αcxcl10  (R&D Systems)

Journal: Cancer Immunology, Immunotherapy

Article Title: Cxcr3 constrains pancreatic cancer dissemination through instructing T cell fate

doi: 10.1007/s00262-022-03338-7

Figure Lengend Snippet: Cxcr3 is dispensable for intratumoral antigen-specific T cell accumulation. A Orthotopic KPC 2a tumor and spleen weights on 14 or 21 days posttumor in Cxcr3 + / + and Cxcr3 −/− mice. Each dot is an independent animal. Data are mean ± S.E.M. * p < 0.05, ** p < 0.005, ANOVA with a Tukey’s posttest. B Frequency and number of CD8 + T cells per gram spleen or tumor from KPC 2a-bearing Cxcr3 + / + and Cxcr3 −/− mice on day 14 (d14) or day 21 (d21) posttumor. Each dot is an independent animal. Data are mean ± S.E.M. n = 3–4 mice per group. * p < 0.05, ** p < 0.005, ANOVA with a Tukey’s posttest. C Representative H-2D b :CB 101-109 tetramer staining gated on live CD8 + T cells infiltrating tumors from KPC 2a-bearing Cxcr3 + / + and Cxcr3 −/− mice on day 14 or day 21 posttumor. D Frequency of tetramer + T cells of CD8 + T cells (left) and number of CD8 + tetramer + T cells per gram tissue. E Immunofluorescent detection of CD8 T cells infiltrating orthotopic tumors isolated from Cxcr3 + / + and Cxcr3 −/− mice on day 14. Scale bar, 50 μm. n = 3 mice per group. F Spleen and tumor weight from mice bearing orthotopic (Orth) or subcutaneous (S.C.) KPC 2a tumors on day 14 posttumor. Each dot is an independent animal. Data are mean ± S.E.M. ** p < 0.005, *** p < 0.0005, Student’s T test. G Representative IF for Cxcl9/Cxcl10 from normal mouse pancreas, KPC 2 (CB-) parental tumors, KPC 2a (CB +) tumors, or KPC 2a tumors from mice treated with αPD-L1 on day 14. H Intensity units of Cxcl9/Cxcl10 + staining from samples in G. Each dot is an independent mouse. NP, normal pancreas. I Number of CD8 + T cells per field of view (FOV) from samples in G. J Correlation between CD8 + T cells and Cxcl9/Cxcl10 intensity from mice bearing KPC 2a orthotopic tumors from G - I

Article Snippet: Sections were rehydrated with PBS + 1% bovine serum albumin (BSA) and incubated for 1 h at room temperature (rt) with 1:200 αCD8α-PerCp-Cy5.5 (53–6.7, BD), 1:500 αThy1.1-BV510 (OX-7, Biolegend), 1:200 αF4/80-PE (BM8, Biolegend), 1:200 αpanCK-FITC (F3418, Sigma-Aldrich), 1:100 Cxcl9-EF660 (MIG-2F5.5, eBioscience) and 1:100 αCxcl10 (goat polyclonal, R&D Systems) diluted in PBS + 1% BSA.

Techniques: Staining, Isolation

Journal: Cancer Immunology, Immunotherapy

Article Title: Cxcr3 constrains pancreatic cancer dissemination through instructing T cell fate

doi: 10.1007/s00262-022-03338-7

Figure Lengend Snippet: CD40 agonist increases Cxcl9 and Cxcl10 in a cell- and tissue-specific manner. A Schematic of immunotherapy in REX3 orthotopic KPC 2a tumor-bearing mice. On day 7 post orthotopic tumor implantation, cohorts received isotype (-), αCD40 (100 μg), αPD-L1 (200 μg), or the combination as we described (28). αPD-L1 (200 μg) was also administered on day 10 and 12 posttumor for a total of 3 doses. B Flow cytometric gating strategy for analysis of indicated immune cells in spleen and tumor of mice depicted in (A). C Flow cytometric gating strategy for detecting Cxcl9 and/or Cxcl10 in non-tumor cells of REX3 mice depicted in (A). D Representative flow cytometry plots of cDC1s isolated from pancreatic draining lymph node (LN), spleen and tumor (or normal pancreas, e.g., no tumor) on day 14 posttumor. E Mean frequency of cDC1s expressing Cxcl9 and/or Cxcl10. F Representative flow cytometry plots of cDC2s isolated from pancreatic draining LN, spleen, and tumor (or normal pancreas, e.g., no tumor) on day 14 posttumor. G Mean frequency of cDC2s expressing Cxcl9 and/or Cxcl10. H Representative IF staining of CD8, B cells (B220) and Cxcl9 and Cxcl10 reporter expression in REX3 spleen and tumor from mice treated with the indicated therapies as in (A). Scale bar, 50 µm

Article Snippet: Sections were rehydrated with PBS + 1% bovine serum albumin (BSA) and incubated for 1 h at room temperature (rt) with 1:200 αCD8α-PerCp-Cy5.5 (53–6.7, BD), 1:500 αThy1.1-BV510 (OX-7, Biolegend), 1:200 αF4/80-PE (BM8, Biolegend), 1:200 αpanCK-FITC (F3418, Sigma-Aldrich), 1:100 Cxcl9-EF660 (MIG-2F5.5, eBioscience) and 1:100 αCxcl10 (goat polyclonal, R&D Systems) diluted in PBS + 1% BSA.

Techniques: Tumor Implantation, Flow Cytometry, Isolation, Expressing, Staining

Journal: Cancer Immunology, Immunotherapy

Article Title: Cxcr3 constrains pancreatic cancer dissemination through instructing T cell fate

doi: 10.1007/s00262-022-03338-7

Figure Lengend Snippet: Subcellular Cxcr3 ligand distribution is altered by immunotherapy and Cxcr3 promotes GzmB + engineered T cells that mitigate metastasis. A Apical Cxcl9/10 by cytokeratin + (CK) ductal tumor cells from KPC GEMM tumors from untreated mice. Scale bar, 50 μm. B Percentage of CK + tumor cells that express apical Cxcl9/Cxc10. * p <0.05, ** p <0.005, *** p <0.0005, one-way ANOVA with a Tukey's post test. C Number of tumor cells that express cytoplasmic Cxcl9/Cxc10 following immunotherapy. ** p <0.005, one-way ANOVA with a Tukey's post test. D Cxcl9/Cxcl10 in KPC GEMM tumors on day 8 post Thy1.1 + 1045 T cells. Scale bar, 50 μm. D Representative IF of tumor sections from KPC mice treated with 1045 T cell therapy (1045 T cells) at 8 days post treatment. IF overlays of Cxcl9/Cxcl10, Thy1.1 expressed on engineered T cells, macrophage marker F4/80, tumor cell marker cytokeratin (CK) and DAPI nuclear stain. E Frequency of Thy1.1 + engineered T cells adjacent to Cxcl9/Cxc10 + cells in PDA from tissues depicted in D. F Experimental schematic for mice receiving orthotopic KPC2 (CB-) PDA cells, adoptive transfer of 1045 Thy1.1 + T cells and anti-Cxcr3 treatment days 6, 9 and 12, or untreated controls. G Representative histograms of Cxcr3 staining by adoptively transferred 1045 T cells in untreated (-) and anti-Cxcr3 treated mice compared to non-T cells (top panel) in mice depicted in F . H Proportion of splenic and intratumoral transferred Thy1.1 + CD8 + T cells in untreated (-) and anti-Cxcr3 treated mice depicted in F . I Proportion of total CD8 + T cells and total Thy1.1 + cell numbers per gram tissue of mice depicted in F . J Proportion of mice depicted in F which developed metastasis, n = 3 mice per group. K Representative flow cytometric plots of Klrg1 and Granzyme B (GzmB) staining on splenic and intratumoral Thy1.1 + CD8 + adoptively transferred 1045 CD8 T cells isolated from untreated (-) or anti-Cxcr3 treated mice 2 weeks following orthotopic tumor implantation. L Proportion of splenic and intratumoral Granzyme B + adoptively transferred Thy1.1 + CD8 T cells 2 weeks following orthotopic tumor implantation. n = 3 mice per group, significance determined by Student’s T test. *, p <0.05. M Simplified model depicting the role for Cxcr3 in promoting effector T cell differentiation in the spleen and exhausted T cell differentiation in the tumor microenvironment

Article Snippet: Sections were rehydrated with PBS + 1% bovine serum albumin (BSA) and incubated for 1 h at room temperature (rt) with 1:200 αCD8α-PerCp-Cy5.5 (53–6.7, BD), 1:500 αThy1.1-BV510 (OX-7, Biolegend), 1:200 αF4/80-PE (BM8, Biolegend), 1:200 αpanCK-FITC (F3418, Sigma-Aldrich), 1:100 Cxcl9-EF660 (MIG-2F5.5, eBioscience) and 1:100 αCxcl10 (goat polyclonal, R&D Systems) diluted in PBS + 1% BSA.

Techniques: Marker, Staining, Adoptive Transfer Assay, Isolation, Tumor Implantation, Cell Differentiation