BARF1 AS A NEW THERAPEUTIC TARGET FOR EBV-ASSOCIATED MALIGNANCIES.

While Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) have been used successfully for the prophylaxis and treatment of the highly immunogenic post-transplant lymphoproliferative disorders, the clinical experience for other EBV-associated malignancies, such as Hodgkin's lymphoma and undifferentiated nasopharyngeal carcinoma (NPC), is limited and the results obtained so far indicate that EBV-CTLs are less effective in these settings. Decreased CTL efficacy most likely reflects immune evasion strategies by tumor cells, including down-regulation of immunodominant EBV proteins and the weak immunogenicity of the viral proteins expressed. One of the possible approaches to overcome these limitations is the identification of additional immunogenic viral proteins expressed by tumor cells that may serve as tumor-associated antigens to be targeted by improved CTL induction and expansion protocols. We have recently demonstrated that NPC patients show strong spontaneous CD4+ and CD8+ T cell responses specific for the EBV-encoded oncogenic protein BARF1. We also showed that BARF1 provides immunogenic HLA-A*0201-restricted epitopes, suggesting that exploitation of the immunogenic features of this viral antigen may help improve the current immunotherapeutic strategies for EBV-associated malignancies. On these grounds, we characterized more extensively the immunogenic properties of BARF1 with the final goal to develop improved protocols of adoptive immunotherapy based on the use of EBV-CTLs enriched in BARF1-specific effectors. In particular, we identified and validated additional BARF1 CTL epitopes presented in the context of common HLA class I alleles. These results strictly correlate to those deriving from a high-resolution HLA genotyping of a large series of NPC, giving a precise estimate of the immunogenicity of BARF1 in relation to the HLA class I profile of Italian NPC patients. To fully exploit the immunologic properties of BARF1, we are also developing and characterizing BARF1-specific monoclonal antibodies that may be of both diagnostic and therapeutic usefulness in these clinical settings. In future perspective, the proposed research may provide a strong rationale for the clinical application of improved adoptive immunotherapy protocols for the treatment of EBV-associated malignancies, particularly the less immunogenic forms, such as NPC and, possibly, Hodgkin’s lymphoma

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

High serum levels of soluble CD40-L in patients with undifferentiated nasopharyngeal carcinoma: pathogenic and clinical relevance

BACKGROUND: Engagement of CD40 promotes survival of undifferentiated nasopharyngeal carcinoma (UNPC) cells and similar effects are induced by the EBV oncoprotein LMP-1 that is expressed in a fraction of cases. Considering that CD40 may be activated also by the soluble isoform of CD40L (sCD40L), we investigated the serum levels of sCD40L in a series of 61 UNPC patients from Italy, a non-endemic area for this disease. RESULTS: At diagnosis, serum samples of UNPC patients contained significantly higher levels of sCD40L than age-matched healthy controls (p < 0.001). High levels of sCD40L (i.e., >18 ng/ml) were more frequently found in patients <40 years of age (p = 0.03) and with distant metastases at presentation (p = 0.03). Serum levels of sCD40L were inversely associated with the expression of the EBV oncoprotein LMP-1 (p = 0.03), which mimics a constitutively activated CD40. The amount of sCD40L decreased in a fraction of patients treated with local radiotherapy alone. Moreover, CD40L(+ )lymphoid cells admixed to neoplastic UNPC cells were detected in cases with high serum levels of sCD40L, suggesting that sCD40L is probably produced within the tumor mass. CONCLUSION: sCD40L may contribute to CD40 activation in UNPC cells, particularly of LMP-1-negative cases, further supporting the crucial role of CD40 signalling in the pathogenesis of UNPC. sCD40L levels may be useful to identify UNPC patients with occult distant metastases at presentation

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)

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-1β, 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

BARF1 AS A NEW THERAPEUTIC TARGET FOR EBV-ASSOCIATED MALIGNANCIES.

While Epstein-Barr virus-specific cytotoxic T lymphocytes (EBV-CTLs) have been used successfully for the prophylaxis and treatment of the highly immunogenic post-transplant lymphoproliferative disorders, the clinical experience for other EBV-associated malignancies, such as Hodgkin's lymphoma and undifferentiated nasopharyngeal carcinoma (NPC), is limited and the results obtained so far indicate that EBV-CTLs are less effective in these settings. Decreased CTL efficacy most likely reflects immune evasion strategies by tumor cells, including down-regulation of immunodominant EBV proteins and the weak immunogenicity of the viral proteins expressed. One of the possible approaches to overcome these limitations is the identification of additional immunogenic viral proteins expressed by tumor cells that may serve as tumor-associated antigens to be targeted by improved CTL induction and expansion protocols. We have recently demonstrated that NPC patients show strong spontaneous CD4+ and CD8+ T cell responses specific for the EBV-encoded oncogenic protein BARF1. We also showed that BARF1 provides immunogenic HLA-A*0201-restricted epitopes, suggesting that exploitation of the immunogenic features of this viral antigen may help improve the current immunotherapeutic strategies for EBV-associated malignancies. On these grounds, we characterized more extensively the immunogenic properties of BARF1 with the final goal to develop improved protocols of adoptive immunotherapy based on the use of EBV-CTLs enriched in BARF1-specific effectors. In particular, we identified and validated additional BARF1 CTL epitopes presented in the context of common HLA class I alleles. These results strictly correlate to those deriving from a high-resolution HLA genotyping of a large series of NPC, giving a precise estimate of the immunogenicity of BARF1 in relation to the HLA class I profile of Italian NPC patients. To fully exploit the immunologic properties of BARF1, we are also developing and characterizing BARF1-specific monoclonal antibodies that may be of both diagnostic and therapeutic usefulness in these clinical settings. In future perspective, the proposed research may provide a strong rationale for the clinical application of improved adoptive immunotherapy protocols for the treatment of EBV-associated malignancies, particularly the less immunogenic forms, such as NPC and, possibly, Hodgkin’s lymphoma

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

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

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