Production of virus in SCC-F1 cells.

<p><b>A)</b> Infected SCC-F1 cells constantly produce infectious particles to the supernatant. <b>B)</b> Virus released to the supernatant by infected SCCF1 cells was able to re-infect both A549 and SCC-F1 cells. <b>C)</b> Viral particles released to the supernatant were completely neutralized with anti-VV antibody.</p

Vaccinia virus replication and maturation in A549 cells.

<p><b>A-B</b>: After enter A549 cells, at 48 hours post infection, vaccinia viral particles begin their normal life cycle quickly: form replication centre (viral factory) in juxta-nuclear region manifested as a homogeneous, low-to-middle electron density, void of any organelles. The replication begins with formation of crescent (Cr), then immature virus (IV), immature virus with nucleoids (IVN), mature virus (MV). Many MV are wrapped by intracellular membrane from Trans-Golgi cistern, form intracellular enveloped virus (IEV). IEVs move towards cell periphery, fuss with cell membrane and obtained another envelope from cell membrane, finally they are released to extracellular space to form extracellular enveloped virus (EEV). Some EEV remain attached on cell membrane as cell associated extracellular enveloped virus (CEV). <b>C-D</b> Different intermediate particles from cells other than cells in a-b in high magnification.</p

Electron microscopy of negative stained, purified vaccinia viral particles released from cultured cells.

<p>Supernatant from infected SCCF1 or A549 cells was collected, purified and negative staining electron microscopy was performed. <b>A-C:</b> viral particles produced by A549 cells: mature particles of brick-shaped, with outer envelope (OE) derived from cell membrane. <b>D-E</b>: immature vaccinia viral particles produced by SCCF1 cells: the particles are irregular in shape and in electron density. Their internal contents are not tightly packed, no clear boundary or membranous outer envelope is visible.</p

Abortive vaccinia virus replication and maturation in SCCF1 cells.

<p><b>A)</b> At 48 hours after infection, the replication centre (viral factory) is inconspicuous, only a few immature viruses (IV) were found around it. <b>B)</b> Two IVs were visible, one IV showed nucleoid inside thus can be identified as IVN. Neither mature viruses nor enveloped viruses were detected throughout all infected cells. <b>C)</b> there are two small immature viruses (IV) located within cell and in the edge of an abnormal granular virosome (VS) region. No MVs were visible there. <b>D)</b> The immature viruses (IV) migrate to peripheral of the cell, underneath cell membrane. They still fail to get membranous envelope even at this late stage of nearly being released out.</p

In vitro cytotoxicity and and transduction of vvdd in SCCF1 cells.

<p><b>A)</b> SCCF1 cells were infected with vvdd with 0.01, 0.1 or 1 pfu/cell. Viability of cells was measured on day 3. <b>B)</b> Compared to A549 cells, SCCF1 cells maintained their viability significantly better on day 3 <b>C)</b> When SCCF1 cells formed a tight monolayer before infection, the cells were still alive 10 days after infection. <b>D)</b> SCCF1 cell were infected with 0.04, 0.2, 1 or 5 pfu per cell and luciferase expression was measured 4 h postinfection in relative light units.</p

Intra-tumor accumulation of antigen-presenting cells (APCs) is increased by GM-CSF and IL-2.

<p>B16.OVA bearing mice were treated with adoptive transfer of 2x10⁶ CD8a⁺ enriched OT-I lymphocytes intraperitoneally and tumors were either injected with PBS or recombinant cytokine in PBS or left non-injected (n = 5). (a) Levels of CD11c⁺ dendritic cells and (b) proportion of dendritic cells expressing maturation marker CD86 on cell surface were analyzed on day 14 post-transfer from tumors. Data presented as mean ± SEM. *P ≤ 0.05 and **P≤ 0.01 by one-way ANOVA followed by Tukey’s post-hoc test.</p

IFN-α2, IFN-γ and IL-2 treatment leads to changes in CD8+ TIL phenotypes.

<p>Mice harboring subcutaneous B16.OVA tumors were treated intraperitoneally with 2x10⁶ CD8a⁺ enriched OT-I lymphocytes and injected intratumorally with either PBS or recombinant cytokine in PBS or left non-injected (n = 5). (a) Levels of tumor-infiltrating endogenous (non-OVA) CD8⁺ T-cells and (b) count of central memory (T_CM) and effector memory (T_EM) T-cells were assessed from tumors on day 14 post-transfer by flow cytometry. (c) Activation status of tumor-infiltrating CD8+ T-cells was evaluated on day 14 by expression of CD69 and IFN-γ following PMA/Ionomycin stimulation <i>ex vivo</i>. Data presented as mean ± SEM. *P ≤ 0.05 and **P≤ 0.01 by one-way ANOVA followed by Tukey’s post-hoc test.</p

List of antibodies used.

<p>*ND = not determined</p><p>List of antibodies used.</p

IFN-α2, IFN-γ and IL-2 augment anti-tumor efficacy but do not increase tumor-accumulation of transferred cells.

<p>Mice bearing syngeneic B16.OVA tumors were adoptively transferred with 2x10⁶ CD8a⁺ enriched, polyclonally activated OT-I lymphocytes intraperitoneally and tumors were either left non-injected or injected with PBS or recombinant cytokine in PBS (n = 10). (a) Tumor growth was monitored every 2–3 days with an electronic caliper. Due to variation in tumor sizes at the beginning of the experiment, the results are represented as relative change compared to day 0 volume, which was set at 100%. (b-c) Levels of OT-I cells in tumors were quantified on days 4 (b) and 14 (c) post-transfer by pentamer staining and flow cytometry. (d) Proportion of major histocompatibility complex (MHC) class I molecules presenting OVA-derived peptide SIINFEKL and (e) mean fluorescence intensity (MFI) of mouse MHC class I H-2kb from tumor samples was assessed by flow cytometry on day 14 post-transfer (n = 5). Data presented as mean ± SEM. *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001, ****P≤ 0.0001 by repeated measures ANOVA (a) or one-way ANOVA followed by Tukey’s post-hoc test (b-e).</p

Doses of recombinant cytokines.

<p>*N/A = not assessed</p><p>Doses of recombinant cytokines.</p