Role for IRF-8 in the HDACi-mediated antitumor response.

<p>Engagement of the IFN-γ receptor (IFN-γR) by IFN-γ and/or the uptake of HDACi, such as TSA, induce IRF-8 transcription. While activation of IRF-8 in both cases is STAT1-dependent, the mechanisms by which this is achieved may be distinct and involve phosphorylated and unphosphorylated modifications. In regard to the latter, the precise nature of interactions remains to be fully detailed. IRF-8 expression, in turn, is known to affect apoptosis by regulating genes associated with both extrinsic and intrinsic pathways of cell death (not illustrated).</p

TSA-mediated IRF-8 transcription is STAT1-dependent.

<p>(<i>A</i>) STAT1 mRNA levels in CMS4 or CMS4.met.sel cells after the indicated treatments, as in Fig. 1. <i>(A)</i> Top, real-time PCR. Data presented as fold-change, as in Fig. 1. (<i>A</i>) Bottom, RT-PCR. *<i>P</i><0.05, based on comparing the single agent treatment to the vehicle-treated control. **<i>P</i><0.05, based on comparing the combination regimen to the single treatment counterparts. (<i>B</i>) CMS4 or CMS4.met.sel cells were transfected with an IRF-8 promoter reporter construct, followed by treatment with the indicated agents for 6 hr. Results are reported as the mean ± SEM of the fold-change relative to the vehicle-treated cells from three separate experiments. *<i>P</i><0.05, based on comparing treatment to matched vehicle control. No activity was observed using the pGL3 vector lacking a promoter. (<i>C</i>) Similar to <i>B</i>, except that CMS4 cells were silenced for STAT1 expression. *<i>P</i><0.05, based on comparing the indicated treatment group to the matched vehicle-treated control. **<i>P</i><0.05, based on comparing the STAT1-deficient groups to their matched STAT1-expressing vector controls. (<i>D</i>) Phosphorylated STAT1 (pSTAT1) and total STAT1 protein levels in CMS4.met.sel cells after treatment with the indicated treatments (TSA, 500 nM; IFN-γ, 200 U/ml) for 15 min, as measured by Western blot. This experiment is representative of one of three with similar results. (<i>E</i>) Similar to <i>D</i>, except that acetylated STAT1 and total STAT1 levels were measured by IP-Western blot (i.e., IP with anti-STAT1 antibody, followed by Western blot with anti-acetyl-lysine antibody) after treatment with or without TSA (500 nM for 6 hr). Band intensities were quantified, and the data presented as fold-change of TSA-treated vs. untreated samples (mean ± SEM of triplicate experiments). *<i>P</i><0.05, based on the TSA-treated group relative to the matched vehicle-treated control.</p

TSA-mediated antitumor activity requires tumor expression of IRF-8.

<p>CMS4 (<i>A</i>) or IRF-8-deficient CMS4-shRNA (<i>B</i>) tumor cells were injected SQ into BALB/c mice. When tumor size was palpable (∼30 mm³), mice were treated with six daily peritumoral injections of TSA or vehicle control. * <i>P</i><0.01 or ** <i>P</i><0.001, based on comparing the control to TSA-treated mice at the indicated time points in panel <i>A</i>. Data were not significant in panel <i>B</i>.</p

HDACi enhances IRF-8 expression in tumor cells.

<p>(<i>A</i>) CMS4 cells were treated with TSA, IFN-γ (100 U/ml) or a combination of both at the indicated concentrations and then analyzed by real-time PCR (top). Representative RT-PCR is shown in the bottom panel, which shares the same treatment labels. Data in top panel are presented as fold-change (shown above each bar) of the treated samples relative to the vehicle-treated controls. (<i>B</i>) Similar to <i>A</i>, except that CMS4 cells were treated with DP (25 ng/ml) instead of TSA. (<i>C</i>) CMS4-met.sel cells were treated with TSA (500 nM), IFN-γ or a combination of both and then analyzed real-time PCR (top) or RT-PCR (bottom), as in <i>A</i>. (<i>D</i>) Similar to <i>C</i>, except that CMS4 cells were treated with DP instead of TSA. All data are expressed as the mean ± SEM of triplicate determinations (shown above each bar). *<i>P</i><0.05, based on comparing the single agent treatment to the vehicle-treated control. **<i>P</i><0.05, based on comparing the combination regimen to the single treatment counterparts.</p

TSA treatment enhances Fas-mediated tumor cell death through an IRF-8-dependent mechanism.

<p>(<i>A</i>) Control or IRF-8-deficient CMS4-shRNA cells were exposed to recombinant mouse FasL (100 ng/ml) after treatment with TSA (100 nM), IFN-γ (200 U/ml), a combination of both or a vehicle control, and cell death measured by a flow-based assay. (<i>B</i>) IRF-8-mutant CMS4-K79E and CMS4-vector control cells were exposed to FasL after treatment with TSA (20 nM) and/or IFN-γ as in <i>A</i>. Data in <i>A</i> and B are expressed as mean ± SEM of six or three independent experiments, respectively. *<i>P</i><0.05, based on comparing the indicated treatment group to the FasL only control. **<i>P</i><0.05, based on comparing the IRF8-deficient to its matched IRF-8-expressing vector controls.</p

TSA enhances IRF-8 expression in a human tumor cell line model of varying malignant potential.

<p>SW480 (<i>A</i>) or SW620 (<i>B</i>) cells were treated with TSA (500 nM), IFN-γ (100 U/ml) or a combination of both and then analyzed by real-time PCR, as in Fig. 1. Data in <i>B</i> are presented as fold-change of the treated samples relative to the vehicle-treated controls. Data expressed as the mean ± SEM of triplicate determinations. *<i>P</i><0.05, based on comparing the single agent treatment to the vehicle-treated control. **<i>P</i><0.05, based on comparing the combination regimen to the single treatment counterparts.</p

Soluble T-Cell Receptors Produced in Human Cells for Targeted Delivery

<div><p>Recently, technology has become available to generate soluble T-cell receptors (sTCRs) that contain the antigen recognition part. In contrast to antibodies, sTCRs recognize intracellular in addition to extracellular epitopes, potentially increasing the number of applications as reagents for target detection and immunotherapy. Moreover, recent data show that they can be used for identification of their natural peptide ligands in disease. Here we describe a new and simplified expression method for sTCRs in human cells and show that these sTCRs can be used for antigen-specific labeling and elimination of human target cells. Four different TCRs were solubilized by expression of constructs encoding the TCR alpha (α) and beta (β) chains lacking the transmembrane and intracellular domains, linked by a ribosomal skipping 2A sequence that facilitates equimolar production of the chains. Cell supernatants containing sTCRs labeled target cells directly in a peptide (p)-human leukocyte antigen (HLA)-specific manner. We demonstrated that a MART-1p/HLA-A*02:01-specific sTCR fused to a fluorescent protein, or multimerized onto magnetic nanoparticles, could be internalized. Moreover, we showed that this sTCR and two sTCRs recognizing CD20p/HLA-A*02:01 could mediate selective elimination of target cells expressing the relevant pHLA complex when tetramerized to streptavidin-conjugated toxin, demonstrating the potential for specific delivery of cargo. This simple and efficient method can be utilized to generate a wide range of minimally modified sTCRs from the naturally occurring TCR repertoire for antigen-specific detection and targeting.</p></div

DMF5 sTCR is internalized upon specific ligand binding.

<p>(a) HeLa cells transfected with either SCT-M1-mCherry or SCT-CD20-mCherry (red) were incubated with His-tagged DMF5 sTCR labeled with an anti-His antibody and visualized using anti-mouse AF488 (green). Co-localization of the sTCR and SCT-M1 is shown in yellow (arrow). (b) Sup-T1 cells expressing SCT-M1 were incubated with DMF5 supernatants containing monomeric sTCR mCherry-His at 37°C for 30 minutes. The cells were subsequently put on ice to block endocytosis and stained with anti-His-AF647 (His). The nucleus was visualized by Hoechst stain (blue). (c) Sup-T1 cells expressing SCT-M1 were incubated with biotinylated DMF5 sTCR-mCherry bound to SA-Miltenyi nanobeads at 37°C.</p

Multimerization of the sTCR increases sensitivity of antigen detection by flow cytometry.

<p>(a) SupT1 cells expressing SCT-CD20 were incubated with increasing concentrations of CD20 swap-sTCR monomer or tetramer, as indicated, and indirectly labeled with anti-FLAG AF647. Results shown are representative of two experiments, and error bars represent SD of duplicates (b) HLA-A2⁺ SupT1 cells were loaded with indicated concentrations of peptide and subsequently labeled with saturating amounts of either sTCR tetramer or nanobeads conjugated to sTCR monomers, followed by staining with anti-FLAG-AF647. Results shown are representative of two experiments. SupT1 cells expressing SCT-CD20 were used as a positive control. The scale shows the calculation of Arcsinh ratio of the Median. (c) SupT1 cells were loaded with increasing concentrations of CD20p (left panel), MART-1p or WT MART-1p (right panel), followed by labeling with saturating amounts of indicated sTCR tetramer, and indirectly stained with anti-FLAG AF647. Irrelevant peptide control (irr peptide) was used at 100 μM (disconnected symbols). Results shown are from one experiment representative of 3 (CD20p and MART-1p) or 2 (WT MART-1p) experiments. Error bars indicate SD of duplicates. (d) SupT1 cells expressing SCT-M1 were incubated with increasing concentrations of PE-conjugated sTCR tetramer.</p

Design of the sTCR and its use as a labeling reagent.

<p>(a) TCRα/β chains in full-length (upper part) and in soluble form (lower part). V: Variable domain, C: Constant domain, TM: transmembrane, CYTO: cytosolic domain. Interchain cystein bridges are indicated with S. (b) Design of the expression construct in which truncated TCRα and β chains are separated by a ribosome skipping sequence (2A) and tagged on their 3’-end. The tags used in this study are listed at their respective position. To increase interchain stability, a high affinity leucine zipper (LZ) was added to some sTCR constructs. (c) HLA-A2^pos T2 cells were loaded O/N with 10 μM MART-1p (red, blue) or 10 μM of an irrelevant peptide (CD20p_188–196) (filled grey). Ten μL of supernatant from HEK293 cells producing the DMF5 sTCR (red), or from mock transfected HEK 293 (blue), was added to the T2 cells and incubated for 15 minutes at RT followed by labeling with anti-His-647 antibodies and flow cytometric analysis (d) SupT1 cells expressing SCT-M1 (red) or SCT-CD20 (filled grey) were incubated with the DMF5 sTCR supernatant as described in (c) and stained using anti-His-647. The results in (c) and (d) are representative of at least two experiments and histograms are gated on viable cells, displayed as FSC^hi, SSC^hi events.</p