Calcitriol-mediated reduction in IFN-γ output in T cell large granular lymphocytic leukemia requires vitamin D receptor upregulation

Abstract

Constitutively activated STAT1 and elevated IFN-γ are both characteristic of T cell large granular lymphocytic leukemia (T-LGLL), a rare incurable leukemia with clonal expansion of cytotoxic T cells due to defective apoptosis. Interferon gamma (IFN-γ) is an inflammatory cytokine that correlates with worse progression and symptomology in multiple autoimmune diseases and cancers. In canonical IFN-γ-STAT1 signaling, IFN-γ activates STAT1, a transcription factor, via phosphorylation of tyrosine residue 701 (p-STAT1). p-STAT1 then promotes transcription of IFN-γ, creating a positive feedback loop. We previously found that calcitriol treatment of the TL-1 cell line, a model of T-LGLL, significantly decreased IFN-γ secretion and p-STAT1 while increasing the vitamin D receptor (VDR) protein. Here we further explore these observations. Using TL-1 cells, IFN-γ decreased starting at 4h following calcitriol treatment, with a reduction in the intracellular and secreted protein levels as well as the mRNA content. A similar reduction in IFN-γ transcript levels was observed in primary T-LGLL patient peripheral blood mononuclear cells (PBMCs). p-STAT1 inhibition followed a similar temporal pattern and VDR upregulation inversely correlated with IFN-γ levels. Using EB1089 and 25(OH)D, which have high or low affinity for VDR, respectively, we found that the decrease in IFN-γ correlated with the ability of EB1089, but not 25(OH)D, to upregulate VDR. However, both compounds inhibited p-STAT1; thus the reduction of p-STAT1 is not solely responsible for IFN-γ inhibition. Conversely, cells treated with VDR siRNA exhibited decreased basal IFN-γ production upon VDR knockdown in a dose-dependent manner. Calcitriol treatment upregulated VDR and decreased IFN-γ regardless of initial VDR knockdown efficiency, strengthening the connection between VDR upregulation and IFN-γ reduction. Our findings suggest multiple opportunities to further explore the clinical relevance of the vitamin D pathway and the potential role for vitamin D supplementation in T-LGLL.

Vitamin D pathway activation selectively deactivates signal transducer and activator of transcription (STAT) proteins and inflammatory cytokine production in natural killer leukemic large granular lymphocytes

“…Neither treatment affected total STAT protein levels at the examined time points, which is consistent with our previous work [18]. One caveat is that our previous work only looked at reduction in activation of STATs 1 and 3 in the TL-1 cell line [5, 18]; in this study, we report the phosphorylation status of STATs 1, 2, 3, 4, 5 and 6 in the NKL cell line and STATs 1 and 3 in NK-LGLL patient PBMC samples.…”

“…One caveat is that our previous work only looked at reduction in activation of STATs 1 and 3 in the TL-1 cell line [5, 18]; in this study, we report the phosphorylation status of STATs 1, 2, 3, 4, 5 and 6 in the NKL cell line and STATs 1 and 3 in NK-LGLL patient PBMC samples. Based on our results, we created a working model (Supplemental Figure 3) that shows how vitamin D pathway activation reduces STAT1 and 3 phosphorylation with decreased cytokine production as the outcome.…”

“…We examined levels of IFN-γ, IL-10, and Flt-3L in 24 h conditioned media from NKL cells treated with calcitriol or EB1089 using a custom Luminex panel. The rationale for the cytokines were from our previous work [5, 18] and published literature. IFN-γ is a pro-inflammatory cytokine that is associated with worse disease state in multiple malignancies and autoimmune disorders [29–31].…”

“…IFN-γ plays a pro-tumorigenic role in many malignancies and is significantly elevated in T-LGLL patient PBMCs [40] likely due to decreased negative regulation [47, 48]. Our previous work in T-LGLL showed potential for calcitriol to reduce IFN-γ levels [5, 18]. Although the NKL cell line produced a 10-fold lower level of extracellular IFN-γ compared to TL-1 [5, 18], we did observe that 100 nM EB1089 caused a significant decrease in IFN-γ output (Figure 8F), suggesting this inflammatory cytokine can be lowered in NK-LGLL.…”

“…This pathway turns off activated T cells by decreasing inflammatory cytokine production and STAT phosphorylation [13–17]. We previously showed this to be the case in T-LGLL patient peripheral blood mononuclear cells (PBMCs) in culture and in the TL-1 cell line, a model of T-LGLL [5, 18]. However, there are few reports about vitamin D effects in NK cells.…”