Th-17/IL-17 Axis in Chronic Lymphocytic Leukemia.

Maintenance of chronic lymphocytic leukemia (CLL) clones is dependent on complex interactions between leukemic cells and soluble factors released by cells within the microenvironment of the blood, bone marrow, lymph node and spleen. Studies in our laboratory demonstrated elevated levels of IL-17, a pro-inflammatory cytokine secreted by a subset of CD4⁺ effector T cells (Th17 cells), in the sera of CLL patients as compared to those of healthy age-matched subjects. We hypothesized that the Th17/IL-17 axis is active in CLL, creating a proinflammatory microenvironment that promotes growth and angiogenesis. To test this hypothesis, we analyzed the expression of both IL-17 and IL-17 receptors in blood, spleen, and lymph nodes of CLL patients and normal control subjects.

All studies involved patients with CLL and healthy subjects matched for age. Constitutive and inducible intracellular IL-17 expression was assessed in cryopreserved peripheral blood mononuclear cells (PBMCs) isolated from untreated CLL patients (n = 27) and healthy individuals (n = 8). Flow cytometric analyses were performed at baseline (Day 0) and after 7-day culture with or without the Th17-polarizing cytokines, IL-1b and IL-23. Surface expression of IL-17 receptors A (IL-17RA) and B (IL-17RB) was analyzed on PBMCs of CLL patients (n = 25) and healthy controls (n = 8) using flow cytometry. Lastly, IL-17 expression was assessed by immunohistochemistry in spleen and lymph nodes from CLL patients and healthy subjects.

Intracellular analysis of IL-17 expression in PBMCs revealed a higher median percentage of CD4⁺ Th17 cells in CLL patients (Median=0.73; range: 0.0-3.6%) compared to healthy subjects (Median=0.13; range: 0.0-0.8%) at baseline; this was a statistically significant difference (P<0.05). While there was no significant difference between the two major patient subgroups, i.e., (Mutated IGHV >2% and CD38^low <30% and unmutated IGHV <2% and CD38^high, >30%). CLL patients in the good prognosis group had significantly higher median percentage of CD4⁺ IL-17⁺ cells compared to healthy controls at baseline (Median=0.73 vs 0.13, P<0.05). Complementary studies also revealed that expression of IL-17 in the CD4⁺ T cell compartment after culturing total PBMC's for 7 days in the absence of Th17-polarizing cytokines was significantly higher in CLL patients than healthy subjects (Median =1.34, P<0.05). When PBMCs were cultured for 7 days in the presence of Th17-polarizing cytokines, the percentage of IL-17-producing CD4⁺ T cells increased in cells from both patients and healthy subjects, however again the percentage of IL-17 expressing CD4⁺cells was higher in cultures of PBMC's from CLL patients as compared to those from healthy subjects (Median= 1.99 vs 0.84). Surface expression of IL-17RA and IL-17RB was observed on monocytes from both patients and healthy subjects. The density of expression of IL-17RA on monocytes was more intense relative to IL-17RB in both patients and healthy subjects; however IL-17RA and IL-17RB expression was not significantly different between patients and healthy subjects with respect to percent of positive cells, mean fluorescence intensity or median fluorescence intensity. Of note, IL-17RA and IL-17RB were expressed on a small fraction of B lymphocytes from patients (Mean for RA and RB= 6.59 and 3.99, respectively) and normal individuals (Mean for RA and RB= 4.31 and 3.43, respectively). For CLL patients, these receptors were detected on CD19⁺CD5⁺ cells, whereas for healthy subjects they were found on CD19⁺CD5⁻ cells. Preliminary immunohistochemical analyses of lymphoid tissues from CLL patients and healthy subjects revealed strong IL-17 expression in tissues from CLL patients but not in healthy age-matched subjects.

Patients with CLL have higher baseline levels of circulating IL-17, as well as increased expression of Th17 cells in the blood and lymphoid tissues, as compared to healthy age-matched subjects. Furthermore, IL-17RA and IL-17RB are expressed on monocytes and on a small population of B cells in CLL patients enabling them to respond to the elevated levels of IL-17 in the microenvironment. Such interactions may stimulate release of pro-survival factors and promote growth and angiogenesis, contributing to the development and evolution of CLL. Further studies are in progress to demonstrate the functional role of IL-17 in CLL.

No relevant conflicts of interest to declare.