Journal: Frontiers in Immunology

Article Title: Phospho-enol pyruvate carboxykinase inhibition limits effector function in inflammatory T cells

doi: 10.3389/fimmu.2026.1706167

Figure Lengend Snippet: The PEPCK inhibitors reduce the TCR-induced upregulation of granzyme B expression. OT-I TCR transgenic T cells were stimulated with cognate peptide SIINFEKL (TCR) ± 3-MP (25–100 μM, as indicated) or iPCK2 (5 μM) for 48 h. (A) Representative histograms of intracellular granzyme (B) The values in histograms are geometric mean fluorescence intensities. (B) The paired biological replicate comparisons of activated control and 3-MP treated OT-I T cells from repeat flow cytometry experiments show the geometric MFI of granzyme B ( n = 7). (C) 3-MP limits TCR-induced Gzmb transcription as determined by qRT-PCR. The values represent means ± SD from biological replicates ( n = 4). (D) Dose-dependent inhibition of granzyme B expression by 3-MP. Each line represents data from an independent experiment with values normalized to no inhibitor control samples. iPCK2 inhibits TCR-induced impede granzyme B expression (E) but not CD71 (F) or cell viability (G) ( n = 4 to 5). In all cases, the dots joined by lines represent paired samples from an independent experiment (B) , ( E – G ). NS, not significant. * p < 0.05, ** p < 0.01, *** p < 0.001 as determined by ratio paired T -test or one-way ANOVA (C) .

Article Snippet: The following Taqman probes were used: Gzmb – Mm00442837_m1; Rpl13a – Mm05910660_g1.

Techniques: Expressing, Transgenic Assay, Fluorescence, Control, Flow Cytometry, Quantitative RT-PCR, Inhibition

Journal: iScience

Article Title: The GPCR Smoothened on cholinergic interneurons modulates dopamine-associated acetylcholine dynamics, learning, and effort management

doi: 10.1016/j.isci.2026.115324

Figure Lengend Snippet: Smo on CIN modulates ACh inhibition following repeated DAN stimulation (A) Left: Viral strategy for simultaneous dopamine neuron (DAN) axon terminal stimulation and G protein-coupled receptor-based sensor for dopamine (GRAB DA) or acetylcholine (GRAB ACh) recording in the dorsolateral striatum. Right: Representative traces across four laser pulse trains with session averages. (B) GRAB sensor and ChrimsonR-tdTomato expression in the dorsolateral striatum and substantia nigra pars compacta. Scale bars = 500μm. (C) Daily stimulation protocol in ChATCre +/− or Smo L/L :ChATCre +/− mice with or without Smoothened agonist (SAG). (D) Dopamine (DA) signals aligned to laser onset on days 1 and 7 in ChATCre +/- mice ( n = 7). A single SAG dose at 20 Mg/Kg was administered on day 8 to assess effects on DA release. (E) Quantification of DA release area under the curve (AUC) from (D) reported as percent of day 1 ( n = 7; repeated-measures one-way ANOVA: F(1.378, 8.269) = 0.03, p > 0.05). (F) ACh signal on days 1 and 7 in ChATCre +/− mice ( n = 7). (G) Same as (F), in Smo L/L :ChATCre +/− mice ( n = 6). (H) Comparison of GRAB ACh AUC on days 1 and 7 in ChATCre +/− mice ( n = 7 per day; paired two-tailed Student’s t test, ∗∗ p < 0.01). (I) Comparison of GRAB ACh AUC on days 1 and 7 in Smo L/L :ChATCre +/− mice ( n = 6 per day; paired two-tailed Student’s t test, p > 0.05). (J–K) ACh signal in SAG-treated mice on days 1 and 7 ( n = 7–8 per genotype). (L) Percent change in ACh dip amplitude in SAG-treated animals relative to untreated control average for each genotype ( n = 6–7; paired two-tailed Student’s t test; ∗∗ p < 0.01). (M) ACh dip AUC across stimulation days with or without SAG treatment in ChATCre +/− ( n = 7–8 per day; two-way repeated-measures ANOVA: day effect, F(2.902, 37.72) = 6.4, ∗∗ p < 0.01; Pharmacology effect, F(1, 13) = 23.47, ∗∗∗ p < 0.001; day × pharmacology interaction, F(4, 52) = 2.03, p > 0.05) and Smo L/L :ChATCre +/− mice ( n = 6–7 per day; two-way repeated-measures ANOVA: day effect, F(2.615, 28.76) = 0.57, p > 0.05; pharmacology effect, F(1, 11) = 1.41, p > 0.05; day × pharmacology interaction, F(4, 44) = 2.18, p > 0.05). (N) ACh dip duration across stimulation days with or without SAG treatment in ChATCre +/− ( n = 7–8 per day; two-way repeated-measures ANOVA: Day effect, F(3.491, 45.39) = 4.81, ∗∗ p < 0.01; Pharmacology effect, F(1, 13) = 5.52, ∗ p < 0.05; day × pharmacology interaction, F(4, 52) = 1.62, p > 0.05) and Smo L/L :ChATCre +/− mice ( n = 6–7 per day; two-way repeated-measures ANOVA: day effect, F(2.785, 30.64) = 0.35, p > 0.05; pharmacology effect, F(1, 11) = 0.57, p > 0.05; day × pharmacology interaction, F(4, 44) = 1.64, p > 0.05).

Article Snippet: ChrimsonR excitation, GRAB sensor excitation, isosbestic excitation (405 nm), and signal collection were all performed through a single patch cord (MFP_200/220/LWMJ-0.37_1m_FCM-MF1.25(F)_LAF; Doric Lenses), connected to the animal’s implanted optical fiber via an interconnect (ADAL3; Thorlabs), and to the sample (S) port of the Mini Cube.

Techniques: Inhibition, Expressing, Comparison, Two Tailed Test, Control

Journal: iScience

Article Title: The GPCR Smoothened on cholinergic interneurons modulates dopamine-associated acetylcholine dynamics, learning, and effort management

doi: 10.1016/j.isci.2026.115324

Figure Lengend Snippet: Smo on CIN modulates ACh inhibition coincident with endogenous DAN bursts (A) Left: Viral strategy for simultaneous G protein-coupled receptor-based acetylcholine and dopamine sensor (GRAB ACh and GRAB DA) recordings in the dorsolateral striatum. Right: Representative traces from a single mouse, with session-averaged GRAB DA event-aligned ACh profiles. (B) GRAB sensor expression in the striatum. Scale bars = 500μm. (C) Recording protocol for control and Smo L/L :ChATCre +/− mice. (D) Frequency of detected DA events with detection threshold set to 2, 3, or 4 MAD ( n = 16 per MAD threshold). (E) Average Spontaneous DA events stratified by amplitude ( top ) and coincident ACh signal ( bottom ) from control and Smo L/L :ChATCre +/− mice ( n = 7–9 per condition). (F) Area under the curve (AUC) for ACh bursts preceding DA events in (E) ( n = 7–9 per condition; two-way repeated measures ANOVA: genotype effect, F(1, 14) = 2.07, p > 0.05; coincident DA effect, F(1.143, 16.01) = 1.02, p > 0.05; genotype × coincident DA interaction, F(2, 28) = 0.84, p > 0.05). (G) AUC for ACh inhibition following DA events in (E) ( n = 7–9 per condition; two-way repeated measures ANOVA: genotype effect, F(1, 14) = 3.22, p > 0.05; coincident DA effect, F(1.555, 21.77) = 5.46, p < 0.05; genotype × coincident DA interaction, F(2, 28) = 5.80, p < 0.01; post hoc Šídák’s multiple comparisons test: ∗∗ p < 0.01). (H) ACh inhibition duration in (E) ( n = 7–9 per condition; two-way repeated measures ANOVA: genotype effect, F(1, 14) = 4.70, ∗ p < 0.05; coincident DA effect, F(1.864, 26.09) = 2.02, p > 0.05; genotype × coincident DA interaction, F(2, 28) = 0.43, p > 0.05). No post hoc tests were performed due to the absence of a significant interaction effect; graph annotations denote significant main effects only. Inhibition duration was quantified on a trial-by-trial basis, capturing variability in event timing not reflected in group averages displayed in (E). (I) ACh inhibition amplitude in (E) ( n = 7–9 per condition; two-way repeated measures ANOVA: genotype effect, F(1, 14) = 2.42, p > 0.05; coincident DA effect, F(1.370, 19.17) = 3.39, p > 0.05; genotype × coincident DA interaction, F(2, 28) = 2.94, p > 0.05).

Article Snippet: ChrimsonR excitation, GRAB sensor excitation, isosbestic excitation (405 nm), and signal collection were all performed through a single patch cord (MFP_200/220/LWMJ-0.37_1m_FCM-MF1.25(F)_LAF; Doric Lenses), connected to the animal’s implanted optical fiber via an interconnect (ADAL3; Thorlabs), and to the sample (S) port of the Mini Cube.

Techniques: Inhibition, Expressing, Control

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 deficiency restricts tumor progression by promoting T PEX cell transition. (A) Schematic diagram of the adoptive transferred tumor model: CD45.1 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells, followed by 2 × 10 6 CD45.2 WT or Mettl8 −/− OT-I cells transfer at 9 dpi. Mice were harvested at 21 dpi. (B) Tumor growth in each group of the mice in A. n = 8 per group. (C) Survival curve in each group of the mice in A. n = 8 per group. (D) The absolute number of tumor infiltrating OT-I cells from the mice in A. n = 8 per group. (E and F) Representative flow cytometry plots and cumulative data show the frequency and absolute number of Tcf1 + Tim3 − T PEX , Tim3 + Tcf1 − T EX (E), and CX3CR1 + Tcf1 − Int-T EX cells (F) gated on tumor-infiltrating OT-I cells. n = 6–8 per group. (G) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of GzmB + , IFN-γ + , and perforin + cells gated on tumor-infiltrating OT-I cells. n = 7 per group. (H) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and MFI of GzmB, IFN-γ, and perforin gated on tumor-infiltrating Tcf1 + Tim3 − T PEX , CX3CR1 + Tcf1 − Int-T EX, and CX3CR1 − Tcf1 − T EX subsets. n = 5–6 per group. Data are representative of three independent experiments. P value was calculated by two-way ANOVA (B), Log-rank test (C), and two-tailed Student’s t test (D−H); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The C57BL/6J (B6, CD45.2), B6.SJL (CD45.1), Cd4 cre , Gzmb cre , P14, and OT-I mice were from the Jackson Laboratory.

Techniques: Injection, Flow Cytometry, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Mettl8 promotes T PEX differentiation without affecting their proliferation and apoptosis. (A and B) Representative flow cytometry plots and cumulative data show the frequency of CD44 (A) and PD-1 (B) OT-I cells infiltrating in tumors. (C) Representative flow cytometry plots and cumulative data show the frequency of caspase and Ki67 in tumor-infiltrating OT-I cells. (D) Cumulative data show the frequency of caspase and Ki67 in Tcf1 + Tim3 − T PEX , CX3CR1 + Tcf1 − Int-T EX , and CX3CR1 − Tcf1 − T EX subsets. (E) Cumulative data show the frequency of GzmB, IFN-γ, and perforin in tumor-infiltrating OT-I subsets mentioned above. (F) Schematic diagram of the classic CRC liver metastases model: Mettl8 fl/fl Cd4 cre and Mettl8 fl/fl mice were intrasplenically injected with 2 × 10 5 MC38 cells (left), and imaging of livers on day 21 after injection (right). (G) Representative flow cytometry plots and cumulative data show the ratio of Tcf1 + Tim3 − T PEX to Tim3 + Tcf1 − T EX cells gated on CD44 hi CD62L lo CD8 + T cells of the livers from mice in F. (H) Representative flow cytometry plots and cumulative data show the frequency of GzmB, IFN-γ, and TNF-α gated on CD44 hi CD62L lo CD8 + T cells from the livers of mice in F. (I) Schematic diagram of the classic melanoma lung metastases model: Mettl8 fl/fl Gzmb cre and Mettl8 fl/fl mice were i.v. injected with 2 × 10 5 B16F10 cells (top) and the survival curve (bottom). (J) Schematic diagram of adoptive transfer model: CD45.1.2 + Mettl8 −/− or WT P14 CD8 + T cells were adoptively transferred into CD45.2 + WT recipients, followed by LCMV-clone 13 (LCMV-C13) infection 24 h later and then analyzed on 30 dpi. (K) Statistical analysis show the absolute number of P14 cells from the spleens of mice in J. (L) Representative flow cytometry plots and cumulative data show Tcf1 + Tim3 − T PEX , Tim3 + Tcf1 − T EX , and CX3CR1 + Tcf1 − Int-T EX cells gated on P14 cells from the spleens of mice in J. n = 4–8 mice per group. Data are representative of two independent experiments. P value was calculated by two-tailed Student’s t test (A–H, K, and L) or Log-rank test (I); *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The C57BL/6J (B6, CD45.2), B6.SJL (CD45.1), Cd4 cre , Gzmb cre , P14, and OT-I mice were from the Jackson Laboratory.

Techniques: Flow Cytometry, Injection, Imaging, Adoptive Transfer Assay, Infection, Two Tailed Test

Journal: The Journal of Experimental Medicine

Article Title: Targeting Mettl8-Tcf1 axis promotes CD8 + T PEX differentiation and antitumor immunity

doi: 10.1084/jem.20250424

Figure Lengend Snippet: Reconstitution of Mettl8 expression in Mettl8 −/− OT-I cells restored their phenotype to that of WT OT-I cells. (A) Schematic diagram of the rescue experiment: CD45.2 mice were subcutaneously injected with 2 × 10 5 EG7-OVA cells. Mettl8 overexpression (OE) or empty vector (EV) retrovirus were transduced to CD45.1.2 WT or Mettl8 −/− OT-I cells. 5 × 10 5 GFP + cells were sorted 48 h after transduction and adoptively transferred into the tumor-bearing mice at 9 dpi. Mice were harvested at 19 dpi. (B) Tumor growth in each group of the mice in A. n = 6 per group. (C) Tumor growth in each group displayed in each replicate. n = 6 per group. (D) Tumor weight (left) and the absolute number of tumor infiltrating OT-I cells (right) from the mice in A. n = 6 per group. (E) Representative flow cytometry plots and cumulative data show the frequency of GFP in OT-I cells. n = 6 per group. (F) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of Tcf1 + Tim3 − T PEX and Tim3 + Tcf1 − T EX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (G) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of CX3CR1 + Tcf1 − Int-T EX cells gated on tumor-infiltrating OT-I cells. n = 6 per group. (H) Representative flow cytometry plots (left) and cumulative data (right) show the frequency and absolute number of GzmB + , IFN-γ + , and perforin + cells gated on tumor-infiltrating OT-I cells. n = 6 per group. Data are representative of two independent experiments. P value was calculated by two-way ANOVA (B) and two-tailed Student’s t test (D to H); *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Article Snippet: The C57BL/6J (B6, CD45.2), B6.SJL (CD45.1), Cd4 cre , Gzmb cre , P14, and OT-I mice were from the Jackson Laboratory.

Techniques: Expressing, Injection, Over Expression, Plasmid Preparation, Transduction, Flow Cytometry, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 promotes tumor immune evasion by upregulating PD-L1 expression within TME (A–C) scRNA-seq analysis of two paired tumor and paratumor samples from LUAD patients showing cell clustering (A), WFDC2 /HE4-expressing cell populations (B), and quantification of WFDC2 /HE4 expression in epithelial/malignant clusters (C). (D) Online dataset analysis of WFDC2 mRNA expression in tumor versus normal LUAD tissues. (E and F) HE4 overexpression (E) or knockout (F) respectively promoted or suppressed the growth of subcutaneous LLC tumors in mice. (G) Survival of mice intraperitoneally inoculated with HE4-overexpressing or control ID8 cells. (H) HE4 knockout attenuated ID8 peritoneal tumor progression, shown by representative abdominal images and ascites volume. (I) SDS-PAGE with Coomassie blue staining showing the purity of Fc and mouse HE4-Fc (mHE4-Fc) recombinant proteins. (J) Administration of mHE4-Fc promoted MC38 subcutaneous tumor growth. (K) mHE4-Fc administration reversed the growth suppression of HE4-KO LLC subcutaneous tumors. (L) mHE4-Fc failed to reverse tumor growth suppression in HE4-KO LLC tumors implanted in Rag1 -deficient mice. (M) Extracellular HE4 did not promote LLC cell proliferation in vitro , as assessed by CCK8 assay. (N) mHE4-Fc administration suppressed CD8 + T cell activation in the TME of HE4-KO LLC tumors, assessed by IFN-γ + and granzyme B + CD8 + T cells. (O) HE4 upregulated PD-L1 expression on macrophages in the microenvironment of LLC, MC38, and HE4-KO LLC tumors. (P–R) mHE4-Fc administration failed to reverse tumor suppression of HE4-KO LLC subcutaneous tumors in Cd274 -deficient mice, with treatment scheme (P), tumor growth (Q), and tumor weight (R). Statistical analyses were performed using Wilcoxon rank-sum test (D), two-way ANOVA (E, F, J–L, and Q), log rank (Mantel-Cox) test (G), and two-tailed paired (C) or unpaired t tests (H, M, N, O, and R). Data represent two independent experiments (E and M).

Article Snippet: Mouse Granzyme B ELISA kit , Elabscience , Cat# E-EL-M0594.

Techniques: Expressing, Over Expression, Knock-Out, Control, SDS Page, Staining, Recombinant, In Vitro, CCK-8 Assay, Activation Assay, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 competes with IFN-γ for IFN-γR binding and modulates downstream gene expression (A and B) Raw264.7 cells were stimulated with HE4-Fc (20 μg/mL) or IFN-γ (100 ng/mL) for 3 h, followed by RNA-seq; volcano plots of HE4- (A) or IFN-γ-regulated genes (B) are shown. (C) Genes commonly upregulated by HE4 and IFN-γ. (D) AlphaFold-3-predicted interfaces of HE4-IFNGR1/2 and IFN-γ-IFNGR1/2 complexes, with shared receptor-contact residues highlighted. (E) Competitive binding assay: His-tagged IFNGR1/2 was incubated with Flag-HE4 in the presence or absence of IFN-γ, followed by Ni-TED pull-down and immunoblotting. (F and G) SPR sensorgrams showing binding of mHE4-Fc (F) or mIFN-γ-Fc (G) to mIFNGR1-His. (H and I) ELISA quantification of HE4 and/or IFN-γ levels in ascites from ID8-tumor-bearing mice (H) and LLC-tumor-conditioned media (I). (J) High concentrations of HE4 reduced IFN-γ binding to IFNGR1/2 in competitive pull-down assays. (K) PCA of RNA-seq profiles from Raw264.7 cells treated with HE4-Fc, HE4-Fc plus IFN-γ, or IFN-γ for 12 h. (L) Expression (TPM) of representative STAT1- or STAT3-associated genes following the indicated treatments. Statistical analyses were performed using two-tailed paired Student’s t tests (H and I). Data in (E) and SPR sensorgrams (F and G) are representative of three independent experiments.

Article Snippet: Mouse Granzyme B ELISA kit , Elabscience , Cat# E-EL-M0594.

Techniques: Binding Assay, Gene Expression, RNA Sequencing, Competitive Binding Assay, Incubation, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 blockade exerts therapeutic efficacy across multiple mouse tumor models (A–C) Mice bearing subcutaneous LLC tumors were treated intraperitoneally with isotype immunoglobulin G (IgG) or anti-mHE4 mAbs (clones #1 or #117). Treatment scheme (A), tumor growth curves (B), and representative tumors with weights (C) are shown. (D and E) Anti-mHE4 (clone #117) treatment suppressed urethane-induced lung tumorigenesis, shown by representative H&E staining and tumor quantification. Scale bar, 1,000 μm. (F and G) HE4 blockade attenuated ID8 intraperitoneal tumor progression, shown by representative abdominal images and ascites volume. (H and I) Therapeutic efficacy of anti-mHE4 (clone #117) in an orthotopic HGS-1 ovarian tumor model, shown by treatment scheme and representative tumors with weights. (J) Serum-free HE4 levels measured by ELISA in LLC-tumor-bearing mice one day after the final antibody treatment. (K and L) Anti-mHE4 (clone #117) reduced PD-L1 expression on tumor-associated macrophages in the LLC subcutaneous and ID8 intraperitoneal models. (M and N) Anti-mHE4 (clone #117) failed to suppress LLC tumor growth in Cd274 -deficient mice. (O) Combination therapy with anti-mHE4 and anti-mCTLA-4 enhanced tumor suppression in the LLC subcutaneous model. (P) Combination therapy with anti-mHE4 and paclitaxel (PTX) enhanced antitumor efficacy in the LLC subcutaneous model. (Q) Serum cytokines and clinical chemistry parameters following treatment with isotype IgG, anti-mHE4 mAb, or anti-mPD-1 mAb. (R and S) Safety assessment showing representative H&E images and incidence of inflammation in heart, liver, lung, and colon after anti-mHE4 or anti-mPD-1 treatment. Statistical analyses were performed using two-way ANOVA (B and N–P), one-way ANOVA (C, K, and N–Q), two-tailed unpaired t tests (E, G–J, and L), and chi-squared test (S). Data in (A–P) are pooled from two independent experiments. Scale bars, 100 μm.

Article Snippet: Mouse Granzyme B ELISA kit , Elabscience , Cat# E-EL-M0594.

Techniques: Drug discovery, Clone Assay, Staining, Enzyme-linked Immunosorbent Assay, Expressing, Two Tailed Test

Journal: Cell Reports Medicine

Article Title: HE4 drives PD-L1 expression in myeloid cells via IFN-γR-JAK-STAT3 signaling to promote tumor immune evasion

doi: 10.1016/j.xcrm.2026.102691

Figure Lengend Snippet: HE4 neutralization exerts therapeutic activity in human cancer models (A–C) PMA-differentiated THP-1 macrophages were stimulated with Fc or hHE4-Fc, and PD-L1 expression was assessed by flow cytometry (A), immunoblotting (B), and RT-qPCR (C); a commercial hHE4-Fc was included as an independent control. (D) Binding of hHE4 to PMA-differentiated THP-1 cells assessed by flow cytometry. (E and F) PMA-differentiated THP-1 cells were pretreated with ruxolitinib, fludarabine, or Stattic, followed by hHE4-Fc stimulation; PD-L1 was quantified by RT-qPCR (E) and flow cytometry (F). (G and H) Anti-hHE4 monoclonal antibodies inhibited hHE4-induced PD-L1 upregulation in PMA-differentiated THP-1 cells, assessed by RT-qPCR (G) and flow cytometry (H). (I) Anti-hHE4 mAb clone #10 blocked hHE4 binding to PMA-differentiated THP-1 cells. (J) Binding of wild-type or epitope-mutant hHE4-Fc to anti-hHE4 mAb clone #10 was quantified by ELISA. (K) Pharmacokinetic analysis of anti-hHE4 mAb clone #10 in C57BL/6 mice following intravenous administration. (L–O) Fresh human LUAD tumor cell suspensions were treated with anti-hHE4 mAb clone #10, followed by flow cytometric analysis of PD-L1 and ELISA measurement of IFN-γ and granzyme B. (P) Recombinant HE4 suppressed IFN-γ production in human LUAD tumor cell suspensions. (Q–T) HE4 blockade enhanced PBMC-mediated antitumor activity in humanized C-NKG mice bearing OVCAR3 or NCI-H358 tumors, shown by treatment scheme, representative tumors, and tumor weights. Schematics (L and Q) were created using BioRender. Statistical analyses were performed using one-way ANOVA (C, E, and G), paired t tests (M–P), or unpaired t tests (S and T). Data in (A–J) are representative of three independent experiments; data in (Q–T) are pooled from two independent experiments.

Article Snippet: Mouse Granzyme B ELISA kit , Elabscience , Cat# E-EL-M0594.

Techniques: Neutralization, Activity Assay, Expressing, Flow Cytometry, Western Blot, Quantitative RT-PCR, Control, Binding Assay, Bioprocessing, Mutagenesis, Enzyme-linked Immunosorbent Assay, Recombinant