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: PLoS ONE

Article Title: Exacerbation of Autoimmune Neuro-Inflammation in Mice Cured from Blood-Stage Plasmodium berghei Infection

doi: 10.1371/journal.pone.0110739

Figure Lengend Snippet: A) C57BL/6 mice (n = 6) were intraperitoneally (i.p.) infected with 1×10 6 P.berghei -infected Red Blood Cells. At the 10 th day of infection, splenocytes were collected and total T cells were isolated using dynabeads (Pan T cell isolation kit). Naïve-derived splenic T cells were used as well. EAE-inflicted mice (n = 6) were killed at the 10 th day after immunization and the total splenic T cell were isolated with the same methodology and CFSE-stained (2,5 µM). As controls, T cells from EAE-inflicted mice were cultured without the presence of other cells. B) Total T cells isolated from naïve and malaria-bearing mice were transferred (1×10 6 cells/mouse) at the onset of EAE (n = 6 mice/group), and clinical course of the disease was evaluated daily. Data was analyzed by Two-Way ANOVA and post-tested with Bonferroni. C) Sorted splenic CD4 + , CD8 + and CD4 + CD8 + T cells from malaria-bearing mice were adoptively transferred (1,5×10 5 cells/mouse) to EAE-inflicted mice (n = 6 mice/group) at disease onset. The clinical score of the disease was evaluated daily. The side panel contains the linear regression lines with the 95% confidence interval (thinner lines). D) Malaria-bearing mice were killed at 10 days after infection and the DP-T cells were sorted and incubated with LPS (1 ng/mL), P. berghei extracts (50 µg/mL) and MOG 35-55 peptide (20 µg/mL) for 72 h, at the end of culture period the supernatants were removed and assayed for the detection of IFN-γ and IL-17. Analyses were conducted using Student's t test. *: p<0,05. **: p<0,01. Representative data of two independent experiments with similar results.

Article Snippet: Total splenic T lymphocytes were isolated using Dynabeads following the manufactureŕs instructions (Mouse Pan T cell isolation kit, Life Technologies, USA).

Techniques: Infection, Isolation, Cell Isolation, Derivative Assay, Staining, Cell Culture, Incubation

Journal: Cell Reports

Article Title: Immunomodulation of Selective Naive T Cell Functions by p110δ Inactivation Improves the Outcome of Mismatched Cell Transplantation

doi: 10.1016/j.celrep.2015.01.002

Figure Lengend Snippet: Inactivation of p110δ Inhibits Allogeneic Naive T Cell Proliferation and Differentiation In Vitro (A and B) B6 sorted naive and memory T cells were labeled with CFSE and cocultured with LPS-matured and irradiated BALB/c BMDCs at a 1:1 ratio with DMSO vehicle control or the indicated doses of IC87114 (IC) or LY294002 (LY). At day 5, proliferation (A) and differentiation (B) of CD8 + T cells were quantified. Differentiation phenotype is based on CD62L and CD44 expression as follows: naive (Tn; CD62L + CD44 lo ), central memory-like (Tcm; CD62L + CD44 hi ), and effector memory-like (Tem; CD62L − CD44 hi ). Results are representative of three independent experiments. (C–F) T cells come from the cultured alloreactive T cells generated in vitro in presence of DMSO or IC. These cells were stimulated first with irradiated BALB/c BMDCs loaded with A20 lysate for 5 days, then expanded with anti-CD3 and anti-CD28 + rmIL-2/rmIL-7 for 2 days and maintained in a low dose of rmIL-2 and rmIL-7 in the presence of DMSO or IC during the whole process. The level of GzmB (D) and P-Akt (E) were assessed at the end of this process. (C) After 5 days of priming, Tn and memory (Tm, CD44 hi ) T cell absolute numbers were evaluated. Tn/Tm ratios were calculated either in DMSO or in IC (5 μM) (left), and DMSO/IC ratios were determined for each subset (right). Results are from four independent experiments (mean ± range, paired t test). (D) GzmB expression was assessed in Tn and in Tm primed and expanded in the presence of DMSO (solid line) or IC (dotted line). Results are representative of three independent experiments. (E) Tn and Tm cultured in the presence of DMSO (solid line) or IC (dotted line) were stained for P-Akt (Ser473) or P-Akt (Thr308) and analyzed by flow cytometry. Results are representative of two independent experiments. (F) Tn (top) or Tm (bottom) cultured in the presence of DMSO (solid line) or IC (dotted line) were stimulated for 5 hr with A20 cells (left) or A20-pulsed BALB/c BMDCs (right). Intracellular IFN-γ was stained and analyzed by flow cytometry. The percentages and geometric mean fluorescence intensities (GMFIs) of IFN-γ + cells are depicted on each histogram. Data are representative of two independent experiments. See also Figure S1 .

Article Snippet: Magnetic beads (CD3 Microbead kit, Pan T Cell Isolation kit II; Miltenyi Biotec) were used to deplete T cells in BMCs and to purify donor T cells from spleens (purity >90%).

Techniques: In Vitro, Labeling, Irradiation, Control, Expressing, Cell Culture, Generated, Staining, Flow Cytometry, Fluorescence