The tumor microenvironment of classical Hodgkin lymphoma (cHL) is clearly responsible for the maintenance of the malignant Hodgkin-Reed-Sternberg (HRS) cells, and Epstein-Barr virus (EBV) has been shown to play a role in this immune evasion. EBV can increase the migration of CD4(+)CD25(+)FOXP3(+) lymphocytes, named regulatory T cells (Tregs). In this study, we assessed the distribution and biological significance of Tregs in patients with cHL. Tissue microarrays were constructed using diagnostic biopsies available in 130 cHL patients and stained with CD4, CD8, CD25, and FOXP3 antibodies. For the present study, only cHL patients whose histology could be confirmed and EBV association established were studied. From the 130 cHL patients selected for this study, 56 were classified as EBV-related and 74 EBV non-related cHL. There were no association between clinical characteristics and the expression of Tregs. However, higher levels of Tregs correlated with EBV presence on HRS cells (p = 0.02), although it did not influence event-free survival (EFS) and overall survival (p = 0.98 and p = 0.59, respectively). This study demonstrates that Tregs expression correlates with EBV presence in HRS cells and has no impact on survival of patients with cHL. Further studies investigating the mechanisms in which EBV recruits Tregs to the tumor microenvironment will contribute not only to our understanding on the pathogenesis of cHL but also to the development of new therapeutic strategies.
Clinical and histological features of classical Hodgkin lymphoma (cHL) are primarily due to the effects of cytokines and enzymes produced by Hodgkin/Reed-Sternberg (HRS) cells and their surrounding inflammatory cells. In EBV-related cancers, the expression of viral latent membrane protein 1 correlates with an increased MMP9 expression. In this study, we evaluated the prognostic relevance of MMP9 expression and EBV status in HRS cells in patients with cHL in Brazil. We selected 97 patients with cHL for EBV and MMP9 detection. EBV was detected in 52.5%, and MMP9 expression positivity was found in 87.6%. Of all cases, there was no correlation between MMP9 expression and EBV status. Response to treatment and relapse rate was independent of MMP9 expression and EBV status. MMP9 positivity did not influence overall survival and event-free survival. The consistent and increased intensity of MMP9 expression in HRS cells make this enzyme a potential target for therapy.
Introduction Although immunosuppression has long been recognized in classical Hodgkin lymphoma (cHL), the underlying basis for the lack of an effective immune response against the tumor remains unclear. Increased frequencies of regulatory CD4+ T lymphocytes in the tumor microenvironment and peripheral blood have been proposed as one of the mechanisms for this anergic state. However, little is known about the disbalance between regulatory and effector CD4+ subpopulations and cytokines in the peripheral blood of cHL patients and how treatment can modify this regulatory/effector ratio. In this study, we analyzed the regulatory and effector CD4+ subpopulations together with pro and anti-inflammatory cytokines in peripheral blood of cHL patients and the impact of treatment on these cells and cytokines. Material and Methods This is an open multicenter study and, so far, we included 54 patients from December 2009 to July 2013. Thirty-four patients have completed therapy on July 2012 and were included in this study. Blood was drawn at diagnosis and after completion of treatment (1 to 4 months). Nineteen healthy blood donors volunteers were recruited as controls. Quantification of regulatory and effector T lymphocytes was done by flow cytometry using CD3, CD8, CD4, CD25, Foxp3, CTLA4, GITR and interleukin-17 (IL17) antibodies. Ten cytokines were studied: IL-2, IL-4, IL-5, IL-6, IL-10, IL-17A, sIL-2Rα, TNF-α, IFN-γ, and VEGF. Cytokine levels were determined by multiplexed immunoassay system. All these parameters were correlated to phenotypic and clinical parameters in uni- and multivariate models pre and post-treatment. In this study, only cHL patients whose histology could be confirmed and EBV association established were studied. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. Results From the 34 cHL patients recruited for this study, 17 (50%) were male, 16 (47%) had Epstein-Barr virus (EBV) related cHL, 27 (79%) patients presented with B symptoms and 18 (53%) patients had advanced diseases at diagnosis. Results of subsets of CD4+ T cells and cytokines are summarized in the following table: After treatment, the percentage of regulatory CD4+CD25highFoxP3+ and effector CD4+IL17+ T lymphocytes were not different from diagnosis (0.9 vs 0.4, p=0.45; 0.6 vs 0.9, p=0.52; respectively) and from controls (0.9 vs 0.3, p=0.22; 0.6 vs 0.7, p=0.84; respectively). Interestingly, increased CD4+CD25highFoxP3+ T lymphocytes were correlated with advanced disease at diagnosis (p=0.03) and an erythrocyte sedimentation rate (ESR) > 30 mmHg (p=0.01). Additionally, we found a negative correlation between soluble IL-2Rα and CD4+GITR+ (p=0.02) and CD4+FOXP3+ (p=0.02). Conclusions In this study, we showed that, after treatment, there was a decrease of some subsets of CD4+ T cells with regulatory phenotype, together with a decrease of IL-6, IL-10 and sIL-2Rα. Understanding cHL associated immunosuppression and the immune reconstitution after treatment maybe the key to develop new treatment strategies, designed to manipulate regulatory activity. Further studies investigating these CD4+ T lymphocytes subpopulations with functional assays are warranted. Given that the incidence of EBV-related cHL, disease presentation and severity are different in developing countries than in developed ones, we emphasize the importance of this ongoing Brazilian multicenter project. Disclosures: No relevant conflicts of interest to declare.
2286 It is known that major surgery and allogeneic blood transfusion mediate immunosuppression by interfering on cytokine secretion, lymphocyte count and cytotoxic response. However, which cytokines and lymphocyte subpopulations participate in such immunosuppressive state remains poorly understood. Regulatory T cells (Tregs) are suppressive CD4(+) cells with a central role in immunosuppression of trauma victims, cancer patients, and transplant recipients. Several markers have been identified with regulatory properties, such as FOXP3, CTLA-4, GITR and the recently described CD69. Recently, allogeneic blood transfusion has been shown capable of inducing regulatory CD4+FOXP3+ T cells in vitro. Purpose: In this study, we aimed to investigate regulatory CD4+ T cells induction and cytokine profile in transfused and non-transfused surgical patients. Patients and Methods: Thirty-five patients undergoing elective hip replacement were recruited for this study and prospectively evaluated. Blood samples were obtained before surgery (D0) and on days 1 (D1) and 4 (D4) after surgery. Quantification of regulatory T lymphocytes was done by flow cytometry using CD4, CD25, FoxP3, CTLA-4, GITR and CD69, while cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-17A, TNF-α, IFN-γ and TGF-β) levels were determined by multiplexed immunoassay system. Results: From the 35 patients recruited for this study, 18 (51.4%) received allogeneic non-leukodepleted red cells transfusion during or after surgery. There were no significant differences in clinical or epidemiological characteristics between transfused and non-transfused groups, except for the duration of surgery, being longer in transfused patients (p<0.001). Results of total lymphocyte count and subsets of CD4+ T cells in the 35 patients are summarized in the table: The total lymphocyte count was significantly decreased only in transfused patients. No significant difference was seen in the percentage of CD4+CD25highFoxp3+ T lymphocytes by comparing days D0, D1 and D4. However, a significant increase of CD4+CD25-CD69+ cells was observed by comparing D1 vs. D4 [0.61(0.23–2.69) vs. 2.27 (0.64–4.45); p=0.001] in transfused patients, but not in the group of non-transfused patients [0.36(0.31–0.81) vs. 1.02(0.42–1.52); D1 vs. D4. Similarly, we also detected a significant increase in the number of CD4+GITR+ T cells in the group of transfused patients [D1 vs. D4; 3.12±2.01 vs. 5.19±3.33; p=0.007). Total hip replacement also led to substantial increase of the following cytokines: IL-6 (p<0.001, D0 vs. D1 and p<0.001, D0 vs. D4), IL-8 (p<0.001, D0 vs. D1 and p=0.017, D0 vs. D4), IL-10 (p<0.001, D0 vs. D1). However, no differences regarding cytokines levels were seen comparing groups of transfused and non-transfused patients. Conclusions: This study demonstrated that although allogeneic non-leukodepleted red cells transfusion is associated with decrease of total lymphocyte count following major surgery, there was a significant increase of total CD4+ T lymphocytes with no difference observed in transfused and non-transfused patients. However, a significant increase of CD4+CD25-CD69+ and CD4+GITR+ T lymphocytes was observed in the transfused group on D4, possibly reflecting the late effect of transfusion on induction of these subsets of CD4+ regulatory cells. The increase of pro-inflammatory IL-6, IL-8 and anti-inflammatory IL-10 levels indirectly highlights the imbalance on immune response after surgical trauma. Further studies, with a larger cohort and functional assays, investigating these CD4+ T cells subpopulations following major surgery and the impact of allogeneic non-leukodepleted red cells transfusion on these cells should be addressed. Disclosures: No relevant conflicts of interest to declare.
4778 Cytotoxic T lymphocyte antigen-4 (CTLA-4) is one of the basic antigens involved in immune responses regulation associated with autoimmune diseases and cancer. Its key role in regulating the immune system has made CTLA-4 an attractive target for cancer. Augmentation of the immune response via blockade of CTLA-4 has shown an improvement in survival for patients with metastatic melanoma, which prompted the Food and Drug Administration (FDA) approval of the CTLA-4 function blocking antibody Ipilimumab for this disease. Objective: The aim of the study was to evaluate the surface expression of CTLA-4 on CD4+ T cells in peripheral blood mononuclear cells (PBMC) of patients with classical Hodgkin lymphoma (cHL) at diagnosis and post-treatment and correlate these findings with clinical and epidemiological aspects. Material and Methods: This is an open study and, so far, we included 35 patients from December 2009 to December 2011. Blood was drawn at diagnosis and post-treatment (1 to 4 months after completion of therapy). The T cell phenotype was evaluated by flow cytometry using CD3, CD4, CD8, CTLA-4 and correlated to phenotypic and clinical parameters in uni- and multivariate models pre and post-treatment. Eighteen healthy blood donors volunteers were recruited as controls. In this study, only cHL patients whose histology could be confirmed and Epstein-Barr (EBV) association established were studied. All patients were HIV negative and received ABVD chemotherapy protocol and radiotherapy if necessary. Three patients relapsed, and blood was also drawn at this time. Results: From the 35 cHL patients, 17 were EBV related and 18 EBV non-related. The percentage of CD4+ T cells with CTLA-4 surface expression was significantly increased in patients with cHL at diagnosis compared with healthy controls (median 7.36 vs 2.73; P<0.001). Additionally, CD4+CTLA-4+ T lymphocytes significantly decreased following treatment and complete response (7.36 vs 4.53; p=0.008), with values similar to healthy controls (4.53 vs 2.73; p=0.07). Interestingly, CD4+CTLA-4+ T lymphocytes on relapse were significantly different from post-treatment values and similar to pre treatment. There was no difference on CD4+CTLA-4+ T lymphocytes in the EBV related and non-related cHL patients. Regarding patient's baseline characteristics, CD4+CTLA-4+ T lymphocytes strongly correlated with erythrocyte sedimentation rate (ESR) values (r=0.67; p=0.002). Conclusions: We showed that CD4+CTLA-4+ T lymphocytes are increased in Brazilian cHL patients at diagnosis compared with post-treatment values and healthy controls. These results suggest a role of CTLA-4 on Hodgkin lymphomagenesis, possibly negatively regulating host anti-tumor immune response. The promising immunotherapy regimen targeting CTLA-4 might be beneficial in classical Hodgkin lymphoma. Disclosures: No relevant conflicts of interest to declare.
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