LGL Disorders: From An Inflammatory-Mediated To a Self-Maintaining Proliferation

T large granular lymphocytes leukemia (T-LGLL) and NK-type chronic lymphoproliferative disorder (CLPD-NK) are rare diseases characterized by the abnormal expansion of large granular lymphocytes (LGLs) with cytotoxic activity, belonging to T and NK lineage, respectively. Currently, the etiology of these diseases is still largely unknown. Several data support the hypothesis that the inciting event is represented by the persistence of antigenic stimulation, maintained by the abnormal release of cytokines (mainly IL-6 and IL-15), establishing an inflammation status not achieving resolution. Recently, we showed that IL-6 and soluble IL-6Rα were highly expressed and released by patients’ LGL-depleted peripheral blood mononuclear cells (PBMC), accounting for a trans-signaling process. IL-6 trans-signaling is critically involved in inflammatory disease and promotes the transition from acute to chronic inflammation. Additionally, LGL proliferation is maintained for an impairment of the apoptotic machinery due to the activation of many survival signaling pathways, including JAK/STAT and RAS/MEK/ERK pathways. In some patients (both T-LGLL and CLPD-NK) STAT3 hot-spot mutations, inducing STAT3 activation, have been demonstrated.

With this as a background, we investigated the IL-15 contribution to sustain IL-6 trans-signaling and in turn inflammation. We analyzed the relationships between STAT3 mutations, IL-6 and IL-15 in disease progression to assess the hypothesis that these findings characterize different stages of LGL disease. Thirty T-LGLL and 15 CLPD-NK patients were included in this study. Patients were subdivided according to the percentage of LGLs in PBMCs (LGL range: 35-90%). By ELISA in patients’ plasma, we showed that IL-6 concentrations were significantly higher in patients characterized by a disease with less than 60% circulating LGLs (35.7 ± 11.4 pg/ml with respect to patients with LGLs >60%: 9.1 ± 2.7 pg/ml; p <0.05). Considering that IL-6 is a mediator of inflammation, we suggest that these low burden disease patients (LGLs <60%) are mostly characterized by an active inflammatory background. By Real Time-PCR, we observed that IL-15 mediated IL-6 expression in patients PBMCs (IL-6 increased 4.7 fold after IL-15) while inhibited IL-6Rα in leukemic LGLs (by 2-fold down), indicating that IL-15 favors IL-6 trans-signalling. By western blotting analysis we showed that both IL-6 and IL-15 were able to activate STAT3 and ERK, then sustaining LGL survival. Interestingly, in the patients showing STAT3 mutations (20% of patients) we demonstrated a significantly lower level of plasma IL-6 (8.9 pg/ml). This feature was found to be associated to a high proliferative disease, with more than 60% circulating LGLs.

These results suggest that an initial step along the development of disease characterized by a low lymphocytosis (LGLs <60% of total PBMCs) is mostly sustained by extrinsic factors contributing to the relevant inflammatory background. A subsequent stage is characterized by a high lymphocytosis (LGLs >60% of total PBMCs) in which LGL disease goes on independently from exogenous stimuli, likely becoming self-maintaining due to the contribution of the emerging STAT3 mutations. It is suggested that these phases can represent two sequential steps in the progression of disease.