in this older-aged patient population with advanced prostate cancer.

Currently three bona fide dendritic cell (DC) types are distinguished in human blood. Herein we focus on type 2 DCs (DC2s) and compare the three defining markers CD1c, CD172, and CD301. When using CD1c to define DC2s, a CD14+ and a CD14− subset can be detected. The CD14+ subset shares features with monocytes, and this includes substantially higher expression levels for CD64, CD115, CD163, and S100A8/9. We review the current knowledge of these CD1c+CD14+ cells as compared to the CD1c+CD14− cells with respect to phenotype, function, transcriptomics, and ontogeny. Here, we discuss informative mutations, which suggest that two populations have different developmental requirements. In addition, we cover subsets of CD11c+CD8− DC2s in the mouse, where CLEC12A+ESAMlow cells, as compared to the CLEC12A−ESAMhigh subset, also express higher levels of monocyte-associated markers CD14, CD3, and CD115. Finally, we summarize, for both man and mouse, the data on lower antigen presentation and higher cytokine production in the monocyte-marker expressing DC2 subset, which demonstrate that the DC2 subsets are also functionally distinct.

Plasmacytoid dendritic cells (pDC) are a pivotal component of the immune system. Here, the authors utilize single-cell microfluidics to interrogate the human pDC compartment and reveal a subset of type I IFN secreting pDCs that is regulated by stochastic gene expression and amplified by microenvironmental cues.

Accumulating evidence demonstrates the decisive role of the gut microbiota in determining the effectiveness of anticancer therapeutics such as immunogenic chemotherapy or immune checkpoint blockade in preclinical tumor models, as well as in cancer patients. In synthesis, it appears that a normal intestinal microbiota supports therapeutic anticancer responses, while a dysbiotic microbiota that lacks immunostimulatory bacteria or contains overabundant immunosuppressive species causes treatment failure. These findings have led to the design of clinical trials that evaluate the capacity of modulation of the gut microbiota to synergize with treatment and hence limit tumor progression. Along the lines of this Trial Watch, we discuss the rationale for harnessing the gut microbiome in support of cancer therapy and the progress of recent clinical trials testing this new therapeutic paradigm in cancer patients.

Vaccination with autologous dendritic cells (DC) loaded ex vivo with melanoma-associated antigens is currently being tested as an adjuvant treatment modality for resected locoregional metastatic (stage III) melanoma. Based on its mechanism of action, DC vaccination might potentiate the clinical efficacy of concurrent or sequential immune checkpoint inhibition (ICI). The purpose of this study was to determine the efficacy of ICI administered following recurrent disease during, or after, adjuvant DC vaccination. To this end, we retrospectively analyzed clinical responses of 51 melanoma patients with either irresectable stage III or stage IV disease treated with first- or second-line ICI following recurrence on adjuvant DC vaccination. Patients were analyzed according to the form of ICI administered: PD-1 inhibition monotherapy (nivolumab or pembrolizumab), ipilimumab monotherapy or combined treatment with ipilimumab and nivolumab. Treatment with first- or second-line PD-1 inhibition monotherapy after recurrence on adjuvant DC vaccination resulted in a response rate of 52%. In patients treated with ipilimumab monotherapy and ipilimumab-nivolumab response rates were 35% and 75%, respectively. In conclusion, ICI is effective in melanoma patients with recurrent disease on adjuvant DC vaccination.

Summary: Plasmacytoid dendritic cells (pDCs) and type 2 conventional dendritic cells (cDC2s) are currently under evaluation for use in cancer vaccines. Although both DC subsets can activate adaptive and innate lymphocytes, their capacity to recruit such cells is rarely considered. Here, we show that pDCs and cDC2s display a striking difference in chemokine secretion, which correlates with the recruitment of distinct types of immune effector cells. Activated pDCs express high levels of CXCR3 ligands and attract more CD8+ T cells, CD56+ T cells, and γδ T cells in vitro, compared to cDC2s. Skin from melanoma patients shows an influx of immune effector cells following intradermal vaccination with pDCs or cDC2s, with pDCs inducing the strongest influx of lymphocytes known to possess cytolytic activity. These findings suggest that combining both DC subsets could unite the preferred chemoattractive properties of pDCs with the superior T cell priming properties of cDC2s to ultimately enhance vaccine efficacy. : Human plasmacytoid dendritic cells and type 2 conventional dendritic cells are currently used in cancer vaccines to induce antitumor immune responses. van Beek et al. compared their chemoattractive properties and identified a striking difference in chemokine secretion, resulting in the attraction of distinct immune effector cells. Keywords: pDC, cDC2, chemokines, migration, DC vaccines

Dendritic cell (DCs) activation by Toll-like receptor (TLR) agonist induces robust metabolic rewiring toward glycolysis. Recent findings in the field identified mechanistic details governing these metabolic adaptations. However, it is unknown whether a switch to glycolysis from oxidative phosphorylation (OXPHOS) is a general characteristic of DCs upon pathogen encounter. Here we show that engagement of different TLR triggers differential metabolic adaptations in DCs. We demonstrate that LPS-mediated TLR4 stimulation induces glycolysis in DCs. Conversely, activation of TLR7/8 with protamine-RNA complex, pRNA, leads to an increase in OXPHOS. Mechanistically, we found that pRNA stimulation phosphorylates BCKDE1α in a PINK1-dependent manner. pRNA stimulation increased branched-chain amino acid levels and increased fatty acid oxidation. Increased FAO and OXPHOS are required for DC activation. PINK1 deficient DCs switch to glycolysis to maintain ATP levels and viability. Moreover, pharmacological induction of PINK1 kinase activity primed immunosuppressive DC for immunostimulatory function. Our findings provide novel insight into differential metabolic adaptations and reveal the important role of branched-chain amino acid in regulating immune response in DC.

Tumor vaccines are an important asset in the field of cancer immunotherapy. Whether prophylactic or therapeutic, these vaccines aim to enhance the T cell-mediated anti-tumor immune response that is orchestrated by dendritic cells. Although promising preclinical and early-stage clinical results have been obtained, large-scale clinical implementation of cancer vaccination is stagnating due to poor clinical response. The challenges of clinical efficacy of tumor vaccines can be mainly attributed to tumor induced immunosuppression and poor immunogenicity of the chosen tumor antigens. Recently, intratumor heterogeneity and the relation with tumor-specific neoantigen clonality were put in the equation.In this perspective we provide an overview of recent studies showing how personalized tumor vaccines containing multiple neoantigens can broaden and enhance the anti-tumor immune response. Furthermore, we summarize advances in the understanding of the intratumor mutational landscape containing different tumor cell subclones and the temporal and spatial diversity of neoantigen presentation and burden, and the relation between these factors with respect to tumor immunogenicity. Together, the presented knowledge calls for the investment in the characterization of neoantigens in the context of intratumor heterogeneity to improve clinical efficacy of personalized tumor vaccines.

Dendritic cells (DCs) play a key role in orchestrating adaptive immune responses. In human blood, three distinct subsets exist: plasmacytoid DCs (pDCs) and BDCA3+ and CD1c+ myeloid DCs. In addition, a DC-like CD16+ monocyte has been reported. Although RNA-expression profiles have been previously compared, protein expression data may provide a different picture. Here, we exploited label-free quantitative mass spectrometry to compare and identify differences in primary human DC subset proteins. Moreover, we integrated these proteomic data with existing mRNA data to derive robust cell-specific expression signatures with more than 400 differentially expressed proteins between subsets, forming a solid basis for investigation of subset-specific functions. We illustrated this by extracting subset identification markers and by demonstrating that pDCs lack caspase-1 and only express low levels of other inflammasome-related proteins. In accordance, pDCs were incapable of interleukin (IL)-1β secretion in response to ATP.