Abstract
T cell large granular lymphocyte leukemia (T-LGLL) is a rare, chronic hematological malignancy characterized by clonal proliferation of cytotoxic CD8+ T cells due to defective apoptosis. Currently there is no cure and the main treatment options are broad immunosuppressant therapies for management of symptoms. The vast majority of T-LGLL patients require treatment at some point and thus there are efforts to identify and evaluate novel and specific therapeutics for T-LGLL. One such area of investigation is the regulation of interferon-gamma signaling. IFN-gamma is an inflammatory cytokine that is associated with worse disease progression and symptomology in multiple cancers and autoimmune diseases. Excess IFN-gamma, in the absence of infection, inhibits proliferation and induces apoptosis of healthy peripheral blood mononuclear cells (PBMCs), contributing to disease state. As a result of this, it is crucial to reduce IFN-gamma production in cancers, including T-LGLL, where patients have significantly elevated circulating levels of this pro-inflammatory cytokine compared to healthy donors.
Calcitriol, the active form of vitamin D, has shown promise as an inhibitor of IFN-gamma production. Thus, we first turned our attention to evaluating calcitriol for use as an IFN-gamma inhibitor in T-LGLL. We initially assessed the effects of 24 h calcitriol treatment on TL-1 cells, the patient-derived cell line model of T-LGLL (Chapter 3). We found that calcitriol significantly decreased IFN-gamma secretion and activation of signal transducer and activator of transcription 1 (STAT1), a transcription factor that becomes activated via phosphorylation of tyrosine residue 701 (p-STAT1) in response to IFN-gamma. STAT1 also typically promotes transcription of IFN-gamma. As a result of calcitriol treatment, p-STAT1 and IFN-gamma inhibition occurred while the vitamin D receptor (VDR), a nuclear receptor and transcription factor, increased on the protein level in TL-1 cells. We next sought to elucidate the mechanism behind calcitriol-mediated reduction in IFN-gamma production and whether VDR upregulation was required for this effect (Chapter 4). We found that calcitriol reduced IFN-gamma intracellular protein and mRNA transcript levels and p-STAT1 protein levels within 4 h. Moreover, calcitriol-mediated IFN-gamma reduction was independent of p-STAT1 levels but required VDR upregulation. Our results suggested that p-STAT1 and IFN-gamma levels were regulated independently of each other, indicating a dysregulation of the canonical IFN-gamma signaling pathway. Thus, in an effort to better understand the regulation of IFN-gamma, we sought to characterize the IFN-gamma-mediated signaling pathway from IFN-gamma signaling to transcription of IFN-gamma (Chapter 5).
Previous studies demonstrated that T-LGLL cells have a deficiency in suppressor of cytokine signaling 1 (SOCS1), a negative regulator of IFN-gamma-mediated signaling. SOCS1 is typically induced in response to IFN-gamma, allowing for a tightly controlled signaling process. However, despite high IFN-gamma output, T-LGLL cells exhibit significantly lower SOCS1 levels compared to normal donor cells. Therefore, T-LGLL cells are likely to be unresponsive to IFN-gamma production, allowing an unchecked production of the inflammatory cytokine as seen in other cancers. We found that TL-1 cells have a significantly lower surface protein and mRNA transcript level of the IFN-gamma receptors (IFNGR) compared to Jurkat T cells, our positive IFN-gamma responsive cell line. IFN-gamma did not induce Janus kinase 2 (JAK2) or STAT1 phosphorylation or established IFN-gamma-mediated gene targets, including IRF-1 and SOCS1, in TL-1 cells. This further demonstrated a lack of responsiveness to IFN-gamma. We found that STAT5b, but not STAT1 or STAT3, played a role in regulating IFN-gamma transcript levels.
Taken together, the decrease in IFNGR levels is a plausible explanation for the excessive IFN-gamma production and lack of negative regulation observed in T-LGLL. This pathway can be targeted effectively using calcitriol to significantly reduce IFN-gamma production. As calcitriol inhibits IFN-gamma independently of STAT1, calcitriol is able to reduce IFN-gamma regardless of its interactions with STAT1, providing a potent therapeutic to reduce inflammation. This thesis is the first study to evaluate IFN-gamma signaling and a treatment aimed at specifically targeting IFN-gamma in T-LGLL. Future studies are needed to assess the efficacy of calcitriol in T-LGLL patients in the clinic.
