Anna Merlo

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.

Reprogramming T Lymphocytes for Melanoma Adoptive Immunotherapy by T-Cell Receptor Gene Transfer with Lentiviral Vectors

T-cell receptor (TCR) gene transfer for cancer immunotherapy is limited by the availability of large numbers of tumor-specific T cells. TCR alpha and beta chains were isolated from a highly lytic HLA-A2-restricted cytotoxic T lymphocyte (CTL) clone recognizing the melanoma-associated Melan-A/MART-1 antigen and inserted into a lentiviral vector carrying a bidirectional promoter capable of robust and coordinated expression of the two transgenes. Lentiviral vector-based gene delivery systems have shown increased transfer efficiency and transgene expression compared with the widely used gamma-retroviral vectors. This vector performed more efficiently than a gamma-retrovirus-based vector containing the same expression cassette, resulting in a T-cell population with 60% to 80% of transgenic TCR expression with mainly CD8(+) intermediate effector phenotype. Transgenic T cells specifically produced cytokine in response to and killed antigen-expressing melanoma cells, retained an overlapping functional avidity in comparison with the TCR donor CTL clone, and exerted significant therapeutic effects in vivo upon adoptive transfer in melanoma-bearing severe combined immunodeficient mice. Optical imaging showed their accumulation in the tumor site. Overall, our results indicate that lentiviral vectors represent a valid tool for stable and high-intensity expression of transgenic TCR and support clinical exploitation of this approach for therapeutic application.

Role of CD4+ Cytotoxic T Lymphocytes in the Control of Viral Diseases and Cancer

Our knowledge on the physiological role of CD4+ T lymphocytes has improved in the last decade: available data convincingly demonstrate that, besides the ‘helper’ activity, CD4+ T cells may be also endowed with lytic properties. The cytotoxic function of these effector cells has a relevant role in the control of pathogenic infections and in mediating antitumor immune responses. On these bases, several immunotherapeutic approaches exploiting the cytotoxic properties of CD4+ T cells are under investigation. This review summarizes available data supporting the functional and therapeutic relevance of cytotoxic CD4+ T cells, with a particular focus on Epstein-Barr virus (EBV)-related disorders.

Virus-Specific Cytotoxic CD4 + T Cells for the Treatment of EBV-Related Tumors

Although adoptive immunotherapy with CD8+ CTL is providing clinically relevant results against EBV-driven malignancies, the effector role of CD4+ T cells has been poorly investigated. We addressed this issue in a lymphoblastoid cell line-induced mouse model of posttransplant lymphoproliferative disease (PTLD) by comparing the therapeutic efficacy of EBV-specific CD4+ and CD8+ T cell lines upon adoptive transfer. CD4+ T cells disclosed a long-lasting and stronger proliferative potential than CD8+ T cells, had a similar activation and differentiation marker profile, efficiently killed their targets in a MHC class II-restricted manner, and displayed a lytic machinery comparable to that of cognate CD8+ T cells. A detailed analysis of Ag specificity revealed that CD4+ T cells potentially target EBV early lytic cycle proteins. Nonetheless, when assessed for the relative therapeutic impact after in vivo transfer, CD4+ T cells showed a reduced activity compared with the CD8+ CTL counterpart. This feature was apparently due to a strong and selective downmodulation of MHC class II expression on the tumor cells surface, a phenomenon that could be reverted by the demethylating agent 5-aza-2′-deoxycytidine, thus leading to restoration of lymphoblastoid cell line recognition and killing by CD4+ T cells, as well as to a more pronounced therapeutic activity. Conversely, immunohistochemical analysis disclosed that HLA-II expression is fully retained in human PTLD samples. Our data indicate that EBV-specific cytotoxic CD4+ T cells are therapeutic in mice bearing PTLD-like tumors, even in the absence of CD8+ T cells. These findings pave the way to use cultures of pure CD4+ T cells in immunotherapeutic approaches for EBV-related malignancies.

Adoptive cell therapy against EBV-related malignancies: a survey of clinical results

Background: Epstein–Barr Virus (EBV) infection is associated with a heterogeneous group of tumors, including lymphoproliferative disorders, Hodgkins disease, nasopharyngeal carcinoma and Burkitts lymphoma. As such neoplastic disorders express viral antigens, they can be treated by adoptive immunotherapy strategies relying mostly on in vitro generation and expansion of virus-specific cytotoxic T lymphocytes (CTL), which can be administered to patients for both prophylaxis and treatment. Objective: We reviewed results obtained in all clinical trials reported thus far employing anti-EBV adoptive immunotherapy for different virus-related malignancies. Methods: ‘PTLD after HSCT’, ‘PTLD after SOT’, ‘NPC’, ‘HD’, ‘SCAEBV’ and ‘extranodal NK/T cell lymphoma’, in combination with ‘Adoptive immunotherapy’ and ‘Adoptive transfer’, were used as search keys for papers in PubMed. Conclusions: Although the heterogeneity of different studies precludes their collection for a meta-analysis, it can be inferred that adoptive therapy with EBV-specific CTL is safe, well tolerated and particularly effective in the case of most immunogenic tumors, like post-transplant lymphoproliferative disease.

Immunotherapy for EBV-associated malignancies

Since 1995 to date, more than 250 patients with EBV-related diseases received virus-specific CTL. Cell therapy proved to be safe and effective, and achieved some complete remissions also in patients who failed all previous standard treatments. The first clinical results with EBV-specific CTL were obtained for both prophylaxis and treatment of post-transplant lymphoproliferative disease arising in stem cell transplant or solid organ transplant recipients. Based on such encouraging results, the same approach was then extended to other EBV-related diseases, namely Hodgkin’s lymphoma, nasopharyngeal carcinoma, and chronic active infection. Nowadays, the modification of the CTL generation protocols and the introduction of new specificities into EBV-specific CTL lines by chimeric antigen receptor transfer allow targeting other viral infections and also non-EBV related malignancies. Aim of this review is to summarize clinical results obtained thus far in adoptive cell therapy approaches with EBV-specific CTL. Moreover, by analyzing ongoing clinical trials, we also provide some insights on the potential future of a successful and paradigmatic history.

Differential expression of constitutive and inducible proteasome subunits in human monocyte-derived DC differentiated in the presence of IFN-α or IL-4

Several studies strongly suggest that DC differentiated in vitro in the presence of type I IFN acquire more potent immune stimulatory properties, compared with DC differentiated in vitro with IL‐4. However, little is known about the molecular mechanisms underlying this phenomenon. To address this question, we compared the Ag‐processing machinery (APM) profile in human DC grown in the presence of IFN‐α (IFNDC) or IL‐4 (IL‐4DC). Using a panel of APM component‐specific mAb in Western blot experiments, we found that IFNDC preferentially express inducible proteasome subunits (LMP2, LMP7, and MECL1) both at immature and mature stages. In contrast, immature IL‐4DC co‐express both constitutive (β1, β2, and β5) and inducible subunits, as shown by Western blotting analysis. In addition, immature IFNDC express higher levels of TAP1, TAP2, calnexin, calreticulin, tapasin, and HLA class I molecules than IL‐4DC. The different proteasome profiles of IFNDC and IL‐4DC were associated with a greater ability of IFNDC to present an immunodominant epitope that requires LMP7 expression for its processing. In general, these data show the impact of cytokines on APM component expression and hence the Ag‐processing ability of DC.

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.

PSMA-Specific CAR-Engineered T Cells Eradicate Disseminated Prostate Cancer in Preclinical Models

Immunology-based interventions have been proposed as a promising curative chance to effectively attack postoperative minimal residual disease and distant metastatic localizations of prostate tumors. We developed a chimeric antigen receptor (CAR) construct targeting the human prostate-specific membrane antigen (hPSMA), based on a novel and high affinity specific mAb. As a transfer method, we employed last-generation lentiviral vectors (LV) carrying a synthetic bidirectional promoter capable of robust and coordinated expression of the CAR molecule, and a bioluminescent reporter gene to allow the tracking of transgenic T cells after in vivo adoptive transfer. Overall, we demonstrated that CAR-expressing LV efficiently transduced short-term activated PBMC, which in turn were readily stimulated to produce cytokines and to exert a relevant cytotoxic activity by engagement with PSMA+ prostate tumor cells. Upon in vivo transfer in tumor-bearing mice, CAR-transduced T cells were capable to completely eradicate a disseminated neoplasia in the majority of treated animals, thus supporting the translation of such approach in the clinical setting.

Survivin Expression and Prognostic Significance in Pediatric Malignant Peripheral Nerve Sheath Tumors (MPNST)

Malignant peripheral nerve sheath tumors (MPNST) are very aggressive malignancies comprising approximately 5–10% of all soft tissue sarcomas. In this study, we focused on pediatric MPNST arising in the first 2 decades of life, as they represent one the most frequent non-rhabdomyosarcomatous soft tissue sarcomas in children. In MPNST, several genetic alterations affect the chromosomal region 17q encompassing the BIRC5/SURVIVIN gene. As cancer-specific expression of survivin has been found to be an effective marker for cancer detection and outcome prediction, we analyzed survivin expression in 35 tumor samples derived from young patients affected by sporadic and neurofibromatosis type 1-associated MPNST. Survivin mRNA and protein expression were assessed by Real-Time PCR and immunohistochemical staining, respectively, while gene amplification was analyzed by FISH. Data were correlated with the clinicopathological characteristics of patients. Survivin mRNA was overexpressed in pediatric MPNST and associated to a copy number gain of BIRC5; furthermore, increased levels of transcripts correlated with a higher FNCLCC tumor grade (grade 1 and 2 vs. 3, p = 0.0067), and with a lower survival probability (Log-rank test, p = 0.0038). Overall, these data support the concept that survivin can be regarded as a useful prognostic marker for pediatric MPNST and a promising target for therapeutic interventions.