monoclonal antibody  (R&D Systems)

Journal: Frontiers in Immunology

Article Title: Tumor-intrinsic interferon signaling drives pancreatic cancer resistance to tumor mucin1-targeted CAR T cell therapy

doi: 10.3389/fimmu.2025.1618415

Figure Lengend Snippet: Suppressing IFN signaling in PDA reduces ICAM-1 and CXCL10 levels. PDA cell lines were plated overnight for attachment. On the following day, PDA cells were treated with JAKi at 10μM and 25μM overnight. After JAKi treatment, it was removed, and tMUC1-CAR T cells or Mock T cells were added at E:T ratio of 5:1 in the absence of JAKi. The E:T ratio was calculated based on the initial number of PDA cells plated. After 24hr, co-culture supernatants were collected and assayed for cytokine levels of (A) ICAM-1 and (B) CXCL10 by ELISA. Data are presented as the mean ± SD of triplicate. Baseline levels of ICAM-1 and CXCL10 were low (<50pg/ml and <10pg/ml, respectively) in either CAR T cell-only or in PDA-only cultures, which were detected by ELISA. The statistical comparison was conducted between CAR T cell treatment in JAKi-pretreated PDA and CAR T cell treatment in PDA without JAKi pretreatment. *p<0.05, ****p<0.0001 (unpaired t test with Welch’s correction).

Article Snippet: On the following day, PDA cells were pre-incubated with PD-L1 blocking antibody (10μg/ml) or its isotype control antibody (10μg/ml; Cat# 400348; BioLegend), anti-ICAM-1 neutralizing antibody (5μg/ml; Cat# AF720; R&D Systems), anti-CXCL10 neutralizing antibody (5μg/ml; Cat# MAB266-100; R&D Systems), or fresh media alone for 2hr.

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Tumor-intrinsic interferon signaling drives pancreatic cancer resistance to tumor mucin1-targeted CAR T cell therapy

doi: 10.3389/fimmu.2025.1618415

Figure Lengend Snippet: Suppressing IFN signaling in CAR T cells slightly decreases ICAM-1 level. PDA cell lines were plated overnight for attachment. On the same day, tMUC1-CAR T cells were treated with or without JAKi at 10μM and 25μM overnight. On the following day, JAKi-pretreated CAR T cells were washed with fresh media to remove JAKi before being added to PDA cells at E:T ratio of 5:1 for co-culture. After 24hr, co-culture supernatants were collected and assayed for cytokine levels of (A) ICAM-1 and (B) CXCL10 by ELISA. Data are presented as the mean ± SD of triplicate. The statistical comparison was conducted between JAKi-pretreated CAR T cell group and media-pretreated CAR T cell group in PDA. *p<0.05, **p<0.01 (unpaired t test with Welch’s correction).

Article Snippet: On the following day, PDA cells were pre-incubated with PD-L1 blocking antibody (10μg/ml) or its isotype control antibody (10μg/ml; Cat# 400348; BioLegend), anti-ICAM-1 neutralizing antibody (5μg/ml; Cat# AF720; R&D Systems), anti-CXCL10 neutralizing antibody (5μg/ml; Cat# MAB266-100; R&D Systems), or fresh media alone for 2hr.

Techniques: Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Comparison

Journal: Frontiers in Immunology

Article Title: Tumor-intrinsic interferon signaling drives pancreatic cancer resistance to tumor mucin1-targeted CAR T cell therapy

doi: 10.3389/fimmu.2025.1618415

Figure Lengend Snippet: Engagement of CAR T cell with PDA induces function loss of CAR T cells and up-regulation of immune checkpoints. (A) Loss of CAR T cell cytotoxicity. CAR T cells or Mock T cells were co-cultured with MiaPaCa-2 or HPAFII cells at E:T ratio of 5:1 or cultured with media alone for overnight. Then the live CAR T cells and Mock T cells were isolated from the first round co-culture, and they were added to the fresh MiaPaCa-2 or HPAFII plates at E:T ratio of 2:1 or 5:1 for 24hr as the second round of co-culture. At the end of culture, tumor cell lysis against MiaPaCa-2 (left panel) or against HPAFII (right panel) was determined using MTT assay. The percentage of lysis was calculated using the formula: [(OD of co-culture with Mock T cells– OD of co-culture with CAR T cells)/OD of co-culture with Mock T cells] ×100. The mock T cells and CAR T cells are from the same pair for calculation. Data are presented as the mean ± SD from quadruplicate. The statistical difference was conducted for cytolysis of CAR T cells retrieved from co-culture with PDA cells when compared with cytolysis of CAR T cells retrieved from culture with media alone. ****p<0.0001 (Multiple unpaired t tests with Welch’s correction). (B) Increase of CD25 expression on CAR T cells after engagement with PDA. CAR T cells were co-cultured with MiaPaCa-2 or HPAFII cells at E:T ratio of 5:1 or cultured with media alone for overnight. Then CAR T cells were stained and analyzed for CD25 expression on CAR-positive and CAR-negative live cells after gating on CD4 + T cells and CD8 + T cells. (C) Expression of ICs on CAR T cells. Cell culture was performed same as in (B) . IC expression on CAR-positive and CAR-negative live cells were displayed after gating on CD4 + T cells and CD8 + T cells. (D) The retaining of tMUC1 and up-regulation of PD-L1 on PDA. After rinsing off suspension cells in co-culture from (B) , adherent MiaPaCa-2 or HPAFII cells were stained and analyzed for tMUC1 and PD-L1. PDA cells cultured in media alone were included as baseline control. (E) Suppressing tumor IFN signaling blocks CAR T cells-induced PD-L1 increase. MiaPaCa-2 or HPAFII cells were pre-treated with JAKi at 25μM overnight. Then JAKi was removed, and CAR T cells were added in the absence of JAKi at E:T ratio of 5:1, calculated based on the initial number of PDA cells plated. After 24hr treatment with CAR T cells, live adherent PDA cells were stained and analyzed for PD-L1 expression. (F) Effectiveness of PD-L1 blocking antibody. CAR T cells-treated HPAFII cells were pre-incubated with PD-L1 blocking antibody at the indicated doses, followed by PD-L1-PE staining. (G) Blocking PD-L1 partially reversed HPAFII resistance to CAR T cell lysis. MiaPaCa-2 or HPAFII cells were treated with CAR T cells or Mock T cells in the presence of PD-L1 blocking antibody or its isotype control for 24hr. PDA cell lysis was determined using MTT assay. Data are presented as the mean ± SD from quadruplicate. The statistical difference was conducted for cytolysis of CAR T cells with PD-L1 blocking antibody when compared with cytolysis of CAR T cells with media alone. ****p<0.0001 (unpaired t tests with Welch’s correction). (H) Involvement of ICAM-1 and CXCL10 in CAR T cell cytotoxicity. MiaPaCa-2 or HPAFII cells were treated with CAR T cells or Mock T cells in the presence of anti-ICAM-1 or anti-CXCL10 antibodies for 24hr. PDA cell lysis was determined using MTT assay. Data are presented as the mean ± SD from quadruplicate. The statistical difference was conducted for cytolysis of CAR T cells with neutralizing antibody when compared with cytolysis of CAR T cells with media alone. **p<0.01 (unpaired t tests with Welch’s correction).

Article Snippet: On the following day, PDA cells were pre-incubated with PD-L1 blocking antibody (10μg/ml) or its isotype control antibody (10μg/ml; Cat# 400348; BioLegend), anti-ICAM-1 neutralizing antibody (5μg/ml; Cat# AF720; R&D Systems), anti-CXCL10 neutralizing antibody (5μg/ml; Cat# MAB266-100; R&D Systems), or fresh media alone for 2hr.

Techniques: Cell Culture, Isolation, Co-Culture Assay, Lysis, MTT Assay, Expressing, Staining, Suspension, Control, Blocking Assay, Incubation

Journal: Cell Reports Medicine

Article Title: Immune targeting of triple-negative breast cancer through a clinically actionable STING agonist-CAR T cell platform

doi: 10.1016/j.xcrm.2025.102198

Figure Lengend Snippet: Sustained tumor cell STING signaling promotes T cell migration (A) Representative IHC images of STING staining of a TMA ( n = 63). 0 = no tumor cell staining; 1+ = faint, 2+ = moderate, 3+ = strong staining in >10% of tumor cells. Scale bar, 50 μm. (B) Tumor cell STING staining in PTEN-null TNBCs. Scale bar, 50 μm. (C) Immunoblot of STING and other proteins across TNBC lines, including ADU-S100 (ADU; 50 μM, 3 h)-treated THP-1 monocytes to distinguish phosphorylated STING (pSTING). (D) Immunoblot of MDAMB231 parental cells, cells expressing scramble (Scr), or Rab7 knockout (KO) vectors (Sg1 and Sg2). (E) CXCL10 ELISA from conditioned medium (CM) of the indicated Scr or Rab7 KO cells treated with ADU at 50 μM for 48 h ( n = 4–8) or PBS control (ctrl). (F) Immunoblot of PTEN-null HCC70 cells treated with ADU at 50 μM for 3 h. (G) Immunoblot of MDAMB231 Scr or Rab7 KO cells treated with 50 μM ADU at the indicated time points. (H) Immunoblot of MDAMB231 Scr or Rab7 KO cells treated with 50 μM ADU for 24 h. (I) Heatmap showing log2 fold change (L2FC) of a cytokine/chemokine panel, normalized to untreated Scr cells ( n = 2–4). Red asterisk indicates values above assay in all conditions, black asterisk indicates above assay in treated conditions, using the upper limit for L2FC calculation. (J) CXCL10 ELISA in CM from the indicated cell lines with or without 50 μM ADU treatment at 48 h ( n = 2–4). (K) T cell migration assay schematic. (L) Representative images of CD8 + T cells (yellow) migrating toward MDAMB231 spheroids (Hoechst). Migrated CD8 + T cells were quantified after 48 h ( n = 9). Scale bar, 100 μm. (M) Representative images of CD8 + T cells (yellow) migrating toward Rab7 KO MDAMB231 spheroids (Hoechst). Migrated CD8 + T cells were quantified after 48 h ( n = 5–6). Scale bar, 100 μm. Quantitative data are represented as mean ± SEM. p values were calculated by one-way (J, L, and M) and two-way (E) ANOVA followed by Tukey’s post hoc test. ns, not significant. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: After collagen polymerization, T cells were stained with CellTrace Yellow (Invitrogen, Cat.# C34567 ) and added to one exterior chamber of the device at a 2:1 ratio of tumor spheroids (1:1 ratio for CAR T migration) in complete media with IL-2 (100 IU/mL) for a culture of 48 h. Antibodies blocking CXCL10 (R&D Systems, Cat.# MAB266-SP) and CCL5 (R&D Systems, Cat.# MAB678-SP) or isotype control (R&D Systems, Cat.# MAB002) were also added to the exterior chamber at 1 μg/mL for neutralization.

Techniques: Migration, Staining, Western Blot, Expressing, Knock-Out, Enzyme-linked Immunosorbent Assay, Control, Cell Migration Assay