Journal: Methods in enzymology

Article Title: Functional Assay to Assess T-cell Inhibitory Properties of Myeloid Derived Suppressor Cells (MDSCs) Isolated from the Tumor Microenvironment of murine Glioma Models

doi: 10.1016/bs.mie.2019.05.047

Figure Lengend Snippet: (A) Schematic shows stereotactic coordinates for injection of syngeneic mouse glioma NS and GL26 cell line as well as the cell number, and expected median survival. AP: Anteroposterior; ML: Mediolateral; DV: Dorsoventral; MS: Median Survival. (B) OT-1 splenocytes are labelled with the CFSE dye and co-cultured with Gr-1high or Gr-1low MDSCs sorted from tumor. When OT-1 splenocytes divide due to SIINFIKLE stimulation, the resulting daughter cells incorporate half the number of cytoplasmic carboxyfluorescein- (CFSE) fluorescent molecules. As cells T cells divide in response to SIINFEKL stimulation, fluorescence intensity decreases exponentially. T cell proliferation can be assessed by measuring the corresponding decrease in the dye intensity via flow cytometry.

Article Snippet: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plate the CFSE labelled splenocytes at a density of 100,000 cells in 50μl in a 96 well round bottom plate (Thermo-fisher, MA) for the following conditions: list-behavior=enumerated prefix-word= mark-type=lower-alpha max-label-size=0 Condition 1: Unstimulated splenocytes (No SIINFEKL) Condition 2: Stimulated OT-1 splenocytes (+ SIINFEKL) Condition 3: Stimulated OT-1 splenocytes + 50μl Gr-1 high MDSCs Condition 4: Stimulated OT-1 splenocyte+ 50μl Gr-1 low MDSCs Prepare 1mL of 200nM SIINFEKL peptide (AnaSpec Inc, CA) in solution 2 to stimulate the OT-1 splenocytes.

Techniques: Injection, Cell Culture, Fluorescence, Flow Cytometry

Journal: Methods in enzymology

Article Title: Functional Assay to Assess T-cell Inhibitory Properties of Myeloid Derived Suppressor Cells (MDSCs) Isolated from the Tumor Microenvironment of murine Glioma Models

doi: 10.1016/bs.mie.2019.05.047

Figure Lengend Snippet: Reagents/Materials used in the protocol

Article Snippet: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plate the CFSE labelled splenocytes at a density of 100,000 cells in 50μl in a 96 well round bottom plate (Thermo-fisher, MA) for the following conditions: list-behavior=enumerated prefix-word= mark-type=lower-alpha max-label-size=0 Condition 1: Unstimulated splenocytes (No SIINFEKL) Condition 2: Stimulated OT-1 splenocytes (+ SIINFEKL) Condition 3: Stimulated OT-1 splenocytes + 50μl Gr-1 high MDSCs Condition 4: Stimulated OT-1 splenocyte+ 50μl Gr-1 low MDSCs Prepare 1mL of 200nM SIINFEKL peptide (AnaSpec Inc, CA) in solution 2 to stimulate the OT-1 splenocytes.

Techniques: Modification, Red Blood Cell Lysis

Journal: Methods in enzymology

Article Title: Functional Assay to Assess T-cell Inhibitory Properties of Myeloid Derived Suppressor Cells (MDSCs) Isolated from the Tumor Microenvironment of murine Glioma Models

doi: 10.1016/bs.mie.2019.05.047

Figure Lengend Snippet: Splenocytes from OT-1 mice were extracted, labelled with CFSE dye, and stimulated in vitro with SIINFEKL peptide in the absence or presence of MDSCs for 4 days. Cells were stained with efluor 780 viability dye, Alexa fluor 700-CD45, Percp cy5.5-CD3, and Pacific Blue-CD8. Percentage of proliferating T cells were gated based on control condition (no SIINFEKL stimulation).

Article Snippet: list-behavior=enumerated prefix-word= mark-type=decimal max-label-size=0 Plate the CFSE labelled splenocytes at a density of 100,000 cells in 50μl in a 96 well round bottom plate (Thermo-fisher, MA) for the following conditions: list-behavior=enumerated prefix-word= mark-type=lower-alpha max-label-size=0 Condition 1: Unstimulated splenocytes (No SIINFEKL) Condition 2: Stimulated OT-1 splenocytes (+ SIINFEKL) Condition 3: Stimulated OT-1 splenocytes + 50μl Gr-1 high MDSCs Condition 4: Stimulated OT-1 splenocyte+ 50μl Gr-1 low MDSCs Prepare 1mL of 200nM SIINFEKL peptide (AnaSpec Inc, CA) in solution 2 to stimulate the OT-1 splenocytes.

Techniques: In Vitro, Staining, Control

Journal: The Journal of Clinical Investigation

Article Title: Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells

doi: 10.1172/JCI40483

Figure Lengend Snippet: (A) Expression of pStat3 in MDSCs from naive and tumor-bearing mice (TB) was determined by Western blotting (upper panel) and FACS (lower panel). (B) CFSE-labeled OT-1 cells loaded with SIINFEKL and cultured alone or with MDSCs isolated from TB or naive mice at different MDSC/OT-1 ratios. Percentage of OT-1 proliferating cells was determined by flow cytometry (n = 3). (C) Peptide-loaded, CFSE-labeled OT-1 cells were cultured alone or with MDSCs isolated from TB mice treated with PBS, JSI124, STA21, or Stat3 siRNA. CFSE dilution was determined by flow cytometry (n = 3). (D) Nude or WT mice were vaccinated or not with frozen/thawed CT26 cells 1 week before i.v. injection of CT26 cells admixed or not with MDSCs isolated from TB and previously treated with STA21 or Stat3 siRNA. 2 weeks later, lung metastasis numbers were evaluated (n = 5 mice per group). For box and whisker plots, bottoms and tops of boxes show the 25th and 75th percentiles, respectively, and middle bands show the median; whiskers show extrema. (E) Peptide-loaded, CFSE-labeled OT-1 cells were cultured alone or with bone marrow–derived MDSCs of naive mice previously treated alone or with tumor cell whole supernatant or TDE or TDSF fractions. CFSE dilution was determined by flow cytometry (n = 3 mice per group). (F) TB mice were injected or not with MDSCs. These MDSCs were either from TB or naive mice and stimulated with PBS, TDEs, or TDSFs. 2 days later, spleen cells were harvested and restimulated in vitro with CD3mAb+ dead tumor cells, then stained for intracellular CD4 and IFN-γ (upper panel) or CD8 IFN-γ (lower panel). *P < 0.05. Error bars represent mean + SD.

Article Snippet: CD8 + cells were cultured with specific peptide SIINFEKL (Bachem) (10 μg/ml) in the presence of MDSCs (1:1, 1:4, 1:8, 1:16 ratios).

Techniques: Expressing, Western Blot, Labeling, Cell Culture, Isolation, Flow Cytometry, Injection, Whisker Assay, Derivative Assay, In Vitro, Staining

Journal: The Journal of Clinical Investigation

Article Title: Membrane-associated Hsp72 from tumor-derived exosomes mediates STAT3-dependent immunosuppressive function of mouse and human myeloid-derived suppressor cells

doi: 10.1172/JCI40483

Figure Lengend Snippet: Purified MDSCs from WT or TLR2-, TLR4-, MyD88-, and Trif-deficient C57BL/6 tumor-free mice were cultured for 24 hours in complete medium supplemented or not with TDEs. IL-6 concentration in the supernatant was determined by ELISA (A), and pStat3 expression in cells was determined by FACS analysis (B). Data represent mean ± SD. (C) WT C57BL/6 mice or TLR2-, TLR4-, MyD88-, and Trif-deficient mice were s.c. injected with 1 × 106 EL4 cells. 3 weeks later, spleen cells were harvested, MDSC percentage was determined in spleen (denoted in left panels), and pStat3 expression was determined by FACS analysis on MDSC gated cells (right panel). (D) 2 × 105 OT-1 cells were labeled with CFSE, loaded with 10 μg/ml of SIINFEKL, and cultured 3 days alone or with different ratios of MDSCs from EL4 tumor-bearing WT mice or from EL4 tumor-bearing TLR2-deficient mice. Percentage of OT-1 proliferating cells was determined by flow cytometry. Each experiment was done in duplicate (n = 3 mice per group). (E) WT or TLR2–/– mice were injected s.c. with EL4 cells, and tumor growth was monitored. *P < 0.05.

Article Snippet: CD8 + cells were cultured with specific peptide SIINFEKL (Bachem) (10 μg/ml) in the presence of MDSCs (1:1, 1:4, 1:8, 1:16 ratios).

Techniques: Purification, Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay, Expressing, Injection, Labeling, Flow Cytometry

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Loss of T Cell RIAM Precludes Conjugate Formation with Antigen-Presenting Cells and Prevents Immune-mediated Diabetes

doi: 10.4049/jimmunol.1601743

Figure Lengend Snippet: OT-1 splenocytes were differentiated to CTL by culture with SIINFEKL peptide and IL-2. (A) RIAM deletion in CTL. RIAM expression in OT-1 CTL was measured by intracellular staining and flow cytometry. (B) In vitro target lysis. CTL were cultured overnight with a mixture of SIINFEKL-pulsed (CFSElo) and unpulsed (CFSEhi) target splenocytes; lysis was detected by flow cytometry; performed twice. (C) In vivo target lysis. CTL were injected i.v. into recipient C57BL/6 mice, which received a mixture of SIINFEKL-pulsed (CFSElo) and unpulsed (CFSEhi) target splenocytes 24h later. Recipient splenocytes were analyzed 4h later by flow cytometry for decrease in the % of pulsed targets; n=4 mice per group; performed twice. (D–E) In vitro degranulation. CTL were cultured with SIINFEKL-pulsed target splenocytes in the presence of anti-CD107a (LAMP-1), followed by staining for CD8 and flow cytometric analysis; performed twice. (F) RIAM localization to CTL immune synapses. CTL were allowed to form conjugates with SIINFEKL-pulsed splenocyte targets, fixed, stained with indicated antibodies, and visualized by confocal microscopy. Scale bar = 5 μm. (G) CTL-Target conjugate formation. CTL were labeled with CFSE and incubated with eFluor 670-labeled target SIINFEKL-pulsed splenocyte targets. The % conjugation was determined by flow cytometry; performed twice.

Article Snippet: SIINFEKL peptide was from Anaspec, and IL-2 from the NCI preclinical repository.

Techniques: Expressing, Staining, Flow Cytometry, In Vitro, Lysis, Cell Culture, In Vivo, Injection, Confocal Microscopy, Labeling, Incubation, Conjugation Assay

Journal: Immunity

Article Title: PIP5 Kinases Regulate Membrane Phosphoinositide and Actin Composition for Targeted Granule Secretion by Cytotoxic Lymphocytes

doi: 10.1016/j.immuni.2018.08.017

Figure Lengend Snippet:

Article Snippet: SIINFEKL (Ovalbumin peptide 257-264) , Anaspec , AS-60193-1.

Techniques: Blocking Assay, Control, Virus, Recombinant, Electron Microscopy, Fractionation, Software