PGE2 inhibits TIL expansion by disrupting IL-2 signalling and mitochondrial function.

Expansion of antigen-experienced CD8+ T cells is critical for the success of tumour-infiltrating lymphocyte (TIL)-adoptive cell therapy (ACT) in patients with cancer1. Interleukin-2 (IL-2) acts as a key regulator of CD8+ cytotoxic T lymphocyte functions by promoting expansion and cytotoxic capability2,3. Therefore, it is essential to comprehend mechanistic barriers to IL-2 sensing in the tumour microenvironment to implement strategies to reinvigorate IL-2 responsiveness and T cell antitumour responses. Here we report that prostaglandin E2 (PGE2), a known negative regulator of immune response in the tumour microenvironment4,5, is present at high concentrations in tumour tissue from patients and leads to impaired IL-2 sensing in human CD8+ TILs via the PGE2 receptors EP2 and EP4. Mechanistically, PGE2 inhibits IL-2 sensing in TILs by downregulating the IL-2Rγc chain, resulting in defective assembly of IL-2Rß-IL2Rγc membrane dimers. This results in impaired IL-2-mTOR adaptation and PGC1α transcriptional repression, causing oxidative stress and ferroptotic cell death in tumour-reactive TILs. Inhibition of PGE2 signalling to EP2 and EP4 during TIL expansion for ACT resulted in increased IL-2 sensing, leading to enhanced proliferation of tumour-reactive TILs and enhanced tumour control once the cells were transferred in vivo. Our study reveals fundamental features that underlie impairment of human TILs mediated by PGE2 in the tumour microenvironment. These findings have therapeutic implications for cancer immunotherapy and cell therapy, and enable the development of targeted strategies to enhance IL-2 sensing and amplify the IL-2 response in TILs, thereby promoting the expansion of effector T cells with enhanced therapeutic potential.

Phase 2b randomized trial of OX40 inhibitor telazorlimab for moderate-to-severe atopic dermatitis.

Background: Telazorlimab is a humanized anti-OX40 monoclonal antibody being studied for treatment of T-cell-mediated diseases. Objective: This randomized, placebo-controlled, phase 2b dose-range finding study investigated efficacy, safety, pharmacokinetics, and immunogenicity of telazorlimab in subjects with atopic dermatitis. Methods: In this 2-part study (NCT03568162), adults (≥18 years) with moderate-to-severe disease were randomized to various regimens of subcutaneous telazorlimab or placebo for 16 weeks' blinded treatment, followed by 38 weeks' open-label treatment and 12 weeks' drug-free follow-up. Telazorlimab treatment groups (following a loading dose) in part 1 were 300 mg every 2 weeks; 300 mg every 4 weeks; or 75 mg every 4 weeks. Part 2 evaluated telazorlimab 600 mg every 2 weeks. The primary end point was percentage change from baseline in Eczema Area and Severity Index (EASI) at week 16. Safety assessments included incidence of treatment-emergent adverse events. Results: The study randomized 313 subjects in part 1 and 149 in part 2. At 16 weeks, the least squares mean percentage change from baseline in EASI was significantly greater in subjects receiving telazorlimab 300 mg every 2 weeks (part 1) and 600 mg every 2 weeks (part 2) versus placebo (-54.4% vs -34.2% for part 1 and -59.0% vs -41.8% for part 2, P = .008 for both). Telazorlimab was well tolerated, with similar distribution of adverse events between telazorlimab- and placebo-treated subjects in both part 1 and part 2. Conclusion: Telazorlimab, administered subcutaneously at 300 mg every 2 weeks or 600 mg every 2 weeks following a loading dose, was well tolerated and induced significant and progressive clinical improvement in adults with moderate-to-severe atopic dermatitis.

Myeloid antigen-presenting cell niches sustain antitumor T cells and license PD-1 blockade via CD28 costimulation.

The mechanisms regulating exhaustion of tumor-infiltrating lymphocytes (TIL) and responsiveness to PD-1 blockade remain partly unknown. In human ovarian cancer, we show that tumor-specific CD8+ TIL accumulate in tumor islets, where they engage antigen and upregulate PD-1, which restrains their functions. Intraepithelial PD-1+CD8+ TIL can be, however, polyfunctional. PD-1+ TIL indeed exhibit a continuum of exhaustion states, with variable levels of CD28 costimulation, which is provided by antigen-presenting cells (APC) in intraepithelial tumor myeloid niches. CD28 costimulation is associated with improved effector fitness of exhausted CD8+ TIL and is required for their activation upon PD-1 blockade, which also requires tumor myeloid APC. Exhausted TIL lacking proper CD28 costimulation in situ fail to respond to PD-1 blockade, and their response may be rescued by local CTLA-4 blockade and tumor APC stimulation via CD40L.

The learning curve for the second generation of laparoscopic surgeons: lesson learned from a large series of laparoscopic adrenalectomies.

BACKGROUND: Laparoscopic adrenalectomy has a well-demonstrated learning curve in the first generation of laparoscopic surgeons. Data about the second generation of laparoscopic surgeons are lacking. METHODS: In this retrospective observational study, data from patients undergoing laparoscopic adrenalectomy from 2000 to 2019 in a high-volume center were collected and analyzed. The cumulative sum of procedures of each surgeon and the operating time were evaluated. A multivariate analysis with backward stepwise logistic regression was carried out to define which factors influenced the operative time. Three surgeons performed the analyzed procedures: a senior surgeon who began his laparoscopic activity without receiving specific training or supervision and two young surgeons, who performed their procedures under the guidance of the "senior" experienced surgeon. The first 38 procedures of the three surgeons were then compared. RESULTS: A total of 244 laparoscopic adrenalectomies were performed. Age, clinical diagnosis, side of the lesion, body mass index, comorbidities, Charlson index, American Society of Anaesthesiologists (ASA) score, and lower abdominal surgery were found to have no significant relationship with the operative time (p > 0.05). Gender, symptoms, previous upper abdominal surgery, size of the lesion, and cumulative sum of procedures were independent predictors of operative time. In the comparison between different surgeons, operative time resulted significantly longer for the senior (165 min; 140-180) than for the two junior surgeons (137.5 min; 115-160; p = 0.003 and 130 min; 120-170; p = 0.001). CONCLUSIONS: The presence of a mentor in operative theater and specific training programs could be useful during the learning period. The cumulative sum of procedures related to the operative time represents a good parameter to measure the acquired expertise of a surgeon.

TIE-2 expressing monocytes in human cancers.

Tumor-associated macrophages (TAM) are well known as a key player in the tumor microenvironment, which support cancer progression. More recently, a lineage of monocytes characterized by the expression of the TIE-2/Tek angiopoietin receptor identified a subset of circulating and tumor-associated monocytes endowed with proangiogenic activity. TIE-2 expressing monocytes (TEM) were found both in humans and mice. Here, we review the phenotypes and functions of TEM reported so far in human cancer and their potential use as markers of cancer progression and metastasis. Finally, we discuss the therapeutic approaches currently used or proposed to target TEM.

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.

TIE-2-expressing monocytes are lymphangiogenic and associate specifically with lymphatics of human breast cancer.

In experimental mouse models of cancer, increasingly compelling evidence point toward a contribution of tumor associated macrophages (TAM) to tumor lymphangiogenesis. Corresponding experimental observations in human cancer remain scarce although lymphatic metastasis is widely recognized as a predominant route for tumor spread. We previously showed that, in malignant tumors of untreated breast cancer (BC) patients, TIE-2-expressing monocytes (TEM) are highly proangiogenic immunosuppressive cells and that TIE-2 and VEGFR signaling pathways drive TEM immunosuppressive function. We report here that, in human BC, TEM express the canonical lymphatic markers LYVE-1, Podoplanin, VEGFR-3 and PROX-1. Critically, both TEM acquisition of lymphatic markers and insertion into lymphatic vessels were observed in tumors but not in adjacent non-neoplastic tissues, suggesting that the tumor microenvironment shapes both TEM phenotype and spatial distribution. We assessed the lymphangiogenic activity of TEM isolated from dissociated primary breast tumors in vitro and in vivo using endothelial cells (EC) sprouting assay and corneal vascularization assay, respectively. We show that, in addition to their known hemangiogenic function, TEM isolated from breast tumor display a lymphangiogenic activity. Importantly, TIE-2 and VEGFR pathways display variable contributions to TEM angiogenic and lymphangiogenic activities across BC patients; however, combination of TIE-2 and VEGFR kinase inhibitors abrogated these activities and overcame inter-patient variability. These results highlight the direct contribution of tumor TEM to the breast tumor lymphatic network and suggest a combined use of TIE-2 and VEGFR kinase inhibitors as a therapeutic approach to block hem- and lymphangiogenesis in BC.

Bone marrow-derived cells are implicated as a source of lymphatic endothelial progenitors in human breast cancer.

Bone marrow-derived endothelial progenitor cells (EPCs) infiltrate into sites of neovascularization in adult tissues and mature into functional blood endothelial cells (BECs) during a process called vasculogenesis. Human marrow-derived EPCs have recently been reported to display a mixed myeloid and lymphatic endothelial cell (LEC) phenotype during inflammation-induced angiogenesis; however, their role in cancer remains poorly understood. We report the in vitro differentiation of human cord blood CD133+CD34+ progenitors into podoplanin+ cells expressing both myeloid markers (CD11b, CD14) and the canonical LEC markers vascular endothelium growth factor receptor 3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and prospero homeobox 1 (PROX-1). These podoplanin+ cells displayed sprouting behavior comparable to that of LECs in vitro and a dual hemangiogenic and lymphangiogenic activity in vivo in an endothelial cell sprouting assay and corneal vascularization assay, respectively. Furthermore, these cells expressed vascular endothelium growth factor (VEGF) family members A, -C, and -D. Thus, bone-marrow derived EPCs stimulate hemangiogenesis and lymphangiogenesis through their ability to differentiate into LECs and to produce angiogenic factors. Importantly, plasma from patients with breast cancer induced differentiation of CD34+ cord blood progenitors into hemangiogenic and lymphangiogenic CD11b+ myeloid cells, whereas plasma from healthy women did not have this effect. Consistent with these findings, circulating CD11b+ cells from breast cancer patients, but not from healthy women, displayed a similar dual angiogenic activity. Taken together, our results show that marrow-derived EPCs become hemangiogenic and lymphangiogenic upon exposure to cancer plasma. These newly identified functions of bone-marrow derived EPCs are expected to influence the diagnosis and treatment of breast cancer.

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.

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.

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.

Differential down-modulation of HLA class I and II molecule expression on human tumor cell lines upon in vivo transfer.

Previous evidence from our laboratory showed that Epstein-Barr virus-immortalized lymphoblastoid B cells undergo a prominent down-modulation of HLA-II molecule expression when injected intraperitoneally in SCID mice, while HLA-I remains almost unaffected. Since this phenomenon can alter the experimental outcome of therapeutic protocols of adoptive cell therapy, we decided to evaluate the behavior of MHC antigens in a panel of cell lines belonging to the B- and T-cell lineages, as well as in epithelial tumor cell lines. Cells were administered in mice either intraperitoneally or subcutaneously and recovered 4 days later for HLA molecule expression analysis. Collected data showed a highly heterogeneous in vivo behavior of the various cell lines, which could alternatively down-modulate, completely abrogate or maintain unchanged the expression of either MHC-I or MHC-II molecules. Moreover, the site of injection impacted differentially on these aspects. Although such phenomena still lack a comprehensive clarification, epigenetic mechanisms are likely to be involved as epigenetic drugs could partially counteract MHC down-modulation in vivo. Nonetheless, it has to be pointed out that careful attention must be paid to the assessment of therapeutic efficacy of translational protocols of adoptive immunotherapy, as modulation of MHC molecules on human target cells when transferred in a mouse environment could readily interfere with the desired and expected therapeutic effects.

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.

Impact of γ-chain cytokines on EBV-specific T cell cultures.

BACKGROUND: Recent preclinical adoptive immunotherapy studies in murine models prompt to employ "proper" rather than "as many as possible" antigen-specific T cells to gain better therapeutic results. Ideally, "proper" T cells are poorly differentiated in vitro, but retain the capacity to fully differentiate into effector cells in vivo, where they can undergo long-term survival and strong proliferation. Such requirements can be achieved by modifying culture conditions, namely using less "differentiating" cytokines than IL-2. METHODS: To evaluate this issue in human T cell cultures, we exploited a well characterized and clinical-grade protocol finalized at generating EBV-specific CTL for adoptive immunotherapy. In particular, we studied the impact of IL-7, IL-15 and IL-21 compared to IL-2 on different aspects of T cell functionality, namely growth kinetics, differentiation/activation marker expression, cytokine production, and short-term and long-term cytotoxicity. RESULTS: Results disclosed that the culture modifications we introduced in the standard protocol did not improve activity nor induce substantial changes in differentiation marker expression of EBV-specific CTL. CONCLUSIONS: Our data indicated that the addition of γ-chain cytokines other than IL-2 for the generation of EBV-specific T cell cultures did not produce the improvements expected on the basis of recent published literature. This fact was likely due to the intrinsic differences between murine and human models and highlights the need to design ad hoc protocols rather than simply modify the cytokines added in culture.

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.

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.

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, Hodgkin's disease, nasopharyngeal carcinoma and Burkitt's 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.