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Both the EBV proteins  and viral ncRNAs  have known oncogenic properties in human cells. Because the immune evasion is an important feature of cancers in general , the modulation of immune responses by the EBV proteins (e.g., LMP1 ) and viral ncRNAs [18,19] can be instrumental for virus-induced carcinogenesis. Expanding our understanding of how the EBV infection hacks the immune system by exploiting the immune checkpoints can sheds new light on the mechanisms of viral carcinogenesis and cancer biology, providing new and important opportunities for better management of these potentially lethal diseases. Therefore, herein, we summarize the state of the knowledge on this topic, aiming to highlight the impact of the immune checkpoints regulation by viral products in the EBV-associated cancers, as well as the consequences considering new therapies based on immune checkpoint blockade. For more details about the interplay between the EBV and immune regulatory molecules, we recommend a recent review by Münz (2021) .
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The HL is divided into the classical form (cHL), most cases, and the rare nodular lymphocyte-predominant form (NLPHL). The tumor has a scarce population of giant neoplastic cells, referred to as Hodgkin or Reed Sternberg (HRS) cells, surrounded by a variable and heterogeneous repertoire of reactive non-neoplastic cells . The EBV is detected in HRS cells in roughly half of all cHL cases, and in all cHL arising in people living with infection by the human immunodeficiency virus (HIV) ; conversely, it is typically absent in NLPHL, except for a few reports [27,28]. The HRS cells have acquired somatic mutations in the variable (V) segment of the IGH gene, which is indicative that the HL clonally arises from GC or post-GC B-cells [29,30].
Currently the literature has more data on some immune regulatory molecules, such as PD-1 and its ligands and CTLA-4, compared to others. Rather than a reliable proxy of the relevance of these molecules in the context of immune checkpoints, this may represent a bias based on the seminal publications about these molecules and their role in the immune system. Furthermore, some immune checkpoints regulators also received more attention from the scientific community after being successfully targeted for immune checkpoint blockade, aiming the development of new protocols in this emerging category of treatment for the management of human cancers.
The association between the EBV infection and the expression of PD-Ls were previously investigated using a humanized mouse model of DLBCL: while mock-infected B-cells had low levels of PD-L1 and low to no expression of PD-L2 (assessed by flow cytometry), both PD-L1 and PD-L2 had increased expression in the same cells when infected by EBV viral strains B95.8 and M81. Interestingly, the authors identified that some neoplastic cells showed EBV EBNA2 and PD-L1 co-expressed by immunohistochemistry (IHC); remarkably, T-cells from the EBV-positive lymphoma model expressed PD-1 and CTLA-4 on their surface, and antibody blockade of these receptors caused a substantial increase of EBV-specific T-cell responses, reducing the lymphoma burden and increasing the survival of mice . This result is consistent with the T-cell mediated control of lymphoma being impaired by the interaction of PD-1 and CTLA-4 with their ligands produced by the EBV-infected B-cells, mostly neoplastic ones.
Encouraging results on the usefulness of PD-1/PD-L1 blockade to treat lymphomas were reported in different clinical trials in the last decade. For instance, the treatment of adult patients with relapsed/refractory cHL with the anti-PD-1 antibody nivolumab was well tolerated and allowed better overall response ("type":"clinical-trial","attrs":"text":"NCT01592370","term_id":"NCT01592370"NCT01592370, "type":"clinical-trial","attrs":"text":"NCT02181738","term_id":"NCT02181738"NCT02181738) [78,79]. Similar results with relapsed/refractory cHL were also observed with another anti-PD-1 drug, pembrolizumab, in a Phase II trial ("type":"clinical-trial","attrs":"text":"NCT02453594","term_id":"NCT02453594"NCT02453594) , and this ICI drug also provided a better overall response for adult patients with relapsed/refractory primary MBCL ("type":"clinical-trial","attrs":"text":"NCT02576990","term_id":"NCT02576990"NCT02576990). Nivolumab was the first ICI against PD-1 approved in the USA for use in hematological malignancies, approved for adult patients with relapsed/refractory cHL Pembrolizumab is approved for adult and pediatric patients with relapsed/refractory cHL, and for relapsed/refractory primary MBCL patients who relapse after two or more lines of primary therapy.
Despite the known or anticipated advantages of using ICIs to treat cancers that express a high level of its regulators, immune checkpoint blockade protocols are not free of limitations and caveats to be considered and better understood. For instance, the use of ICIs drugs can lead to an increased number of hyper-responsive T-cells in the periphery, resulting in inflammatory toxicities, known as immune-related adverse events (irAEs). Most irAEs are light to mild, although, in rare cases, can lead to death. Overall, they are well-tolerated and do not adversely affect the treatment . Besides irAEs, one of the major characteristics that hampers a more successful use of ICIs is therapy resistance, essentially categorized as primary, adaptative, or acquired .
Another important point is that more immunogenic tumors usually have better responses to ICIs, and the composition and humoral profile of the tumor immune microenvironment (TIME) is a key factor affecting the success of immune checkpoint blockade strategies. The TIME can be classified as cold, when the tumor lacks immune cell infiltrates, or hot, on tumors highly inflamed, showing a high number of T-cells and high production of pro-inflammatory cytokines. Hot TIMEs tend to respond better to immunotherapy, including PD-1/PD-L1 blockade . For instance, the TIME in cHL benefits immune checkpoint blockade therapy, as this lymphoma features extensive non-malignant immune infiltrate with CTLA-4+ T-cells , in addition to the very scarce neoplastic HRS cells that commonly express PD-Ls [85,86]. Conversely, BL consists of over 90% of malignant cells, with a very scarce TIME formed by some macrophages and rare T-cells. Furthermore, myc alterations in BL provide growth and survival signals to the neoplastic cells (which could explain the independence of a microenvironment for survival) , and they lack significant PD-L1 expression [85,88]. These features make it very challenging to design therapeutic protocols with immune checkpoint blockade for BL treatment, and there are no approved drugs for this disease so far.
Cancer cells may also not respond to immune checkpoint blockade due to the absence of tumor antigens, or by mechanisms that impact the MHC-mediated antigen presentation, allowing the immune escape of the neoplastic cells. Loss or dysfunctions in genes encoding components of the antigen processing machinery, such as beta-2-microglobulin (B2M) or MHC class I molecules, have been shown to mediate checkpoint blockade resistance . Mutations in the B2M gene or loss of MHC are found in different lymphomas, but they appear to be less frequent in those associated with EBV infection. In tumor biopsies of 111 DLBCL patients, B2M mutations were found in GC-type DLBCL and ABC/NOS subtypes, but not in BL samples . Thus, it is an open question whether the resistance to immune checkpoint blockade due to loss/dysfunctions of antigen processing genes is eventually less frequent in EBV-positive lymphomas.
Cyclin-dependent kinases (CDK) 4 and 6 are important drivers of the cell cycle and are relevant to oncogenesis as they promote the progression of many cancers. In vivo studies of solid cancers have shown that inhibitors of CDK4/6 potentiate antigen presentation of tumor cells, increase expression of IFNγ of effector T-cells, and decrease expression of inhibitory immune checkpoints (e.g., PD-1, PD-L1, TIM-3) in CD8+ T-cells. In this context, treating tumors with CDK4/6 inhibitors greatly enhances the effects of anti-PD-1 therapy [91,92]. In a study published in 2009 , a potential role of B7-H4 was demonstrated in inducing growth-arrest of EBV+ lymphoblastoid cell lines in vitro, by downregulating gene expression of CDKs (including CDK4/6) and cyclins. Although better proof-of-concept data are necessary to conclude whether EBV-associated cancers may behave differently regarding immune checkpoint blockade response, these results provide a hint on whether cancers associated with EBV infection can achieve a more favorable response to ICIs.
Immune checkpoint molecules are promising anticancer targets, among which therapeutic antibodies targeting the PD-1/PD-L1 pathway have been widely applied to cancer treatment in clinical practice and have great potential. However, this treatment is greatly limited by its low response rates in certain cancers, lack of known biomarkers, immune-related toxicity, innate and acquired drug resistance, etc. Overcoming these limitations would significantly expand the anticancer applications of PD-1/PD-L1 blockade and improve the response rate and survival time of cancer patients. In the present review, we first illustrate the biological mechanisms of the PD-1/PD-L1 immune checkpoints and their role in the healthy immune system as well as in the tumor microenvironment (TME). The PD-1/PD-L1 pathway inhibits the anticancer effect of T cells in the TME, which in turn regulates the expression levels of PD-1 and PD-L1 through multiple mechanisms. Several strategies have been proposed to solve the limitations of anti-PD-1/PD-L1 treatment, including combination therapy with other standard treatments, such as chemotherapy, radiotherapy, targeted therapy, anti-angiogenic therapy, other immunotherapies and even diet control. Downregulation of PD-L1 expression in the TME via pharmacological or gene regulation methods improves the efficacy of anti-PD-1/PD-L1 treatment. Surprisingly, recent preclinical studies have shown that upregulation of PD-L1 in the TME also improves the response and efficacy of immune checkpoint blockade. Immunotherapy is a promising anticancer strategy that provides novel insight into clinical applications. This review aims to guide the development of more effective and less toxic anti-PD-1/PD-L1 immunotherapies.