Immune Derangements in Patients with Myelofibrosis- the Role of T_reg, Th17, and sIL2α

Abstract 1763

Primary myelofibrosis (PMF), myelofibrosis post essential thrombocytosis (MF-ET), and myelofibrosis post polycythemia vera (MF-PV) have been reported to be associated with autoimmune phenomena, such as Coombs positive anemia, lupus anticoagulant, positive ANA and the presence of circulating immune complex, etc. Regulatory T cells (T_reg) and IL-17 producing cells (Th17) have been known to play important roles in modulating immune responses. Hence we studied T_reg cells in 38 patients with MF including PMF (25), MF-ET (8), MF-PV (5) and compared them to other MPD patients including ET (7), PV (14), and normal controls (16). Patients on lenalidomide or Pomalidomide were excluded. Blood ( 10⁶ MNCs) were stained for flow cytometry analysis using the T_reg Detection Kit (Miltenyi Biotec). The number of T_reg cells was calculated as the percentage of positive CD4⁺ CD25⁺ FoxP3⁺ T cells from the number of gated CD4⁺ cells. T_reg function was evaluated by XTT cell proliferation assay (Invitrogen) with ratios of T_reg to T-effector cells at 1:1 in the presence of anti-CD3 and CD28 micro-beads (Invitrogen). The results (mean + SE) showed numbers of T_reg cells in MF were 0.79 + 0.080, other MPD were 1.27 + 0.20, and normal controls were 1.21 + 0.30. No significant difference was found among the three groups. T_reg function was evaluated in MF (18 patients). MPD (21) and controls (16). No significant difference was found among the three groups. Th17 cell assays were performed by culturing Blood CD4⁺ cells in IMDM medium and stimulated with PMA (25ng/ml), ionomycin (1 ug/ml ) and monensin ( 500ng/ml) for 4h at 37°C with 5% CO₂. Then Th17 was stained with Th17 Flow Kit (Biolegend) and analyzed by flow cytometry. The results were expressed as % of isolated CD4⁺ cells (mean ± SE) as follows: MF (2.31 ± 0.65) (n=15), MPD (1.31 ± 0.32) (n=7), and controls (1.89 ± 0.44) (n=10). No significant difference was found among the three groups. We further studied the soluble interleukin 2 alpha subunit (sIL2α) levels. sIL2 α were measured in plasma by ELISA kits, results were expressed as (mean± SE ) ( pg/ml) as follows: MF ( 3534± 298) (n=18), MPD ( 2303 ± 171) (n=22) and controls ( 1734 ± 115) (n=16), P values were <0.05 in MF vs MPD and MF vs Controls, and P=NS in MPD Vs controls. These results confirm our previous observation (Br J Haematol,1994), that sIL2 α levels were significantly elevated in patients with MF, compared with MPD patients and controls.

We further studied the effects of the sIL2α on the immune function in MF patients:

A) Effects on the Th1, Th17, and T_reg cells. CD4⁺ cells after isolation were cultured for 7 days in IMDM containing IL-2 (100ug/ml) and anti- CD3CD28 micro-beads, with or without recombinant sIL-2Rα (100ng/ml). The cells then were stimulated with PMA (20ng/ml) ionomycin (1ug/ml) and monensin (1uM) for 4 hours before harvest. Then Th1 Th17 and T_reg were numerated by flow cytometry. The results showed no difference in Th1 and Th17 cells in cultures with and without sIL2α, but sIL2α significantly increased the numbers of T _reg (1.71 +.28,P=0.02) ( fold increase).

B) Effects on the T_reg function. Viable CD4⁺CD25⁻ (10⁶) cells were labeled with CFSE ( Invitrogen), then added unlabeled T_regs at 1:1 (responder: T_reg) ratio and stimulated with 50 μl of anti CD3/CD28 micro-beads with and without sIL2α for 4 –7days at 37 °C in a 5% CO2 incubator. Cell proliferation was measured by counting the percentage of CFSE^dim cells. The results showed that sIL2α significantly increased the CFSE ^dim cells. Therefore sIL2α suppressd the T _reg cell function and increase the T responsive cell proliferation.

C) Identification of cells producing sIL2α. CD4⁺, CD14⁺, CD8⁺, CD20⁺, and T_reg were isolated using isolation kits (Miltenyi ), then cultured either with PHA or anti CD3/CD28 micro-beads. ELISA assay for sIL2α were then performed on the cultured supernatant. The results (mean ± SE) ( pg/ml) showed that CD4⁺ cells produced 342 ±152.6; CD8 ⁺,71.6 ± 19.8; T_reg, 306.9 ± 53.5, while CD14⁺ and CD20⁺ cells produced negligible quantity of sIL2α. Thus T_reg cells were the cells predominantly producing sIL2α in patients with MF. We conclude that in patients with MF, numbers of T_reg and Th17 cells were not different from controls or other MPD patients, but T_reg cells produce significantly increased amount of sIL2α which further inhibits T_reg function and results in autoimmune phenomenon observed in patients with MF.