Journal: bioRxiv

Article Title: HAPI Cells are SIM-A9-related Mouse Microglial Cells Useful for In Vitro Modeling of Microglial Immunometabolism

doi: 10.64898/2026.02.11.705385

Figure Lengend Snippet: HAPI cells undergo morphology changes in response to various pro-inflammatory stimuli. Representative phase-contrast images of unstimulated HAPI cells (control) and HAPI cells following 18 hours of stimulation with the toll-like receptor 4 agonist lipopolysaccharide (LPS, 100 ng/mL), the type I interferon, interferon-alpha (IFN-α, 10 ng/mL), the type II interferon, interferon-gamma (IFN-γ, 10 ng/mL), or combined LPS/IFN-α or LPS/IFN-γ at the same concentrations. The white arrowheads denote examples of protrusions with phagocytic cups.

Article Snippet: Mouse-origin recombinant IFN-γ protein was from Sino Biological (Paoli, PA; Cat# 50709-MNAH).

Techniques: Control

Journal: bioRxiv

Article Title: HAPI Cells are SIM-A9-related Mouse Microglial Cells Useful for In Vitro Modeling of Microglial Immunometabolism

doi: 10.64898/2026.02.11.705385

Figure Lengend Snippet: HAPI cellular bioenergetic function is altered by various pro-inflammatory stimuli. (A) Cell number of the non-activated control (CTRL) group plotted across the five experiments, showing the variability binned into low- and high-cell-density categories. (B) Average HAPI cell oxygen consumption rate (OCR) measurements normalized to cell number following 18 hours of stimulation with lipopolysaccharide (LPS, 100 ng/mL), interferon-gamma (IFN-γ, 10 ng/mL), interferon-alpha (IFN-α, 10 ng/mL), or combined LPS/IFN-γ or LPS/IFN-α at the same concentrations. The results are mean ± SEM of n=5 biological replicates, with each experiment consisting of 2-3 technical replicates. The serial additions of the uncoupler FCCP (4 µM) + pyruvate (pyr, 10 mM), the nitric oxide scavenger cPTIO (200 μM), and the Complex III inhibitor antimycin A (Anti A, 1 μM) are indicated by arrows. (C) Basal mitochondrial OCR normalized to cell number calculated from the data shown in B. (D) Basal extracellular acidification rate (ECAR) normalized to cell number. ECARs were acquired simultaneously to OCR for the experiment shown in B. (E) Maximal mitochondrial OCR normalized to cell number calculated from the data shown in B. The data in panels C-E were analyzed by two-way analysis of variance (ANOVA) with treatment and experiment number as factors, followed by Tukey’s post hoc analysis. Experiment number had a significant effect on basal OCR (p<0.05) and basal ECAR (p<0.0001), while only a strong trend was observed on maximal OCR (p=0.06). (F) OCRs before and after cPTIO addition for the indicated treatment groups for the experiment shown in B. The data in panel F were analyzed by two-way ANOVA with repeated measures and Šídák’s multiple comparison’s test. (G) Linear regression analysis of cPTIO-induced OCR versus cell number for the LPS/IFN-γ and LPS/IFN-α treatment groups, with R 2 and p-values indicated on the graph. The p-values indicate the probability of a non-zero slope of the linear fits. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Article Snippet: Mouse-origin recombinant IFN-γ protein was from Sino Biological (Paoli, PA; Cat# 50709-MNAH).

Techniques: Control

Journal: bioRxiv

Article Title: HAPI Cells are SIM-A9-related Mouse Microglial Cells Useful for In Vitro Modeling of Microglial Immunometabolism

doi: 10.64898/2026.02.11.705385

Figure Lengend Snippet: Eighteen-hour activation of HAPI cells with lipopolysaccharide plus interferon-gamma (LPS/IFN-γ) or LPS plus interferon-alpha (LPS/IFN-α) reduces cell number compared to the non-activated control (CTRL) group. HAPI cells were treated with the following stimuli prior to the determination of cell number by manual cell counting of three representative fields per treatment: LPS (100 ng/mL), IFN-α (10 ng/mL), IFN-γ (10 ng/mL), LPS/IFN-γ (100 ng/mL / 10 ng/mL, respectively), or LPS/IFN-α (100 ng/mL / 10 ng/mL, respectively). Cell number is shown in (A) while the results in (B) express the cell number as a percentage of the CTRL group mean. The results are mean ± SEM of n=5 biological replicates. The data were analyzed by two-way analysis of variance with treatment and experiment number as factors, followed by Tukey’s post hoc analysis. Both the treatment and the experiment number had a significant effect (p<0.0001 each), and there was a significant interaction between the two (p<0.05). ***p<0.001, ****p<0.0001.

Article Snippet: Mouse-origin recombinant IFN-γ protein was from Sino Biological (Paoli, PA; Cat# 50709-MNAH).

Techniques: Activation Assay, Control, Cell Counting

Journal: bioRxiv

Article Title: HAPI Cells are SIM-A9-related Mouse Microglial Cells Useful for In Vitro Modeling of Microglial Immunometabolism

doi: 10.64898/2026.02.11.705385

Figure Lengend Snippet: Respiratory suppression induced by combined lipopolysaccharide/interferon-gamma (LPS/IFN-γ) stimulation is partly attenuated by an inhibitor of inducible nitric oxide synthase (iNOS). (A) HAPI cell oxygen consumption rate (OCR) measurements from an unstimulated control (CTRL) group or following 18 hours of stimulation with LPS/IFN-γ (100 ng/mL / 10 ng/mL, respectively) in the absence or presence of the iNOS inhibitor 1400W (50 µM). The serial additions of FCCP (4 µM) + pyruvate (pyr, 10 mM), cPTIO (200 μM), and antimycin A (Anti A, 1 μM) are indicated by arrows. (B) The data in A expressed as a percentage of the baseline OCR (third measurement) because cell counts were not done for this experiment. Baseline normalization removes the influence of cell number on OCR, illustrating that the recovery of respiratory capacity is incomplete. The results are mean ± SD of n=3 technical replicates and are representative of n=7 similar experiments done in triplicate. Because this experiment was done as part of another study with additional comparisons, only the representative trace is shown, and the results will be reported in more detail elsewhere.

Article Snippet: Mouse-origin recombinant IFN-γ protein was from Sino Biological (Paoli, PA; Cat# 50709-MNAH).

Techniques: Control

Journal: Advanced Science

Article Title: PD‐L1‐Binding Antigen Presenters: Redirecting Vaccine‐Induced Antibodies for Cancer Immunotherapy

doi: 10.1002/advs.202519574

Figure Lengend Snippet: Serum from Herpes Zoster Vaccine (LZ901)‐immunized Mice Enhances PBAP‐Mediated ADCC Against PD‐L1 + Tumor Cells In Vitro. (A) Schematic diagram of the sPD‐1‐gE and PBAP‐gE fusion protein. The sPD‐1‐gE construct consists of sPD‐1 fused to gE. The PBAP‐gE construct comprises sPD‐1‐gE fused with an Fc domain (sPD‐1‐gE‐Fc). (B) Structural modeling of PBAP‐gE with AlphaFold 3. (C) Pharmacokinetic profiles of sPD‐1‐gE and PBAP‐gE following intravenous injection into C57BL/6J mice (n=3 mice/group, 100 µg/mice). Data are presented as the mean ± SD (n = 3). (D) The binding inhibition of PBAP‐gE on PD‐L1/PD‐1 interaction was assessed by ELISA. The absorbance was measured at 450 nm to determine the blocking effect. (E) The fluorescence intensity of the antibody‐cell binding was analyzed using a flow cytometry to assess the blocking effect on the PD‐1/PD‐L1 pathway. Data are presented as the mean ± SD (n = 3). (F) In vitro cytotoxicity assay. KIL C.2 cells were co‐incubated with PBAP‐gE and serum from LZ901‐immunized mice, against 4T1‐IFNγ (IFN‐γ‐induced, PD‐L1 + ) tumor cells and 4T1‐WT cells. KIL C.2 cells, KIL C.2 cells co‐incubated with gE and serum from LZ901‐immunized mice, KIL C.2 cells co‐incubated with PBAP‐gE and serum from saline vaccine‐immunized mice and all groups treated with anti‐FcγRIII blocking antibody were used as controls. Data are presented as mean ± SD of 3 independent experiments, each performed in triplicate. (G) Flow cytometry analysis of perforin, granzyme B, IFN‐γ and CD107a in KIL C.2 cells. Representative of 3 independent experiments.

Article Snippet: The medium was then replaced with fresh complete medium containing recombinant mouse IFN‐ γ (10 μg/mL, Sino Biological, 50709‐MNAH) for 4T1 cells or recombinant human IFN‐ γ (10 μg/mL, Sino Biological, 11725‐HNAS) for MDA‐MB‐231 cells.

Techniques: In Vitro, Construct, Injection, Binding Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Blocking Assay, Fluorescence, Flow Cytometry, Cytotoxicity Assay, Incubation, Saline

Journal: Advanced Science

Article Title: PD‐L1‐Binding Antigen Presenters: Redirecting Vaccine‐Induced Antibodies for Cancer Immunotherapy

doi: 10.1002/advs.202519574

Figure Lengend Snippet: PBAP‐gE Elicits a Robust Antitumor Immune Response, Predominantly Mediated by NK Cells through gE‐Specific Antibody Production by B Cells. (A) Experimental design to assess the contributions of antibody versus CD8 + T cells in PBAP‐gE‐mediated tumor suppression. On day 0, C57BL/6J mice (n=5 mice/group) were initially vaccinated with the LZ901 vaccine (5 µg/dose), followed by a booster immunization on day 21. On day 28, a subcutaneous inoculation of 5×10 5 B16‐Trop2 tumor cells was performed to establish the tumor model. On day 33, immune cell depletion was conducted via intraperitoneal injection of antibodies targeting NK cells (anti‐NK1.1), B cells (anti‐CD19), and CD8 + T cells (anti‐CD8), respectively. On day 34, each mouse received an intratumoral injection of 150 µg PBAP‐gE. Subsequently, tumor growth was monitored via bioluminescence imaging on days 36, 38, 40, and 42. (B) Tumor volumes were measured every 2 days from day 32 to day 42 in all groups, and all mice were euthanized on day 42. Tumor growth curves were plotted for the five experimental groups: untreated, NK cell block (αNK cell), B cell block (αB cell), CD8 + T cell block (αCD8 + T cell), IgG2a isotype block (control). Corresponding changes of tumor volum levels are shown on the right‐down panel. (C) Representative images of excised tumors are displayed in the left panel, and tumor weights (measured at the experimental endpoint on day 42) are presented in the right panel. Mice in the B cell depletion (αB cell) or NK cell depletion (αNK cell) groups exhibited significantly greater tumor weights compared to those in the CD8 + T cell depletion (αCD8 + T cell) group. In contrast, tumor weights in the CD8 + T cell depletion group were comparable to those in the IgG2a isotype control group. Data are presented as the mean ± SD (n = 6). Statistical significance was determined using one‐way ANOVA. ns indicates not significant (p > 0.05). (D) The gE‐specific IgG antibody levels (OD450) were measured every 2‐4 days from day 32 to day 42 across five experimental groups: Untreated, NK cell depletion (αNK cell), B cell depletion (αB cell), CD8 + T cell depletion (αCD8 + T cell), and IgG2a isotype control. Data are presented as the mean ± SD (n = 6). (E) Kinetics of initial body weight changes and serum levels of interferon‐gamma (IFN‐γ), tumor necrosis factor‐α (TNF‐α), and interleukin‐6 (IL‐6) across five experimental cohorts: Untreated, NK cell depletion (αNK cell), B cell depletion (αB cell), CD8 + T cell depletion (αCD8 + T cell), and IgG2a isotype control. Data are presented as the mean ± SD (n = 6).

Article Snippet: The medium was then replaced with fresh complete medium containing recombinant mouse IFN‐ γ (10 μg/mL, Sino Biological, 50709‐MNAH) for 4T1 cells or recombinant human IFN‐ γ (10 μg/mL, Sino Biological, 11725‐HNAS) for MDA‐MB‐231 cells.

Techniques: Injection, Imaging, Blocking Assay, Control

Journal: Advanced Science

Article Title: PD‐L1‐Binding Antigen Presenters: Redirecting Vaccine‐Induced Antibodies for Cancer Immunotherapy

doi: 10.1002/advs.202519574

Figure Lengend Snippet: PBAP Conjugated with Tumor‐Specific Antigens Enhances Synergistic Anti‐Tumor Activity When Combined with Clinical Antibodies and Antibody‐Drug Conjugates (ADCs) In Vitro. A) Schematic representation of the design of sPD‐1‐HER2 and PBAP‐HER2 (sPD‐1‐HER2‐Fc). PBAP‐HER2 was engineered via the fusion of extracellular domain of human PD‐1 (sPD‐1) with Domain IV of HER2 protein, followed by the incorporation of an Fc region to enhance protein stability and prolong in vivo half‐life. vB) Structural modeling of PBAP‐HER2 with AlphaFold 3. (C) Pharmacokinetic profiles of sPD‐1‐HER2 and PBAP‐HER2 following intravenous injection into C57BL/6J mice (n=3 mice/group, 100 µg/mice). Data are presented as the mean ± SD (n = 3). (D) The binding inhibition of PBAP‐HER2 on PD‐L1/PD‐1 interaction was assessed by ELISA. The absorbance was measured at 450 nm to determine the blocking effect. Data are presented as the mean ± SD (n = 3). (E) The fluorescence intensity of the antibody‐cell binding was analyzed using a flow cytometry to assess the blocking effect on the PD‐1/PD‐L1 pathway. (F) Diagram illustrating the mechanism by which PBAP‐HER2 synergizes with Herceptin and Kadcyla to kill PD‐L1‐positive target cells. Created with BioRender.com. (G) ADCC and ADCP activities were assessed using Jurkat‐FcγR reporter systems: ADCC (FcγRIIIa‐V158 variant) and ADCP (FcγRIIa‐R131 variant) in response to PBAP‐HER2/PBAP‐gE combined with Herceptin. PBAP‐HER2 in combination with Herceptin significantly enhanced ADCC and ADCP activities against HER2‐negative MDA‐MB‐231 cells. Representative of 3 independent experiments. Data are presented as mean ± SD (n = 3). (H) NK cells were co‐incubated with PBAP‐Her2 and Herceptin, against MDA‐MB‐231‐IFN‐γ (IFN‐γ induced, PD‐L1 + ) tumor cells and MDA‐MB‐231‐WT cells. NK cells, NK cells co‐incubated with PBAP‐Her2, NK cells co‐incubated with Herceptin, and all groups treated with anti‐FcγRIII blocking antibody were used as controls. Data are presented as mean ± SD of 3 independent experiments, each performed in triplicate. (I) Flow cytometry analysis of perforin, granzyme B, IFN‐γ and CD107a in NK cells. Representative of 3 independent experiments. (J) The CCK8 assay was used to evaluate the cytotoxicity of commercial ADCs (Kadcyla and Adcetris) combined with PBAP‐HER2. MDA‐MB‐231‐PD‐L1‐OE cells were treated with PBAP‐HER2 (10 µg/well) for 4 h, followed by ADC drugs (Kadcyla or Adcetris) at various concentrations (0.1, 1, 10, 100, 1000 ng/mL). After 24 h of incubation, cell viability was measured using the CCK8 assay. PBAP‐HER2 with Adcetris and HER2 protein with Kadcyla were used as controls. Representative of 3 independent experiments. Data are presented as mean ± SD (n = 3).

Article Snippet: The medium was then replaced with fresh complete medium containing recombinant mouse IFN‐ γ (10 μg/mL, Sino Biological, 50709‐MNAH) for 4T1 cells or recombinant human IFN‐ γ (10 μg/mL, Sino Biological, 11725‐HNAS) for MDA‐MB‐231 cells.

Techniques: Activity Assay, In Vitro, In Vivo, Injection, Binding Assay, Inhibition, Enzyme-linked Immunosorbent Assay, Blocking Assay, Fluorescence, Flow Cytometry, Variant Assay, Incubation, CCK-8 Assay