Journal: PLoS ONE

Article Title: Effect of B7.1 Costimulation on T-Cell Based Immunity against TAP-Negative Cancer Can Be Facilitated by TAP1 Expression

doi: 10.1371/journal.pone.0006385

Figure Lengend Snippet: 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 ). 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.

Article Snippet: TAP protein was determined by Western blot using a goat anti-mouse TAP1 polyclonal Ab (for TAP1, Santa Cruz Biotechnology, Santa Cruz, CA) or mouse antiserum (for TAP2, generated in our laboratory ).

Techniques: Expressing, Control, Western Blot, Staining, Negative Control

Journal: PLoS ONE

Article Title: Effect of B7.1 Costimulation on T-Cell Based Immunity against TAP-Negative Cancer Can Be Facilitated by TAP1 Expression

doi: 10.1371/journal.pone.0006385

Figure Lengend Snippet: A) Left: RT-PCR was performed to detect two spliced (long and short) Lass5 gene transcripts in CMT.64, CMT.TAP1,2, cl.2 and RMA-S cells. Only the long transcript encodes a Lass5 epitope . A) Right: Lass5 epitope presentation by TAP-deficient and TAP-proficient CMT.64 cells was detected by 12–16 h 51 Cr-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 in vitro 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 51 Cr-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 51 Cr-labled targets were shown in . B) Right: Standard 51 Cr-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 51 Cr-labled targets were shown in . 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 10 6 splenocytes (IFN-γ-SC/10 6 splenocytes).

Article Snippet: TAP protein was determined by Western blot using a goat anti-mouse TAP1 polyclonal Ab (for TAP1, Santa Cruz Biotechnology, Santa Cruz, CA) or mouse antiserum (for TAP2, generated in our laboratory ).

Techniques: Reverse Transcription Polymerase Chain Reaction, Generated, Irradiation, In Vitro, Infection, Enzyme-linked Immunospot

Journal: PLoS ONE

Article Title: Effect of B7.1 Costimulation on T-Cell Based Immunity against TAP-Negative Cancer Can Be Facilitated by TAP1 Expression

doi: 10.1371/journal.pone.0006385

Figure Lengend Snippet: 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 51 Cr-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.

Article Snippet: TAP protein was determined by Western blot using a goat anti-mouse TAP1 polyclonal Ab (for TAP1, Santa Cruz Biotechnology, Santa Cruz, CA) or mouse antiserum (for TAP2, generated in our laboratory ).

Techniques: Infection, Expressing, Negative Control, Western Blot, Control, Generated, Irradiation

Journal: PLoS ONE

Article Title: Effect of B7.1 Costimulation on T-Cell Based Immunity against TAP-Negative Cancer Can Be Facilitated by TAP1 Expression

doi: 10.1371/journal.pone.0006385

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

Article Snippet: TAP protein was determined by Western blot using a goat anti-mouse TAP1 polyclonal Ab (for TAP1, Santa Cruz Biotechnology, Santa Cruz, CA) or mouse antiserum (for TAP2, generated in our laboratory ).

Techniques: