Fluorescence spectra of WT and cysteine mutants of Viperin in folded and unfolded condition.

<p>Fluorescence emission spectra of (a) the WT Viperin, (b) the triple mutant (c) C83A mutant, (d) C87A mutant and (e) C90A mutant (f) NATA in the absence (void circle) and presence (closed circle) of 10 M urea. Fluorescence experiments have been carried out in 20 mm phosphate buffer at pH 7.5. A red shift in the emission spectra is shown by a double headed arrow for the WT protein.</p

UV-visible absorption spectra of WT and cysteine mutants of Viperin.

<p>UV-visible absorption spectra of the WT Viperin (black), the triple cysteine mutant (cyan), C83A mutant (red), C87A mutant (blue), and C90A mutant (dark green). The experiments are performed in 20 mM sodium phosphate buffer at ph7.5. The WT protein exhibits two peaks at 325 nm and 410 nm (shown by arrows) which are characteristic of Fe-S cluster. They are found to be absent in the case of cysteine mutants.</p

Thermodynamic parameters of the equilibrium unfolding transitions of WT and cysteine mutants of Viperin.

<p>Thermodynamic parameters of the equilibrium unfolding transitions of WT and cysteine mutants of Viperin.</p

Unfolding transitions of WT and cysteine mutants of Viperin monitored by far- UV CD.

<p>Urea induced unfolding of the WT (black), the triple mutant (red), C83A mutant (blue), C87A mutant (dark green), and C90A mutant (magenta) of Viperin. The ellipticity measured at 223 nm is plotted against urea concentration. The data for WT and the cysteine mutants are fit to equation 1 assuming two state unfolding transitions. The experiments have been carried out in 20 mM phosphate buffer at pH 7.5.</p

Far-UV circular dichroism spectra of WT and cysteine mutants of Viperin.

<p>Far-UV CD spectra of the WT Viperin (____), the triple cysteine mutant (-.-.-.), C83A mutant (_ . . . _), C87A mutant (- - - -), C90A mutant (. . . .). The CD experiments have been carried out in 20 mM sodium phosphate buffer at ph7.5.</p

Transitory increase in stress-associated antigens on TMZ-resistant cell line U87 after exposure to TMZ.

<p>U87 cells were cultured to confluence and incubated in media containing 100 µM TMZ. Stress antigens were assessed at the time intervals noted on the x axis by flow cytometry and the increase in median fluorescence intensity over isotype control were calculated. Data are shown as percentage increase over unmanipulated U87 cells. SD of 3 experiments are shown.</p

Transduction of γδ T cells with lentivirus vector was performed on day 6, 7 and 8 of expansion culture (see text) with increasing MOI.

<p>(a) On day +14 cells were incubated in media supplemented with 400 µM TMZ and viable cell counts were obtained for each MOI. Two separate experiments are shown. (b) Quantitative PCR analysis to measure P140KMGMT copy numbers of the bioengineered γδ T cells in the presence of increasing concentrations of TMZ, which are indicated in the figure.</p

Genetic modification of expanded/activated γδ T cells by HIV (a) and SIV-GFP (b) lentiviral vectors 6 days following transduction with an MOI of 15.

<p>Mean fluorescence intensity (MFI) is approximately the same for both vectors, but transduction efficiency and expression from the SIV-derived vector is higher when measured on day+6. Quadrant values are noted to the right of each plot.</p

Proliferation of Modified vs. Transduced γδ T cells in Culture.

*<p>Cell dose is extrapolated to final volume of unmodified cells based on starting volume removed for transfection.</p

Expanded/activated γδ T cells were manufactured as described in the text.

<p>Flow cytometry from two separate donors shown from (<i>a</i>) unmanipulated and (<i>b</i>) P140KMGMT-transduced γδ T cells. For both panels (a) and (b) quadrant 2 (Q2) represents γδ T cells. As discussed in the text, the yield of γδ T cells was slightly lower than control due to loss of cells during the transduction procedure; however, purity of the final product was not affected as both products from a single donor show >90% purity of γδ T cells. (c and d) Cytotoxicity assays from two separate expansions (panel c and d, respectively) of unmodified γδ T cells (solid line) versus TMZ P140KMGMT transduced γδ T cells (dashed line) against the TMZ-resistant glioma cell line U87 were conducted to determine if genetic modification impairs γδ T cell function. Cytolytic activity of γδ T cells against U87 cells was nearly equivalent at all E:T ratios, verifying that P140KMGMT transduced γδ T cells function is equivalent to that of unmodified γδ T cells.</p