Phd in IINNOVATION IN IMMUNO-MEDIATED AND HEMATOLOGICAL DISORDERS

Thesis title: LYMPHOCYTOSIS CHARACTERIZATION IN ARTHROPATHIES PATIENTS FOLLOWING TNFα BLOCKING AGENTS

Introduction: Anti-TNFα drugs (TNFi) were the first biological agents used in treatment of Rheumatoid Arthritis (RA) and Spondylarthritis (SpA). Since their use in clinical practice, lymphocytosis has been described. Pathogenesis of this phenomenon was not well investigated. Lymphocytosis would be composed mainly by CD4+ cells, with reversibility after drug discontinuation. TNF probably limits T cells expansion, induces memory and naïve T cells apoptosis, acting on TNFR2. TNFi could lead to dysregulation, resulting in clonal expansion and prolonged T cells survival. Aim: The aim of the project was to analyze lymphocytes characteristics in RA and SpA patients, treated with TNFi and presenting lymphocytosis (L>4000/l), Tregs and TNFR2 role, and to identify predictive factors. Methods and results: We performed a cross-sectional study enrolling RA and SpA patients, treated with TNFi and with lymphocytosis (LC+), referring to Rheumatology Unit, Sapienza University Rome. Severe neutropenia and active viral infections (CMV, EBV) were exclusion criteria. At recruitment, clinical and disease activity data were collected. 11 RA pts, 15 PsA pts and 11 SA pts (F/M 23/14) were enrolled; lymphocytosis was about 4260/l (IQR 4000-4400/l), developed after 4 months median period of TNFi therapy (IQR 4-12). A control group, composed by patients with the same diseases and treatment, without lymphocytosis (LC-) was enrolled. Higher lymphocyte count and use of glucocorticoids and lower Minimal Disease Activity (MDA) achievement were observed in LC+ group (p<0.05). We collected blood samples from subgroups of 5 pts LC+, 5 LC-, with homogeneous features regarding sex (female), age (menopausal status), disease (psoriatic arthritis) and TNFi (adalimumab), and from 5 healthy donors (HD). Peripheral blood mononuclear cells (PBMCs) were obtained, and flow cytometry studies were performed. Lymphocytes were analyzed using a FACSCalibur instrument. Combinations of antibodies conjugated to different fluorochromes (fluorescein, phycoerythrin, allophycocyanin and peridinin-chlorophyll-protein) were used for direct staining. CD4+ cell markers were: CD4, CD25, CD127, CD45RA, ox40, Ki67, FoxP3, TNFR2. We focused analysis on Tregs, their proliferation and regulation. We observed no differences in percentage of Tregs/CD4 among LC-, LC+ and HD groups (median% 6.52, 3.35 and 4.47, respectively) with a trend for higher Treg percentage in LC- pts. Tregs activated (41.2%) and resting (30.6%) subsets contributed to the global Treg expansion in LC- patients, determined by both proliferation [higher Ki67 expression in Tregs than in Tconv (22% vs 5.3%, p<0.05)] and regulation [upregulation of TNFR2 in Treg than Tconv (8.1% vs 2.4%)]. Higher disease activity in patients with axial involvement (BASDAI) was related to higher Treg resting (p 0.034, r 0.67). We observed also that interval time between TNFi administration and sampling was correlated, with a directly trend with Treg resting (p 0.05, r 0.661) and inversely with Treg activated (p 0.003, r -0.865). TNF blockade could lead in some pts TNFR2 upregulation promoting Treg proliferation with lymphocytosis control while in other pts to lymphocytosis with cytokines (IL-2) consume and thus Treg not expansion. Conclusions: No differences in rheumatological (therapy response) and hematological (lymphoma, leukemia development) features were observed between patients treated with TNFi, with and without lymphocytosis. A longest follow-up and larger patients’ number are needed to better analyze this phenomenon effect, but with this pilot study, we could confirm a role of Treg and TNFR2 in lymphocytosis control in these patients.