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Purpose: Immunotherapy for hepatocellular carcinoma (HCC) shows considerable promise in improving clinical outcomes. HepaVac-101 represents a single-arm, first-in-human phase I/II multicenter cancer vaccine trial for HCC (NCT03203005). It combines multipeptide antigens (IMA970A) with the TLR7/8/RIG I agonist CV8102. IMA970A includes 5 HLA-A*24 and 7 HLA-A*02 as well as 4 HLA-DR restricted peptides selected after mass spectrometric identification in human HCC tissues or cell lines. CV8102 is an RNA-based immunostimulator inducing a balanced Th1/Th2 immune response. Patients and Methods: A total of 82 patients with very early- to intermediate-stage HCCs were enrolled and screened for suitable HLA haplotypes and 22 put on study treatment. This consisted in a single infusion of low-dose cyclophosphamide followed by nine intradermal coadministrations of IMA970A and CV8102. Only patients with no disease relapse after standard-of-care treatments were vaccinated. The primary endpoints of the HepaVac-101 clinical trial were safety, tolerability, and antigen-specific T-cell responses. Secondary or exploratory endpoints included additional immunologic parameters and survival endpoints. Results: The vaccination showed a good safety profile. Transient mild-to-moderate injection-site reactions were the most frequent IMA970A/CV8102-related side effects. Immune responses against ≥1 vaccinated HLA class I tumor-associated peptide (TAA) and ≥1 vaccinated HLA class II TAA were respectively induced in 37% and 53% of the vaccinees. Conclusions: Immunotherapy may provide a great improvement in treatment options for HCC. HepaVac-101 is a first-in-human clinical vaccine trial with multiple novel HLA class I– and class II–restricted TAAs against HCC. The results are initial evidence for the safety and immunogenicity of the vaccine. Further clinical evaluations are warranted.

Patients with glioblastoma currently do not sufficiently benefit from recent breakthroughs in cancer treatment that use checkpoint inhibitors1,2. For treatments using checkpoint inhibitors to be successful, a high mutational load and responses to neoepitopes are thought to be essential3. There is limited intratumoural infiltration of immune cells4 in glioblastoma and these tumours contain only 30–50 non-synonymous mutations5. Exploitation of the full repertoire of tumour antigens—that is, both unmutated antigens and neoepitopes—may offer more effective immunotherapies, especially for tumours with a low mutational load. Here, in the phase I trial GAPVAC-101 of the Glioma Actively Personalized Vaccine Consortium (GAPVAC), we integrated highly individualized vaccinations with both types of tumour antigens into standard care to optimally exploit the limited target space for patients with newly diagnosed glioblastoma. Fifteen patients with glioblastomas positive for human leukocyte antigen (HLA)-A*02:01 or HLA-A*24:02 were treated with a vaccine (APVAC1) derived from a premanufactured library of unmutated antigens followed by treatment with APVAC2, which preferentially targeted neoepitopes. Personalization was based on mutations and analyses of the transcriptomes and immunopeptidomes of the individual tumours. The GAPVAC approach was feasible and vaccines that had poly-ICLC (polyriboinosinic-polyribocytidylic acid-poly-l-lysine carboxymethylcellulose) and granulocyte–macrophage colony-stimulating factor as adjuvants displayed favourable safety and strong immunogenicity. Unmutated APVAC1 antigens elicited sustained responses of central memory CD8+ T cells. APVAC2 induced predominantly CD4+ T cell responses of T helper 1 type against predicted neoepitopes.

Summary Background In a phase 2 study in patients with metastatic renal cell carcinoma, overall survival was associated with T-cell responses against IMA901, a vaccine consisting of ten tumour-associated peptides. In this phase 3 trial, we aimed to determine the clinical effect of adding IMA901 to sunitinib, the standard first-line treatment in metastatic renal cell carcinoma with postulated favourable immunomodulatory effects. Methods The IMPRINT study is an open-label, randomised, controlled, phase 3 trial done at 124 clinical sites in 11 countries. HLA-A*02 -positive patients (aged ≥18 years) with treatment-naive, histologically confirmed metastatic or locally advanced (or both) clear-cell renal cell carcinoma were randomly assigned (3:2) to receive sunitinib plus up to ten intradermal vaccinations of IMA901 (4·13 mg) and granulocyte macrophage colony-stimulating factor (75 μg), with one dose of cyclophosphamide (300 mg/m 2 ) 3 days before the first vaccination, or to receive sunitinib alone. Sunitinib (50 mg) was given orally once daily, with each cycle defined as 4 weeks on treatment followed by 2 weeks off treatment, until progression of disease as determined by the investigator, death, or withdrawal of consent. Block randomisation (block size five) was done centrally using an interactive web response system, stratified by prognostic risk, geographical region, and previous nephrectomy. Patients and investigators were not masked to treatment allocation. The primary endpoint was overall survival from randomisation until death of any cause as determined by the investigator, analysed by intention to treat. This study is registered with ClinicalTrials.gov, number NCT01265901. Findings Between Dec 22, 2010, and Dec 15, 2012, we screened 1171 patients, of whom 339 were randomly assigned to receive sunitinib plus IMA901 (n=204) or sunitinib monotherapy (n=135). Patients had a median follow-up of 33·27 months (IQR 29·92–35·64). Median overall survival did not differ significantly between the groups (33·17 months [95% CI 27·81–41·36] in the sunitinib plus IMA901 group vs not reached [33·67–not reached] in the sunitinib monotherapy group; hazard ratio 1·34 [0·96–1·86]; p=0·087). 116 (57%) of 202 patients in the sunitinib plus IMA901 group and 62 (47%) of 132 in the sunitinib group had grade 3 or worse adverse events, the most common of which were hypertension, neutropenia, and anaemia in both groups, and mild-to-moderate transient injection-site reactions (eg, erythema, pruritus) were the most frequent IMA901-related side-effect in the sunitinib plus IMA901 group. Serious adverse events leading to death occurred in four (2%) patients (one respiratory failure and circulatory collapse [possibly related to sunitinib], one oesophageal varices haemorrhage [possibly related to sunitinib], one cardiac arrest [possibly related to sunitinib], and one myocardial infarction) and eight (6%) patients in the sunitinib group (one case each of renal failure, oesophageal varices haemorrhage, circulatory collapse, wound infection, ileus, cerebrovascular accident [possibly treatment related], and sepsis). Interpretation IMA901 did not improve overall survival when added to sunitinib as first-line treatment in patients with metastatic renal cell carcinoma. The magnitude of immune responses needs to be improved before further development of IMA901 in this disease is indicated. Funding Immatics Biotechnologies.

Purpose: To perform a two-cohort, phase I safety and immunogenicity study of IMA950 in addition to standard chemoradiotherapy and adjuvant temozolomide in patients with newly diagnosed glioblastoma. IMA950 is a novel glioblastoma-specific therapeutic vaccine containing 11 tumor-associated peptides (TUMAP), identified on human leukocyte antigen (HLA) surface receptors in primary human glioblastoma tissue. Experimental Design: Patients were HLA-A*02–positive and had undergone tumor resection. Vaccination comprised 11 intradermal injections with IMA950 plus granulocyte macrophage colony-stimulating factor (GM-CSF) over a 24-week period, beginning 7 to 14 days prior to initiation of chemoradiotherapy (Cohort 1) or 7 days after chemoradiotherapy (Cohort 2). Safety was assessed according to NCI CTCAE Version 4.0 and TUMAP-specific T-cell immune responses determined. Secondary observations included progression-free survival (PFS), pretreatment regulatory T cell (Treg) levels, and the effect of steroids on T-cell responses. Results: Forty-five patients were recruited. Related adverse events included minor injection site reactions, rash, pruritus, fatigue, neutropenia and single cases of allergic reaction, anemia and anaphylaxis. Two patients experienced grade 3 dose-limiting toxicity of fatigue and anaphylaxis. Of 40 evaluable patients, 36 were TUMAP responders and 20 were multi-TUMAP responders, with no important differences between cohorts. No effect of pretreatment Treg levels on IMA950 immunogenicity was observed, and steroids did not affect TUMAP responses. PFS rates were 74% at 6 months and 31% at 9 months. Conclusions: IMA950 plus GM-CSF was well-tolerated with the primary immunogenicity endpoint of observing multi-TUMAP responses in at least 30% of patients exceeded. Further development of IMA950 is encouraged. Clin Cancer Res; 22(19); 4776–85. ©2016 AACR. See related commentary by Lowenstein and Castro, p. 4760

In addition to genomic mutations, RNA editing is another major mechanism creating sequence variations in proteins by introducing nucleotide changes in mRNA sequences. Deregulated RNA editing contributes to different types of human diseases, including cancers. Here we report that peptides generated as a consequence of RNA editing are indeed naturally presented by human leukocyte antigen (HLA) molecules. We provide evidence that effector CD8+ T cells specific for edited peptides derived from cyclin I are present in human tumours and attack tumour cells that are presenting these epitopes. We show that subpopulations of cancer patients have increased peptide levels and that levels of edited RNA correlate with peptide copy numbers. These findings demonstrate that RNA editing extends the classes of HLA presented self-antigens and that these antigens can be recognised by the immune system.

A variety of different mRNA-based drugs are currently in development. This became possible, since major breakthroughs in RNA research during the last decades allowed impressive improvements of translation, stability and delivery of mRNA. This article focuses on antigen-encoding RNA-based vaccines that are either directed against tumors or pathogens. mRNA-encoded vaccines are developed both for preventive or therapeutic purposes. Most mRNA-based vaccines are directly administered to patients. Alternatively, primary autologous cells from cancer patients are modified ex vivo by the use of mRNA and then are adoptively transferred to patients. In the EU no regulatory guidelines presently exist that specifically address mRNA-based vaccines. The existing regulatory framework, however, clearly defines that mRNA-based vaccines in most cases have to be centrally approved. Interestingly, depending on whether RNA-based vaccines are directed against tumors or infectious disease, they are formally considered gene therapy products or not, respectively. Besides an overview on the current clinical use of mRNA vaccines in various therapeutic areas a detailed discussion of the current regulatory situation is provided and regulatory perspectives are discussed.

Every cancer is different and cancer cells differ from normal cells, in particular, through genetic alterations. HLA molecules on the cell surface enable T lymphocytes to recognize cellular alterations as antigens, including mutations, increase in gene product copy numbers or expression of genes usually not used in the adult organism. The search for cancer-associated antigens shared by many patients with a particular cancer has yielded a number of hits used in clinical vaccination trials with indication of survival benefit. Targeting cancer-specific antigens, which are exclusively expressed on cancer cells and not on normal cells, holds the promise for much better results and perhaps even a cure. Such antigens, however, may specifically appear in very few patients or may be mutated appearing just in one patient. Therefore, to target these in a molecularly defined way, the approach has to be individualized.

Non-specific immunotherapy has been for a long time a standard treatment option for patients with metastatic renal cell carcinoma but was redeemed by specific targeted molecular therapies, namely the VEGF and mTOR inhibitors. After moving treatment for mRCC to specific molecular agents with a well-defined mode of action, immunotherapy still needs this further development to increase its accuracy. Nowadays, an evolution from a rather non-specific cytokine treatment to sophisticated targeted approaches in specific immunotherapy led to a re-launch of immunotherapy in clinical studies. Recent steps in the development of immunotherapy strategies are discussed in this review with a special focus on peptide vaccination which aims at a tumor targeting by specific T lymphocytes. In addition, different combinatory strategies with immunomodulating agents like cyclophosphamide or sunitinib are outlined, and the effects of immune checkpoint modulators as anti-CTLA-4 or PD-1 antibodies are discussed. peerReviewed

Peptides presented at the cell surface reflect the protein content of the cell; those on HLA class I molecules comprise the critical peptidome elements interacting with CD8 T lymphocytes. We hypothesize that peptidomes from ex vivo tumour samples encompass immunogenic tumour antigens. Here, we uncover >6000 HLA-bound peptides from HLA-A*02(+) glioblastoma, of which over 3000 were restricted by HLA-A*02. We prioritized in-depth investigation of 10 glioblastoma-associated antigens based on high expression in tumours, very low or absent expression in healthy tissues, implication in gliomagenesis and immunogenicity. Patients with glioblastoma showed no T cell tolerance to these peptides. Moreover, we demonstrated specific lysis of tumour cells by patients' CD8(+) T cells in vitro. In vivo, glioblastoma-specific CD8(+) T cells were present at the tumour site. Overall, our data show the physiological relevance of the peptidome approach and provide a critical advance for designing a rational glioblastoma immunotherapy. The peptides identified in our study are currently being tested as a multipeptide vaccine (IMA950) in patients with glioblastoma.

The tyrosine kinase inhibitors sorafenib and sunitinib are approved for the treatment of patients with malignant diseases. To analyze the possible use of these compounds in combination with immunotherapeutic approaches, we analyzed the effects of both inhibitors on the immunostimulatory capacity of human dendritic cells (DCs) and the induction of primary immune responses in vivo. Sorafenib, but not sunitinib, inhibits function of DCs, characterized by reduced secretion of cytokines and expression of CD1a, major histocompatibility complex, and costimulatory molecules in response to TLR ligands as well as by their impaired ability to migrate and stimulate T-cell responses. These inhibitory effects are mediated by inhibition of PI3 and MAP kinases and NFκB signaling. In contrast, sorafenib had no influence on the phenotype and proliferation of T cells. To analyze the effects of both TKIs on cytotoxic T-cell induction in vivo, C57BL/6 mice were pretreated with sorafenib or sunitinib and immunized with OVA257-264 peptide. Sorafenib, but not sunitinib, application significantly reduced the induction of antigen-specific T cells. Numbers of regulatory T cells were reduced in peripheral blood mononuclear cells from mice treated with sunitinib. These results indicate that sunitinib, but not sorafenib, is suitable for combination with immunotherapeutic approaches for treatment of cancer patients.

Human cytomegalovirus (HCMV) has evolved a multitude of molecular mechanisms to evade the antiviral immune defense of the host. Recently, using soluble recombinant molecules, the HCMV UL16 glycoprotein was shown to interact with some ligands of the activating immunoreceptor NKG2D and, therefore, may also function as a viral immunomodulator. However, the role of UL16 during the course of HCMV infection remained unclear. Here, we demonstrate that HCMV infection of fibroblasts induces expression of all known NKG2D ligands (NKG2DL). However, solely MICA and ULBP3 reach the cellular surface to engage NKG2D, whereas MICB, ULBP1 and ULBP2 are selectively retained in the endoplasmic reticulum by UL16. UL16-mediated reduction of NKG2DL cell surface density diminished NK cytotoxicity. Thus, UL16 functions by capturing activating ligands for cytotoxic lymphocytes that are synthesized in response to HCMV infection.

Heat shock proteins (HSPs) like glycoprotein (gp)96 (glucose-regulated protein 94 [grp94]) are able to induce specific cytotoxic T lymphocyte (CTL) responses against cells from which they originate. Here, we demonstrate that for CTL activation by gp96-chaperoned peptides, specific receptor-mediated uptake of gp96 by antigen-presenting cells (APCs) is required. Moreover, we show that in both humans and mice, only professional APCs like dendritic cells (DCs), macrophages, and B cells, but not T cells, are able to bind gp96. The binding is saturable and can be inhibited using unlabeled gp96 molecules. Receptor binding by APCs leads to a rapid internalization of gp96, which colocalizes with endocytosed major histocompatibility complex (MHC) class I and class II molecules in endosomal compartments. Incubation of gp96 molecules isolated from cells expressing an adenovirus type 5 E1B epitope with the DC line D1 results in the activation of E1B-specific CTLs. This CTL activation can be specifically inhibited by the addition of irrelevant gp96 molecules not associated with E1B peptides. Our results demonstrate that only receptor-mediated endocytosis of gp96 molecules leads to MHC class I–restricted re-presentation of gp96-associated peptides and CTL activation; non–receptor-mediated, nonspecific endocytosis is not able to do so. Thus, we provide evidence on the mechanisms by which gp96 is participating in the cross-presentation of antigens from cellular origin.

IMA901 is the first therapeutic vaccine for renal cell cancer (RCC) consisting of multiple tumor-associated peptides (TUMAPs) confirmed to be naturally presented in human cancer tissue. We treated a total of 96 human leukocyte antigen A (HLA-A)*02(+) subjects with advanced RCC with IMA901 in two consecutive studies. In the phase 1 study, the T cell responses of the patients to multiple TUMAPs were associated with better disease control and lower numbers of prevaccine forkhead box P3 (FOXP3)(+) regulatory T (T(reg)) cells. The randomized phase 2 trial showed that a single dose of cyclophosphamide reduced the number of T(reg) cells and confirmed that immune responses to multiple TUMAPs were associated with longer overall survival. Furthermore, among six predefined populations of myeloid-derived suppressor cells, two were prognostic for overall survival, and among over 300 serum biomarkers, we identified apolipoprotein A-I (APOA1) and chemokine (C-C motif) ligand 17 (CCL17) as being predictive for both immune response to IMA901 and overall survival. A randomized phase 3 study to determine the clinical benefit of treatment with IMA901 is ongoing.

Dendritic cells (DC) play a key role in regulating immune responses and are the best professional antigen-presenting cells. Two major DC populations are defined in part according to cell surface CD11c expression levels. Unexpectedly, we observed that mouse DC strongly down-regulate the typical DC marker CD11c upon activation. To better characterize DC responses, we have analyzed CD11c expression on mouse and human myeloid DC after Toll-like receptor (TLR) triggering. Here we show that mouse bone marrow-derived DC (BMDC) as well as spleen DC down-regulate cell surface CD11c upon activation by TLR3/4/9 agonists. In all cases, full DC activation was reached, as determined by cytokine secretion, cell stimulation in mixed leukocyte reactions (MLR), and CD40/CD86/major histocompatibility complex (MHC) up-regulation. Interestingly, membrane CD11c down-regulation correlated with increased cytoplasmic pools of CD11c. In contrast to the up-regulation of CD40 and MHC class II molecules, lipopolysaccharide (LPS)-induced CD11c down-regulation was MyD88-dependent. Polyinosinic-polycytidylic acid (poly I:C), which does not signal through MyD88, also induced cell surface CD11c down-regulation. Notably, CD11c down-regulation was not observed upon activation of human DC, either through TLR-dependent or -independent cell activation. Thus, activated mouse DC may be transiently CD11c-negative in vivo, hampering the identification of those cells. On the other hand, cell surface CD11c down-regulation may serve as a new activation marker for mouse DC.

Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer. © 2011 Fox et al; licensee BioMed Central Ltd.

Heat shock proteins (Hsps) are able to induce protective immune responses against pathogens and tumors after injection into immunocompetent hosts. The activation of components of the adaptive immune system, including cytotoxic T lymphocytes specific for pathogen- or tumor-derived peptides, is crucial for the establishment of immuno- protection. Hsps acquire these peptides during intracellular protein degradation and when released during necrotic cell death, facilitate their uptake and Minor Histocompatibility Complex (MHC)-restricted representation by professional antigen-presenting cells (APCs). In addition, the interaction of Hsps with APCs, including the Endoplasmatic Reticulum (ER)-resident chaperone glycoprotein 96 (Gp96), induces the maturation of these cells by Toll-like receptor (TLR)- mediated signaling events. We now provide evidence that in contrast to lipopolysaccharides (LPS)-mediated dendritic cell (DC) maturation, the interaction of Gp96 with DCs leads to the preferential expansion of antigen-specific CD8- positive T cells in vitro and in vivo. This CD8 preference induced by mouse and human DCs did not correlate with enhanced levels of interleukin-12 secretion. Thus, despite the fact that both LPS and Gp96 activate DCs in a TLR4- dependent manner, the experiments of this study clearly demonstrate qualitative differences in the outcome of this maturation process, which preferentially favors the expansion of CD8-positive T cells.

Nucleosome is the major autoantigen in systemic lupus erythematosus. It is found as a circulating complex in the sera of patients and seems to play a key role in disease development. In this study, we show for the first time that physiologic concentrations of purified nucleosomes directly induce in vitro dendritic cell (DC) maturation of mouse bone marrow-derived DC, human monocyte-derived DC (MDDC), and purified human myeloid DC as observed by stimulation of allogenic cells in MLR, cytokine secretion, and CD86 up-regulation. Importantly, nucleosomes act as free complexes without the need for immune complex formation or for the presence of unmethylated CpG DNA motifs, and we thus identified a new mechanism of DC activation by nucleosomes. We have clearly demonstrated that this activation is nucleosome-specific and endotoxin-independent. Particularly, nucleosomes induce MDDC to secrete cytokines known to be detected in high concentrations in the sera of patients. Moreover, activated MDDC secrete IL-8, a neutrophil chemoattractant also detected in patient sera, and thus might favor the inflammation observed in patients. Both normal and lupus MDDC are sensitive to nucleosome-induced activation. Finally, injection of purified nucleosomes to normal mice induces in vivo DC maturation. Altogether, these results strengthen the key role of nucleosomes in systemic lupus erythematosus and might explain how peripheral tolerance is broken in patients.

There is substantial need for molecularly defined tumor antigens to prime cytotoxic T cells in vivo for cancer immunotherapy, especially in the case of tumor entities for which only a few tumor antigens have been defined so far. In this review, we present the “Tubingen approach” to identify, select, and validate large numbers of MHC/HLA class I–associated peptides derived from tumor-associated antigens. Step 1 is the identification of naturally presented HLA-associated peptides directly from primary tumor cells. Step 2 is selection of tumor-associated peptides from step 1 by differential gene expression analysis and data mining. Step 3 is validation of selected candidates by monitoring in vivo T-cell responses in the context of patient-individualized immunizations. Our approach combines methods from genomics, proteomics, bioinformatics, and T-cell immunology. The aim is to develop effective immunotherapeutics consisting of multiple tumor-associated epitopes in order to induce a broad and specific immune response against cancer cells.

The endoplasmic reticulum (ER)–resident heat shock protein Gp96 is involved in protein folding and is released into the extracellular space after necrotic cell death. In this context, Gp96 has immunostimulatory properties: it activates dendritic cells or macrophages and delivers associated peptides into the antigen presentation pathway, resulting in the induction of specific T-cell responses. The inflammatory response after necrotic tissue damage leads to the recruitment of polymorphonuclear neutrophils (PMNs) and monocytes, allowing them to make their first encounter with Gp96. We therefore investigated whether PMNs and monocytes interact with Gp96. We were able to show that PMNs and monocytes specifically bind fluorescein isothiocyanate (FITC)–conjugated Gp96. The binding of Gp96-FITC was competed by lipopolysaccharide (LPS) or fucoidan, a known inhibitor of scavenger receptors. Interestingly, the binding of LPS-FITC was also competed not only by fucoidan, but by Gp96, suggesting that LPS and Gp96 share a common receptor on PMNs. One important effector function of PMNs is the clearance of an inflammatory site by phagocytosis. We therefore assessed the influence of Gp96 on phagocytic activity using fluorochrome-labeled polystyrene beads. We found a marked enhancement of phagocytosis in the presence of Gp96 and concluded that PMNs not only bind Gp96, but are also activated by it. Additionally, Gp96-stimulated PMNs and especially monocytes release large amounts of interleukin-8, a potent neutrophil-attracting chemokine. In conclusion, we demonstrate that Gp96 specifically binds to and activates PMNs and monocytes, extending the function of Gp96 as a danger signal to additional members of the innate immune system.

Immunizations using the endoplasmic reticulum–resident heat shock protein Gp96 induce specific immune responses. Specificity is based on the major histocompatibility complex class I–restricted cross-presentation of Gp96-associated peptides derived from endogenous proteins. Initiation of the immune response depends on the ability of Gp96 to induce the production of proinflammatory cytokines by macrophages and dendritic cells (DCs) and of their maturation in a fashion presumably independent of associated peptide. Both events are mediated by Gp96 receptors on antigen-presenting cells. It is known that Gp96 is released from cells at necrosis induced, for example, by virus infection. Although this event supports the efficient induction of immune responses, it might also interfere with processes that are susceptible to chronic inflammation, such as wound healing after tissue damage. Therefore, Gp96-mediated stimulation of the immune system requires tight regulation. Here we show that human thrombocytes specifically interact with Gp96 and that binding of Gp96 to platelets is enhanced more than 10-fold on activation by thrombin. Gp96 interferes with neither thrombin-induced platelet activation nor platelet aggregation. However, the presence of platelets during Gp96-mediated DC activation reduces the secretion of proinflammatory cytokines and the activation of DCs. This effect is independent of soluble platelet factors and cell-to-cell contact between DCs and thrombocytes. Thus, we provide evidence for a regulatory mechanism that neutralizes Gp96 molecules systemically, especially in the blood. This effect might be of significance in wounds in which chronic inflammation and immune responses against autoantigens have to be prevented.

The heat shock protein Gp96 has been shown to induce specific immune responses. On one hand, this phenomenon is based on the specific interaction with CD91 that mediates endocytosis and results in major histocompatibility complex class I-restricted representation of the Gp96-associated peptides. On the other hand, Gp96 induces activation of professional antigen-presenting cells, resulting in the production of pro-inflammatory cytokines and up-regulation of costimulatory molecules by unknown mechanisms. In this study, we have analyzed the consequences of Gp96 interaction with cells expressing different Toll-like receptors (TLRs) and with bone marrow-derived dendritic cells from mice lacking functional TLR2 and/or TLR4 molecules. We find that the Gp96-TLR2/4 interaction results in activation of nuclear factor kappaB-driven reporter genes and mitogen- and stress-activated protein kinases and induces IkappaBalpha degradation. Bone marrow-derived dendritic cells of C3H/HeJ and more pronounced C3H/HeJ/TLR2(-/-) mice fail to respond to Gp96. Interestingly, activation of bone marrow-derived dendritic cells depends on endocytosis of Gp96 molecules. Our results provide, for the first time, the molecular basis for understanding the Gp96-mediated activation of antigen-presenting cells by describing the simultaneous stimulation of the innate and adaptive immune system. This feature explains the remarkable ability of Gp96 to induce specific immune responses against tumors and pathogens.

Heat shock proteins (HSPs) have been described as potent tumor vaccines in animal models and are currently studied in clinical trials. The underlying immune response relies on immunogenic peptides that the HSPs have acquired intracellularly by interfering with the classical antigen processing pathways. There have been numerous reports shedding light on how HSPs are able to gain this function and a number of important requirements for HSP-mediated specific immunity have been described: first, the ability of HSPs to bind immunogenic peptides. Second, the acquisition of HSPs by specialized antigen presenting cells with efficient antigen processing pathways capable of inducing cellular immune responses. Third, the existence of specific receptors on the surfaces of antigen presenting cells, allowing efficient and rapid uptake of HSP-peptide complexes from the extracellular fluid. And fourth, the ability of heat shock proteins to activate antigen presenting cells, enabling the latter to prime cytotoxic T cell responses against the peptides associated to HSPs.

Heat shock proteins like gp96 (grp94) are able to induce specific cytotoxic T-cell (CTL) responses against cells from which they originate and are currently studied in clinical trials for use in immunotherapy of tumors. We have recently demonstrated that gp96 binds to at least one yet unidentified receptor restricted to antigen-presenting cells (APCs) like dendritic cells (DCs) but not to T cells. Moreover we have shown, that for CTL activation by gp96-chaperoned peptides receptor-mediated uptake of gp96 by APCs is required. Lately, we have discovered a second function of gp96 when interacting with professional APCs. Gp96 is able to mediate maturation of DCs as determined by upregulation of MHC class II, CD86 and CD83 molecules, secretion of pro-inflammatory cytokines IL-12 and TNF-alpha and enhanced T-cell simulatory capacity. Furthermore, the gp96 receptor(s) are down-regulated on mature DCs, suggesting that the gp96 receptor(s) behave similar to other endocytic receptors like CD36, mannose receptor etc. Our findings now provide additional evidence for the remarkable immunogenicity of gp96: first, the existence of specific gp96 receptors on APCs and second, the capacity to activate dendritic cells which is strictly required to enable these highly sophisticated APCs to prime CTL responses.

Background Therapeutic vaccination directed to induce an anti-tumoral T-cell response is a field of extensive investigation in the treatment of melanoma. However, many vaccination trials in melanoma failed to demonstrate a correlation between the vaccine-specific immune response and therapy outcome. This has been mainly attributed to immune escape by antigen loss, rendering us in the need of new vaccination targets. Patients and methods This phase-II trial investigated a peptide vaccination against survivin, an oncogenic inhibitor-of-apoptosis protein crucial for the survival of tumor cells, in HLA-A1/-A2/-B35-positive patients with treatment-refractory stage-IV metastatic melanoma. The study endpoints were survivin-specific T-cell reactivity (SSTR), safety, response, and survival (OS). Results Sixty-one patients (ITT) received vaccination therapy using three different regimens. 55 patients (PP) were evaluable for response and survival, and 41/55 for SSTR. Patients achieving progression arrest (CR + PR + SD) more often showed SSTRs than patients with disease progression (p = 0.0008). Patients presenting SSTRs revealed a prolonged OS (median 19.6 vs. 8.6 months; p = 0.0077); multivariate analysis demonstrated SSTR as an independent predictor of survival (p = 0.013). The induction of SSTRs was associated with gender (female vs. male; p = 0.014) and disease stage (M1a/b vs. M1c; p = 0.010), but not with patient age, HLA type, performance status, or vaccination regimen. Conclusion Survivin-specific T-cell reactivities strongly correlate with tumor response and patient survival, indicating that vaccination with survivin-derived peptides is a promising treatment strategy in melanoma. Electronic supplementary material The online version of this article (doi:10.1007/s00262-012-1266-9) contains supplementary material, which is available to authorized users.

International audience; Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the 'Immunoscore' into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).