Variation of T-Reg and CD 200+ T- Lymphocytes After in Vitro Treatment with Active Drugs against CLL.

Purine analogues, in particular fludarabine, are considered the gold standard of treatment of CLL. However, fludarabine therapy is sometimes complicated by autoimmune haemolytic anemia (AHA). The mechanism of this side effect is not clear but it is conceivable that a fludarabine-induced suppression of some regulatory systems, including T-reg, is responsible for this phenomenon. In addition, we have observed that patients affected by autoimmune diseases such as AHA or PTI have a reduced number of T lymphocytes bearing the CD200 antigen that is considered a tolerogenic molecule. In this perspective, we evaluated the variation of T-reg and CD200+ T lymphocytes induced by incubating in vitro peripheral blood mononuclear cells (PBMC) of CLL patients and normal subjects with purine analogues and other drugs active against CLL.

PBMC obtained from patients with chronic lymphocytic leukaemia (CLL) (n=9) and from normal adult (n=6) were isolated by density gradient and cultured in RPMI supplemented with 10% FBS and 1% of penicillin streptomicyn. Cells were then incubated for 24 hours with drugs at two concentrations: bendamustine (1 and 50 μg/ml) campath (1 and 5 μg/ml) prednisone ( 1 and 10 nM), fludarabine (0,25 and 10 μg/ml) pentostatin (3 and 60 μg/ml). The cytotoxicity was evalutated after 24 hours by trypan Blue and flow cytometry. T-reg cells were identified as CD4+/CD25+/FoxP3+ T cells and expressed as a percentage of the CD4+T-cell population.

Although all of these drugs induced lymphocytes cytotoxicity, fludarabine, prednisone, and campath reduced also the percentage of T-reg and CD200+ T –lymphocytes, while bendamustin and especially pentostatin induced the same cytotoxicity but spared T-reg populations and CD200+ T-lymphocytes. Table I indicates results obtained with the highest concentration of drugs.

In conclusion, pentostatin and bendamustine seems to be active drugs against CLL and their usage shouldn't be complicated by autoimmune phenomena.

No relevant conflicts of interest to declare.