We analysed the clinical features of patients with lymph node-reactive hyperplasia or Hodgkin's lymphoma and found no differences between the two groups. Therefore, we raised questions about why people in the same area and same living environment with the same population attributes would have different disease outcomes after infection with Epstein‒Barr virus[9, 10].We suspected that this difference in outcome was the result of an interaction between the body's own immune system and external antigenic stimulation, which would be related to differences in the antigens recognized by he immune cells on the one hand and the microenvironment of the tumour cells on the other hand.
Differences in immune cell activity after EBV infection
As basic components of the immune system, lymphocytes are widely distributed in the body and mainly include T lymphocytes, B lymphocytes, NK cells, etc. These cells are the first line of defence against external antigens[11].The process of the immune response depends on recognition between immune cells. A functional imbalance in these cells usually means disorder in the immune system and is an obstacle to an immune response[12].Functional molecules on the cell surface are the basis for mutual recognition among immune cells[13], and the antigenic determinants cluster of differentiation (CD) molecules are one type of functional molecule on the cell surface. CD3 is expressed on the surface of all T cells, making it a surface hallmark of T cells. CD4 and CD8 can be used to distinguish T-lymphocyte subsets. Helper T cells mainly express CD4, while cytotoxic T cells mainly express CD8[14];CD19 is a key auxiliary receptor for B-cell antigen signal transduction and can be used to identify B cells. CD56 is expressed in all natural killer (NK) cells[15], which are an important part of the innate immune system. NKT cells are a special subset of T cells with both T-cell receptors and NK-cell receptors on the cell surface. Under normal circumstances, the ratio of CD4+/CD8 + T lymphocytes in the body is in a state of dynamic balance to maintain stable immune function in the body. Our results showed that compared with the RLH group, the HL group exhibited a decreased proportion of CD4 + T cells in lymphocytes, an increased proportion of CD8 + T cells, and a decreased ratio of CD4+/CD8 + T cells, at the same time, the proportion of NK cells was decreased (P < 0.05), suggesting that immune function was disrupted after EBV infection in patients with Hodgkin's lymphoma, making these patients unable to effectively initiate an immune response[16],and indicating that the immunoregulatory function of NK cells and the ability to clear virally infected cells were also reduced[17].Compared with single-cell sequencing, the dominant cell cannot be identified, but it can indicate the defect of partial immune function.
B lymphocytes, as the most important infection target of EBV, are also important antigen-presenting cells. Live Epstein‒Barr virus adheres to the surface of B lymphocytes in the form of the HLA-DPB1:EBV complex and enters B lymphocytes through the cell membrane with the assistance of various glycoproteins to establish a latent infection[8]. Once the external stimulus or living environment changes, the recognition ability of CD4 + T cells and the clearance ability of NK cells decrease, which not only prevents effective clearance of the complex containing HLA-DPB1 and EBV but can also assist EBV in progressing from the latent infection stage to the lytic infection stage, and viral replication and cytotoxicity are enhanced accordingly. These changes promote the continuous malignant proliferation of B lymphocytes, which leads to Hodgkin's lymphoma[18].
Inhibitors of the immune checkpoint Processed Death-1/Programmed Death Ligand-1 (PD-1/PD-L1) have shown relatively high efficacy and low toxicity in the treatment of melanoma, non-small cell lung cancer, and renal cell carcinoma[19], and this checkpoint pathway is currently a focus in research and the development of antitumour therapies. PD-1 is widely expressed in activated B lymphocytes, T lymphocytes and myeloid cells, high rates of PD-1 expression can be detected in most tumours but are rarely detected in normal human tissues[20].PD-L1 is mainly expressed in helper T lymphocytes, highly expressed PD-1 can bind with PD-L1 produced by immune cells in the microenvironment to induce immune tolerance, killing effect of T lymphocytes and assisting tumour cells in avoiding immune surveillance and achieving immune escape[21, 22]. It has been suggested that overexpression of PD-L1 can support the survival of HRS cells and interactions with PD-1-expressing T lymphocytes. It leads to reduced T-lymphocyte activation, proliferation inhibition, and cytokine production and plays an important role in shaping the Hodgkin lymphoma microenvironment. The current data mostly define high expression of PD-L1/PD-1 as positive staining of greater than 50% cells and low expression as positive staining of less than 25%[23].Süleyman et al. showed that the positive rate of PD-L1 expression in HRS cells in Hodgkin's lymphoma was as high as 89.2%, while in our test, PD-L1 exhibited low expression (20.22%) while PD-1 exhibited high expression (51.14%). This is a distinct feature of EBV-positive Hodgkin's lymphoma in Xinjiang compared to that in other regions, and it also suggests that Hodgkin's lymphoma patients in Xinjiang may be more inclined to benefit from anti-PD-1 inhibitors. Most studies have shown that people with PD-L1/PD-1 overexpression can benefit from treatment with immune checkpoint inhibitors[24], there are also some limitations: patients who respond to PD-1/PD-L1 blockers achieve obvious effects, but some patients do not respond. To achieve efficacy with PD-1/PD-L1 blockers, corresponding biomarkers need to be found[25].
For example, some studies suggest that the expression of MHC-I on tumour cells is a prerequisite for successful anti-PD-1/PD-L1 therapy and that the expression of MHC-I may be an important predictive biomarker.Approximately 88% of Hodgkin's lymphoma cells exhibit surface MHC-I, MHC-II, or HLA-DM expression, all of which may impact immune checkpoint-mediated suppression[26].One study also noted that an increase in the Epstein‒Barr virus load might stimulate the expression of HLA-DPB1 molecules.HLA-DPB1 is an HLA-Ⅱ antigen with high genetic polymorphism.How anti-PD-1/PD-L1 therapy play? In addition to allowing activation of cytotoxic T lymphocytes, inhibition of the receptor PD-1 can also directly kill tumour cells producing major histocompatibility complex class I (MHC-I) molecules.There is no significant difference was found in HLA-DPB1, PD-1 or PD-L1 between two groups in our study, alought both HLA-DPB1 and PD-1 were overexpressed,we also cannot deny the correlation between HLA-DPB1 and the strength of the immune response. We completed an HLA-I/II typing test in Xinjiang previously,and found HLA-DPB1*1701are responsible for the differences in immune responses.That is why we focus on HLA-DPB1 and PD-1.
Differences in the expression of inflammatory factors
In the early stage of cancer, inflammation is usually associated with cancer development and progression[27][28].Exogenous inflammation in tumours includes bacterial, viral and autoimmune diseases,these inflammatory factors can provide feedback and regulate the inherent inflammation in tumours. Both endogenous inflammation and exogenous inflammation can lead to immune suppression, produce conditions conducive to the occurrence of tumours, and provide a supportive environment for the development of tumours[29].Among inflammatory factors, macrophages are an important bridge between inflammation and the occurrence of cancer. Tumour-associated macrophages (TAMs) can provide various cytokines, including TNF-α, IL-6, IL-8, and IL-1, which tend to accumulate at the site of inflammation and play immune functions in a variety of acute and chronic inflammatory diseases[30].In our study, above factors measured were significantly different, with most significantly increased factors being IL-18, IL-33, MCP-1, IL-8, IL-6 and TNF-α, which suggested that TAMs played an important role in the activated inflammatory response after EBV infection. In addition, activated B cells also produce a large number of cytokines involved in immune regulation, inflammation and the haematopoietic response. CD4 + T cells expand the immune response[31]and promote the proliferation and differentiation of immune cells through cytokines such as TNF-α, IFN-γ, IL-2 and IL-4. These factors participate in tumor cell differentiation, create various growth conditions for HRS cell survival and prevent the occurrence of various antitumour immune response processes of external origin.
Studies have shown that HRS cells, in addition to their inherent malignant characteristics, secrete factors such as TGF-B, IL-10 and the chemokine TARC, which support the response by Th2 cells. IL-10 and TGF-β are common in the Hodgkin's lymphoma environment and can not only reduce the production of IFN-γ but also facilitate the aggregation of NK cells. HRS cells also secrete soluble CD25, which binds to IL-2 and blocks interactions with NK cells expressing IL-2 receptors.
There were significant differences the expression of various inflammatory factors between the two groups. The expression of these inflammatory factors was higher in the Hodgkin lymphoma group than in the RLH group. In some cancers, inflammatory conditions precede the development of malignancy, or promote inflammatory environmental changes. We believe that inflammatory responses, especially chronic inflammatory responses, play a supporting role in the pathogenesis of Hodgkin's lymphoma.
Differences in response products produced by tumor cells
After invading the host, EBV produces viral proteins with the help of the host and can also produce downstream metabolites by regulating signaling pathways. These proteins are isohexoproteins recognized by the host immune system, which can induce an immune response. LMP1, CD30, and PD-L1 are detected on the surface of only tumor cells[32], they are not expressed or only rarely detected in normal cells. They are usually synchronous with the growth of tumor cells and are believed to regulate the growth and expansion of tumor cells. In multiple studies, they have been shown to be produced after EBV infection and have been identified as future targets for immunotherapy [33].In our study, these three proteins were highly expressed in the Hodgkin's lymphoma group(P < 0.05), so these three proteins have specific diagnostic value in this disease.
EBV-encoded LMP-1 is a recognized oncogene and an EBV product. LMP1 transduces a unique durable activation signal[34], binding to tumor necrosis factor (TNF) receptor-associated factor (TRAF) and initiating a signaling cascade leading to the activation of the transcription factors NF-κB, AP-1 and STAT[35], LMP-1 promotes the expression of PD-L1 by regulating the AP-1 and JAK/STAT pathways[36–38].An immunosuppressive microenvironment can also be maintained by inducing the production of immunosuppressive factors and the expression of PD-1[39].At present, there are many studies on LMP-1, and its role has been relatively clearly elucidated, but there are few studies on the correlation between LMP-1 and PD-L1 in Hodgkin's lymphoma.
CD30, a member of the tumor necrosis factor (TNF) receptor superfamily, is the most specific biomarker for classical Hodgkin's lymphoma and is overexpressed on HRS cells[40], in which it drives signal transduction in a ligand-independent manner. The regulatory effect of CD30 on tumor cells is mediated by regulating cell growth and lymphoblastic transformation through the NF-κB pathway. High expression of CD30 in HRS cells activates the downstream JAK/STAT signaling pathway and PI3K/Akt/mTOR signaling pathway [41],which are involved in the development of Hodgkin's lymphoma. In tumour cell lines and Hodgkin's lymphoma xenograft models, administration of bortezomib, a proteasome inhibitor that inhibits NK-kB activation, has been shown to help overcome apoptosis resistance and achieve synergy with cytotoxic T cells[32].Given that CD30 is described in more than 50% of Hodgkin's lymphoma patients and has been explicitly used as an indicator for differential diagnosis, clinical trials testing antibodies against CD30 have been made great progress in the treatment of refractory and relapsing Hodgkin's lymphoma[42]: In a phase I trial assessing brentuximab vedotin (BV, SSN-35), 86% of refractory/relapsing Hodgkin's lymphoma cases were effectively controlled, and in a phase II trial, the 3-year overall survival (OS) and progression-free survival (PFS) rates were 73% and 57%, respectively. Drugs targeting CD30 do not have the same variety of side effects as chemotherapeutic drugs, and patients with a generally poor condition can still benefit[2, 43].In addition, CD30 has been explored as a target antigen for CAR-T cells, resulting in a complete response in 2 patients (8%), a partial response in 30% of patients, and stable disease in 35% of patients[44, 45].It seems necessary to continue to explore targeted therapies against CD30 overexpression in future studies.
As mentioned above, both LMP-1 and CD30 can be involved in the pathogenesis of Hodgkin's lymphoma through the regulation of the NF-κB signaling pathway, and some studies have suggested that dysregulation of NF-κB activity is a common feature of Hodgkin's lymphoma. In Xinjiang, EBV-positive Hodgkin's lymphoma also exhibited a similar feature. We detected downstream products of the NF-κB signaling pathway [46],the expression rates of P65, IKBα and IKKα were significantly increased in the disease group, while the expression of RIP2 was significantly decreased, indicating that the NF-κB signaling pathway was activated in the pathogenesis of Epstein Barr virus-positive Hodgkin's lymphoma.
Tumor Microenvironment composed of immune cells, inflammatory factors and tumor cells
Stephen Paget proposed the "seed and soil theory", stating that the growth and colonization of tumor cells require compatibility between the intrinsic properties of tumor cells and their microenvironment[47–49].Traditional cancer studies isolate cancer cells from their environment, but this often fails to reflect the real situation, as it ignores the interactions between tumor cells and their coevolved microenvironment. The interactions between cancer cells and neighbouring cells (including immune cells) determine the main characteristics of a tumor. Aleksandra et al. proposed that the cellular composition and functional properties of the tumor microenvironment can be regarded as the result of the interactions between tumor cells and immune cells, which are mediated by a large network of cytokines and chemokines[26, 50].The differences in the activities of immune cells, inflammatory factors, and tumour metabolites together constitute the microenvironment affecting the growth of Hodgkin's lymphoma cells[51].That is, when tumor cells change their own metabolism to adapt to the environment, they further change the microenvironment in which they live[52], different components play multiple roles in tumour and immune system. The activation of key intracellular signalling pathways regulates cell expansion and limits cell-mediated immune effects, leading to the persistence of malignant tumours. As shown in Fig. 7, following infection with EB virus, T-lymphocyte immune function is disrupted, and NK cells cannot effectively play a role in clearance [52]. EB virus combines with HLA-DPB1 on the cell surface to enter B lymphocytes in the form of a complex. At the same time, immune cells produce response products and inflammatory factors through different pathways, which change the tumour microenvironment and provide a growth environment for lymphoma cells. The above steps are involved in the development of Hodgkin's lymphoma. It is precisely because of the diversity of microenvironmental components and functional complexity that immunotherapy for Hodgkin's lymphoma has not made further progress. The complexity of the microenvironment can also be explained by the different disease outcomes observed with the same EBV strain and the same population with the same attributes.
At present, studies on the correlations between tumor microenvironment components or blood-based biomarkers and patient prognosis are relatively comprehensive, but these correlations are only partially understood and are not used to guide the selection of treatment strategies in clinical practice. With the progress in molecular technology, combined with various enrichment strategies, key pathogenic concepts and characteristic pathways have been discovered, helping us continue to explore specific indicators in the microenvironment to select the best approach for patients before the start of treatment to avoid treatment failure and unnecessary toxicity[53].