Journal: Oncoimmunology

Article Title: Local endothelial complement activation reverses endothelial quiescence, enabling t-cell homing, and tumor control during t-cell immunotherapy

doi: 10.1080/2162402X.2017.1326442

Figure Lengend Snippet: Complement C3 is required for the homing of effective (T) cells and tumor suppression. (A) C3 mRNA is significantly overexpressed in human tumor endothelial cells sorted from ovarian cancers with tumor-infiltrating lymphocytes (TILs), when compared with ovarian cancers lacking TILs ( n = 6/group). (B) Detection of complement C3b/iC3b/C3c activation fragments (red) on tumor vasculature (CD31 in green) after adoptive transfer of 5 × 10 6 E7-primed CD8 + T cells. Arrows indicate areas of juxtaposition of complement fragments and CD31. The right panel depicts the quantification of C3 fragments co-localized with CD31. (C–F) Mouse chimeras were generated by transferring wild-type bone marrow from B6.SJL- Ptprc a Pep3 b /BoyJ mice to lethally irradiated c3 +/+ and c3 −/− mice. The chimeras were inoculated s.c with TC-1 tumors, followed by i.v. administration of 5 × 10 6 E7-primed CD45.2 CD3 + T cells. (C and D) Flow cytometry analysis showing the number of donor E7-specific CD8 + T cells and total CD8 + T cells in the tumors. (E and F) TC-1 tumor growth in BM-transplanted c3 +/+ and c3 −/− mice in the absence of treatment (CTRL) or after transfer of 5 × 10 6 E7-primed CD3 + T-cells.

Article Snippet: In adoptive transfer experiments, CD3 + splenocytes were sorted using a Miltenyi CD3 pan-T cell purification kit and inoculated i.v. into recipient tumor-bearing mice 1 week after the last immunization.

Techniques: Activation Assay, Adoptive Transfer Assay, Generated, Transferring, Irradiation, Flow Cytometry

Journal: Oncoimmunology

Article Title: Local endothelial complement activation reverses endothelial quiescence, enabling t-cell homing, and tumor control during t-cell immunotherapy

doi: 10.1080/2162402X.2017.1326442

Figure Lengend Snippet: Triggering of the C5a-C5aR1 axis is required for T-cell extravasation and tumor suppression. Tumor-bearing mice received an adoptive transfer of 5 × 10 6 E7-primed T cells, and were then treated with the C5aR1 antagonist (C5aR1A) or a control peptide (CTRLpept). (A and B) The number of E7-specific CD8 + T cells and total CD3 + CD45 + T cells recruited to the tumor was determined by flow cytometry. (C) TC-1 tumor growth was measured over time. (D and E) Tumor-bearing mice were vaccinated with pConE6E7, followed by treatment with C5aR1A or control peptide. (D) The number of E7-specific CD8 + T cells recruited to the tumor was determined by flow cytometry. (E) TC-1 tumor growth was measured over time.

Article Snippet: In adoptive transfer experiments, CD3 + splenocytes were sorted using a Miltenyi CD3 pan-T cell purification kit and inoculated i.v. into recipient tumor-bearing mice 1 week after the last immunization.

Techniques: Adoptive Transfer Assay, Control, Flow Cytometry

Journal: Oncoimmunology

Article Title: Local endothelial complement activation reverses endothelial quiescence, enabling t-cell homing, and tumor control during t-cell immunotherapy

doi: 10.1080/2162402X.2017.1326442

Figure Lengend Snippet: C5aR1-mediated signaling in the tumor stroma is required for effective T-cell infiltration. (A–E) Tumor-bearing C5aR1-deficient ( c5ar1 −/− )mice and C5aR1-sufficient littermate controls ( c5ar1 +/+ ) were given an effective dose (5 × 10 6 ) of E7-primed T cells. (A and B) Flow cytometry showing spleen expansion and tumor recruitment of total and E7-specific donor CD8 + cells. (C) Immunohistochemical staining for CD3 of TC-1 tumor sections. (D and E) TC-1 tumor growth in c5ar1 +/+ (blue) and c5ar1 −/− (red) mice in the absence of treatment (CTRL) or after transfer of 5 × 10 6 E7-primed CD3 + T-cells. (F–I) Chimeras were generated by transferring wild-type bone marrow from B6.SJL- Ptprc a Pep3 b /BoyJ mice to c5ar1 −/− and c5ar1 +/+ littermatemice. Mice were inoculated in the back with TC-1 tumors and then given an effective dose of E7 vaccine-primed adoptive CD45.2 T cells (5 × 10 6 CD3 + cells/mouse). Flow cytometry analysis showing the number of CD8 + (F) and E7-specific CD8 + (G) engrafting TC-1 tumors in c5ar1 +/+ and c5ar1 −/− mice reconstituted with bone marrow cells from WT mice. (H and I) TC-1 tumor growth in BM-transplanted c5ar1 +/+ (blue) and c5ar1 −/− (red) mice in the absence of treatment (CTRL) or after the transfer of 5 × 10 6 E7-primed CD3 + T-cells.

Article Snippet: In adoptive transfer experiments, CD3 + splenocytes were sorted using a Miltenyi CD3 pan-T cell purification kit and inoculated i.v. into recipient tumor-bearing mice 1 week after the last immunization.

Techniques: Flow Cytometry, Immunohistochemical staining, Staining, Generated, Transferring

Journal: Oncoimmunology

Article Title: Local endothelial complement activation reverses endothelial quiescence, enabling t-cell homing, and tumor control during t-cell immunotherapy

doi: 10.1080/2162402X.2017.1326442

Figure Lengend Snippet: Th1 cytokines activate the endothelium through endothelial complement activation. (A) HUVEC cells were treated with TNF-α or IFNγ alone or in combination, or with the supernatant of T cells activated with anti-CD3/CD28, and the levels of C3 were measured in the culture supernatants by ELISA. (B) HUVECs activated by TNF-α or medium from anti-CD3/CD28-co-stimulated human T cells (T medium) were stained for deposition of the C3 activation fragments C3b, iC3b, and C3c. (C) HUVEC cells were treated with TNF-α or IFNγ alone or in combination, and the levels of C5a were measured in the culture supernatants by ELISA. (D) Adhesion of activated T cells to HUVECs was determined after treatment of HUVECs with supernatants of activated T cells (T medium) in the presence of C5a receptor 1 antagonist (C5aR1A), control peptide (CTRLPept), or antibody neutralizing ICAM-1 or VCAM-1. After addition of CFSE-labeled T cells, cell adhesion was measured by detecting total fluorescence using a fluorocounter microplate reader. CTRL indicates the adhesion of activated T cells on HUVECs in the absence of T-cell medium or any of the above factors. (E) Expression levels of VCAM-1 in response to the treatment with IFNγ in the presence of C5aR1 antagonist (C5aR1A) or the C3 inhibitor Compstatin were measured by flow cytometry. (F) Primary mouse lung micro vascular endothelial cells from wild-type, c5ar1 −/− ,or c3 −/− mice were treated with IFNγ or TNF-α alone or in combination, and the expression levels of VCAM-1 were measured by flow cytometry. Right panels depict quantification of VCAM-1 expression on the different mouse endothelial cells. * p < 0.05; ** p < 0.02; *** p < 0.0002.

Article Snippet: In adoptive transfer experiments, CD3 + splenocytes were sorted using a Miltenyi CD3 pan-T cell purification kit and inoculated i.v. into recipient tumor-bearing mice 1 week after the last immunization.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Staining, Control, Labeling, Fluorescence, Expressing, Flow Cytometry

Journal: Oncoimmunology

Article Title: Local endothelial complement activation reverses endothelial quiescence, enabling t-cell homing, and tumor control during t-cell immunotherapy

doi: 10.1080/2162402X.2017.1326442

Figure Lengend Snippet: Complement hyperactivation enhances T-cell engraftment in tumors and facilitates tumor rejection by suboptimal numbers of tumor-specific (T)cells. (A) Mice were given an ineffective dose of T cells (2.5 × 10 6 CD3 + T cells/mouse) by adoptive transfer, and the frequency of E7-specific CD8 + cells engrafted in the tumors of daf1 +/+ and daf1 −/− mice was determined by flow cytometry. (B and C) TC-1 tumor growth curve in daf1 +/+ and daf1 −/− mice in the absence of treatment (CTRL) or after the transfer of 2.5 × 10 6 E7 primed CD3 + T cells. (D) Some daf1 −/− mice also received C5aR1 antagonist (C5aR1A), and the number of E7-specific CD8 + cells engrafted in the tumors was determined by flow cytometry. (E) TC-1 tumor growth curve in daf1 −/− mice treated with C5aR1 antagonist (C5aR1A) after the transfer of 2.5 × 10 6 CD3 + T cells/mouse harvested from spleens of donor mice vaccinated with pConE6-E7 DNA.

Article Snippet: In adoptive transfer experiments, CD3 + splenocytes were sorted using a Miltenyi CD3 pan-T cell purification kit and inoculated i.v. into recipient tumor-bearing mice 1 week after the last immunization.

Techniques: Adoptive Transfer Assay, Flow Cytometry

Journal: Oncoimmunology

Article Title: Local endothelial complement activation reverses endothelial quiescence, enabling t-cell homing, and tumor control during t-cell immunotherapy

doi: 10.1080/2162402X.2017.1326442

Figure Lengend Snippet: Pioneering CD4 + and CD8 + cells are both required for local complement activation, T-cell homing, and tumor suppression. (A and B) c5ar1 +/+ or c5ar1 −/− C57BL/6 tumor-bearing mice were treated with 5 × 10 6 CD3 + T cells isolated from the spleen of pConE6E7-vaccinated B6.SJL- Ptprc a Pep3 b /BoyJ mice, and the expansion of the T cells was determined over time in the spleen and tumor. (C–G) C57BL/6 mice were injected s.c. with the TC-1 tumor, and after 1 week they were given total CD3 + (1 × 10 7 /mouse), CD8 + (4 × 10 6 /mouse) or CD4 + (6 × 10 6 /mouse) T cells isolated from pConE6E7 vaccinated B6.SJL- Ptprc a Pep3 b /BoyJ mice by adoptive transfer. (C) Frequency of total CD3 + CD45.1 cells in the spleens of mice receiving a transfer of CD3 + , CD8 + , or CD4 + T cells. Numbers are normalized for millions of analyzed cells. (D) Frequency of CD3 + CD45.1 cells (left) and E7-specific CD8 + CD45.1 cells (right) in tumors from mice that received CD3 + or CD8 + cells, followed by treatment with C5aR1 antagonist (C5aR1A). (E) Tumor growth curves of C57BL/6 mice that received CD3 + , CD4 + , or CD8 + cells. (F) Immunohistochemical staining for the endothelial cell marker CD31 and C3 activation fragments in TC-1 tumor sections from mice given the CD3 + , CD4 + , or CD8 + cell populations by adoptive transfer. (G) Tumor growth curves of C57BL/6 mice that received CD3 + T cells and treated with C5aR1 antagonist (C5aR1A). CTRL- control mice did not receive any T- cell therapy.

Article Snippet: In adoptive transfer experiments, CD3 + splenocytes were sorted using a Miltenyi CD3 pan-T cell purification kit and inoculated i.v. into recipient tumor-bearing mice 1 week after the last immunization.

Techniques: Activation Assay, Isolation, Injection, Adoptive Transfer Assay, Immunohistochemical staining, Staining, Marker, Control

Journal:

Article Title: Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR

doi: 10.1073/pnas.171076798

Figure Lengend Snippet: Enhanced S6 kinase activation in PTEN null tumor lines. (A) Phosphorylated (Ser-235/236) and total S6 protein and actin were measured by immunoblot in 9L and U87MG cells treated with vehicle or doses of 0.1, 1.0, and 10 nM CCI-779 for 7 h. Serum challenge was for 15 min. Equal amounts of protein were loaded per lane as determined by Bio-Rad DC protein assay. (B) S6 kinase activity was measured by in vitro kinase assay (31) in MDCK cells stably transfected with vector or wild-type PTEN (Left) and in LAPC-4 prostate cancer cells stably infected with retrovirus expressing myr-Akt or vector control (Right). MDCK cells were serum-starved overnight, pretreated with vehicle (open bars) or 10 nM of CCI-779 (solid bars), and then challenged with serum for 15 min. LAPC-4 cells were treated identically except that no serum challenge was given. Results from two experiments are plotted relative to serum-starved, untreated cells. Expression of PTEN and Akt in the transfectants was confirmed by immunoblot (data not shown). (C) Levels of total and activated Akt and MAPK were measured by immunoblot in lysates of PTEN wild-type (DU145) and PTEN null (PC3) cells by using specified antibodies after 2 h of pretreatment with vehicle or 10 nM CCI-779.

Article Snippet: Phosphorylated (Ser-235/236) and pan-S6 antibodies were provided by Cell Signaling Technology (Beverly, MA).

Techniques: Activation Assay, Western Blot, DC Protein Assay, Activity Assay, In Vitro, Kinase Assay, Stable Transfection, Transfection, Plasmid Preparation, Infection, Expressing

Journal:

Article Title: Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR

doi: 10.1073/pnas.171076798

Figure Lengend Snippet: PTEN null cells have enhanced sensitivity to mTOR inhibition in vivo. (A) PTEN+/+ or PTEN−/− ES cells were injected s.c. into nude mice at a dose of 5 × 105 cells per mouse (n = 20). When tumor volume reached 200 mm3, mice were randomized to treatment with vehicle or 40 mg/kg CCI-779. The fold change in tumor volume in response to treatment was plotted. (B) Single-cell suspensions of LAPC-4 or LAPC-9 prostate cancer xenografts were injected s.c. into male SCID mice (n = 80) at a dose of 106 cells per mouse. When tumors became palpable, mice were randomized (arrow) to treatment with vehicle, 0.1 mg/kg, 4 mg/kg, or 40 mg/kg CCI-779. The fold change in tumor volume from two independent experiments is plotted. (C) Tumors were harvested from mice after 5 days of treatment with vehicle or 0.1 mg/kg CCI-779 and lysed by boiling in 2% SDS buffer. Immunoblots were performed by using antibodies for phosphorylated S6 (Ser-235/236), total S6, and actin.

Article Snippet: Phosphorylated (Ser-235/236) and pan-S6 antibodies were provided by Cell Signaling Technology (Beverly, MA).

Techniques: Inhibition, In Vivo, Injection, Western Blot

Journal:

Article Title: Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR

doi: 10.1073/pnas.171076798

Figure Lengend Snippet: Analysis of S6, 4E-BP1, and cyclin D1 in PTEN+/+ and PTEN−/− MEFs. PTEN+/+ or PTEN−/− MEFs were treated with the indicated concentrations of CCI-779 for 6 h, lysed by three freeze/thaw cycles in 50 mM Tris, pH 7.5/150 mM KCl mM EDTA/1 mM EGTA/1 mM DTT/50 mM 2-mercaptoethanol, supplemented with protease and phosphatase inhibitors, and probed with antibodies to phosphorylated S6, total S6, total 4E-BP1, or actin. The level of 4E-BP1 bound to eIF4E was measured by precipitation of eIF4E by using 7methyl-GTP Sepharose, followed by 4E-BP1 immunoblot. Comparable precipitation of eIF4E was confirmed by immunoblot.

Article Snippet: Phosphorylated (Ser-235/236) and pan-S6 antibodies were provided by Cell Signaling Technology (Beverly, MA).

Techniques: Western Blot