Journal: Advanced Science

Article Title: Targeting DAP5 Disrupts Alternate Mode of Translational Initiation in Tregs and Potentiates Antitumor Immunity

doi: 10.1002/advs.202520625

Figure Lengend Snippet: Mice with heterozygous Dap5 deletion in Tregs displayed unaltered peripheral immune homeostasis but enhanced Teff response against subcutaneous tumors. a) WB results showing intermediate expression level of Dap5 in pTregs from HE‐ Dap5 ΔFoxp3 mice. b) Bar plot displaying unaltered splenic Treg frequencies in HE‐ Dap5 ΔFoxp3 mice. c) Curves of body weight changes for HE‐ Dap5 ΔFoxp3 and Dap5 flox mice under DSS‐induced colitis modeling. d) Curves of body weight changes for Rag1 −/− mice receiving adoptive transfer of naïve CD4 + T cells or naïve CD4 + T cells mixed with Tregs purified from HE‐ Dap5 ΔFoxp3 mice or Dap5 flox mice. e) Suppressed MC38 tumor growth in HE‐ Dap5 ΔFoxp3 mice. f) Curves monitoring MC38 tumor growth in HE‐ Dap5 ΔFoxp3 mice or Dap5 flox mice. g) Suppressed Panc02 tumor growth in HE‐ Dap5 ΔFoxp3 mice. h) Curves monitoring Panc02 tumor growth in HE‐ Dap5 ΔFoxp3 mice or Dap5 flox mice. i) Violin plots comparing gene expressions between tumor‐infiltrating CD4 + Tconv from HE‐ Dap5 ΔFoxp3 and Dap5 flox mice. j) Bar plots comparing infiltrations of IFN‐γ + CD4 + T cells in the MC38 tumors that grew in HE‐ Dap5 ΔFoxp3 and Dap5 flox mice. k) RNA velocity streamlines projected onto the UMAP‐based embedding. Cells were grouped according to their annotations. l) The directed Partition‐based Graph Abstraction (PAGA) graph showing the connectivity of these CD8 + T subpopulations. The edge weights quantify the connectivity between cell groups. m) Projection of RNA velocity streamlines on UMAP, grouped by cell type. n) GSEA plots displaying enriched signaling pathways in tumor‐infiltrating CD8 + T cells from HE‐ Dap5 ΔFoxp3 mice in contrast to that from Dap5 flox mice. o) Bar plots comparing infiltrations of IFN‐γ + CD8 + T and GZMB + CD8 + T cells between HE‐ Dap5 ΔFoxp3 and Dap5 flox mice. p‐ values were determined by two‐tailed student's T ‐test (b‐d, f, h, j and o), * p <0.05, ** p <0.01, **** p <0.001, *** p <0.001.

Article Snippet: The HeLa (RRID: CVCL_0030, ATCC Cat# CCL‐2), MC38 (RRID: CVCL_B288, Cytion Cat# 305223), and Panc02 (RRID: CVCL_D627, Cytion Cat# 300501) cell lines were purchased from the indicated suppliers in 2020, 2022, and 2022, respectively.

Techniques: Expressing, Adoptive Transfer Assay, Purification, Protein-Protein interactions, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma

doi: 10.1016/j.xcrm.2023.101212

Figure Lengend Snippet: Low or absence of cell surface FGFR4 in primary human cells does not induce cytokine release when cocultured with 3A11 CAR T cells (A) Representative flow cytometry plots show low or absence of FGFR4 cell surface expression on human cardiomyocytes, renal epithelial cells, renal cortical epithelial cells, renal proximal epithelial cells, HEK293, cholangiocytes, and hepatocytes pooled from 10 individuals using 3A11 scFvFc. MFI of 3A11 scFvFc or huIgG1 isotype control staining on these primary cells is shown in the top right table. Relative FGFR4 expression levels on these primary cells compared with RH30 cells are calculated as R e l a t i v e e x p r e s s i o n = [ M F I ( s c F v F c ) − M F I ( h u I g G i s o t y p e ) ] P r i m a r y c e l l [ M F I ( s c F v F c ) − M F I ( h u I g G i s o t y p e ) ] R H 30 . (B) Log 2 ratio of cytokine (IFN-γ, IL-2, and TNF-α) release in the supernatant, by 3A11 CAR T cells cocultured with primary cells, as indicated, in their respective media, compared with the RMS cell RH30. Cell line 7250 serves as a FGFR4 − control. Values represent n = 3 independent experiments with n = 3 individual donors. Values represent mean ± SEM (error bars).

Article Snippet: Human cardiomyocytes (Cat# 36044-15), cholangiocytes (Cat# 36755-12), and pancreatic epithelial primary cells (Cat# 36099-25) were obtained from Cellprogen; human coronary artery smooth muscle cells (CASMC, Cat# CC-2583), small airway epithelial cells (SAEC, Cat# CC-2547), bronchial/tracheal epithelial cells (NHBE, Cat# CC-2541), human renal epithelial cells (HRE, Cat# CC-2556), renal cortical epithelial cells (HREC, Cat# CC-2554), renal proximal tubule epithelial cells (RPTEC, Cat# CC-2553), hepatocytes (Cat# HUCPG), and 10 Donors pooled hepatocytes (Cat# HUCS10P) were purchased from Lonza; HEK 293 cells (Cat# CRL-1573) was purchased from ATCC.

Techniques: Flow Cytometry, Expressing, Control, Staining

Journal: Cell Reports Medicine

Article Title: Preclinical development of a chimeric antigen receptor T cell therapy targeting FGFR4 in rhabdomyosarcoma

doi: 10.1016/j.xcrm.2023.101212

Figure Lengend Snippet:

Article Snippet: Human cardiomyocytes (Cat# 36044-15), cholangiocytes (Cat# 36755-12), and pancreatic epithelial primary cells (Cat# 36099-25) were obtained from Cellprogen; human coronary artery smooth muscle cells (CASMC, Cat# CC-2583), small airway epithelial cells (SAEC, Cat# CC-2547), bronchial/tracheal epithelial cells (NHBE, Cat# CC-2541), human renal epithelial cells (HRE, Cat# CC-2556), renal cortical epithelial cells (HREC, Cat# CC-2554), renal proximal tubule epithelial cells (RPTEC, Cat# CC-2553), hepatocytes (Cat# HUCPG), and 10 Donors pooled hepatocytes (Cat# HUCS10P) were purchased from Lonza; HEK 293 cells (Cat# CRL-1573) was purchased from ATCC.

Techniques: Purification, Control, Recombinant, Labeling, Virus, Microarray, Cytokine Assay, Transfection, Chromatin Immunoprecipitation, ChIP-sequencing, Plasmid Preparation, Software, Imaging

Journal: Journal for Immunotherapy of Cancer

Article Title: Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

doi: 10.1136/jitc-2020-001250

Figure Lengend Snippet: NAM+GEM significantly improved clinical parameters, including survival, in mouse PDAC. Orthotopic Panc-02 model. Tumors and metastases were generated and treatments with NAM+GEM and controls were performed as outlined in . At the conclusion of the experiment tumor weight (A) and number of metastases (B) was measured, as well as the grade of ascites (T0–5) (C). Average of two experiments with n=10 mice per group. Orthotopic KPC model. Tumors and metastases were generated and treatments with NAM+GEM and controls were performed as outlined in . At the conclusion of the experiment tumor weight (D) and number of metastases (E) was measured, as well as the grade of ascites (T0–5) (F). Average of two experiments with n=10 mice per group. Significant differences were determined by Mann-Whitney U test. *P<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Error bars represent SE of the mean (SEM). Survival study orthotopic Panc-02 model. Tumors and metastases were generated and treatments with NAM+GEM and controls were performed as outlined in . At the end of treatments mice were monitored until death, and survival was assessed (G) when defined clinical endpoints were reached. The results of 2 experiments were averaged with n=10 mice per group. Significant differences were determined by the Mantel-Cox test. GEM, gemcitabine; NAM, nicotinamide; PDAC, pancreatic ductal adenocarcinoma.

Article Snippet: Immune cells from spleens or tumors were isolated from treated and control Panc-02 mice for ELISPOT (BD Biosciences, cat# 551083) analysis, as described previously.

Techniques: Generated, MANN-WHITNEY

Journal: Journal for Immunotherapy of Cancer

Article Title: Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

doi: 10.1136/jitc-2020-001250

Figure Lengend Snippet: CD4 T cells significantly reduced pancreatic cancer by NAM+GEM in immune competent mice, while little effect of NAM+GEM was observed in nude mice. Orthotopic Panc-02 tumors were generated in immune competent C57BL/6 mice and depleted for CD4 and CD8 T cells during NAM+GEM treatment as outlined in . Antibodies to CD4 T cells (300 µg/200 µL) or to CD8 T cells (300 µg/200 µL) or isotype control were administered IP every 3rd day for 2 weeks. At the conclusion of the experiment the average tumor weight (A) and number of metastases (B) was determined in the NAM+GEM-treated/T cell-depleted compared with NAM+GEM-alone treated mice, with n=5 mice per group. Mann-Whitney U test. *P<0.05, **p<0.01 is significant. depletion of CD4 and CD8 T cells was confirmed by flow cytometry in the spleen (C). Orthotopic Panc-02 tumors were generated in nude mice (Fox n1nu/J) and treated with NAM+GEM as outlined in . At the conclusion of the experiment the average tumor weight (D) and number of metastases (E) was determined in the NAM+GEM-treated compared with the saline mice with n=5 mice per group. Mann-Whitney U test. GEM, gemcitabine; NAM, nicotinamide; ns, non-significant.

Article Snippet: Immune cells from spleens or tumors were isolated from treated and control Panc-02 mice for ELISPOT (BD Biosciences, cat# 551083) analysis, as described previously.

Techniques: Generated, MANN-WHITNEY, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

doi: 10.1136/jitc-2020-001250

Figure Lengend Snippet: NAM+GEM decreased the production of collagen I and HABP, and increased αSMA and CD31, in pancreatic tumors of orthopic Panc-02 model. Tumors were analyzed for collagen I by trichrome staining (A) and quantified (B) and shown in more detail (C). n=5 mice per group and the results of 5 fields were averaged. HABP fibrils was analyzed by RT-PCR (D). αSMA protein was by analyzed by IHC (E), and quantified (F), and shown in more detail (G). For both trichrome and αSMA the percentage of positive areas were determined and the results of n=5 mice per group was averaged. The presence of blood vessels in the pancreatic tumors was analyzed by IHC using anti-CD31 antibodies (H) and quantified (I) and shown in more detail (J). n=5 mice per group and the results of 10 fields were averaged. Mann-Whitney U test. *P<0.05, **p<0.01 is significant. Error bars represent SEM. αSMA, α-smooth muscle actin; GEM, gemcitabine; HABP, hyaluronic acid binding protein; IHC, immunohistochemistry; SEM, SE of the mean.

Article Snippet: Immune cells from spleens or tumors were isolated from treated and control Panc-02 mice for ELISPOT (BD Biosciences, cat# 551083) analysis, as described previously.

Techniques: Staining, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY, Binding Assay, Immunohistochemistry

Journal: Journal for Immunotherapy of Cancer

Article Title: Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

doi: 10.1136/jitc-2020-001250

Figure Lengend Snippet: Peritumoral and intratumoral LNS in pancreatic tumors of orthotopic Panc-02 model. Detail of a peritumoral LNS (A). Peritumoral LNS, characterized by well-developed follicular structures with occasional germinal centers. CD4 + and CD8 + T cells are concentrated in the deep cortical zone/paracortex, where they were arranged in dense sheets, and are scattered within follicles and surrounding tissue. The peritumoral LNS is surrounded by a fibroblast layer (red arrows). For more detail about peritumoral and intratumoral LNS (see ). Immunostaining for CD31 highlights the presence of vessels within peritumoral and intratumoral LNS in pancreatic tumors (black arrows) (B), which may allow the T cells to migrate to the tumor areas. LNS, lymph node-like structures.

Article Snippet: Immune cells from spleens or tumors were isolated from treated and control Panc-02 mice for ELISPOT (BD Biosciences, cat# 551083) analysis, as described previously.

Techniques: Immunostaining

Journal: Journal for Immunotherapy of Cancer

Article Title: Nicotinamide combined with gemcitabine is an immunomodulatory therapy that restrains pancreatic cancer in mice

doi: 10.1136/jitc-2020-001250

Figure Lengend Snippet: NAM+GEM increased the expression of genes involved in T cell migration and activation, and reduced the expression of genes involved in invasion of tumor cells. Orthotopic Panc-02 tumors of the different treatment groups were analyzed by Nanostring through gene expression profiles involved in T cell migration, activation, epitope spreading and invasion of tumor cells. A heatmap of relevant genes is shown (A), and the function of each gene and p values (B). Gene expression levels in the NAM+GEM group were compared with the saline group and analyzed by ANOVA p<0.05 is significant. To confirm the Nanostring data, sections of tumors in the different treatment groups were analyzed for the expression of Ccl21a and Fcer by RNAscope (C D), and quantified (E F). Whole tissues were quantified using HALO Image Analysis; the number of cells with red dot(s) was determined. Images were acquired using ×40 magnification. Data are presented as SE of the mean (SEM); statistical analysis was performed using a one-way ANOVA. *p<0.05 is significant. ANOVA, analysis of variance; GEM, gemcitabine; NAM, nicotinamide.

Article Snippet: Immune cells from spleens or tumors were isolated from treated and control Panc-02 mice for ELISPOT (BD Biosciences, cat# 551083) analysis, as described previously.

Techniques: Expressing, Migration, Activation Assay