mouse igg2α fitc  (Miltenyi Biotec)

Journal: bioRxiv

Article Title: PD-L2 regulates natural antibody and IL-10 secretion by B-1 cells

doi: 10.64898/2025.12.11.693708

Figure Lengend Snippet: (A) Schematic showing the structure of PD-L2 (left) containing the extracellular IgV and IgC domains, the TM (transmembrane) domain and a short cytoplasmic domain. Sequences of the last 7 amino acids of the TM domain and the whole cytoplasmic domain are shown for the mouse C57BL/6J (B6) and CAST/EiJ ( Mus musculus castaneus ) strains, and several other mammals, indicating amino acid numbering and amino acids conserved between multiple mammalian species in green. Mutation of the stop codon in B6 mice to tryptophan (W248) in the corrected mouse B6 strain (X248W) results in a cytoplasmic domain very similar to that in the CAST/EiJ mouse. (B) Graphs of percentage of B-1a, B-1b and B-2 cells among all peritoneal cavity CD19 + B cells (gated as in ) in PD-L2ko, WT and X248W mouse strains (n=10). (C-H) Flow cytometric analysis of peritoneal cavity B-1a cells from the indicated strains, gated as in , showing representative plots for expression of PC1 (C), CD73 (E) and IgG3 (G) and graphs showing percentage of B-1a cells positive for the same three proteins (D, F, H). Gates set using B-2 cells which are largely negative for both proteins . Sample numbers: PC1, PD-L2ko (n=6), WT (n=9) and X248W (n=9); CD73, PD-L2ko (n=5), WT (n=7) and X248W (n=9); IgG3, PD-L2ko (n=8), WT (n=5) and X248W (n=6). (I, J) Flow cytometric analysis of splenocytes from indicated strains showing IgG1 expression on B-1a cells (I). Graph shows percentage of IgG1 + B-1a cells (J). Sample numbers: PD-L2ko (n=7), WT (n6), X248W (n=7). Numbers indicate percentage of cells in gates. Each dot represents one mouse. Horizontal line indicates mean. Data pooled from 3 (B, D, F, H) or 2 (J) independent experiments. Mann-Whitney U test was used for statistical analysis; * 0.01 < p < 0.05 ** 0.001 < p < 0.01, *** 0.0001 < p < 0.001.

Article Snippet: Intracellular antibodies used, indicating antigen and fluorophore (clone): Blimp-1 APC (5E7) and IRF4 PE (IRF4.3E4) (both BioLegend), CstF-64 CL488 (rabbit polyclonal) (Proteintech); Isotype controls: Rat IgG2a APC isotype control (G013C12), rat IgG1 PE isotype control (G0114F7) (both BioLegend), rabbit IgG1 AF488 (EPR25A; BD Biosciences).

Techniques: Mutagenesis, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: PD-L2 regulates natural antibody and IL-10 secretion by B-1 cells

doi: 10.64898/2025.12.11.693708

Figure Lengend Snippet: (A) Comparison of sequences of the last 7 amino acids of the transmembrane (TM) domain and the whole cytoplasmic domain of PD-L2 for the indicated mouse strains, including C57BL/6J (B6), the corrected C57BL/6J (B6) strain with the X248W mutation and CAST/EiJ ( Mus musculus castaneus ), indicating amino acid numbering and amino acids identical between strains in green. (B) Diagram showing the amino acid sequence of the last 7 amino acids of the TM domain and the reconstituted cytoplasmic domain of PD-L2 X248W with residues conserved between different mammals in green (from ) and numbers showing amino acid positions. Upper panel: predicted phosphorylation sites Y252 and S257 are indicated in red with kinases that may phosphorylate these listed below. Lower panel: predicted binding partners to these two residues and P259 indicated below. Confidence scores of predicted kinases (from NetPhos 3.1) and binding partners (from Scansite 4.0): INSR (0.521), PKA (0.770); ITK (0.580), NEK10 (0.655), SHC1 (0.691), PKA (0.693), NEK2 (0.910), 14-3-3 (0.733), AURKA (0.703), SRC (0.619). (C) Histograms showing PD-L2 expression on B-1a (CD11b + CD5 + ) and B-1b (CD11b + CD5 - ) cells from peritoneal cavity of WT and PD-L2 X248W mice, pre-gated on live CD19 + cells. (D) Graphs of PD-L2 expression on B-1a and B-1b cells (n=10). (E, F) Flow cytometric analysis of PC1 (E) and CD73 (F) expression on peritoneal B-2 cells (CD19 + CD11b - ) showing example flow plots and graphs of frequencies of PC1 + and CD73 + B-2 cells from mice of the indicated genotypes. These gates were used to set gates for PC1 + and CD73 + B-1 cells in . Sample numbers: n=4 for PC1 in PD-L2ko and all CD73 analyses, 5 for PC1 in X248W and 6 for PC1 in WT. (G) Graphs showing frequency of IgG3-secreting B-1 cells (spot forming units, SFU) and amount of IgG3 production (spot size) determined using ELISpot assays on peritoneal B-1 cells from mice of the indicated genotypes incubated for 48 h (n=4 for PD-L2ko and WT, 5 for X248W). (H) Flow cytometric analysis of intracellular Blimp-1 detected with an antibody in B-2, B-1a and B-1b peritoneal cavity cells from a mouse expressing Blimp-1-GFP. Numbers indicate percentage of cells in gates. Each dot represents one mouse. Horizontal line indicates mean. Data pooled from 4 (D) or 2 (E-G) independent experiments. Mann-Whitney U test was used for statistical analysis; * 0.01 < p < 0.05 ** 0.001 < p < 0.01.

Article Snippet: Intracellular antibodies used, indicating antigen and fluorophore (clone): Blimp-1 APC (5E7) and IRF4 PE (IRF4.3E4) (both BioLegend), CstF-64 CL488 (rabbit polyclonal) (Proteintech); Isotype controls: Rat IgG2a APC isotype control (G013C12), rat IgG1 PE isotype control (G0114F7) (both BioLegend), rabbit IgG1 AF488 (EPR25A; BD Biosciences).

Techniques: Comparison, Mutagenesis, Sequencing, Phospho-proteomics, Binding Assay, Expressing, Enzyme-linked Immunospot, Incubation, MANN-WHITNEY

Journal: Frontiers in Veterinary Science

Article Title: The subunit vaccine of Lumpy skin disease virus elicits significant humoral and cell-mediated immune responses in mice

doi: 10.3389/fvets.2025.1731692

Figure Lengend Snippet: Protein expression, purification, and assessment of the immunization regimen in mice. (A) SDS-PAGE analysis of ORF073, ORF075, ORF090, ORF110: Lane M, protein labeling; Lane 1, uninduced pCold II BL21; Lane 2, uninduced; Lane 3, induced; Lane 4, supernatant of cells induced by ultrasound treatment; Lane 5, inducing cell precipitation after ultrasound treatment; Lane 6, purified protein. (B) Western blot analysis. (C) Timeline for immunization, blood, and tissue sampling schedules. (D) Changes of serum IgG1 and IgG2a antibody levels in mice. (E) Neutralizing antibody levels. Data were analyzed using two-way ANOVA to evaluate significant differences. *** P < 0.001; ns, no significant difference.

Article Snippet: The coating antigen (0.25 μg/ml, 1 μg/ml, 2 μg/ml, 4 μg/ml), test serum (1:50, 1:100, 1:500, 1:1,000), and HRP-conjugated goat anti-mouse IgG, IgG1, and IgG2a antibodies (Proteintech, CN; 1:10,000, 1:20,000, 1:50,000).

Techniques: Expressing, Purification, SDS Page, Labeling, Western Blot, Sampling

Journal: Journal of Translational Medicine

Article Title: A novel immunocytokine promotes T cell– and cytokine-induced killer cell-mediated antitumor immunity via a non-MHC-restricted mechanism in glioblastoma

doi: 10.1186/s12967-025-07321-5

Figure Lengend Snippet: Experimental design of immunocytokine αBC-IL15. GBM patients showed limited lymphocyte infiltration in tumor, particularly CD3 + T cells, and reduced peripheral immune cells compared to healthy donors. Based on these characterizations, we developed αBC-IL15, a novel immunocytokine with robust tumor-binding ability. (A) Representative immunostaining showing CD3 + T cell infiltration in both the tumor core and tumor periphery of GBM patients. Scale bar, 100 μm. (B) The CD3 + cells per field in different areas. Data are presented as mean values ± SD and were analyzed by Student’s t-test; **** P < 0.0001. n = 10 independent experiments. (C) Proportions of lymphocytes, T cells, and NK cells in tumor tissue derived from GBM patients. Cells were analyzed by flow cytometry. Data are presented as mean. n = 10 independent experiments. (D) Proportions of T cells and NK cells in PBMCs derived from GBM patients and healthy donors. PBMCs were isolated from peripheral blood and analyzed by flow cytometry. T cell, CD3 + CD56-; NK cell, CD3-CD56+. Data are presented as mean ± SD. Two-way ANOVA with Tukey multiple comparison was performed, and mean values are indicated as lines. n = 10 independent experiments; * P < 0.05. (E) Schematic of the αBC-IL15 fusion protein. (F) αBC-hIL15 was analyzed by SDS-PAGE. M: Marker, NR: Non-reduced; R: Reduced. (G) The binding affinities of αBC-hIL15 or αBC to B7-H3-His fusion proteins were assayed by ELISA. Isotype mIgG2a was used as a negative control. Data represent three independent experiments. (H) Detection of purified αBC-hIL15 bound to U87-MG, U251 and A172 human GBM cell lines, as measured by flow cytometry after staining αBC-hIL15-incubated tumor cells with anti-Fc-FITC. mIgG2a isotype served as control. n = 3 independent experiments. (I) After co-culturing U87-MG or U251 cells with αBC-hIL15 or αBC, immunofluorescence staining was performed on cells incubated with CoraLite594 – conjugated Goat Anti-Mouse IgG(H + L) (Red). Cell nuclei were stained with DAPI(Blue) for visualization. mIgG2a isotype served as the negative control. Scale bar, 20 μm

Article Snippet: 96-well ELISA plates were coated with 2 μg/mL of B7-H3-6×His fusion proteins overnight, then 5% blocking solution of nonfat powdered milk (Thermo Fisher Scientific, USA) was added into each well and incubated at 37 °C for 1 h. After blocking, αBC-h(m)IL15 was added to the ELISA plate and incubated for 2 h. The unconjugated antibodies were washed off, and the bound antibodies were detected using horseradish peroxidase (HRP)-coupled goat anti-mouse IgG2a antibody (Proteintech, USA).

Techniques: Binding Assay, Immunostaining, Derivative Assay, Flow Cytometry, Isolation, Comparison, SDS Page, Marker, Enzyme-linked Immunosorbent Assay, Negative Control, Purification, Staining, Incubation, Control, Immunofluorescence

Journal: Journal of Translational Medicine

Article Title: A novel immunocytokine promotes T cell– and cytokine-induced killer cell-mediated antitumor immunity via a non-MHC-restricted mechanism in glioblastoma

doi: 10.1186/s12967-025-07321-5

Figure Lengend Snippet: αBC-hIL15 induces immune activation. PBMCs were co-cultured with 100 ng/ml of αBC, αBC-hIL15, or mIgG2a isotype, with PBS as the control. αBC-hIL15 promotes PBMC proliferation and activation, specifically expands T cells, drives differentiation toward CD8 + and TEM phenotypes, reduces Tregs, and does not increase T cell exhaustion. A . Representative images of PBMC treated with mIgG2a, αBC(h), or αBC-hIL15. Media was used as a negative control. Scale bar, 100 μm. B . Fold expansion of T cells. Data are mean ± SD. two-way ANOVA with Tukey’s multiple comparisons test; **** p < 0.0001. C . Cell viability was measured by trypan blue exclusion test. Data are mean ± SD. Two-way ANOVA with Tukey multiple comparison was performed, n = 3 independent experiments; **** p < 0.0001. D-L. Flow cytometry was used to determine the function of αBC(h), αBC-hIL15, or mIgG2a isotype on PBMCs, with PBS as the control. The data are presented as mean values ± SD; n = 3 independent experiments. D . The proportions of CD3 + T cells were evaluated on days 0, 1, 7, and 14. Additionally, PBMCs treated with PBS or mIgG2a were completely depleted by day 14. One-way ANOVA with Tukey’s correction; * P < 0.05, ** P < 0.01. The proportions of T cells, NK cells, and CIK cells in PBMCs were evaluated on days 0, 1, 7, and 14. Two-way ANOVA with Tukey’s multiple comparisons test, Student’s t-test; * p < 0.05, ** p < 0.01, *** P < 0.001, **** p < 0.0001. E . The proportions of CD3 + CD69 + T cells were evaluated at 1,12, 24 and 48 h. Two-way ANOVA with Tukey’s multiple comparisons test; **** p < 0.0001. F . The proportions of CD3 + CD25 + T cells were evaluated on days 0, 1, 7, and 14. G . The ratio of CD8+/CD4 + T cells in PBMCs was evaluated on day 7. One-way ANOVA with Tukey’s correction; ** p < 0.01, *** P < 0.001. H . The proportion of T EM of T cells was evaluated on day 7. One-way ANOVA with Tukey’s correction; * p < 0.05, ** p < 0.01. I . The proportion of T SCM of T cells was evaluated on day 7. One-way ANOVA with Tukey’s correction; **** p < 0.0001. J . The proportion of Treg of CD4 + T cells was evaluated on day 7. One-way ANOVA with Tukey’s correction; **** p < 0.0001. K . Pie charts were used to represent the proportions of four subpopulations within CD3 + cells on days 1, 7, and 14: PD-1 + TIM-3+ (blue), PD-1–TIM-3+ (orange), PD-1 + TIM-3– (gray), and PD-1–TIM-3– (yellow). Data are presented as mean. L . The expression of PD-1 and TIM-3 was evaluated on day 7. One-way ANOVA with Tukey’s correction; ns, non-significant. * p < 0.05

Article Snippet: 96-well ELISA plates were coated with 2 μg/mL of B7-H3-6×His fusion proteins overnight, then 5% blocking solution of nonfat powdered milk (Thermo Fisher Scientific, USA) was added into each well and incubated at 37 °C for 1 h. After blocking, αBC-h(m)IL15 was added to the ELISA plate and incubated for 2 h. The unconjugated antibodies were washed off, and the bound antibodies were detected using horseradish peroxidase (HRP)-coupled goat anti-mouse IgG2a antibody (Proteintech, USA).

Techniques: Activation Assay, Cell Culture, Control, Negative Control, Comparison, Flow Cytometry, Expressing

Journal: Journal of Translational Medicine

Article Title: A novel immunocytokine promotes T cell– and cytokine-induced killer cell-mediated antitumor immunity via a non-MHC-restricted mechanism in glioblastoma

doi: 10.1186/s12967-025-07321-5

Figure Lengend Snippet: αBC-hIL15 mediates effective cytotoxicity in vitro. A . Different E: T ratios were similarly analyzed at a fixed αBC-hIL15 concentration where each bullet point represents the final U87-MG or U251 viable cell count at the end of the 72 h cytotoxicity study. Two-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05, ** p < 0.01, **** p < 0.0001. B . U87-MG and U251 were incubated with two-fold serial dilutions of αBC(h), αBC-hIL15, and coculture with PBMCs at effector/targe = 2:1. After 2 days, the cells were incubated with CCK8. * p < 0.05, ** p < 0.01, **** p < 0.0001, two-way ANOVA with Šídák’s multiple comparisons test. C . Detection of CD3 + Granzyme B + cells and CD3 + TNF-α + cells by flow cytometry. The data are presented as mean values ± SD and were analyzed by one-way ANOVA with Tukey’s correction. ** P < 0.01, *** P < 0.001, **** P < 0.0001. D . U87 spheroids treated with αBC(h), αBC-hIL15, or mIgG2a were cocultured with human T cells, respectively. Tumor spheroids were stained with CytoTell™ Orange (Red), cell nuclei were stained with Hoechst (Blue) for visualization, and T cells were labeled with CFSE(Green). Pictures were captured by a confocal microscope (Zeiss 880), and the scale bars are 100 μm

Article Snippet: 96-well ELISA plates were coated with 2 μg/mL of B7-H3-6×His fusion proteins overnight, then 5% blocking solution of nonfat powdered milk (Thermo Fisher Scientific, USA) was added into each well and incubated at 37 °C for 1 h. After blocking, αBC-h(m)IL15 was added to the ELISA plate and incubated for 2 h. The unconjugated antibodies were washed off, and the bound antibodies were detected using horseradish peroxidase (HRP)-coupled goat anti-mouse IgG2a antibody (Proteintech, USA).

Techniques: In Vitro, Concentration Assay, Cell Counting, Incubation, Flow Cytometry, Staining, Labeling, Microscopy

Journal: Journal of Translational Medicine

Article Title: A novel immunocytokine promotes T cell– and cytokine-induced killer cell-mediated antitumor immunity via a non-MHC-restricted mechanism in glioblastoma

doi: 10.1186/s12967-025-07321-5

Figure Lengend Snippet: αBC-hIL15 demonstrates effective T cell infiltration and cytotoxicity in primary GBM cells and tumor spheroids. Primary GBM cells isolated from clinical patient tissues were cultured into tumor spheroids. αBC-hIL15 exerts effective cytotoxicity in both primary cells and tumor spheroids. A . Detection of purified αBC-hIL15 bound to GBM cells, as measured by flow cytometry after staining αBC-hIL15-incubated tumor cells with anti-Fc-FITC, with mIgG2a isotype served as control. n = 3 independent experiments. B . After co-culturing GBM cells with αBC-hIL15 or αBC, immunofluorescence staining was performed on cells incubated with CoraLite594 – conjugated Goat Anti-Mouse IgG(H + L) (Red). Cell nuclei were stained with DAPI(Blue) visualization. mIgG2a isotype served as the negative control. Scale bar, 20 μm. n = 3 independent experiments. C . After co-culturing GBM cells with αBC-hIL15 or αBC, immunofluorescence staining was performed on GBM spheroids incubated with CoraLite594–conjugated Goat Anti-Mouse IgG(H + L) (Red). Cell nuclei were stained with Hoechst (Blue) for visualization. IgG2a isotype served as the negative control. scale bar, 100 nm. n = 3 independent experiments. D . GBM spheroids treated with αBC(h), αBC-hIL15, or PBS control were cocultured with human T cells, respectively. Tumor spheroids were stained with CytoTell™ Orange (Red), and T cells were labeled with CFSE(Green). Pictures were captured by a confocal microscope (Zeiss 880), and the scale bars are 100 μm. n = 3 independent experiments. E . The ratio of T cells to tumor cells was calculated by analyzing the integrated fluorescence signal using ImageJ. Data are presented as mean ± SD and analyzed by one-way ANOVA with Tukey’s correction for multiple comparisons. * p < 0.05, **** p < 0.0001. n = 3 independent experiments. F . GBM spheroids treated with αBC(h), αBC-hIL15, or saline control were cocultured with T cells. After 24 h, GBM spheroids were stained with Calcein/PI. Pictures were captured by a confocal microscope (Zeiss 880), and the scale bars are 100 μm. n = 3 independent experiments. G . The ratio of the integrated fluorescence signal of PI-stained cells to Calcein-stained cells was measured using ImageJ. The data are presented as mean values ± SD and were analyzed by one-way ANOVA with Tukey’s correction for multiple comparisons. ** p < 0.01, **** p < 0.0001 n = 3 independent experiments

Article Snippet: 96-well ELISA plates were coated with 2 μg/mL of B7-H3-6×His fusion proteins overnight, then 5% blocking solution of nonfat powdered milk (Thermo Fisher Scientific, USA) was added into each well and incubated at 37 °C for 1 h. After blocking, αBC-h(m)IL15 was added to the ELISA plate and incubated for 2 h. The unconjugated antibodies were washed off, and the bound antibodies were detected using horseradish peroxidase (HRP)-coupled goat anti-mouse IgG2a antibody (Proteintech, USA).

Techniques: Isolation, Cell Culture, Purification, Flow Cytometry, Staining, Incubation, Control, Immunofluorescence, Negative Control, Labeling, Microscopy, Fluorescence, Saline

Journal: Journal of Translational Medicine

Article Title: A novel immunocytokine promotes T cell– and cytokine-induced killer cell-mediated antitumor immunity via a non-MHC-restricted mechanism in glioblastoma

doi: 10.1186/s12967-025-07321-5

Figure Lengend Snippet: Optimizing the dose of αBC-mIL15 improved anti-tumor efficacy. A . Detection of purified αBC-mIL15 bound to GL261-hB7 cell lines, as measured by flow cytometry after staining αBC-mIL15-incubated tumor cells with anti-Fc-FITC. mIgG2a isotype served as a control. Experiments are representative of n = 3 independent experiments with similar results. B . Representative images showing T cells (5 × 10 3 ) co-cultured with GL261-hB7 cells (1 × 10 4 ) 24 h, with or without αBC-mIL15. C . GL261-hB7 were incubated with two-fold serial dilutions of αBC(m), αBC-mIL15, and coculture with murine T cells at effector/target = 2:1. After 2 days, the cells were incubated with CCK8. two-way ANOVA with Šídák’s multiple comparisons test. D . 1 × 10 5 GL261-hB7 cells were stereotactically injected into the brain’s right frontal lobe (2 mm lateral and 1 mm anterior to bregma at a depth of 3 mm). For survival studies, 7 days after tumor implantation, mice were randomly divided into four groups ( n = 5 per group) that were intracerebroventricular (i.c.v.) injected with anti-B7-H3/mCD3-mIL15 (αBC-m15, 0.1 mg/kg,0.5 mg/kg,1.0 mg/kg) in 5µl PBS or with PBS alone as control. E . Survival times of tumor-bearing mice were analyzed using the Kaplan-Meier method with the log rank test ( n = 5). F . Body weight was measured every two days from treatment for each experimental mouse. G . H&E staining of sections of brains harvested from mice on 3 days after treatment. Representative images are shown. The delineated areas, marked with dashed lines, represent the presence of tumors. Paraffin tumor sections were stained with CD3. Enumeration of positive cells per square millimeter of tissue was calculated to provide a density of positive cells across the entire tissue section. H . The CD3 + cells per field in groups with different treatments. Data are presented as mean values ± SD and were analyzed by Student’s t-test. * P < 0.05, *** P < 0.001, **** P < 0.0001. I . The CD3-NK1.1 + cells per field in groups with different treatments. Data are presented as mean values ± SD and were analyzed by Student’s t-test. ** P < 0.01. J . The levels of TNF-α and Granzyme B of treated tumors were detected by ELISA. Results were expressed as mean ± SD, statistical analyses were performed by two-way ANOVA test followed by Tukey’s post-test analysis performed for all comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001

Article Snippet: 96-well ELISA plates were coated with 2 μg/mL of B7-H3-6×His fusion proteins overnight, then 5% blocking solution of nonfat powdered milk (Thermo Fisher Scientific, USA) was added into each well and incubated at 37 °C for 1 h. After blocking, αBC-h(m)IL15 was added to the ELISA plate and incubated for 2 h. The unconjugated antibodies were washed off, and the bound antibodies were detected using horseradish peroxidase (HRP)-coupled goat anti-mouse IgG2a antibody (Proteintech, USA).

Techniques: Purification, Flow Cytometry, Staining, Incubation, Control, Cell Culture, Injection, Tumor Implantation, Enzyme-linked Immunosorbent Assay

Journal: Journal of Translational Medicine

Article Title: A novel immunocytokine promotes T cell– and cytokine-induced killer cell-mediated antitumor immunity via a non-MHC-restricted mechanism in glioblastoma

doi: 10.1186/s12967-025-07321-5

Figure Lengend Snippet: αBC-mIL15 improved anti-tumor efficacy in GL261-hB7 tumors. A . 1 × 10 5 GL261-hB7 cells were stereotactically injected into the brain’s right frontal lobe (2 mm lateral and 1 mm anterior to bregma at a depth of 3 mm). For survival studies, 7 days after tumor implantation, mice were randomly divided into five groups ( n = 5 per group) that were intracerebroventricular (i.c.v.) injected with mIgG2a (0.5 mg/kg), anti-CD19/mCD3-mIL15 (α19C-mIL15, 0.5 mg/kg), anti-B7-H3/mCD3-mIL15 (αBC-mIL15, 0.5 mg/kg), anti-B7-H3/mCD3 (αBC(m), 0.5 mg/kg) in 5µl PBS or with PBS alone as control. B . Survival times of tumor-bearing mice were analyzed using the Kaplan-Meier method with the log rank test ( n = 5). C . Body weight was measured every two days from treatment for each experimental mouse. D . The CD3 + cells per field in groups with different treatments. Data are presented as mean values ± SD and were analyzed by Student’s t-test. ** P < 0.01; **** P < 0.0001. The CD3-NK1.1 + cells per field in groups with different treatments. Data are presented as mean values ± SD and were analyzed by Student’s t-test. *** P < 0.001; **** P < 0.0001 E . H&E staining of sections of brains harvested from mice on 3 days after treatment. Representative images are shown. The delineated areas, marked with dashed lines, represent the presence of tumors. Paraffin tumor sections were stained with CD3. Enumeration of positive cells per square millimeter of tissue was calculated to provide a density of positive cells across the entire tissue section. F . H&E staining was performed on heart, liver, spleen, lung, and kidney specimens following the administration of αBC-mIL15(i.c.v.) treatment. The scale bar is 100 μm. G . The levels of TNF-α of the treated tumors were detected by ELISA. Results were expressed as mean ± SD, statistical analyses were performed by two-way ANOVA test followed by Tukey’s post-test analysis performed for all comparisons. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. H . The levels of Granzyme B of treated tumors were detected by ELISA. Results were expressed as mean ± SD, statistical analyses were performed by two-way ANOVA test followed by Tukey’s post-test analysis performed for all comparisons. * P < 0.05, *** P < 0.001, **** P < 0.0001

Article Snippet: 96-well ELISA plates were coated with 2 μg/mL of B7-H3-6×His fusion proteins overnight, then 5% blocking solution of nonfat powdered milk (Thermo Fisher Scientific, USA) was added into each well and incubated at 37 °C for 1 h. After blocking, αBC-h(m)IL15 was added to the ELISA plate and incubated for 2 h. The unconjugated antibodies were washed off, and the bound antibodies were detected using horseradish peroxidase (HRP)-coupled goat anti-mouse IgG2a antibody (Proteintech, USA).

Techniques: Injection, Tumor Implantation, Control, Staining, Enzyme-linked Immunosorbent Assay