Journal: Frontiers in Immunology

Article Title: Generation and Functional In Vitro Analysis of Semliki Forest Virus Vectors Encoding TNF-α and IFN-γ

doi: 10.3389/fimmu.2017.01667

Figure Lengend Snippet: Vector-derived TNF-α induces cell death in L929 fibroblasts. L929 cells were cultured for 24 h until they reached 90% confluency. The cells were then (A) left untreated for 24 h and used as a negative control or (B) treated with 1 µM staurosporine for 24 h at 37°C and used as a positive control. The remaining cells were treated with rTNF-α or vdTNF-α for 24 h at the following concentrations: (C,D) 2.2 ng/mL, (E,F) 6.7 ng/mL, (G,H) 20 ng/mL or (I,J) 60 ng/mL. The resulting cell monolayers were analyzed using bright-field microscopy (scale bar, 50 µM). Cell death was determined using flow cytometry analysis after cell staining with annexin V-FITC (depicted on the x -axis) and propidium iodide (PI, depicted on the y -axis). Annexin V-positive/PI-negative cells were regarded as apoptotic, whereas annexin V-positive/PI-positive cells were regarded as necrotic. Two independent experiments were performed in duplicates with standard error not exceeding 10% between independent experiments. Data from one representative experiment are shown, where the percentages of the four distinct cell populations represent the averages of duplicates with SEM <10%.

Article Snippet: The human lung carcinoma cell line A549 (Cat. No. CCL-185TM) and murine fibrosarcoma cell line L929 (Cat. No. CCL-1TM) were obtained from American Type Culture Collection (ATCC/LGC Standards GmbH, Wesel, Germany).

Techniques: Plasmid Preparation, Derivative Assay, Cell Culture, Negative Control, Positive Control, Microscopy, Flow Cytometry, Staining

Journal: Frontiers in Immunology

Article Title: Generation and Functional In Vitro Analysis of Semliki Forest Virus Vectors Encoding TNF-α and IFN-γ

doi: 10.3389/fimmu.2017.01667

Figure Lengend Snippet: Interferon (IFN)-γ in combination with TNF-α induces cell death in mouse lung carcinoma cells. L929 fibroblasts and Lewis lung carcinoma (LLC) cancer cells were cultured for 24 h before starting treatment with recombinant cytokines. Both L929 and LLC cells were (A,B) left untreated for 48 h and used as negative controls or (C) L929 cells were treated with 50 ng/mL TNF-α for 48 h and used as a positive control. LLC cell viability was retained after treatment with (D) 50 ng/mL TNF-α or (E) 100 ng/mL IFN-γ and after (F) simultaneous treatment with both 50 ng/mL TNF-α and 100 ng/mL IFN-γ for 24 h. Cell death was induced in LLC cells after (G) simultaneous treatment with 100 ng/mL of IFN-γ in combination with 50 ng/mL of TNF-α for 48 h and after (H) LLC pretreatment with 100 ng/mL of IFN-γ for 24 h followed by treatment with TNF-α for 24 h. The resulting cell monolayers were analyzed using bright-field microscopy (scale bar, 50 µM). Cell death was determined using flow cytometry analysis after cell staining with annexin V-FITC (depicted on the x -axis) and propidium iodide (PI, depicted on the y -axis). Annexin V-positive/PI-negative cells were regarded as apoptotic, whereas annexin V-positive/PI-positive cells were regarded as necrotic. Experiment was performed in duplicates and repeated two times with standard error not exceeding 10% between independent experiments. One representative experiment is shown, where the percentages of the four distinct cell populations are averages of duplicates with SEM <5%.

Article Snippet: The human lung carcinoma cell line A549 (Cat. No. CCL-185TM) and murine fibrosarcoma cell line L929 (Cat. No. CCL-1TM) were obtained from American Type Culture Collection (ATCC/LGC Standards GmbH, Wesel, Germany).

Techniques: Cell Culture, Recombinant, Positive Control, Microscopy, Flow Cytometry, Staining

Journal: The Journal of Experimental Medicine

Article Title: In vivo labeling reveals continuous trafficking of TCF-1 + T cells between tumor and lymphoid tissue

doi: 10.1084/jem.20210749

Figure Lengend Snippet: Limited intratumoral T cell proliferation prevents rapid loss of Kaede Red protein. (A) Gating strategy for identification of TILs within MC38 tumors. (B) Proportion of Kaede Green + CD4 T cells, CD8 T cells, Tregs, and NK cells at 24, 72 and 120 h after photoconversion. (C) Total number of Kaede Green + and Kaede Red + CD4 T cells, CD8 T cells, Tregs, and NK cells at 24, 72 and 120 h after photoconversion. For B and C, CD4 T cells: n = 39 at 24 h, n = 17 at 72 h, n = 16 at 120 h; for CD8 T cells: n = 41 at 24 h, n = 18 at 72 h, n = 16 at 120 h; for Tregs: n = 24 at 24 h, n = 10 at 72 h, n = 9 at 120 h; for NK cells: n = 9 at 24 h, n = 5 at 72 h, n = 3 at 120 h) pooled from six independent experiments. Bars represent the median values. (D) Quantification of Kaede Red + and Kaede Green + TILs in CT26 tumors over 5 d after photoconversion, CD8 T cells (24 h, n = 25; 72 h, n = 14; 120 h, n = 5), CD4 T cells (24 h, n = 25; 72 h, n = 13; 120 h, n = 5) and Tregs (24 h, n = 12; 72 h, n = 10; 120 h n = 5) pooled from three independent experiments. Data presented as mean values ± SEM. (E) Quantification of Kaede Red + and Kaede Green + TILs in MCA205-OVA tumors over 5 d after photoconversion, CD8 T cells (24 h, n = 9; 72 h, n = 12), CD4 T cells (24 h, n = 9; 72 h, n = 12) and Tregs (24 h, n = 6; 72 h, n = 6) pooled from two independent experiments. (F) Representative expression of Ki-67 versus Kaede Red in CD8 T cells isolated from MC38 tumors at 24, 72, and 120 h after photoconversion. Values on flow cytometry plots represent percentages. (G) Proportion of Kaede Green + and Kaede Red + CD8 T cells expressing Ki-67 over the time course, n = 34 at 24 h, n = 19 at 72 h, and n = 6 at 120 h pooled from six independent experiments. Data was presented as mean values ± SEM. (H) Enumeration (%) of Ki-67 expression amongst MC38 neo-Ag specific CD8 T cells in the tumor and dLN ( n = 19 pooled from five independent experiments). Bars represent mean values. (I) Detection of bound CD45 Ab on hematopoietic cells isolated from tumor or blood following i.v. injection. (J) Proportion of hematopoietic cells labeled with i.v. administered anti-CD45 Ab in blood and tumor ( n = 5). Two independent experiments were performed. Data was presented as mean values ± SEM. Statistical significance was tested using paired t test (H and J): ****, P ≤ 0.0001.

Article Snippet: MC38 murine colon adenocarcinoma cells (kindly provided by Dr. Gregory Sonnenberg; Weill Cornell Medicine, New York, NY), MC38-Ova murine colon adenocarcinoma cells (obtained from AstraZeneca), MCA205-Ova murine fibrosarcoma cells (obtained from AstraZeneca), and CT26 murine colorectal carcinoma cells (kindly provided by Professor Tim Elliott, University of Oxford, Oxford, UK) were cultured in RPMI supplemented with 2 mM L-glutamine (21875034; Thermo Fisher Scientific), 10% FBS (F9665; Sigma-Aldrich), and penicillin–streptomycin (P4333; Sigma-Aldrich) at 37°C and with 5% CO 2 .

Techniques: Expressing, Isolation, Flow Cytometry, Injection, Labeling

Journal: The Journal of Experimental Medicine

Article Title: In vivo labeling reveals continuous trafficking of TCF-1 + T cells between tumor and lymphoid tissue

doi: 10.1084/jem.20210749

Figure Lengend Snippet: Anti–PD-L1 Abs drive reinvigoration of exhausted effector cells alongside sustained activation of newly arrived populations in multiple tumor models. MC38-Ova tumors treated with three doses of anti–PD-L1 ( n = 11) or isotype control Abs ( n = 13; A–E and T–V) pooled from two independent experiments, or one dose anti–PD-L1 ( n = 5) or isotype control Abs ( n = 5) from one independent experiment (F–J and W–X); MCA205-Ova tumors treated with three doses of anti–PD-L1 ( n = 4) or isotype control Abs ( n = 4) from one independent experiment (K–O); CT26 tumors treated with three doses of anti–PD-L1 ( n = 6 including three responders and three non-responders) or isotype control Abs ( n = 8) from one independent experiment (P–S). The TCF-1 + PD-1 + CD8 T cell compartment was assessed in T–X. (A) Individual tumor growth curves. (B) Tumor mass (mg) upon tissue harvest. (C) Proportion of total, Kaede Green + and Kaede Red + neo-Ag–specific CD8 T cells expressing both IFNγ and Granzyme B. (D) Proportion of neo-Ag–specific cells amongst the total, Kaede Green + , and Kaede Red + CD8 T cell compartment. (E) Proportion of Kaede Green + cells amongst neo-Ag–specific CD8 T cells. (F) Individual tumor growth curves. (G) Tumor mass (mg) upon tissue harvest. (H) Proportion of total, Kaede Green + , and Kaede Red + neo-Ag–specific CD8 T cells expressing both IFNγ and Granzyme B. (I) Proportion of neo-Ag–specific cells amongst the total, Kaede Green + and Kaede Red + CD8 T cell compartment. (J) Proportion of Kaede Green + cells among neo-Ag–specific CD8 T cells. (K) Individual tumor growth curves. (L) Tumor mass (mg) upon tissue harvest. (M) Proportion of total, Kaede Green + and Kaede Red + CD8 T cells expressing both IFNγ and Granzyme B. (N) Proportion of Kaede Green + cells amongst CD8 T cells. (O) Proportion of Ova-specific T cells expressing both IFNγ and Granzyme B amongst the total, Kaede Green + , and Kaede Red + CD8 T cell compartment. (P) Individual tumor growth curves. (Q) Tumor mass (mg) upon tissue harvest. (R) Proportion of total, Kaede Green + and Kaede Red + CD8 T cells expressing both IFNγ and Granzyme B. (S) Proportion of Kaede Green + cells amongst CD8 T cells. (T) The proportion of TCF-1 + PD-1 + amongst the total, Kaede Green + , and Kaede Red + CD8 T cell compartment. (U) The number per mg tumor of TCF-1 + PD-1 + CD8 T cells, TCF-1 + PD-1 + Kaede Green + CD8 T cells, and TCF-1 + PD-1 + Kaede Red + CD8 T cells. (V) The number per mg tumor of TCF-1 + PD-1 + Ova-specific T cells, TCF-1 + PD-1 + Kaede Green + Ova-specific T cells, and TCF-1 + PD-1 + Kaede Red + Ova-specific T cells. (W) The number per mg tumor of TCF-1 + PD-1 + CD8 T cells, TCF-1 + PD-1 + Kaede Green + CD8 T cells and TCF-1 + PD-1 + Kaede Red + CD8 T cells. (X) The number per mg tumor of TCF-1 + PD-1 + Ova-specific T cells, TCF-1 + PD-1 + Kaede Green + Ova-specific T cells, and TCF-1 + PD-1 + Kaede Red + Ova-specific T cells. All bars on graphs represent the mean. Statistical significance was tested with unpaired t test (B, G, J, L, N, Q, and S) and unpaired multiple t test (C, D, H, M, O, R, and U–X): *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.

Article Snippet: MC38 murine colon adenocarcinoma cells (kindly provided by Dr. Gregory Sonnenberg; Weill Cornell Medicine, New York, NY), MC38-Ova murine colon adenocarcinoma cells (obtained from AstraZeneca), MCA205-Ova murine fibrosarcoma cells (obtained from AstraZeneca), and CT26 murine colorectal carcinoma cells (kindly provided by Professor Tim Elliott, University of Oxford, Oxford, UK) were cultured in RPMI supplemented with 2 mM L-glutamine (21875034; Thermo Fisher Scientific), 10% FBS (F9665; Sigma-Aldrich), and penicillin–streptomycin (P4333; Sigma-Aldrich) at 37°C and with 5% CO 2 .

Techniques: Activation Assay, Expressing