Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma

doi: 10.1101/2021.02.23.432584

Figure Lengend Snippet: (A) ELISpot for IFN γ release by CD8 + T cells using parental YUMM1.7 and CCN4-KO YUMM1.7 (KO1) cells as targets and different amount of in vivo activated CD8 + T cells. (B) ELISpot for IFN γ release by in vivo activated CD8 + T cells with CCN4-inducible cells as targets in the presence or absence of 0.5 mg/ml doxycycline. (C) CD8+ T cells isolated from the spleens of C57BL/6 mice that rejected YUMM1.7 tumors were assayed by in vitro ELISpot using variants of the YUMM1.7 cell line as targets (WT YUMM1.7 (Ym1.7) -yellow, CCN4-KO YUMM1.7 (Ym1.7-KO1)-light green, CCN4-KO YUMM1.7 with a blank inducible expression vector (Ym1.7-KO1-IDvector) -dark green and blue, CCN4-KO YUMM1.7 with a CCN4 inducible expression vector (Ym1.7-KO1-IDmCCN4) -purple and red). Variants containing the inducible expression vector were also cultured in the absence (dark green and purple) or presence of 0.5 µ g/ml doxycycline (blue and red). CD8+ T cells expressing IFN γ and TNF α were quantified following 24 hour coculture (bar graph). Results shown as mean ± S.D. for three biological replicates.

Article Snippet: 50 μ l (5 ×10 4 ) of the YUMM1.7-reactive CD8+ T cells were aliquoted into 96-well plates for ELISpot assay using Mouse IFN γ /TNF α Double-Color ELISpot kit (Cellular Technology Limited) following manufacturer’s instructions.

Techniques: Enzyme-linked Immunospot, In Vivo, Isolation, In Vitro, Expressing, Plasmid Preparation, Cell Culture

Journal: bioRxiv

Article Title: Cell Communication Network factor 4 promotes tumor-induced immunosuppression in melanoma

doi: 10.1101/2021.02.23.432584

Figure Lengend Snippet: (A) Average tumor volumes of mice bearing YUMM1.7-WT (squares and triangles) or CCN4-KO (KO1, circles and inverted triangles) tumors (n = 4/group). Groups were treated with either α PD1 (triangles and inverted triangles) or isotype control (squares and circles) antibodies when the tumors reached 100 mm 3 . (B) Expression of H-2K b (top panel) and PD-L1 (bottom panel) were assayed by flow cytometry in WT (red curves) and CCN4-KO (KO1 blue curves) YUMM1.7 cells with (dotted curves) and without (solid curves) preconditioning with IFN γ . Unstained cells were used as a negative control (shaded curve). (C) CD45 − fraction isolated from WT and CCN4-KO YUMM1.7 tumors in a time-matched experiment were assayed for H-2K b and PD-L1 expression by flow cytometry. Contour curves enclose 90% (dotted curve) and 50% (solid curves) of CD45 − events obtained from WT (red) and CCN4-KO (blue) YUMM1.7 tumors. CD8+ T cells expressing PD1 within the tumor (D) and spleen (E) were assayed by flow cytometry in mice bearing WT and CCN4-KO YUMM1.7 tumors (F). Results representative of at least three biological replicates.

Article Snippet: 50 μ l (5 ×10 4 ) of the YUMM1.7-reactive CD8+ T cells were aliquoted into 96-well plates for ELISpot assay using Mouse IFN γ /TNF α Double-Color ELISpot kit (Cellular Technology Limited) following manufacturer’s instructions.

Techniques: Control, Expressing, Flow Cytometry, Negative Control, Isolation

Journal: Cell reports

Article Title: Alpha synuclein, the culprit in Parkinson disease, is required for normal immune function

doi: 10.1016/j.celrep.2021.110090

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: V-plex for mouse cytokines IFNγ, IL-4, IL-5 and TNFα , Meso Scale Discovery , Cat# K152A0H-2.

Techniques: Recombinant, Saline, Stripping Membranes, Reverse Transcription, SYBR Green Assay, RNAscope, Multiplex Assay, Polymer, Software, Flow Cytometry, Control, Light Microscopy

Journal: Molecular Therapy Oncolytics

Article Title: Therapeutic effects of mesenchymal stem cells loaded with oncolytic adenovirus carrying decorin on a breast cancer lung metastatic mouse model

doi: 10.1016/j.omto.2022.01.007

Figure Lengend Snippet: MSCs.Null and rAd.Null enhanced antitumor responses in peripheral blood (A) The CD4 + CD44 High CD62L + memory T cells were analyzed by flow cytometry on days 14 and 19. (B) The statistical results of flow cytometry detection. (C) The protein concentrations of mouse interleukin-6 (IL-6), TNF-α, and IFN-γ were measured using the CBA Mouse Flex Set for IL-6, IFN-γ, and TNF-α (BD Biosciences) and analyzed by flow cytometry on days 10 and 17. Data are the mean ± SEM. ∗p < 0.05 and ∗∗p < 0.01 versus the corresponding group.

Article Snippet: The concentrations of IL-6, IFN-γ, and TNF-α were measured using a CBA Mouse Flex Set for IL-6, IFN-γ, and TNF-α (BD Biosciences).

Techniques: Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Evaluating mesothelin as an immunotherapeutic target for endogenous T cells

doi: 10.3389/fimmu.2026.1853409

Figure Lengend Snippet: Effector functions of MSLN-specific T cells. (A) representative donor data (left panel) and summary results (median ± IQR, n=21; right panel) showing CD8 + /IFNγ + and CD4 + /IFNγ + T cells, as assessed by ICS. (B) representative donor data (left panel) and summary results (median ± IQR; n=21; right panel) showing dual (IFNγ/TNFα)-producing CD4 + and CD8 + MSLN-specific T cells, as assessed by ICS. Box plots show the median and 25th and 75th data quartiles. (C) polyfunctionality of MSLN-specific T cells as measured by FluoroSpot (IFNγ/Granzyme B/TNFα) (n=6). Top panel shows the total frequency (SFC/2x105 cells) of specific T cells for each of the individual effector molecules, while the bottom panel shows the proportion of cells that are single, dual or triple analyte-producing. All data represent biological replicates (i.e. independent donors). TNFα, tumor necrosis factor alpha; ICS, intracellular cytokine staining; GrB, Granzyme B.

Article Snippet: For the quantitation of polyfunctional cells simultaneously secreting IFNγ, GrB and/or TNFα a commercial FluoroSpot assay was used (Human IFNγ/Granzyme B/TNFα FluoroSpot Plus, Mabtech, Inc.).

Techniques: Staining