Phospholipid scramblase 1 is involved in immunogenic cell death and contributes to dendritic cell-based vaccine efficiency to elicit antitumor immune response in vitro.

BACKGROUND AIMS: Whole tumor cell lysates (TCLs) obtained from cancer cells previously killed by treatments able to promote immunogenic cell death (ICD) can be efficiently used as a source of tumor-associated antigens for the development of highly efficient dendritic cell (DC)-based vaccines. Herein, the potential role of the interferon (IFN)-inducible protein phospholipid scramblase 1 (PLSCR1) in influencing immunogenic features of dying cancer cells and in enhancing DC-based vaccine efficiency was investigated. METHODS: PLSCR1 expression was evaluated in different mantle-cell lymphoma (MCL) cell lines following ICD induction by 9-cis-retinoic acid (RA)/IFN-α combination, and commercial kinase inhibitor was used to identify the signaling pathway involved in its upregulation. A Mino cell line ectopically expressing PLSCR1 was generated to investigate the potential involvement of this protein in modulating ICD features. Whole TCLs obtained from Mino overexpressing PLSCR1 were used for DC loading, and loaded DCs were employed for generation of tumor antigen-specific cytotoxic T lymphocytes. RESULTS: The ICD inducer RA/IFN-α combination promoted PLSCR1 expression through STAT1 activation. PLSCR1 upregulation favored pro-apoptotic effects of RA/IFN-α treatment and enhanced the exposure of calreticulin on cell surface. Moreover, DCs loaded with TCLs obtained from Mino ectopically expressing PLSCR1 elicited in vitro greater T-cell-mediated antitumor responses compared with DCs loaded with TCLs derived from Mino infected with empty vector or the parental cell line. Conversely, PLSCR1 knock-down inhibited the stimulating activity of DCs loaded with RA/IFN-α-treated TCLs to elicit cyclin D1 peptide-specific cytotoxic T lymphocytes. CONCLUSIONS: Our results indicate that PLSCR1 improved ICD-associated calreticulin exposure induced by RA/IFN-α and was clearly involved in DC-based vaccine efficiency as well, suggesting a potential contribution in the control of pathways associated to DC activation, possibly including those involved in antigen uptake and concomitant antitumor immune response activation.

Vesicle-bound EBV-BART13-3p miRNA in circulation distinguishes nasopharyngeal from other head and neck cancer and asymptomatic EBV-infections.

Cell-free microRNA (miRNA) in biofluids released by tumors in either protein or vesicle-bound form, represent promising minimally-invasive cancer biomarkers. However, a highly abundant non-tumor background in human plasma and serum complicates the discovery and detection of tumor-selective circulating miRNAs. We performed small RNA sequencing on serum and plasma RNA from Nasopharyngeal Carcinoma (NPC) patients. Collectively, Epstein Barr virus-encoded miRNAs, more so than endogenous miRNAs, signify presence of NPC. However, RNAseq-based EBV miRNA profiles differ between NPC patients, suggesting inter-tumor heterogeneity or divergent secretory characteristics. We determined with sensitive qRT-PCR assays that EBV miRNAs BART7-3p, BART9-3p and BART13-3p are actively secreted by C666.1 NPC cells bound to extracellular vesicles (EVs) and soluble ribonucleoprotein complexes. Importantly, these miRNAs are expressed in all primary NPC tumor biopsies and readily detected in nasopharyngeal brushings from both early and late-stage NPC patients. Increased levels of BART7-3p, BART9-3p and particularly BART13-3p, distinguish NPC patient sera from healthy controls. Receiver operating characteristic curve analysis using sera from endemic NPC patients, other head and neck cancers and individuals with asymptomatic EBV-infections reveals a superior diagnostic performance of EBV miRNAs over anti-EBNA1 IgA serology and EBV-DNA load (AUC 0.87-0.96 vs 0.86 and 0.66 respectively). The high specificity of circulating EBV-BART13-3p (97%) for NPC detection is in agreement with active secretion from NPC tumor cells. We conclude EV-bound BART13-3p in circulation is a promising, NPC-selective, biomarker that should be considered as part of a screening strategy to identify NPC in endemic regions.

In-depth analysis of compartmentalization of HIV-1 matrix protein p17 in PBMC and plasma.

HIV-1 p17 plays an important role in the virus life-cycle and disease pathogenesis. Recent studies indicated a high heterogeneity of p17. A high number of insertions in the p17 carboxy-terminal region have been more frequently detected in patients with non-Hodgkin lymphoma (NHL), suggesting a role of altered p17 in lymphomagenesis. Based on p17 heterogeneity, possible PBMC/plasma compartmentalization of p17 variants was explored by ultra-deep pyrosequencing in five NHL patients. The high variability of p17 with insertions at the carboxy-terminal region was confirmed in plasma and observed for the first time in proviral genomes. Quasispecies compartmentalization was evident in 4/5 patients. Further studies are needed to define the possible role of p17 quasispecies compartmentalization in lymphomagenesis.

A natural HIV p17 protein variant up-regulates the LMP-1 EBV oncoprotein and promotes the growth of EBV-infected B-lymphocytes: implications for EBV-driven lymphomagenesis in the HIV setting.

Human immunodeficiency virus p17 matrix protein is released by infected cells and may accumulate within lymphoid tissues where it may deregulate the biological activities of different cell populations by binding to CXCR1 and CXCR2 cellular receptors. S75X, a natural p17 variant, was recently shown to enhance the malignant properties of lymphoma cells. We investigated a reference p17 protein and the S75X variant for their ability to bind to Epstein-Barr virus (EBV)-infected primary and fully transformed B-lymphocytes and trigger downstream effects of potential pathogenic relevance. We demonstrate that EBV infection of primary B-lymphocytes or the ectopic expression of the latent membrane protein-1 viral oncoprotein in EBV-negative B-cells up-regulates CXCR2, but not CXCR1. Multispectral imaging flow cytometry showed that EBV-infected primary B-cells more efficiently bind and internalize p17 proteins as compared with activated B-lymphocytes. The S75X variant bound more efficiently to EBV-infected primary and fully transformed B-lymphocytes compared with reference p17, because of a higher affinity to CXCR2, and enhanced the proliferation of these cells, an effect associated with cyclin D2 and D3 up-regulation and increased interleukin-6 production. Notably, the S75X variant markedly up-regulated latent membrane protein-1 expression at both mRNA and protein levels and enhanced the activation of Akt, ERK1/2 and STAT3 signaling, thereby contributing to EBV(+) B-cell growth promotion. These results indicate that EBV infection sensitizes B-lymphocytes to CXCR2-mediated effects of p17 proteins and provide evidence supporting a possible contribution of natural p17 variants to EBV-driven lymphomagenesis in the human immunodeficiency virus setting.

Simian immunodeficiency virus and human immunodeficiency virus type 1 matrix proteins specify different capabilities to modulate B cell growth.

UNLABELLED: Exogenous HIV-1 matrix protein p17 (p17) deregulates the function of different cells after its N-terminal loop (AT20) binding to the chemokine receptors CXCR1 and CXCR2. One site within AT20 has been recently found to be the major determinant of viral fitness following transmission of simian immunodeficiency virus (SIV) to the human host. Therefore, we sought to determine whether SIV matrix protein (MA) was already capable of interacting with CXCR1 and CXCR2 and mimic p17 biological activities rather than this being a newly acquired function during host adaptation. We show here that SIV MA binds with the same affinity of p17 to CXCR1 and CXCR2 and displays both p17 proangiogenic on human primary endothelial cells and chemotactic activity on human primary monocytes and B cells. However, SIV MA exhibited a higher degree of plasticity than p17 in the C terminus, a region known to play a role in modulating B cell growth. Indeed, in contrast to p17, SIV MA was found to activate the phosphatidylinositol 3-kinase/Akt signaling pathway and strongly promote B cell proliferation and clonogenic activity. Interestingly, we have recently highlighted the existence of a Ugandan HIV-1 strain-derived p17 variant (S75X) with the same B cell growth-promoting activity of SIV MA. Computational modeling allowed us to hypothesize an altered C terminus/core region interaction behind SIV MA and S75X activity. Our findings suggest the appearance of a structural constraint in the p17 C terminus that controls B cell growth, which may help to elucidate the evolutionary trajectory of HIV-1. IMPORTANCE: The HIV-1 matrix protein p17 (p17) deregulates the biological activities of different cells after binding to the chemokine receptors CXCR1 and CXCR2. The p17 functional domain responsible for receptors interaction includes an amino acid which is considered the major determinant of SIV replication in humans. Therefore, we sought to determine whether SIV matrix protein (SIV MA) already had the ability to bind to both chemokine receptors rather than being a function newly acquired during host adaptation. We show here that SIV MA binds to CXCR1 and CXCR2 and fully mimics the p17 proangiogenic and chemokine activity. However, it differs from p17 in its ability to signal into B cells and promote B cell growth and clonogenicity. Computational analysis suggests that the accumulation of mutations in the C-terminal region may have led to a further SIV MA adaptation to the human host. This finding in turn sheds light on the evolutionary trajectory of HIV-1.

Interplay among viral antigens, cellular pathways and tumor microenvironment in the pathogenesis of EBV-driven lymphomas.

Epstein-Barr virus (EBV) is a ubiquitous human γ-herpes virus that has established an elegant strategy to persist as a life-long asymptomatic infection in memory B lymphocytes. EBV has potent transforming properties for B lymphocytes and it is pathogenically associated with a variety of lymphomas of B or NK/T cell origin. The viral latency programs expressed can hijack or deregulate cellular pathways critical for cell proliferation and survival, while impairing anti-viral immune responses. Similar effects may also be induced by EBV-encoded micro-RNAs, which may have a pathogenic role particularly in lymphomas showing a restricted expression of viral proteins. Of note, recent data have challenged the view that only the EBV latency is relevant for lymphomagenesis, suggesting that lytic EBV replication may also contribute to the development of EBV-associated lymphoproliferations. The recent advances in the elucidation of the mechanisms underlying EBV-induced cell transformation and immune evasion are providing the rationale for innovative and tailored treatment approaches for EBV-driven lymphomas.

Virologic and immunologic evidence supporting an association between HHV-6 and Hashimoto's thyroiditis.

Hashimoto's thyroiditis (HT) is the most common of all thyroid diseases and is characterized by abundant lymphocyte infiltrate and thyroid impairment, caused by various cell- and antibody-mediated immune processes. Viral infections have been suggested as possible environmental triggers, but conclusive data are not available. We analyzed the presence and transcriptional state of human herpesvirus 6 (HHV-6) in thyroid fine needle aspirates (FNA) and peripheral blood mononuclear cells (PBMCs) from 34 HT patients and 28 controls, showing that HHV-6 DNA prevalence (82% vs. 10%, p≤0.001) and viral load were significantly increased in FNA from HT patients, and thyrocytes from HT FNA displayed a 100-fold higher HHV-6 DNA load compared to infiltrating lymphocytes. In addition, while HHV-6 was strictly latent in positive samples from controls, a low grade acute infection was detected in HT samples. HHV-6 variant characterization was carried out in 10 HT FNA samples, determining that all specimens harbored HHV-6 Variant A.The tropism of HHV-6 for thyroid cells was verified by infection of Nthy-ori3-1, a thyroid follicular epithelial cell line, showing that thyrocytes are permissive to HHV-6 replication, which induces de novo expression of HLA class II antigens. Furthermore, HHV-6-infected Nthy-ori3-1 cells become targets for NK-mediated killing, NK cells from HT patients show a significantly more efficient killing of HHV-6 infected thyroid cells than healthy controls, and HT patients have increased T-cell responses to HHV-6 U94 protein, associated to viral latency. These observations suggest a potential role for HHV-6 (possibly variant A) in the development or triggering of HT.

Anthracycline-free neoadjuvant therapy induces pathological complete responses by exploiting immune proficiency in HER2+ breast cancer patients.

BACKGROUND: Neoadjuvant Chemotherapy (NC) including trastuzumab induces a high rate of pathological Complete Responses (pCR) in patients with locally advanced HER2-overexpressing Breast Cancer (BC), but is penalized by a severe cardiotoxicity when combined with anthracyclines. A phase II study was designed to assess whether an anthracycline-free NC regimen based on the early addition of trastuzumab to paclitaxel may increase the pCR rate without inducing severe cardiotoxicity in patients with locally advanced HER2-overexpressing BC. Immunomonitoring was performed to assess the contribution of patients' immunological background to the induction of clinical responses. METHODS: Stage II-III HER2-positive BC patients received 24 weeks paclitaxel and trastuzumab NC, followed by 1 year adjuvant trastuzumab ± hormonal and/or radio-therapy. Assessment of pCR rate was the primary endpoint. A group of HER2-negative BC patients treated with neoadjuvant taxanes and anthracyclines was included. Serum levels of 10 cytokines and the efficiency of trastuzumab-mediated antibody-dependent cell cytotoxicity (ADCC) were monitored in vitro every 3 months. RESULTS: From July 2006 to February 2013, we enrolled 109 patients including 46 evaluable HER2-positive cases. A pCR rate of 50% was reached and no severe cardiotoxicity occurred. Serum cytokine profiling revealed only an IL-10 decrease (P = 0.02) in patients achieving a partial response, while HER2-negative patients disclosed marked cytokines changes. Compared to the unfavourable F/F genotype, patients carrying the V allele in the FcγRIIIa-158 polymorphism showed a higher efficacy of trastuzumab-ADCC throughout treatment (P ≤0.05). CONCLUSIONS: In the absence of anthracyclines, trastuzumab and paclitaxel induced a high rate of pCR, exploiting the synergy between the immunomodulating properties of these drugs and the retained immunological proficiency of patients with HER2-overexpressing BC. TRIAL REGISTRATION: Trial registration number: NCT02307227, registered on ClinicalTrials.gov (http://www.clinicaltrials.gov, November 26, 2014).

Remodeling of the epitope repertoire of a candidate idiotype vaccine by targeting to lysosomal degradation in dendritic cells.

The generation of efficacious vaccines against self-antigens expressed in tumor cells requires breakage of tolerance, and the refocusing of immune responses toward epitopes for which tolerance may not be established. While the presentation of tumor antigens by mature dendritic cells (mDC) may surpass tolerance, broadening of the antigenic repertoire remains an issue. We report that fusion of the candidate idiotype vaccine IGKV3-20 to the Gly-Ala repeat (GAr) of the Epstein-Barr virus nuclear antigen (EBNA)-1 inhibits degradation by the proteasome and redirects processing to the lysosome. mDCs transduced with a recombinant lentivirus encoding the chimeric idiotype efficiently primed CD4+ and CD8+ cytotoxic T-cell (CTL) responses that lysed autologous blasts expressing IGKV3-20 or pulsed with IGKV3-20 synthetic peptides, and HLA-matched IGKV3-20-positive tumor cell lines. Comparison of the cytotoxic response of CD4+ and CD8+ T lymphocytes activated by mDCs expressing the wild-type or chimeric IGKV3-20 reveled largely non-overlapping epitope repertoires in both CD4+ and CD8+ effectors. Thus, fusion to the GAr may provide an effective means to broaden the immune response to an endogenous protein by promoting the presentation of antigenic epitopes that require a lysosome-dependent processing step.

Exploiting the interplay between innate and adaptive immunity to improve immunotherapeutic strategies for Epstein-Barr-virus-driven disorders.

The recent demonstration that immunotherapeutic approaches may be clinically effective for cancer patients has renewed the interest for this strategy of intervention. In particular, clinical trials using adoptive T-cell therapies disclosed encouraging results, particularly in the context of Epstein-Barr-virus- (EBV-) related tumors. Nevertheless, the rate of complete clinical responses is still limited, thus stimulating the development of more effective therapeutic protocols. Considering the relevance of innate immunity in controlling both infections and cancers, innovative immunotherapeutic approaches should take into account also this compartment to improve clinical efficacy. Evidence accumulated so far indicates that innate immunity effectors, particularly NK cells, can be exploited with therapeutic purposes and new targets have been recently identified. We herein review the complex interactions between EBV and innate immunity and summarize the therapeutic strategies involving both adaptive and innate immune system, in the light of a fruitful integration between these immunotherapeutic modalities for a better control of EBV-driven tumors.

A different immunologic profile characterizes patients with HER-2-overexpressing and HER-2-negative locally advanced breast cancer: implications for immune-based therapies.

INTRODUCTION: The clinical efficacy of trastuzumab and taxanes is at least partly related to their ability to mediate or promote antitumor immune responses. On these grounds, a careful analysis of basal immune profile may be capital to dissect the heterogeneity of clinical responses to these drugs in patients with locally advanced breast cancer undergoing neoadjuvant chemotherapy. METHODS: Blood samples were collected from 61 locally advanced breast cancers (36 HER2- and 25 HER2+) at diagnosis and from 23 healthy women. Immunophenotypic profiling of circulating and intratumor immune cells, including regulatory T (Treg) cells, was assessed by flow cytometry and immunohistochemistry, respectively. Serum levels of 10 different cytokines were assessed by multiplex immunoassays. CD8+ T cell responses to multiple tumor-associated antigens (TAA) were evaluated by IFN-γ-enzyme-linked immunosorbent spot (ELISPOT). The Student's t test for two tailed distributions and the Wilcoxon two-sample test were used for the statistical analysis of the data. RESULTS: The proportion of circulating immune effectors was similar in HER2+ patients and healthy donors, whereas higher percentages of natural killer and Treg cells and a lower CD4+/CD8+ T cell ratio (with a prevalence of naïve and central memory CD8+ T cells) were observed in HER2- cases. Higher numbers of circulating CD8+ T cells specific for several HLA-A*0201-restricted TAA-derived peptides were observed in HER2+ cases, together with a higher prevalence of intratumor CD8+ T cells. Serum cytokine profile of HER2+ patients was similar to that of controls, whereas HER2- cases showed significantly lower cytokine amounts compared to healthy women (IL-2, IL-8, IL-6) and HER2+ cases (IL-2, IL-1ß, IL-8, IL-6, IL-10). CONCLUSIONS: Compared to HER2- cases, patients with HER2-overexpressing locally advanced breast cancer show a more limited tumor-related immune suppression. This may account for the clinical benefit achieved in this subset of patients with the use of drugs acting through, but also promoting, immune-mediated effects.

Biological Predictors of De Novo Tumors in Solid Organ Transplanted Patients During Oncological Surveillance: Potential Role of Circulating TERT mRNA.

BACKGROUND: De novo tumors are a major cause of morbidity and mortality after long-term solid organ transplantation. Chronic immunosuppression strongly affects solid organ transplanted (SOT) patients' immune system by promoting immune evasion strategies and reactivations of viruses with oncogenic potential, ultimately leading to cancer onset. In this scenario, an oncological Surveillance Protocol integrated with biobanking of peripheral blood samples and evaluation of immunovirological and molecular parameters was activated for SOT patients at CRO-IRCCS Aviano, with the aim of identifying suitable biomarkers of cancer development. METHODS: An exploratory longitudinal study was designed based on two serial peripheral blood samples collected at least three months apart. Forty nine SOT patients were selected and stratified by tumor onset during follow-up. Spontaneous T-cell responses to EBV, CMV and tumor associated antigens, EBV-DNA and CMV-DNA loads, and circulating TERT mRNA levels were investigated. RESULTS: Significantly higher levels of circulating TERT mRNA were observed 3.5-23.5 months before and close to the diagnosis of cancer as compared to tumor-free patients. Plasmatic TERT mRNA levels >97.73 copies/mL at baseline were significantly associated with the risk of developing de novo tumors (HR=4.0, 95%C.I. = 1.4-11.5, p=0.01). In particular, the risk significantly increased by 4% with every ten-unit increment in TERT mRNA (HR=1.04, 95%C.I. = 1.01-1.07, p=0.01). CONCLUSIONS: Although obtained in an exploratory study, our data support the importance of identifying early biomarkers of tumor onset in SOT patients useful to modulate the pace of surveillance visits.

Immune signatures in human PBMCs of idiotypic vaccine for HCV-related lymphoproliferative disorders.

Hepatitis C virus (HCV) is one of the major risk factors for chronic hepatitis, which may progress to cirrhosis and hepatocellular carcinoma, as well as for type II mixed cryoglobulinemia (MC), which may further evolve into an overt B-cell non-Hodgkin's lymphoma (NHL). It has been previously shown that B-cell receptor (BCR) repertoire, expressed by clonal B-cells involved in type II MC as well as in HCV-associated NHL, is constrained to a limited number of variable heavy (VH)- and light (VL)-chain genes. Among these, the VK3-20 light chain idiotype has been selected as a possible target for passive as well as active immunization strategy. In the present study, we describe the results of a multiparametric analysis of the innate and early adaptive immune response after ex vivo stimulation of human immune cells with the VK3-20 protein. This objective has been pursued by implementing high-throughput technologies such as multiparameter flow cytometry and multiplex analysis of cytokines and chemokines.

The interplay between Epstein-Barr virus and the immune system: a rationale for adoptive cell therapy of EBV-related disorders.

The Epstein-Barr virus has evolved a plethora of strategies to evade immune system recognition and to establish latent infection in memory B cells, where the virus resides lifelong without any consequence in the majority of individuals. However, some imbalances in the equilibrium between the inherent virus transforming properties and the host immune system can lead to the development of different tumors, such as lymphoproliferative disorders, Hodgkin's lymphoma, Burkitt's lymphoma, and nasopharyngeal carcinoma. The expression of viral antigens in malignant cells makes them suitable targets for immunotherapeutic approaches, which are mainly based on the ex vivo expansion of EBV-specific T cells. Indeed, the infusion of virus-specific cytotoxic T lymphocytes has proved not only to be safe and effective, but also capable of restoring or inducing a protective anti-virus immunity, which is lacking, albeit to a different extent, in every EBV-driven malignancy. The purpose of this review is to summarize the results of adoptive immunotherapy approaches for EBV-related malignancies, with particular emphasis on the immunological and virological aspects linked to the clinical responses obtained. Data collected confirm the clinical relevance of the use of EBV-specific cytotoxic T lymphocytes in the field of adoptive immunotherapy and suggest the increasing importance of this approach also against other tumors, concurrent with the increasing knowledge of the intimate and continuous interplay between the virus and the host immune system.

Undifferentiated nasopharyngeal carcinoma from a nonendemic area: protective role of HLA allele products presenting conserved EBV epitopes.

The role of genetic factors involved in the development of undifferentiated nasopharyngeal carcinoma (UNPC) in nonendemic areas has been poorly investigated. High-resolution human leukocyte antigen (HLA) class I genotyping carried out in 82 Italian UNPC patients and 286 bone marrow donors born in the same province showed that A*0201, B*1801, and B*3501, known to efficiently present Epstein-Barr virus (EBV)-derived epitopes, were significantly under-represented in UNPC patients. Moreover, the A*0201/B*1801 haplotype was significantly less frequent in UNPC cases, with a 90% reduced risk (odds ratio [OR] 0.1, 95% confidence interval [CI] = 0.0-0.5) to develop UNPC, suggesting an additive effect. Notably, all 5 BARF1 epitopes and 7 of the 8 LMP-2 epitopes known to bind A*0201 showed a fully conserved sequence in all the 31 Italian EBV isolates investigated. The 4 amino acid changes affecting the 436-447 LMP-2 epitope do not reduce, but rather increase in two cases, the predicted ability of "variant" epitopes to bind the HLA-A*0201 allele, as shown by immunoinformatic analysis. Moreover, a significantly increased risk for UNPC was associated with A*2601 (OR 2.4, 95% CI = 1.1-4.9) and B*4101 (OR 9.2, 95% CI = 2.5-34.3). These findings indicate that Italian UNPC patients have a distinct HLA-A and -B genotypic profile and suggest that the decreased risk for UNPC conferred by definite HLA class I molecules is probably related to their ability to efficiently present LMP-2 and BARF1 epitopes that are highly conserved in EBV isolates from this geographic region. These results have practical implications for the immunotherapy of UNPC.

Clinical and Antitumor Immune Responses in Relapsed/Refractory Follicular Lymphoma Patients after Intranodal Injections of IFNα-Dendritic Cells and Rituximab: a Phase I Clinical Trial.

PURPOSE: This study was aimed at evaluating the feasibility, safety, immunologic and clinical responses in patients with follicular lymphoma treated with monocyte-derived dendritic cells generated in the presence of IFNα and GM-CSF (IFN-DC) in combination with low doses of rituximab. PATIENTS AND METHODS: Firstly, we analyzed in vitro and in vivo the immunologic properties of IFN-DC against follicular lymphoma. Thus, we performed a phase I trial in 8 patients with refractory and relapsed follicular lymphoma based on sequential intranodal injections of low-dose of rituximab and unloaded IFN-DC and report the safety, clinical, and immunologic results of the enrolled patients. RESULTS: Preclinical studies indicated that IFN-DC can synergize with rituximab leading to increased cytotoxicity and T-cell tumor infiltration. The clinical evaluation showed that the combined treatment was totally safe. The overall response rate was 50%, PET-negative complete response rate 37%, and remission is still ongoing in 2/4 of responding patients (median follow-up 26 months, range 11-47). Notably, following the combined therapy all patients showed induction/enhancement of T-cell responses by CD107 degranulation or IFNγ ELISPOT assay against patient-specific tumor IGHV sequences. CONCLUSIONS: These results represent the proof-of-principle on the effectiveness of unloaded IFN-DC in inducing durable clinical responses and promoting induction of tumor-specific peripheral T cells, thus suggesting the occurrence of an effective endogenous antitumor vaccination. The overall findings indicate that some unique properties of IFN-DC can be successfully exploited to induce/enhance antitumor responses, thus representing a valuable antitumor strategy for novel and more effective combination therapies in patients with cancer.

Spontaneous T cell responses to Epstein-Barr virus-encoded BARF1 protein and derived peptides in patients with nasopharyngeal carcinoma: bases for improved immunotherapy.

Immunotherapy approaches targeting Epstein-Barr virus (EBV)-encoded antigens induce objective clinical responses only in a fraction of patients with undifferentiated nasopharyngeal carcinoma (UNPC). In the present study, we have characterized the immunogenicity of the EBV-encoded BARF1 oncogene with the aim to assess whether this protein could constitute a new target antigen for immunotherapy in this setting. Spontaneous CD4+ and CD8+ T cell responses specific for the recombinant p29 BARF1 protein were detected by IFNgamma-ELISPOT in both EBV-seropositive donors and UNPC patients, but not in EBV-seronegative individuals. Using immunoinformatic prediction tools, we have selected 5 different candidate BARF1 T cell epitopes presented by HLA-A*0201. Although only one of these peptides was able to bind HLA-A2 with low affinity in the T2 stabilization assay, all 5 BARF1 nonamers readily elicited specific CD8+ T cell responses in EBV-seropositive HLA-A*0201+ donors and UNPC patients. Notably, the magnitude of CD8+ T cell responses to the whole BARF1 protein and derived A*0201 peptides was significantly higher in UNPC patients than in healthy donors. Moreover, cytotoxic T lymphocytes specific for the p2-10, p23-31, or p49-57 BARF1 peptides were easily obtained from HLA-A*0201+ donors. These cultures were not only able to lyse autologous targets loaded with the antigenic peptide, but also recognized tumor cells endogenously expressing BARF1 in an antigen-specific and HLA-A2-restricted manner. These findings, indicate that BARF1 is a particularly attractive antigen with immunogenic properties in most UNPC patients and provide valuable information to develop new strategies to improve the efficacy of EBV-targeting immunotherapy of UNPC patients.

Radical radiation therapy for oligometastatic breast cancer: Results of a prospective phase II trial.

BACKGROUND AND PURPOSE: We conducted a prospective phase II multicentric trial to determine if radical radiation therapy to all metastatic sites might improve the progression-free survival (PFS) in oligometastatic breast cancer patients. Secondary endpoints were local control (LC), overall survival (OS) and toxicity. METHODS AND MATERIALS: Inclusion criteria were the following: oligometastatic breast cancer with ≤5 metastatic sites, FDG-PET/CT staging, no brain metastases, primary tumor controlled. Radiotherapy could be delivered using stereotactic body radiotherapy (SBRT) technique or fractionated intensity modulated radiotherapy (IMRT). SBRT consisted of 30-45Gy in 3 fractions, while IMRT was delivered to a total dose of 60Gy in 25 fractions. We hypothesized that radical radiation therapy could increase the PFS from 30% (according to the published literature) to 50% at two years. RESULTS: 54 Patients with 92 metastatic lesions were enrolled. Forty-four were treated with SBRT, and 10 with IMRT. Forty-eight (89%) patients received a form of systemic therapy concomitantly to radiation therapy. Sites of metastatic disease were the following: bones 60 lesions, lymph nodes 23 lesions, lung 4 lesions, liver 5 lesions. After a median follow-up of 30months (range, 6-55months), 1- and 2-year PFS was 75% and 53%, respectively. Two-year LC and OS were 97% and 95%, respectively. Radiation therapy was well tolerated, and no Grade ≥3 toxicity was documented. Grade 2 toxicity were pain and fatigue in 2 cases. CONCLUSIONS: Patients with oligometastatic breast cancer treated with radical radiotherapy to all metastatic sites may achieve long-term progression-free survival, without significant treatment-related toxicity. While waiting for data from randomized trials, the use of radical radiation therapy to all metastatic sites in patients with oligometastatic breast cancer should be considered a valuable option, and its recommendation should be individualized.

Fighting Viral Infections and Virus-Driven Tumors with Cytotoxic CD4+ T Cells.

CD4+ T cells have been and are still largely regarded as the orchestrators of immune responses, being able to differentiate into distinct T helper cell populations based on differentiation signals, transcription factor expression, cytokine secretion, and specific functions. Nonetheless, a growing body of evidence indicates that CD4+ T cells can also exert a direct effector activity, which depends on intrinsic cytotoxic properties acquired and carried out along with the evolution of several pathogenic infections. The relevant role of CD4+ T cell lytic features in the control of such infectious conditions also leads to their exploitation as a new immunotherapeutic approach. This review aims at summarizing currently available data about functional and therapeutic relevance of cytotoxic CD4+ T cells in the context of viral infections and virus-driven tumors.

Local High-Dose Radiotherapy Induces Systemic Immunomodulating Effects of Potential Therapeutic Relevance in Oligometastatic Breast Cancer.

Local irradiation of cancer through radiotherapy can induce spontaneous regression of non-directly irradiated lesions, suggesting the involvement of systemic antitumor immune responses. In oligometastatic breast cancer (BC) patients, the use of stereotactic body radiotherapy (SBRT) favors the local control of treated lesions and may contribute to break local tolerance and release tumor-associated antigens (TAAs), improving host antitumor immunity. We performed a detailed immunomonitoring of BC patients undergoing SBRT to verify its ability to "switch on" the anti-tumor immunity both systemically, in peripheral blood, and locally, employing in vitro BC models. Twenty-one BC patients with ≤6 metastases were treated with 3 daily doses of 10 Gy with SBRT. Blood samples for immune profiling were collected before and after treatment. One month after treatment a third of patients displayed the boosting or even the de novo appearance of polyfunctional CD4+ and CD8+ T cell responses against known BC TAAs (survivin, mammaglobin-A, HER2), through intracellular staining in flow cytometry. Half of patients showed increased numbers of activated natural killer (NK) cells, measured with multispectral flow cytometry, immediately after the first dose of SBRT. Interestingly, high levels of activated NK cells at diagnosis correlated with a longer progression-free survival. BC in vitro models, treated with the same SBRT modality, showed enhanced expression of MHC class-I and class-II, major histocompatibility complex class I-related chain A/B, and Fas molecules, and increased release of pro-inflammatory cytokines, such as IL-1ß and TNF-α. Consistently, we noticed enhanced production of perforin by CD4+ T cells when patients' lymphocytes were cultured in the presence of irradiated BC cell line, compared to untreated targets. Besides immunogenic effects, SBRT also enhanced the percentages of circulating regulatory T cells, and increased indoleamine 2,3 dioxygenase and PD-L1 expression in BC in vitro models. These results suggest that SBRT may boost host antitumor immune responses also in an advanced disease setting such as oligometastatic BC, by inducing immunomodulating effects both locally and systemically. However, the concomitant induction of immunosuppressive pathways suggests that a combination with immunotherapy could further enhance the in situ vaccination ability of radiotherapy, possibly further improving the curative potential of SBRT in this subset of patients.