Journal: PLoS ONE

Article Title: Rapid Maturation of Effector T Cells in Tumors, but Not Lymphoid Organs, during Tumor Regression

doi: 10.1371/journal.pone.0000821

Figure Lengend Snippet: 3×10 6 CMS5 tumor cells were injected s.c. and were allowed to grow for 8 days prior to transfer of 30×10 6 in vitro activated DUC18 T cells. This transfer protocol was repeated for all subsequent experiments. A) Tumor sizes for 6 DUC18 T cell + recipient and 4 control mice are shown. Data are representative of 3 experiments. B) Mean tumor sizes +/− SEM for 30 DUC18 T cell + recipient mice.

Article Snippet: DUC18 TCR transgenic mice are on a BALB/c background and were bred to Thy1.1 BALB/c mice (provided by Dr. Hyam Levitsky, Johns Hopkins) to generate Thy1.1 + DUC18 mice.

Techniques: Injection, In Vitro, Control

Journal: PLoS ONE

Article Title: Rapid Maturation of Effector T Cells in Tumors, but Not Lymphoid Organs, during Tumor Regression

doi: 10.1371/journal.pone.0000821

Figure Lengend Snippet: Thy1.1 DUC18 T cells were activated for four days in vitro and transferred i.v. into Thy1.2 CMS5-tumor bearing mice. A) Prior to transfer, some cells were stained for CD25, CD27 and CD62L. Remaining cells were left in vitro without further stimulation, and were stained for the same markers on days 2 and 6. Data from one experiment, representative of three, are shown. B) Following DUC18 T cell transfer, dLNs, spleens, and tumors were harvested from mice and stained on days 2,4, and 6. All analyses were done by gating on live, Thy1.1 + Vβ8.3 + DUC18 T cells. Shown are mean fluorescence intensity +/− SEM for combined results from 3 independent experiments, n = 8–10 mice at each time point.

Article Snippet: DUC18 TCR transgenic mice are on a BALB/c background and were bred to Thy1.1 BALB/c mice (provided by Dr. Hyam Levitsky, Johns Hopkins) to generate Thy1.1 + DUC18 mice.

Techniques: In Vitro, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Rapid Maturation of Effector T Cells in Tumors, but Not Lymphoid Organs, during Tumor Regression

doi: 10.1371/journal.pone.0000821

Figure Lengend Snippet: DUC18 T cells were transferred as in . A) On day 4, 5×10 6 each TAMRA + control cells and CFSE + targets were injected i.v. Spleens and dLNs were harvested and analyzed on day 5. Percentages of cells within the indicated gates are shown. Data are representative of 3 experiments. B) CMS5 cells were injected s.c. and allowed to grow for 8 days. At this time, 3×10 6 each TAMRA + Meth A reference cells and CFSE + CMS5 targets were injected i.t. Tumors were harvested and analyzed on day 9; the percentages of TAMRA + and CFSE + cells in individual tumors were determined by flow cytometry. C) The mean ratio +/− SEM of CFSE + CMS5 to TAMRA + Meth A cells was calculated using data from 13 individual mice. D) Tumor areas were measured on day 8 after CMS5 challenge. Four days later, fluorescently labeled tumor cells were injected as in B. Tumor sizes were measured just prior to i.t. injections and again the following day. Data represent the means +/− s.d. for 12 tumors from 4 independent experiments. No statistical difference is present in the size of control versus FL+ tumors at day 13 (p = 0.11) or in the change in control versus FL+ tumor sizes from day 12 to day 13 (p = 0.40). E) DUC18 T cells were transferred into tumor-bearing mice as in A. On day 4 post-T cell transfer, fluorescently labeled tumor cells were injected as in B. Tumors were harvested and analyzed on day 5. Percentages of cells within the indicated gates are shown. Data from 1 experiment are shown; representative of 10.

Article Snippet: DUC18 TCR transgenic mice are on a BALB/c background and were bred to Thy1.1 BALB/c mice (provided by Dr. Hyam Levitsky, Johns Hopkins) to generate Thy1.1 + DUC18 mice.

Techniques: Control, Injection, Flow Cytometry, Labeling

Journal: PLoS ONE

Article Title: Rapid Maturation of Effector T Cells in Tumors, but Not Lymphoid Organs, during Tumor Regression

doi: 10.1371/journal.pone.0000821

Figure Lengend Snippet: A) ITC assays were performed beginning on days 2, 4, or 6. For tumors, each point represents the mean percent lysis, derived from 2–3 individual mice, for one experiment. Results from 17 experiments are shown, with overall mean values indicated by bars. For dLN and spleen data, each point represents killing from one mouse, with overall means indicated by bars. B) Thy1.1 + DUC18 T cells were transferred on day 0, and organs harvested and analyzed on indicated days. Numbers of live Thy1.1 DUC18 T cells are plotted (for tumors, p values for day 2 versus day 4 = .03, for day 2 versus day 6 = .05; for dLNs, p = 0.03 for day 2 versus day 4, and p = 0.04 for day 2 versus day 6). Points represent individual mice (n = 13–20) from 3 experiments. C) Normalized killing values were calculated by dividing the mean % specific killing by the mean number of live DUC18 T cells present in tumors for each time point. D) Activated DUC18 T cells were transferred into tumor bearing mice on day 0. Organs were harvested on days 2 and 6, and the percentage of cells expressing IFNγ ex vivo was determined by intracellular cytokine staining, after gating on Thy1.1 + Vβ8.3 + DUC18 T cells. E) Linear regression analyses for % specific killing in tumors versus % Meth A reference cells present for all individual mice used in ITC assays shown in A. The slopes were not statistically different from 0; day 2 p = 0.83, day 4 p = .90, day 6 p = .16).

Article Snippet: DUC18 TCR transgenic mice are on a BALB/c background and were bred to Thy1.1 BALB/c mice (provided by Dr. Hyam Levitsky, Johns Hopkins) to generate Thy1.1 + DUC18 mice.

Techniques: Lysis, Derivative Assay, Expressing, Ex Vivo, Staining

Journal: PLoS ONE

Article Title: Rapid Maturation of Effector T Cells in Tumors, but Not Lymphoid Organs, during Tumor Regression

doi: 10.1371/journal.pone.0000821

Figure Lengend Snippet: Thy1.1 DUC 18 effector T cells were transferred into tumor-bearing recipients on day 0. On the indicated days, organs were harvested and stained for Thy1.1/Vβ8.3 + DUC18 T cells. A) The percentage of DUC18 T cells that were dead was determined by gating on the DUC18 T cell population and analyzing the PI bright percentage. Data were pooled from 9 mice in 3 independent experiments. B) Organs were harvested on day 6 post-T cell transfer, and samples were stained with Thy1.1, Vβ8.3, Annexin V Alexa 488 and 7AAD. Histograms are shown after gating on the DUC18 T cell population, and represent data from a single mouse. C) The percentage of Annexin V + DUC18 T cells is shown, based on the region shown in the histogram in B (p value for % apoptotic DUC18 T cells at day 6 in tumors versus dLNs = 0.0002 and for tumors versus spleens = 0.0007). Cumulative data from 3 independent experiments are shown.

Article Snippet: DUC18 TCR transgenic mice are on a BALB/c background and were bred to Thy1.1 BALB/c mice (provided by Dr. Hyam Levitsky, Johns Hopkins) to generate Thy1.1 + DUC18 mice.

Techniques: Staining

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Collective action of hematopoietic cell subsets mediates anti-IL10R1 and CpG tumor immunity

doi: 10.1007/s00262-011-1175-3

Figure Lengend Snippet: CpG stimulation and IL-10 inhibition potentiate dendritic cell maturation in vitro. a CpG stimulation in combination with IL-10 blockade induced increased IL-12p40 and IL-6 secretion by DCs. DCs were in vitro stimulated with either (10 μg/ml) isotype control, anti-IL-10 or anti-IL-10R1 antibody in the presence or absence of 0.3 mM CpG. Culture supernatants were harvested after 48 h of stimulation and analyzed for IL-12p40 and IL-6 production via Luminex analysis. b Cell surface activation markers B7.1 and B7.2 were also analyzed by flow cytometry. Data are the mean of triplicates. Error bars represent SEM. c TCR transgenic Thy1.1+ Pmel CD8+ T cells were adoptively transferred into wild-type C57BL/6 mice. These mice were immunized the next day as indicated with 100 μg gp100 peptide, 250 μg anti-IL-10R1, 5 μg CpG 1668, or 20 μg LPS. Draining lymph nodes were harvested 3 days after immunization and analyzed for the frequency of Thy1.1+ CD8+ T cells and IFN-γ+ Thy1.1+ CD8+ T cells (*P < 0.05, n ≥ 4). The results are representative of two experiments

Article Snippet: Rag2 − / − mice were purchased from Taconic. il10r1 −/− , Myd88 − / − , TCR transgenic Pmel mice were bred and maintained in specific pathogen-free conditions in the animal facility at Genentech in accordance with the institutional guidelines.

Techniques: Inhibition, In Vitro, Control, Luminex, Activation Assay, Flow Cytometry, Transgenic Assay