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BackgroundPrimary and secondary resistance is a major hurdle in cancer immunotherapy. Therefore, a better understanding of the underlying mechanisms involved in immunotherapy resistance is of pivotal importance to improve therapy outcome.MethodHere, two mouse models with resistance against therapeutic vaccine-induced tumor regression were studied. Exploration of the tumor microenvironment by high dimensional flow cytometry in combination with therapeuticin vivosettings allowed for the identification of immunological factors driving immunotherapy resistance.ResultsComparison of the tumor immune infiltrate during early and late regression revealed a change from tumor-rejecting toward tumor-promoting macrophages. In concert, a rapid exhaustion of tumor-infiltrating T cells was observed. Perturbation studies identified a small but discernible CD163himacrophage population, with high expression of several tumor-promoting macrophage markers and a functional anti-inflammatory transcriptome profile, but not other macrophages, to be responsible. In-depth analyses revealed that they localize at the tumor invasive margins and are more resistant to Csf1r inhibition when compared with other macrophages.In vivostudies validated the activity of heme oxygenase-1 as an underlying mechanism of immunotherapy resistance. The transcriptomic profile of CD163himacrophages is highly similar to a human monocyte/macrophage population, indicating that they represent a target to improve immunotherapy efficacy.ConclusionsIn this study, a small population of CD163hitissue-resident macrophages is identified to be responsible for primary and secondary resistance against T-cell-based immunotherapies. While these CD163hiM2 macrophages are resistant to Csf1r-targeted therapies, in-depth characterization and identification of the underlying mechanisms driving immunotherapy resistance allows the specific targeting of this subset of macrophages, thereby creating new opportunities for therapeutic intervention with the aim to overcome immunotherapy resistance.

Introduction: 5-fluorouracil, folinic acid, irinotecan and oxaliplatin (FOLFIRINOX) is promising in treating patients with pancreatic ductal adenocarcinoma. However, many patients and physicians are reluctant to start FOLFIRINOX due to its high toxicity and limited clinical response rates. In this study, we investigated the effect of a single FOLFIRINOX cycle, in combination with a granulocyte colony-stimulating factor, on the blood immune transcriptome of patients with pancreatic ductal adenocarcinoma. We aimed to iden-ify an early circulating biomarker to predict the lack of FOLFIRINOX response. Methods: Blood samples of 68 patients from all disease stages, who received at least four FOLFIRINOX cycles, were collected at baseline and after the first cycle. The response to treatment was radiologically evaluated following the Response Evaluation Criteria in Solid Tumours criteria 1.1. Targeted immune-gene expression profiling (GEP) was performed using Nano-String technologies. To predict the lack of FOLFIRINOX response, we developed a FOLFIRINOX delta GEP (FFX-Delta GEP) score. Results: A single FOLFIRINOX cycle significantly altered 395 genes, correlating to 30 significant alterations in relative immune cell abundances and pathway activities. The eight-gene (BID, FOXP3, KIR3DL1, MAF, PDGFRB, RRAD, SIGLEC1 and TGFB2) FFX-Delta GEP score predicted the lack of FOLFIRINOX response with a leave-one-out cross-validated area under the curve (95% confidence interval) of 0.87 (0.60-0.98), thereby outperforming the predictiveness of absolute and proportional Delta carbohydrate antigen19-9 values. Conclusions: A single FOLFIRINOX cycle, combined with granulocyte colony-stimulating factor, alters the peripheral immune transcriptome indisputably. Our novel FFX-Delta GEP is, to our knowledge, the first multigene early circulating biomarker that predicts the lack of FOLFIRINOX response after one cycle. Validation in a larger independent patient cohort is crucial before clinical implementation. (c) 2022 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

BackgroundThe complete response rate of cervical high-grade squamous intraepithelial lesion (cHSIL) patients to imiquimod immunotherapy is approximately 60%. Consequently, many patients are exposed to unnecessary adverse effects of imiquimod. On the other hand, conventional surgical large loop excision therapy is associated with increased risk of premature births in subsequent pregnancies. An in-depth analysis of the cHSIL immune microenvironment was performed in order to identify and develop a predictive biomarker for response to imiquimod, to maximize therapy efficacy and to avoid adverse effects in patients unlikely to respond.MethodsBiopsies of 35 cHSIL patients, before and 10 weeks on imiquimod treatment, were analyzed by two multispectral seven-color immunofluorescence panels for T cell and myeloid cell composition in relation to treatment response. Based on these results a simplified immunohistochemical detection protocol was developed. Samples were scanned with the Vectra multispectral imaging system and cells were automatically identified using machine learning.ResultsThe immune microenvironment of complete responders (CR) is characterized by a strong and coordinated infiltration by T helper cells (activated PD1+/type 1 Tbet+), M1-like macrophages (CD68+CD163-) and dendritic cells (CD11c+) prior to imiquimod. The lesions of non-responders (NRs) displayed a high infiltration by CD3+FOXP3+regulatory T cells. At 10 weeks on imiquimod, a strong influx of intraepithelial and stromal CD4+T cells was observed in CR but not NR patients. A steep decrease in macrophages occurred both in CR and NR patients, leveling the pre-existing differences in myeloid cell composition between the two groups. Based on the pre-existing immune composition differences, the sum of intraepithelial CD4 T cell, macrophage and dendritic cell counts was used to develop a quantitative simplified one color immunohistochemical biomarker, the CHSIL immune biomarker for imiquimod (CIBI), which can be automatically and unbiasedly quantified and has an excellent predictive capacity (receiver operating characteristic area under the curve 0.95, pConclusionThe capacity of cHSIL patients to respond to imiquimod is associated with a pre-existing coordinated local immune process, fostering an imiquimod-mediated increase in local T cell infiltration. The CIBI immunohistochemical biomarker has strong potential to select cHSIL patients with a high likelihood to experience a complete response to imiquimod immunotherapy.

Myeloid immune cells are frequently present in the tumor environment, and although they can positively contribute to tumor control they often negatively impact anticancer immune responses. One way of inhibiting the positive contributions of myeloid cells is by signaling through the cluster of differentiation 47 (CD47)/signal regulatory protein alpha (SIRP alpha) axis. The SIRP alpha receptor is expressed on myeloid cells and is an inhibitory immune receptor that, upon binding to CD47 protein, delivers a 'don't eat me' signal. As CD47 is often overexpressed on cancer cells, treatments targeting CD47/SIRP alpha have been under active investigation and are currently being tested in clinical settings. Interestingly, the CD47/SIRP alpha axis is also involved in T cell-mediated antitumor responses. In this perspective we provide an overview of recent studies showing how therapeutic blockade of the CD47/SIRP alpha axis improves the adaptive immune response. Furthermore, we discuss the interconnection between the myeloid CD47/SIRP alpha axis and adaptive T cell responses as well as the potential therapeutic role of the CD47/SIRP alpha axis in tumors with acquired resistance to the classic immunotherapy through major histocompatibility complex downregulation. Altogether this review provides a profound insight for the optimal exploitation of CD47/SIRP alpha immune checkpoint therapy.

High serum levels of interleukin‐6 (IL‐6) correlate with poor prognosis and chemotherapy resistance in several cancers. The underlying mechanisms and its effects on immunotherapy are largely unknown. To address this, we developed a human papillomavirus type 16 (HPV16)‐associated tumor model expressing IL‐6 to investigate the impact of tumor‐expressed IL‐6 during cisplatin chemotherapy and HPV16 synthetic long peptide vaccination as immunotherapy. The effects of tumor‐produced IL‐6 on tumor growth, survival and the tumor microenvironment were analyzed. Our data demonstrated that tumor‐produced IL‐6 conferred resistance to cisplatin and therapeutic vaccination. This was not caused by a changed in vitro or in vivo growth rate of tumor cells, or a changed sensitivity of tumor cells to chemotherapy or T‐cell‐mediated killing. Furthermore, no overt differences in the frequencies of tumor‐infiltrating subsets of T cells or CD11b+ myeloid cells were observed. IL‐6, however, affected the systemic and local function of myeloid cells, reflected by a strong reduction of major histocompatibility complex (MHC) class II expression on all major myeloid cell subtypes. Resistance to both therapies was associated with a changed intratumoral influx of MHC class II+ myeloid cells toward myeloid cells with no or lower MHC class II expression. Importantly, while these IL‐6‐mediated effects provided resistance to the immunotherapy and chemotherapy as single therapies, their combination still successfully mediated tumor control. In conclusion, IL‐6‐mediated therapy resistance is caused by an extrinsic mechanism involving an impaired function of intratumoral myeloid cells. The fact that resistance can be overcome by combination therapies provides direction to more effective therapies for cancer., What's new? Interleukin‐6 (IL‐6) cytokine has multiple effects on hematopoiesis and immune function and typically circulates at low levels. In cancer, however, IL‐6 serum levels are significantly elevated, with suspected impacts on tumor behavior. In this study, using a mouse model of human papillomavirus‐induced cancer with IL‐6 expression, the authors show that tumor‐produced IL‐6 confers resistance to both chemotherapy and immunotherapy. Resistance was associated with impaired myeloid cell maturation, with no evidence of involvement of mechanisms intrinsic to tumor cells. Resistance was overcome by combining chemotherapy and immunotherapy, providing insight into a potentially effective therapeutic approach for cancers with IL‐6‐mediated resistance.

Immunotherapy of vulvar high‐grade squamous intraepithelial lesion (vHSIL) is investigated as an alternative for surgery, because of high comorbidity and risk of recurrence. Limited evidence exists on the role and composition of the immune microenvironment in current immunotherapeutic approaches for vHSIL. The vHSIL of 29 patients biopsied before treatment with imiquimod were analyzed by two multiplex seven‐color immunofluorescence panels to investigate the pre‐existing T‐cell and myeloid cell composition in relation to treatment response. The samples were scanned with the Vectra multispectral imaging system. Cells were automatically phenotyped and counted with inForm advanced image analysis software. Cell counts and composition were compared to that of vHSIL patients before therapeutic vaccination (n = 29) and to healthy vulva (n = 27). Our data show that the immune microenvironment of complete responders (CR) to imiquimod resembled the coordinated infiltration with type 1 CD4+ and CD8+ T cells and CD14+ inflammatory myeloid cells also found in healthy vulva. However, more CD8+ T cells and FoxP3+ regulatory T cells were present in CR. The lesions of partial responders (PR) lacked such a coordinated response and displayed an impaired influx of CD14+ inflammatory myeloid cells. Importantly, complete responses after imiquimod or therapeutic vaccination showed the same dependency on a pre‐existing coordinated type 1 T‐cell and CD14+ myeloid cell infiltration. In conclusion, a good clinical outcome after two different forms of immunotherapy for vHSIL is associated with the presence of a primary inflammatory process resulting in the coordinated influx of several types of immune cells which is then amplified., What's new? Premalignant vulvar high‐grade squamous intraepithelial lesions (vHSIL) are predominantly induced by human papilloma virus infection. The immune microenvironment in vHSIL and its role in immunotherapeutic approaches remain largely unknown. This study is the first to show that a complete clinical response in patients is associated with a pre‐existent coordinated influx of type 1 T cells and CD14+ myeloid cells, irrespective of the type of successful immunotherapy given (topical imiquimod therapy or therapeutic vaccination). This coordinated immune microenvironment closely resembles that of healthy vulvar tissue, suggesting that an impaired primary inflammatory process acts as an immune resistance mechanism in non‐complete responders.

BackgroundThe composition of the tumor immune microenvironment (TIME) associated with good prognosis generally also predicts the success of immunotherapy, and both entail the presence of pre-existing tumor-specific T cells. Here, the blueprint of the TIME associated with such an ongoing tumor-specific T-cell response was dissected in a unique prospective oropharyngeal squamous cell carcinoma (OPSCC) cohort, in which tumor-specific tumor-infiltrating T cells were detected (immune responsiveness (IR+)) or not (lack of immune responsiveness (IR−)).MethodsA comprehensive multimodal, high-dimensional strategy was applied to dissect the TIME of treatment-naive IR+ and IR− OPSCC tissue, including bulk RNA sequencing (NanoString), imaging mass cytometry (Hyperion) for phenotyping and spatial interaction analyses of immune cells, and combined single-cell gene expression profiling and T-cell receptor (TCR) sequencing (single-cell RNA sequencing (scRNAseq)) to characterize the transcriptional states of clonally expanded tumor-infiltrating T cells.ResultsIR+ patients had an excellent survival during >10 years follow-up. The tumors of IR+ patients expressed higher levels of genes strongly related to interferon gamma signaling, T-cell activation, TCR signaling, and mononuclear cell differentiation, as well as genes involved in several immune signaling pathways, than IR− patients. The top differently overexpressed genes included CXCL12 and LTB, involved in ectopic lymphoid structure development. Moreover, scRNAseq not only revealed that CD4+ T cells were the main producers of LTB but also identified a subset of clonally expanded CD8+ T cells, dominantly present in IR+ tumors, which secreted the T cell and dendritic cell (DC) attracting chemokine CCL4. Indeed, immune cell infiltration in IR+ tumors is stronger, highly coordinated, and has a distinct spatial phenotypical signature characterized by intratumoral microaggregates of CD8+CD103+ and CD4+ T cells with DCs. In contrast, the IR− TIME comprised spatial interactions between lymphocytes and various immunosuppressive myeloid cell populations. The impact of these chemokines on local immunity and clinical outcome was confirmed in an independent The Cancer Genome Atlas OPSCC cohort.ConclusionThe production of lymphoid cell attracting and organizing chemokines by tumor-specific T cells in IR+ tumors constitutes a positive feedback loop to sustain the formation of the DC–T-cell microaggregates and identifies patients with excellent survival after standard therapy.

BackgroundMany solid tumors do not respond to immunotherapy due to their immunologically cold tumor microenvironment (TME). We and others found that oncolytic viruses (OVs), including reovirus type 3 Dearing, can enhance the efficacy of immunotherapy by recruiting CD8+ T cells to the TME. A significant part of the incoming CD8+ T cells is directed toward reovirus itself, which may be detrimental to the efficacy of OVs. However, here we aim to exploit these incoming virus-specific T cells as anticancer effector cells.MethodsWe performed an in-depth characterization of the reovirus-induced T-cell response in immune-competent mice bearing pancreatic KPC3 tumors. The immunodominant CD8+ T-cell epitope of reovirus was identified using epitope prediction algorithms and peptide arrays, and the quantity and quality of reovirus-specific T cells after reovirus administration were assessed using high-dimensional flow cytometry. A synthetic long peptide (SLP)-based vaccination strategy was designed to enhance the intratumoral frequency of reovirus-specific CD8+ T cells.ResultsReovirus administration did not induce tumor-specific T cells but rather induced high frequencies of reovirus-specific CD8+ T cells directed to the immunodominant epitope. Priming of reovirus-specific T cells required a low-frequent population of cross-presenting dendritic cells which was absent in Batf3-/- mice. While intratumoral and intravenous reovirus administration induced equal systemic frequencies of reovirus-specific T cells, reovirus-specific T cells were highly enriched in the TME exclusively after intratumoral administration. Here, they displayed characteristics of potent effector cells with high expression of KLRG1, suggesting they may be responsive against local reovirus-infected cells. To exploit these reovirus-specific T cells as anticancer effector cells, we designed an SLP-based vaccination strategy to induce a strong T-cell response before virotherapy. These high frequencies of circulating reovirus-specific T cells were reactivated on intratumoral reovirus administration and significantly delayed tumor growth.ConclusionsThese findings provide proof of concept that OV-specific T cells, despite not being tumor-specific, can be exploited as potent effector cells for anticancer treatment when primed before virotherapy. This is an attractive strategy for low-immunogenic tumors lacking tumor-specific T cells.

BackgroundExpression of killer cell lectin-like receptor B1 (KLRB1), the gene encoding the cell surface molecule CD161, is associated with favorable prognosis in many cancers. CD161 is expressed by several lymphocyte populations, but its role and regulation on tumor-specific CD4+ T cells is unknown.MethodsWe examined the clinical impact of CD4+CD161+ T cells in human papillomavirus (HPV)16+ oropharyngeal squamous cell carcinoma (OPSCC), analyzed their contribution in a cohort of therapeutically vaccinated patients and used HPV16-specific CD4+CD161+ tumor-infiltrating lymphocytes and T cell clones for in-depth mechanistic studies.ResultsCentral and effector memory CD4+ T cells express CD161, but only CD4+CD161+ effector memory T cells (Tem) are associated with improved survival in OPSCC. Therapeutic vaccination activates and expands type 1 cytokine-producing CD4+CD161+ effector T cells. The expression of CD161 is dynamic and follows a pattern opposite of the checkpoint molecules PD1 and CD39. CD161 did not function as an immune checkpoint molecule as demonstrated using multiple experimental approaches using antibodies to block CD161 and gene editing to knockout CD161 expression. Single-cell transcriptomics revealedKLRB1expression in many T cell clusters suggesting differences in their activation. Indeed, CD4+CD161+ effector cells specifically expressed the transcriptional transactivatorSOX4,known to enhance T cell receptor (TCR) signaling via CD3ε. Consistent with this observation, CD4+CD161+ cells respond more vigorously to limiting amounts of cognate antigen in presence of interleukin (IL)-12 and IL-18 compared to their CD161- counterparts. The expression of CD161/KLRB1andSOX4was downregulated upon TCR stimulation and this effect was boosted by transforming growth factor (TGF)β1.ConclusionHigh levels of CD4+CD161+ Tem are associated with improved survival and our data show that CD161 is dynamically regulated by cell intrinsic and extrinsic factors. CD161 expressing CD4+ T cells rapidly respond to suboptimal antigen stimulation suggesting that CD161, similar to SOX4, is involved in the amplification of TCR signals in CD4+ T cells.

BackgroundExpression of CD103 and CD39 has been found to pinpoint tumor-reactive CD8+T cells in a variety of solid cancers. We aimed to investigate whether these markers specifically identify neoantigen-specific T cells in colorectal cancers (CRCs) with low mutation burden.Experimental designWhole-exome and RNA sequencing of 11 mismatch repair-proficient (MMR-proficient) CRCs and corresponding healthy tissues were performed to determine the presence of putative neoantigens. In parallel, tumor-infiltrating lymphocytes (TILs) were cultured from the tumor fragments and, in parallel, CD8+T cells were flow-sorted from their respective tumor digests based on single or combined expression of CD103 and CD39. Each subset was expanded and subsequently interrogated for neoantigen-directed reactivity with synthetic peptides. Neoantigen-directed reactivity was determined by flow cytometric analyses of T cell activation markers and ELISA-based detection of IFN-γ and granzyme B release. Additionally, imaging mass cytometry was applied to investigate the localization of CD103+CD39+cytotoxic T cells in tumors.ResultsNeoantigen-directed reactivity was only encountered in bulk TIL populations and CD103+CD39+(double positive, DP) CD8+T cell subsets but never in double-negative or single-positive subsets. Neoantigen-reactivity detected in bulk TIL but not in DP CD8+T cells could be attributed to CD4+T cells. CD8+T cells that were located in direct contact with cancer cells in tumor tissues were enriched for CD103 and CD39 expression.ConclusionCoexpression of CD103 and CD39 is characteristic of neoantigen-specific CD8+T cells in MMR-proficient CRCs with low mutation burden. The exploitation of these subsets in the context of adoptive T cell transfer or engineered T cell receptor therapies is a promising avenue to extend the benefits of immunotherapy to an increasing number of CRC patients.

Simple Summary Immune checkpoint inhibitors (ICIs) have revolutionized treatment of advanced melanoma and survival of melanoma patients has radically improved since. However, as durable responses after ICIs are only observed in 30–50% of melanoma patients, there is an unmet need to identify predictive biomarkers for response. This systematic review demonstrates the substantial number of publications that have studied a wide variety of possible biomarkers. Covering 177 publications that investigated 128 unique biomarkers, we provide an overview of all studied biomarkers in correlation with response or survival. We highlight blood, tumor and fecal biomarkers that were associated with response to ICIs in multiple studies. Of these, only T-cell inflamed gene expression profiling was predictive for response in a large clinical trial and validated in other studies, thus representing a promising biomarker for clinical practice. Large validation studies are warranted to confirm the predictive utility of other biomarkers, thereby further personalizing immunotherapy treatment. Abstract Immune checkpoint inhibitors (ICIs) have strongly improved the survival of melanoma patients. However, as durable response to ICIs are only seen in a minority, there is an unmet need to identify biomarkers that predict response. Therefore, we provide a systematic review that evaluates all biomarkers studied in association with outcomes of melanoma patients receiving ICIs. We searched Pubmed, COCHRANE Library, Embase, Emcare, and Web of Science for relevant articles that were published before June 2020 and studied blood, tumor, or fecal biomarkers that predicted response or survival in melanoma patients treated with ICIs. Of the 2536 identified reports, 177 were included in our review. Risk of bias was high in 40%, moderate in 50% and low in 10% of all studies. Biomarkers that correlated with response were myeloid-derived suppressor cells (MDSCs), circulating tumor cells (CTCs), CD8+ memory T-cells, T-cell receptor (TCR) diversity, tumor-infiltrating lymphocytes (TILs), gene expression profiling (GEP), and a favorable gut microbiome. This review shows that biomarkers for ICIs in melanoma patients are widely studied, but heterogeneity between studies is high, average sample sizes are low, and validation is often lacking. Future studies are needed to further investigate the predictive utility of some promising candidate biomarkers.

The surface inhibitory receptor NKG2A forms heterodimers with the invariant CD94 chain and is expressed on a subset of activated CD8 T cells. As antibodies to block NKG2A are currently tested in several efficacy trials for different tumor indications, it is important to characterize the NKG2A+ CD8 T cell population in the context of other inhibitory receptors. Here we used a well-controlled culture system to study the kinetics of inhibitory receptor expression. Naïve mouse CD8 T cells were synchronously and repeatedly activated by artificial antigen presenting cells in the presence of the homeostatic cytokine IL-7. The results revealed NKG2A as a late inhibitory receptor, expressed after repeated cognate antigen stimulations. In contrast, the expression of PD-1, TIGIT and LAG-3 was rapidly induced, hours after first contact and subsequently down regulated during each resting phase. This late, but stable expression kinetics of NKG2A was most similar to that of TIM-3 and CD39. Importantly, single-cell transcriptomics of human tumor-infiltrating lymphocytes (TILs) showed indeed that these receptors were often coexpressed by the same CD8 T cell cluster. Furthermore, NKG2A expression was associated with cell division and was promoted by TGF-β in vitro, although TGF-β signaling was not necessary in a mouse tumor model in vivo. In summary, our data show that PD-1 reflects recent TCR triggering, but that NKG2A is induced after repeated antigen stimulations and represents a late inhibitory receptor. Together with TIM-3 and CD39, NKG2A might thus mark actively dividing tumor-specific TILs.

BackgroundOropharyngeal squamous cell carcinoma (OPSCC) is the most prevalent type of head and neck cancer. The survival of patients with OPSCC is tightly linked to the intratumoral presence of tumor-specific CD4+ and CD8+ T cells. Yet, immunotherapy is currently far from effective in OPSCC partly due to our limited understanding of its immune microenvironment.MethodsHere a multi-modal, high-dimensional approach was used to dissect the immune landscape in a unique cohort of pre-therapy OPSCC patient samples (n=20) in which intratumoral tumor-specific T cells were either detected (immune response positive, IR+) or not (IR-). This included imaging mass cytometry (Hyperion) for high-dimensional phenotyping, spatial localization and interaction analyses of the cells in the tumor mircoenvironment with our newly developed imaging processing pipeline employing machine learning, Nanostring PanCancer IO360 panel analysis of immune signaling pathways, and combined single-cell gene expression profiling and T cell receptor sequencing (scRNAseq) to characterize the transcriptional states of clonally expanded tumor-infiltrating T cells.ResultsImmune cell infiltration in IR+ tumors is stronger and highly coordinated, with a distinct spatial phenotypic signature characterized by microaggregates of tumor-resident (CD103+) CD8+ and CD4+ T cells and dendritic cells within the tumor cell beds, which retained after permutation based correction for differences in cell frequencies. Furthermore, the increased expression of CXCL12 and LTB produced by CD4+ T cells, both involved in the spatial organization of immune cell infiltration, and the clonal expansion of CD8+ T cells producing the DC-attracting chemokines CCL4 or XCL1 in IR+ OPSCC, indicate that tumor-reactive T cells act as a positive feedback loop in the formation of these aggregates. The impact of these chemokines on local immunity and clinical outcome was confirmed in an independent TCGA OPSCC cohort. In contrast, the IR- OPSCC signature comprised spatial interactions between lymphocytes and different subpopulations of immunosuppressive myeloid cells.ConclusionsOur study reveals that the chemokine-driven spatial immune signature of OPSCC has strong potential as a prognostic and predictive biomarker. While the immune signature of IR+ OPSCC suggests potential benefit from neoadjuvant immunotherapeutic approaches to limit the side effects of current radio(chemo)therapy, that of IR- OPSCC calls for strategies focused on stimulating T cells and counteracting immune suppressive mechanisms.

Simple Summary Chemoradiation with photon radiotherapy is very effective as a locally advanced cervical cancer (LACC) treatment. However, the majority of women with LACC experience treatment-related toxicity involving the gastrointestinal and urogenital tracts and the immune system. Compared to that of photon therapy, proton therapy substantially reduces undesired dose to the organs around the tumor, leading to a decrease in radiotherapy-related side-effects. At present, few studies on proton therapy in patients with LACC will be conducted. The PROTECT trial aims to evaluate the differences in side effects between photon therapy and proton therapy, both combined with chemotherapy, for LACC. Fifteen patients will be enrolled per treatment group. Information will be collected on the differences in dose to the organs around the tumor, treatment-related side effects, and the impact on the immune system. This information will be used to assess the potential of proton therapy as an innovative treatment for LACC. Abstract External beam radiation therapy (EBRT) with concurrent chemotherapy followed by brachytherapy is a very effective treatment for locally advanced cervical cancer (LACC). However, treatment-related toxicity is common and reduces the patient’s quality of life (QoL) and ability to complete treatment or undergo adjuvant therapies. Intensity modulated proton therapy (IMPT) enables a significant dose reduction in organs at risk (OAR), when compared to that of standard intensity-modulated radiation therapy (IMRT) or volumetric-modulated arc therapy (VMAT). However, clinical studies evaluating whether IMPT consequently reduces side effects for LACC are lacking. The PROTECT trial is a nonrandomized prospective multicenter phase-II-trial comparing clinical outcomes after IMPT or IMRT/VMAT in LACC. Thirty women aged >18 years with a histological diagnosis of LACC will be included in either the IMPT or IMRT/VMAT group. Treatment includes EBRT (45 Gy in 25 fractions of 1.8 Gy), concurrent five weekly cisplatin (40 mg/m2), and 3D image (MRI)-guided adaptive brachytherapy. The primary endpoint is pelvic bones Dmean and mean bowel V15Gy. Secondary endpoints include dosimetric parameters, oncological outcomes, health-related QoL, immune response, safety, and tolerability. This study provides the first data on the potential of IMPT to reduce OAR dose in clinical practice and improve toxicity and QoL for patients with LACC.

BackgroundA profound insight into the immune landscape of vulvar squamous cell carcinoma (VSCC) is lacking. Here, an in-depth interrogation of T cell infiltration, local immune contexture, signaling pathways and checkpoint molecule expression was performed in early-stage and late-stage VSCC.MethodsThe type, location, and infiltration pattern of T cells were studied in 109 patients with primary VSCC FIGO stage I–III. RNA expression of genes involved in immune oncology and oncogenic signaling pathways was analyzed in 40 VSCC, matched for prognostic clinicopathological variables, analyzed for HPV and p53 status, and selected based on T cell infiltration.ResultsHigh intraepithelial infiltration with CD4 or CD8 T cells was associated with longer overall and recurrence-free survival and formed an independent prognostic factor, outperforming molecular subtype and stage of the disease. Strong T cell infiltrated VSCC displayed a coordinated immune response reflected by a positive association between T cells and different lymphocyte and myeloid cell subsets. The expression of genes involved in the migration of T cells and myeloid cells, T cell activation and costimulation, interferon (IFN)-γ signaling, cytotoxicity and apoptosis was higher than in low infiltrated tumors. An active immune signaling profile was observed in all inflamed, part of the altered-excluded and not in altered-immunosuppressed or deserted VSCC. While several checkpoint molecules were overexpressed, only PD-L1 expression displayed discriminatory ability and clinical usefulness. High PD-L1 expression was detected in all inflamed and ~60% of the altered-excluded VSCC.ConclusionAn active immune signaling profile is present in 35% of primary FIGO I–III VSCCs, suggesting potential responsiveness to neoadjuvant PD-1/PD-L1 immunotherapy.

Cancer cells frequently display defects in their antigen-processing pathway and thereby evade CD8 T cell immunity. We described a novel category of cancer antigens, named TEIPP, that emerge on cancers with functional loss of the peptide pump TAP. TEIPPs are non-mutated neoantigens despite their ‘self’ origin by virtue of their absence on normal tissues. Here, we describe the development of a synthetic long peptide (SLP) vaccine for the most immunogenic TEIPP antigen identified thus far, derived from the TAP-independent LRPAP1 signal sequence. LRPAP121–30-specific CD8 T cells were present in blood of all tested healthy donors as well as patients with non-small cell lung adenocarcinoma. SLPs with natural flanking, however, failed to be cross-presented by monocyte-derived dendritic cells. Since the C-terminus of LRPAP121–30 is an unconventional and weakly binding serine (S), we investigated if replacement of this anchor would result in efficient cross-presentation. Exchange into a valine (V) resulted in higher HLA-A2 binding affinity and enhanced T cell stimulation. Importantly, CD8 T cells isolated using the V-variant were able to bind tetramers with the natural S-variant and respond to TAP-deficient cancer cells. A functional screen with an array of N-terminal and C-terminal extended SLPs pointed at the 24-mer V-SLP, elongated at the N-terminus, as most optimal vaccine candidate. This SLP was efficiently cross-presented and consistently induced a strong polyclonal LRPAP121–30-specific CD8 T cells from the endogenous T cell repertoire. Thus, we designed a TEIPP SLP vaccine from the LRPAP1 signal sequence ready for validation in clinical trials. Supplementary Information The online version contains supplementary material available at 10.1007/s00262-021-02984-7.

BackgroundMany cancer patients do not obtain clinical benefit from immune checkpoint inhibition. Checkpoint blockade targets T cells, suggesting that tyrosine kinase activity profiling of baseline peripheral blood mononuclear cells may predict clinical outcome.MethodsHere a total of 160 patients with advanced melanoma or non-small-cell lung cancer (NSCLC), treated with anti-cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) or anti-programmed cell death 1 (anti-PD-1), were divided into five discovery and cross-validation cohorts. The kinase activity profile was generated by analyzing phosphorylation of peripheral blood mononuclear cell lysates in a microarray comprising of 144 peptides derived from sites that are substrates for protein tyrosine kinases. Binary grouping into patients with or without clinical benefit was based on Response Evaluation Criteria in Solid Tumors V.1.1. Predictive models were trained using partial least square discriminant analysis (PLS-DA), performance of the models was evaluated by estimating the correct classification rate (CCR) using cross-validation.ResultsThe kinase phosphorylation signatures segregated responders from non-responders by differences in canonical pathways governing T-cell migration, infiltration and co-stimulation. PLS-DA resulted in a CCR of 100% and 93% in the anti-CTLA-4 and anti-PD1 melanoma discovery cohorts, respectively. Cross-validation cohorts to estimate the accuracy of the predictive models showed CCRs of 83% for anti-CTLA-4 and 78% or 68% for anti-PD-1 in melanoma or NSCLC, respectively.ConclusionBlood-based kinase activity profiling for response prediction to immune checkpoint inhibitors in melanoma and NSCLC revealed increased kinase activity in pathways associated with T-cell function and led to a classification model with a highly accurate classification rate in cross-validation groups. The predictive value of kinase activity profiling is prospectively verified in an ongoing trial.

Oropharyngeal squamous cell carcinoma (OPSCC) develops as a consequence of several mutations in the tumor suppressor pathways or after a progressive infection with high risk human papillomavirus (HPV). The dismal side effects of the current standard of care and the clear involvement of the immune system has led to a surge in clinical trials that aim to reinforce the tumor-specific immune response as a new treatment option. In this review, we have focused on the most recent literature to discuss the new findings and insights on the role of different immune cells in the context of OPSCC and its etiology. We then applied this knowledge to describe potential biomarkers and analyzed the rationale and outcomes of earlier and ongoing immunotherapy trials. Finally, we describe new developments that are still at the preclinical phase and provide an outlook on what the near future may bring, now that several new and exciting techniques to study the immune system at the single cell level are being exploited.

Objective. There is great need for better risk stratification in vulvar squamous cell carcinoma (VSCC). Our aim was to define the prognostic significance of stratifying VSCC based on p16 and p53 immunohistochemistry (IHC) as surrogate markers for HPV and TP53 mutations.Methods. A large retrospective cohort of surgically treated women with primary VSCC was used. VSCC were classified into three subtypes: HPV-positive (HPVpos), HPV-negative/p53 mutant (HPVneg/p53mut), and HPVnegative/p53 wildtype (HPVneg/p53wt). Overall survival (OS), relative survival (RS), and recurrence-free period (RFP) were depicted using the Kaplan-Meier method and survival curves for relative survival; associations were studied using univariable and multivariable Cox proportional hazard models.Results. Of the 413 VSCCs, 75 (18%) were HPVpos, 63 (15%) HPVneg/p53wt, and 275 (66%) HPVneg/p53mut VSCC. Patients with HPVneg/p53mut VSCC had worse OS and RS (HR 3.43, 95%CI 1.80-6.53, and relative excess risk (RER) of 4.02; 95%CI 1.48-10.90, respectively, and worse RFP (HR 3.76, 95%CI 2.02-7.00). HPVpos VSCC patients showed most favorable outcomes. In univariate analysis, the molecular subtype of VSCC was a prognostic marker for OS, RS and RFP (p = 0.003, p = 0.009, p Conclusions. Stratification of VSCC by p16and p53-IHC has potential to be used routinely in diagnostic pathology. It results in the identification of three clinically distinct subtypes and may be used to guide treatment and follow-up, and in stratifying patients in future clinical trials. (C) 2020 Elsevier Inc. All rights reserved.

BackgroundCross-presentation of exogenous antigens in HLA-class I molecules by professional antigen presenting cells (APCs) is crucial for CD8+ T cell function. Recent murine studies show that several non-professional APCs, including cancer-associated fibroblasts (CAFs) also possess this capacity. Whether human CAFs are able to cross-present exogenous antigen, which molecular pathways are involved in this process and how this ultimately affects tumor-specific CD8+ T cell function is unknown.MethodsIn this study, we investigated the ability of human colorectal cancer (CRC)-derived CAFs to cross-present neoantigen-derived synthetic long peptides (SLPs), corresponding to tumor-derived mutant peptides, and how this affects tumor-specific T-cell function. Processing of the SLP was studied by targeting components of the cross-presentation machinery through CRISPR/Cas9 and siRNA-mediated genetic ablation to identify the key molecules involved in fibroblast-mediated cross-presentation. Multispectral flow cytometry and killing assays were performed to study the effect of fibroblast cross-presentation on T cell function.ResultsHere, we show that human CRC-derived CAFs display an enhanced capacity to cross-present neoantigen-derived SLPs when compared with normal colonic fibroblasts. Cross-presentation of antigens by fibroblasts involved the lysosomal protease cathepsin S. Cathepsin S expression by CAFs was detected in situ in human CRC tissue, was upregulated in ex vivo cultured CRC-derived CAFs and showed increased expression in normal fibroblasts after exposure to CRC-conditioned medium. Cognate interaction between CD8+ T cells and cross-presenting CAFs suppressed T cell function, reflected by decreased cytotoxicity, reduced activation (CD137) and increased exhaustion (TIM3, LAG3 and CD39) marker expression.ConclusionThese data indicate that CAFs may directly suppress tumor-specific T cell function in an antigen-dependent fashion in human CRC.

BackgroundHigh serum interleukin (IL-6) levels may cause resistance to immunotherapy by modulation of myeloid cells in the tumor microenvironment. IL-6 signaling blockade is tested in cancer, but as this inflammatory cytokine has pleiotropic effects, this treatment is not always effective.MethodsIL-6 and IL-6R blockade was applied in an IL-6-mediated immunotherapy-resistant TC-1 tumor model (TC-1.IL-6) and immunotherapy-sensitive TC-1.control. Effects on therapeutic vaccination-induced tumor regression, recurrence and survival as well on T cells and myeloid cells in the tumor microenvironment were studied. The effects of IL-6 signaling in macrophages under therapy conditions were studied in Il6rafl/fl×LysMcre+ mice.ResultsOur therapeutic vaccination protocol elicits a strong tumor-specific CD8+ T-cell response, leading to enhanced intratumoral T-cell infiltration and recruitment of tumoricidal macrophages. Blockade of IL-6 signaling exacerbated tumor outgrowth, reflected by fewer complete regressions and more recurrences after therapeutic vaccination, especially in TC-1.IL-6 tumor-bearing mice. Early IL-6 signaling blockade partly inhibited the development of the vaccine-induced CD8+ T-cell response. However, the main mechanism was the malfunction of macrophages during therapy-induced tumor regression. Therapy efficacy was impaired in Il6rafl/fl×LysMcre+ but not cre-negative control mice, while no differences in the vaccine-induced CD8+ T-cell response were found between these mice. IL-6 signaling blockade resulted in decreased expression of suppressor of cytokine signaling 3, essential for effective M1-type function in macrophages, and increased expression of the phagocytic checkpoint molecule signal-regulatory protein alpha by macrophages.ConclusionIL-6 signaling is critical for macrophage function under circumstances of immunotherapy-induced tumor tissue destruction, in line with the acute inflammatory functions of IL-6 signaling described in infections.

IntroductionTreatment with anti-PD-1 immunotherapy does not lead to long-lasting clinical responses in approximately 60% of patients with metastatic melanoma. These refractory patients, however, can still respond to treatment with tumour infiltrating lymphocytes (TIL) and interferon-alpha (IFNa). A combination of TIL, pegylated-interferon-alpha (PEG-IFNa) and anti-PD-1 is expected to provide a safe, feasible and effective therapy for patients with metastatic melanoma, who are refractory to standard of care treatment options.Methods and analysisPatients are treated in two phases. In phase I, the safety of the combination TIL and anti-PD-1 is assessed (cohort 1) according to CTCAE 4.03 criteria. Subsequently, the safety of cotreatment with PEG-IFNa is tested in cohort 2. The efficacy will be evaluated in the second phase of the trial. Efficacy is evaluated according to RECIST 1.1 and immune-related response criteria. Clinical and immunological parameters will be evaluated for their relation with clinical responsiveness.Ethics and disseminationEthical approval of the trial was obtained from the Central Committee on Research Involving Human Subjects in the Netherlands. The trial results will be shared with the scientific community at (inter)national conferences and by publication in a peer-reviewed journal.Trial registration numberNCT03638375.

BackgroundNeutrophils have been reported to have protumor, antitumor or neutral effects in cancer progression. The underlying causes for this functional variability are not clear.MethodsWe studied the role of neutrophils in six different mouse tumor models by intratumoral injection of antimicrobial peptides or vaccination. Changes in systemic and intratumoral immune cells were analyzed by flow-cytometry and mass-cytometry. The role of neutrophils was studied by antibody-mediated neutrophil depletion. Neutrophils from different mouse strains were compared by RNA sequencing.ResultsThe antimicrobial peptide Omiganan reduced the growth of TC-1 tumors in BL/6 mice and CT26 tumors in BALB/c mice. No significant effects were observed in B16F10, MC38 and 4T1 tumors. Growth delay was associated with increased abundance of neutrophils in TC-1 but not CT26 tumors. Systemic neutrophil depletion abrogated Omiganan efficacy in TC-1 but further reduced growth of CT26, indicating that neutrophils were required for the antitumor effect in TC-1 but suppressed tumor control in CT26. Neutrophils were also required for a therapeutic vaccine-induced T-cell mediated control of RMA tumors in BL/6 mice. Clearly, the circulating and intratumoral neutrophils differed in the expression of Ly6G and CD62L, between TC-1 and CT26 and between blood neutrophils of tumor-naïve BL/6 and BALB/c mice. RNA-sequencing revealed that neutrophils from BL/6 mice but not BALB/c mice displayed a robust profile of immune activation, matching their opposing roles in TC-1 and RMA versus CT26.ConclusionsNeutrophil functionality differs strongly between mouse strains and tumor types, with consequences for tumor progression and therapy.

BackgroundHuman papillomavirus (HPV)-associated oropharyngeal squamous cell carcinoma (OPSCC) is a distinct clinical entity with a much better prognosis after (chemo)radiotherapy than HPV-negative OPSCC, especially in patients with a concomitant intratumoral HPV-specific and type-1 cytokine-oriented T cell response. However, knowledge on the type of myeloid cells and their coordination with intratumoral T cells and influence on patient outcome in OPSCC is lacking.MethodsWe analyzed the presence of intratumoral myeloid cells and their relationship to tumor-infiltrating T cells and patient outcome in a well-described cohort of HPV16+ patients with OPSCC using multispectral immunofluorescence, flow cytometry and functional analyses.ResultsWe show that the tumor microenvironment of HPV16+ OPSCC tumors with such an ongoing HPV16-specific T cell response is highly infiltrated with a newly defined CD163+ cytokine-producing subset of conventional dendritic cell type 2 (cDC2), called DC3. These CD163+ cDC2 predominantly stimulated type 1 T cell polarization and produced high levels of interleukin-12 (IL-12) and IL-18, required for IFNγ and IL-22 production by T cells after cognate antigen stimulation. Tumor-infiltration with these CD163+ cDC2 positively correlated with the infiltration by Tbet+ and tumor-specific T cells, and with prolonged survival.ConclusionsThese data suggest an important role for intratumoral CD163+ cDC2 in stimulating tumor-infiltrating T cells to exert their antitumor effects.

BackgroundAdoptive cell therapy (ACT) with tumor-reactive T cells has shown consistent clinical efficacy. We evaluated the response to ACT in combination with interferon alpha (IFNa) preconditioning in patients with stage IV metastatic melanoma, most of which were progressive on cytotoxic T-lymphocyte-associated protein 4 and/or programmed cell death protein 1 checkpoint blockade therapy.MethodsThirty-four patients were treated with ex vivo expanded tumor reactive T cells, derived from mixed lymphocyte autologous tumor cultures, or with autologous tumor-infiltrating lymphocytes and evaluated for clinical response. Clinical and immunological parameters associated with response were also evaluated.ResultsBest overall response defined as clinical benefit, comprising either complete response, partial response or stable disease >6 months, was observed in 29% of the patients. Forty-three per cent of the 14 immunotherapy-naïve patients and 20% of the 20 patients progressive on prior immunotherapy benefited from ACT. The overall survival (OS) was 90% versus 28.6% at 1 year and 46.7% versus 0% at 3 years follow-up, of responder and non-responder patients, respectively. Median OS was 36 versus 7 months, respectively. IFNa pretreatment resulted in leukopenia, neutropenia and lymphopenia, which was sustained during the treatment in clinical responders and associated with response. Differences in antigen specificity, but not in phenotype, cytokine profile or CD8+ T cell number of the ACT products correlated with clinical response. Cross-reactivity of the ACT products to one or more allogeneic human leukocyte antigen-matched melanoma cell lines was associated with short OS after treatment while the ACT products of very long-term survivors showed no cross-reactivity but recognized patient-specific neoantigens.ConclusionThis study demonstrates that ACT in combination with a mild IFNa preconditioning regimen can induce clinical benefit even in immunotherapy pretreated patients, although with lower success than in immunotherapy-naïve patients. ACT products comprising neoantigen reactivity may be more effective.

Objectives A minority of NSCLC patients benefit from anti-PD1 immune checkpoint inhibitors. A rational combination of biomarkers is needed. The objective was to determine the predictive value of tumor mutational load (TML), CD8+ T cell infiltration, HLA class-I and PD-L1 expression in the tumor. Materials and methods Metastatic NSCLC patients were prospectively included in an immune-monitoring trial (NTR7015) between April 2016-August 2017, retrospectively analyzed in FFPE tissue for TML (NGS: 409 cancer-related-genes) and by IHC staining to score PD-L1, CD8+ T cell infiltration, HLA class-I. PFS (RECISTv1.1) and OS were analyzed by Kaplan–Meier methodology. Results 30 patients with adenocarcinoma (67%) or squamous cell carcinoma (33%) were included. High TML was associated with better PFS (p = 0.004) and OS (p = 0.025). Interaction analyses revealed that patients with both high TML and high total CD8+ T cell infiltrate (p = 0.023) or no loss of HLA class-I (p = 0.026), patients with high total CD8+ T cell infiltrate and no loss of HLA class-I (p = 0.041) or patients with both high PD-L1 and high TML (p = 0.003) or no loss of HLA class-I (p = 0.032) were significantly associated with better PFS. Unsupervised cluster analysis based on these markers revealed three sub-clusters, of which cluster-1A was overrepresented by patients with progressive disease (15 out of 16), with significant effect on PFS (p = 0.007). Conclusion This proof-of-concept study suggests that a combination of PD-L1 expression, TML, CD8+ T cell infiltration and HLA class-I functions as a better predictive biomarker for response to anti-PD-1 immunotherapy. Consequently, refinement of this set of biomarkers and validation in a larger set of patients is warranted.

Cancer immunotherapies, including checkpoint inhibitors, adoptive T cell transfer and therapeutic cancer vaccines, have shown promising response rates in clinical trials. Unfortunately, there is an increasing number of patients in which initially regressing tumors start to regrow due to an immunotherapy-driven acquired resistance. Studies on the underlying mechanisms reveal that these can be similar to well-known tumor intrinsic and extrinsic primary resistance factors that precluded the majority of patients from responding to immunotherapy in the first place. Here, we discuss primary and secondary immune resistance and point at strategies to identify potential new mechanisms of immune evasion. Ultimately, this may lead to improved immunotherapy strategies with improved clinical outcomes.

Therapeutic cancer vaccines have effectively induced durable regressions of premalignant oncogenic human papilloma virus type 16 (HPV16)-induced anogenital lesions. However, the treatment of HPV16-induced cancers requires appropriate countermeasures to overcome cancer-induced immune suppression. We previously showed that standard-of-care carboplatin/paclitaxel chemotherapy can reduce abnormally high numbers of immunosuppressive myeloid cells in patients, allowing the development of much stronger therapeutic HPV16 vaccine (ISA101)-induced tumor immunity. We now show the clinical effects of ISA101 vaccination during chemotherapy in 77 patients with advanced, recurrent, or metastatic cervical cancer in a dose assessment study of ISA101. Tumor regressions were observed in 43% of 72 evaluable patients. The depletion of myeloid suppressive cells by carboplatin/paclitaxel was associated with detection of low frequency of spontaneous HPV16-specific immunity in 21 of 62 tested patients. Patients mounted type 1 T cell responses to the vaccine across all doses. The group of patients with higher than median vaccine-induced immune responses lived longer, with a flat tail on the survival curve. This demonstrates that chemoimmunotherapy can be exploited to the benefit of patients with advanced cancer based on a defined mode of action.

The success of checkpoint blockade therapy revolutionized cancer treatment. However, we need to increase the fraction of responding patients and overcome acquired resistance to these therapies. Recently, the inhibitory receptor NKG2A received attention as a new kid on the block of immune checkpoints. This receptor is selectively expressed on cytotoxic lymphocytes, including natural killer cells and CD8 T cells, and NKG2A+ T cells are preferentially residing in tissues, like the tumor microenvironment. Its ligand, histocompatibility leucocyte antigen E (HLA-E), is a conserved nonclassical HLA class I molecule that binds a limited peptide repertoire and its expression is commonly detected in human cancer. NKG2A blockade as a standalone therapy appears poorly effective in mouse tumor models, however, in the presence of activated T cells, for example, induced by PD-1/PD-L1 blockade or cancer vaccines, exerts strongly enhanced efficacy. Clinical trials demonstrated safety of the humanized NKG2A-blocking antibody, monalizumab, and first results of phase II trials demonstrate encouraging durable response rates. Further development of this axis is clearly warranted.

BackgroundVulvar high-grade squamous intraepithelial lesion (vHSIL) is predominantly induced by high-risk human papilloma virus type 16 (HPV16). In two independent trials, therapeutic vaccination against the HPV16 E6 and E7 oncoproteins resulted in objective partial and complete responses (PRs/CRs) in half of the patients with HPV16+vHSIL at 12-month follow-up. Here, the prevaccination and postvaccination vHSIL immune microenvironment in relation to the vaccine-induced clinical response was investigated.MethodsTwo novel seven-color multiplex immunofluorescence panels to identify T cells (CD3, CD8, Foxp3, Tim3, Tbet, PD-1, DAPI) and myeloid cells (CD14, CD33, CD68, CD163, CD11c, PD-L1, DAPI) were designed and fully optimized for formalin-fixed paraffin-embedded tissue. 29 prevaccination and 24 postvaccination biopsies of patients with vHSIL, and 27 healthy vulva excisions, were stained, scanned with the Vectra multispectral imaging system, and automatically phenotyped and counted using inForm advanced image analysis software.ResultsHealthy vulvar tissue is strongly infiltrated by CD4 and CD8 T cells expressing Tbet and/or PD-1 and CD14+HLA-DR+inflammatory myeloid cells. The presence of such a coordinated pre-existing proinflammatory microenvironment in HPV16+vHSIL is associated with CR after vaccination. In partial responders, a disconnection between T cell and CD14+myeloid cell infiltration was observed, whereas clinical non-responders displayed overall lower immune cell infiltration. Vaccination improved the coordination of local immunity, reflected by increased numbers of CD4+Tbet+T cells and HLA-DR+CD14+expressing myeloid cells in patients with a PR or CR, but not in patients with no response. CD8+T cell infiltration was not increased after vaccination.ConclusionA prevaccination inflamed type 1 immune contexture is required for stronger vaccine-induced immune infiltration and is associated with better clinical response. Therapeutic vaccination did not overtly increase immune infiltration of cold lesions.

Immune therapy is a promising field within oncology but has been unsuccessful in ovarian cancer (OC). Still, there is rationale and evidence supporting immune therapy in OC. We investigated the potential for adoptive cell therapy (ACT) from in vitro expanded tumor-infiltrating lymphocytes (TILs) in combination with checkpoint inhibitors (ICI) and conducted immunological testing of ex vivo expanded TILs (REP-TILs). Six patients with late-stage metastatic high-grade serous OC were treated with immune therapy consisting of ipilimumab followed by surgery to obtain TILs and infusion of REP-TILs, low-dose IL-2 and nivolumab. One patient achieved a partial response and 5 others experienced disease stabilization for up to 12 months. Analysis of the REP-TILs with flow- and mass-cytometry show primarily activated and differentiated effector memory T cells. REP-TILs showed in vitro reactivity and expression of inhibitory receptors, such as LAG-3 and PD-1. Furthermore, our data indicate that addition of ipilimumab therapy improves the T cell fold expansion during production, increase the level of CD8 T cell tumor reactivity, and favorably affect the T cell phenotype. We show that the combination of ICI and ACT is feasible and safe. With one partial response and one long-lasting SD, we demonstrated the potential of ACT in OC.

BackgroundPancreatic ductal adenocarcinoma (PDAC) is notoriously resistant to treatment including checkpoint-blockade immunotherapy. We hypothesized that a bimodal treatment approach consisting of dendritic cell (DC) vaccination to prime tumor-specific T cells, and a strategy to reprogram the desmoplastic tumor microenvironment (TME) would be needed to break tolerance to these pancreatic cancers. As a proof-of-concept, we investigated the efficacy of combined DC vaccination with CD40-agonistic antibodies in a poorly immunogenic murine model of PDAC. Based on the rationale that mesothelioma and pancreatic cancer share a number of tumor associated antigens, the DCs were loaded with either pancreatic or mesothelioma tumor lysates.MethodsImmune-competent mice with subcutaneously or orthotopically growing KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) PDAC tumors were vaccinated with syngeneic bone marrow-derived DCs loaded with either pancreatic cancer (KPC) or mesothelioma (AE17) lysate and consequently treated with FGK45 (CD40 agonist). Tumor progression was monitored and immune responses in TME and lymphoid organs were analyzed using multicolor flow cytometry and NanoString analyzes.ResultsMesothelioma-lysate loaded DCs generated cross-reactive tumor-antigen-specific T-cell responses to pancreatic cancer and induced delayed tumor outgrowth when provided as prophylactic vaccine. In established disease, combination with stimulating CD40 antibody was necessary to improve survival, while anti-CD40 alone was ineffective. Extensive analysis of the TME showed that anti-CD40 monotherapy did improve CD8 +T cell infiltration, but these essential effector cells displayed hallmarks of exhaustion, including PD-1, TIM-3 and NKG2A. Combination therapy induced a strong change in tumor transcriptome and mitigated the expression of inhibitory markers on CD8 +T cells.ConclusionThese results demonstrate the potency of DC therapy in combination with CD40-stimulation for the treatment of pancreatic cancer and provide directions for near future clinical trials.

BackgroundT-cell-engaging CD3-bispecific antibodies (CD3-bsAbs) are promising modalities for cancer immunotherapy. Although this therapy has reached clinical practice for hematological malignancies, the absence of sufficient infiltrating T cells is a major barrier for efficacy in solid tumors. In this study, we exploited oncolytic reovirus as a strategy to enhance the efficacy of CD3-bsAbs in immune-silent solid tumors.MethodsThe mutant p53 and K-ras induced murine pancreatic cancer model KPC3 resembles human pancreatic ductal adenocarcinomas with a desmoplastic tumor microenvironment, low T-cell density and resistance to immunotherapy. Immune-competent KPC3 tumor-bearing mice were intratumorally injected with reovirus type 3 Dearing strain and the reovirus-induced changes in the tumor microenvironment and spleen were analyzed over time by NanoString analysis, quantitative RT-PCR and multicolor flow cytometry. The efficacy of reovirus in combination with systemically injected CD3-bsAbs was evaluated in immune-competent mice with established KPC3 or B16.F10 tumors, and in the close-to-patient human epidermal growth factor receptor 2 (HER2)+ breast cancer model BT474 engrafted in immunocompromised mice with human T cells as effector cells.ResultsReplication-competent reovirus induced an early interferon signature, followed by a strong influx of natural killer cells and CD8+ T cells, at the cost of FoxP3+ Tregs. Viral replication declined after 7 days and was associated with a systemic activation of lymphocytes and the emergence of intratumoral reovirus-specific CD8+ T cells. Although tumor-infiltrating T cells were mostly reovirus-specific and not tumor-specific, they served as non-exhausted effector cells for the subsequently systemically administered CD3-bsAbs. Combination treatment of reovirus and CD3-bsAbs led to the regression of large, established KPC3, B16.F10 and BT474 tumors. Reovirus as a preconditioning regimen performed significantly better than simultaneous or early administration of CD3-bsAbs. This combination treatment induced regressions of distant lesions that were not injected with reovirus, and systemic administration of both reovirus and CD3-bsAbs also led to tumor control. This suggests that this therapy might also be effective for metastatic disease.ConclusionsOncolytic reovirus administration represents an effective strategy to induce a local interferon response and strong T-cell influx, thereby sensitizing the tumor microenvironment for subsequent CD3-bsAb therapy. This combination therapy warrants further investigation in patients with non-inflamed solid tumors.

BackgroundImmunotherapy of cancer is successful but tumor regression often is incomplete and followed by escape. Understanding the mechanisms underlying this acquired resistance will aid the development of more effective treatments.MethodsWe exploited a mouse model where tumor-specific therapeutic vaccination results in tumor regression, followed by local recurrence and resistance. In depth studies on systemic, local and tumor intrinsic changes were performed with flow and mass cytometry, immunohistochemistry, transcriptomics and several perturbation studies with inhibitors or agonistic antibodies in mice. Main findings were recapitulated in vaccinated patients.ResultsFull tumor regression and cure of tumor-bearing mice is dependent on the magnitude of the vaccine-induced T-cell response. Recurrence of tumors did not involve classical immune escape mechanisms, such as antigen-presentation alterations, immune checkpoint expression, resistance to killing or local immune suppression. However, the recurrent tumors displayed a changed transcriptome with alterations in p53, tumor necrosis factor-α and transforming growth factor-β signaling pathways and they became immunologically cold. Remarkably, ex vivo cell-sorted recurrent tumors, directly reinjected in naïve hosts retained their resistance to vaccination despite a strong infiltration with tumor-specific CD8+ T cells, similar to that of vaccine-responsive tumors. The influx of inflammatory mature myeloid effector cells in the resistant tumors, however, was impaired and this turned out to be the underlying mechanisms as restoration of inflammatory myeloid cell infiltration reinstated the sensitivity of these refractory tumors to vaccination. Notably, impaired myeloid cell infiltration after vaccination was also associated with vaccine resistance in patients.ConclusionAn immunotherapy-induced disability of tumor cells to attract innate myeloid effector cells formed a major mechanism underlying immune escape and acquired resistance. These data not only stresses the importance of myeloid effector cells during immunotherapy but also demands for new studies to harness their tumoricidal activities.

BackgroundEpithelial ovarian cancer (EOC) is considered an immunogenic tumor, as illustrated by the clear correlation between T-cell infiltration and overall survival. This suggests that patients with EOC may be eligible for immunotherapy including adoptive cell therapy with autologous Tumor Infiltrating Lymphocytes (TIL). However, immunosuppressive cells including myeloid derived suppressor cells an regulatory T cells are also abundant in EOC and may need to be targeted simultaneously to achieve the full potential of the infused TIL. Carboplatin-paclitaxel chemotherapy (CPC) reduces the number of immunosuppressive cells in cervical cancer patients,1 creating a window-of-opportunity for TIL to exert their full effector function. Interferon-alpha further supports infused TIL. A phase I/II trial (NCT04072263) was initiated to study the feasibility and safety of TIL during CPC with or without additional interferon-alpha in patients with recurrent platinum-sensitive EOC.MethodsFifteen patients with recurrent platinum-sensitive EOC received 6 cycles of CPC intravenously every 3 weeks and TIL intravenously 2 weeks after the 2nd,3rd and 4th CPC cycle. Pegylated-interferon-alpha was added in the second cohort for 12 weeks, starting one week before the first TIL infusion. Patients who received 3 TIL infusions were evaluable. The primary endpoint was feasibility and safety of TIL administration during CPC with or without interferon-alpha. As secondary endpoints signs of activity, underlying mechanisms, immunomodulation, and T-cell reactivity were studied.ResultsThirteen patients were available for analysis. Median age 63 years (range, 29–77). TIL could be successfully expanded for all patients. Treatment with TIL during CPC was safe and did not add toxicity. Addition of IFNα resulted in grade 3 leucopenia and grade 3 trombocytopenia in the first 2 patients and was therefore omitted in subsequent patients. CPC alleviated the immunosuppressive status, reflected by reduced plasma IL-6 levels and circulating myeloid-cell numbers, while lymphocytes numbers are not affected. This was most prominently at 1–2 weeks after the 2nd CPC and is suggested to reflect improved conditions promoting intra-tumoral T-cell reactivity. Objective responses were observed in 10/13 (77%) patients and 3 patients had stable disease. Interestingly, in at least one patient the ongoing platinum-free interval of 25 months far exceeds the first platinum-free interval of 8 months after similar CPC. In depth studies on immune modulation by chemotherapy and by TIL/Interferon-alpha, and correlations between TIL phenotype and clinical outcome are ongoing and will be presented.ConclusionsCombined treatment with CP chemotherapy and properly timed TIL may result in clinical benefit for patients with EOC.AcknowledgementsThe unrestricted funding of the trial by Ovacure is greatly acknowledged.Trial RegistrationThe trial is registered at www.clinicaltrials.gov under number NCT04072263.ReferenceWelters MJ, van der Sluis TC, van Meir H, Loof NM, van Ham VJ, van Duikeren S, Santegoets SJ, Arens R, de Kam ML, Cohen AF, van Poelgeest MI, Kenter GG, Kroep JR, Burggraaf J, Melief CJ, van der Burg SH. Vaccination during myeloid cell depletion by cancer chemotherapy fosters robust T cell responses. Sci Transl Med 2016;8(334):334ra52. doi: 10.1126/scitranslmed.aad8307Ethics ApprovalThis study was approved by Leiden University Medical Center‘s Ethics Board; approval number L18-012 and the Central Committee on Research Involving Human Subjects; approval number NL63434.000.17.

Adoptive T cell transfer (ACT) with ex vivo-expanded tumor-reactive T cells proved to be successful for the treatment of metastatic melanoma patients. Mixed lymphocyte tumor cell cultures (MLTC) can be used to generate tumor-specific T cells for ACT; however, in a number of cases tumor-reactive T cell, expansion is far from optimal. We hypothesized that this is due to tumor intrinsic and extrinsic factors and aimed to identify and manipulate these factors so to optimize our clinical, GMP-compliant MLTC protocol. We found that the tumor cell produced IDO and/or galectin-3, and the accumulation of CD4+CD25hiFoxP3+ T cells suppressed the expansion of tumor-specific T cells in the MLTC. Strategies to eliminate CD4+CD25hiFoxP3+ T cells during culture required the depletion of the whole CD4+ T cell population and were found to be undesirable. Blocking of IDO and galectin-3 was feasible and resulted in improved efficiency of the MLTC. Implementation of these findings in clinical protocols for ex vivo expansion of tumor-reactive T cells holds promise for an increased therapeutic potential of adoptive cell transfer treatments with tumor-specific T cells. Electronic supplementary material The online version of this article (doi:10.1007/s00262-017-1995-x) contains supplementary material, which is available to authorized users.

The implementation of immune checkpoint inhibitors to the oncology clinic signified a new era in cancer treatment. After the first indication of melanoma, an increasing list of additional cancer types are now treated with immune system targeting antibodies to PD-1, PD-L1 and CTLA-4, alleviating inhibition signals on T cells. Recently, we published proof-of-concept results on a novel checkpoint inhibitor, NKG2A. This receptor is expressed on cytotoxic lymphocytes, including NK cells and subsets of activated CD8+ T cells. Blocking antibodies to NKG2A unleashed the reactivity of these effector cells resulting in tumor control in multiple mouse models and an early clinical trial. Monalizumab is inhibiting this checkpoint in human beings and future clinical trials will have to reveal its potency in combination with other cancer treatment options.

Background Vulvar squamous cell carcinoma (VSCC) has been suggested to consist of three subtypes; HPV-positive, HPV-negative mutated TP53 or HPV-negative TP53 wildtype, with different clinical courses. To analyze the immune infiltrate in these molecular subtypes and its impact on clinical outcome, an in-depth study of the tumor immune microenvironment was performed. Methods Sixty-five patients with invasive VSCC matched for age, FIGO stage and treatment modality, were grouped according to the presence of HPV and p53 protein expression status. Archived tissues were analyzed for intraepithelial and stromal expression of CD3, CD8, Foxp3, PD-1, and pan-keratin in randomly selected areas using immunofluorescence. Additional phenotyping of T cells was performed ex-vivo on VSCC (n = 14) and blood samples by flow cytometry. Healthy vulvar samples and blood served as controls. Results Based on T-cell infiltration patterns about half of the VSCC were classified as inflamed or altered-excluded while one-third was immune-deserted. High intraepithelial helper T cell infiltration was observed in 78% of the HPV-induced VSCC, 60% of the HPVnegVSCC/p53wildtype and 40% of the HPVnegVSCC with abnormal p53 expression. A high intraepithelial infiltration with activated (CD3+PD-1+), specifically helper T cells (CD3+CD8−Foxp3−), was associated with a longer recurrence-free period and overall survival, irrespective of HPV and p53 status. Flow cytometry confirmed the tumor-specific presence of activated (CD4+PD-1++CD161−CD38+HLA-DR+ and CD8+CD103+CD161−NKG2A+/−PD1++CD38++HLA-DR+) effector memory T cells. Conclusion This is the first study demonstrating an association between intraepithelial T cells and clinical outcome in VSCC. Our data suggest that abnormal p53 expressing VSCCs mostly are cold tumors whereas HPV-driven VSCCs are strongly T-cell infiltrated. Electronic supplementary material The online version of this article (10.1186/s40425-019-0712-z) contains supplementary material, which is available to authorized users.

One of the characteristics of prognostically infaust uveal melanoma (UM) is an inflammatory phenotype, which is characterized by high numbers of infiltrating T cells and macrophages, and a high HLA Class I expression. We wondered how this inflammation is regulated, and considered that one of the most important regulators of inflammation, the NFkB pathway, might play a role. We analyzed 64 UM samples for expression of HLA Class I, its regulators, and of members of the NFkB transcription family, using an Illumina HT12V4 array. HLA Class I expression and infiltrating immune cells were also determined by immunohistochemical staining. Information was obtained regarding chromosome status by Affymetrix Nsp array. Our analysis shows that expression of NFkB1, NFkB2 and RELB positively correlates with the level of HLA Class I expression and the number of infiltrating T cells and macrophages, while SPP1 and PPAR&gamma, are negatively correlated. Increased levels of NFkB1 and NFkB2 and decreased levels of SPP1 and PPAR&gamma, are seen in Monosomy 3/BAP1-negative tumors. This is also the case in non-inflammatory UM, indicating that our observation not only involves infiltrating leukocytes but the tumor cells themselves. We report that the NFkB pathway is associated with inflammation and HLA Class I expression in UM, and is upregulated when BAP1 expression is lost.

The intracellular peptide pump TAP feeds antigenic peptides for loading onto HLA class I molecules, and its down-modulation is a frequent immune evasion mechanism in human cancers. Two recent papers describe which ‘hidden‘ antigens we might exploit to target these escaped cancer variants by CD8 T cells. These unTAPped peptides are now ready for clinical testing.

Background T-cell mediated immunotherapy brought clinical success for many cancer patients. Nonetheless, downregulation of MHC class I antigen presentation, frequently occurring in solid cancers, limits the efficacy of these therapies. Unraveling the mechanisms underlying this type of immune escape is therefore of great importance. We here investigated the immunological effects of metabolic stress in cancer cells as a result of nutrient deprivation. Methods TC1 and B16F10 tumor cell lines were cultured under oxygen- and glucose-deprivation conditions that mimicked the tumor microenvironment of solid tumors. Presentation of peptide antigens by MHC class I molecules was measured by flow cytometry and via activation of tumor-specific CD8 T cell clones. The proficiency of the IFNy-STAT1 pathway was investigated by Western blots on phosphorylated proteins, transfection of constitutive active STAT1 constructs and qPCR of downstream targets. Kinase inhibitors for PI3K were used to examine its role in IFNy receptor signal transduction. Results Combination of oxygen- and glucose-deprivation resulted in decreased presentation of MHC class I antigens on cancer cells, even in the presence of the stimulatory cytokine IFNy. This unresponsiveness to IFNy was the result of failure to phosphorylate the signal transducer STAT1. Forced expression of constitutive active STAT1 fully rescued the MHC class I presentation. Furthermore, oxygen- and glucose-deprivation increased PI3K activity in tumor cells. Pharmacological inhibition of this pathway not only restored signal transduction through IFNy-STAT1 but also improved MHC class I presentation. Importantly, PI3K inhibitors also rendered tumor cells sensitive for recognition by CD8 T cells in culture conditions of metabolic stress. Conclusions These data revealed a strong impact of metabolic stress on the presentation of tumor antigens by MHC class I and suggest that this type of tumor escape takes place at hypoxic areas even during times of active T cell immunity and IFNy release. Electronic supplementary material The online version of this article (10.1186/s40425-019-0627-8) contains supplementary material, which is available to authorized users.

Importance In recurrent human papilloma virus (HPV)–driven cancer, immune checkpoint blockade with anti–programmed cell death 1 (PD-1) antibodies produces tumor regression in only a minority of patients. Therapeutic HPV vaccines have produced strong immune responses to HPV-16, but vaccination alone has been ineffective for invasive cancer. Objective To determine whether the efficacy of nivolumab, an anti–PD-1 immune checkpoint antibody, is amplified through treatment with ISA 101, a synthetic long-peptide HPV-16 vaccine inducing HPV-specific T cells, in patients with incurable HPV-16–positive cancer. Design, Setting, and Participants In this single-arm, single-center phase 2 clinical trial, 24 patients with incurable HPV-16–positive cancer were enrolled from December 23, 2015, to December 12, 2016. Duration of follow-up for censored patients was 12.2 months through August 31, 2017. Interventions The vaccine ISA101, 100 μg/peptide, was given subcutaneously on days 1, 22, and 50. Nivolumab, 3 mg/kg, was given intravenously every 2 weeks beginning day 8 for up to 1 year. Main Outcomes and Measures Assessment of efficacy reflected in the overall response rate (per Response Evaluation Criteria in Solid Tumors, version 1.1). Results Of the 24 patients (4 women and 20 men; 22 with oropharyngeal cancer; median age, 60 years [range, 36-73 years]), the overall response rate was 33% (8 patients; 90% CI, 19%-50%). Median duration of response was 10.3 months (95% CI, 10.3 months to inestimable). Five of 8 patients remain in response. Median progression-free survival was 2.7 months (95% CI, 2.5-9.4 months). Median overall survival was 17.5 months (95% CI, 17.5 months to inestimable). Grades 3 to 4 toxicity occurred in 2 patients (asymptomatic grade 3 transaminase level elevation in 1 patient and grade 4 lipase elevation in 1 patient), requiring discontinuation of nivolumab therapy. Conclusions and Relevance The overall response rate of 33% and median overall survival of 17.5 months is promising compared with PD-1 inhibition alone in similar patients. A randomized clinical trial to confirm the contribution of HPV-16 vaccination to tumoricidal effects of PD-1 inhibition is warranted for further study. Trial Registration ClinicalTrials.gov identifier:NCT02426892

Background The capacity of cytomegalovirus (CMV) to elicit long-lasting strong T cell responses, and the ability to engineer the genome of this DNA virus positions CMV-based vaccine vectors highly suitable as a cancer vaccine platform. Defined immune thresholds for tumor protection and the factors affecting such thresholds have not well been investigated in cancer immunotherapy. We here determined using CMV as a vaccine platform whether critical thresholds of vaccine-specific T cell responses can be established that relate to tumor protection, and which factors control such thresholds. Methods We generated CMV-based vaccine vectors expressing the E7 epitope and tested these in preclinical models of HPV16-induced cancer. Vaccination was applied via different doses and routes (intraperitoneal (IP), subcutaneous (SC) and intranasal (IN)). The magnitude, kinetics and phenotype of the circulating tumor-specific CD8+ T cell response were determined. Mice were subsequently challenged with tumor cells, and the tumor protection was monitored. Results Immunization with CMV-based vaccines via the IP or SC route eliciting vaccine-induced CD8+ T cell responses of > 0.3% of the total circulating CD8 T cell population fully protects mice against lethal tumor challenge. However, low dose inoculations via the IP or SC route or IN vaccination elicited vaccine-induced CD8+ T cell responses that did not reach protective thresholds for tumor protection. In addition, whereas weak pre-existing immunity did not alter the protective thresholds of the vaccine-specific T cell response following subsequent immunization with CMV-based vaccine vectors, strong pre-existing immunity inhibited the development of vaccine-induced T cells and their control on tumor progression. Conclusions This study highlight the effectiveness of CMV-based vaccine vectors, and shows that demarcated thresholds of vaccine-specific T cells could be defined that correlate to tumor protection. Together, these results may hold importance for cancer vaccine development to achieve high efficacy in vaccine recipients. Electronic supplementary material The online version of this article (10.1186/s40425-019-0500-9) contains supplementary material, which is available to authorized users.

Purpose: The tumor immune microenvironment determines clinical outcome. Whether the original tissue in which a primary tumor develops influences this microenvironment is not well understood. Experimental Design: We applied high-dimensional single-cell mass cytometry [Cytometry by Time-Of-Flight (CyTOF)] analysis and functional studies to analyze immune cell populations in human papillomavirus (HPV)–induced primary tumors of the cervix (cervical carcinoma) and oropharynx (oropharyngeal squamous cell carcinoma, OPSCC). Results: Despite the same etiology of these tumors, the composition and functionality of their lymphocytic infiltrate substantially differed. Cervical carcinoma displayed a 3-fold lower CD4:CD8 ratio and contained more activated CD8+CD103+CD161+ effector T cells and less CD4+CD161+ effector memory T cells than OPSCC. CD161+ effector cells produced the highest cytokine levels among tumor-specific T cells. Differences in CD4+ T-cell infiltration between cervical carcinoma and OPSCC were reflected in the detection rate of intratumoral HPV-specific CD4+ T cells and in their impact on OPSCC and cervical carcinoma survival. The peripheral blood mononuclear cell composition of these patients, however, was similar. Conclusions: The tissue of origin significantly affects the overall shape of the immune infiltrate in primary tumors.

Cancers frequently evade immune-recognition by lowering peptide:MHC-I complexes on their cell surface. Limited peptide supply due to TAP-deficiency results in such MHC-Ilow immune-escape variants. Previously, we reported on a category of TAP-independent self-peptides, called TEIPP, with selective presentation by these tumors. Here we demonstrate that in contrast to T cells specific for conventional tumor antigens, TEIPP-directed T cells remain naïve in mice bearing immune-escaped tumors. This unaffected state was caused by low levels of MHC-I on the tumors and the failure to cross-present low levels of antigenic protein by host APCs. Importantly, increased levels of MHC-I, antigen or co-stimulation resulted in potent activation of TEIPP-specific T cells via direct presentation. Genetic knockdown by CRISPR/Cas9 technology of the relevant MHC-I allele in tumor cells indeed abrogated T cell activation. Vaccine-mediated priming of TEIPP-specific T cells induced efficient homing to MHC-Ilow tumors and subsequently protected mice against outgrowth of their MHC-Ilow tumor. Thus, our data open up the search of TEIPP-specific T cells in cancer patients to explore their application against MHC-Ilow tumor cells.

Uveal melanoma progression can be predicted by gene expression profiles enabling a clear subdivision between tumors with a good (class I) and a poor (class II) prognosis. Poor prognosis uveal melanoma can be subdivided by expression of immune-related genes; however, it is unclear whether this subclassification is justified; therefore, T cells in uveal melanoma specimens were quantified using a digital PCR approach. Absolute T-cell quantification revealed that T-cell influx is present in all uveal melanomas associated with a poor prognosis. However, this infiltrate is only accompanied by differential immune-related gene expression profiles in uveal melanoma with the highest T-cell infiltrate. Molecular deconvolution of the immune profile revealed that a large proportion of the T-cell–related gene expression signature does not originate from lymphocytes but is derived from other immune cells, especially macrophages. Expression of the lymphocyte-homing chemokine CXCL10 by activated macrophages correlated with T-cell infiltration and thereby explains the correlation of T-cell numbers and macrophages. This was validated by in situ analysis of CXCL10 in uveal melanoma tissue with high T-cell counts. Surprisingly, CXCL10 or any of the other genes in the activated macrophage-cluster was correlated with reduced survival due to uveal melanoma metastasis. This effect was independent of the T-cell infiltrate, which reveals a role for activated macrophages in metastasis formation independent of their role in tumor inflammation. Implications: The current report uses an innovative digital PCR method to study the immune environment and demonstrates that absolute T-cell quantification and expression profiles can dissect disparate immune components.

e14532 Background: Growth and differentiation factor 15 (GDF-15) is a divergent member of the TGF-β superfamily with low to absent expression in healthy tissue. GDF-15 has been linked to feto-maternal immune tolerance, to prevention of excessive immune cell infiltration during tissue damage, and to anorexia. Various major tumor types secrete high levels of GDF-15. In cancer patients, elevated GDF-15 serum levels correlate with poor prognosis and reduced overall survival (OS). Methods: Impact of a proprietary GDF-15 neutralizing antibody (CTL-002) regarding T cell trafficking was analyzed by whole blood adhesion assays, a HV18-MK melanoma-bearing humanized mouse model and a GDF-15-transgenic MC38 model. Additionally, patient GDF-15 serum levels were correlated with clinical response and overall survival in oropharyngeal squamous cell carcinoma (OPSCC) and melanoma brain metastases. Results: In whole blood cell adhesion assays GDF-15 impairs adhesion of T and NK cells to activated endothelial cells. Neutralization of GDF-15 by CTL-002 rescued T cell adhesion. In HV18-MK-bearing humanized mice CTL-002 induced a strong increase in TIL numbers. Subset analysis revealed an overproportional enrichment of T cells, in particular CD8+ T cells. As immune cell exclusion is detrimental for checkpoint inhibitor (CPI) therapy, a GDF-15-transgenic MC38 model was tested for anti-PD-1 therapy efficacy. In GDF-15 overexpressing MC38 tumors response to anti PD-1 therapy was reduced by 90% compared to wtMC38 tumors. Combining aPD-1 with CTL-002 resulted in 50% of the mice rejecting their GDF-15 overexpressing tumors. Clinically, inverse correlations of GDF-15 levels with CD8+ T cell infiltration were shown for HPV+ OPSCC and for melanoma brain metastases. GDF-15 serum levels were significantly higher in HPV- than in HPV+ OPSCC patient (p < 0.0001). Low GDF-15 levels corresponded to longer OS in both HPV- and HPV+ OPSCC. In two independent melanoma patient cohorts treated with nivolumab or pembrolizumab low baseline serum GDF-15 levels were predictive for clinical response to anti-PD1 treatment and superior OS. Bivariate analysis including LDH indicates that GDF-15 independently predicts poor survival in aPD-1 treated melanoma patients. Conclusions: Taken together our in vitro and in vivo data show that elevated GDF-15 levels block T-cell infiltration into tumor tissues. Neutralizing GDF-15 with CTL-002 restores the ability of T cells to extravasate blood vessels and enter tumor tissue both in vitro and in vivo. In melanoma, patients with higher GDF-15 levels have significantly shorter survival and are less likely to respond to anti-PD1 therapy. GDF-15 may thus serve as a new predictive biomarker for anti-PD1 response, but most importantly also represents a novel target for cancer immunotherapy to improve tumor immune cell infiltration and response to anti-PD1 therapy.

Background: Therapeutic vaccines based on synthetic long peptides (SLPs) have a great potential for immunotherapy of cancer patients as these SLPs include both human leukocyte antigen (HLA) class I and II epitopes and no patient selection for HLA types is required. The antigen-induced immune response can be strengthened with immune stimulating additives. Amplivant (AV) is a synthetic Toll-like receptor 2 ligand which can be directly conjugated to tumor peptide antigens. In preclinical studies, AV-conjugation to antigens led to both enhanced antigen presentation by dendritic cells and T-cell priming and caused superior induction of effective anti-tumor responses. Moreover, AV-conjugated SLPs showed a 100 times higher immune response compared to unconjugated SLP. The current study is a first-in-human trial to investigate safety and immunogenicity of AV-conjugated human papillomavirus (HPV)16-SLPs. Methods: A dose escalation phase I trial was performed in 24 patients with HPV16 positive (pre-) malignant lesions. AV was conjugated to two SLPs derived from the most immunodominant regions of the HPV16 E6 oncoprotein. Four dose groups (1, 5, 20 or 50 μg of each peptide) in 6 patients each were studied. The vaccine was injected three times intradermally in DMSO / water with a three-week interval. Adverse events (AE) were collected according to CTCAE v4.0 up to 26 weeks. Peptide-specific T-cell immune responses were determined in blood samples taken before and after vaccination using complementary immunological assays (proliferation assay, IFNγ-ELISPOT and cytokine bead array). Results: Toxicity after three AV-conjugated HPV16-SLP vaccinations was limited to CTCAE grade 1 or 2, with predominantly inflammation at the vaccination site and sometimes flu-like symptoms, which generally resolved within one day. Dose increase resulted from no AE in the lowest dose group to mild/moderate AE in all vaccinated persons in the highest dose group. In the lowest dose group, minor vaccine-induced T-cell responses were observed in three of six vaccinated persons. In the highest dose group, all patients displayed a strong HPV16-specific T-cell response after vaccination. The induced T-cell response against HPV16 lasted until the end of the trial. Conclusions: This first-in-human study showed that AV conjugated to SLPs can safely be used as an intradermal therapeutic vaccine. AV-conjugated HPV16-SLP was able to induce robust HPV16-specific T-cell immunity in patients treated for HPV16 positive (pre-) malignancies without any other vaccine adjuvant or formulation. Increase in dose resulted in both a higher number of mild adverse events as well as stronger T-cell immunity. Clinical trial information: NCT02821494.

Adoptive transfer of tumor-specific T cells, expanded from tumor-infiltrating lymphocytes or from peripheral blood, is a promising immunotherapeutic approach for the treatment of cancer. Here, we studied whether the tumor-draining lymph nodes (TDLN) of patients with human papillomavirus (HPV)-induced cervical cancer can be used as a source for ACT. The objectives were to isolate lymph node mononuclear cells (LNMC) from TDLN and optimally expand HPV-specific CD4+ and CD8+ T cells under clinical grade conditions. TDLN were isolated from 11 patients with early-stage cervical cancer during radical surgery. Isolated lymphocytes were expanded in the presence of HPV16 E6 and E7 clinical grade synthetic long peptides and IL-2 for 22 days and then analyzed for HPV16 specificity by proliferation assay, multiparameter flow cytometry and cytokine analysis as well as for CD25 and FoxP3 expression. Stimulation of LNMC resulted in expansion of polyclonal HPV-specific T cells in all patients. On average a 36-fold expansion of a CD4+ and/or CD8+ HPV16-specific T cell population was observed, which maintained its capacity for secondary expansion. The T helper type 1 cytokine IFNγ was produced in all cell cultures and in some cases also the Th2 cytokines IL-10 and IL-5. The procedure was highly reproducible, as evidenced by complete repeats of the stimulation procedures under research and under full good manufacturing practice conditions. In conclusion, TDLN represent a rich source of polyclonal HPV16 E6- and E7-specific T cells, which can be expanded under clinical grade conditions for adoptive immunotherapy in patients with cervical cancer. Electronic supplementary material The online version of this article (doi:10.1007/s00262-016-1892-8) contains supplementary material, which is available to authorized users.