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SUPPLEMENTARY DATA
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Results
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The recombinant Lmdd-MPFG strain was stable in vivo after 20 generations
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The in vivo stability of the Lmdd-MPFG strain was examined according to the
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number of viable bacteria at every five passages, by plating the bacteria on both
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selective and nonselective media. We observed no significant differences (P=0.1085)
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in colony forming unit (CFU) counts after 20 generations by plating under each
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condition, suggesting that the recombinant bacteria are extremely stable, with no loss
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or modification of the transgene during sequential replications in vivo (Supplementary
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Figure S1a). The expression of the segments was retained by the bacteria recovered
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from the spleens 3 days post-injection after every 5 passages, showing the in-vivo
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stability and the capacity to retain the chromosomal transgene of the strains
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(Supplementary Figure S1b).
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Materials and methods
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Western blot
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The culture supernatants from which Listeria strains grew were trichloroacetic acid
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(TCA) precipitated and resuspended in 0.1 N NaOH + 2% SDS as described. Blots
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were stained with an anti-flag monoclonal antibody (Zymed, San Diego, CA) and
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alkaline phosphatase-labeled anti-rabbit goat antibody (Kirkegaard and Perry,
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Gaithersburg, MD) was used for detection of bound anti-flag antibody as
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recommended by the manufacturer. Blots were developed with Super Signal
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WestDura chemiluminescent substrate (Pierce, Rockford, IL) and visualized using a
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CCD camera.
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In vivo stability studies and expression of Lmdd-MPFG
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The stability of the recombinant L. monocytogenes integrants was determined after
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20 passages in-vivo as described by Peters et al (Peters and Paterson, 2003). Briefly, a
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dose of 5×107 CFU of Lmdd-MPFG was injected intravenously (i.v.) into 6–8 week
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old mice. Single cell suspensions were prepared from the spleens three days after the
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injection and plated on Brain-Heart Infusion (BHI) plates in the absence of D-alanine
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to select for Lmdd-MPFG growth. Following every five passages, 25 colonies were
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tested for growth on BHI plates containing 100 μg/ml D-alanine. At the end of the
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stability test, one colony recovered was tested for MPFG expression by Western-blot
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with anti-flag antibody.
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Detection of anti-MPFG Abs by enzyme-linked immunosorbent assay (ELISA)
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Sera were collected at week four following the last vaccination and assayed for
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antibodies specific to HBc-Ag and MPFG fusion peptides by ELISA. For detection of
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anti-HBc Abs, an ELISA kit from Diagnostic Automation Incorporation (Calabasas,
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CA) was used according to the instructions. The ELISA-Peptide assay was used to
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detect MPFG fusion peptides HBx52-60, HBx140-148, AFP158-166 and MAGE271-279.
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Briefly, ninety-six-well plates were coated with different MPFG peptides overnight at
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4°C and then blocked by incubation with 10 mg/ml BSA. Mouse sera were diluted
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1:10 in PBS and applied to duplicate wells, followed by goat anti–mouse IgG
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horseradish peroxidase (HRP) conjugates (Sigma, St. Louis, MO) as the secondary Ab
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at a dilution of 1:5000. ABTS 1 Component Microwell Substrate (BioFX
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Laboratories, Owings Mills, MD) was added subsequently and the assay results were
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analyzed at OD 405.
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In vivo depletion of CD4 or CD8 T lymphocyte subsets
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Immunized mice were injected with 500 µg i.p. rat IgG (Abcam, Cambridge, MA),
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anti-CD4 (clone GK1.5), and/or anti-CD8 (clone GK 2.43) on day-3 and day-1 prior
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to challenge with Lmdd-MPFG. The depletion was verified 24 h following mAbs
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injection by analyzing the CD8 and CD4 T cell populations in the blood. The in-vivo
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cytotoxicity assay was assessed following depletion by flow cytometric (FC) analysis
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of spleen.
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T-cell proliferation assay
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Responses to the six peptides were assessed in vitro by measuring proliferation
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after antigen exposure. Splenocytes were established from immunized mice in culture
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at 5×105 cells/well in flat-bottom 96-well plates and stimulated with 5 µg/mL, 20
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µg/mL, or 50 µg/mL peptides in the presence of IL-2 (20 U/mL) (Protech, London,
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UK). Splenocytes were also established without peptide or with 5 µg/mL Con A. Then
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200 µl final culture volumes were incubated at 37°C for 5 days and pulsed with the
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addition of [3H] thymidine at 0.4 µCi/well 16 h before harvesting. Proliferation was
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measured using a Wallac 1450 scintillation counter. Results were expressed using the
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stimulation index (SI), which is the ratio of counts per min (CPM) from peripheral
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blood mononuclear cells (PBMCs) exposed to peptide stimulation to the CPM from
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PBMC cultured in medium only.
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IFN-γ ELISA
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Splenocytes from immunized mice (three per group) were cultured in 24-well
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plates at 5×106 cells/well in-vitro in the presence of 5 µg/L of the various peptides and
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IL-2 (20 U/mL) in 1 mL of complete RPMI medium. Samples from supernatants were
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collected on day 3 and tested for the presence of IFN-γ using a mouse IFN-γ ELISA
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kit (BD Biosciences) according to the manufacturer’s recommendations.
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Cell staining and flow cytometry
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Splenocytes harvested 5 days after the last immunization were stimulated with 20
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µg/mL various peptides in the presence of Golgiplug and then stained with
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PE-Cy5–conjugated
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PE-conjugated IFN-γ (clone XMG1.2; eBioscience). Treg cell staining, followed the
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isolation of TIL populations from the tumor-bearing mice was as follows: cells were
mAb
against
CD8
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(clone
53-6.7;
eBioscience)
and
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stained with CD4-FITC, CD25-APC, CD4-PE, anti-GITR-FITC, anti-CD62L-PE,
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anti-CD127-PE,
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anti-CCR4-PE, and anti-IL-10-PE and assayed on a FACScan flow cytometer (Becton
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Dickinson). Antibodies and their respective isotypes, used as a negative control for
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surface and intracellular staining, were all purchased from BD PharMingen. The
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mouse regulatory T cell staining kit (eBioscience) was used for intracellular cell
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staining for Foxp3 according to the instructions.
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Cytotoxicity assay
anti-CCR7-PE,
anti-TGF-β-PE,
anti-CD152-PE
(CTLA-4),
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For the measurement of cytolytic activity in vivo, target cells were pooled with
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splenocytes from naive mice and pulsed with 20 µg/mL HBx 140-148 peptide and
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labeled with 6 μM CFSE (CFSEhigh) (Molecular Probes) fluorescence intensity.
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Uncoated splenocytes (control target cells) were labeled with 0.3 μM low CFSE
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(CFSElow) fluorescence intensity. The mixed target cells (2107), at a ratio of 1:1,
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were adoptively transferred into the immunized mice. At 24 h after injection of the
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CFSE-labeled targets cells, spleens were removed and the ratio of CFSElow to
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CFSEhigh cells was determined by flow cytometry. The percentage of cytotoxicity was
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determined as follows: (1-CFSEhigh/CFSElow) ×100%. Due to the limited amounts of
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TILs, the cytotoxic activity of TILs and MPFG-specific CTL against various targets
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was assayed by LDH release assays, as described previously (Chen et al., 2009).
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Interferon-γ (IFN-γ), IL-4, and IL-10 ELISPOT assay
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Detection of antigen-specific IFN-γ-secreting T cells from TILs in response to
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peptide stimulation was performed using ELISPOT kits (BioSource International,
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Camarillo, CA). Briefly, 50 µl of cell suspensions from immunized or tumor burden
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mice at 107 cells/ml were added to each well and incubated in the presence of 5 µg/ml
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various peptides/stimulators plus IL-2 (5 U/ml) overnight at 37°C. Splenocytes
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without peptide or with 2.5 µg/ml Concanavalin A (Con A) were used as control. For
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the IL-4 and IL-10 Elispot assays, splenocytes from naive or immunized mice were
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plated with 1ng/ml PMA and 0.5 ng/ml Inomycine (Sigma-Aldrich). IFN-γ, IL-4 and
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IL-10 spots in the wells were then developed according to the manuscript. Results
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were expressed as spot-forming units (SFU)/106 cells after subtracting background
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spots.
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Isolation of tumor infiltrating lymphocytes (TIL)
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At selected time-points after treatment, tumors were removed from mice and
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single-cell suspensions were prepared by enzymatic digestion. Resected tumors were
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weighed, minced into small (1–2 mm3) pieces with a scalpel, and immersed in 10 mL
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of digestion mixture [5% FBS in RPMI 1640, 0.5 mg/ml collagenase A (Roche
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Diagnostic), 0.2 mg/ml hyaluronidase, type V (Sigma-Aldrich), and 0.02 mg/ml
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DNase I (Sigma-Aldrich)] per 0.25 g of tumor tissue. The resulting cell suspensions
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were filtered sequentially through 70- and 40-µm cell strainers (BD Falcon) and
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washed with 5% FBS in RPMI 1640. Red blood cells (RBC) were lysed by brief
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incubation in 0.15 M ammonium chloride solution, and cell debris was removed by
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centrifugation using a Lympholyte-M gradient as recommended by the manufacturer
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(Cedarlane).
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Immunohistochemistry
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Immunoperoxidase staining of CD4+ or foxP3 T cells was done on 4-μm sections of
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formalin-fixed paraffin-imbedded tumors. Sections were deparaffinized and subjected
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to heat-induced antigen retrieval using DAKO's target retrieval solution for 25 min.
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Following endogenous peroxidase removal using 3% hydrogen peroxide in methanol,
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the samples were incubated 2×15 min with avidin-biotin blocking reagent (Vector,
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Burlingame, CA) and protein-blocking reagent (DAKO, Carpinteria, CA) for 15 min.
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Slides were incubated with a 1:80 dilution of primary antibody (anti-CD4 or Foxp3)
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or isotype control (BD PharMingen, San Diego, CA) overnight at 4°C.
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Immunodetection was done using a secondary biotinylated-polyclonal anti-Rat IgG
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(Sigma, St. Louis, MO). Diaminobenzidine/hydrogen peroxidase was used as the
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chromogen substrate. Serial sections were stained by routine H&E.
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Supplementary Figure legends
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Figure S1. In vivo stability and expression studies of Lmdd-MPFG strain. A, In
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vivo stability was examined by immunizing mice with 5×107 CFU of the
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p1565-MPFG strain intravenously in the tail vein. The CFUs were determined in the
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homogenized spleens after 24, 48, and 72 h. Viable CFUs were determined after
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plating on both selective and nonselective media. No colonies were recovered at the
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time points of 72 h. Columns, mean number of CFU from each mouse; bars, SD. B,
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Expression of MPFG fusion protein by Lmdd-MPFG by passaging in vivo. Protein
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extracts were prepared by precipitating culture supernatants with 10% trichloroacetic
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acid (TCA) at 4°C. The Western blot represents the expression of MPFG in the total
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proteins secreted by the Lmdd-MPFG strains. The blot was stained with a 1:1,000
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dilution of anti-flag antibodies.
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Figure S2. Frequency of total CD8+ and CD4+ cells in the PBL following the
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indicated antibody treatment. 500 µg rat IgG, anti-CD4, or anti-CD8 (clone GK 2.43)
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were injected intraperitoneally (i.p.) to immunized mice on day-3 and day-1 prior to
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challenge with Lmdd-MPFG. The CD8 and CD4 T cell populations in the blood 24
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hours after mAbs injection were analyzed by flow cytometry to verify complete
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depletion.
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Figure S3. Detection of Th2/antibody mediated responses. Splenocytes from
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immunized or control mice were harvested and cultured in the presence of PMA and
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Inomycine. A, HBc-specific Abs elicited in HLA-A2 Tg mice were measured by
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enzyme-linked immunosorbent assay (ELISA). B, The number of cytokine-secreting
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cells for IL-4, and IL-10 was determined as described in the Supplementary Materials
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and Methods.
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