Abstract

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:

 Pre-treatment Post -treatment Controls P value 
 2.2 0.8 0.8 Pre vs Post (p = 0.01) 
CD4+GITR+(%)*     
 (0.8 - 9.9) (0.5 - 10.5) (0.2 - 3.2) Pre vs Control (p < 0.001) 
 8.7 3.9 2.5 Pre vs Post (p = 0.01) 
CD4+CTLA4+     
 (0.8 - 30.3) (0.8 - 10.3) (0.7 - 11.2) Pre vs Control (p < 0.001) 
 5.6 0.4 0.4 Pre vs Post (p = 0.01) 
IL-6( pg/dL)*     
 (0.8 - 110.1) (0.1 - 20.4) (0.1 - 9.1) Pre vs Control (p < 0.001) 
 7.7 0.3 0.3 Pre vs Post (p < 0.001) 
IL-10     
 (0.8 - 109.1) (0.1 - 29.5) (0.1 - 8.2) Pre vs Control (p < 0.001) 
 575.4 93.5 7.5 Pre vs Post (p < 0.001) 
sIL-2Rα     
 (10.5 – 1,200.4) (7.5 - 733.4) (5.0 - 150.8) Pre vs Control (p < 0.001) 
 Pre-treatment Post -treatment Controls P value 
 2.2 0.8 0.8 Pre vs Post (p = 0.01) 
CD4+GITR+(%)*     
 (0.8 - 9.9) (0.5 - 10.5) (0.2 - 3.2) Pre vs Control (p < 0.001) 
 8.7 3.9 2.5 Pre vs Post (p = 0.01) 
CD4+CTLA4+     
 (0.8 - 30.3) (0.8 - 10.3) (0.7 - 11.2) Pre vs Control (p < 0.001) 
 5.6 0.4 0.4 Pre vs Post (p = 0.01) 
IL-6( pg/dL)*     
 (0.8 - 110.1) (0.1 - 20.4) (0.1 - 9.1) Pre vs Control (p < 0.001) 
 7.7 0.3 0.3 Pre vs Post (p < 0.001) 
IL-10     
 (0.8 - 109.1) (0.1 - 29.5) (0.1 - 8.2) Pre vs Control (p < 0.001) 
 575.4 93.5 7.5 Pre vs Post (p < 0.001) 
sIL-2Rα     
 (10.5 – 1,200.4) (7.5 - 733.4) (5.0 - 150.8) Pre vs Control (p < 0.001) 
*

Results reported as median (P25-P75)

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.

Author notes

*

Asterisk with author names denotes non-ASH members.