Blood immune cells as potential biomarkers predicting relapse-free survival of stage III/IV resected melanoma patients treated with peptide-based vaccination and interferon-alpha.

Introduction: Despite the recent approval of several therapies in the adjuvant setting of melanoma, tumor relapse still occurs in a significant number of completely resected stage III-IV patients. In this context, the use of cancer vaccines is still relevant and may increase the response to immune checkpoint inhibitors. We previously demonstrated safety, immunogenicity and preliminary evidence of clinical efficacy in stage III/IV resected melanoma patients subjected to a combination therapy based on peptide vaccination together with intermittent low-dose interferon-α2b, with or without dacarbazine preconditioning (https://www.clinicaltrialsregister.eu/ctr-search/search, identifier: 2008-008211-26). In this setting, we then focused on pre-treatment patient immune status to highlight possible factors associated with clinical outcome. Methods: Multiparametric flow cytometry was used to identify baseline immune profiles in patients' peripheral blood mononuclear cells and correlation with the patient clinical outcome. Receiver operating characteristic curve, Kaplan-Meier survival and principal component analyses were used to evaluate the predictive power of the identified markers. Results: We identified 12 different circulating T and NK cell subsets with significant (p ≤ 0.05) differential baseline levels in patients who later relapsed with respect to patients who remained free of disease. All 12 parameters showed a good prognostic accuracy (AUC>0.7, p ≤ 0.05) and 11 of them significantly predicted the relapse-free survival. Remarkably, 3 classifiers also predicted the overall survival. Focusing on immune cell subsets that can be analyzed through simple surface staining, three subsets were identified, namely regulatory T cells, CD56dimCD16- NK cells and central memory γδ T cells. Each subset showed an AUC>0.8 and principal component analysis significantly grouped relapsing and non-relapsing patients (p=0.034). These three subsets were used to calculate a combination score that was able to perfectly distinguish relapsing and non-relapsing patients (AUC=1; p=0). Noticeably, patients with a combined score ≥2 demonstrated a strong advantage in both relapse-free (p=0.002) and overall (p=0.011) survival as compared to patients with a score <2. Discussion: Predictive markers may be used to guide patient selection for personalized therapies and/or improve follow-up strategies. This study provides preliminary evidence on the identification of peripheral blood immune biomarkers potentially capable of predicting the clinical response to combined vaccine-based adjuvant therapies in melanoma.

Type I interferons induce peripheral T regulatory cell differentiation under tolerogenic conditions.

The type I interferons are central to a vast array of immunological functions. The production of these immune-modulatory molecules is initiated at the early stages of the innate immune responses and, therefore, plays a dominant role in shaping downstream events in both innate and adaptive immunity. Indeed, the major role of IFN-α/ß is the induction of priming states, relevant for the functional differentiation of T lymphocyte subsets. Among T-cell subtypes, the CD4+CD25+Foxp3+ T regulatory cells (Tregs) represent a specialized subset of CD4+ T cells with a critical role in maintaining peripheral tolerance and immune homeostasis. Although the role of type I interferons in maintaining the function of thymus-derived Tregs has been previously described, the direct contribution of these innate factors to peripheral Treg (pTreg) and induced Treg (iTreg) differentiation and suppressive function is still unclear. We now show that, under tolerogenic conditions, IFN-α/ß play a critical role in antigen-specific and also polyclonal naive CD4+ T-cell conversion into peripheral antigen-specific CD4+CD25+Foxp3+ Tregs and inhibit CD4+ T helper (Th) cell expansion in mice. While type I interferons sustain the expression and the activation of the transcription master regulators Foxp3, Stat3 and Stat5, these innate molecules reciprocally inhibit Th17 cell differentiation. Altogether, these results indicate a new pivotal role of IFN-α/ß on pTreg differentiation and induction of peripheral tolerance, which may have important implications in the therapeutic control of inflammatory disorders, such as of autoimmune diseases.

Nicotinamide inhibits melanoma in vitro and in vivo.

BACKGROUND: Even though new therapies are available against melanoma, novel approaches are needed to overcome resistance and high-toxicity issues. In the present study the anti-melanoma activity of Nicotinamide (NAM), the amide form of Niacin, was assessed in vitro and in vivo. METHODS: Human (A375, SK-MEL-28) and mouse (B16-F10) melanoma cell lines were used for in vitro investigations. Viability, cell-death, cell-cycle distribution, apoptosis, Nicotinamide Adenine Dinucleotide+ (NAD+), Adenosine Triphosphate (ATP), and Reactive Oxygen Species (ROS) levels were measured after NAM treatment. NAM anti-SIRT2 activity was tested in vitro; SIRT2 expression level was investigated by in silico transcriptomic analyses. Melanoma growth in vivo was measured in thirty-five C57BL/6 mice injected subcutaneously with B16-F10 melanoma cells and treated intraperitoneally with NAM. Interferon (IFN)-γ-secreting murine cells were counted with ELISPOT assay. Cytokine/chemokine plasmatic levels were measured by xMAP technology. Niacin receptors expression in human melanoma samples was also investigated by in silico transcriptomic analyses. RESULTS: NAM reduced up to 90% melanoma cell number and induced: i) accumulation in G1-phase (40% increase), ii) reduction in S- and G2-phase (about 50% decrease), iii) a 10-fold increase of cell-death and 2.5-fold increase of apoptosis in sub-G1 phase, iv) a significant increase of NAD+, ATP, and ROS levels, v) a strong inhibition of SIRT2 activity in vitro. NAM significantly delayed tumor growth in vivo (p ≤ 0.0005) and improved survival of melanoma-bearing mice (p ≤ 0.0001). About 3-fold increase (p ≤ 0.05) of Interferon-gamma (IFN-γ) producing cells was observed in NAM treated mice. The plasmatic expression levels of 6 cytokines (namely: Interleukin 5 (IL-5), Eotaxin, Interleukin 12 (p40) (IL12(p40)), Interleukin 3 (IL-3), Interleukin 10 (IL-10) and Regulated on Activation Normal T Expressed and Secreted (RANTES) were significantly changed in the blood of NAM treated mice, suggesting a key role of the immune response. The observed inhibitory effect of NAM on SIRT2 enzymatic activity confirmed previous evidence; we show here that SIRT2 expression is significantly increased in melanoma and inversely related to melanoma-patients survival. Finally, we show for the first time that the expression levels of Niacin receptors HCAR2 and HCAR3 is almost abolished in human melanoma samples. CONCLUSION: NAM shows a relevant anti-melanoma activity in vitro and in vivo and is a suitable candidate for further clinical investigations.

Tumor-Intrinsic or Drug-Induced Immunogenicity Dictates the Therapeutic Success of the PD1/PDL Axis Blockade.

Immunotherapy with immune checkpoint inhibitors (ICIs) has revolutionized cancer treatment providing unprecedented clinical benefits. However, many patients do not respond to ICIs as monotherapy or develop resistance. Combining ICI-based immunotherapy with chemotherapy is a promising strategy to increase response rates, but few rationale-driven chemo-immunotherapy combinations have reached the clinical arena thus far. In the present study, we show that combined anti-PDL1 and anti-PDL2 antibodies optimally synergize with cyclophosphamide but not with cisplatin, and that the magnitude and duration of the therapeutic response is dependent on the immunogenic potential of the drug and of the tumor itself. Hallmarks of successful therapeutic outcomes were the enhanced infiltration by myeloid (mainly cross-presenting dendritic cells, eosinophils, and monocytic myeloid cells) and T lymphocytes into the tumor tissue and the expansion of circulating memory pools. Overall, our results suggest that immunomodulating chemotherapy can be exploited to increase the efficacy of PD1/PDL axis inhibitors in vivo, and that the magnitude of the synergic therapeutic response is affected by tumor-intrinsic immunogenicity.

Clinical and Immunological Outcomes in High-Risk Resected Melanoma Patients Receiving Peptide-Based Vaccination and Interferon Alpha, With or Without Dacarbazine Preconditioning: A Phase II Study.

Clinical studies based on novel rationales and mechanisms of action of chemotherapy agents and cytokines can contribute to the development of new concepts and strategies of antitumor combination therapies. In previous studies, we investigated the paradoxical immunostimulating effects of some chemotherapeutics and the immunoadjuvant activity of interferon alpha (IFN-α) in preclinical and clinical models, thus unraveling novel rationales and mechanisms of action of chemotherapy agents and cytokines for cancer immunotherapy. Here, we carried out a randomized, phase II clinical trial, in which we analyzed the relapse-free (RFS) and overall survival (OS) of 34 completely resected stage III-IV melanoma patients, treated with peptide-based vaccination (Melan-A/MART-1 and NY-ESO-1) in combination with IFN-α2b, with (arm 2) or without (arm 1) dacarbazine preconditioning. All patients were included in the intention-to-treat analysis. At a median follow-up of 4.5 years (interquartile range, 15.4-81.0 months), the rates of RFS were 52.9 and 35.3% in arms 1 and 2, respectively. The 4.5-year OS rates were 68.8% in arm 1 and 62.7% in arm 2. No significant differences were observed between the two arms for both RFS and OS. Interestingly, the RFS and OS curves remained stable starting from 18 and 42 months, respectively. Grade 3 adverse events occurred in 5.9% of patients, whereas grade 4 events were not observed. Both treatments induced a significant expansion of vaccine-specific CD8+ T cells, with no correlation with the clinical outcome. However, treatment-induced increase of polyfunctionality and of interleukin 2 production by Melan-A-specific CD8+ T cells and expansion/activation of natural killer cells correlated with RFS, being observed only in nonrelapsing patients. Despite the recent availability of different therapeutic options, low-cost, low-toxic therapies with long-lasting clinical effects are still needed in patients with high-risk resected stage III/IV melanoma. The combination of peptide vaccination with IFN-α2b showed a minimal toxicity profile and resulted in encouraging RFS and OS rates, justifying further evaluation in clinical trials, which may include the use of checkpoint inhibitors to further expand the antitumor immune response and the clinical outcome. Clinical Trial Registration: https://www.clinicaltrialsregister.eu/ctr-search/search, identifier: 2008-008211-26.

Antigen-specificity and DTIC before peptide-vaccination differently shape immune-checkpoint expression pattern, anti-tumor functionality and TCR repertoire in melanoma patients.

We have recently described that DNA-damage inducing drug DTIC, administered before peptide (Melan-A and gp100)-vaccination, improves anti-tumor CD8+ Melan-A-specific T-cell functionality, enlarges the Melan-A+ TCR repertoire and impacts the overall survival of melanoma patients. To identify whether the two Ags employed in the vaccination differently shape the anti-tumor response, herein we have carried out a detailed analysis of phenotype, anti-tumor functionality and TCR repertoire in treatment-driven gp100-specific CD8+ T cells, in the same patients previously analyzed for Melan-A. We found that T-cell clones isolated from patients treated with vaccination alone possessed an Early/intermediate differentiated phenotype, whereas T cells isolated after DTIC plus vaccination were late-differentiated. Sequencing analysis of the TCRBV chains of 29 treatment-driven gp100-specific CD8+ T-cell clones revealed an oligoclonal TCR repertoire irrespective of the treatment schedule. The high anti-tumor activity observed in T cells isolated after chemo-immunotherapy was associated with low PD-1 expression. Differently, T-cell clones isolated after peptide-vaccination alone expressed a high level of PD-1, along with LAG-3 and TIM-3, and were neither tumor-reactive nor polyfunctional. Blockade of PD-1 reversed gp100-specific CD8+ T-cell dysfunctionality, confirming the direct role of this co-inhibitory molecule in suppressing anti-tumor activity, differently from what we have previously observed for Melan-A+CD8+ T cells, expressing PD-1 but highly functional. These findings indicate that the functional advantage induced by combined chemo-immunotherapy is determined by the tumor antigen nature, T-cell immune-checkpoints phenotype, TCR repertoire diversity and anti-tumor T-cell quality and highlights the importance of integrating these parameters to develop effective immunotherapeutic strategies.

Disruption of IFN-I Signaling Promotes HER2/Neu Tumor Progression and Breast Cancer Stem Cells.

Type I interferon (IFN-I) is a class of antiviral immunomodulatory cytokines involved in many stages of tumor initiation and progression. IFN-I acts directly on tumor cells to inhibit cell growth and indirectly by activating immune cells to mount antitumor responses. To understand the role of endogenous IFN-I in spontaneous, oncogene-driven carcinogenesis, we characterized tumors arising in HER2/neu transgenic (neuT) mice carrying a nonfunctional mutation in the IFNI receptor (IFNAR1). Such mice are unresponsive to this family of cytokines. Compared with parental neu+/- mice (neuT mice), IFNAR1-/- neu+/- mice (IFNAR-neuT mice) showed earlier onset and increased tumor multiplicity with marked vascularization. IFNAR-neuT tumors exhibited deregulation of genes having adverse prognostic value in breast cancer patients, including the breast cancer stem cell (BCSC) marker aldehyde dehydrogenase-1A1 (ALDH1A1). An increased number of BCSCs were observed in IFNAR-neuT tumors, as assessed by ALDH1A1 enzymatic activity, clonogenic assay, and tumorigenic capacity. In vitro exposure of neuT+ mammospheres and cell lines to antibodies to IFN-I resulted in increased frequency of ALDH+ cells, suggesting that IFN-I controls stemness in tumor cells. Altogether, these results reveal a role of IFN-I in neuT-driven spontaneous carcinogenesis through intrinsic control of BCSCs. Cancer Immunol Res; 6(6); 658-70. ©2018 AACR.

Engineered exosomes emerging from muscle cells break immune tolerance to HER2 in transgenic mice and induce antigen-specific CTLs upon challenge by human dendritic cells.

We recently described a novel biotechnological platform for the production of unrestricted cytotoxic T lymphocyte (CTL) vaccines. It relies on in vivo engineering of exosomes, i.e., nanovesicles constitutively released by all cells, with full-length antigens of choice upon fusion with an exosome-anchoring protein referred to as Nefmut. They are produced upon intramuscular injection of a DNA vector and, when uploaded with a viral tumor antigen, were found to elicit an immune response inhibiting the tumor growth in a model of transplantable tumors. However, for a possible application in cancer immunotherapy, a number of key issues remained unmet. Among these, we investigated: (i) whether the immunogenic stimulus induced by the engineered exosomes can break immune tolerance, and (ii) their effectiveness when applied in human system. As a model of immune tolerance, we considered mice transgenic for the expression of activated rat HER2/neu which spontaneously develop adenocarcinomas in all mammary glands. When these mice were injected with a DNA vector expressing the product of fusion between Nefmut and the extracellular domain of HER2/neu, antigen-specific CD8+ T lymphocytes became readily detectable. This immune response associated with a HER2-directed CTL activity and a significant delay in tumor development. On the other hand, through cross-priming experiments, we demonstrated the effectiveness of the engineered exosomes emerging from transfected human primary muscle cells in inducing antigen-specific CTLs. We propose our CTL vaccine platform as part of new immunotherapy strategies against tumors expressing self-antigens, i.e., products highly expressed in oncologic lesions but tolerated by the immune system. KEY MESSAGES: We established a novel, exosome-based method to produce unrestricted CTL vaccines. This strategy is effective in breaking the tolerance towards tumor self-antigens. Our method is also useful to elicit antigen-specific CTL immunity in humans. These findings open the way towards the use of this antitumor strategy in clinic.

Role of interferon regulatory factor 1 in governing Treg depletion, Th1 polarization, inflammasome activation and antitumor efficacy of cyclophosphamide.

The antitumor effectiveness of cyclophosphamide (CTX) and other chemotherapeutics was shown to rely not only on direct cytotoxicity but also on immunogenic tumor cell death and systemic immunomodulatory mechanisms, including regulatory T cell (Treg) depletion, Th1 cell polarization, type I interferon (IFN) and proinflammatory cytokine production. IFN regulatory factor (IRF)-1 is a transcriptional regulator of IFNs and IFN-inducible genes, involved in the control of Th1 and Treg differentiation and in sterile inflammation. Aim of this study was to explore the role of IRF-1 in CTX-induced antitumor effects and related immune activities. This study shows for the first time that IRF-1 is important for the antitumor efficacy of CTX in mice. Moreover, experiments in tumor-bearing C57BL/6 mice showed that Irf1 gene expression in the spleen was transiently increased following CTX administration and correlated with the induction of Th1 cell expansion and of Il12p40 gene expression, which is the main Th1-driving cytokine. At the same time, CTX administration reduced both Foxp3 expression and Treg cell percentages. These effects were abrogated in Irf1-/- mice. Further experiments showed that the gene and/or protein expression of caspase-1, iNOS, IL-1ß, IL-6 and CXCL10 and the levels of nitric oxide were modulated following CTX in an IRF-1-direct- or -indirect-dependent manner, and highlighted the importance of caspase-1 in driving the sterile inflammatory response to CTX. Our data identify IRF-1 as important for the antitumor efficacy of CTX and for the regulation of many immunomodulatory activities of CTX, such as Th1 polarization, Treg depletion and inflammation.

The added value of type I interferons to cytotoxic treatments of cancer.

Type I interferons (IFNs) exert anti-proliferative, antiviral and immunomodulatory activities. They are also involved in cell differentiation and anti-tumor defense processes. A growing body of literature indicates that the success of conventional chemotherapeutics, epigenetic drugs, targeted anticancer agents and radiotherapy (RT) relies, at least in part, on the induction of type I IFN signaling in malignant cells, tumor-infiltrating antigen presenting cells or other immune cells within lymphoid organs or blood. The mechanisms underlying type I IFN induction and the clinical consequences of these observations are only beginning to be elucidated. In the present manuscript, we reviewed the recent advances in the field and provided our personal view on the role of type I IFNs induced in the context of cytotoxic anticancer treatments and on its possible exploitation as a complement in cancer therapy.

Goals and objectives of the Italian Network for Tumor Biotherapy (NIBIT).

The explosion in the immuno-oncology field, exemplified by the clinical implementation of immune checkpoint inhibitor blockade and other immunotherapeutic strategies was quickly recognized by the Italian biomedical community, thanks to the networking activities of the Italian Network for Tumor Biotherapy (NIBIT), which has been active since 2004 in the diffusion of new scientific and clinical findings in the fields of tumor immunology and immunotherapy. Numerous activities of NIBIT have also helped to overcome the hurdles associated with the clinical implementation of cancer immune-biotherapeutic strategies at the national and international levels. Looking forward, a concerted interaction of NIBIT with existing European networks focused on cancer bio-immunotherapy will further contribute to the development of improved therapies in the immuno-oncology field. This Introduction briefly summarizes the history and objectives of NIBIT, as well as the current activities of the Network.

Chemo-immunotherapy induces tumor regression in a mouse model of spontaneous mammary carcinogenesis.

Tumor-specific immune tolerance represents an obstacle for the development of effective anti-tumor immune responses through cancer vaccines. We here evaluated the efficacy of chemo-immunotherapy in breaking tumor-specific immune tolerance in an almost incurable mouse model of spontaneous carcinogenesis.Transgenic HER-2/neu mice bearing large mammary tumors received the adoptive transfer of splenocytes and serum isolated from immune donors, with or without pre-conditioning with cyclophosphamide. Treatment efficacy was assessed by monitoring tumor growth by manual inspection and by magnetic resonance imaging. The same chemo-immunotherapy protocol was tested on tumor-free HER-2/neu mice, to evaluate the effects on tumor emergence.Our data show that chemo-immunotherapy hampered carcinogenesis and caused the regression of large mammary tumor lesions in tumor-bearing HER-2/neu mice. The complete eradication of a significant number of tumor lesions occurred only in mice receiving cyclophosphamide shortly before immunotherapy, and was associated with increased serum anti HER-2/p185 antibodies and tumor leukocyte infiltration. The same protocol significantly delayed the appearance of mammary tumors when administered to tumor-free HER-2/neu mice, indicating that this chemo-immunotherapy approach acted through the elicitation of an effective anti-tumor immune response. Overall, our data support the immune-modulatory role of chemotherapy in overcoming cancer immune tolerance when administered at lymphodepleting non-myeloablative doses shortly before transfer of antigen-specific immune cells and immunoglobulins. These findings open new perspectives on combining immune-modulatory chemotherapy and immunotherapy to overcome immune tolerance in cancer patients.

Intratumoral injection of IFN-alpha dendritic cells after dacarbazine activates anti-tumor immunity: results from a phase I trial in advanced melanoma.

BACKGROUND: Advanced melanoma patients have an extremely poor long term prognosis and are in strong need of new therapies. The recently developed targeted therapies have resulted in a marked antitumor effect, but most responses are partial and some degree of toxicity remain the major concerns. Dendritic cells play a key role in the activation of the immune system and have been typically used as ex vivo antigen-loaded cell drugs for cancer immunotherapy. Another approach consists in intratumoral injection of unloaded DCs that can exploit the uptake of a wider array of tumor-specific and individual unique antigens. However, intratumoral immunization requires DCs endowed at the same time with properties typically belonging to both immature and mature DCs (i.e. antigen uptake and T cell priming). DCs generated in presence of interferon-alpha (IFN-DCs), due to their features of partially mature DCs, capable of efficiently up-taking, processing and cross-presenting antigens to T cells, could successfully carry out this task. Combining intratumoral immunization with tumor-destructing therapies can induce antigen release in situ, facilitating the injected DCs in triggering an antitumor immune response. METHODS: We tested in a phase I clinical study in advanced melanoma a chemo-immunotherapy approach based on unloaded IFN-DCs injected intratumorally one day after administration of dacarbazine. Primary endpoint of the study was treatment safety and tolerability. Secondary endpoints were immune and clinical responses of patients. RESULTS: Six patients were enrolled, and only three completed the treatment. The chemo-immunotherapy was well tolerated with no major side effects. Three patients showed temporary disease stabilization and two of them showed induction of T cells specific for tyrosinase, NY-ESO-1 and gp100. Of interest, one patient showing a remarkable long-term disease stabilization kept showing presence of tyrosinase specific T cells in PBMC and high infiltration of memory T cells in the tumor lesion at 21 months. CONCLUSION: We tested a chemo-immunotherapeutic approach based on IFN-DCs injected intratumorally one day after DTIC in advanced melanoma. The treatment was well tolerated, and clinical and immunological responses, including development of vitiligo, were observed, therefore warranting additional clinical studies aimed at evaluating efficacy of this approach. TRIAL REGISTRATION: Trial Registration Number not publicly available due to EudraCT regulations: https://www.clinicaltrialsregister.eu/doc/EU_CTR_FAQ.pdf.

Twenty-five years of type I interferon-based treatment: a critical analysis of its therapeutic use.

The clinical exploitation of type I interferon (IFN) as an antiviral and antineoplastic agent is based on the properties originally attributed to this cytokine family, with schedules reflecting only their antiviral and antiproliferative activities. Nevertheless, type I IFN has emerged as a central activator of the innate immunity. As current schedules of treatment for chronic hepatitis C and for hematological and solid tumors, based on the continuous administration of recombinant type I IFN or pegylated formulations, disregard viral resistance, host genetic variants predicting treatment outcome and mechanisms of refractoriness, new administration schedules, the combination of type I IFN with new drugs and the increased monitoring of patients' susceptibility to type I IFN are expected to provide a new life to this valuable cytokine.

Cancer cell-autonomous contribution of type I interferon signaling to the efficacy of chemotherapy.

Some of the anti-neoplastic effects of anthracyclines in mice originate from the induction of innate and T cell-mediated anticancer immune responses. Here we demonstrate that anthracyclines stimulate the rapid production of type I interferons (IFNs) by malignant cells after activation of the endosomal pattern recognition receptor Toll-like receptor 3 (TLR3). By binding to IFN-α and IFN-ß receptors (IFNARs) on neoplastic cells, type I IFNs trigger autocrine and paracrine circuitries that result in the release of chemokine (C-X-C motif) ligand 10 (CXCL10). Tumors lacking Tlr3 or Ifnar failed to respond to chemotherapy unless type I IFN or Cxcl10, respectively, was artificially supplied. Moreover, a type I IFN-related signature predicted clinical responses to anthracycline-based chemotherapy in several independent cohorts of patients with breast carcinoma characterized by poor prognosis. Our data suggest that anthracycline-mediated immune responses mimic those induced by viral pathogens. We surmise that such 'viral mimicry' constitutes a hallmark of successful chemotherapy.

The Janus face of cyclophosphamide: A sterile inflammatory response that potentiates cancer immunotherapy.

Cyclophosphamide, within well-defined therapeutic regimens, increases the antineoplastic effects of immunotherapy. We have recently identified multiple factors and mechanisms that underlie the paradoxical synergy between these two treatment modalities. In particular, we found that cyclophosphamide stimulates anticancer immune responses upon the perception by the immune system of inflammatory danger signals associated with the death of leukocytes, via p53 and type I interferon-related mechanisms.

Immune monitoring in cancer vaccine clinical trials: critical issues of functional flow cytometry-based assays.

The development of immune monitoring assays is essential to determine the immune responses against tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs) and their possible correlation with clinical outcome in cancer patients receiving immunotherapies. Despite the wide range of techniques used, to date these assays have not shown consistent results among clinical trials and failed to define surrogate markers of clinical efficacy to antitumor vaccines. Multiparameter flow cytometry- (FCM-) based assays combining different phenotypic and functional markers have been developed in the past decade for informative and longitudinal analysis of polyfunctional T-cells. These technologies were designed to address the complexity and functional heterogeneity of cancer biology and cellular immunity and to define biomarkers predicting clinical response to anticancer treatment. So far, there is still a lack of standardization of some of these immunological tests. The aim of this review is to overview the latest technologies for immune monitoring and to highlight critical steps involved in some of the FCM-based cellular immune assays. In particular, our laboratory is focused on melanoma vaccine research and thus our main goal was the validation of a functional multiparameter test (FMT) combining different functional and lineage markers to be applied in clinical trials involving patients with melanoma.

Exploiting dendritic cells in the development of cancer vaccines.

Due to their central role in priming and modulating the immune response, dendritic cells (DCs) represent an ideal instrument for the design of effective immunotherapeutic strategies for cancer patients. Recent advancement on the knowledge of the numerous DC subtypes, their functions and T-cell polarizing abilities has led to the development of several protocols for the ex vivo differentiation of autologous DCs and their loading with tumor-associated antigens. Moreover, novel strategies for the in vivo targeting of tumor antigens and adjuvants to natural DC subsets have been developed. Despite the large number of clinical studies carried out in cancer patients, a consensus on the optimal treatment modalities has not been reached yet. In this review, we summarize our current knowledge on DC biology and on DC use in clinical trials. Special attention is given to the many open issues regarding DC-based vaccination to sensitize researchers in the field to the compelling need of conducting comparative studies systematically addressing the still unresolved problems.

Cyclophosphamide induces a type I interferon-associated sterile inflammatory response signature in cancer patients' blood cells: implications for cancer chemoimmunotherapy.

PURPOSE: Certain chemotherapeutics, particularly cyclophosphamide, can enhance the antitumor efficacy of immunotherapy. A better understanding of the cellular and molecular basis of cyclophosphamide-mediated immunomodulation is needed to improve the efficacy of chemoimmunotherapy. EXPERIMENTAL DESIGN: Transcript profiling and flow cytometry were used to explore cyclophosphamide-induced immunoadjuvanticity in patients with hematologic malignancies. RESULTS: A single high-dose treatment rapidly (1-2 days) induced peripheral blood mononuclear cell (PBMC) transcriptional modulation, leading to reduction of cell-cycle and biosynthetic/metabolic processes and augmentation of DNA damage and cell death pathways (p53 signaling pathway), death-related scavenger receptors, antigen processing/presentation mediators, T-cell activation markers and, noticeably, a type I IFN (IFN-I) signature (OAS1, CXCL10, BAFF, IFITM2, IFI6, IRF5, IRF7, STAT2, UBE2L6, UNC93B1, ISG20L1, TYK2). Moreover, IFN-I-induced proinflammatory mediators (CXCL10, CCL2, IL-8, and BAFF) were increased in patients' plasma. Accordingly, cyclophosphamide induced the expansion/activation of CD14(+)CD16(+) monocytes, of HLA-DR(+), IL-8RA(+), and MARCO(+) monocytes/dendritic cells, and of CD69(+), OX40(+), and IL-8RA(+) lymphocytes. CONCLUSIONS: Altogether, these data identify the cyclophosphamide-induced immunomodulatory factors in humans and indicate that preconditioning chemotherapy may stimulate immunity as a consequence of danger perception associated with blood cell death, through p53 and IFN-I-related mechanisms.