Ultra-Low Dose IL-2 Expands Natural Regulatory T Cells and CD56bright NK Cells in Patients and Healthy Donors and Is Associated with Clinical Improvement in Chronic Graft Versus Host Disease

Low dose interleukin-2 (IL-2) can increase regulatory T cells (T_reg) and has therapeutic benefit in steroid refractory cGVHD. To further define immunological changes underlying the response to IL-2 and to determine tolerability, we performed phenotypic characterization and functional analysis of T_reg and natural killer (NK) cells in individuals given IL-2. Six healthy volunteers received subcutaneous ultra low dose IL-2 (0.2MIU/m²/day) for 5 days without significant side effects. Two patients with steroid and calcineurin-refractory cGVHD received the same dose of IL-2 daily for 4 or 8 weeks, after HLA identical myeloablative stem cell transplant (SCT) for AML. Patient 1 (50 year old male, 3 yr post SCT) had skin and fascial GVHD affecting the lower limbs. Patient 2 (44 year old female, 2 yr post SCT) had upper gastrointestinal cGVHD. Both patients had a prompt and durable partial response by NIH cGVHD criteria. Immunosuppression was subsequently withdrawn in patient 1 and reduced (budesdonide from 15 to 5mg) in patient 2. Mononuclear cells were analyzed by flow cytometry before and after IL-2 in patients and healthy donors. T_reg subsets were determined within the CD4+ T cell population to identify thymus-derived natural T_reg (nT_reg) and induced T_reg (iT_reg). NK cell subsets were determined within CD56+CD3- population to identify CD56^bright, CD56^dimNKG2A+KIR-, and CD56^dim KIR+CD57+ NK cells. Functional analysis was performed by isolating T_reg (CD3+CD4+CD25^hiCD127^dim), conventional T cells (T_con: CD3+CD4+CD25^loCD127^hi) and NK cells (CD3-CD56+). T_reg function was measured by suppression of CD154 expression on T_con stimulated with CD3/CD28 beads. NK cell function was measured by K562 killing. Healthy volunteers showed a significant increase in both nT_reg and iT_reg by 2-3 fold (p=0.0003) and CD56^bright NK cells by 1.2-1.8 fold in 7 days after the initial dose of IL-2 (p=0.02). In patients, the fraction of nT_reg increased above baseline by 1.2-1.4 fold while iT_reg decreased. NK cells showed marked expansion of CD56^bright NK cells within 2 weeks of treatment, with no change in CD56^dimNKG2A+KIR-, and CD56^dimKIR+CD57+ NK cells. Functional assays demonstrated increased T_reg suppression of autologous T_con and NK cell killing of K562 after IL-2 treatment in both patients. These findings indicate that ultra low dose IL-2 causes expansion of natural T_reg and NK cells resulting in greater suppressive activity and K562 killing activity correlating with an improvement in cGVHD for steroid refractory patients.