Elevated IL-17 Producing Cells (Th17 and Non-Th17) In Different CLL Microenvironments: Correlation with Overall Survival, Prognostic Relevance and Phenotypic Heterogenity

Inflammation may provide pro- or anti-tumor effects. Th17 cells that produce IL-17 cytokines have proinflammatory functions, and they can be relevant in some cancer microenvironments. However the clinical and biological relevance of Th17 cells within the context of hematological malignancies is still in question. Furthermore, it is now recognized that IL-17 can be produced by “Non-Th17” cells in autoimmune diseases. Therefore we have analyzed the types and numbers of IL-17-producing cells in patients with CLL and compared this information with clinical parameters and overall survival [OS]. In this regard, we now report that Th17 cells are elevated in (peripheral blood mononuclear cells) PBMCs of untreated CLL patients compared to those of healthy age-matched subjects. Furthermore, we show that CLL patients with high Th17 levels have significantly longer OS compared to those with low Th17 cells.

All studies involved untreated CLL patients and healthy subjects matched for age. Constitutive intracellular IL-17A expression was assessed in CD4⁺ cells from cryopreserved PBMCs isolated from 42 CLL patients and 15 healthy subjects using specific mAbs and flow cytometry. Immunohistochemistry (IHC) and immunofluoresence techniques in paraffin sections of spleen and lymph nodes from 4 CLL patients and 4 healthy subjects were also analyzed.

The median percentage of CD4⁺ Th17 cells was higher in CLL patients (Median=0.64; range: 0.0–3.6%) compared to healthy subjects (median=0.16; range: 0.0–0.8%); this difference was statistically significant (p=0.007). Furthermore, CLL patients in the good prognosis group (IGHVmutated/CD38^low) had significantly higher median CD4⁺Th-17⁺ (0.5–3.5%) than healthy controls (median=0.73 versus 0.16, p=.004). CLL patients with Th17% ≥1.2% (n=11) had significantly higher OS (p=0.023) as compared to subset of patients with lower (<1.2) percentages of Th17 cells (n=30). This difference was independent of IGHV mutation status in patients with high Th17%, i.e., both IGHV mutated and unmutated patients had higher survival if they had high Th17%. Pairwise comparisons with respect to IGHV mutation status and Th17% also showed a significant difference among the 4 groups (p=0.007), and CLL patients with mutated IGHVs and high Th17% had significantly higher OS than those with unmutated IGHVs and low Th17%. Furthermore, CLL patients with high Th17% exhibited better prognostic markers (IGHVmutated/CD38^low; n=6) than those with low Th17%, who had markers associated with poor prognosis (IGHVunmutated/CD38^high; n=15). Finally, CLL patients with high Th17% also tended to have other prognostic markers associated with better outcome (normal or solely 13q⁻ karyotypes and low Rai stages) as compared to patients with low Th17%.

To determine whether lymphoid tissue microenvironments also have IL-17 expressing cells, we analyzed CLL and normal spleens (n=4) and LNs (n=4) for IL-17-producing cells. Large IL-17 and CD13+, CD13+CD15+, or CD15+ (myeloid cell differentiation markers) co-expressing cells were found only in the CLL spleens and not in healthy spleens. The majority of these IL-17 cells were MPO⁺ and Ki67⁺ with interpatient variability. A lesser number of IL-17-producing cells in CLL spleens were mast cells, defined by CD117 and mast cell tryptase expression. Inversely, in CLL LNs, majority of IL-17 producing cells were mast cells and were localized to subcapsular areas. Such IL-17A producing mast cells were also present in normal reactive LNs.

CLL patients have significantly higher levels of Th17 cells in the blood than healthy subjects. Elevated Th17 cells (≥1.2%) correlates with good prognostic markers and patients in this subset have a significantly higher OS; the higher OS is apparently independent of IGHV mutation status. Furthermore, non-Th17 IL-17A producing myeloid cells and mast cells were identified in CLL spleens and LNs. It is possible that IL-17 expression in CLL is microenvironment-specific in contrast with any other hematological cancers where IL-17 producing cells are reported so far. Thus this study not only highlights a growing clinical significance of Th17 cells in hemato-oncology, but also opens new directions to understand the heterogeneity among CLL patients based on multiple sources of IL-17 and possible roles of lymphoid and non-lymphoid IL-17 producing cells in CLL microenvironments.

No relevant conflicts of interest to declare.