Fitness assays of the engineered <i>Sc/Su</i> hybrids carrying different type of <i>TRP2/TRP3</i> chimeric complexes.

<p><i>Sc/Su</i> hybrids were genetically modified to carry either the two different chimeric complexes, Trp2p^Su/Trp3p^Sc and Trp2p^Sc/Trp3p^Su, or the two parental hemizygous controls, Trp2p^Su/Trp3p^Su and Trp2p^Sc/Trp3p^Sc (panel A). The growth curves of <i>S. cerevisiae</i>, <i>S. uvarum</i>, the hybrid <i>Sc/Su</i> and the engineered hybrids shows that Trp2p^Su/Trp3p^Sc grows better than the other combinations in SD media lacking tryptophan (panel B). The fitness competition assay between <i>Sc/Su</i> hybrids with different combination of the <i>TRP2/TRP3</i> complex and the GFP reference strain shows again that Trp2p^Su/Trp3p^Sc grows faster (panel C). The competitive fitness coefficient Sg represents the difference between the ln of the ratio of hybrid and reference strain between final and initial time points, normalized for the number of generations. An equal fitness between hybrid and reference strains would be indicated by a value of zero (see Method section).</p

Growth assays of <i>Sc/Su</i> hybrids carrying different types of MBF chimeric complexes.

<p><i>Sc/Su</i> hybrids were genetically modified either to carry the two different chimeric complexes, Mbp1^Su/Swi6^Sc and Mbp1^Sc/Swi6^Su, or the two uni-parental controls, Mbp1^Su/Swi6^Su and Mbp1^Sc/Swi6^Sc (Panel A). The growth spot assay of the engineered hybrids in rich YPD and YP-glycerol media are shown in Panel B. The strain carrying the <i>S. uvarum</i> homologous Mbp1^Su and Swi6^Su is the only one that performs respiratory growth and grows normally in the presence of glycerol a sole carbon source.</p

TAP-strategy for recovery and identification of hybrid protein complexes.

<p><i>S. cerevisiae</i> strains with the TAP cassette inserted into the C-terminal of one member of the complex (TAP-tag A) were crossed with <i>S. mikatae</i> and <i>S. uvarum</i> species. The complexes that freely formed in the hybrids were then isolated and the interacting members identified via MS analysis. A', B' and C' represent the orthologs of the <i>S. cerevisiae</i> A, B, C proteins, respectively.</p

A Whole-site First-assessment Toolkit for combined Mineral Resource and Archaeological assessment in Sand and Gravel deposits.

A Whole-site First-assessment Toolkit for combined Mineral Resource and Archaeological assessment in Sand and Gravel deposits

Increased immune protection of mice from TAP-negative tumor challenge by VV-B7.1+VV-TAP1-infected CMT.64 cell immunization.

<p>C57BL/6 mice (each group, n = 10) were immunized i.p with CMT.64 cells (5×10⁶ cells/mouse) infected overnight with VV-GFP+VV-GFP, VV-B7.1+VV-GFP, or VV-B7.1+VV-TAP1 at MOI of 3 for each VV. After a 20-day immunization, mice were challenged i.p. with live CMT.64 tumor cells (2.5×10⁵ cells/mouse), and the time of morbidity was recorded.</p>#<p>Experiments ended at day 120 of CMT.64 cell challenge.</p>*<p>Indicated that the tested mouse groups compared with a control group (immunized with VV-GFP+VV-GFP-infected CMT.64 cells) for statistical analysis (ANOVA).</p

TAP-deficient tumor cells present TAP-independent antigen for T cell priming.

<p>A) Left: RT-PCR was performed to detect two spliced (long and short) Lass5 gene transcripts <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006385#pone.0006385-vanHall1" target="_blank">[13]</a> in CMT.64, CMT.TAP1,2, cl.2 and RMA-S cells. Only the long transcript encodes a Lass5 epitope <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006385#pone.0006385-vanHall1" target="_blank">[13]</a>. A) Right: Lass5 epitope presentation by TAP-deficient and TAP-proficient CMT.64 cells was detected by 12–16 h ⁵¹Cr-release assays. Lass5 specific T cells were generated by immunization i.p with γ-irradiated RMA-S/B7.1 cells pulsed with 5 µM Lass5 peptide. The immunized splenocytes were re-stimulated <i>in vitro</i> with Lass5 peptide-pulsed γ-irradiated RMA-S/B7.1 cells for 5 days. One of three experiments is shown. B) Left: 12–16 h ⁵¹Cr-release assays were conducted to confirm that an NP_366–374 eptitope specific T-cell population generated by immunization with γ-irradiated RMA-S/B7.1 cells pulsed with 5 µM NP_366–374 peptide did not contain T-cell sub-populations recognizing TAP-independent epitopes presented by CMT.64 and its transfectants. The ⁵¹Cr-labled targets were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006385#pone-0006385-g004" target="_blank">figure 4B</a>. B) Right: Standard ⁵¹Cr-release assays were conducted to confirm that TAP-deficient CMT.64 transfectants did not present TAP-dependent NP_366–374 epitope when the cells were infected overnight with VV carrying NP_366–374 minigene at multiplicity of infection (MOI) of 3. The ⁵¹Cr-labled targets were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006385#pone-0006385-g004" target="_blank">figure 4B</a>. The NP_366–374 epitope specific T-cells were generated by immunization with γ-irradiated RMA-S/B7.1 cells pulsed with 5 µM NP_366–374 peptide and re-stimulated with 5 µM NP_366–374 peptide in vitro. C) An ELISPOT assay was performed to detect Lass5 specific IFN-γ-secreting precursors. Mice were immunized with γ-irradiated CMT.64/pp, CMT.TAP1,2, CMT.B7.1/p and CMT.TAP1/B7.1 tumor cells. The immunized splenocytes were stimulated with or without Lass5 peptide. The number of Lass5 antigen-specific, IFN-γ-secreting precursors was determined. Precursor frequency is reported as IFN-γ-secreting cells per 10⁶ splenocytes (IFN-γ-SC/10⁶ splenocytes).</p

Expression of TAP1, B7.1 and MHC-I molecules in transfectants.

<p>TAP, B7.1, K^b and D^b expression in CMT.64 transfectants was determined. A) TAP1 and TAP2 proteins were detected in CMT.64 transfectants and control RMA cells by Western blots using anti-TAP1 and TAP2 polyclonal antibodies respectively (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006385#s4" target="_blank">Material and Methods</a>). Levels of expression of GAPDH protein were detected in each sample as the loading control. B) B7.1 expression in CMT.B7.1/p and CMT.TAP1/B7.1 cells was detected by FACS assays using FITC-conjugated B7.1-specific mAb 16-10A1. Control is CMT.64 cells stained with mAb 16-10A1. C) K^b or D^b expression was detected by FACS assays using primary mAbs Y-3 against H-2K^b or 28-14-8S against H-2D^b followed by staining with a FITC-conjugated goat anti-mouse IgG secondary Ab. CMT.64 cells stained with a primary mAb 15-5-5S (against H-2D^k) followed by staining with a FITC-conjugated goat anti-mouse IgG secondary Ab were used as a negative control. a: negative control; b: CMT.64; c: CMT.64/pp; d: CMT.B7.1/p; e: CMT.TAP1/p; f: CMT.TAP1/B7.1; and g: CMT.TAP1,2 cl.21.</p

Deduction or abolishment of immune response against CMT.64 tumor by depleting CD4⁺ or CD8⁺ T cell sub-population in C57BL/6 mice.

<p>Mice (each group, n = 10) were injected i.p. with mAbs GK1.5 for CD4⁺ T cells or 2.43 for CD8⁺ T cells (0.1 ml per mouse) every other day for the first week and once per week afterward to deplete relevant T-cell sub-population. A) Before γ-irradiated tumor cell immunization, depletion of CD8⁺ or CD4⁺ T cell subsets was assessed in blood by FACS assays using FITC-conjugated anti-mouse CD8a (5H1-1) or FITC-conjugated anti-mouse CD4 (RM4-4) together with PE/Cy5-conjugated anti-mouse CD3 (145-2C11). Frequencies of CD8⁺ or CD4⁺ T cells were detected before mAb treatment (indicated as normal) and after first week mAb treatment and before γ-irradiated tumor cell immunization (indicated as mAb-treatment). B) After a 20-day immunization with γ-irradiated tumor cells (5×10⁶ cells per mouse) the mice were challenged i.p. with live CMT.64 tumor cells (2.5×10⁵ cells per mouse). The time of morbidity was recorded.</p

Influence of TAP-independent tumor antigen presentation in virally infected TAP-deficient tumor cells.

<p>CMT.64 cells were infected overnight with a combination of two VVs at MOI of 3 for each VV. A) B7.1 expression was detected by FACS assay using FITC-conjugated B7.1-specific mAb 16-10A1. CMT.64 cells without viral infection were used as a negative control. TAP1 expression was detected by Western blot using a goat anti-mouse TAP1 polyclonal Ab. RMA-S cells were used as a control. B) Standard ⁵¹Cr-release assays were conducted to detect if virally infected tumor cells affected presentation of endogenous tumor antigens. CMT.64 tumor cells were infected overnight with VV-GFP+VV-GFP, VV-B7.1+VV-GFP, or VV-B7.1+VV-TAP1, and used as target cells. Control cell lines were CMT.64, CMT.B7.1/p and CMT.TAP1/B7.1. Tumor antigen-specific T cells were generated by immunization with γ-irradiated CMT.B7.1/p cells. Target and effector ratio was used at 1∶100. a: CMT.64; b: CMT.B7.1/p; c: CMT.TAP1/B7.1; d: CMT.64+VV-GFP+VV-GFP; e: CMT.64+VV-B7.1+VV-GFP and f: CMT.64+VV-B7.1+VV-TAP1. ** indicates statistical significance (P≪0.05) using ANOVA analysis.</p

Decrease in tumorigenicity and increase in immune response by B7.1 and TAP1 co-expressing tumor cells.

<p>The time of morbidity was recorded for mice (each group, n = 10) inoculated with live tumor cells or immunized with γ-irradiated cells and followed by challenge with CMT.64 cells. Statistics for mouse survival were obtained using the Kaplan–Meier log rank survival test and differences were considered significant at P<0.05. A) Tumorigenicity was detected by injection of C57BL/6 mice i.p. with live CMT.64 cells or their transfectants (5×10⁴ cells per mouse). B) Nude mice were used for determination of tumorigenicity with conditions of tumor injection the same as shown in A), P>0.05 for all comparisons. C) C57BL/6 mice were immunized i.p. with different γ-irradiated tumor cells or PBS at a high-dose (left, 5×10⁶ cells per mouse) or a low-dose (right, 5×10⁵ cells per mouse). After a 20-day immunization, the mice were challenged i.p. with live CMT.64 tumor cells (2.5×10⁵ cells per mouse). Different survival rates and/or times were observed.</p