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.

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.

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.

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.

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.

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.

A BARF1-specific mAb as a new immunotherapeutic tool for the management of EBV-related tumors.

The use of monoclonal antibodies (mAb) for the diagnosis and treatment of malignancies is acquiring an increasing clinical importance, thanks to their specificity, efficacy and relative easiness of use. However, in the context of Epstein-Barr virus (EBV)-related malignancies, only cancers of B-cell origin can benefit from therapeutic mAb targeting specific B-cell lineage antigens. To overcome this limitation, we generated a new mAb specific for BARF1, an EBV-encoded protein with transforming and immune-modulating properties. BARF1 is expressed as a latent protein in nasopharyngeal (NPC) and gastric carcinoma (GC), and also in neoplastic B cells mainly upon lytic cycle induction, thus representing a potential target for all EBV-related malignancies. Considering that BARF1 is largely but not exclusively secreted, the BARF1 mAb was selected on the basis of its ability to bind a domain of the protein retained at the cell surface of tumor cells. In vitro, the newly generated mAb recognized the target molecule in its native conformation, and was highly effective in mediating both ADCC and CDC against BARF1-positive tumor cells. In vivo, biodistribution analysis in mice engrafted with BARF1-positive and -negative tumor cells confirmed its high specificity for the target. More importantly, the mAb disclosed a relevant antitumor potential in preclinical models of NPC and lymphoma, as evaluated in terms of both reduction of tumor masses and long-term survival. Taken together, these data not only confirm BARF1 as a promising target for immunotherapeutic interventions, but also pave the way for a successful translation of this new mAb to the clinical use.

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.

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.

Broadening Specificity and Enhancing Cytotoxicity of Adoptive T Cells for Nasopharyngeal Carcinoma Immunotherapy.

Although promising, clinical responses to adoptive immunotherapy for nasopharyngeal carcinoma (NPC) are still limited by the restricted number of Epstein-Barr virus (EBV) antigens that can be targeted and their poor immunogenicity. Our previous work indicated that the immunogenic features of the NPC-associated viral antigen BARF1 may be exploited for immunotherapeutic purposes. Nevertheless, T-cell lines obtained with current protocols include only negligible numbers of BARF1-specific cytotoxic T lymphocytes, pointing to the need to enrich these effectors in BARF1 specificities. Considering that in B lymphocytes BARF1 is mainly a lytic EBV antigen, we tested different EBV lytic-cycle inducers (TPA/butyric acid, doxorubicin, and cisplatin) used at suboptimal concentrations for their ability to upregulate BARF1 expression in lymphoblastoid B-cell lines (LCL), the commonly used antigen-presenting cells, without compromising their survival. The LCLs treated with doxorubicin (DX-LCL) can reproducibly and efficiently generate EBV-specific effectors enriched in BARF1 specificities from both healthy donors and NPC patients. These DX-LCLs also had more pronounced immunogenic properties, including HLA class I upregulation and expression of immunogenic cell death markers, such as enhanced calreticulin exposure and HMGB1 release. In particular, doxorubicin triggers an HMGB1 autocrine/paracrine loop with its receptor, TLR4, which is also upregulated in DX-LCLs and is responsible for NF-κB activation and a delayed apoptosis that allows a prolonged stimulation of EBV-specific T-cell precursors. This protocol may thus constitute a valid alternative to the use of engineered LCLs to generate EBV-specific T-cell lines for adoptive immunotherapy, being relatively simple, easily upgradable to Good Manufacturing Practice standards, and therefore more broadly applicable. Cancer Immunol Res; 4(5); 431-40. ©2016 AACR.

Detection of nasopharyngeal carcinoma in Morocco (North Africa) using a multiplex methylation-specific PCR biomarker assay.

BACKGROUND: Silencing of tumor suppressor genes (TSGs) or activation of oncogenes by, e.g., aberrant promoter methylation, may be early events during carcinogenesis. The methylation status of such genes can be used for early detection of cancer. We are pursuing this approach in our efforts to develop markers for early detection and follow-up of nasopharyngeal carcinoma (NPC). We set out to develop this approach to allow identification of NPC from Morocco and then also compared with NPC samples from different geographical locations and different ethnicity with different NPC incidences, Epstein-Barr virus (EBV) prevalence, and environments. RESULTS: By multiplex methylation-specific PCR (MMSP), multiple relevant genes can be detected simultaneously, to achieve high sensitivity and specificity. The strong association of EBV with NPC is also very useful in such an approach. We have initially screened for 12 potential marker genes including EBV genes coding for EBV nuclear antigen 1 (EBNA1) and latent membrane protein-1 (LMP1) and ten potential TSGs obtained from previously published data. The resulting assay included EBNA1, LMP1, and three cellular TSGs: ITGA9, RASSF1A, and P16. We evaluated this assay on 64 NPC patient biopsies from Morocco, Italy, and China compared to deoxyribonucleic acid (DNA) from 20 nasopharyngeal control tissues. In the Moroccan NPC cohort (n = 44), prevalence of the EBNA1 gene showed the highest sensitivity (36/44; 82 %) with 94 % specificity. Out of eight (18 %) EBNA1 negative Moroccan samples, only three were positive for at least one methylated cellular gene. By detection of cellular marker genes, the sensitivity increased from 82 to 89 % (39/44). In the whole material of 64 biopsies from three geographical locations, at least any one marker (viral or cellular) could be detected in 91 % of biopsies with 90 % specificity. In a pilot evaluating assay performance on serum DNA from NPC and controls including samples from Italy (n = 11) and China (n = 5), at least any one marker from the MMSP assay could be detected in 88 %, but the specificity was only 50 %. CONCLUSIONS: An MMSP assay has the potential for detection of NPC by screening in high-risk populations. Serum-derived DNA seems not as good as earlier published NPC swab DNA for screening purpose.

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.

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).

A CXCR1 haplotype hampers HIV-1 matrix protein p17 biological activity.

OBJECTIVE: Monocyte inflammatory processes are fundamental events in AIDS pathogenesis. HIV-1 matrix protein p17, released from infected cells, was found to exert an interleukin (IL)-8 chemokine-like activity on human monocytes, promoting their trafficking and sustaining inflammatory processes, after binding to CXCR1. A haplotype of the CXCR1 gene (CXCR1_300_142) has been associated with slow HIV disease progression. Here, we determine how CXCR1 genetic variations impact on p17 biological activity. DESIGN/METHODS/RESULTS: Our results show that Jurkat cells overexpressing CXCR1 or the receptor carrying single polymorphism CXCR1_300 or CXCR1_142 are able to adhere and migrate in response to both IL-8 and p17. On the contrary, Jurkat cells overexpressing CXCR1_300_142 and monocytes of individuals with such CXCR1 polymorphisms lose the capacity to adhere and migrate in response to p17, but not to their physiological ligand IL-8. Surface plasmon resonance (SPR) and multispectral imaging flow cytometry showed that p17 bound with similar affinity to CXCR1 and CXCR1_300_142. Moreover, whereas p17 was able to activate CXCR1, it was incapable of functionally interacting with CXCR1_300_142 by phosphorylating extracellular signal-regulated kinase 1/2, which regulates chemokine-induced cellular responses. Finally, mutagenesis studies showed that, unlike IL-8, p17 does not use Glu-Leu-Arg-like motifs to activate CXCR1. CONCLUSIONS: Our results, showing the inability of p17 to activate CXCR1_300_142, a receptor found to be expressed on immune cells of patients with a low progression of HIV disease, point to a crucial role of p17 in AIDS pathogenesis. Our findings herein call for an exploration of the therapeutic potential of blocking the p17/CXCR1 axis in HIV infection.

Detection of HIV-1 matrix protein p17 quasispecies variants in plasma of chronic HIV-1-infected patients by ultra-deep pyrosequencing.

BACKGROUND: The HIV-1 matrix protein p17 (p17MA) is a pleiotropic protein that plays a key role in the HIV-1 life cycle. It has been long believed to have a highly conserved primary amino acid sequence and a well-preserved structural integrity to avoid severe fitness consequences. However, recent data revealed that the carboxy (COOH)-terminus of p17MA possesses high levels of predicted intrinsic disorder, which would subtend to at least partially unfolded status of this region. This finding pointed to the need of investigating p17MA heterogeneity. METHODS: The degree of intrapatient variations in the p17MA primary sequence was assessed on plasma viral RNA by using ultra-deep pyrosequencing. RESULTS: Data obtained support a complex nature of p17MA quasispecies, with variants present at variable frequency virtually in all patients. Clusters of mutations were scattered along the entire sequence of the viral protein, but they were more frequently detected within the COOH-terminal region of p17MA. Moreover, deletions and insertions also occurred in a restricted area of the COOH-terminal region. CONCLUSIONS: On the whole, our data show that the intrapatient level of sequence diversity in the p17MA is much higher than predicted before. Our results pave the way for further studies aimed at unraveling possible correlations between the presence of distinct p17MA variants and peculiar clinical evolutions of HIV-1 disease. The presence of p17MA quasispecies diversity may offer new tools to improve our understanding of the viral adaptation during the natural history of HIV-1 infection.

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.

Successes, failures and new perspectives of idiotypic vaccination for B-cell non-Hodgkin lymphomas.

The idiotype of B-cell non-Hodgkin lymphomas has been intensively investigated for its proven immunogenicity as a promising cancer vaccine. Indeed, available data clearly indicate that these vaccines are able to induce tumor-specific immune responses and molecular remissions in patients with follicular lymphoma. However, only one of the three phase III trials performed so far demonstrated a prolonged disease-free survival in vaccinated patients. The observed failures have been mainly ascribed to defects in the study design and not to the limited efficacy of idiotype vaccines per se. Therefore, innovative and optimized idiotype-based vaccine formulations are being developed in order to overcome current limitations and improve the clinical benefit of this immunotherapeutic strategy. Among the most promising advances, the development of "off-the-shelf" vaccines appears of particular relevance, being potentially able to overcome the limitations related to the complex, time-consuming and expensive production of the individualized idiotypic vaccines currently used. Moreover, there is a pressing need to identify biomarkers suitable for the identification of the subset of patients who are most likely to benefit from vaccination. Recent findings also indicate that idiotypic vaccines may be safely and successfully used in additional clinical settings, including lymphoma patients after high-dose chemotherapy and autologous stem cell transplantation.

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.

IGKV3 proteins as candidate "off-the-shelf" vaccines for kappa-light chain-restricted B-cell non-Hodgkin lymphomas.

PURPOSE: An increasing set of B-cell non-Hodgkin lymphomas (B-NHL) show a biased usage of IGKV3-20 and IGKV3-15 immunoglobulin genes, a feature that could be exploited for the development of ready-to-use, broadly applicable cancer vaccines. EXPERIMENTAL DESIGN: The immunogenic properties of clonal IGKV3-20 and IGKV3-15 proteins were analyzed with particular focus on their ability to elicit cross-reactive responses against molecularly related IGKV proteins expressed by different B-cell lymphoproliferative disorders. RESULTS: IGK+ lymphoma patients show humoral and T-cell responses to IGKV3-20 and IGKV3-15 proteins and IGKV3-specific cytotoxic T lymphocytes (CTL) can be easily induced ex vivo. IGKV3-20-specific CTLs cross-react against different IGKV3 proteins, an effect mediated by the presence of 21 shared, sometimes promiscuous, T-cell epitopes, presented by common HLA class I allele products, thus assuring a broad HLA coverage of IGKV3-based vaccines. Many natural epitope variants are carried by IGK light chains expressed by a broad spectrum of B-NHLs and we show that IGKV3-20-specific CTLs cross-react also against several of these variant epitopes. Both humoral and CTL-specific responses were induced by KLH-conjugated IGKV3-20 protein in HLA-A2-transgenic mice and coinjection of IGKV3-20-specific CTLs with IGKV3-20+ or IGKV3-15+ lymphoma cells into SCID mice totally prevented tumor growth, thus confirming the ability of these effectors to mediate efficient and cross-reactive cytotoxic responses also in vivo. CONCLUSIONS: These results provide the rationale to exploit IGKV3 proteins as "off-the-shelf" vaccines for a large fraction of lymphoma patients.