Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: Demographics of colorectal cancer patients and the statistical association with FSTL1/DIP2A expressions in the tumor tissues

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques:

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: Apoptotic T cells increase in the mice with FSTL1+ colorectal cancer. A. Establishment of FSTL1 transfectants (TR1-3) using murine colorectal cancer (CRC) MC38 cells. Photos show morphological changes (round types to spindle types) after fstl1 transduction (scale = 1,000 µm). FSTL1 in the cultured supernatants was measured by ELISA, and cellular functions were tested (n = 3). B. Retardation of the TR tumor growth after subcutaneous implantation in mice (n = 5). C. Increase of PD1+ annexin V+ apoptotic T cells within the TR tumors (day 14; n = 5). D. Increase of PD1+ annexin V+ apoptotic T cells in spleen of the TR-implanted mice (day 14; n = 5). E. Expansion of a DIP2A+LAG3+ subset in the CD11b+ cells of the TR-implanted mice (day 14; n = 5). Graphs show means ± SDs except scatter plots (open circles, individual data; closed circles, means). *P < 0.01, **P < 0.05 versus mock control. Representative data of three independent experiments.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Transduction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: The T-cell apoptosis is caused by the FSTL1+ tumor-induced CD11b+DIP2A+LAG3+ cells. (A) FSTL1 upregulates LAG3 expression in CD11b+ cells. Splenic CD11b+ cells obtained from naive mice were stimulated with FSTL1 in the presence or absence of anti-FSTL1 mAb, anti-LAG3 mAb, or isotype control for 3 days, and were analyzed by flow cytometry. (B, C) The TR-stimulated CD11b+ cells suppress CD8+ T-cell proliferation partly via LAG3. Splenic CD11b+ cells obtained from tumor-implanted mice (day 14) were cocultured (2:1) with CD8+ T cells (2:1) in the presence of anti-CD3 mAb and/or anti-LAG3 mAb for 5 days (n = 3). CFSE-labeled CD8+ T cells were used for tracking cell division (B). The cocultured CD8+ T cells were analysed for expression of immune checkpoint molecules by flow cytometry (C). (D) In vivo T-cell exclusion caused by the TR-stimulated CD11b+ cells partly via the LAG3. MC38 cells were s.c. coinjected (1:1) with the CD11b+ cells in mice, and 4 days later, the mice were treated with anti-LAG3 mAb or isotype control at 10 mg/kg (n = 5). Twenty days after coinjection, tumors and spleens were harvested for flow cytometric analysis. *P < 0.01, **P < 0.05. Graphs show means ± SDs. Representative data of three independent experiments.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Expressing, Flow Cytometry, Labeling, In Vivo

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: Blocking LAG3 interferes the FSTL1-induced T-cell suppression in human system. A. Establishment of FSTL1 transfectants (TR1-3) using human CRC HCT116 cells. Photos show morphological changes (round types to spindle types) and DIP2A enhancement after fstl1 transduction (scale = 100 µm). FSTL1 in the cultured supernatants was measured by ELISA, and cellular functions were tested (n = 3). *P < 0.01 versus mock control. B. The TR-induced CD11b+ cells suppress T-cell proliferation via LAG3. CD11b+ were sorted from human PBMCs, and were stimulated with tumor supernatant fluids for 5 days followed by flow cytometric analysis for LAG3 and DIP2A expressions. CD3+ T cells were cocultured (2:1) with the stimulated CD11b+ cells in the presence of anti-CD3 mAb and/or anti-LAG3 mAb for 5 days (n = 3). **P < 0.05. Graphs show means ± SDs. Representative data of two independent experiments.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Blocking Assay, Transduction, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: Blocking FSTL1 and LAG3 synergizes in treatment of mouse CRC models. (A-C) Blocking LAG3 successfully elicits anti-tumor effects by suppressing CD8+ T-cell apoptosis in the TR-implanted mice. Mice were s.c. implanted with tumor cells, and were i.p. injected with anti-PD1 mAb, anti-LAG3 mAb, or rat IgG as a control (10 mg/kg) on days 4 and 7 after tumor implantation (n = 5). Tumor growth was measured (A), and tumors and spleen were harvested for flow cytometric analysis (B) and cytotoxic assay (E:T ratio = 20:1; C) on day 18. (D) Blocking LAG3 synergizes with blocking FSTL1 in the treatment of CRC metastasis models. Mice were both s.c. and intravenously (i.v.) implanted with FSTL1+ Colon26 cells, and were i.p. injected with anti-LAG3 mAb, anti-FSTL1 mAb, and/or rat IgG (5 mg/kg) on days 4 and 7 (n = 5). *P < 0.01, **P < 0.05 versus control group. Graphs show means ± SDs. Representative data of three independent experiments.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Blocking Assay, Injection, Tumor Implantation

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: FSTL1 positivity in tumor tissues significantly correlates with poor prognosis of CRC patients. Tumor tissues obtained from CRC patients were immunohistochemically analyzed for FSTL1 and DIP2A expressions (n = 306). A. Representative photos (scale = 100 µm). B. Significant correlation between FSTL1 and DIP2A expressions. The molecular expression patterns were categorized into 4 levels according to the immunofluorescence intensity depicted as pixel counts: Level 0, no expression; Level 1, weak expression; Level 2, moderate expression; and Level 3, strong expression. C. FSTL1 positivity is reversely correlated with overall survival of the patients.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Expressing, Immunofluorescence

Journal: American Journal of Cancer Research

Article Title: CD11b + DIP2A + LAG3 + cells facilitate immune dysfunction in colorectal cancer

doi:

Figure Lengend Snippet: CD11b+DIP2A+LAG3+ cells are expanded in peripheral blood of metastatic CRC patients. PBMCs were obtained from healthy donors (n = 4) and patients with stage IV metastatic CRC (n = 11). A. FSTL1 concentration measured by ELISA (means ± SDs). B. Gating strategy and representative dot plot data in flow cytometric analysis. C. Significant correlation between CD11b+DIP2A+LAG3+ cell expansion and CD3+CD8+Ki67+GZMB+ T-cell reduction. Open circles, healthy donors. Closed circles, CRC patients. The P value in the scatter plot was analyzed by the nonparametric Spearman’s rank test.

Article Snippet: Human FSTL1 -/low HCT116 cells were transfected with a plasmid vector pCMV6-ENTRY (Origene Technologies) encoding human human fstl1 (GenBank accession number: {"type":"entrez-nucleotide","attrs":{"text":"NM_007085","term_id":"1519241778","term_text":"NM_007085"}} NM_007085 ), or the empty vector as mock transfection by electroporation (0.4 kV, 25 µFD).

Techniques: Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Biological Chemistry

Article Title: Wolbachia Lipoprotein Stimulates Innate and Adaptive Immunity through Toll-like Receptors 2 and 6 to Induce Disease Manifestations of Filariasis *

doi: 10.1074/jbc.M901528200

Figure Lengend Snippet: The inflammatory stimuli of BMFE are lipoproteins that primarily signal via TLR2/6. A , HEK-TLR2 cells were transfected with plasmids encoding small interfering (psi) RNA specific for TLR1 or TLR6 before being stimulated with BMFE or control stimuli (doses stated are in micrograms/ml). Accumulations of IL-8 secreted by HEK-psiTLR1 or -psiTLR6 triplicate cultures 20 h post-stimulation are plotted as mean (±1 S.E.) percentages of corresponding HEK-TLR2 responses (mean ± 1 S.E. max IL-8 concentrations as follows: TNFα = 7859 ± 98 pg/ml, PAM 3 CSK = 9807 ± 175 pg/ml, FSL-1 = 2001 ± 345 pg/ml, and BMFE = 9495 ± 137 pg/ml). Significant differences compared with HEK-TLR2 responses are indicated: ***, p < 0.001; **, p < 0.01. B , peritoneal macrophages from WT, TLR1 −/− , TLR6 −/− were stimulated with BMFE in triplicate (doses stated are micrograms/ml), and production of TNFα after 20 h is plotted as mean ± 1 S.E. percentages of WT response to 400 μg/ml BMFE (mean ± 1S.E. max TNFα concentration = 188.4 ± 10.36 pg/ml). Significant differences compared with WT are indicated ***, p < 0.001; **, p < 0.01. C , triplicate HEK-TLR2 cultures were stimulated with BMFE or control stimuli (doses stated are micrograms/ml) before or following Cleanascite TM or BindPro TM treatment. Data plotted are mean IL-8 ± 1S.E. All data are representative of three independent experiments.

Article Snippet: Ultra-pure LPS, PAM 3 CSK 4 , FSL-1 (Autogen BioClear), and rTNFα (R&D Systems) were used at the doses stated.

Techniques: Transfection, Concentration Assay

Journal: The Journal of Biological Chemistry

Article Title: Wolbachia Lipoprotein Stimulates Innate and Adaptive Immunity through Toll-like Receptors 2 and 6 to Induce Disease Manifestations of Filariasis *

doi: 10.1074/jbc.M901528200

Figure Lengend Snippet: Synthetic diacyl-lipopeptide analogue of wBmPAL (Diacyl WoLP) replicates BMFE-TLR2/6-specific activation of inflammation. A , HEK-TLR2 cells were transfected with plasmids encoding small interfering (psi) RNA specific for TLR1 or TLR6 before being stimulated with Diacyl WoLP or Triacyl WoLP (doses stated are in micrograms/ml). Accumulations of IL-8 secreted by HEK-psiTLR1 or -psiTLR6 triplicate cultures 20 h post-stimulation are plotted as mean ± 1 S.E. percentages of corresponding HEK-TLR2 responses (mean ± 1S.E. max IL-8 concentrations are as follows: PAM 3 CSK = 9807 ± 175 pg/ml, FSL-1 = 2001 ± 345 pg/ml, Diacyl WoLP = 8195 ± 199 pg/ml, Triacyl WoLP 571 ± 27 pg/ml, and BMFE = 9495 ± 137 pg/ml). Significant differences compared with HEK-TLR2 responses are indicated: ***, p < 0.001; **, p < 0.01; and *, p < 0.05. B , peritoneal macrophages from WT, TLR1 −/− , and TLR6 −/− were stimulated with Diacyl WoLP or Triacyl WoLP and control TLR1/6 ligands PAM 3 CSK 4 and FSL-1 (doses stated are in nanograms/ml) in triplicate, and production of TNFα after 20 h is plotted as mean ± 1S.E. All data are representative of three independent experiments.

Article Snippet: Ultra-pure LPS, PAM 3 CSK 4 , FSL-1 (Autogen BioClear), and rTNFα (R&D Systems) were used at the doses stated.

Techniques: Activation Assay, Transfection

Journal: The Journal of Biological Chemistry

Article Title: Wolbachia Lipoprotein Stimulates Innate and Adaptive Immunity through Toll-like Receptors 2 and 6 to Induce Disease Manifestations of Filariasis *

doi: 10.1074/jbc.M901528200

Figure Lengend Snippet: DC maturation and activation by Wolbachia and Diacyl WoLP requires MyD88, TLR2, and TLR6 but not TLR4. A , increase in CD80 or CD86 surface molecules following 20-h exposure to LPS, FSL-1, Diacyl WoLP, or BMFE in DC derived from WT, MyD88 −/− , TLR2 −/− , TLR4 −/− ,or TLR6 −/− mice. Doses stated are in micrograms/ml. Bars represent mean fold increase in MFI ± S.E. compared with unstimulated cells of triplicate labeling reactions. B , stimulation of TNFα, IL-12/IL-23p40, and IL-12p70 by LPS, FSL-1, Diacyl WoLP, or BMFE (doses stated are in micrograms/ml) from DC derived from WT, MyD88 −/− , TLR2 −/− , TLR4 −/− , or TLR6 −/− mice. Bars are mean ± 1S.E. cytokine production from triplicate cultures. C , Diacyl WoLP mediates an expansion of mature CD11c + DC in vivo . Increases in CD86 surface expression on CD11c + splenocytes 6 h following intraperitoneal inoculation with 50 μg of Diacyl WoLP were compared with sham inoculated WT mice. Numbers are percentages of splenocytes in the upper left and right quadrants. D , significant differences in CD80 and CD86 MFI on CD11c + splenocytes derived from WT mice were compared with TLR2 −/− -deficient mice 6 h following inoculation with 50 μg of Diacyl WoLP intraperitoneally. Bars are mean MFI from groups of three mice. E , significant differences in levels of IL-12/IL-23 p40 measured in spleen extracts from WT mice compared with TLR2 −/− -deficient mice 6 h following inoculation with 50 μg of Diacyl WoLP intraperitoneally. Bars are mean cytokine levels from groups of three mice. Significant reductions compared with WT are indicated: ***, p < 0.001; **, p < 0.01; and *, p < 0.05. All data are representative of two independent experiments.

Article Snippet: Ultra-pure LPS, PAM 3 CSK 4 , FSL-1 (Autogen BioClear), and rTNFα (R&D Systems) were used at the doses stated.

Techniques: Activation Assay, Derivative Assay, Labeling, In Vivo, Expressing