Autocrine/paracrine cytokine stimulation of leukemic cell proliferation in smoldering and chronic adult T-cell leukemia.

Adult T-cell leukemia (ATL), a heterogeneous disease, can be divided into smoldering, chronic, lymphoma, and acute types clinically. In addition to different clinical manifestations, different stages of ATL have different molecular signatures. Here, we demonstrated that smoldering/chronic ATL peripheral blood mononuclear cells spontaneously proliferated ex vivo in a cytokine (interleukin-12 [IL-12]/IL-9/IL-15)-dependent manner, while acute-type ATL peripheral blood mononuclear cells did not proliferate or proliferated independent of cytokines. Smoldering/chronic ATL cells produced IL-2 and IL-9 in 6-day ex vivo cultures. Interestingly, the addition of an anti-IL-2R-α monoclonal antibody profoundly inhibited IL-9 expression, suggesting optimal expression of IL-9 was dependent on IL-2 signaling in these patients. To determine whether there would be autonomous proliferation of ATL leukemic cells, we purified leukemic cells from patients with smoldering/chronic ATL. Purified leukemic cells cultured alone produced IL-2/IL-9, and the downstream Janus kinase/signal transducer and activator of transcription pathway was activated. However, the leukemic cells did not proliferate independently, but required coculture with autologous monocytes to induce proliferation. Moreover, interaction between leukemic cells and monocytes was contact dependent, and major histocompatibility complex class II expression may have contributed to this interaction. In conclusion, our data provide evidence that there is autocrine/paracrine cytokine stimulation of leukemic cell proliferation in patients with smoldering/chronic ATL that could be targeted for treatment.

A new domain in the Toll/IL-1R domain-containing adaptor inducing interferon-ß factor protein amino terminus is important for tumor necrosis factor-α receptor-associated factor 3 association, protein stabilization and interferon signaling.

Toll/IL-1R domain-containing adaptor inducing interferon-ß (IFN-ß) factor (TRIF) is a key adaptor for Toll-like receptor (TLR) 3 and TLR4 signaling. Using a novel cDNA isolate encoding a TRIF protein with a 21-residue deletion (Δ160-181) from its amino-terminal half, we investigated the impact of this deletion on TRIF functions. Transfection studies consistently showed higher expression levels of the (Δ160-181) TRIF compared to wild-type (wt) TRIF, an effect unrelated to apoptosis, cell lines or plasmid amplification. Colocalization of wt and (Δ160-181) TRIF proteins led to a dramatic reduction of their respective expressions, suggesting that wt/(Δ160-181) TRIF heterocomplexes are targeted for degradation. We demonstrated that wt TRIF associates with tumor necrosis factor-α receptor-associated factor 3 (TRAF3) better than (Δ160-181) TRIF, culminating in its greater ubiquitination and proteolysis. This explains, in part, the differential expression levels of the two TRIF proteins. Despite higher expression levels in transfected cells, (Δ160-181) TRIF inefficiently transactivated the IFN pathway, whereas the nuclear factor-κB (NF-κB) pathway activation remained similar to that by wt TRIF. In coexpression studies, (Δ160-181) TRIF marginally contributed to the IFN pathway activation, but still enhanced NF-κB signaling with wt TRIF. Therefore, this 21 amino acid sequence is crucial for TRAF3 association, modulation of TRIF stability and activation of the IFN pathway.

Affinity of Stat2 for the subunits of the interferon alpha receptor.

The interferon alpha receptor is composed of two subunits: IFNaR1 and IFNaR2. Interferon alpha binding to the receptor induces phosphorylation of tyrosine 466 on IFNaR1, which in turn binds the SH2 domain of the latent transcription factor Stat2 to initiate signaling. Stat2 also binds to IFNaR2 in a constitutive, phosphorylation-independent manner. To explore the function of the Stat2-IFNaR2 interaction and its possible relationship to the SH2-dependent docking of Stat2 to phosphorylated IFNaR1, the affinity of Stat2 for each of the receptor subunits was determined. Recombinant proteins corresponding to the cytoplasmic domains of the receptor subunits and the central core region of Stat2 were partially purified and used in affinity precipitation experiments to demonstrate that Stat2 binds more avidly to IFNaR2 than to phosphorylated IFNaR1. Surface plasmon resonance based biosensor analysis confirmed this finding; Stat2 bound IFNaR2 (K(d) = 45 nM) approximately 6-fold stronger than it bound tyrosine 466-phosphorylated IFNaR1 (K(d) = 245 nM). Affinity precipitation experiments involving all three proteins (Stat2, phosphorylated IFNaR1, and IFNaR2) indicated that the Stat2-receptor interactions are independent of one another. The relevance of these data to possible models of interferon alpha signal transduction is discussed.

Stat2 binding to the interferon-alpha receptor 2 subunit is not required for interferon-alpha signaling.

The interferon-alpha (IFNalpha) receptor consists of two subunits, the IFNalpha receptor 1 (IFNaR1) and 2 (IFNaR2) chains. Following ligand binding, IFNaR1 is phosphorylated on tyrosine 466, and this site recruits Stat2 via its SH2 domain. In contrast, IFNaR2 binds Stat2 constitutively. In this study we have characterized the Stat2-IFNaR2 interaction and examined its role in IFNalpha signaling. Stat2 binds the major IFNaR2 protein but not a variant containing a shorter cytoplasmic domain. The interaction does not require a STAT SH2 domain. Both tyrosine-phosphorylated and non-phosphorylated Stat2 bind IFNaR2 in vitro; however, relatively little phosphorylated Stat2 associates with IFNaR2 in vivo. In vitro binding assays defined IFNaR2 residues 418-444 as the minimal interaction domain and site-specific mutation of conserved acidic residues within this domain disrupted in vitro and in vivo binding. An IFNaR2 construct carrying these mutations was either (i) overexpressed in 293T cells or (ii) used to complement IFNaR2-deficient U5A cells. Unexpectedly, the activity of an IFNalpha-dependent reporter gene was not reduced but, instead, was enhanced up to 2-fold. This suggests that this particular IFNaR2-Stat2 interaction is not required for IFNalpha signaling, but might act to negatively inhibit signaling. Finally, a doubly truncated recombinant fragment of Stat2, spanning residues 136-702, associated with IFNaR2 in vitro, indicating that the interaction with IFNaR2 is direct and occurs in a central region of Stat2 marked by a hydrophobic core.