PB2049 DETECTION OF TREGS BY FLOW CYTOMETRY IN THE DIAGNOSIS OF HODGKIN LYMPHOMA
Authors: Callegari B. , Di Gaetano R. , De Benedetto M.A. , Belvini D. , Sartori R. 
Published: June 2019

Background:Regulatory T (Tregs) cells constitute a subset of CD4+ lymphocytes that play a key role in the regulation of self‐tolerance and the maintenance of tissue homeostasis. It seems well established that in cancer patients, accumulation of Tregs both in tissue and peripheral blood (PB) is associated with the suppression of anti‐tumor immune effector functions, tumor progression and poor prognosis. Flow cytometry (FC) is an effective and rapid tool to quantify Tregs and to evaluate the immunological characteristics of the individual patient.Aims:We wanted to verify the usefulness of FC in quantifying Tregs in the diagnosis of the Hodgkin lymphoma (HL), where the contribution of the FC is limited, and to demonstrate if this subset T CD4+ could help to define the microenvironment of the HL characterized by a low number of malignant cells surrounded by an abundant reactive infiltrate of immune cells rich of T lymphocyte.Methods:We investigated Tregs in the involved lymph nodes (LN) of 15 HL patients and 15 patients with reactive lymph‐nodes. Peripheral presence of Tregs was analysed in PB obtained from the same patients and 15 age‐matched healthy donors (controls). Analyses were performed using FACSCanto flow cytometer (BD Biosciences) and FACSDiva software. The Tregs were calculated both as percentage of total CD4 T lymphocytes. Tregs were analyzed according to the commonly used Treg definitions FOXP3+ CD4highCD25high, characterized by low expression of CD127 (IL‐7 receptor) and negativity for CD45RA (to discriminate activate cells from naïve cells). To improve the specificity of the analysis, the gating strategy considered Tregs positive for CD39 (marker of homing to inflamed regions), positive for CD62L (L‐selectin) and negative for CD26. Differences were considered statistically significant at with p‐value of < 0.05. Statistical analysis was performed using PRISM‐Graph Pad software version 6.3.Results:The percentage of Tregs was significantly higher in biopsy of HL patients compared with reactive lymph nodes’ patients: median (3,400 ± 0,8800) vs (1,130 ± 0,1647) p = 0,0430. Instead no statistically significant differences were found comparing circulating Tregs values, between the HL patients group vs patients with reactive lymph‐nodes and vs healthy controls: median (0,2857 ± 0,05728) vs (0,192 ± 0,02215) vs (0,2500 ± 0,052) p = 0,3722. To identify potential differences in proportions of Tregs between patients with HL, reactive and healthy controls, we included detailed analysis of Treg immunophenotypes.Summary/Conclusion:: Up to now, Tregs were evaluated mostly in solid cancer, while studies evaluating the numbers of Tregs in patients with lymphomas are few and with controversial results in fact it may be associated with either negative or positive outcome. Our work was focused on patients with HL where we found an increased number of Tregs that seems to be context‐dependent. In fact in HL lymph nodes the number of Tregs were higher compared to reactive ones. Otherwise no statistical differences were found in percentages of Treg among circulating CD4+ lymphocytes in patients compared with the control group. So, detecting Tregs during diagnosis in LN can help to discriminate HL from reactive ones. Our data could suggest a routine application of the FC in the diagnosis of HL to characterize specifically the microenvironment. Moreover we highlight the need to request a confirmation in larger patient population to confirm our data in PB.image