Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD95 , REA738 , 50 , 130-113-004 , PE (FITC) , Miltenyi Biotec.

Techniques: Imaging

Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: T cell subset classification and spatial distribution in an HCC sample. (A) Bar graph quantifications of gated T cell CD3 + T cells subpopulations (CD4 + T h , CD8 + T c , FoxP3 + /CD25 + T reg ) as well as T cell differentiation subsets (T N , T SCM , T CM , T EM , T EMRA , T TE ) for HCC tumor margin and tumor core. (B) Subclassification criteria used for T cell differentiation subset gating. Detailed gating scheme see Supplementary Figures S5A–D Spatial distribution of CD3+ T cell subsets as well as CD4+ and CD8+ differentiation subsets for tumor margin (C) and tumor core (D) . Arrowheads in (C, D) highlight the spatial distribution of regulatory T cells (T reg ). (E) Violin plots demonstrating expression levels of markers used for the definition of T cell differentiation subsets in tumor margin and tumor core: CD25, FoxP3, CD45RA, CD45RO, CD27, CD95, CD11a. Depicted markers and annotated cell types as indicated by the color code. ROI sizes: Tumor margin (ROI15) and tumor core (ROI16): 975 x 769 µm.

Article Snippet: CD95 , REA738 , 50 , 130-113-004 , PE (FITC) , Miltenyi Biotec.

Techniques: Cell Differentiation, Expressing

Journal: Cancer Immunology Research

Article Title: Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand–Expressing Solid Tumors

doi: 10.1158/2326-6066.cir-22-0115

Figure Lengend Snippet: Figure 1. CAR T cells are susceptible to suppression by tumor-expressed FasL. T cells from healthy donor PBMCs were transduced with SFG-based retrovirus engineered to express a second-generation CAR specific for human PSCA. A, Transduction efficiency was determined by flow cytometry on day 5 after transduction. Shown in the histogram are data representative of a single donor and in the bar graph are summary data (mean SEM, n ¼ 8). Statistical significance was calculated using an unpaired t test. B, Cytolytic activity of nontransduced control T cells (NT) and CAR PSCA T cells (CAR) was tested in a 51Cr-release assay using 5103 PSCA– 293T cells and PSCAþ CAPAN-1 and CFPAC-1 pancreatic cancer cells (mean SEM, n ¼ 3). Statistical difference was calculated using an unpaired t test. C and D, Expression of Fas (CD95) on T cells at baseline, upon activation, and after CAR transduction was determined by flow cytometry. Expression by T cells from a representative donor (C) and summary data (mean SEM, n ¼ 5; D). Significance was determined using one-way ANOVA. E, The viability of CAR PSCA T cells was determined by flow cytometry using Annexin V and 7-AAD staining after exposure to the indicated concentrations of recombinant FasL. The % viable cells (Annexin V–7-AAD–) under each condition is shown (mean SEM, n ¼ 3). Significance was determined using one-way ANOVA. F, FasL expression on CAPAN-1 and CFPAC-1 pancreatic cancer cell lines was assessed by flow cytometry during normal culture conditions and following 24-hour exposure to IFNg. Shown in the histograms are data representative of 3 independent analyses. G, CAPAN-1 tumor cells engineered to express PSCA-GFP were cultured with CAR PSCA T cells in the absence or presence of a Fas- blocking antibody(ZB4) before analysisby flow cytometry. Shown in the FACS plots are data representativeof a single sample and in the bar graph are summary data (mean SEM, n ¼ 4). Significance was determined using one-way ANOVA. H, Illustration demonstrating the susceptibility of CAR T cells to suppression by solid tumors via the Fas/FasL signaling axis. P > 0.05 ¼ nonsignificant (ns); , P ≤0.05; , P ≤0.01; , P ≤0.001, as indicated in the figure panels.

Article Snippet: 200 ng/mL recombinant FasL (BioLegend; catalog no. 589406) was first incubated in different media conditions (fresh IMDM, conditioned media obtained from 293T cells, or conditioned media obtained from 293T-FD cells) at 37 C for 1 hour and subsequently used in a FasL ELISA (R&D Systems; catalog no. DY126).

Techniques: Transduction, Cytometry, Activity Assay, Control, Release Assay, Expressing, Activation Assay, Staining, Recombinant, Cell Culture, Blocking Assay

Journal: Cancer Immunology Research

Article Title: Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand–Expressing Solid Tumors

doi: 10.1158/2326-6066.cir-22-0115

Figure Lengend Snippet: Figure 2. Engineering a soluble Fas decoy receptor to sequester FasLCAR PSCA T cells were engineered to express Fas decoy (FD) by retroviral transduction and used for functional assessments. A, Diagram illustrating FD secreted by CAR T cells enhancing T-cell antitumor activity by blocking inhibitory FasL signaling. B, FD construct schematic is shown on top and CAR and FD coexpression in T cells from a representative donor assessed by flow cytometry on day 5 after transduction (density plots). C, Summary transduction data indicating expression of CAR PSCA and CAR.FD in T cells (mean SEM, n ¼ 8). D, Phenotype of CAR PSCA and CAR.FD T cells was compared 10 days after transduction by flow cytometry (mean SEM, n ¼ 3). No significant difference was observed between CAR PSCA and CAR.FD based on unpaired t test. E, Expansion of CAR PSCA and CAR.FD T cells stimulated with irradiated K562-PSCA tumor cells was monitored for six days using a flow cytometer (mean SEM, n ¼ 4). No significant difference was observed between CAR PSCA and CAR.FD based on an unpaired t test on days 3 and 6. F, Secretion of effector cytokines IFNg) and TNFa by CAR PSCA and CAR.FD T cells after 48-hour culture with K562 or K562-PSCA cells was measured using a multiplex cytokine assay (mean SEM, n ¼ 3). No significant difference was observed between CAR PSCA and CAR.FD based on an unpaired t test. G, Cytolytic function of CAR PSCA and CAR.FD T cells was compared in a chromium release assay using 293T, CAPAN-1, and CFPAC-1 cells as targets at indicated E:T (mean SEM, n ¼ 3). Unpaired t test indicated no significant difference between CAR PSCA and CAR.FD in tumor killing. H, FD secretion by CAR PSCA and CAR.FD T cells in the absence or presence of recombinant PSCA stimulation was measured in the supernatant by soluble Fas ELISA 48 hours after culture initiation (mean SEM, n ¼ 4). Statistical significance was calculated using an unpaired t test. P > 0.05 ¼ nonsignificant (ns); , P ≤0.05; , P ≤0.01; , P ≤0.001.

Article Snippet: 200 ng/mL recombinant FasL (BioLegend; catalog no. 589406) was first incubated in different media conditions (fresh IMDM, conditioned media obtained from 293T cells, or conditioned media obtained from 293T-FD cells) at 37 C for 1 hour and subsequently used in a FasL ELISA (R&D Systems; catalog no. DY126).

Techniques: Retroviral, Transduction, Functional Assay, Activity Assay, Blocking Assay, Construct, Cytometry, Expressing, Irradiation, Multiplex Assay, Cytokine Assay, Release Assay, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology Research

Article Title: Secreted Fas Decoys Enhance the Antitumor Activity of Engineered and Bystander T Cells in Fas Ligand–Expressing Solid Tumors

doi: 10.1158/2326-6066.cir-22-0115

Figure Lengend Snippet: Figure 3. FD-engineered T cells exhibit superior function in the presence of FasL. FasL-neutralizing and T-cell function–enhancing properties of FD were assessed using in vitro immunoassays. A, FasL neutralization by FD was measured by incubating recombinant human FasL with fresh media, conditioned media obtained from 233T cells, or conditioned media obtained from 293T-FD cells and subsequently used in a human FasL ELISA (mean SEM, n ¼ 4). Statistical significance was calculated using one- way ANOVA. B and C, Suppression of FasL-mediated T-cell apoptosis by FD was assessed by measuring the viability of CAR PCSA T cells in different culture conditions—no FD or FasL [Control sup. (no FasL)]; no FD, in the presence of recombinant FasL [Control sup. (þ FasL)]; no FD in the presence of recombinant FasL and FasL-blocking antibody NOK-2 [Control sup. (þ FasL, þ NOK-2)]; recombinant FasL in the presence of FD [FD sup. (þ FasL)]. T-cell viability was measured by flow cytometry by Annexin V and 7-AAD staining after overnight culture. Flow cytometry plots demonstrating the viability of CAR PSCA T cells for a representative donor (B) and summary data (mean SEM, n ¼ 3) (C). Statistical significance was determined using one-way ANOVA. D and E, Antitumor activity of CAR PSCA and CAR.FD T cells was measured against CAPAN-1–PSCA tumor cells in a coculture assay (E:T of 1:10) and tumor as well as T-cell numbers were quantified by flow cytometry. Starting cell numbers (day 0) were used as reference to calculate fold change in cell numbers for all time points. Line graphs demonstrating fold change in tumor cell numbers (D) and fold change in T-cell numbers (mean SEM, n ¼ 4; E). Statistical significance was calculated using an unpaired t test. F, Pie chart depicting the percentage of polyfunctional (defined as cells producing 2 or more cytokines) CAR PSCA and CAR.FD T cells for a representative donor after 5-hour stimulation with CAPAN-1-PSCA cells (E:T of 1:4). Stimulated T cells were analyzed using IsoPlexis single-cell cytokine assay after stimulation. Numbers next to the pie charts indicate the number of cytokines produced by each fraction of T cells specified in the pie charts. Data representative of three independent donors. P > 0.05, nonsignificant (ns); , P ≤0.05; , P ≤0.01; , P ≤0.001.

Article Snippet: 200 ng/mL recombinant FasL (BioLegend; catalog no. 589406) was first incubated in different media conditions (fresh IMDM, conditioned media obtained from 293T cells, or conditioned media obtained from 293T-FD cells) at 37 C for 1 hour and subsequently used in a FasL ELISA (R&D Systems; catalog no. DY126).

Techniques: Cell Function Assay, In Vitro, Neutralization, Recombinant, Enzyme-linked Immunosorbent Assay, Control, Blocking Assay, Cytometry, Staining, Flow Cytometry, Activity Assay, Co-culture Assay, Cytokine Assay, Produced