HB1.F3.eGFP cell movement and encirclement of U251.dsRed target cells.

<p>Frames are excised from larger wide-field sequences taken at 30 min intervals. <b><i>A</i></b>, Approach and contact formation by one HB1.F3.eGFP cell (filled arrow) and progression of contact formation by another (open arrow). Stages of HB1.F3.eGFP cell (filled arrow) approach and contact formation were typical: extension of a leading process making contact with the U251.dsRed cell (3.5 h) followed by translocation of the cell body (5.0–7.0 h) and then envelopment of the glioma cell (7.5 h). The other HB1.F3.eGFP cell (open arrow), initially in contact with one of two adjacent U251.dsRed cells, over time extended forward and laterally to move off the surface of both cells. A third HB1.F3.eGFP cell (lower right, not marked) appeared to contact the U251.dsRed cell of interest to the second HB1.F3.eGFP cell, begin to move over the U251.dsRed cell surface (1.0–3.0 h), stall and regress (3.5–7.0 h), and then begin to advance at the end of the sequence (7.5 h). <b><i>B</i></b>, Process of HB1.F3.eGFP cell encirclement of an U251.dsRed.dsRed target. Lamellipodia extend from the initial contact site (1.0–3.0 h) over almost the entire surface of the U251.dsRed glioma cell (6.0–12.0 h) to eventually encircle the U251.dsRed cell. Scale bars  = 15 µm.</p

Analyses of HB1.F3.eGFP::target cell contacts.

<p>Formation of contacts, and the extent of target cell encirclement, did not vary with types of cell encountered, including multiple human malignant lines, primary human glioma cell lines, primary mouse astroglia, and human dermal fibroblasts (adult and new born) and astrocytes. <b><i>A</i></b>, Proportions of total HB1.F3.eGFP cells contacting target cells. U251 cells were one of several malignant brain and breast cancer lines analyzed, and in addition served as a reference cell line for each additional group (marked by either dsRed or CMRA as indicated). No significant differences were observed (all <i>p</i>>0.05 within each group of target cells and across multiple groups; ANOVA with Bonferroni's multiple comparisons test, or <i>t</i> test, as appropriate). <b><i>B</i></b>, Proportions of HB1.F3::target cell pairs displaying each of the thee morphologies defined as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051859#pone-0051859-g003" target="_blank">Figure 3</a> and illustrated in the legend (showing only the HB1.F3.eGFP cell). No significant differences were observed (all <i>p</i>>0.05) with the exception of HB1.F3 homotypic interactions (see text). Data are mean ± s.d. for 3 or more volumes within each of 2–4 independent experiments.</p

HB1.F3.eGFP cells will form contacts with, but not encircle, other HB1.F3 cells.

<p><i>Above</i>, Images of HB1.F3.eGFP::HB1.F3.eGFP and HB1.F3.eGFP::HB1.F3.CD (CMRA) contacting pairs. Cell boundaries are outlined using phalloidin_Alexa647 to show the cortical F-actin cytoskeleton; contact zones are marked by arrows. While the HB1.F3 cells showed filopodial extensions over their hydrogel-exposed surfaces, the contact zone was smooth and clearly delineated by the phalloidin_Alexa647. <i>Below</i>, Heterotypic HB1.F3.eGFP::U251.dsRed pairs were characterized by extension of actin-rich (arrows) lamellipodia from the HB1.F3.eGFP cell over the target glioma cell. These images illustrate the data presented quantitatively in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051859#pone-0051859-g005" target="_blank">Figure 5</a>.</p

Stages of HB1.F3 contact formation and encirclement of U251.dsRed cells.

<p>Images are Z-axis projections of 28–44 optical sections taken at 1 µm intervals. <b><i>A</i></b>, Solitary HB1.F3.eGFP cell extending filopodia-tipped process into the hydrogel matrix. <b><i>B</i></b>, Initial HB1.F3.eGFP contact with a U251.dsRed cell (<b>Type I</b>). Note the morphology of the process contacting the target cell, which has shorter stubbier extensions resembling a holdfast. <b><i>C</i></b>, Loss of processes, apposition of the HB1.F3.eGFP cell body to the U251.dsRed cell, and the beginnings of lamellipodia extension (<b>Type II</b>). <b><i>D</i></b>, Complete HB1.F3.eGFP cell encirclement of a U251.dsRed cell with the cell body and lamellipodia covering much of the glioma cell surface (<b>Type III</b>).</p

Spatial relationships between HB1.F3.eGFP NSCs (green) and U251.dsRed glioma cells (red).

<p>Cells were co-cultured for 18 h in 3-dimensional Puramatrix hydrogel. Shown is the Z-axis projection of 34 optical sections of 900×900 µm spanning 102 µm. Expanded X-Z projections of individual contacting cell pairs confirm true contacts rather than superposition. Of a total of 157 HB1.F3.eGFP cells in this image, 38 (36.9%) were contacting U251.dsRed cells. Of the remaining solitary cells, 94 (59.9%) were roughly spherical or ellipsoid with minimal or no process extension, and 25 (15.9%) showed extended filopodia-tipped processes.</p

Disruption of F-actin filaments with cytochalasin D and myosin II by blebbistatin and consequences for motor protein distributions and HB1.F3.eGFP interaction with target U251.dsRed cells.

<p><b><i>A</i></b>, Images illustrating the sensitivities of cell morphology in Puramatrix (<i>a1, b1, c1</i>) and of the distributions of myosin II heavy chain B (by antibody immunofluorescence) (<i>a2, b2, c2</i>) and F-actin (by phalliodin_Alexa647 fluorescence) (<i>a3, b3, c3</i>). <b><i>a1</i></b>, Normal pattern of HB1.F3.eGFP::U251.dsRed cell interactions (inset shows U251.dsRed cell encirclement by a HB1.F3.eGFP cell). <b><i>a2, a3</i></b>, Normal distributions of MHC-IIB and F-actin. <b><i>b1</i></b>, Disruption of target cell encirclement after MHC-IIB inhibition by blebbistatin (50 µM; 18 h), contraction of MHC-IIB (<b><i>b2</i></b>) and formation of long actin-lined tubes emanating from both tumor cells and NSCs (<b><i>b3</i></b>). <b><i>c1</i></b>, Loss of lamellipodia and truncation of target cell encirclement (inset) after F-actin inhibition by cytochalasin D (1 µM; 18 h), and loss of filamentous structure and appearance of multiple aggregates by both MHC-IIB (<b><i>c2</i></b>) and F-actin (<b><i>c3</i></b>) and loss of spike-like filopodial structures emerging from both HB1.F3.eGFP and U251.dsRed cells (<b><i>c3</i></b>). <b><i>B</i></b>, <i>Above</i>, Proportions of HB1.F3.eGFP:: target cell pairs were not significantly affected by actin or myosin inhibition (<i>p</i>>0.05). <i>Below</i>, After actin disruption or myosin inhibition, encirclement was blocked and did not progress beyond type II (<i>p</i><0.05).</p

Primary human T cells transduced to express huEGFRt/DHFR^FS/IMPDH2^IY can be selected <i>in vitro</i> with MTX and MPA.

<p>(<b>a</b>), Representative flow cytometric evaluation of EGFRt transgene expression (grey histograms) on transduced T cells over 10 days of culture in 0.1 µM MTX, 1 µM MPA, or 0.05 µM MTX +0.75 µM MPA. Percentage of positive cells above control staining (open histograms) is indicated in each histogram. (<b>b</b>), Graphical depiction of the percentages of EGFRt⁺ cells shown in (a). Equal numbers of three different gene-modified T cell lines were each plated in a 6-well plate at the indicated drug concentrations. The data represent means ± S.D. There was a significant difference between the cells on D0 vs. D10 at either 0.1 µM MTX, 1 µM MPA, or 0.05 µM MTX +0.75 µM MPA (****, p≤0.0001; *, p≤0.05).</p

Experimental system for evaluating selection of gene-modified T cells in NSG mice.

<p>(<b>a</b>), Plasmid map of construct containing drug resistance genes DHFR^FS and IMPDH2^IY, huEGFRt and T2A gene sequences (black) that was used to genetically alter primary human T cells. Lentiviral vector backbone (epHIV7) - related sequences are depicted in grey. (<b>b</b>), Schematic for isolation, genetic modification and selection of primary human T cells. (<b>c</b>), CD45RA and CD14 staining of mononuclear cells after sorting from PBMC (top), and CD62L and CD45RO staining of T cells after enriching from PBMC (bottom). The percent positive cells in each quadrant are indicated. (<b>d</b>), Primary human T cells were transduced with above lentiviral vector; transduced T cells and immunomagnetic sorted EGFRt⁺ T cells were stained for EGFRt expression and analyzed for transduction efficiency by flow cytometry. The percent EGFRt⁺ T <i>(</i>grey histogram; isotype control-dark line) cells are depicted.</p

Establish non-toxic MTX and MMF dose regimens for <i>in vivo</i> selection.

<p>6–8 week old NSG mice (n = 6) were (<b>a</b>), administered MTX by i.p. injection at 25 mg/kg/day twice a week the first week (days 1 and 4) and only once the second week (day 8), and/or (<b>b</b>), fed 0.563% MMF medicated feed for 2 weeks. Serum MTX and MPA levels in each case were analyzed by HPLC. Drug toxicity was then monitored by measuring (<b>c</b>), white blood cell counts, (<b>d</b>), body weight, (<b>e</b>), Hemoglobin, (<b>f</b>), ALT, and (<b>g</b>), creatinine levels. (c–g), Mean levels of each measurement (± S.D) after the last treatment (i.e., at day 14) are depicted. There was no significant difference between control and treatment mice (p≥0.05). Data were evaluated between control and treatment mice using an unpaired, two-tailed Student’s t - test.</p

Primary human T cells transduced to DHFR^FS/IMPDH2^IY transgenes are resistant to MTX and MPA.

<p>Non-transduced T cells (non-Txd; grey line/bar) and immunomagnetically-enriched EGFRt⁺ T cells (99.5% EGFRt⁺; Txd+Enr; black line/bar) were plated on day 8 at the indicated concentrations of MTX (<b>a</b>), MPA (<b>b</b>), and a combination of MTX+ MPA (<b>c</b>), cells were followed for total viable cell number, percentage of viable cells, and fold expansion for12 days. Equal numbers of cells were plated in triplicate wells of 24-well plates. The data represent the mean ± S.D. There was a significant difference in viability and fold expansion at day 12 between the non-transduced (Non-Txd) and the transduced, EGFRt-enriched T cells (Txd+Enr) at each drug concentration. ***, p≤0.0002; **, p≤0.001; *, p≤0.01. The data are representative of three separate experiments.</p